Treatment of lupus nephritis consists of six months of induction immunosuppression followed by years of maintenance immunosuppression. The aim of present study was to find the prevalence of remission after six months of induction immunosuppressive treatment with induction therapy in patients with lupus nephritis.A descriptive cross-sectional study was conducted in the nephrology unit of department of internal medicine of a tertiary care hospital from September 2018 to September 2020. The study was approved by institutional review committee of same institution (reference number: 184/2018). Convenience sampling method was used and Statistical Package for Social Sciences version 26 was used for statistical analysis. Point estimate at 90% Confidence Interval was calculated along with frequency and proportion for binary data.Out of 24 patients, overall remission was seen in 21 patients (87.4%) . Complete remission and partial remission were seen in 16 (66.6%) and 5 (20.8%) patients respectively resulting in an at the end of six months of induction immunosuppressive treatment. The most common class of lupus nephritis was class IV, 7 patients, followed by class IV+V, and class V, 6 patients in each respectively. The mean 24-hour urinary total protein, serum albumin and serum creatinine were 2492±1051 mg, 2.1±0.4 g/dl, and 0.9±0.1 mg/dl respectively. Adverse events were observed in 6 (25%) patients.Our study shows that good proportions of patients with lupus nephritis achieve clinical remission at the end of six months of induction immunosuppressive treatment with induction therapy, however, at the cost of some tolerable side effects. Either cyclophosphamide (CYC) or mycophenolate mofetil (MMF) along with prednisolone (PNL) are being used as an induction regimen in the treatment of class III, IV V, and mixed LN.5Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease and lupus nephritis (LN) is seen in about 40-50 % of the patients.7There has been scarcity of data from Nepal regarding the clinical outcomes after six months of induction immunosuppressive treatment in patients with LN.Hence present study was conducted to find the prevalence of remission after six months of induction immunosuppressive treatment with mycophenolate mofetil and PNL in patients with lupus nephritis.8 Patients were excluded if they had already received immunosuppressive therapy before enrolling in the study, if they disagreed to give the consent and when they failed to come monthly for at least six months. Convenience sampling method was used. The sample size was calculated using the formula:A descriptive cross-sectional study was conducted in the nephrology unit of the department of internal medicine of Nobel Medical College Teaching Hospital (NMCTH), Biratnagar, Nepal. All patients with LN from September 2018 to September 2020, who received six months of induction immunosuppression with MMF were included in the study. The study was approved by the Institutional Review Committee of the hospital (NMCTH IRC reference number 184/2018). Written informed consent was taken from all patients. The inclusion criterion was newly diagnosed patients of SLE with renal biopsy proven class III, IV, V and mixed LN. Lupus nephritis (LN) was classified according to the International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification system.2 × p × q / e2n = Z2 × (0.88) × (0.12) / (0.11)2  = (1.645)  = 23.6  = 24where,n = sample size,Z= 1.645 at 90% Confidence Interval,6p = prevalence, 88%,q = 1-pe = margin of error, 11%The calculated sample size was 24.The baseline characteristics of the study population were digitally recorded at the time of enrollment in the study. After enrollment patients were started on induction immunosuppressive treatment for six months with MMF and PNL. Patients received MMF with a starting dose of 500 mg twice a day, which was increased after 2 weeks to 1000 mg twice a day for 6 months. Oral PNL was given with a starting dose of 0.5mg/kg for 2 months which was gradually tapered by 5 mg/every 2 weeks to reach 10mg/day. Injection methyl prednisolone (MP) 500 mg intravenous, once a day for three days was given at the start of induction regimen only if the serum creatinine was raised. Besides the immunosuppressive treatment, all patients also received angiotensin receptor blockers (ARBs) to titrate