By analyzing data from NHANES, we aimed to evaluate the prevalence, characteristics, and associated factors of wrist fractures in Americans aged 50 and above.Wrist fractures, whose prevalence increases with age, are one of the most common fractures in the United States. However, epidemiological studies on the prevalence of wrist fractures of certain ages were limited.The data of Americans aged 50 or above from 2013–2014 and 2017–2018 in NHANES were extracted and analyzed.p = 0.267). Among those who had experienced their first wrist fracture, 17.8% of the population experienced a second wrist fracture. The top two causes of the first wrist fracture were a fall from a standing height (56%) or a hard fall (34.8%). The prevalence of wrist fractures was higher in men than in women  aged < 60, but higher in women than in men aged ≥ 60 . Multivariate analysis showed that obesity, frequent drinking, current smoking, high serum phosphate level, non-Hispanic white women, and osteoporosis were independently associated with wrist fractures. Stratified by race, osteoporosis, frequent drinking, and high serum phosphate level were risk factors for wrist fractures in all races. As for Mexican Americans, non-Hispanic whites, and other races including multi-racial, current smoking was a risk factor of wrist factures. Furthermore, obesity was positively associated with wrist fractures in Mexican Americans, other Hispanics, and non-Hispanic whites.The prevalence of wrist fractures among Americans whose age was 50 or above was 12%, which was similar between men and women (men 12.8% vs. women 11.4%, The prevalence of wrist fractures in Americans aged 50 and above was 12%. Falling from a standing height was the main cause of the first wrist fracture. Frequent drinking, current smoker, high serum phosphate level, osteoporosis, obesity, and non-Hispanic women were more likely to experience wrist fractures. Wrist fractures are one of the most common fractures in the United States, accounting for about one-sixth of all fractures treated in emergency departments. The prevalence of wrist fractures increases with age –4. Studin = 239; female subjects, n = 315).The National Health and Nutrition Examination Survey (NHANES) is a cross-sectional study of the American population collected every 2 years by the Center for Disease Control and Prevention (CDC), whose data are publicly available for use worldwide. NHANES was approved by the CDC for written informed consent from each participant. The data of 5,846 Americans aged 50 or above from 2013–2014 and 2017–2018 NHANES were extracted to estimate the prevalence of wrist fractures among the U.S. civilian population aged ≥ 50. All study subjects were examined physically and evaluated at medical centers. In this study, 4 subjects who had poor memory of their own past wrist fractures were excluded, while a total of 5,842 remaining subjects were included into the cohort, which contained 554 wrist fracture cases  a fall from a standing height or less, for example, tripped, slipped, fell out of bed; (2) a hard fall, such as falling off a ladder or step stool, or down the stairs; and (3) a car accident or other severe trauma], race , education , marital status, smoking history, drinking history, obesity, hypertension, diabetes, osteoporosis, medication history of prednisone or cortisone, wrist fracture history of parents, and serum calcium and phosphorus level. Participants were defined as high blood pressure patients if they complied with at least one of the following conditions: (1) currently taking antihypertensive medications; (2) previously diagnosed by a physician; (3) four blood pressure measurements indicated a mean systolic blood pressure ≥ 130 mmHg; (4) diastolic blood pressure ≥ 80 mmHg (2 (2) = 175 × standardized Scr−1.154 × age−0.203 × 1.212 [if black] × 0.742 [if female] (2 was defined as chronic kidney disease (CKD), indicated by the Modification of Diet in Renal Disease (MDRD) Study  was considered statistically significant.Statistical analysis was performed using R version 4.1.0. Considering the complex design of study of NHANES, we applied the survey package in R to calculate sample-weighted prevalence and other statistics to study the population characteristics of American wrist fracture patients. In addition, generalized linear model was used to analyze variables including age, gender, race, education, marital status, smoking, drinking, hypertension, diabetes mellitus, heart failure, obesity, chronic kidney diseases, osteoporosis, medication history of prednisone or cortisone, history of parental wrist fracture, and univariate and multivariate relationships with wrist fractures. The covariate with Clinical characteristics of the study population are displayed in via standard questionnaire described above. The prevalence of wrist fractures was 12%, with similarities in men and women  (p = 0.023). However, after the age ≥ 60 years, the prevalence in women was higher than in men .Among the total 5,842 US participants ≥ 50 years of age, 554 patients with wrist fractures were identified = 0.267) Table 2.Univariate analysis of the wrist fractures and demographic characteristics of American men and women aged ≥ 50 are shown Table 3:Multivariate analysis of the wrist fractures and demographic characteristics of American men and women aged ≥ 50 are shown Table 3:The analysis of sex and race subgroups was conducted. Non-Hispanic white women were positively associated with the occurrence of wrist fractures  or a hard fall (34.8%). Among those who had experienced their first wrist fracture, 17.8% had experienced a second wrist fracture, while 1.7% had experienced four or more fractures. The difference in the prevalence of wrist fractures between male and female was related to the age at which the first fracture occurred. Before the age of 60, men had a higher prevalence of their first wrist fracture than women. However, the situation became the opposite after the age of 60. Obesity, frequent drinking, current smoking, high serum phosphate level, non-Hispanic white women, and osteoporosis in women in the wrist fracture population were positively associated with wrist fractures. To the best of our knowledge, our study conducted the first research on patients with wrist fractures above the age of 50 based on data extracted from the NHANES, which enlightens the understanding of the disease as well as lends insight into prevention and improving the prognosis.In 2000, WHO reported that the prevalence of wrist fracture in the total population whose ages ≥ 50 was 18.5% . Hye-YouIn addition, this study also showed that a fall from a standing height was a major contributing factor to forearm fractures (56.0%), which was in line with the results reported by Norma J Mac Intyre and K C Chung , 30. PeoObesity, smoking, drinking alcohol, high serum phosphate level, usage of corticosteroids, and osteoporosis are common factors associated with fractures , 38, 39.2 (The study conducted by Jason Lacombe et al. showed that high BMI was associated with a reduced risk of forearm and wrist neck fractures compared to women with an ideal BMI of 20.0 to 24.9 kg/m2 . The fac2 .Some limitations in this study should be addressed. First, this study could not avoid reporting bias and memory bias, such as the history of wrist fractures, medication history of prednisone or cortisone, and smoking and drinking history. Second, due to the design of a cross-sectional study, relative conclusions of causality could not be obtained. Finally, some variables that were closely related to fracture, such as activity level, use of vitamin D and/or calcium in dietary supplements, and BMD, were not considered as covariates because they involved too many missing values. Despite the discussed limitations, the overall data collected from the NHANES database were reliable and had undergone sufficient validation to filter accurate individual samples to determine the prevalence.In conclusion, our study demonstrated that the prevalence of wrist fractures in Americans aged 50 and above was 12%. The prevalence was similar between male and female patients. Moreover, falling from a standing height was the main cause of the first wrist fracture. Frequent drinking, current smoker, high serum phosphate level, osteoporosis, and obesity were the risk factors for wrist fractures. In women, non-Hispanic whites are more likely to experience wrist fractures. Stratified by race, smoking was negatively associated with wrist fracture in other Hispanics and non-Hispanic blacks, while obesity was a risk factor of wrist fractures in Mexican Americans, other Hispanics, and non-Hispanic whites.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was designed by JN. JY extracted the associated data from NHANES. QL performed statistical analysis. JY and QL completed the composition of the manuscript, helped supervised the analysis, and revised and approved the manuscript. All authors contributed to the article and approved the submitted version.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.


