IntroductionBone tumors are a common pathology of the musculoskeletal system being frequently encountered by clinicians. Radiological workup is a mainstay in the diagnostic workup of bone tumors. This study aimed to highlight the importance of plain radiography and MRI in the diagnosis of bone tumors keeping histopathology as a gold standard. It is a descriptive validation study conducted in the Radiology Department of Pakistan Institute of Medical Sciences Islamabad.MethodologyThe study included 92 patients with suspected bone lesions. After taking a complete history and receiving informed written consent. X-rays radiographs and magnetic resonance imaging were performed. X-ray radiograph and magnetic resonance imaging parameters were recorded and compared with the histopathology of lesions as a standard.ResultsThe mean age of patients was 30.50 ± 8.95 years. Of 92 patients examined on X-ray, 51 (55.4%) had lytic lesions, 34 (37.0%) had sclerotic lesions, and seven (7.6 %) had mixed lesions. MRI revealed the location of the lesion. There were 25 (27.2%) bone lesions in diaphysis, 19 (20.7%) in metaphysis, nine (9.8%) at meta-diaphysis, and 32 (34.8 %) in the meta-epiphyseal region. These findings were later on confirmed with histopathological results.ConclusionMRI can differentiate soft-tissue components and periosteal reactions. An X-ray radiograph can provide information about bony matrix and calcifications within tumors. After analysis of imaging findings and histopathological results, it is concluded that these modalities can be used to diagnose bone tumors with high diagnostic accuracy. Bone tumors are caused by either abnormal growth of bone-like tissue or soft tissue in the bones. Tumors are classified into benign or malignant and primary or secondary. Globally malignant bony lesions have two age incidences (between 10-20 years and 40-80 years). They also have gender predilection, with the incidence in males 1.5% more than in females [The diagnostic evaluation of focal bone lesions involves information from the patient’s medical history , examination of the lesion, radiographic examination of the margins, degree of cortical expansion, periosteal reaction, and previous imaging. Primary bone tumors are overshadowed by metastatic cancers such as carcinoma and hematologic malignancies. MRI helps differentiate malignant bony lesions from benign lesions. In small lesions, MRI is helpful as small lesions can be picked up on a diffusion-weighted image (DWI) sequence, on which diffusion restriction indicates more toward malignancy. MRI is also helpful in imaging to follow up after treatment .Radiological assessment of bone lesions is difficult if specialized centers are not available. Conventional radiography plays a major role in bone tumor diagnosis. X-rays play their specific role in imaging diagnostic workups. Bone tumor aggressiveness is calculated by interpretation of its bony matrix on the radiograph. For local staging, MRI is considered the most effective method .An accurate diagnosis requires the patient’s age, location of tumor, pattern of their destruction/margins, aggressiveness, growth speed, matrix formation, periosteal reaction (inside or outside), cortical involvement, size, number, and appearance on MR imaging ,6,7. BenOur study compared imaging features of X-rays and MRI in bone tumors with histopathological findings taken as the gold standard.The study conducted was according to the Helsinki Declaration. It was a human study approved by the Ethical Review Board of Shaheed Zulfiqar Ali Bhutto Medical University (SZABMU)-approval F 1.1/2015/ERB/SZABMU/951. All adult participants provided written informed consent to participate in this study from July 2021 to October 2021. The inclusion criteria of this study are patients of all ages with mass lesions planned for an MRI scan. The exclusion criteria of this study are patients with a history of surgeries/known for any other malignancies or already taking chemotherapy.The sample size was calculated as 92 cases with the WHO sample size calculator using the following parameters: sensitivity 92%; specificity 87%; expected prevalence 50%; precision level 8%, and confidence level of 95% .After taking a complete history and informed written consent X-ray radiographs were conducted. Patients were positioned on the examination table; X-ray radiographs were taken with at least two projections anteroposterior and lateral views.Once the imaging was completed, X-ray findings were interpreted by a consultant radiologist (at least three years of post-fellowship experience) and looked for the presence or absence of a bony lesion. X-ray findings were compared with MRI.Data were recorded and analyzed through SPSS  (V. 25). For continuous variables, mean ± SD were calculated, and categorical data were presented as frequency(percentage). Using a 2*2 table diagnostic accuracy, sensitivity, and specificity were calculated.After acquiring informed consent, 92 participants were enrolled in the study. Their mean age was 31.3±9.61 years. There was a minimum age of 20 years and a maximum of 59 years. Of the 90 participants, 43 (46.7 %) were males, and 49 (53.3 %) were females. All 92 patients underwent X-rays and MRI, and results were recorded. Primary symptoms were recorded for all patients. Out of 92 patients, 42 (45.7 %) complained about pain, 24 (26.1 %) complained about swelling of joints, 15 (16.3 %) complained about weight loss, and 11 (12.0 %) complained about restricted movement, as shown in Table Diabetes was reported in 38 (41.3%) patients, hypertension in 21 (22.8%) patients, arthritis in 12 (13.1%) patients. All patients had their vital signs recorded; 70% of them had a mild or moderate fever. X-rays of 92 patients revealed 51 (55.4%) lytic lesions, 34 (37.0%) sclerosis, and only seven (7.6%) mixed lesions.In 92 patients, 25 (27.2%) had bone lesions in diaphysis, 19 (20.7%) in metaphysis, nine (9.8%) in meta-diaphysis, and 32 (34.8 %) in meta-epiphyseal region as shown in Table In all 92 patients who had histopathology performed, 81 were found to have malignant bone tumors, and 11 were found benign. MRI and X-rays have almost equal diagnostic accuracy. Through X-ray imaging, osteoporosis, periosteal reaction, sclerotization of lesions, calcifications, and ossification of bone can be evaluated  or biopsy with high diagnostic accuracy.


This study is aimed at investigating the correlation between lumbar spinal stenosis (LSS) severity, ligamentum flavum hypertrophy, and the upregulation of inflammatory markers. α, TGF-β1, and IL-1α were compared between the study group and the control group. The levels of TNF-α, TGF-β1, and IL-1α that were found to be positively expressed were compared between the mild, moderate, and severe groups. Patients with LSS had their ligamentum flavum thickness and their positive expression rates of TNF-α, TGF-β1, and IL-1α analyzed using Spearman correlation analysis. We evaluated the diagnostic utility of the positive expression rates of IL-α1, TGF-β1, and TNF-α and ligamentum flavum thickness in distinguishing the severity of LSS using a receiver operating characteristic (ROC) curve.  From March 2019 and May 2022, eighty-five inpatients with LSS were enlisted as the study's research group, while sixty-five patients hospitalized for lumbar intervertebral disc herniation over the same time period served as the study's control group. Moreover, mild, moderate, and severe subgroups of patients were created within the research population based on their LSS severity. The ligamentum flavum thickness and the positive expression rates of TNF-P < 0.05). However, no substantial disparity was observed in left lower limb pain, right lower limb pain, low back pain, lower limb sensation, muscle strength, and reflex abnormalities between the two groups (P > 0.05). Positive expressions of TGF-β1, TNF-α, and IL-1α and thicker ligamentum flavum were more prevalent in the LSS group than in the lumbar intervertebral disc herniation group. All indexes were significantly (P < 0.05) higher in the moderate stenosis group than in the severe stenosis group. Additionally, the thickness of the ligamentum flavum and the positive expression rates of TNF-α, TGF-β1, and IL-1α were higher in the mild and moderate stenosis groups than in the severe stenosis group. The expression levels of TNF-α, TGF-β1, and IL-1α were favorably linked with ligamentum flavum thickness (P < 0.05). ROC curve analysis showed that the thickness of ligamentum flavum, the expression of IL-1α, the expression of TGF-β1, and the expression of TNF-α could effectively diagnose mild, moderate, and severe LSS (P < 0.05).  The rates of both lower limb pain (40.00%) and intermittent claudication (80.00%) in the LSS group were higher than those in the lumbar disc herniation group , with statistical significance (α, TGF-β1, and TNF-α are closely linked to LSS, which can effectively identify mild, moderate, and severe LSS. Ligamentum flavum hypertrophy and positive expression rates of IL-1 Lumbar spinal stenosis (LSS) can cause lumbago and leg pain or lower extremity motor dysfunction in middle-aged and elderly people. The pathogenesis is mainly intervertebral disc bulge, protrusion or degenerative calcification, vertebral joint hyperplasia and ossification, ligamentum flavum hypertrophy, and calcification, which can lead to stenosis of the spinal canal, lateral recess, nerve root canal, or foramina, thus causing a series of symptoms such as compression of cauda equina nerve, blood vessels, and nerve roots in the dural sac or inflammatory reactions , 2. AnatP > 0.05). Eventually, MRI and CT scans were performed on every patient. Patients with LSS were categorized into those with mild stenosis (23 cases), moderate stenosis (45 cases), and severe stenosis (17 cases), depending on the number of stenotic segments in their arteries. The hospital's ethics board gave its stamp of approval to this research. Inclusion criteria include ① patients diagnosed with LSS or lumbar disc herniation by imaging examination, ② central stenosis, ③ complete lumbar spine imaging data, and ④ all patients who voluntarily participated in this study. Exclusion criteria include ① those who have suffered a spinal fracture or spinal cord damage, ② patients with developmental lumbar spinal stenosis, ③ patients complicated with spinal tumor, spinal deformity, and other spinal diseases, ④ patients with osteoarticular tuberculosis, osteomyelitis, and severe senile osteoporosis, and ⑤ patients with severe organ dysfunction.The selection included 85 LSS patients who received care at our facility between March 2019 and May 2022, and 65 patients with lumbar intervertebral disc herniation who were hospitalized during the same period were selected. There were 51 men and 34 females in the LSS group, with ages ranging from 40 to 75 (56.96 ± 7.05 years) and disease durations averaging 29.22 ± 9.46 months (range = 10 to 55). There were 40 men and 25 females in the lumbar disc herniation cohort; the age range was from 42 to 72 years old, with a mean age of 55.97 ± 7.38 years; the duration of disease was from 6 to 45 months, with a mean of 30.74 ± 8.01 months. Overall, there was comparable difference between the two groups' data  α, and interleukin- (IL-) 1α in the two groups were observed with Olympus light microscope (Japan) by two-person double-blind method. The sections of each group were placed under the same intensity light, and 5 visual fields were randomly selected from each section under a high-power microscope (×40). Judgment of immunohistochemical results: if there were brown or dark brown particles in the cytoplasm or membrane of the ligamentum flavum tissue and the staining intensity was higher than the background nonspecific staining, the cells were considered positive. If the nucleus was stained blue and there was no brown-yellow reactant in the membrane or cytoplasm, the cells were considered negative. Five complete and nonoverlapping high magnification fields were selected at the tissue edge of ligamentum flavum in each section, and the expression rate of positive cells in each field was measured. The average expression rate of 5 fields was taken as the final measurement value. Positive cell expression rate: 0 indicates no expression (-); <25% indicates slight expression (+); ≥25% indicates obvious expression (++). The expression degree and positive expression rate of three inflammatory factors including TGF-β1, TNF-α, and IL-1α in the ligamentum flavum of the two groups were compared.