blood pressure below 125/75 mmHg, hydroxychloroquine 5mg/kg/day (maximum 400 mg/day), and a prophylactic antibiotic cotrimoxazole double strength (DS) ½ tablet once a day.9 Complete remission (CR) was defined as return of serum creatinine to previous baseline, plus a decline in the urine protein creatinine ratio (uPCR) to <500 mg/g (<50 mg/mmol). Partial remission (PR) was defined as stabilization (±25%), or improvement of serum creatinine, but not to normal, plus a ≥50% decrease in uPCR.Clinical outcomes at the end of six months were assessed in terms of complete remission (CR), partial remission (PR) and no remission (NR) or deterioration, defined as per the Kidney Disease Improving Global Outcomes (KDIGO) guidelines.Statistical analysis was done using Statistical Package for Social Sciences version 26. Point estimate at 90% Confidence Interval and descriptive statistics were done.Out of 24 patients, overall remission was seen in 21 patients (87.4%) . Complete remission and partial remission were seen in 16 (66.6%) and 5 (20.8%) patients at the end of six months of induction immunosuppressive treatment. No response was seen in 3 (12.5%) patients .The mean age of the patients was 23.6±7.0 years, and 23 (95.9%) of them were females. The most common class of lupus nephritis was class IV, 6 (29.16%) patients, followed by class IV+V and class V, 6 (25%) patients in each class respectively .The mean 24-hour UTP, serum albumin and serum creatinine were 2492±1051 mg, 2.1 ±0.4 g/dl, and 0.9±0.1 mg/dl respectively. Baseline characteristics of the patients are shown in .Adverse events were observed in 6 (25%) patients during the six months of induction immunosuppression .None of the patients died because of the adverse events.In the present study we analyzed the clinical remission rates and adverse events after six months of induction immunosuppression with mycophenolate mofetil and prednisolone in patients with lupus nephritis. We found that good proportions of patients achieve complete or partial remission at the cost of some adverse events.7 These are the only two published studies we could found from Nepal on extensive literature search, where the clinical outcomes were studied after six months of induction immunosuppressive treatment in patients with LN. Ours is the third study in the row.The study done by Sedhai et al in Nepalese population had used NIH regimen 1.5 gm of mycophenolate mofetil along with prednisolone as an induction regimen.11Mycophenolate mofetil (MMF) as an alternative induction regimen in LN was introduced by Chen et al. in the year 2000; and the Aspreva Lupus Management Study (ALMS) established its role as a standard of care in the induction phase treatment of LN.7-11In our study we used MMF in all our patients at a dose of 2 g/day (1 gm in two divided doses), which was less than the dose used in ALMS trial (3 g/day) but slightly more than the dose used in the Nepalese study (1.5 g/day).11 In our study 66.6% of patients achieved complete remission and 20.8% of patients achieved partial remission. Overall remission rate was 87.4%. A higher rate of renal remission in our study as compared to western studies was also seen in previous studies done in Nepal.7 In another Nepalese study by Sedai et al,7 primary outcome  and secondary outcome (complete response) were 28.6% and 66.7% in the mycophenolate mofetil group making an overall remission rate of 95.3 % in the MMF.7In the ALMS studies the response rate was 56.2% in the MMF group.13 In the study done by Sahay et al. from India the overall remission rate was 72.9% in MMF group.12 In another Indian study by Rathi et al. total response rate was 74% in the low dose of MMF group; the complete remission rate was 54% in MMF group.13Indeed, studies done in other South Asian populations have also shown better remission rates as compared to western studies.In our study adverse events were observed in 25% of the patients, and they were herpes zoster and diarrhea in two patients each and tubercular plural effusion and leucopenia in one patient each. None of our patients died due to adverse events in our study.The limitation of present study was that it was a single center study with a small number of participants. Furthermore, we only included patients who received MMF as an induction regimen and did not compared the outcomes with patients who received CYC. We recommend multicenter randomized control studies in Nepal comparing the two different induction immunosuppressive regimens.Our study demonstrates that good proportions of patients with lupus nephritis achieve clinical remission at the end of six months of induction immunosuppressive treatment with mycophenolate mofetil and prednisolone, however, at the cost of some tolerable adverse events.