Background: Infection with COVID-19 and its control entailing steroids and immunomodulatory medications disrupted normal immune function. The ensuing immunological disorder led to the rise of another infection—Black Fungus (Mucormycosis). However, the spread of Black Fungus can be minimized through proper knowledge, informed attitude, and conscious preventive practice. This study aimed to assess students’ knowledge, attitude, and practice (KAP) regarding Black Fungus amid the COVID-19 pandemic in Bangladesh. Methods: This cross-sectional study was carried out among Bangladeshi students from June to July 2021. Using Google Forms, an e-questionnaire was developed for this web-based survey, and the participants were selected through a snowball sampling approach. Results: Out of the 2009 participants, more than half were female (53.5%), and the majority were at an age between 18 and 25 years (31.5%) and had received higher secondary (HSC) schooling (77.8%), while around 61% resided in urban areas. Findings revealed that most of the students (63.8%) spent around 2 h on electronic and social media to become informed about COVID-19 and Black Fungus. Approximately 33% of the students showed low KAP scores (32.9%), whereas around 26% had high KAP scores. Our results show a significant association between KAP and sex, schooling, living status, residence, and media exposure. Conclusion: The knowledge of Black Fungus considerably varies among Bangladeshi students considering the place of residence, age, sex, living arrangement, and media exposure. Policymakers should emphasize awareness among people focusing on the results of this study to increase deterrent attitudes and protective practices to minimize the risks of being infected. Following the outbreak of the novel coronavirus disease 2019 (COVID-19), also known as SARS-CoV-2, from Wuhan, China . LikewisMucormycosis—a fungal infection, popularly known as Black Fungus—infected COVID-19 patients in India [Mucorales, affects people, particularly diabetes patients, with a reduced ability to fight the infection, which helps the fungi to flourish more [The strict implementation of non-therapeutic measures ,6 togethin India . During ish more . Althougish more . As of 2ish more ,23. Hencish more .Bangladesh—another South Asian country and a neighbour to India—reported its first two cases of Black Fungus on 8 May 2021, of which one patient died on 23 May 2021 . Hence, This cross-sectional web-based survey was conducted among Bangladeshi students from June to July 2021. The countrywide lockdown and “social distancing” during the second wave of COVID-19 made it impossible to conduct a nationwide face-to-face survey; therefore, a web-based survey was conducted to reach out to students in Bangladesh. Moreover, a web-based survey during an infectious disease has already been proven as an efficient and effective way to understand the social dynamics of different infectious diseases ,42,43,44For this study, a snowball sampling approach was followed, and the participants were selected based on some specifications: (i) the participant must be a student; (ii) must have an active account on social media, e.g., Facebook, WhatsApp, Messenger, Viber; (iii) must have a capacity to read and understand the e-questionnaire written in English; (iv) must be a Bangladeshi citizen; and (v) stayed in Bangladesh during the COVID-19 outbreak. Considering the criteria mentioned above, the researchers communicated with teachers at different schools, colleges, and universities through email as well as social media, including Facebook, Messenger, and WhatsApp. The teachers were requested to forward the e-questionnaire to their students in Bangladesh. The student participants were further requested to forward the e-questionnaire to their school/college/university classmates and other students connected through different social media platforms. The data were collected for a month, from June 2021 to July 2021. From an initial response of over 2150, a total of 2009 responses were retained for this study, as some of the participants did not complete the e-questionnaire correctly, while there were some others who did not fulfill the criteria set by the researchers.This research was ethically approved by the Khulna University Ethical Clearance Committee (Reference No. KUECC-2021/06/24). The e-questionnaire contained a consent form detailing the rights of the participants, i.e., the anonymity and confidentiality of the participants and their responses, respectively, and a right to withdraw without justification within a stipulated timeframe.An e-questionnaire in English was developed for this web-based survey after a careful review of relevant studies ,46,47,48Data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 27 and STATA (version 14.2) computer software. A Chi-squared test was carried out to investigate the association between sociodemographic characteristics and knowledge, attitude, practice, and total KAP scores regarding Black Fungus. Shapiro–Wilk and Kolmogorov–Smirnov tests were conducted to determine the normality of numeric data about the total knowledge, attitude, practice, and total KAP scores. As the data were not normally distributed, therefore, non-parametric tests, such as the Kruskal–Wallis statistic test or Mann–Whitney U tests, were conducted to analyze the association between sociodemographic characteristics and knowledge, attitude, practice, and total KAP scores. A post hoc analysis was also executed through Bonferroni and Scheffé corrections for adjusting the significance value. In addition, descriptive statistics  were also estimated. Finally, we utilized the generalized, ordered logistic regression model for predicting the knowledge, attitude, and practice level of students in Bangladesh . For allMost participants (80.9%) reported that keeping updated with the current situation is essential for individual and collective well-being from Black Fungus; 77.3% of the students stated that the information shared by government agencies is vital to control the disease and rumors. p > 0.05 for all). Most participants demonstrated a moderate knowledge score (41.3%) and a low knowledge score (38.3%). Female students showed relatively better knowledge; 23.7% had moderate knowledge, while 12% had high knowledge, compared to their male equivalents (17.2% moderate and 8.2% high knowledge score). Regarding religion, Muslim students showed moderate (38.6%) knowledge about Black Fungus. Students living with families—with or without children—also had better knowledge about Black Fungus (19.9% high and 39.3% moderate knowledge). In addition, students with greater exposure to information through radio or television had high knowledge (10.0%) score, and exposure from other sources of information, such as social media, exhibited a high (7.6%) knowledge score on Black Fungus. Again, p > 0.05 for all). Findings suggest that most of the students showed low (40.8%) to moderate (50.8%) attitude scores. Female students (29.4%) and those living with families (49.0%) and in urban areas (32.0%) attained moderate attitude scores compared to male students (21.1%) and those living away from families (1.8%) and in rural or suburban areas (18.8%). It was also found that students with greater exposure to information through radio and television showed moderate (24.5%) attitude to high (4.4%) attitude scores regarding Black Fungus.p > 0.05 for all). A quarter of the students (25%) were reluctant to take preventive measures to reduce the risk of Black Fungus infection, whereas 38.4% took all possible measures and scored higher regarding practice to prevent Black Fungus infection. It is apparent that female students (23.3%), as well as those living in urban areas (24.7%) and with families (37%), took necessary actions to reduce the risk of Black Fungus infection compared to male students and those living in rural or suburban areas (13.7%) and living alone (1.4%). It is also observed that older students were relatively unwilling to follow the non-therapeutic measures compared to younger students. In addition, students with better exposure to information, mainly through radio and television (20.2%), were more interested in following protective measures. Moreover, Post hoc analysis further showed that students with good and excellent health status exhibited significantly better scores than students with poor health status. Moreover, students not affected by COVID-19 had higher scores in KAP than those infected or at risk of infection with COVID-19. Students with greater media exposure had significantly higher knowledge, practice, and total KAP scores than students with low or no media exposure. Moreover, students who received information from radio and television reportedly had significantly higher attitude, practice, and total KAP scores than students receiving news from social media and other internet sources. Students who spent less time (≤2 h) seeking information showed significantly higher attitude, practice, and total KAP scores than those who spent more than five hours a day. Increased exposure to media compared to pre-COVID-19 situation among students demonstrated significantly higher scores in all sections than the students with decreased exposure to media from pre-COVID-19.In this model, sex identity, living location, history of COVID-19, source of information, time spent (hours) on media, and media exposure were found to be significantly associated with knowledge level among students during the COVID-19 pandemic towards Black Fungus. Compared to male students, female students were 1.49 times  more likely to be in the moderate and higher knowledge group for Black Fungus than the lower knowledge group. In the case of the higher knowledge group compared to the lower and moderate knowledge group, the female students were 1.27 times  more likely to be in the higher knowledge group for Black Fungus compared to male students. When compared to students who never used media to gather information, students who always used media were 2.05 times  more likely to be in the moderate and higher knowledge group for Black Fungus than the lower knowledge group. Similarly, comparing the students who never used media, the observed knowledge for the students who always used media to obtain information about Black Fungus was 2.11 times more  likely to be in the higher knowledge group than in the lower and moderate knowledge group.Again, considering attitude as a dependent variable, the sex identity, religious identity, age (years), marital status, media exposure, source of information, and exposure to media compare to pre-COVID-19 were found to be significantly associated with attitude levels among students during the COVID-19 pandemic towards Black Fungus. Compared to male students, female students were 1.58 times  more likely to have a higher and moderate attitude level than a lower attitude level. Students belonging to the Hindu religion were 1.55 times more likely  to be in the higher and moderate attitude group than in the lower attitude group compared to Muslim students concerning Black Fungus. Similarly, compared to the lower attitude group students, students in the ≥26 age group were 2.21 times more  likely be to in the higher and moderate level regarding Black Fungus compared to those who were in ≤17 age group.Students who acquired their information from YouTube and online news were 2.39 times more  likely to be in the higher attitude group versus lower and moderate attitude than those who receive their information from social media. Students who had the same exposure to media compared to pre-COVID-19 situation were 2.39 times more  likely to exist in the higher attitude group compared to those students who had decreased exposure to COVID-19 concerning Black Fungus.Moreover, in the model with the dependent variable practice, we found that sex identity, age (years), living location, history of COVID-19, media exposure, source of information, time spent (hours) on media, and exposure to media compared to pre-COVID-19 situation were found significantly associated with practice level among students during the COVID-19 pandemic towards Black Fungus. It shows that female students were 1.57 times  more likely to be in the higher practice group than those in the lower and moderate practice group concerning Black Fungus than male students. Compared to the students in ≤17 age group, students in the ≥26 age group are 3.13 times more  likely to be in the higher practice level compared to the lower and moderate practice level regarding Black Fungus. Compared to the students who never used to obtain information from media, students who always used media were 3.80 times  more likely to be in the moderate and higher practice group than in the lower practice group. Moreover, students who acquired their information from radio and television were 1.39 times  more likely to be in the moderate and higher practice group than those who receive their information from social media. Furthermore, comparing the students who had decreased exposure to COVID-19 concerning Black Fungus, students who had increased exposure to COVID-19 were 1.56 times more  likely to be in the higher practice group than in the lower and moderate practice group.This study assessed students’ knowledge, attitude, and practice regarding Black Fungus. It is noteworthy that no previous research on KAP analysis of Black Fungus has been conducted in Bangladeshi students. Our results indicate that six out of ten students (61.7%) had proper knowledge  scores about Black Fungus, implying that nearly 40% of Bangladeshi students (38.3%) had low to no knowledge about Black Fungus. The knowledge score (61.2%) in a prior study of young adults in Bangladesh regarding COVID-19  was almoIn addition, our study also found that sex identity, living, history of COVID-19, media exposure, source of information, and time spent on social media were all significantly linked to the knowledge score of students regarding Black Fungus. Our findings corroborate that of a prior COVID-19 study in Bangladesh . It is eSimilar to the knowledge score, this study’s findings showed a significant association of sex identity, religious identity, age, marital and living status, history of COVID-19, media exposure, source of information, time spent on media, and exposure to media compared to pre-COVID-19 situation with the attitude score of the students regarding Black Fungus. Previous studies show similar results that age, gender, media exposure, and marital status have a significant association with the attitude score of pandemic diseases ,60,61. FThis study found that sex identity, religion, age, and media exposure were significantly correlated with a positive attitude, and the majority of participants (80.9%) were aware of the risk of Black Fungus. Our findings also revealed that approximately 77.3% of the students believed that information provided by government agencies is critical to controlling the disease. This study discovered that a higher proportion of female students had a more positive (29.4%) attitude than their male equivalents (21.1%). Likewise, it was reported that students who had more exposure to information via radio and television had attitudes toward Black Fungus that ranged from moderate (24.5%) to high (24.5%). On the other hand, a study in Thailand showed that attitudes were subject to gender and age . Again, At the beginning of COVID-19, an extensive amount of misinformation spread across the world via modern technologies, and students were the main victims of this misinformation. It is crucial to investigate the current knowledge, attitude, and practice of the students toward Black Fungus in order to guide policymakers and public health professionals in ways in which the best outcomes can be achieved through public health interventions. To prevent the further rise in Black Fungus incidence and potential health hazards where early diagnosis is essential in accomplishing a positive outcome, proper knowledge among students is a must.This study is the first to investigate students’ knowledge, attitude, and practice regarding Black Fungus (Mucormycosis) amid the COVID-19 pandemic in Bangladesh. This research used a web-based investigation to maintain “social distancing” and to avoid the risk of infection—a universalized and validated approach to carry out research during an outbreak of infectious disease. Some limitations may apply to this research. Considering that this study was cross-sectional, it may be challenging to draw any strong conclusions about causality. Self-reporting has several drawbacks, such as different biases, in comparison to face-to-face interviews. The COVID-19 pandemic has shown how inadequately equipped the Least Developed Countries (LDCs) are for the technological environment despite the Sustainable Development Goal (SDG) to increase access to communications technology and reasonable access to the internet in developing countries by 2020, which has not been fulfilled yet. Consequently, this study failed to ensure a representative student sample, as this study only collected data from those with internet access in Bangladesh. We utilized a restricted number of questions to assess knowledge, attitude, and practice. Thus, extensive and inclusive studies would be necessary to ascertain the true prevalence of KAP regarding Black Fungus among the general population.The findings of this study suggest that knowledge, attitude, and practice toward Black Fungus significantly differs among Bangladeshi students with regards to sex, living arrangement, age, places of residence, and media exposure. It indicates that we could control and anticipate Black Fungus and relevant infectious diseases by maintaining safe practices and encouraging favorable attitudes with the cooperation of the authorities, health policymakers, and the general population. This study suggests maintaining a precautionary behavior to increase safe practices toward Black Fungus. Specific interventions need to be designed and implemented for the students who live in rural areas to increase their knowledge and awareness about Black Fungus. Increased media exposure is required to reach male students as well as respondents who are living in rural areas. Critical and timely insights from this research may also guide policymakers in improving strategic planning for COVID-19 and Black Fungus outbreaks in developing countries, including Bangladesh.