① Thickness measurement of ligamentum flavum: all patients underwent CT scanning at different lesion segments . The oblique diameter of the spinal canal was measured and compared to the thickness of the ligamentum flavum of both joint capsules to determine the disc height ratio at the superior vertebral arch notch. ② Lamina ligamentum flavum was removed during posterior spinal canal and lateral recess decompression, rinsed in 0.9% sodium chloride solution, fixed in 4.0% paraformaldehyde, embedded in paraffin, and cut into 5-8 pieces for immunohistochemistry staining. Immunohistochemical staining sections of transforming growth factor- (TGF-) β1, TNF-α, and IL-1α between the LSS group and the lumbar disc herniation group; ② comparison of ligamentum flavum thickness and expression quantity of TGF-β1, TNF-α, and IL-1α in mild, moderate, and severe stenosis groups; ③ correlation analysis between the severity of LSS and the expression quantity of TGF-β1, TNF-α, and IL-1α and the ligamentum flavum thickness; ④ the ROC curve analysis of TGF-β1, TNF-α, and IL-1α and ligamentum flavum thickness in differentiating the severity of LSS.The indicators are as follows: ① comparison of ligamentum flavum thickness and expression quantity of TGF-x ± s) format for our measurement data. When the data follows a normal distribution with consistent variance, the t-test is used. n and % were utilized to characterize the count data, and the chi-square test was performed to compare the two groups. All were two-sided tests. The relationship between the thickness of the ligamentum flavum and the levels of TGF-β1, TNF-α, and IL-1α in individuals with LSS was examined using Spearman correlation analysis. P < 0.05 was regarded as a statistically significant difference when analyzing the diagnostic value of ligamentum flavum thickness and the positive expression rates of TGF-β1, TNF-α, and IL-1α in determining the degree of LSS using the receiver operating characteristic (ROC) curve.Data for this study were entered into an Excel spreadsheet by two researchers working independently of one another and then analyzed and processed using SPSS 24.0. In this study, we used a mean ± SD (P < 0.05) between the two groups. Left lower extremity pain, right lower extremity pain, low back pain, lower extremity sensation, muscle strength, and reflex abnormalities were all similar across the two groups (P > 0.05). See Both lower extremity pain (40.00%) and intermittent claudication (80.00%) were more prevalent in the LSS group than in the lumbar disc herniation group, with a statistically significant difference (P < 0.05) increase in the TGF-β1, TNF-α, and IL-1α positive expression when compared to the group with lumbar intervertebral disc herniation . There were statistically significant (P < 0.05) increases in ligamentum flavum thickness, TGF-β1, TNF-α, and IL-1α positive expression, and each index in the moderate stenosis group compared to the severe stenosis group, as shown in Figures There were significant differences in the ligamentum flavum thickness and the TGF-α, TGF-β1, and TNF-α (P < 0.05). See The ligamentum flavum thickness was positively correlated with the expressions of IL-1β1, TNF-α, and IL-1α expression could effectively diagnose mild, moderate, and severe LSS (P < 0.05). The AUC values of ligamentum flavum thickness and expression quantity of TNF-α, TGF-β1, and IL-1α in the identification of mild to moderate LSS were 0.986, 0.825, 0.765, and 0.976, respectively, and the cut-off values were 4.67, 89.18, 73.93, and 42.14, respectively. The AUC values of ligamentum flavum thickness and expression quantity of TNF-α, TGF-β1, and IL-1α in the identification of moderate to severe LSS were 0.835, 0.742, 0.890, and 0.891, and the cut-off values were 6.62, 94.66, 80.89, and 50.55, respectively. See Tables ROC curve analysis showed that the ligamentum flavum thickness and TGF-S P < 0.0. The AUCS P < 0.0. The AUCα can promote the formation of inflammation and cause pain. IL-1α is a predisposed factor for the thickening and ossification of ligamentum flavum, which can affect ligamentum flavum by stimulating nerves, degrading collagen, and mediating cycloxygenase-2 metalloproteinase inhibitors. The positive expression rate of IL-1α in the LSS group was substantially greater than that in the control group in this research, which may be related to chronic mechanical effects and increased pain in patients with LSS, and pain is caused by stimulation of nerve fibers. The ligamentum flavum was thicker and fibrotic in the LSS group, with fibroblasts serving as the predominant cell type, so the positive expression of IL-1α was increased. According to relevant studies, IL-1α is highly expressed in the intervertebral disc and lumbar facet joint and patients with lumbar stenosis tend to have a more active ligamentum flavum, and this activity is positively connected with lumbar region and leg pain [β1 is closely related to fibroblast proliferation [β1 can change the growth characteristics of fibrocytes and reduce the contact inhibition effect in the process of fibrocyte growth. It can not only promote cell proliferation but also promote the synthesis of extracellular matrix. Since TGF-β1 can cause a variety of tissue hypertrophy, which is closely related to a variety of fibrotic diseases, the mechanism of TGF-β1 promoting ligament flavum hypertrophy lies in inducing fibroblast differentiation into myofibroblasts, which can promote the fibrosis of ligament flavum tissue by promoting the production of type I and III collagen [β1 in inducing ligamentum flavum hypertrophy is to promote the proliferation and differentiation of fibroblasts and the formation of collagen fibers. TNF-α mainly affects the metabolism of collagen fibers in the ligamentum flavum matrix. TNF-α is an inflammatory factor secreted by macrophages, which is an initiating factor of inflammatory cascade reaction and can reflect the severity of inflammation [α content is closely related to the effect of macrophage migration inhibitory factor (MIF), which is an upstream factor of TNF-α and can promote the expression of TNF-α in fibroblasts and stroma. In addition, MIF can indirectly promote the expression of TNF-α by inhibiting the migration of macrophages [α due to its ability to stimulate the production of downstream metalloproteinases through positive feedback. Ligamentum flavum has both type I and type III collagen fibers. Hypertrophy of the ligamentum flavum is associated with TNF-α because TNF-α may increase the production of type III collagen.Hypertrophy and ligamentum flavum hypertrophy may cause a narrowing of the spinal canal , as well as hyperplasia and cohesiveness of the bilateral facet joints and the superior facet joints, all of which increase the chances of LSS –11. Patileg pain , 15. Hypleg pain , 17. It feration , 19. TGFcollagen . Therefoammation , 22. Accrophages , 24. In β1, TNF-α, and IL-1α positive expression. Each indicator was greater in the moderate stenosis group than the severe stenosis group, and the differences between the groups were statistically significant (P < 0.05). Positive expression rates of TNF-α, TGF-β1, and IL-1α increased with increasing stenosis severity, and the findings demonstrated that these cytokines were strongly expressed in the LSS group. The degree of LSS was correlated with an increase in ligamentum flavum thickness, which was considerably greater in the LSS group than in the lumbar disc herniation group. Clinical studies have shown that corticosteroids and nonsteroidal analgesic and anti-inflammatory drug COX-2 inhibitors can relieve clinical symptoms by inhibiting inflammatory response, and patients with LSS often have ligamentum flavum hypertrophy. In this work, we found that LSS was closely linked with the TGF-β1, TNF-α, and IL-1α positive expression rate as assessed by Spearman's correlation analysis (P < 0.05). Previous research has shown that TNF-α and IL-1α are both substantially expressed in the intervertebral discs, lumbar facet joints, and ligamentum flavum of individuals with LSS and that this expression is connected with neurological dysfunction and lumbar and leg pain [α can enhance the synthesis of pain mediators prostaglandin and 5-hydroxytryptamine. Prostaglandin E2 is an important transmitter of inflammatory response, which can enhance the sensitivity of nerve terminal receptors, thus enhancing the pain effect. IL-1α induces nerve root pain through mediating other inflammatory mediators, eventually leading to lumbar and leg pain. TNF-α can induce inflammatory reactions such as endoneurial edema, decreased blood flow, intravascular coagulation, and medulla decomposition, which can play a role in the regulation of nerve root sensitivity and cause lumbar and leg pain. Lumbar and leg pain is the main symptom of LSS, which can also explain that the expression of inflammatory factors increases with the degree of LSS.In this study, in patients with LSS, the ligamentum flavum was thicker than in those with lumbar disc herniation, and the LSS group also had higher levels of TGF-leg pain , 26. IL-α, TGF-β1, and IL-1α could effectively diagnose mild, moderate, and severe LSS (P < 0.05). The AUC values of ligamentum flavum thickness and expression quantity of TNF-α, TGF-β1, and IL-1α in identifying mild to moderate LSS were 0.986, 0.825, 0.765, and 0.976, respectively, and the cut-off values were 4.67, 89.18, 73.93, and 42.14, respectively. The AUC values of ligamentum flavum thickness and expression quantity of IL-1α, TGF-β1, and TNF-α in identifying moderate to severe LSS were 0.835, 0.742, 0.890, and 0.891, respectively, and the cut-off values were 6.62, 94.66, 80.89, and 50.55, respectively. For patients with mild LSS, rest, medication, and relevant symptomatic treatment are required, while surgery may be required for patients with moderate and severe LSS. Early identification of patients after admission is beneficial to quickly determine the treatment plan, and appropriate treatment plan can promote the rational utilization of medical resources and improve the prognosis of patients, suggesting that the thickness of ligamentum flavum can be measured in patients with LSS after admission. In this study, the TGF-β1, TNF-α, and IL-1α positive expression rate was determined by postoperative immunohistochemistry, which can reveal the expression patterns of inflammatory factors in patients with LSS.ROC curve analysis results of this study showed that the thickness of ligamentum flavum and the expression quantity of TNF-α, TGF-β1, and IL-1α. Ligamentum flavum hypertrophy and the TGF-β1, TNF-α, and IL-1α positive expression rate can effectively distinguish the LSS severity.In conclusion, the severity of LSS is closely correlated with ligamentum flavum hypertrophy and the expression quantity of TNF-