Subvalvular aortic stenosis (SAS) can occur as discrete or tunnel-like obstruction of the left ventricular outflow tract and as progressive disease often leads to aortic valve regurgitation. We report our 30-year single-center experience after surgical repair of SAS.A retrospective chart review of all patients aged < 18 years, who underwent surgical repair of SAS from May 1985 to April 2020, was conducted. Mortality was cross-checked with the national health insurance database . Survival and competing risks analysis were used to analyze the primary endpoints survival and incidence of reoperations.From May 1985 until April 2020 103 patients (median age 5.5 years) underwent surgical repair of SAS. Survival was 90.8% at 10 years and 88.7% at 20 and 30 years. Age < 1 year at time of surgery, Shone’s complex, mitral stenosis and concomitant mitral valve surgery were associated with mortality. The cumulative incidence of reoperation for SAS was 21.6% at 10 years, 28.2% at 20 and 30 years. The incidence of reoperation for SAS did not differ between the myectomy, membrane resection and combined myectomy and membrane resection groups. The cumulative incidence of reoperation on the aortic valve was 13.5% at 20 years.Recurrence rate of SAS is not to be neglected, though surgical repair of subaortic stenosis has good long-term results. Patients who needed a combined membrane resection and septal myectomy are not more prone to recurrence than patients who underwent solitaire myectomy or membrane resection. Subvalvular aortic stenosis (SAS) has a broad disease spectrum and a progressive disease nature. SAS can occur as a solitaire subvalvular membrane in the left ventricular outflow tract (LVOT) or a minor fibrous muscular ridge on the subvalvular ventricular septum (discrete SAS). In severe cases SAS occurs as a narrow fibromuscular tunnel-like obstruction of the LVOT , 2. The A chart review of all patients less than 18 years of age at time of surgery who underwent SAS repair between May 1985 and April 2020 was conducted. The study was approved by the institutional review board of the center (Ethics committee submission number: 1414/2019) and patient consent was waived due to the retrospective study design. Patients, who underwent myectomy and/or membrane resection for SAS were included. Patients undergoing modified Konno procedure were excluded from this study. In the mentioned study period 11 pediatric patients underwent modified Konno procedure. A mortality cross-check with the national health insurance database was conducted. Mortality follow-up is available until April 2020. Six patients , who were transferred and followed-up at foreign countries could not be looked up in the database. In the conducted survival analysis patients, who could not be cross-checked were censored at the last cardiac follow-up at the center. Median follow-up time was 10.5 years  with a longest follow-up of 34.6 years. Early mortality included patients, who died within the first 30 days after the procedure or in-hospital during the index hospitalization of the procedure. Reoperation was evaluated for SAS reoperation as well as for aortic valve reoperation . Modified Konno procedure and Ross-Konno procedure were analyzed as reoperation for subvalvular aortic stenosis and in case of the Ross-Konno procedure for aortic valve reoperation respectively. Cumulative incidences of SAS reoperation were compared between the following surgical cohorts: myectomy, membrane resection, combined myectomy, and subvalvular membrane resection.via an oblique aortotomy, the aortic valve is retracted, allowing for resection of the membrane and septal myectomy in the setting of myocardial hypertrophy  and SAS 9.5 .Continuous data are expressed as mean ± standard deviation, whilst skewed continuous data are expressed as median with interquartile range (IQR). Categorical variables were shown as frequencies and percentages. The