Thyroid angiosarcoma is an extremely rare malignancy, which occurs more frequently in the alpine region, likely associated with iodine deficiency and endemic goiter. This is an aggressive neoplasm that usually harbors a poor prognosis. We report the case of a 49-year-old Portuguese female patient presenting with a large nodule in the anterior neck region, with rapid growth and associated dysphonia. The neck ultrasound showed a hypoechogenic and heterogeneous thyroid nodule, with a larger axis of 44 mm. The fine needle aspiration cytology was not conclusive, and a biopsy of the lesion was performed. The result was suggestive of a mesenchymal tumor constituted by spindle cells and vascular clefts, showing positivity for endothelial markers and negativity for thyroglobulin, calcitonin and TTF1. The chest CT scan performed before surgery showed multiple pulmonary nodules suggestive of secondary lesions. The patient was submitted to total thyroidectomy and lymph node dissection in order to relieve compressive symptoms. A diagnosis of thyroid angiosarcoma was made after histologic examination of the surgical specimen. Despite undergoing multiple lines of palliative chemotherapy, the pulmonary lesions increased in size and number. The patient died due to respiratory failure 29 months after the diagnosis. Thyroid angiosarcoma is a rare malignancy, generally with poor prognosis. In our case, the patient presented with pulmonary metastases at diagnosis, which is a negative prognostic factor. Due to its rarity, data regarding management and treatment of this disease are scarce. Thyroid angiosarcoma is an extremely rare and highly aggressive malignant tumor. This entity constitutes less than 1% of all sarcomas, with a higher prevalence in alpine regions . This ge2). At the second cycle (about 2 months after surgery), a nodular cervical lesion was detected, with a larger axis of 37 mm and involving the jugular vein, compatible with a local recurrence that was considered non-resectable. The patient was submitted to local radiation therapy  with a dimensional reduction of the mass (from a larger axis of 37 mm to 22 mm). The CT scan performed after nine cycles of paclitaxel was compatible with partial response, showing a considerable dimensional reduction in most pulmonary metastases. Despite the initial favorable response, the CT scan performed after 25 cycles of weekly paclitaxel (about 6 months after the start of chemotherapy) was compatible with disease progression, with dimensional increase on the previously known lung metastases. Second-line palliative treatment with pegylated liposomal doxorubicin 40 mg/m2 every 4 weeks was started. The patient completed only three cycles of therapy before stopping the treatment due to pulmonary disease progression. Third-line palliative treatment was started with gemcitabine 1,000 mg/m2 once weekly for 3 weeks of each 28-day cycle, which was stopped after six cycles, again due to progression on the lung metastases. Fourth-line treatment was started with docetaxel 50 mg/m2 weekly, with no response on the CT scan after five cycles. At this stage it was decided to start treatment with pazopanib, a tyrosine kinase inhibitor targeting VEGFR. Once again, the drug was stopped after only 3 months due to pulmonary disease progression. Taking into consideration the deterioration of the patient’s performance status, a shared decision was reached to stop chemotherapy and focus on optimal supportive care. The patient died due to respiratory failure, secondary to pulmonary disease progression, 29 months after the diagnosis.We describe the case of a 49-year-old female patient who was a smoker (40 packs/year) with no other relevant personal or family history. The patient was born in Portugal, never left the country and was referred to our department due to a 2-month history of a painless, rapidly growing mass on her anterior neck, as well as associated hoarseness. In the initial consultation, the patient presented with a palpable 4 cm nodule with hard consistency, located in the left thyroid lobe, which moved when swallowing. A cervical ultrasound was performed, showing an hypoechogenic nodule in the left thyroid lobe, with bosselated limits, measuring 44 x 43 mm. Thyroid function was normal and anti-thyroglobulin, and anti-thyroperoxidase antibodies were negative. A fine needle aspiration cytology (FNAC) was performed, revealing inflammatory cells, apoptotic nuclei and abundant fusiform cells, seemingly fibroblasts, with polymorphic nuclei and small nucleolus. These findings were regarded as inconclusive, so we obtained a needle biopsy of the lesion. The result was suggestive of a mesenchymal tumor, with fusiform cells and vascular slits, showing positivity in the immunohistochemical examination for CD31, CD34 and factor VIII. The sample was negative for thyroglobulin, calcitonin, thyroid transcription factor 1 (TTF-1) and cytokeratin AE1/AE3. Due to a high index of suspicion of malignancy, we opted to perform a cervical and thoracic computerized tomography (CT) scan in order to determine the best therapeutic approach. The CT scan showed a voluminous thyroid nodule in the left lobe, which was 52 x 50 x 66 mm, heterogeneous and had no calcifications. This lesion was causing lateral displacement of the left internal jugular vein and trachea but with no alterations in the tracheal lumen diameter. There were multiple disperse nodular lesions in the pulmonary parenchyma, suggestive of secondary lesions, the largest with a diameter of 22 mm. Due to the rapid growth of the tumor and the associated compressive symptoms, the patient was submitted to total thyroidectomy and left lymph nodes’ dissection of compartments IIA, III and IV. Gross examination revealed a 75 mm tumor, occupying most of the left thyroid lobe, with infiltrative, ill-defined borders and foci of necrosis . MicroscThyroid angiosarcoma is a rare malignancy that occurs more frequently in females, mostly over the age of 60 . Most of


The accurate estimation of acute ischemic stroke (AIS) using diffusion-weighted imaging (DWI) is crucial for assessing patients and guiding treatment options. This study aimed to propose a method that estimates AIS volume in DWI objectively, quickly, and accurately. We used a dataset of DWI with AIS, including 2159 participants  with various types of AIS. We constructed algorithms using 3D segmentation (direct estimation) and 2D segmentation (indirect estimation) and compared their performances with those annotated by neurologists. The proposed pretrained indirect model demonstrated higher segmentation performance than the direct model, with a sensitivity, specificity, F1-score, and Jaccard index of 75.0%, 77.9%, 76.0, and 62.1%, respectively, for internal validation, and 72.8%, 84.3%, 77.2, and 63.8%, respectively, for external validation. Volume estimation was more reliable for the indirect model, with 93.3% volume similarity (VS), 0.797 mean absolute error (MAE) for internal validation, VS of 89.2% and a MAE of 2.5% for external validation. These results suggest that the indirect model using 2D segmentation developed in this study can provide an accurate estimation of volume from DWI of AIS and may serve as a supporting tool to help physicians make crucial clinical decisions. Acute ischemic stroke (AIS) is a major cause of disability, and an accurate and rapid decision for treatment strategies is critical to improving patient outcomes ,2. Time Various high-performance automatic segmentation methods have been developed for DWI of AIS ,10,11, bThis study aimed to propose a method that estimates AIS volume in DWI objectively, quickly, and accurately. The proposed method performs segmentation on DWI and estimates the volume of the segmented AIS.b-value of 1000 s/mm2 showed no lesions, whereas a b-value of 0 s/mm2 might show some old lesions. This retrospective study was approved by the Institutional Review Boards of HUCSHH and KNUH, which waived the requirement for informed consent .Consecutive patients who underwent DWI for AIS were retrospectively recruited from two hospitals  and Kangwon National University Hospital (KNUH)). The AIS cases in each hospital were as follows: from January 2011 to December 2019 for HUCSHH, and from July 2014 to October 2019 for KNUH. Eventually, 2159 DWI images of AIS  were included. Additionally, 121 DWI images of control subjects  were included in the evaluation of the developed segmentation algorithm. These DWIs were of healthy participants or those whose b-value, 1000 s/mm2.MRI was performed with various machines, including a 1.5T  and a 3T scanner . The parameters for the DWI sequences were as follows: repetition time, 3000 ms–8000 ms; echo time, 56 ms–103 ms; flip angle, 90°; matrix, 256 × 256–512 × 512; field of view, 220 × 220–256 × 256 mm; the number of excitations, 1–5; the number of slices, 20–50; slice thickness, 3 mm; http://www.itksnap.org. accessed on 30 December 2020). All images with masks were saved in Digital Imaging and Communications in Medicine (DICOM) format. All lesions on DWI were manually segmented by one neurologist (C.K) in HUCSHH and by two neurologists (S.H.K and J.-W.J) in KNUH using ITK-Snap, an open-source software application used to segment structures in medical images Direct volume estimation was executed by a 3D U-Net , an archIndirect Volume Estimation (Using 2D Segmentation)Indirect volume estimation measures the volume of a slice image after changing the volumetric image to a slice image. By dividing the volumetric image into each cross-sectional image and using it as an input for the model, the amount of data increases, and the overall brain cross-sectional information can be viewed, mitigating hierarchical imbalances and data shortages. Indirect volume estimation is executed by 2D U-Net, which has the same model architecture as 3D U-Net but uses 2D convolutions instead of 3D convolutions. In the previous method , one chaTo estimate the volume indirectly, we used the pixels of the mask predicted as AIS. The detailed algorithm for estimating the volume using the predicted mask is shown in To obtain the actual volume, the scale factors, By letting The patient’s AIS volume was obtained by multiplying the volume, The Dice coefficient  is used We trained a model with Dice loss, and the model trained knowledge, such as DWI of AIS. Where To evaluate the segmentation models, we adopted the sensitivity, specificity, F1-score, and Jaccard index as follows:TP). In Equations (4)–(7), TP is the number of outcome pixels correctly predicted as AIS. False-positive (FP) is the number of outcome pixels mispredicted as AIS, and false-negative (FN) is the number of outcome pixels mispredicted as non-AIS.Because our study focused on the importance of accurate AIS segmentation, we mainly set metrics for true-positive (VS) [MAE):To evaluate the volumes, we adopted the volume similarity (VS)  and meanVS estimated the volume of the predicted AIS to indicate its similarity with the labeled AIS. MAE is the average of the absolute values of the errors that are the difference between the actual and predicted values, i.e., it estimates the error between the predicted AIS and labeled AIS. In Equations (8) and (9), the measurement is the region of the predicted AIS (TP), and the ground truth is the region of the labeled AIS.All methods were implemented using the Pytorch framework 1.10 in Python 3.8.10 on Ubuntu, using four NVIDIA RTX 3090s, and have been included as core algorithms in AI-based medical solutions . Internal validation was performed using data acquired from HUCSHH. We randomly split the dataset into training, validation, and testing sets at a ratio of 8:1:1. Data acquired from KNUH were used for external validation. Loading the image and spacing information of the dataset is time-consuming. Considering that it takes a long time to load the