Background and Objectives: The Greek Society of Migraine and Headache Patients conducted, in 2020, its second online survey, titled “Migraine in Greece—2020”, after publication of the first similar online survey conducted in 2018. To compare the current findings with the corresponding data obtained in 2018, we herein release the second part of results obtained from the 2020 survey on the efficacy of preventive and symptomatic anti-migraine medications and the patients’ reported satisfaction with these treatments. Materials and Methods: We surveyed 2105 migraine patients from all over Greece with the use of a 151-questions specific migraine-focused questionnaire in Greek language, which was distributed through the online research software “SurveyMonkey”. Results: Triptans were mostly used with efficacy for the symptomatic relief of migraine attacks. About 2 of 3 surveyed patients had received various prophylactic oral medications and the majority of them discontinued these prophylactic medications as a result of inefficacy/safety issues. BoNTA was reported to be effective only when administration was commenced by a trained neurologist/headache specialist, while our current findings are generally comparable to those obtained in our 2018 pre-COVID-19 survey and the pandemic has not imposed any significant attitudes on migraine therapies and corresponding patients’ satisfaction. Conclusion: Although a market change is anticipated with the evolving widespread use of anti-CGRPs monoclonal antibodies or gepants in the symptomatic and prophylactic treatment of migraine, it is of great interest to review published results of larger longitudinal population-based studies to further ascertain the satisfaction of patients to migraine therapies. Migraine, clinically classified as either episodic  or chronic migraine , is a common primary headache disorder imposing a significant burden on patients , there iThere are significant unmet clinical needs in relation to migraine acute treatment, but mostly with its prophylaxis. Several oral non-specific preventative medications are currently used as first line treatments in both EM and CM, including antihypertensives, antiepileptic drugs, beta and calcium channel blockers, and various antidepressants . HoweverAlthough previous population-based observational studies conducted with the use of Internet- or telephone-led surveys from USA or Europe ,12,13,14Globally, there are associations of patients with migraine that aim to raise public and state awareness on headaches and treatment options through publication of results from population web-based surveys. Towards the latter view, the non-profit Greek Society of Migraine and Headache Patients (GSMHP), which was established in 2017, conducted in 2020 (year of COVID-19 pandemic) its second online survey, titled “Migraine in Greece—2020”, after publication of the first similar online survey in 2018. The 2020 web-based survey included 2105 migraine patients, who were called to answer 151 questions, covering the whole spectrum of demographics, clinical phenotype, and treatment of patients with migraine.We recently published the first part of results concerning the demographic and clinical characteristics of participants, the severity and effects of migraine on their quality of life, and the effects of the coronavirus pandemic on the course of migraine . In an aThe data collection occurred over a period of two months from June 1 to July 31 2020. The methods for conducting this survey are described in detail in our recent publication containing the first part of its results concerning the demographic and clinical characteristics of participants, the severity and effects of migraine on their quality of life, and the effects of the coronavirus pandemic on the course of migraine . The samBriefly, candidate participants from all over Greece  were randomly called to reply to a 151-questions specific migraine-focused questionnaire in Greek language, which was distributed through the online research software “SurveyMonkey”. The target group included GSMHP members , but not exclusively, as the survey was promoted through GSMHP’s social media accounts . In 2020, GSMHP formally had 1812 members and 10,000 followers in its social media accounts. Eventually, this survey included adult patients with a definite diagnosis of migraine in a two-stage procedure, i.e., (i) the screening interview (first stage), wherein demographics and residency were first collected and then participants had to reply to a screening question expressed as “do you have a definite diagnosis of migraine established by a physician or do you have clinical symptoms resembling migraine but not formally diagnosed by a physician?”, whereas afterwards, they completed key clinical questions to ascertain whether the diagnostic criteria for a definite migraine diagnosis were fulfilled .The study was performed in accordance with the principles of the Declaration of Helsinki, while its protocol was approved from the ethics committee of Euromedica General Clinic, Thessaloniki, Greece. At the beginning of the study procedure, each patient had to read a mandatory consent form so as to be informed about the objectives, which was in agreement with regulations of data protection, and clearly note her/his agreement in order to be further allowed to anonymously complete the web-based survey.p-value were used to compare proportions. Significance was set at the p < 0.05 level.Descriptive statistics, performed with the SPSS for Windows , generated categorical variables (observed counts and weighted percentages) and continuous variables (mean or median with the corresponding standard error or range), depending on their nature. The Mann–Whitney U test for two samples was used for non-parametric comparisons, whereas the Chi-square one-sample test and Chi-square with Yates corrected N = 286; 58.8%), followed by hypo/hyperthyroidism . A total of 1248 (83.5%) participants had EM, either very low, low or high frequency, while 247 (16.5%) were with CM. Among the total of 2105 participants, 1550 of them had been diagnosed by a physician (group A), and 555 cases were allocated to group B as they were at baseline with clinical migraine symptoms but lacked a formal migraine diagnosis established by a medical professional. Group B patients were without any other evidence of suffering from another type of headache disorder.As previously mentioned, the study enrolled 2105 patients who after completion of the first diagnostic part of the questionnaire fulfilled the diagnostic criteria for migraine, according to both the ICHD-3 symptom criteria  and physWe obtained the following data regarding the efficacy of preventive and symptomatic anti-migraine medications and the patients’ reported satisfaction to these treatments.Among patients with established migraine diagnosis by a physician, 815 patients (57.23%) had received various prophylactic treatments. The answers to the multiple-choice question “Which prophylactic treatment(s) have you received at least once are shown (drug class)?” is described in Nonetheless, the majority of participants (64.2%) prioritized safety over effectiveness in medications prescribed for their migraine prophylaxis.A total of 490 (60.20%) of the responders had received antiepileptics for their migraine prophylaxis, either topiramate and/or valproic acid. Of those, 139 (28.95%) were not happy at all with this particular prophylaxis, 151 (30.81%) were modestly happy, 113 (23.06%) were fairly happy, 43 (8.77%) were very happy, and 28 (5.71%) were extremely happy. When asked “Did you stop taking antiepileptics for some reason?”, 345 (70.4%) answered positively. Of those, 227 (65.8%) stopped due to ineffectiveness and 190 (55.07%) because of side effects such as weight loss or gain, agitation, dizziness, somnolence, numbness, and clumsiness or unsteadiness.In total, 503 (61.8%) participants had been treated with antidepressants as a preventive treatment, 182 (36.3%) with SSRIs or SNRIs  and 128 (25.5%) with tricyclics (amitriptyline). Of those, 113 (22.46%) were not happy at all with the corresponding effects, 151 (30.02%) were modestly happy, 104 (20.67%) were fairly happy, 45 (8.94%) were very happy, and 19 (3.77%) were extremely happy. To the question “have you stop antidepressants for some reason?”, 273 (54.27%) answered positively. Among them, 190 (63.59%) stopped the antidepressants due to ineffectiveness and 146 (53.47%) due to side effects such as weight gain, agitation, dizziness, somnolence, and indigestion/stomach aches.Prophylactic treatments with either beta blockers (propranolol) or calcium channel blockers (flunarizine) were commenced in 221 (27.07%) and 329 (40.42%) patients, respectively. The vast majority (90%) of propranolol-treated patients discontinued treatment due to side effects, such as bradycardia, irritability, and mood disorders, while ineffectiveness was the main cause of flunarizine intake, coupled with weight gain as a side effect.Among the 158 (19.41%) responders with CM who had been treated with BoNTA, only 99 (62.65%) answered that their treatment was performed with the approved fixed site-fixed dose PREEMPT protocol by a trained neurologist. The remaining 59 patients received BoNTA by dermatologists or ENT physicians at arbitrary and unapproved injection sites, i.e., just in forehead, and dosage. To the question “How happy are you with the effect of BoNTA treatment in migraine prophylaxis”, 42 (26.58%) were not happy at all (37 of them were treated by a non-neurologist physician), 57 (36.07%) were modestly happy, 30 (18.98%) were fairly happy, 15 (9.49%) were very happy, and 12 (7.59%) were extremely happy. The question “Have you stopped your treatment with BoNTA for any reason?” was answered positively by 87 (55.06%) of the responders. When asked (multiple choice) “Why did you stop your treatment with BoNTA?”, 83 (95.4%) responded “due to ineffectiveness” and 4 (4.6%) responded “due to side effects”, including neck pain and eyelid ptosis.® , the question “How happy are you with the way you are treating your migraine with Cefaly®” was answered as “not happy at all” by 23 (45.1%), “modestly happy” by 13 (25.49%), “fairly happy”, by 14 (27.45%), “very happy” by 0 (0%), and “extremely happy” by 1 (1.96%). When asked “Have you stopped treatment with Cefaly® for any reason” 38 (74.5%) answered positively. A total of 35 (92.10%) of those discontinued “due to lack of effectiveness” and 3 (7.9%) “due to side effects” .Among migraine responders that had been treated with the only readily available in Greece external neurostimulation device CefalyA relative low number of patients  had received anti-calcitonin gene related peptide (CGRP) monoclonal antibodies (anti-CGRP MAbs), because in Greece, fremanezumab was approved for reimbursement by the Greek National Health System (NHS) and social services on July 2021 and erenumab followed on February 2022 . Anti-CGRP MAbs-treated participants were questioned (multiple choice) “How did you learn about the new anti-CGRP MAbs treatment?”. The majority of them  responded “by my doctor”, 6 (24.0%) “by the GSMHP” and 2 (8.0%) “through the Internet”. When asked “How happy are you with the way you are treating your migraine with the anti-CGRP MAbs?” 2 (8%) answered “not happy at all”, 7 (28%) “modestly happy”, 4 (16%) “fairly happy”, 4 (16%) “very happy”, and 8 (32%) “extremely happy”. The question “Have you stopped treatment with monoclonal antibodies for any reason?” was answered negatively by 15 (60.0%), and positively by 10 (40.0%). When asked (multiple choice) “Why did you early stop taking anti-CGRP MAbs” 5 (50.00%) answered “due to high cost”, 4 (40.00%) “due to lack of effectiveness” and 1 (10.00%) “due to side effects”.When participants of both groups A and B were asked “Have you tried at least once some kind of prophylactic complementary and alternative medicine treatment ?”, 1032 (49.02%) of them answered negatively and 1073 (50.98%) answered positively. Of those 1073 participants, 404 (37.65%), 337 (31.4%), 254 (23.66%), and 78 (7.27%) had tried acupuncture, homeopathy, dietary supplements, and reflexology, respectively. (Question (multiple choice): “Which types of prophylactic CAMT treatment have you tried at least once?”).A total of 165 (40.84%), 118 (29.20%), 80 (19.80%), 26 (6.43%), and 15 (3.71%) participants were “not happy at all”, “modestly happy”, “fairly happy”, “very happy” and “extremely happy”, respectively, with acupuncture. (Question “How happy are you with the way you are treating your migraine with acupuncture?”). A total of 335 (82.92%) participants stopped acupuncture due to ineffectiveness. (Question “Have you stopped your acupuncture treatment for any reason?”).In all, 120 (35.60%), 124 (36.79%) 49 (14.54%), 27(8.01%), and 17 (5.04%) participants were “not happy at all”, “modestly happy”, “fairly happy”, “very happy”, and “extremely happy”, respectively, with homeopathy. Question: “How happy are you with the way you are treating your migraine with homeopathy?”