median follow-up time was estimated by the inverse Kaplan-Meier method . Survivap = 0.571) between myectomy, membrane resection and combined myectomy, and membrane resection groups with 67 mmHg (IQR 37–95 mmHg), 62 mmHg (IQR 47–78 mmHg) and 67.5 mmHg (IQR 55–84 mmHg). Median age at time of surgery was 5.5 years (IQR 1.6–10.8 years) and 43 patients  had already undergone previous cardiac surgery before SAS repair. Ten patients  had undergone previous repair of atrioventricular canal with a mean time from corrective surgery to SAS repair of 10.6 ± 3.8 years. Two patients  had been treated with radiofrequency ablation for SAS earlier. Four patients  had undergone surgery on the aortic valve . Concomitant aortic valve procedures at time of SAS repair were performed in 18  cases and are listed in From May 1985 until April 2020, 103 patients  underwent repair of SAS. Demographic and operative data is seen in In p = 0.003), Shone’s complex , mitral stenosis  and concomitant mitral valve surgery  were statistically significantly associated with mortality.In addition to seven early deaths, three late deaths occurred, and Kaplan-Meier estimated survival was 90.8% (95% CI 83.0–95.1) at 10 years and 88.7% (95% CI 79.4–93.9) at 20 and 30 years . Two patp = 0.845) between the myectomy, membrane resection and combined myectomy, and membrane resection groups with 26.3% (95% CI 10.2–45.9), 27.9% (95% CI 13.5–44.4) and 31.2% (95% CI 8.3–58) at 20 years (p = 0.669) between patients, who underwent concomitant aortic valve procedures at SAS repair and patients without aortic valve procedures at SAS repair with 21.3% (95% CI 4.7–45.8) and 29.8% (95% CI 17.4–43.2) at 20 years respectively (p = 0.479) between patients aged < 1 year at SAS repair and patients aged > 1 year at SAS repair with 32.1% (95% CI 8.7–59) and 27.4% (95% CI 15.8–40.2) at 20 years respectively (p = 0.015). Year of surgery did not correlate as factor for SAS reoperation . Ten patients underwent concomitant or solitaire reoperation on the aortic valve. The predominant indication for surgery on the aortic valve was valve regurgitation . The cumulative incidence of any reoperation on the aortic valve and aortic valve replacement were 13.5% (95% CI 6.8–22.5) and 7% (95% CI 2.5–14.7) at 20 years respectively (p = 0.236) between patients, who underwent concomitant aortic valve procedures at SAS repair and patients without aortic valve procedures at SAS repair with 23.5% (95% CI 5–49.5) and 11.3% (95% CI 4.9–20.6) at 20 years respectively  and tunnel-like SAS (n = 50) reported an early mortality rate of 4% (7/190) and 10 late deaths. Actuarial survival including operative mortality of patients with discrete and tunnel-like SAS was 94 and 84% at 40 years (p = 0.14) respectively. In our cohort Kaplan-Meier estimated survival was 88.7% at 30 years. Etnel et al. . The study cohort is within the larger cohorts observed with this disease spectrum. This allows to assess the risk for reoperation over lifetime in the setting of SAS recurrence. As a typical limitation of a retrospective study design, it is possible that the variability regarding indication and an evolvement of perioperative management over the years are not fully accounted for.Surgical repair of subaortic stenosis in pediatric patients has good long-term outcomes, though recurrence rate of subaortic stenosis is not to be neglected and progression of aortic regurgitation might require aortic valve surgery. The incidence of reoperation for SAS did not differ between the myectomy, membrane resection and combined myectomy, and membrane resection groups. Patients who needed a combined membrane resection and septal myectomy are not more prone to recurrence than patients who underwent solitaire myectomy or membrane resection.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Ethics Committee Medical University of Vienna, submission number: 1414/2019. Written informed consent from the participants or their legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.JS: conceptualization, investigation, data curation, formal analysis, visualization, and writing—original draft. FW: investigation, data curation, and writing—review and editing. AK: conceptualization, formal analysis, visualization, and writing—review and editing. DW, HG, and SH: writing—review and editing. EB: validation and writing—review and editing. IM-B and GL: resources, validation, and writing—review and editing. DZ: supervision, resources, validation, and writing—review and editing. All authors contributed to the article and approved the submitted version.