DICOM file, the image and spacing information extracted from the DICOM file were saved in HDF5.We conducted an experiment to evaluate the methods for the segmentation performance of DWI in AIS. Through VS and MAE, we confirmed whether the result of estimating the AIS volume with the proposed model is reliable. In addition, to check for FP errors, our best model (indirect pretrained) was validated in the normal group that had no AIS volume at all. For internal and external validation, the MAE values were 0.028 cc and 0.009 cc, respectively.We proposed a novel method to estimate AIS volume and help doctors make quick decisions accurately. The proposed method is an indirect volume estimation method that estimates AIS volume by converting a volumetric image into a slice image; the volume is estimated using pixels predicted as AIS in each slice. This approach is an effective and attractive method with the advantage of low computational cost. We conducted internal and external validations to confirm this remarkable performance. First, the image information was different for each image. Heterogeneity exists because of the devices used in each hospital and the option settings in the devices , noise, etc., which can make it difficult to train models for indirect volume estimation and direct volume estimation. Additionally, the volume shape of patients depends on the number of slices, pathological lesions and slice width . To solvAs mentioned in 2.4 Method, patch-based 3D segmentation was performed on direct volume estimation. This patch-based 3D segmentation trains a model by transforming an input 3D image into 3D subsamples of arbitrary size. However, patch-based 3D segmentation may not be able to extract global features considering the actual image volume, so the size of the AIS to be segmented like ours is relatively small compared to the size of the brain and may show poor results when located infrequently. In addition, class imbalance may occur if an inappropriate patch size is used. Due to these limitations of path-based 3D segmentation, it can be inferred that the training result of direct volume estimation is worse than indirect volume estimation.In the case of direct segmentation, there was no significant performance difference in the F1-score between the estimated scratch model and the pretrained model. In the case of indirect volume estimation, the pretrained model had a higher F1-score than the scratch model. The reasons for these results are as follows:First, what the pretrained model predicts is different. In direct volume estimation, the pretrained model for auto implants predicts the shape and size of an implant through an image of a defective skull, unlike ours, which segments lesions through brain images. Moreso, the number of modalities and classes used in the pretrained model is different from ours. In direct volume estimation, brain tumor segmentation of the pretrained model is for segmenting brain tumors composed of multiple labels, like activators and edema, by inputting multimode MRI, such as FLAIR (fluid-attenuated inversion recovery), T1, and T2. This is different from our study, which involves segmenting AIS lesions by inputting only DWI. Therefore, the result of the F1-score seems poor. However, in indirect volume estimation, the pretrained model that segments gliomas by inputting only FLAIR is similar to our study, which segments only one lesion. Hence, the pretrained model outperformed the scratch model in terms of transfer learning effects.In AIS, estimating infarction volume is crucial for properly managing patients regarding clinical decisions and predicting outcomes ,23. DelaIn conclusion, indirect volume estimation showed better results than direct volume estimation. However, indirect volume estimation may encounter difficulty in segmenting sparse and small-sized AIS because the small-sized AIS does not provide many axial slices useful for learning segmentation modes . In futu


Our results showed that high expression of sCD8, iCD8, and tCD8 correlated with higher Ki67, TILs density, ER/PgR negativity, and higher histological grade. We confirmed that patients with high iCD8+ and tCD8+ TILs had longer DFS and OS, as compared to those with low counts/mm2. Survival benefit was retained when adjusting for classical clinical and pathological characteristics, but was not correlated to specific BC subtype. More data are needed to empower the prognostic role of TILs and establish their use in clinical practice.Tumor-infiltrating lymphocytes (TILs) have been reported to contribute to breast cancer (BC) prognosis. The aim of our study was to investigate the prognostic impact of CD8+ TILs and their subtypes in patients with early breast cancer treated with sequential, dose-dense adjuvant chemotherapy. Tumors of 627 patients were examined for total (t), stromal (s), and intratumoral (i) CD8 lymphocyte density (counts/mm2) on tissue-microarray cores. The impact of CD8+ TILs counts on DFS and OS, and its correlation with breast cancer subtypes and standard clinicopathological parameters, were investigated, along with efficacy and safety data. Among the 928 eligible patients, 627 had available CD8+ data. Of which, 24.9% had a high expression of sCD8, iCD8, and total CD8, which were correlated with higher Ki67, TILs density, ER/PgR negativity, and higher histological grade. The 5year DFS and OS rates were 86.1% and 91.4%, respectively. Patients with high iCD8 and tCD8 had longer DFS and OS compared to those with low counts/mm2 . Upon adjustment for clinicopathological parameters, iCD8 and tCD8 retained their favorable prognostic significance for DFS and OS, whereas high sCD8 was only prognostic for DFS. Menopausal status, tumor size, and nodal status retained their prognostic significance in all examined multivariate models. CD8+ TILs, and especially their intratumoral subset, represent a potential favorable prognostic factor in early BC.Tumor-infiltrating lymphocytes (TILs) contribute to breast cancer (BC) prognosis. We investigated the prognostic impact of CD8+ TILs in patients with early breast cancer treated with adjuvant chemotherapy in a large observational clinical trial. Along with a 10 year follow-up, considering the efficacy and safety, we report the results of the translational part of our study. We examined the patients’ tumors for total (t), stromal (s), and intratumoral (i) CD8 lymphocyte density (counts/mm Female breast cancer (BC) is the most commonly diagnosed cancer globally and represents the leading cause of cancer death among women . SurgeryThe tumor immune microenvironment has been reported to play a vital role in cancer spread, progression, and response to treatment , and sevBeyond prognosis, there has been a large research effort to establish TILs as predictive markers and establish their role in daily clinical practice. Losurdo et al. showed that low TILs negatively impact prediction of outcome in TNBC treated with immunotherapy. This information may suggest a need for induction chemotherapy before PD1/PD-L1  inhibitoDespite these meaningful advantages in the field, the prognostic or predictive value of TILs in BC remains debatable, due to the significant amount of heterogeneity in the experimental design. In particular, the variability of the methods and criteria used to quantify TILs and the interobserver differences between pathologists when evaluating iTILs constitute obstacles to the integration of TILs into clinical practice. In addition, the heterogeneity of BC makes the use of TILs much more complex ,23,24.The aim of the present study was to evaluate the prognostic role of CD8+ TILs within a Hellenic Cooperative Oncology Group study, along with a final report on the efficacy and safety data after a 10 year follow-up period.A total of 990 women were enrolled in the study between November 2007 and December 2010. Eleven patients were deemed ineligible and were excluded from the analysis , leadingSelected patient and tumor characteristics are presented in The relative dose intensities of all drugs were assessed in the 964 patients (98.5%) with available data who received the study treatment. Of note, five patients received docetaxel instead of paclitaxel, while one additional patient was treated with adriamycin instead of epirubicin. Lastly, another patient received epirubicin followed by the administration of doxorubicin.The median relative dose intensity (RDI) administered for epirubicin and paclitaxel was 0.997 and 1.00, respectively. This was similar to previously published results by our group ,26,27. T2 was 113.7 and 3.7, respectively  had higher Ki67 levels  and higher TILs density . Additionally, tumors with high counts/mm2 of sCD8, iCD8, and tCD8 were more frequently ER/PgR-negative  and of higher histological grade  (p < 0.001), iCD8 , and tCD8 . Additionally, a significant positive correlation was observed between TILs density and Ki67, which, however, was not considered strong .The median number of sCD8 and iCD8 counts/mmectively . Of 627 ectively A–D. Pati< 0.001) . TILs deAt a median follow-up of 132.5 months (range 6.8–158.8), 253 DFS events (25.8%) had been recorded and 201 deaths (20.5%) had occurred. Information on the cause of death was available for 140 of 148 patients (94.6%) who died upon disease progression following adjuvant E-T-CMF chemotherapy, with 96.4% of them (n = 135) dying of their disease. No deaths were recorded during adjuvant chemotherapy. The median DFS and OS had not yet been reached at the data cut-off date for the analysis (15 June 2021). The 5-year DFS and OS rates were 86.1% (95% CI 84–88%) and 91.4% (95% CI 90–93%), respectively. Four patients (0.4%) developed myelodysplastic syndrome and 33 patients (3.4%) developed other cancers, including colorectal cancer (n = 1), endometrial cancer (n = 3), gastric cancer (n = 2), lung cancer (n = 5), breast cancer (n = 10), ovarian cancer (n = 3), pancreatic cancer (n = 3), melanoma of the skin (n = 3), sarcoma (n = 1), non-Hodgkin lymphoma (n = 1), and multiple myeloma (n = 1). In addition, two patients (0.2%) developed in situ breast carcinoma, seven months and 10 years post-completion of adjuvant chemotherapy, respectively.Data regarding the site of progression were available for 163 of 185 patients (88.1%) who experienced disease progression. Distant relapses were noted in 151 patients , while 19 patients  presented locoregional relapses. Of note, six patients experienced both locoregional and distant relapses .As expected, increasing age, postmenopausal status, radical mastectomy, increased tumor size and higher nodal status were associated with worse patient outcomes in terms of both DFS and OS. In addition, patients treated with adjuvant radiotherapy were at a significantly higher risk of death compared to those who did not receive radiation .2 of iCD8 and tCD8 had longer DFS and OS compared to those with low counts/mm2  (Patients with high counts/mmctively) .2 of sCD8   .2 of sCD8 were significantly associated with prolonged DFS , both iCD8 and tCD8 retained their favorable prognostic significance for DFS and OS, whereas high counts/mmnged DFS . Of note2 of sCD8, iCD8, and tCD8, the evaluation of the impact of CD8 expression on DFS and OS was only feasible among patients with luminal B tumors, but neither CD8 nor TIL density showed prognostic significance in this subpopulation . Among these, 226 patients reported a total of 325 grade 3–4 events . No fataIn the present study, we investigated the impact of CD8+ TIL density on the survival of patients with early BC treated with adjuvant chemotherapy and its correlation with classical clinicopathological characteristics. Simultaneously, we documented the first report of a HeCOG clinical study, regarding the efficacy and safety results after a 10-year follow-up period.Patients enrolled in this study were treated with an