. In total, 273 (81.0%) participants had stopped homeopathy due to ineffectiveness. Question: “Have you stopped your homeopathy treatment for any reason?”.Among 254 patients using dietary supplements and nutrients, 180 (70.87%), 41 (16.10%), 29 (11.40%), and 4 (1.57%) used magnesium, vitamin complexes, cannabidiol products, or another supplement, respectively. (Question (multiple choice) “Which dietary supplements do you use?”). In the responses, 69 (27.16%), 110 (43.3%), 54 (21.25%), 19 (7.48%), and 2 (0.78%) participants were “not happy at all”, “modestly happy”, “fairly happy”, “very happy”, and “extremely happy” with a dietary supplement, respectively. (Question “How happy are you with the way you are treating your migraine with dietary supplements?”). A total of 130 (51.18%) participants had stopped dietary supplements due to ineffectiveness. .In the sample, 1428 (95.71%) participants from group A and 450 (90.36%) from group B systematically used symptomatic treatment. (Question “Do you receive symptomatic treatment (painkillers) during crisis?”). The answers to the multiple-choice question “During migraine attacks, what symptomatic treatment do you receive?” are shown in When asked “How long after the onset of migraine crisis do you receive your symptomatic treatment?”, 883 (62.45%), 373 (26.38%), and 158 (11.17%) group A patients as well as 216 (48.65%), 117 (26.35%), and 111 (25.00%) group B patients answered “20 min”, “1 h”, and “over 1 h”, respectively. To the question “How many days per week, on average, have you received any acute symptomatic treatment during the last trimester?”, 812 (57.43%), 487 (34.44%), and 115 (8.13%) group A patients and 339 (76.35%), 93 (20.95%), and 12 (2.70%) group B patients answered “6–7 times”, “0–2 times” and “3–5 times” per week, respectively. When asked “How many pills per week did you take, on average, to treat migraine during the last trimester?”, 517 (36.56%), 456 (32.25%), 288 (20.37%), and 153(10.82%) group A patients and 221 (49.77%), 135 (30.41%), 63 (14.19%), and 25 (5.63%) group B patients answered “0–2”, “3–4” “5–8” and “over 8” pills per week, respectively.In all, 618 (43.99%) patients of group A and 281 (63.43%) patients of group B reported systematic consumption of various simple analgesics and NSAIDs, such as paracetamol, ibuprofen, and naproxen. When asked “How happy are you with the way you are treating your migraine with simple analgesics and NSAIDs”, 87 (14.12%), 285 (46.27%), 204 (33.12%), 32 (5.19%), and 8 (1.30%) group A patients and 23 (8.16%), 114 (40.43%), 118 (41.84%) 20 (7.09%), and 7 (2.48%) group B patients were “not happy at all”, “modestly happy”, “fairly happy”, “very happy”, and “extremely happy”, respectively. Nonetheless, 365 (59.25%) group A patients and 199 (70.57%) group B patients continued taking simple analgesics and NSAIDs for symptomatic relief of acute migraine attacks. . When asked (multiple choice) “Which was the reason(s) for discontinuing intake of simple analgesics and NSAIDs” 232 (92.06%) group A patients as well as 76 (93.83%) group B patients answered “due to ineffectiveness”.A total of 660 (47.76%) patients from group A and 255 (59.03%) patients from group B reported using some combination of paracetamol and caffeine. When asked “How happy are you with the way you are treating your migraine with a paracetamol/caffeine combination?”, 28 (4.24%), 220 (33.28%) 322 (48.71%), 64 (9.68%), and 27 (4.08%) group A patients and 5 (1.98%), 64 (25.40%), 137 (54.37%), 34 (13.49%), and 12 (4.76%) group B patients answered “not happy at all”, “modestly happy”, “fairly happy”, “very happy”, and “extremely happy” with simple analgesics, respectively. When asked “Have you stopped taking an analgesic/caffeine combination for any reason?”, 510 (77.16%) group A patients and 202 (80.16%) group B patients answered negatively, respectively. When asked (multiple choice) “Why did you stop taking a paracetamol/caffeine combination?” 111 (73.51%) and 50 (33.11%) group A patients and 35 (70.0%) and 20 (40.0%) group B patients answered “due to ineffectiveness” and “due to side effects”, respectively.p < 0.001). When asked “How happy are you with the way you are treating your acute migraine with triptans?”, 20 (2.35%), 96 (11.28%), 359 (42.19%), 247 (29.02%), and 129 (15.16%) group A patients and 1 (1.25%), 13 (16.25%), 38 (47.50%), 14 (17.50%), and 14 (17.50%) group B patients answered, “not happy at all”, “modestly happy”, “fairly happy”, “very happy”, and “extremely happy” with simple analgesics, respectively. When asked “Have you stopped taking triptans for any reason?” 658 (77.32%) group A and 74 (92.50%) group B patients answered negatively. To the multiple-choice question “Why did you stop taking your triptan?”, 118 (61.14%) and 97 (50.26%) group A patients and 4 (57.14%) and 3 (42.86%) group B patients answered “due to side effects” and “due to ineffectiveness”, respectively.Triptans use was reported by 850 patients from group A and 79 patients from group B , compared to pharmacological approaches. Age effects were also noted in relation to the acute and prophylactic pharmacological medication intake, which was much lower in the 18–35 age group, compared to older patients, especially to those in the 45–59 age group . We were not able to detect any other notable effect of demographic and baseline clinical characteristics on treatment preferences.When we examined potential associations of demographic and baseline clinical characteristics with both acute and prophylactic treatment preferences, we found gender effects with males favoring CAM approaches three times more than females (odds ratio (OR) 2.9, With the release of the second part of results from the current population web-based survey conducted by GSMHP in 2020, we primarily aimed to trace potential treatment differences of patients with migraine in Greece, according to study groups , focusing on the assessment of efficacy of preventive and symptomatic anti-migraine medications as also the satisfaction of patients to these treatments, in terms of safety/efficacy. In addition, we sought to compare the relevant results of the 2020 survey with those previously obtained in 2018 .Our results, overall, point towards the view that patients whose migraine was diagnosed by a neurologist/headache expert (group A) had higher disability and were more difficult to treat than patients belonging to group B, a fact which most likely contributed to ask specialized consultation. Amongst formally diagnosed migraine patients (group A), 815 (57.23%) have received prophylactic anti-migraine treatment with orally and/or injectable-administered medications, a percentage that is comparable to the corresponding findings of the 2018 survey in which 58.2% of participants received various prophylactic treatments . This coSpecifically for migraine prophylaxis with the use of antiepileptic or antidepressant medications, it was evident that about 60% of 815 participants from group A had received either topiramate/valproic acid or SSRIs/SNRIs/tricyclics, with modest efficacy/safety as the majority of them (61%) remained null to minimally satisfied from these medications due to inefficacy (about 70%) or evidence of side effects (about 55%). Very or quite satisfied from antiepileptics/antidepressants only represented 15% of our interviewed patients. About 65% (2 out of 3) of our participants discontinued these prophylactic medications as a result of inefficacy/safety issues, findings which are in close agreement with data demonstrating that up to 55% of migraine patients discontinue oral pharmacological preventive approaches after 12 months of treatment , becauseOnly a small percentage of group A participants was treated with BoNTA (19.4%) and even fewer patients with anti-CGRP MAbs (3%). The satisfaction with these treatments was rather modest, although tellingly, these findings were strongly biased to allow generalization. Specifically, for BoNTA, it was evident that about 40% of exposed patients were treated by other medical specialties, instead of neurologists/headache experts, with schemes not adhering to the approved fixed-site, fixed-dose (155–195 UI) PREEMPT protocol . As suchThe findings on the exposure in CAMT are rather surprising to us, as it was evident that a rather high percentage (about 45%) of our participants received nutrients or was treated with homeopathy or acupuncture, although 80% of them discontinued these unapproved approaches due to ineffectiveness.Regarding the use of acute treatments for the symptomatic relief of migraine attacks, it was evident that the majority of patients who were diagnosed and regularly followed by a neurologist/headache expert (group A) used triptans, as opposed to the much lower percentage of triptans use in group B patients (61.06% in group A vs. 18.08% in group Β). Moreover, group A patients seem to have comprehended the clinical benefits of early triptan use for the adequate symptomatic relief of migraine attack  as a sigAs previously reported, although the data for this survey were collected during the COVID-19 pandemic, the majority of participants mentioned that they did not experience any effect on their migraine, including in treatment attitudes . This isWe should acknowledge as a limitation of our study the sampling process we applied to target advocacy group members with diagnosed migraine (group A) via a web-based survey and a questionnaire which is not validated or subjected to any form of psychometric evaluation, instead of a prospective follow up in our headache outpatients’ clinics. This approach restricted us from collecting more objective data. Moreover, the cross-sectional study design and the inclusion of participants with a tentative diagnosis of migraine at baseline (group B) may also further bias the interpretation of our findings.Taken together, our results overall demonstrate that about 2 of 3 surveyed patients had received various prophylactic oral medications and the majority of them discontinued these prophylactic medications as a result of inefficacy/safety issues. BoNTA was reported to be effective only when administration was commenced by a trained neurologist/headache specialist, while our current findings are generally comparable to those obtained in our 2018 pre-COVID-19 survey  and the