Early diagnosis of ovarian cancer is challenging due to vague symptoms and a lack of screening tools. We aimed to evaluate two simple, non-invasive urine biomarkers, CA125 and HE4, for the diagnosis of ovarian cancer in symptomatic women. Paired urine and serum samples were collected from women undergoing treatment for ovarian cancer (cases) or investigations for gynaecological symptoms (controls) and were tested for CA125 and HE4. This study found urine CA125 and HE4 levels to be higher in women with cancer compared to controls, and that a combination of urine CA125 and HE4 had a sensitivity that was similar to the currently used serum CA125 for the detection of ovarian cancer. This suggests that urine CA125 and HE4 may be useful non-invasive diagnostic tools to triage women for formal ovarian cancer investigations and could be used as point-of-care testing in the community or low resource settings.p = 0.01. HE4: 12.0 nmol/L (IQR: 10.3–23.1) vs. 6.7 nmol/L (IQR: 3.4–13.6), p = 0.006]. Urine CA125 and HE4 detected ovarian cancer with an AUC of 0.69 (95% CI: 0.55–0.82) and 0.71 (95% CI: 0.69–0.82), respectively (p = 0.73). A combination of urine CA125 and HE4 at optimal thresholds had a sensitivity of 82.4% (95% CI: 56.6–96.2) and was comparable to the sensitivity of serum CA125 [88.2% (95% CI: 63.6–98.5)]. Larger studies are required to confirm our findings, standardise urine collection, and evaluate optimal biomarker thresholds. Urine CA125 and HE4 may be useful non-invasive diagnostic tools to triage women for formal ovarian cancer investigations.The symptoms of ovarian cancer are vague, and current risk assessment tools such as serum CA125 and transvaginal ultrasound scan fail to reliably detect the disease early. This study aimed to evaluate urine CA125 and HE4 as diagnostic biomarkers for ovarian cancer in symptomatic women. Paired urine and serum samples were collected from women undergoing treatment for ovarian cancer (cases) or investigations for gynaecological symptoms (controls). Biomarkers were measured using an automated chemiluminescent enzyme immunoassay analyser. Standard diagnostic accuracy metrics were calculated. In total, 114 women were included, of whom 17 (15%) were diagnosed with an epithelial ovarian malignancy. Levels of urine CA125 and HE4 were significantly elevated in women with ovarian cancer compared to controls [CA125: 8.5 U/mL (IQR: 2.4–19.5) vs. 2.3 U/mL (IQR: 1.0–6.4),  Ovarian cancer affects 7495 women annually in the United Kingdom (UK) and has the highest mortality rate of the gynaecological malignancies . OvarianThe symptoms of ovarian cancer are vague, have an insidious onset, and are extremely common presentations. In the UK, women with suspected ovarian cancer are recommended to undergo a serum cancer antigen 125 (CA125) and transvaginal ultrasound scanning (TVS) in the community, with a referral to secondary care if both results are abnormal or if clinical features prompt suspicion of a pelvic mass or ascites . ReasonsAlternative sources of biomarkers, particularly urine, have gained interest over recent years. Urine is an attractive alternative to blood as it can be self-collected at home, is non-invasive, has a simpler proteome and is not under the influence of homeostatic mechanisms in the same way as blood . CA125 aThe aim of this study is to evaluate urine CA125 and HE4 as diagnostic biomarkers of ovarian cancer in symptomatic women presenting to secondary care. We hypothesised that urine CA125 and HE4 would accurately differentiate between ovarian cancer cases and controls.We recruited women who were attending St. Mary’s Hospital, Manchester, UK for the investigation of suspected gynaecological malignancy or management of presumed ovarian cancer. Women were identified from referrals to the cancer exclusion clinic and from gynaecological oncology multidisciplinary team (MDT) meetings. Women were excluded if they had previously undergone a hysterectomy or had received neo-adjuvant chemotherapy.Women were recruited either from the cancer exclusion clinic, outpatient clinic or on the morning of their surgery. Women undergoing surgical management were recruited pre-operatively. The surgical management of suspected ovarian cancer was in line with current guidance , aiming Research samples were collected prior to any clinical procedures. Women were asked to produce a self-collected urine sample in a dry pot. Serum samples were taken by routine venepuncture.