E-T-CMF regimen followed as indicated by one year of trastuzumab treatment. This regimen was used in several studies by our group ,26,27; vAfter a 10 year follow-up, it was found that a DFS event occurred in 25.8% of patients and that 20.5% of patients died of various reasons. The 5 year DFS and OS rates were 86.1% and 94.1%, respectively. These results were consistent with the efficacy data of other reported adjuvant trials by our research group ,27 and wAs mentioned above, in this trial, we investigated TIL density and the presence of CD8+ TILs in a series of BC patients, their correlation with specific clinicopathological parameters, and their correlation with patient outcomes. High CD8+ TIL counts, regardless of subtype, were associated with tumors demonstrating higher TIL density, higher ki67, and higher tumor grade. Κi67 index is not T cell specific but an indicator of proliferation activity of tumor cells. Therefore, a high Ki67 index provides an estimation of the growth fraction of tumor cells and reflects the ability of neoplastic cells to highly proliferate. In breast cancer, Ki67 immunohistochemical expression has been extensively investigated and is applied to assess the proliferative activity of cancer cells, taking account of breast cancer molecular subtyping. As it seems that a favorable prognostic factor correlates with poor prognostic parameters, our findings may, in fact, reflect that, in tumors with similar ki67 or the same histological grade, higher TILs can play a favorable prognostic role, if present. In the statistical analysis of this study, ki67 was not examined regarding survival and after multivariate regression, and histological grade was related with improved OS in high CD8+ tumors. The methodology we followed, in combination with the small size of CD8 positive tumors in our study, suggest that the prognostic significance of TILs between Ki67-high and Ki67-low breast cancers remains to be elucidated in a large series of patients. Similarly, negative hormone receptors were associated with higher counts of CD8+ TILs, reflecting the known distinct immunogenic properties of triple-negative and HER2-enriched breast cancers. These findings are consistent with the majority of the literature regarding correlations between TILs and clinicopathological parameters ,39,40.2, reflecting their widely reported positive impact on patient outcomes [Regarding efficacy, this study confirmed the favorable role of high CD8 TILs in BC prognosis. The prognostic role of TILs in BC has been widely reported ,42,43, woutcomes ,16.2 of iCD8 demonstrated DFS and OS benefits. This finding is in line with several published studies, including one by our research group, which studied BC patients in adjuvant settings [2 of sCD8, despite the fact that in most published studies, this lymphocytic subset has been associated with a statistically significant survival benefit. These contradictory results can be explained by a variety of reasons, such as cancer heterogeneity, treatment setting, different methodology, and variability in patients’ populations between studies, along with interobserver discrepancies. In contrast to another published study of our group by Koletsa et al. [In recent years, research efforts have focused on establishing the specific population of TILs that could more precisely, and with repeatability, predict clinical outcomes, as the cell type and location of TILs seem to be of great interest. Two TIL locations, stromal and intratumoral, are being investigated separately. This research field is where most discrepancies and ambiguous results appear. In the present study, patients with high counts/mmsettings ,48,49 ansettings . On the a et al. , in the 2, which led us and other researchers to use binary instead of continuous variables; the cell heterogeneity within a tumor specimen; the selection of tumor areas for evaluation; and the fact that TMAs represent a static snapshot of TILs, while the immune response in tumor stroma is a dynamic situation, are the main limitations of this study that should be considered.In addition to the above-mentioned factors, the wide distribution of TILs counts/mmRM (T cells with features of tissue -resident memory) was associated with improved survival of patients with breast cancer and specifically in those with a triple-negative phenotype. According to these factors, an explanation of the aforementioned findings could be the gradient degree of immunosurveillance of T cells on prognosis over time. Nevertheless, it could be due to other unidentified reasons or chance, given the relatively small number of patients. Another limitation of this study, which obstructed our attempt to assess the impact of the TIL subset on patient survival according to BC molecular subtypes, was the small number of events of interest in the category of patients with high counts/mm2 of sCD8, iCD8, and total CD8. Only the luminal B subgroup could be analyzed, but without statistically significant results. However, given the strong impact on DFS and OS that we found in the entire patient cohort, we could indirectly suggest that HER2-enriched and triple-negative subgroups were, to a large extent, present in these results. This conjecture is consistent with the known prognostic role of TILs in these subgroups [An interesting observation of our study was that high CD8+ TILs are not prognostic in the first two to four years, and their favorable impact on DFS and OS appears later, as demonstrated in the Kaplan–Meier curves in ubgroups ,9,13,17.Although the molecular subtypes did not differ in survival in our study, specific clinicopathological characteristics were associated with improved outcomes in favor of patients with high CD8+ TIL counts and regardless of subsets. These were predominantly tumor size, nodal status, and histological grade, likely following the reported knowledge that the more advanced and aggressive tumors offer a biological background for neoantigen production. This, in turn, enhances a tumor’s immunogenicity, and intratumoral/stromal lymphocytic infiltration is able to predict better responses ,53. The As intensified CMF chemotherapy is obsolete in early breast cancer, the regimen used represents another limitation of this study, along with its non-randomized design. On the other hand, we consider the long-term follow-up and the fact that all samples were prospectively collected and centrally assessed as strong points of our study.In regards to safety, 93.6% of patients completed study treatment with multiple reasons for discontinuation, and no treatment-related deaths were reported. Despite the prophylactic use of G-CSF after every chemotherapy cycle, neutropenia was the most common adverse event, followed by peripheral neuropathy and fatigue. These toxicity data were similar to those previously reported from other randomized adjuvant breast cancer clinical trials conducted by our group ,26,55.Eligible patients were at least 18 years old with node-positive early BC, or node-negative disease of intermediate risk according to the 2005 St. Gallen criteria . The patBefore enrollment, each patient signed a written informed consent form for the use of their biological material for future research purposes.All investigations conducted in this study complied with the principles expressed in the Declaration of Helsinki.The study protocol was approved by the Bioethics Committee of the Aristotle University of Thessaloniki, School of Medicine (77/10.6.14), by the Institutional Review Board of Papageorgiou Hospital (180/15.7.13), and by the Institutional Review Board of Thermi Clinic (307/2.3.16). The study was registered in the Australian New Zealand Clinical Trials Registry (ANZCTR) and allocated the following Registration Number: ACTRN12615000161527.Pre-treatment evaluation included medical history, physical examination, imaging examinations according to international guidelines, complete blood count (CBC), and comprehensive biochemistry. Blood tests were obligatory before each chemotherapy cycle and EF, after the completion of chemotherapy, and then every four months during treatment with trastuzumab. Additionally, blood examinations were performed whenever clinically indicated .2) every 2 weeks, followed by 3 cycles of paclitaxel  every 2 weeks, followed by 3 cycles of intensified CMF  every 2 weeks (E-T-CMF) with G-CSF support. According to the treatment protocol, no patient received preoperative treatment.The chemotherapeutic regimen of the present study consisted of three cycles of epirubicin  was planned for all patients who had undergone partial mastectomy or those with tumor size ≥5 cm and/or more than 4 positive lymph nodes, regardless the type of surgery , 3–4 weeks following the completion of chemotherapy.Hormonal therapy was administered to all patients with hormone receptor-positive tumors. Details regarding the administration of hormonotherapy were previously reported .The immunohistochemical staining of all markers was performed on 3 μm ΤΜA sections using a Bond Max autostainer , as previously described  and showIHC for estrogen receptor (ER), progesterone receptor (PgR), HER2, Ki67 labelling index, cytokeratin 5 (CK5), and epidermal growth factor receptor (EGFR) was performed centrally at the Laboratory of Molecular Oncology of the Hellenic Foundation of Cancer Research, Aristotle University of Thessaloniki , as previously described . The resTumors were classified according to well-known immunohistochemical markers using the IHC4 model ,59. TheyImmunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) protocols for ER, PgR, HER2, and Ki67 assessments have previously been described . BrieflyCD8+ lymphocytes were evaluated by two Pathologists without knowledge of the clinical, pathological, and survival data.Immunostaining for CD8 was estimated in the tumor stroma (sCD8), as well as in the intratumoral/intraepithelial compartment where CD8+ cells were attached to malignant cells (iCD8). In addition, CD8+ cells within the tumor area ) defined as the sum of sCD8 and iCD8 was also examined.2 surface. Therefore, the stromal tumor area, the tumor area occupied by the malignant cells and the total tumor area were recorded as % increments of the total core area on matched Hematoxylin and Eosin TMA sections. The surface of each compartment in mm2 was calculated based on the percentage of the recorded area % and the total core surface (1.76625 mm2 for 1.5 mm cores). Then, the stromal CD8+ cell count per core was divided by the respective stromal surface, the intratumoral CD8+ cell count by the respective malignant cell surface, and the total tumor cell count by the total tumor surface, to assess the density of CD8 positive cells [2.For each core (1.5 mm of diameter), CD8+ cells were counted in four fields of magnification (×200) covering the entire area of the core. Therefore, four values per core were recorded. In cases in which the distinction of sCD8 and iCD8 was ambiguous, CD8 positivity was estimated at higher magnification (×400). The density of CD8+ cells in each tumor compartment was assessed as the ratio of cell counts per mmve cells ,68. AverWhole sections of hematoxylin and eosin stained slides were used to evaluate stromal TILs (sTILs) in accordance to the criteria proposed by the International TILs BC Working Group. The percentage of all mononuclear cells (including lymphocytes and plasma cells) in the stromal tumor component within the border of invasive carcinomas was recorded. Stromal TILs density was assessed as an average of all evaluated ×100 fields per tumor.All patients were followed up at study entry, every six months for the first five years and every year thereafter, with clinical examinations, CBC, biochemistry panels, serological markers, chest X-rays, and abdominal ultrasonography . Mammography and ultrasonography of the patients’ breasts were performed annually. Bone scans were not routinely performed after the third year, except when clinically indicated.Disease-free survival (DFS), calculated from the date of study treatment initiation to the first locoregional/distant relapse, contralateral breast cancer, secondary neoplasm, death from any cause, or last contact, whichever occurred, was the primary endpoint of the study. Secondary endpoints included assessment of overall survival (OS), estimated as the time interval from treatment initiation until death (from any cause) or last contact; the safety profile of the study treatment; and the prognostic significance of the biomarkers of interest for the patient’s outcome.2. Descriptive statistics with counts (%) and medians  values were used to summarize patient and tumor characteristics and the biomarker distributions. Associations between sCD8, iCD8, and tCD8 with selected clinicopathological parameters were assessed using a chi-square test  and the Wilcoxon rank-sum test (for continuous variables). Spearman correlations were used to evaluate the associations of TILs with Ki67, sCD8, iCD8, and tCD8.Due to the wide range of the distribution of stromal CD8 (sCD8), intratumoral (iCD8), and total CD8 (tCD8) and the presence of multiple natural outliers that could not be excluded from the analysis (to avoid misinterpretation of the results), the third quartiles of the respective distributions were used as the cut-off points to dichotomize tumors into high and low counts/mmp of 0.050.OS and DFS survival rates were obtained via Kaplan–Meier analyses and compared between groups with a two-sided log-rank test. Univariate Cox regression models were applied to evaluate the effect of clinicopathological parameters of interest, CD8 and TILs on DFS and OS. In the multivariate analysis, the effect of each lymphocytic subset marker that was univariately associated with patient outcomes was adjusted for menopausal status , nodal status , histological grade , and adjuvant radiotherapy . Age and the type of surgery were not included in the multivariate models, due to their correlation with menopausal status and nodal status, respectively. All statistical analyses were performed using the SAS software . Statistical significance was set at a two-sided In conclusion, the present trial showed that dose-dense E-T-CMF is a tolerable chemotherapy regimen for adjuvant treatment of intermediate/high risk breast cancer, with an efficacy comparable to more recent dose-dense schemes. CD8+ TILs, and especially their intratumoral subset, represent a potential favorable prognostic factor and impact survival rates. Although quantifying TILs using histopathology and immunohistochemistry is the most used technique, a variety of methodological factors may confound the results and, therefore, the impact of TILs in prognosis. In addition, the quantification of TILs does not take into account the dynamics and functionality of the tumor microenvironment. Studies in various histological subtypes of breast cancer, along with strict laboratory procedures and the use of novel approaches, such as automated computational assessments, are needed, in order to better understand the role of TILs and their subsets in BC prognosis and treatment.


Between acute RRDs (n = 16) and controls, FGF2 (p = 0.0001), IP10 (p = 0.0027), MCP-1 (p = 0.0015), MIP-1β (p = 0.0004), IL-8 (p = 0.0146), and IL-6 (p = 0.0031) were elevated. Determining alterations in inflammatory cytokine profiles may aid in understanding their impact on RRD development, clinical course, and complications before and after surgical repair.The purpose of this study is to characterize the inflammatory cytokine profile in rhegmatogenous retinal detachments (RRDs) compared to surgical controls. Vitreous humor was collected from patients undergoing vitrectomy for RRD and noninflammatory vitreoretinal diseases. A quantitative immunoassay was used to measure the levels of 36 cytokine markers. Linear regression analysis with the duration of detachment as the predictor and log-transformed cytokine levels as the outcome was conducted for normally distributed cytokines as determined by the Shapiro–Wilk test. The analysis was adjusted for age, sex, and race. The Kruskal–Wallis test was used for cytokines not normally distributed. Twenty-seven RRD cases and thirteen control cases were studied. Between all RRDs and controls, fibroblast growth factor 2 (FGF2) ( Rhegmatogenous retinal detachment (RRD) is a vision-threatening condition in which mechanical forces at the vitreoretinal interface lead to a break in the retina, allowing for the passage of fluid into the subretinal space, separating the retina from the retinal pigment epithelium (RPE) and choroid . This coThe goal of treatment of RRDs is to reattach the retina to the RPE with surgical techniques such as pneumatic cryopexy, scleral buckle, or pars plana vitrectomy . In genePrevious studies have found elevated levels of inflammatory cytokines in patients with RRD; however, the reported cytokine profiles are variable, and the lack of consistency in the data warrants further study  18,19,2,31,32,33The aim of this study is to further characterize the nature of inflammation in patients with RRD by evaluating their vitreous cytokine profile compared to controls, with results stratified based on the duration of detachment.Inclusion CriteriaThis study was approved by the Institutional Review Board of Boston Medical Center (BMC) and Boston University Medical Campus Institutional Review Board and adheres to the tenets of the Declaration of Helsinki. Patients enrolled in this prospective, cross-sectional study were 18 years or older with a primary language of English or Spanish and scheduled for a pars plana vitrectomy in at least one eye. For patients included in this study, the following demographic variables were collected from their medical charts: age, sex, and race. Self-reported racial categories included White, Black or African American, American Indian or Alaska Native, Asian, and Native Hawaiian or Other Pacific Islander according to the U.S. Census Bureau guidelines . The stuCases were grouped based on the duration of detachment, either less than 2 weeks or greater than 2 weeks, defined as the onset of symptoms such as flashes, floaters, decreased vision, and peripheral visual field loss. Symptoms were used as a proxy for the duration of detachment since this was the only way to clinically estimate RRD duration. We stratified the RRD cases at 2 weeks because it is at this point that a prior study defined RRD as chronic:  (1) Lessb.Biospecimen Collection and AnalysisPrior to starting the infusion during pars plana vitrectomy, 0.5–1.0 mL of undiluted vitreous fluid was aspirated by the vitrector into an attached 3 mL syringe ,38,39,40c.Statistical AnalysisStatistical analysis was conducted using SAS v 9.4. The Shapiro–Wilk test was used to determine the normality of log-transformed cytokine levels. If the cytokine levels were normally distributed, we used a linear regression model controlling for age, race, and sex to compare mean cytokine levels between groups. We did not adjust for lens status since it has been found that lens status does not significantly impact cytokine levels . If the p ≤ 0.05) and within an FDR of 10%. We provided effect size and standard error for normally distributed cytokines analyzed with a linear regression model. We were not able to report effect sizes for cytokines analyzed with the Kruskal–Wallis test due to the fact that the SAS does not report effect sizes for this nonparametric test.In the primary analysis, we compared mean cytokine levels between RRD cases and controls. In the secondary analysis, we compared mean cytokine levels between acute RRD cases (≤2 weeks duration) and controls. We did not complete a subgroup analysis comparing chronic RRD cases against controls or acute cases due to large variation in duration (3 weeks to 8 months), smaller sample size, and greater likelihood of recall bias by patients as the length of time from symptom onset and the chronicity of the retinal detachment increased. To correct for multiple comparisons, the p-values of cytokines were adjusted to account for potential type 1 errors using the false discovery rate (FDR), and we focused on the cytokines that were statistically significant , loss to follow-up (n = 3), surgery cancellation (n = 4), mislabeled specimens (n = 2), and withdrawal of consent (n = 1). An additional forty subjects (out of eighty) were excluded as their surgical indication was not RRD or did not meet the criteria for the control group. In total, 40 patients were included in this study, comprising 27 subjects with RRD (11 subjects with chronic RRD and 16 with acute RRD) and 13 controls without RRD . p = 0.0926). White patients comprised just under 50% of cases and controls. RRD cases and controls were similar with respect to gender distribution (p = 0.1862). Four of the eleven chronic cases had preoperative PVR. Demographic information of our subjects is listed in This study found that certain inflammatory markers were significantly increased in RRDs, including FGF2, IP10, MCP-1, IL-16, MIP-1α, MIP-1β, IL-8, Eotaxin, TARC, and IL-6. These findings are consistent with previously published studies . AdditioSome of the cytokines analyzed in this study have been implicated in the inflammatory causes of photoreceptor death. MCP-1, released by Muller cells, induces resident microglia migration and subsequent microglia activation and secretion of cytotoxic factors . ActivatAs previously mentioned, inflammation contributes to the formation of PVR, the most common cause of surgical treatment failure. While this study identifies a subset of cytokines upregulated within the first two weeks of detachment, correlating cytokine levels within this subset with those involved in the later development of PVR may provide insight into the biochemical pathways associated with PVR. Understanding the cytokines that trigger the cascade of fibrosis in some postoperative eyes but not others will help further work in preventing PVR in order to achieve better surgical and visual outcomes.Among the interleukins tested, IL-8 and IL-6 were found to be upregulated between RRD cases and controls. IL-6 is known to stimulate B-cells, hematopoiesis, and the production of acute-phase proteins ,52. IL-6Non-interleukin growth factor cytokines significantly upregulated in acute RRD includes FGF2. FGF2 stimulates endothelial cells, promotes angiogenesis and wound healing, and leads to the proliferation of Müller cells, retinal astrocytes, and retinal pigment epithelial cells ,54. MultIP10, MCP-1, and MIP-1β are non-interleukin cytokines involved in monocyte chemotaxis and activation and were upregulated in this study. Yang et al. found that MCP-1 can activate monocytes that induce RPE apoptosis and increase levels of intracellular calcium and reactive oxygen species . MCP-1 mThe strength of this study lies in isolating acute RRD cases by the duration of detachment and analyzing cytokine profiles while adjusting for demographic variables, particularly patient race. Because most previous studies use nonparametric tests that cannot adjust for demographic covariates, adjusting for demographic variables, such as race and sex, has rarely been performed in most prior studies  18,22,2,24,30. FIn conclusion, we corroborated the findings of elevated cytokines in RRD and identified a subset of inflammatory markers that may be early markers of RRD. Our findings may be foundational for future studies aiming to elucidate the effects of these cytokines on visual and anatomic outcomes after surgical repair, understand the pathogenesis of long-term consequences, such as PVR, and identify potential targets for the prevention of those complications as well as therapeutic interventions.