Therapeutic options for advanced salivary gland cancer (SGC) are rare. Therefore, it was the aim of this study to investigate the extent and intensity of Mucin-1 (MUC1), Mucin-16 (MUC16), and Mucin-5AC (MUC5AC) as potential molecular targets using immunohistochemistry. The medical records of all patients who underwent primary surgery for salivary gland cancer with curative intent in a tertiary referral center between 1990 and 2018 were reviewed. Immunohistochemical staining for MUC1, MUC16, and MUC5AC was performed for all patients with sufficient formalin-fixed paraffin-embedded material, and a semi-quantitative combined score derived from the H-score for the cytoplasmatic, the membranous and the apical membrane was built for the most common entities of SGC. 107 patients with malignancies of the parotid (89.7%) and the submandibular gland (10.3%) were included. The most common entities were mucoepidermoid carcinoma , adenoid cystic carcinoma , and salivary duct carcinoma . The highest mean MUC1 combined score was found in SaDu with 223.6 (±91.7). The highest mean MUC16 combined score was found in MuEp with 177.0 (±110.0). The mean MUC5AC score was low across all entities. A higher MUC1 combined score was significantly associated with male gender (p = 0.03), lymph node metastasis (p < 0.01), lymphovascular invasion (p = 0.045), and extracapsular extension (p = 0.03). SaDu patients with MUC16 expression showed a significantly worse 5-year progression-free survival than those without MUC16 expression (p = 0.02). This is the first study to give a comprehensive overview of the expression of MUC1, MUC16, and MUC5AC in SGC. Since advanced SGCs lack therapeutic options in many cases, these results warrant in vitro research on therapeutic targets against MUC1 in SaDu cell lines and xenograft models. Salivary gland carcinomas (SGC) are rare, representing 5–8% of all malignant tumors in the head and neck region , 2. MostGenerally, the therapy of choice for SGC in absence of distant metastasis is surgical tumor resection. Furthermore, an ipsilateral neck dissection is recommended in advanced-stage carcinomas (T3–4), in high-grade carcinomas, and in carcinomas with clinical, sonographic, or radiological suspicion of loco-regional nodal involvement , 8. In aMucins are a family of glycosylated proteins produced by epithelial tissues in humans. Mucin-1 (MUC1), also known as CA 15–3, is a type I transmembrane glycoprotein with a cytoplasmatic tail serving as an adaptor protein connected with kinases and further cell signaling proteins leading to cell proliferation, infiltration into the extracellular matrix, deregulation of apoptosis and changes in the adhesion state of the cell . It has Mucin-16 (MUC16), also known as CA-125, is another type 1 transmembrane glycoprotein, routinely used as a tumor marker in ovarian cancer and found in the epithelia of several organs such as the trachea, the ocular surface, the abdominal cavity, and the female reproductive tract . MUC16 wIn opposite to type 1 transmembrane MUC1 and MUC16, Mucin-5AC (MUC5AC) is a type 2 secreted mucin mainly found in the mucus of the respiratory tract. A significant association between MUC5AC and a worsened survival in adenocarcinoma of the lung has been reported in multiple studies , 26. In The aim of the current study was to investigate the expression of the potential molecular targets MUC1, MUC16, and MUC5AC in different entities of SGCs using a combined score of apical, membranous, and cytoplasmatic expression in immunohistochemistry.All patients with primary salivary gland carcinoma of the parotid or submandibular gland who had undergone primary surgery with curative intent at the Department of Otorhinolaryngology, Head and Neck Surgery, University of Cologne between 1990 and 2018 with sufficient formalin-fixed paraffin-embedded (FFPE) material of the primary tumors were included in the analysis. The study was performed according to the regulations of the Ethics Committee of the University of Cologne.MYB, MYBL1, PRKD1, PRKD2, PRKD3, EWSR1, MAML2 and ETV6 genes as well as Sanger sequencing of PRKD1 hotspot mutations [Demographic characteristics and histopathological data were retrieved from clinical records and histopathological reports with respect to tumor characteristics including the stage of disease at the time of diagnosis according to the AJCC TNM staging system  . Four tiutations .Briefly, tissue slides were stained with antibodies against MUC1 , MUC16 , and MUC5AC . All IHC stainings were carried out with a Leica BOND-MAX stainer  in accordance with the manufacturer’s protocol. Counterstaining was done using haematoxylin and bluing reagent.First, all TMA sections were screened to explore different staining patterns. Cytoplasmatic, membranous and apical  staining patterns were observed. Then, two pathologists with special expertise in the field of SGC  assesed the expression of each pattern using the Histo-score (H-score) , which wStatistical analyses were performed using SPSS software version 28.0 . Distribution was tested using the Shapiro–Wilk-Test. Spearman’s rank correlation coefficient was used to measure the strength of correlation between non-normally distributed variables. The Mann–Whitney–U-test was used to compare differences between independent groups for metric, non-normally distributed variables. Kaplan–Meier method with 95% confidence intervals was used to test for progression-free survival (PFS) probability rates. For this, statistical significance was tested by using the log-rank test. PFS was defined as the time interval between the end of treatment and the date of progression of the specific disease. A p-value < 0.05 was considered statistically significant. R studio (version 2021.09.1) was used for visualization of box plots (ggplot2 package).Overall, 107 patients with primary cancer of the parotid-  and submandibular gland  were included , followed by adenoid cystic carcinoma , salivary duct carcinoma , acinic cell carcinoma , adenocarcinoma NOS , epithelial-myoepithelial carcinoma , and secretory carcinoma . Other rare entities  were basal cell carcinoma , myoepithelial carcinoma , oncocytic carcinoma , carcinoma ex pleomorphic carcinoma , carcinosarcoma , and polymorphous adenocarcinoma . Additional histopathological data is presented in Table A significantly positive correlation was found between cytoplasmatic and membranous staining in MUC1, MUC 16, and MUC5AC . Further, a significantly positive correlation was found between cytoplasmatic and apical staining in MUC1 and MUC 16 . A significantly positive correlation was found between apical and membranous staining in MUC16 . No significant correlation was found between apical and membranous staining in MUC1 . Due to the low apical expression of MUC5AC no analyses were performed for the correlation between apical and cytoplasmatic or apical and membranous staining in MUC5AC. Since most of the correlations between the apical, membranous, and cytoplasmatic H-scores were significant, the mean combined scores are presented in the following to display a comprehensive expression pattern of the whole cell.The protein expression of MUC1, MUC16, and MUC5AC is showed for exemplary cases in Fig. The distribution of the combined scores for MUC1, MUC16, and MUC5AC is displayed in Fig. The mean combined score for MUC1 among all included entities was 109.1 (± 107.4). The highest mean MUC1 combined score was found in SaDu being as high as 223.6 (± 91.7), followed by MuEp (146 ± 80.1), SecC (140.7 ± 110.4), ANOS (136.7 ± 106.3), EpMy (73.6 ± 78.2), ACC (29.1 ± 60.9), and AdCy (26.9 ± 45.1). In total, 81 SGC (75.7%) showed a MUC1 expression.The mean combined score for MUC16 among all included entities was 74.6 (± 104.4). The highest mean MUC16 combined score was found in MuEp with a score of 177.0 (± 101.0), followed by ANOS (85.3 ± 134.0), ACC (81.1 ± 110.9), SaDu (63.0 ± 112.5), AdCy (33.6 ± 44.7), EpMy (27.4 ± 37.7), and SeC (8.4 ± 9.6). In total, 80 SGC (74.8%) showed a MUC16 expression.The mean combined score for MUC5AC among all included entities was 5.3 (± 23.3). The mean combined score for MUC5AC was generally low compared to the MUC1 and MUC16 combined scores. MUC5AC was detected in MuEp with a mean combined score of 24.0 (± 46.2), in SaDu with a very low mean score of 0.6 (± 2.6), and in ANOS with 0.6 (± 1.7). MUC5AC was not found in MuEp, ACC EpMy, and SeC. In total, 10 SGC (9.3%) showed a MUC5AC expression.Table After a mean follow-up of 50.1 months (± 18.9) the 5-year progression-free survival (PFS) among all patients was 76.4%. PFS did not differ significantly between patients with and without MUC1 expression among all entities , Mucin-16 (MUC16), and Mucin-5AC (MUC5AC) in salivary gland carcinomas (SGC) using immunohistochemistry. To the best of our knowledge, this is the first study to display the expression of MUC1, MUC16, and MUC5AC among various entities of SGC using a semi-quantitative combined score derived from the H-score.The most frequent entities in this series were mucoepidermoid carcinoma (MuEp) , followed by adenoid cystic carcinoma (AdCy) , salivary duct carcinoma (SaDu) , and acinic cell carcinoma (ACC) . This is partly in line with the histopathological distribution of SGC found in population-based studies showing MuEp, AdCy, and ACC to be the most frequent entities , whereasThe highest mean MUC1 combined score among all included entities was found in SaDu being as high as 223.6 (± 91.7), followed by MuEp (146.3 ± 80.1), and SecC (140.7 ± 110.4). This is of particular interest as SaDu shows distant metastases in 52–82% of cases , 13 withThe present data shows that in our cohort male gender (p = 0.03), the presence of lymph node metastasis (p < 0.01), lymphovascular invasion (p = 0.045), and extracapsular extension (p = 0.03) were significantly associated with a higher mean MUC1 combined score among all SGC. The results are in line with a study from Liu et al. who showed a statistically significant association between a high expression of MUC1 and male gender as well as lymph node metastasis in a series of MuEp  as well The highest mean MUC16 combined score was found in MuEp with 177.0 (± 101.0), followed by ANOS (85.3 ± 134.0), ACC (81.1 ± 110.9), SaDu (63.0 ± 112.5), and AdCy (33.6 ± 44.7). MUC16 is mainly known as a routinely used tumor marker CA-125 in ovarian cancer. However, it was also shown to be associated with growth and metastasis  of canceA higher mean MUC16 combined score was statistically significantly associated with the female gender. This is most likely due to the finding that MuEp showed the highest mean MUC16 combined score and most patients with MuEp in this series (73.9%) were female. No statistically significant association between a higher MUC16 combined score and histopathological data or localization was found.The mean MUC5AC combined score was generally low compared to the MUC1 and MUC16 combined scores. MUC5AC was detected in MuEp with a mean combined score of 24.0 (± 9.6) and in SaDu with a very low mean score of 0.6 (± 0.6), and in ANOS with 0.6 (± 1.7). MUC5AC was not found in MuEp, ACC, EpMy, and SeC. The most likely explanation is that MUC5AC is, compared to MUC1 and MUC16, a type 2 secreted Mucin. MUC5AC seems not to qualify as a molecular target for salivary gland cancer therapy. As for MUC16, a significant association between the MUC5AC score and the female gender was found, most likely due to the gender distribution of MuEp and the MUC5AC combined score being the highest among MuEp compared to the other entities.To date, in vitro data on therapeutic targets against MUC1 in SaDu cell-lines and xenograft models, MUC1 gene expression levels, and mechanisms for overexpression of MUC1 in SaDu are lacking. Therefore, the results of this study warrant further research regarding the abovementioned therapeutic targets against MUC1 in cell lines and animal models. Further, the limitation of retrospective collection of histopathological and clinical data must be considered when interpreting the results of this study.Overall, the present study displays the extent and intensity of MUC1, MUC16, and MUC5AC among different entities of SGC. The results show that MUC1 is intensely expressed in salivary duct carcinoma, which is known for its aggressive growth and low survival rates while MUC16 shows the highest intensity in mucoepidermoid carcinoma.