® G600II  automated analyser, which uses a chemiluminescent enzyme immunoassay technique. In brief, this is a two-step sandwich immunoassay method. The luminescent signal generated by the final enzyme reaction is read by the analyser and reflects the level of analyte in the sample. The immunoreaction cartridges used were the Lumipulse® G CA125-II and Lumipulse® G HE4 . Analyte concentration was calculated by the analyser from the calibration curve. Calibration of the analyser for CA125 and HE4 was performed every 28 days, when a new batch of immunoreaction cartridges was used or if the control was out of range, as per the manufacturer’s advice.Serum and urine samples were processed without fixing and stored at −80 °C in the MFT Biobank until analysis. Urine samples were centrifuged at 1000 g for 10 min to remove debris, and supernatant fractions used for CA125 and HE4 analysis. Samples were analysed for CA125 and HE4 using the Fujirebio Lumipulse® G600II analyser , and so underwent serial dilutions to ascertain the optimal dilution for analysis and achieved good dilutional linearity. As a result of serial dilutions, urine samples were tested undiluted for CA125 and diluted to 1:100 for HE4. Samples were diluted with diluent  as recommended by the manufacturer. Serum samples were analysed undiluted.Urine samples had not been previously analysed for CA125 and HE4 on the LumipulseSamples were thawed to room temperature prior to testing and were analysed in accordance with the manufacturers protocol. Quality controls  were run before and after each batch of patient samples. Where the analyte level was at the maximum detection limit (1000 U/L for CA125 and 1500 pmol/L for HE4), the sample was further diluted and re-tested. Urine samples were tested in triplicate and the mean taken. The median intra-assay coefficient of variation (CV) for the urinary triplicates was 15.45% (IQR: 12.64–20.66) for CA125 and 4.15% (IQR: 2.24–7.11) for HE4. The total inter-assay CV ranged from 5.60 to 7.52%.Continuous data were reported as medians with an interquartile range as they were non-parametric. Comparison between groups was performed using the Mann U Whitney test and chi squared test for continuous and categorical data, respectively. Correlations between the biomarkers and clinico-pathological features were calculated using Spearman’s rank. Due to the large quantities of HE4 in urine, levels were presented as nmol/L rather than pmol/L as used for serum HE4.To evaluate the diagnostic performance of urine CA125 and HE4 for the detection of ovarian cancer, receiver operator characteristic (ROC) curves were constructed, and area under the curve (AUC) was calculated. Univariable and multivariable logistic regression models predicting ovarian cancer were calculated for urine CA125 and HE4 as continuous variables, both alone and in combination. As there is limited evidence in the literature regarding thresholds for urine CA125 and HE4, the optimal threshold for each biomarker was calculated from the ROC curves using the method of Liu . The diap-value of < 0.05 was considered significant. Data analyses were performed using STATA .The study was conducted in accordance with the STARD 2015 guidelines for diagnostic test reporting. A 2 (IQR: 24–33), respectively. Of these, 17 (15%) were diagnosed with an ovarian malignancy, 51 (45%) had a benign gynaecological pathology, and 46 (40%) had no demonstrable pathology  and 27 kg/mathology .p = 0.37) or BMI (p = 0.77) between those with ovarian cancer and those without.The histological subtypes of the ovarian malignancies included serous , mucinous , endometrioid , clear cell  and mixed . Around half were high grade (47%), and the majority were early-stage (FIGO stages I+II) disease (80%). The commonest benign ovarian cyst was a serous cystadenoma . There was no significant difference in age (p < 0.001). There was no significant correlation between urine CA125 and age  or BMI ; however, there was weak evidence of correlations