Tinnitus is a phantom sound perceived in the absence of external acoustic stimulation. It is described in a variety of ways  and can be a single sound or a combination of different sounds. Our study evaluated associations between audiological parameters and the presence or severity of tinnitus, to improve tinnitus diagnosis, treatment, and prognosis. Our sample included 122 older participants (63 women and 59 men), aged 55–75 years from the Portuguese population, with or without sensory presbycusis and with or without tinnitus. All participants underwent a clinical evaluation through a structured interview, Ear, Nose, and Throat observation, and audiological evaluation . The Tinnitus Handicap Inventory was used to measure tinnitus symptom severity. Our data confirmed that the odds of developing tinnitus were significantly higher in the presence of noise exposure and hearing loss. Also, participants who had abrupt tinnitus onset and moderate or severe hyperacusis featured higher odds of at least moderate tinnitus. However, it was in the ABR that we obtained the most exciting and promising results, namely, in wave I, which was the common denominator in all findings. The increase in wave I amplitude is a protective factor to the odds of having tinnitus. Concerning the severity of tinnitus, the logistic regression model showed that for each unit of increase in the mean ratio V/I of ABR, the likelihood of having at least moderate tinnitus was 10% higher. Advancing knowledge concerning potential tinnitus audiological biomarkers can be crucial for the adequate diagnosis and treatment of tinnitus. Tinnitus is a phantom sound perceived in the absence of external acoustic stimulation that can be described in a variety of ways  and can be a single sound or a combination of different sounds . It can Tinnitus has a variety of etiological factors and may be associated with other diseases, as it is usually viewed only as a symptom. It often accompanies hearing loss (HL) or hyperacusis, but neither is necessary for its presence , 2015. MThe causes and pathogenesis of tinnitus remain unclear, and there are no objective audiological or non-audiological tests for the diagnosis of tinnitus. Currently, tinnitus presence and impact are established using self-report and subjective measures, such as questionnaires, e.g., Tinnitus Handicap Inventory (THI) , or the Psychoacoustic assessment of tinnitus can also be performed. Even though participants with similar psychoacoustic measurements may report very different impacts on their lives, it may be useful to interpret neurophysiological mechanisms of tinnitus.There are two main theories regarding tinnitus pitch prediction in cases where the tinnitus is accompanied by HL, particularly in sloping configurations. On the one hand, several authors argue that tinnitus pitch should be associated with the audiogram’s edge frequency, corresponding to the boundary between a region of normal or near-normal hearing and a region of more significant HL . On the Abnormal synchronous neural activity can be identified by specialized clinical tests, namely, auditory-evoked potentials (AEP). Previous studies have used AEP measures to study abnormal neuronal activity in tinnitus participants . The mosDifferences in ABR traces can be seen depending on the type of stimulus used to evoke the response, type of HL, the degree of HL, and the presence of tinnitus, among others. Concerning the degree of HL and the type of stimulus used, elevated hearing thresholds reduce wave V amplitude to click stimuli, so using tone burst ABR when the tone burst characteristic frequency falls within the frequency region of the HL may provide higher sensitivity . AccordiAlthough there is a lack of consensus regarding the use of AEP as a diagnostic tool of tinnitus, mostly because of the lack of homogeneity in participant groups and methodologies, AEP measures may contribute to the clarification of the origin of tinnitus and provide objective diagnostic indicators . FurtherConsidering the models of pathological enhanced neural synchrony and the potential cortical influence on subcortical tuning functions, it is hypothesized that if there are unique ABR features in tinnitus they will become more pronounced if the ABR parameters are adjusted from click stimuli to tone burst stimuli matched to the tinnitus pitch. However, there are recognized difficulties in tinnitus pitch and loudness matching, and these difficulties occur even in the same individual due to intrinsic or even extrinsic variabilities . MoreoveGiven the likely role of cochlear function in the generation of tinnitus, it is essential to assess the inner ear. Otoacoustic emissions (OAE) are sound signals produced by the cochlea and reflect the activity of the outer hair cells (OHC). Through OAE, the cochlear function can be tested in an objective and non-invasive way . StudiesIn the literature, there are conflicting results regarding the levels of DPOAE in tinnitus participants. Some studies report decreased DPOAE levels in tinnitus participants compared to controls , whereasIdentifying reliable audiological biomarkers in participants with tinnitus will allow us to improve the diagnosis, treatment, and prognostic of tinnitus . The preThe study considered a sample of 122 older participants (63 women and 59 men). According to World Health Organization (WHO), aging is categorized as middle age (45–59 years), elderly (60–74 years), elder (75–90 years), and extreme old age (90 years upward). Since we aimed to study older individuals, we decided that the inclusion criterion would be being in the age group of 55–75 years, which would give us a good appreciation of the aging process regarding tinnitus and related comorbidities. Participants were consecutively recruited from Ear, Nose, and Throat consultations at CUF Infante Santo Hospital from March 2016 to December 2018.Exclusion criteria were tinnitus from the disease of the outer ear , Ménière’s disease, chronic otitis media, otosclerosis, history of ototoxic drug use, exposure to massive noise, history of previous malignancy with chemotherapy, history of autoimmune disorders, neurodegenerative or demyelinating disease, uncompensated medical disorder, or a severe psychiatric disorder. All participants were subjected to immittance audiometry to rule out middle ear pathology .Additionally, participants unable to comprehend and sign the informed consent or with cognitive impairment were also excluded.Data were collected from all participants concerning their personal clinical history (past and present), family history, and audiological assessment, including a tinnitus intensity rating on a scale from 0 to 10 (10 being the loudest possible) . Clinica® Assens, Denmark audiometer  and TDH39/HDA300 headphones fitted with noise-excluding headset ME70 and bone conductor B-71 were used.Pure tone audiometry (air and bone) was conducted to evaluate hearing thresholds according to ISO 8253 and 389. Standard tonal and extended high-frequency audiometry (250 Hz to 16K kHz) was performed in a soundproof booth employing an Interacoustics,The category of HL was defined according to the recommendations of the Bureau International d’Audiophonologie as follows: normal or subnormal hearing (below 20 dB), mild HL (21–40 dB), moderate HL (41–70 dB), severe HL (71–90 dB), very severe HL (91–119 dB), or total HL-cophosis (over 120 dB) . Pure toThe presence or absence of presbyacusis and the presence of tinnitus were recorded. Presbycusis was defined as bilateral sensorineural deafness in a downslope audiometric pattern, above 1,000 Hz, with poor speech discrimination (Speech Recognition Threshold >40 dB SPL and 100% discrimination to 60 dB or worse) .Several tests for measurement and evaluation of tinnitus were performed on all the participants having this complaint.The same experimenter performed all the audiological tests in a standardized protocol.® Assens, Denmark audiometer  and TDH39/HDA300 headphones fitted with noise-excluding headset ME70. First, it was established whether the tinnitus percept was more similar to a pure tone or a narrow-band noise. Both sounds were presented to the participant who was asked which of the two had the most resemblance to their tinnitus.Tinnitus evaluation was performed after audiometric testing in a soundproof booth using an Interacoustics,Estimation of tinnitus frequency was then performed using frequencies from 125 to 16 kHz (pure tones or narrow-band noise centered on the same frequencies). The procedure for determining tinnitus pitch was a forced choice between two presented stimuli. Stimuli were presented to the participant who identified which most closely resembled their tinnitus. The test continued until a correspondence between the tinnitus and the presented stimulus was found. For the estimation of tinnitus loudness (intensity), the determined frequency (from the previous step) was presented at an intensity similar to the individual’s hearing threshold and gradually increased (5 dB steps) until it reached the closest matching to the participant’s tinnitus percept.The collection of the discomfort thresholds was performed for each ear individually on the frequencies tested in the tonal audiogram and the frequency at which the tinnitus was identified using pure tones, beginning at the hearing threshold, using an ascendant process with 5 dB increments. The patient was instructed to signal when the sound becomes uncomfortable, not only loud but also uncomfortable. Three tests should be carried out to investigate the thresholds to ensure the test’s reliability .The difference between the auditory threshold and the discomfort thresholds gave the dynamic auditory field . Once thThis test was performed at the frequencies where standard tonal audiometry was tested, using narrow-band noises or pure tones (where narrow-band noises did not mask tinnitus). The sound was presented in 5 dB steps (1–2 s stimulation), from hearing thresholds, until the participant reported that they could no longer hear their tinnitus. According to the spatial relationship of the resulting curves from hearing thresholds and tinnitus masking, Feldmann’s masking curves were categorized as follows: 1, Convergent; 2, Divergent; 3, Congruent; 4 Distant; and 5, Persistent .Residual inhibition or residual excitation was tested by presenting participants with a narrow-band noise centered at their tinnitus pitch, at 10 dB above the tinnitus loudness, for 1 min. RI was categorized as follows: 1, complete (tinnitus is not audible); 2, partial (tinnitus became quieter); 3, negative (no change at tinnitus percept); and 4, “rebound” effect (tinnitus became louder). In categories 1, 2, and 4, we measured the duration of time that tinnitus was abolished or diminished in seconds or minutes, the time that it takes for the tinnitus percept to come back to basal characteristics in terms of loudness .Self-reported tinnitus severity was measured using the Portuguese validated version of the THI . This inAuditory brainstem response examination was performed in a soundproofed electrically insulated room. The participant was placed in a comfortable position in order to ensure proper relaxation of cervical muscles. The Vivosonic audiometer system  was used to collect ABR and determine electrophysiological thresholds. The earphones used were the ER-3A, calibrated according to ANSI S3.6-1996, and a 4,000 Hz tone burst was used to evoke ABR, calibrated in decibel peak equivalent to the sound pressure level . We usedThe distortion product otoacoustic emissions were performed using a Vivosonic