Mangifera indica L.) fruit was studied to investigate the potential role of membrane integrity, energy charge, and ripening-related changes in the development of CI, and its management by melatonin. ‘Langra’ and ‘Gulab Jamun’ cultivar mango fruit was immersed in 100 μM of melatonin before storage for 28 days at 5°C with weekly transfers to shelf life at 25°C. CI symptom development was associated with compositional and enzymatic aspects of textural changes, cell membrane deterioration, and chemical energy status. Melatonin-treated ‘Langra’ fruit exhibited very low CI (5 vs. 21%) while ‘Gulab Jamun’ fruit exhibited higher CI (36 vs. 38%) during 28 days of storage at 5 ± 1°C. Higher chilling tolerance in melatonin-treated ‘Langra’ was associated with lower softening, ascribed to lower cell wall degrading exo- and endo-polygalacturonase, pectinesterase, and endo-1,4-β-D-glucanase. In addition, lower membrane deteriorating-phospholipase D and lipoxygenase activity in melatonin-treated ‘Langra’ corresponded to lower palmitic and stearic acids and higher oleic, linoleic, and linolenic acids accumulation, thus, higher unsaturated/saturated fatty acids ratio. Additionally, there was a higher intracellular energy supply with melatonin, represented by a higher adenylate energy charge (AEC) arising from higher ATP and ADP and lower AMP accumulation, related to higher H+-ATPase, Ca2+-ATPase, succinate dehydrogenase, and cytochrome c oxidase activities. This study for the first time provides evidence, suggesting that melatonin alleviation of CI is related to the preservation of membrane integrity, thereby protecting the intracellular energy supply, and preserving cell wall integrity via impeding cell wall degrading enzyme activities.The efficacy of the signaling molecule melatonin for alleviating chilling injury (CI) in mango ( Mangifera indica) is economically one of the most important fruits, possessing high nutritive value, pleasant flavor, and attractive color and aroma ; the cultivars ‘Langra’ and ‘Dashehari’ are exceptions to some extent, tolerating exposure to 8–9 C. Symptoms of CI include abnormal (uneven) softening and color development, peel darkening and pitting, poor flavor due to lack of aroma, and lenticel prominence –7. Therevia membrane fatty acids peroxidation, resulting in a lower unsaturated to saturated fatty acids (unSFA/SFA) ratio   ratio . Besides (EGase) –13. TherN-acetyl-5-methoxytryptamine) not only serves as a receptor-dependent signaling molecule but also exhibits a powerful receptor-independent ROS-scavenging cascade  enhancing ROS protection and scavenging systems activity, (4) preserving membrane unsaturation status by higher expression of fatty acid desaturase (FADs) genes, along with (5) lower expression of phospholipase D (PLD) and lipoxygenase (LOX) genes and enzyme activities, (6) promoting oxidative pentose phosphate, shikimic acid and phenylpropanoid pathways activity, (7) enhancing arginase pathway activity by activating the ZAT2/6/12-CBF1 signaling pathway for triggering endogenous nitric oxide, polyamines, proline, and γ-aminobutyric acid (GABA) accumulation, (8) enhancing intracellular energy adenosine triphosphate (ATP) and reducing power of NADPH supply, and (9) activating GABA shunt pathway activity.In plants, melatonin  cell wall modification enzymes, (ii) membrane lipid metabolism, and (iii) energy metabolism of mango fruit stored at the chilling temperature of 5 ± 1°C for up to 28 days.Mangifera indica L.) cultivars ‘Langra’ and ‘Gulab Jamun’ were used in this study. The fruit was physiologically mature but pre-ripe, having 29 ± 1 N firmness at harvest time; however; the data presented here is after 3 days of shelf life. The fruit was hand-harvested from an orchard located in Sonepat, Haryana, India. The harvested fruit was transported to the laboratory and fruit with any injury or defects was sorted out from the main lot. The sorted fruit was disinfected with 1% (v/v) sodium hypochlorite for 2 min followed by a random division of 300 fruit from each cultivar into six groups, consisting of 50 fruits in each group. The first three groups were immersed in distilled water (control) in three different buckets, and the other three groups were immersed in 100 μM MT (treated) for 2 h at 25 ± 2°C under the low light conditions in three different buckets  with 1.5 mm sexo-PG (EC 3.2.1.82), endo-PG (EC 3.2.1.15), pectin esterase , and EGase (EC 3.2.1.4) were done according to Khan and Singh (3) and 12% (w/v) polyethylene-glycol (PEG). The homogenate was centrifuged  at 12,000 × g at 4°C for 30 min, and the pellets were each washed with 4 ml of 0.2% (w/v) of NaHSO3 solution. The pellets obtained after re-centrifuging  at 12,000 × g for 40 min were stored at −80°C and were used to determine the peel and pulp activities of exo-PG, endo-PG, PE, and EGase. For exo-PG and endo-PG, a pellet immersed in 50 mM sodium acetate buffer (pH 5.0) containing 0.5 M sodium chloride was incubated for 1 h at 4°C on an incubator shaker. Incubation was followed by centrifugation  at 12,000 × g at 4°C for 15 min and the supernatant was diluted with 50-mM sodium acetate buffer (pH 5.0). The diluted supernatant was used as a crude extract for the determination of the activities of exo-PG and endo-PG.The extraction and assessment of activities of nd Singh  with somexo-PG and endo-PG activities, mixtures consisting of 0.15 ml of 50 ml sodium acetate buffer (pH 4.4) containing 0.5% (w/v) of polygalacturonic acid (PGA) and 0.15 ml of an enzyme extract were incubated for 18 h at 30°C. An assay tube consisting of the above mixture, 0.1 M borate buffer (pH 9.0), and 1% (w/v) cyanoacetamide was boiled for 10 min, cooled, and the absorbance of the reaction mixture was measured at 274 nm with a spectrophotometer . The activity of the exo-PG enzyme was expressed in terms of μg of galacturonic acid mg–1 protein h–1 according to the standard curve of D-galacturonic acid. For endo-PG, 3 ml of an enzyme extract and chilled 2% (w/v) PGA in a 50-mM sodium acetate buffer (pH 4.4) were measured for viscosity change during 18 h of incubation period at 30°C with a viscometer . The activity of endo-PG was expressed as a change in viscosity, i.e., Δ viscosity (mg–1 protein h–1) in triplicates with three fruits in each replication.For the g for 15 min. A total of 20 m of a citrus pectin solution  was mixed with 5 ml of the supernatant obtained as above, followed by titration with 0.01 N sodium hydroxide (NaOH). The activity of PE was estimated in triplicates (with three fruits in each replication) and was calculated from the total volume of NaOH solution required to maintain the pH of the reaction mixture up to 7.4 and expressed as mM NaOH mg–1 protein h–1.For PE activity, the pellet obtained through the above-mentioned extraction process was re-suspended in 15 ml of chilled 7.5% (w/v) sodium chloride, 0.75% (w/v) ethylenediaminetetraacetic acid (EDTA) solution at pH 6.5. After re-suspension, the mixture was incubated for 10 min at 4°C followed by centrifugation  at 12,000 × g for 15 min, and the supernatant obtained was mixed with 6 ml of 0.2% (w/v) carboxymethyl cellulose in citrate-phosphate buffer (pH 6.0). The activity of EGase was measured in triplicates with three fruits in each group. Estimation was done as a change in viscosity during 18 h of incubation at 30°C with a viscometer  and was expressed as Δ viscosity (mg–1 protein h–1).The EGase activity was determined by stirring the extraction pellet in 15 ml of 0.1 M citrate-phosphate buffer (pH 6.0) consisting of 1 M NaCl followed by incubation for 1 h. The mixture was centrifuged  at 12,000 × g at 4°C for 15 min. The supernatant obtained was used for PLD estimation. First, the reaction substrate was prepared by the addition of 0.4 g of lecithin (phosphatidylcholine) to 50 ml of ether followed by its evaporation using a rotary evaporator  at 35°C. The dried solution was re-dissolved in 1 L of 100 mM of acetate buffer (pH 5.5) consisting of 5 mM of dithiothreitol (DTT) and 25 mM of calcium chloride. In an assay tube, 1 ml of enzyme extract was mixed with 3 ml of reaction substrate, followed by 1 h shaking at 28°C. The reaction substrate was washed with petroleum ether and the aqueous layer was collected. Furthermore, 2 g of Reinecke salt (NH4[Cr(NCS)4(NH3)2]⋅H2O) was dissolved in 100 ml of methanol and added to the reaction mixture in order to get a precipitate after centrifugation  at 12,000 × g for 10 min. The precipitate was re-dissolved in 3 ml acetone, followed by spectrophotometric  analysis at 520 nm. The standard curve of choline chloride in 100-mM acetate buffer (pH 5.6) was used for estimation of PLD activity from triplicate samples (in which each replication consisted of three fruit), where 1 U was defined as mM of choline produced min–1.The activity of PLD (EC 3.1.1.4) was determined according to Liu et al.  involving at 4°C for 20 min. The LOX activity was assayed according to the method of Lin et al.  enzyme, 1 g of fruit tissue was homogenized from triplicate samples (three fruit in each replication) with 5 ml of Tris-HCl buffer , followed by centrifugation  at 10,000 × n et al.  with somg for 10 min. The organic phase was dried, and methylation of fatty acids was done at boiling temperature with the addition of 1 ml of 140 M boron trifluoride dissolved in methanol. Furthermore, extraction of methylated fatty acids was done with hexane, again taken to dryness, followed by re-dissolving in 200 μl of chloroform prior to injection. Separation and quantification of fatty acids were done according to Mirdehghan et al. (The extraction of fatty acids was done in triplicates (three fruit in each replicate), according to Cao et al. , with son et al.  with som2+-ATPase (EC 7.2.2.10) and H+-ATPase (EC 3.6.3.6) activities was done in triplicates (each replication consisting of three fruit), according to Jin et al.  with 10 ml of pre-cooled extraction buffer. The homogenate was centrifuged  at 5,000 × g at 4°C for 10 min, and the obtained supernatant was collected. The remaining sediment was washed with washing buffer prepared from 10-mM Tris-HCl, 0.25-M sucrose, 1-mM EDTA, and 0.3 M mannite. Finally, the sediment in dissolution with washing buffer was used as an enzyme extract.The preparation of the extract for determination of Can et al. , incorpo2+-ATPase and H+-ATPase, the reaction mixtures were prepared and the procedure followed was as per Jin et al. , 3 mM magnesium sulfate (Mg2SO4), 0.1 mM sodium orthovanadate (Na3VO4), 50 mM sodium nitrate (NaNO3), 0.1 mM ammonium molybdate, 3 mM calcium nitrate [Ca(NO3)2], and an enzyme extract. On the other hand, the H+-ATPase reaction mixture consists of all the above mentioned constituents except 3 mM Ca(NO3)2. An assay tube consisting of a reaction mixture was added with 100 μl of 30-mM ATP-Tris (pH 8.0), followed by 20 min incubation at 37°C. Afterward, the reaction was terminated with the addition of 30 mM trichloroacetic acid (TCA), and absorbance was recorded