with serum CA125 , serum HE4  and urine HE4 . No significant difference was found in median urine CA125 levels in those aged over 55 years [2.8 U/mL (IQR: 1.0–7.1)] compared to those under 55 years [4.0 U/mL (IQR: 1.4–11.2) p = 0.38]. In women with a BMI ≥30 kg/m2, the median urine CA125 was 3.8 U/mL (IQR: 1.4–9.0) compared to 2.7 U/mL (IQR: 1.1–10.6) in women with a BMI < 30 kg/m2 (p = 0.39).The median urine CA125 of the total population was 3.3 U/mL (IQR: 1.1–10.0) and values ranged from 0.3 to1607.0 U/mL. Levels of CA125 were found to be significantly higher in serum samples [14.3 U/mL (IQR: 10.1–25.1)] compared to urine samples (p = 0.01]. There was no evidence of an association between urine CA125 levels and cancer grade (p = 0.85). Whilst median urine CA125 levels were observed to be higher in those with advanced stage disease [11.6 U/mL (IQR: 5.7–16.9)] compared to those with early stage disease [8.1 U/mL (IQR: 1.9–25.7)], this did not reach statistical significance (p = 0.65), likely due to small numbers.p < 0.001], necessitating the conversion of values from pmol/L to nmol/L for simplicity. The values of urine HE4 ranges from 0.6 nmol/L to 1717.0 nmol/L, with a median of 8.41 nmol/L (IQR: 3.84–14.33). Whilst urine HE4 correlated with serum CA125 , serum HE4  and urine CA125 , no significant correlation was seen with age  or BMI . In women over 55 years, median urine HE4 was 6.7 nmol/L (IQR: 3.4–11.8) compared to 10.2 nmol/L (IQR: 4.1–21.1) in women under 55 years (p = 0.07). There was no significant difference in median urine HE4 levels between women with a BMI < 30 kg/m2 [7.9 nmol/L (IQR: 4.0–15.6)] and those with a BMI ≥ 30 kg/m2 .Unlike CA125, the HE4 concentration was found to be significantly higher in urine compared to serum . There was no evidence of a difference between those with low- and high-grade disease , or those with early- and advanced-stage disease . Urine HE4 levels were significantly higher in women with ovarian cancer [12.0 nmol/L (IQR: 10.3–23.1)] compared to those without  compared to those without [13.6 U/mL (IQR: 9.3–19.0)]; however, there was no significant difference in biomarker levels between low- and high-grade disease  or early- and advanced-stage disease .No significant correlation was observed between serum CA125 and age , or BMI . Serum HE4 levels were significantly elevated in women with ovarian cancer compared to those without . In addition, serum HE4 levels were elevated in those with high grade disease [291.6 pmol/L (IQR: 258.5–620.2)] compared to those with low grade disease . There was no significant difference in the serum levels of HE4 between those with early stage disease and those with advanced stage disease .There was no significant correlation between serum HE4 and age  and 0.71 , respectively (p = 0.73). When combined, urine CA125 did not significantly add to urine HE4 alone to predict ovarian cancer .Urine CA125 and HE4 were able to predict epithelial ovarian cancer with a model AUC of 0.69 . Urine HE4 was positive in 12/17 (71%) of women with ovarian cancer compared to 40/97 (41%) of those without (p = 0.03)  had a urine CA125 ≥ 6.2 U/mL compared to 25/97 (26%) of those without ( = 0.03) .Urine CA125 and HE4 were able to detect early stage disease (FIGO I+II) with an AUC 0.67 (95% CI: 0.51–0.83) and AUC 0.68 (95% CI: 0.56–0.81), respectively . CombiniIn this study, we evaluated urine CA125 and HE4 for the detection of ovarian cancer. Urine CA125 and HE4 levels were significantly higher in women with ovarian cancer compared to those without and were able to detect disease with moderate accuracy. At optimal thresholds, a combination of urine CA125 and HE4 where either was positive had a sensitivity similar to that of serum CA125 and, in our cohort, missed one additional cancer compared with serum CA125. If confirmed in larger studies, a combination of urine CA125 and HE4 could be of use as a non-invasive point-of-care triage tool to identify those requiring formal investigation for ovarian cancer; however, our numbers are small and confidence intervals wide. Large prospective studies are needed to validate our findings and optimise urine biomarker thresholds.To our knowledge, this is the first study investigating the combined diagnostic performance of urine CA125 and HE4 for the detection of ovarian cancer. Major strengths of our