audiometer system in a soundproofed room. We tested the DPOAE for the frequencies of 500, 750, 1,000, 1,500, 2,000, 2,500, 3,000, 3,200, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 7,000, and 8,000 Hz, with a 1.22 F2/F1 ratio and with an intensity of 65 dB SPL and 55 dB SPL for L1 and L2, respectively. The presence of OAE was considered when the signal-to-noise ratio was equal to or above 6 dB. For all variables analyzed, an average for both ears was used.An exploratory analysis of all registered variables was carried out initially, followed by a data modeling phase. Categorical data were presented as frequencies and percentages, and continuous variables as median and inter-quartile range (25th percentile and 75th percentile), as they presented asymmetric distributions and deviations from normality.The nonparametric chi-square test or Fisher’s exact test was used for qualitative variables, and for the continuous variables, the Mann–Whitney U-test was applied.When clinically relevant, some of the variables were recorded. Additionally, the self-reported tinnitus severity score was recoded into a binary variable: Grades 1, 2, and 3 (Low THI score) vs. Grades 4 and 5 (High THI score).To assess the association between the presence of tinnitus or tinnitus severity and the demographic and audiological variables, univariable logistic regression analyses were performed. Odds ratios estimates ell test .Additionally, to evaluate the discriminative ability (tinnitus vs. non-tinnitus groups) of some of the audiological parameters, the area under the ROC (receiver operating characteristic) curve (AUC) was reported.The level of significance α = 0.05 was considered. Statistical analyses were performed in SPSS .n = 63, 51.6%), we obtained a median age of 63.0  years, while in men , the median age was 63.0  years.In this study, 122 participants were recruited with a median age of 63.0  years. In women   . In subgp = 0.033)  . When anp = 0.014), and for the 3,500 Hz (p = 0.049), 4,000 Hz (p = 0.013), 4,500 Hz (p = 0.017), 5,500 Hz (p = 0.014), and 6,000 Hz (p = 0.047)  . In moren = 92) are presented in The clinical characteristics of participants with tinnitus , followed by 22 with a moderate handicap (23.9%), 17 with slight or no handicap (18.5%), severe handicap in 14 participants (15.2%), and finally, 1 participant had a catastrophic handicap score.Although several variables were identified in the univariable study as potential candidates for the multivariable models of both “presence of tinnitus” and “tinnitus severity” outcomes, no multiple models were obtained because all of those identified variables became statistically non-significant when simultaneously considered.Six univariable logistic regression analyses were performed for the presence or absence of tinnitus, and the results are presented in p = 0.036), mean PTA thresholds (p = 0.009), HL (p = 0.014), and mean “HF” PTA thresholds (p = 0.001) increased the odds of tinnitus. However, some other variables represented lower odds of having tinnitus, including, the mean DPOAE between 500 and 800 Hz (p = 0.023) and the amplitude of ABR wave I (p = 0.016). HL was highly associated with tinnitus; “HF” PTA attained an AUC = 0.72, with 95% CI:0.61, 0.83.From this analysis, we found several variables that were associated with tinnitus. Noise exposure (p = 0.017), hyperacusis (p = 0.030), and residual inhibition (p = 0.035).A univariable analysis considering the severity of tinnitus as the outcome variable was performed. Two subgroups were considered, lower THI (slight or no handicap and mild handicap) and higher THI scores . Only the significant results pertaining to tinnitus severity are presented in Still considering participants’ characteristics , particiConcerning ABR evaluation, the logistic regression model showed that for each unit of increase in the mean ratio V/I of ABR, the probability of having at least moderate tinnitus was 10% higher This study identifies associations between audiological parameters and the presence of tinnitus. Additionally, variables characterizing these participants were analyzed, and associations with tinnitus severity were identified.We found statistically significant differences for both the mean PTA thresholds and “HF” PTA thresholds when we compared participants with and without tinnitus. Thus, there is a possible association between the development of HL and the appearance of tinnitus. These results are in agreement with the literature where it has been hypothesized that tinnitus is an epiphenomenon of a neuronal process to attempt normalizing impaired hearing thresholds, that is, a central compensation for peripheral damage . The agevia lateral olivocochlear efferent, which terminates on their endings, or there is a diffuse loss of ANFs sufficient to manifest a reduction in mean wave I amplitude. Concerning the severity of tinnitus, the logistic regression model showed that for each unit of increase in the mean ratio V/I of ABR, the likelihood of having at least moderate tinnitus was 10% higher. Since no statistically significant differences were found in the amplitude of wave V, we can infer that this finding is exclusively due to the values of the amplitude of wave I, thus also corroborating the various possibilities described above.Regarding the ABR, one of the findings was the reduction of the amplitude in wave I in tinnitus participants. The reduction of the wave I amplitude is in accordance with the published studies on tinnitus participants . From anAnother finding regarding the ABR is the statistical difference concerning the interpeak latency I-III when comparing Subgroup 1 (no HL or tinnitus) and Subgroup 2 (tinnitus but no HL). We can see a diminished interval interpeak I-III in the group with normal hearing with tinnitus. Interestingly, we did not find similar results in our literature review. However, although we did not find significant differences in absolute latency of wave I in our sample when we compared both subgroups, in Subgroup 2, wave I started later than in Subgroup 1. This could explain the difference in the interpeak latencies I-III when we compared both groups, since, according to several authors, in tinnitus participants, wave I has a significant prolongation . It has When we compared the four subgroups, considering HL and tinnitus, no significant differences were found in the levels of the DPOAE. However, when we compared participants without tinnitus and tinnitus, not considering the presence or absence of HL, we found significant differences . One of When we analyzed these findings in more detail, we noticed a decrease in DPOAE in the group with tinnitus compared to the group without tinnitus for all the frequencies where the results were statistically significant. According to In the participants of our sample, tinnitus frequency (pitch) ranged from 2,000 to 8,000 Hz, with 4,000 Hz being the most often found. This could be explained by the expected localization of the tinnitus pitch in the “edge” frequencies or within the lowest regions in participants presenting both HL, particularly sloping configurations as observed in our sample, and tinnitus . The tinRegarding Feldmann’s curves, the convergent (47.6%) and distant types (29.8%) are the most frequent, considering the sloping configuration in our sample. Our results are in accordance with the studies conducted by Regarding the severity of tinnitus, participants with an abrupt tinnitus onset were more likely to have at least moderate tinnitus. This immediate interpretation of the result is that people who have a gradual tinnitus onset develop natural habituation processes effortlessly . In analOur study confirms that in older people, tinnitus is positively associated with HL and noise exposure. Indeed, HL and noise exposure are risk factors for tinnitus.Nowadays, tinnitus is considered a symptom involving a network of peripheral and central pathways of the nervous system. Due to its complex nature, tinnitus should be approached in a multidisciplinary fashion involving various health professionals specialized in dealing with each of the dimensions encompassed within this symptom .Our study puts in evidence some interesting findings, especially concerning audiological tinnitus characteristics or its development. Our data may contribute to defining the patient’s odds of developing a severe or catastrophic grade of tinnitus.It was in the ABRs that we obtained the most exciting and promising results, namely, in the diminished I-III interval in participants without HL and no tinnitus compared to participants without HL and tinnitus, and the reduction of the amplitude in wave I in tinnitus participants compared with participants without tinnitus. Also, the increased amplitude of wave I has a protective factor to the odds of having tinnitus. Conversely, the increased ratio of V/I showed higher odds of developing at least moderate tinnitus. It should also be noted that there is a common denominator in all the findings in ABR, which is wave I. Future studies should be carried out with the main target of studying wave I in participants with tinnitus. If confirmed in more extensive population studies, these findings may be candidates as audiological biomarkers of tinnitus severity/presence. These are, indeed, the most original contributions of this study, since we have documented the relevant audiological tinnitus severity biomarkers.Regarding DPOAE, findings highlight the correlation between HL and tinnitus. We can say that participants with tinnitus and relevant noise exposure have lower DPOAE between 500 and 8,000 Hz than participants without tinnitus, and participants with higher DPOAE have a lower risk of developing tinnitus.Lastly, participants who had abrupt tinnitus onset and moderate or severe hyperacusis featured higher odds of at least moderate tinnitus.Notable highlights of our findings that could serve as potential audiological biomarkers, in particular, wave I amplitude, wave I absolute latency, and interwave latency interval I-III, suggest the necessity to have appropriate tinnitus subtyping to understand the most probable underlying mechanisms and consequently the most appropriate diagnosis and treatment strategies.Future research should be designed to improve the sensitivity of non-invasive electrophysiological measures of cochlear synaptopathy in humans and examine the broader neurophysiological impacts of noise exposure and devise a clear distinction between mechanisms more specific to tinnitus or HL. Advancing knowledge concerning potential tinnitus audiological biomarkers can be crucial for the adequate diagnosis and treatment of tinnitus.The original contributions presented in this study are included in the article/The studies involving human participants were reviewed and approved by Ethical Committee of CUF Infante Santo Hospital (26th November 2014), NOVA Medical School (no 65/2014/CEFCM). The patients/participants provided their written informed consent to participate in this study.HH conceived and designed this study and had contributions to all its stages. IP and AP performed the statistical analysis. HH, DR, SR, DH, NT, LB, and HC contributed equally to all other stages of the manuscript development, drafted, and revised the manuscript. DR worked with HH on the interpretation of results and created appendices. DR created all figures, abbreviations, references, and conceived the final version of the manuscript. JP, NT, and DH provided consultative advice and revised the final manuscript. All authors contributed to the article and approved the submitted version.