at 660 nm with a spectrophotometer , and 1 unit of activity was expressed as a release of 1 μM phosphorus min–1.For the assessment of Can et al.  with somg at 4°C for 20 min. The supernatant volume was immediately adjusted to have a pH between 6.5–6.8 using potassium hydroxide solution. The final volume of supernatant was maintained at 4 ml and passed through a 0.45-μm filter . Furthermore, AMP, ADP, and ATP were examined by high-performance liquid chromatography (HPLC)  using a reserved-phase C18 column , accompanied by ultraviolet detector at wavelength 254 nm. Mobile phase A consisted of 0.04 M potassium dihydrogen phosphate and 0.06 M dipotassium hydrogen phosphate dissolved in distilled water and maintained at pH 7.0 using 0.1 M potassium hydroxide, whereas acetonitrile was used as mobile phase B. Programming consisted of a linear gradient program with 75–100% and 0–25% of mobile phase A and B, respectively, for 7 min at a flow rate 1.2 ml min–1. Lastly, the sample (10 μl) was injected into the HPLC for analysis of AMP, ADP, and ATP as per the external standard program. The energy charge was calculated from triplicates (consisting of three fruits in each replication) as ATP+1/2 ADP divided by the summation of AMP, ADP, and ATP.The measurement of AMP, ADP, ATP, and AEC was done according to Yi et al.  with somg at 4°C for 15 min and the supernatant obtained was used as the extract for protein determination. Furthermore, 0.1 ml of extract was mixed with 5 ml of Bradford reagent and was examined spectrophotometrically  at 595 nm after 5 min of incubation at ambient conditions. Bovine serum albumin (BSA) was used as the standard to estimate protein content in terms of mg g–1 fresh weight (FW).Protein estimation was done using the Bradford  method wAnalysis of variance (ANOVA) was used for interpretation of the experimental results using a completely randomized design in triplicates. Additionally, the means were further compared using Duncan’s Multiple Range Test using SPSS (Version 20) with a 0.05 level of significance.The visual symptoms of CI, such as pitting, blackening, wrinkling, and browning over the peel of mango fruit, were regarded as CI visual signs, which were observed on the third day of observation at ambient conditions after removal from cold storage (5 ± 1°C) at every 7-day interval. ‘Langra’ mangoes without MT treatment exhibited CI symptoms just after 7 days of cold storage. However, MT-treated ‘Langra’ mangoes exhibited the first signs of CI on 14 days of storage .Decreasing trends in fruit firmness were seen with increasing storage duration. However, MT treatment exerted a positive effect in maintaining higher firmness of ‘Langra’ mangoes . Whereasexo-PG  suppression in exo-PG  is a primary enzyme involved in phospholipid degradation and is thereby involved in the integrity and functioning of biomembranes during environmental stress. However, no definite trend was observed in the activity of the PLD enzyme throughout the duration of 28 days at low-temperature storage . The actp < 0.05) in ‘Langra’ mangoes. On the contrary, palmitoleic acid, oleic acid, and linoleic acid showed decreasing trends in ‘Langra’ mangoes in contrast to ‘Gulab Jamun’ mangoes. Additionally, ‘Langra’ mangoes treated with MT maintained significantly (p < 0.05) higher palmitoleic, oleic, and linoleic acid accumulation in their peel or pulp. Furthermore, the unsaturated to saturated fatty acid (unSFA/SFA) ratio was prominently higher in the peel and the pulp of MT-treated ‘Langra’ mangoes in contrast to ‘Gulab Jamun’ mangoes  was observed in the peel and pulp ADP and ATP accumulation of MT-treated ‘Langra’ mangoes only. However, a relatively higher AMP and AEC accumulation were noted with MT application in ‘Langra’ mangoes  and ACC oxidase (ACO) gene expressions. During low temperature storage, ACS is suppressed, resulting in lower production of ethylene. However, when fruit was reconditioned at room temperature, a notable rise in ethylene production occurred due to subsequent ACC production, which led to severe signs of CI  and EGase lead to mango fruit softening in line with the studies done by Zaharah et al.  genes (PI3K) gene expression and enzyme activity could be responsible for supplying phosphatidylinositol 3-phosphate (PI3P) for PLD enzyme activity. PLD enzyme activity produces phosphatidic acid (PA), which could trigger cytosolic Ca2+ accumulation and promote NADPH oxidase enzyme activity  could be responsible for promoting shikimate and phenylpropanoid pathway activities for phenol accumulation, which could be responsible for pulp browning in mango fruit during the cold storage via polyphenol oxidase (PPO) enzyme activity  genes . By trigactivity –54. By psduction .via either LOX enzyme activity or non-enzymatic peroxidation by ROS, which would give rise to the malondialdehyde (MDA) accumulation  reduced PLD activity  enzyme activity would provide free fatty acid substrate for the linolenic acid oxidation pathway mulation , 55. Themulation , 54. Theactivity , LOX actactivity , and MDAactivity  in contrPLD and LOX gene expressions and enzyme activities, which have been found to be beneficial for alleviating CI in tomato (CaPLD and CaLOX gene expressions (p < 0.05) lower MDA accumulation , whi, whiPLD 2–. and H2O2) accumulation, along with lower ATP biosynthesis  kinases, accompanied by H+-ATPase, Ca2+-ATPase, and Mg2+-ATPase enzymes activity, are also responsible for alleviating stresses, delaying senescence, and preserving quality in fruits and vegetables during postharvest life , along with fatty acid unsaturation of activity . In mangoftening . In addiorcement . During est life .+-ATPase, Ca2+-ATPase, SDH, and CCO enzyme activities. A similar alleviation with MT application is seen in the present study but with variability between two mango cultivars. This cultivar-dependent CI variability is in accordance with the difference in ATP and ADP accumulation, where ‘Langra’ mangoes treated with MT maintained the energy units in significantly (p < 0.05) higher amounts than ‘Gulab Jamun’ mangoes  application. MT-treated mangoes of the ‘Langra’ cultivar were found to be CI-tolerant. This chilling tolerance with MT treatment in response to the chilling temperature of 5 ± 1°C in ‘Langra’ mangoes was found to be associated with lower activity of cell wall-modifying enzymes, better-maintained cell membrane, and unsaturated-to-saturated fatty acid ratio, in addition to maintenance of higher cellular energy status in both peel and pulp of the fruit. On the contrary, ‘Gulab Jamun’ mangoes failed to maintain the same preservative metabolisms neither in peel nor in pulp in response to MT application and there was no development of chilling tolerance.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.RB and SP: conceptualization, methodology, investigation, and writing—original draft preparation. RB: software, validation, and formal analysis. SP: resources, visualization, supervision, and project administration. RB, SP, and MSA: data curation. SP, MSA, MBA, JB, and OF: writing—review and editing. SP and OF: funding acquisition. All authors have read and agreed to the submitted version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


To investigate the clinical characteristics of patients with multiple endocrine neoplasia type 1 (MEN1)-related insulinoma and their relationship with specific biochemical changes and to summarize the features of treatment options for the Chinese population with this disease and the impact on long-term prognosis.“MEN1” and “insulinoma” were used when searching the Peking Union Medical College Hospital (PUMCH) medical record retrieval system to obtain clinical information about patients. We identified patients diagnosed with MEN1-associated insulinoma based on endocrinological, radiological, and pathological examinations, and subsequently analyzed their clinical data.p < 0.001) during hypoglycemic episodes. However, serum C-peptide level at 0.5 h and serum insulin level at 1 h was higher in patients with hypercalcemia than in patients with normal serum Ca levels in the oral glucose tolerance test (OGTT), although the differences were not statistically significant. Multifocal pancreatic neuroendocrine tumors (pNETs) were present in 38 (69.1%) patients; most of them  underwent multiple enucleations, and 45% (9/20) had a postoperative recurrence. Five patients (10%) who underwent distal pancreatectomy developed pancreatic insufficiency after an average of seven years. patients who underwent genetic testing, 23 (95.8%) were positive for MEN1 mutation, with mutations most commonly found in exons 2 (21.7%) and 3 (13%).A total of 55 patients with MEN1-associated insulinoma were included, including 29 (52.7%) men and 26 (47.3%) women. The parathyroid gland was the most commonly affected (78.2%), followed by the pituitary gland (69.1%) and adrenal gland (16.4%). Insulinoma was the first manifestation of MEN1 in at least 23.6% (13/55) of patients. Nineteen (34.5%) patients presented with initial symptoms of hypoglycemia before the age of 22 years. Among the 24 Patients with high serum calcium (Ca) had significantly lower serum insulin levels than those with normal serum Ca levels (In our study, the rates of postoperative recurrence and long-term complications in patients with MEN1 with multifocal pNETs were significantly different from those in other international centers and might be related to the choice of surgical method. In addition, elevated serum Ca levels in patients with primary hyperparathyroidism may affect insulin secretion.The online version contains supplementary material available at 10.1186/s13023-022-02370-1. Multiple endocrine neoplasia type 1 (MEN1) is a rare  inherited multi-tumor syndrome that affects specific neuroendocrine organs and non-endocrine tissues, principally the parathyroid glands, gastro-entero-pancreatic (GEP) tracts, and anterior pituitary . InsulinParathyroid adenoma is the first clinical presentation in approximately 90% of cases, with a mean age of onset of 20–25 years and approximately 100% penetrance by the age of 50 years , manifesThis study aimed to analyze the clinical data of patients with MEN1-associated insulinoma treated at the Peking Union Medical College Hospital (PUMCH) from January 2002 to August 2021 and to explore the impact of biochemical changes caused by the presence of hypercalcemia on their clinical features to provide insights into screening, management, and follow-up strategies for this rare disease.The data were retrieved from the PUMCH Medical Record Retrieval System. A total of 310 patients with a discharge diagnosis of MEN1 between January 2002 and August 2021 were identified using ICD-9/10 codes; 255 without insulinoma were excluded. Ultimately, 55 eligible patients were included in the analysis.MEN1 gene.The diagnosis of MEN1 was established based on one of the following criteria , 6: (1) The presence of an insulinoma was based on randomly observed signs of hypoglycemia and the Whipple triad with concomitant biochemical endogenous hyperinsulinemic hypoglycemia (HH) according to clinical practice guidelines . LocalizMEN1 gene mutation, pathological information, and results of oral glucose tolerance test (OGTT) performed with the presence of insulinoma in 51 patients requiring assessment of glucose metabolism.We retrospectively analyzed the detailed clinical information of the 55 patients and collected the following data: sex, age at hypoglycemia onset, age at diagnosis of MEN1 and insulinoma, family history, all MEN1-associated endocrine tumors, clinical signs, laboratory test results, imaging findings, pharmacological and surgical management regimens, post-treatment outcomes, type of Matsuda-insulin sensitivity index (ISI) was defined as 10,000/square root of [(mean plasma insulin × mean plasma glucose during OGTT) × (fasting plasma glucose × fasting plasma insulin)] .The glucose oxidase assay was used to determine serum glucose levels. Serum insulin and C-peptide levels were examined using radioimmunoassay  up to 1991 and chemiluminescence assays  after 1991. Biochemical indices, including serum Ca, phosphorus (P), and albumin, were measured using a Beckman Automatic Biochemical analyzer . Serum ionized-Ca levels were measured using a blood gas analyzer radiometer . Serum parathyroid hormone (PTH) levels were measured using chemiluminescence . The following were the normal reference ranges for indices: fasting serum glucose, 3.9–6.1 mmol/L; fasting serum insulin, 5.2–17.2 μIU/mL; fasting serum C-peptide, 0.8–4.2 ng/mL; serum Ca, 2.13–2.70 mmol/L; serum iCa, 1.08–1.28 mmol/L; serum P, 0.81–1.45 mg/mL; serum albumin, 35–51 g/L; PTH, 13–65 pg/mL. Ca was corrected using the following formula: corrected CA =  + serum Ca [P < 0.05 was considered significant.Continuous data are expressed as mean ± standard deviation when normally distributed and as median (interquartile range) when non-normally distributed. Categorical variables are expressed as absolute numbers and percentages. The t-test was performed on the mean of normally distributed continuous data, whereas the Mann–Whitney U test was used for non-normally distributed continuous data. The chi-squared test was used to compare data between groups. Multiple linear regression analysis was used to explore the association between serum Ca and insulin/glucose ratio during hypoglycemic episodes. Adjustment for body mass index (BMI) or ISI, respectively, were conducted simultaneously. Statistical analyzes were performed using IBM SPSS Statistics for Windows version 26 . A two-tailed Among the 55 patients with MEN1-related insulinoma, the mean age at the first episode of HH was 30.76 ± 14.03 years (range 9–75 years), and that at the time of diagnosis of insulinoma and MEN1 was 34.49 ± 14.99 and 36.20 ± 14.08 years, respectively (Table All 55 subjects experienced recurrent hypoglycemic symptoms manifesting as neuroglycopenic symptoms 100%) and/or autonomic symptoms (81.8%). The most common neuroglycopenic symptoms included loss of consciousness or coma (85.5%) and seizures (10.9%). The most common autonomic symptoms were sweating (63.6%), palpitations (50%), hunger (34.2%), and dizziness (21.8%). hypoglycemia occurred most often during periods of fasting (98.2%), whereas it was rare after meals (9.1%). The mean minimum serum glucose level during hypoglycemic episodes was 2.25 ± 0.56 mmol/L, the median insulin level was 23.77 μIU/ml, and the mean C-peptide level was 3.38 ± 1.79 ng/mL  patients with insulinoma only as their first presentation developed other manifestations of MEN1 after a median of 3 years (range 1–26 years). PHPT or PA was found concurrently with the onset of insulinoma in 15 (27.3%) and one (1.8%) patient, respectively, and the above three adenomas were discovered simultaneously in one (1.8%) patient. There was definite evidence of gastrinoma or PA only as of the first manifestation of MEN1 in one (1.8%) and three (5.5%) patient, respectively. The order of manifestations of adenomas was unclear in 21 (38.2%) patients .As the disease progressed, all 55 patients developed adenomas of other glands, and the distribution of their clinical manifestations is depicted in detail in Fig. Among the patients with PA, 25 (65.8%) had non-functioning PA, 12 (31.6%) had prolactinomas, and one (2.63%) had both prolactin and growth hormone secretion. Seven (18.4%) patients with PA underwent surgery, and four (10.5%) received dopamine agonists for prolactinomas. Ten (18.2%) patients had adrenocortical tumors, and all adenomas in this group were non-functioning adenomas.P < 0.001) and insulin/glucose ratio  during hypoglycemic episodes. According to multiple linear regression analysis, serum Ca was negatively associated with insulin/glucose ratio after adjustment for BMI  or ISI . Patients with hypercalcemia had a tendency for older mean age at diagnosis and a larger diameter of the primary lesion, although no statistical difference was reached. After a median follow-up of 7 (range 1–19) years, 3 and 8 patients in the normocalcaemia and hypercalcemia groups, respectively, developed recurrent hypoglycemia. Four patients in the hypercalcemia group developed insulin-producing liver metastases, and the remaining seven had new insulinomas.After excluding the 13 patients with missing serum Ca values when the insulinoma was untreated, compared with the 18 42.9%) patients with normal serum Ca, no significant differences were found in serum glucose levels during hypoglycemic episodes in the 24 (57.1%) patients with hypercalcemia , the serum insulin, C-peptide, insulin/glucose ratio, and C-peptide/glucose ratio were lower at 0 h, 2 h, 3 h, 4 h, and 5 h in those with high serum Ca than in those with normal serum Ca, while serum C-peptide at 0.5 h and serum insulin at 1 h was higher in the former group, but the differences were not statistically significant  patients underwent surgery Table ; three  years, 13 (26%) had recurrent hypoglycemia at a mean of 7.6 ± 4.27 years after surgery, nine (69.2%) of them had a new insulinoma and four (30.8%) developed insulin‐producing liver metastases, most of these patients  had had the multifocal disease and had undergone multiple enucleations. Eleven (84.6%) of these patients required a second operation, and two patients (15.4%) rejected the operation with extra meals every day. Of the 13 patients aged less than 22 years at the time of surgery, six developed recurrent insulinoma. In terms of complications, five patients who underwent distal pancreatectomy developed abnormal glucose tolerance or new-onset diabetes mellitus after an average of seven years.Histopathological analysis was performed on samples following surgical resection in all patients. Immunohistochemical reports were not available for retrieval in three patients, and details of insulin staining and mitotic figures were absent in nine patients. Among the other 38 patients with complete pathological information, 13 (34.2%) patients had no insulin-positive pNET, three of whom had recurrent hypoglycemia 8.33 ± 7.02 years after initial surgical cure.In our cohort, 19 34.5%) patients presented with initial symptoms of hypoglycemia before the age of 22 years, and the mean age at diagnosis of insulinoma and MEN1 was 19.89 ± 5 and 22.95 ± 5.79 years, respectively , which is different from previous MEN1 studies, which had more female patients (64.2%) . FurtherIn addition, the incidence of PHPT was only 78.2%, which is difficult to compare because of the lack of data related to MEN1 patients with insulinoma from other centers, and 32.7% of affected patients were symptomatic. Moreover, insulinoma was the first manifestation in 13 (23.6%) patients, and it was detected simultaneously with PHPT or PA in 17 (30.9%) patients. These results emphasize that PHPT is not always the first biological or clinical abnormality to emerge in the course of MEN1 disease , 21, 22 Previous studies have found that Ca levels in the circulation were elevated in patients with insulinoma following a selective arterial calcium injection test and may stimulate insulinoma secretion  and its However, during the OGTT, serum C-peptide level at 0.5 h and serum insulin level at 1 h was higher in those with high serum Ca than in those with normal serum Ca. This could be related to the mechanism by which serum Ca promotes insulin secretion in response to nutrient stimulation. Extracellular iCa influx through voltage-dependent iCa channels leads to an increase in cytoplasmic iCa concentration and initiates iCa-dependent cell division containing insulin-secreting granules . GlucoseIn our cohort, 19 (34.5%) patients developed hypoglycemic symptoms before the age of 22 years. The mean size of resected tumors in our study was similar compared to other cohorts of young patients with MEN1-associated insulinomas , 28, butSurgical resection is the only curative treatment for insulinoma and is recommended regardless of tumor size. Current guidelines recommend an individualized treatment approach depending on the location and size of the suspected insulinoma . For isoMEN1 gene mutations, which were observed in exons 2, 3, 4, 5, 6, 7, 8, 9, 10, and introns 3 and 4, with the most frequently observed mutation site being exon 2. The distribution of mutant loci in our patients was similar to that reported in a previous study in Japan [Genetic testing was conducted in 24 of our patients after the onset of the disease, and it was possible to identify 23 carriers of in Japan , althougin Japan . Unfortuin Japan , 34–36. The present study has several limitations. First, the study involved a single institution, and the number of patients (55) was insufficient to draw firm conclusions from the results. Second, the follow-up period was relatively short. Third, the long enrolment period of nearly two decades (January 2002 to August 2021) implies a lack of uniformity in the diagnostic criteria, imaging resources, and treatment options. Fourth, only < 50% of patients underwent genetic testing, which limited further analysis of genotype–phenotype correlations. Fifth, the information on insulin staining of samples after surgical resection was limited, whereas it would be more reliable in confirming the removal of insulinoma using insulin staining.In conclusion, we established an insulinoma cohort and differed from other similar studies in that our patients had a higher rate of postoperative recurrence, which may be related to the selection of the surgical method. In addition, the incidence of PHPT in our patients is probably lower, and similar analyzes in other study centers are needed for comparison. Moreover, individuals with high serum Ca may obscure the symptoms of hypoglycemia by producing less insulin during fasting than those with normal serum Ca; hence, they should be more alert to the occurrence of HH.Additional file 1: Table S1. The site and distribution of the MEN1 mutation; Table S2. First manifestation of MEN1 patients with insulinoma.