study include its prospective design and that we had matched urine and serum samples, allowing direct comparison of diagnostic performance. Our study is limited by the small sample size, and we recognise that there are a small number of ovarian cancer cases. The number of ovarian cancer cases in our study population is lower (15%) than those in many other diagnostic biomarker studies in the literature, which often report a prevalence of between 20 to 50% . Our stuUrine has gained increasing interest as a source of biomarkers and is cheap, acceptable to patients, and less influenced by homeostatic mechanisms than serum. Despite this, there are few studies investigating urine CA125 and HE4 for the detection of ovarian cancer, and none evaluating the two markers in combination. Only two studies report on the diagnostic accuracy of urine CA125. Moore et al. reported a disappointing sensitivity of less than 40% in 67 women with ovarian cancer and 166 women with benign adnexal masses . Tay et Serum biomarkers have been much more extensively studied, with CA125 in routine clinical use for the diagnosis of ovarian cancer worldwide. Serum HE4 is a promising diagnostic biomarker and has been evaluated both alone and as part of diagnostic algorithms, of which, ROMA seems to be superior . No studThere is considerable interest in developing alternative biofluids for disease detection. Advances in technology mean that biofluids with lower biomarker concentrations can now be exploited for clinical use. Urine in particular is an attractive biofluid, as it is cheap, readily available, and non-invasive. Our study suggests that a combination of urine biomarkers may be a promising tool for ovarian cancer detection and, at optimal thresholds, may have a comparable sensitivity to serum CA125. The relatively poor specificity of the combined urine biomarker test (40.2%) is offset by its high sensitivity (82.4%), since a triage tool must first seek to not falsely reassure. Whilst our study suggests a comparable accuracy to CA125, urine biomarkers are not intended to replace CA125 and imaging in secondary care; however, if our findings were confirmed in larger populations, urine biomarkers may have a role in triaging symptomatic women for additional testing in the community and in low-resource settings, or as a home-based monitoring tool for women at high risk of developing ovarian cancer.BRCA pathogenic variants and at high risk of developing ovarian cancer, a risk-reducing bilateral salpingo-oophorectomy is recommended following the completion of childbearing [Diagnostic delays in primary care are due to vague symptoms, a low incidence of disease, a lack of recognition of the significance of symptoms, and a failure to investigate and follow up on those who are symptomatic . A urinedbearing . Howeverdbearing . Surveildbearing ,28, and dbearing , and uriIn low-resource healthcare settings and in areas of healthcare inequalities, diagnostic urine biomarkers offer an exciting opportunity to improve ovarian cancer detection. Although mortality rates of ovarian cancer are highest in high income countries, reflecting more incident cases, the mortality/incidence ratios are greatest in low-income countries, due to availability, cost and access to diagnostic investigations . Even wiThis proof-of-concept study, comparing the combination of urine CA125 and HE4 with serum CA125, is the first to be reported, and an important preliminary step in biomarker development, demonstrating the ability of urine CA125 and HE4 to detect ovarian cancer cases from symptomatic controls, the majority of whom had early-stage disease. Biomarker development requires several phases prior to clinical translation, including discovery, analytical validation, clinical validation and clinical utility. These phases have been outlined by the Early Detection Research Network (ERDN) . Our stuThe combination of urine CA125 and HE4 using a strategy where either was positive, shows promise for the detection of ovarian cancer, with a sensitivity comparable to that of serum CA125. Urine biomarkers are cheap and acceptable non-invasive tests, and could be a useful point-of-care tool to triage symptomatic women, or as a remote monitoring tool for women at high risk of ovarian cancer, identifying those requiring further investigations. Larger studies to confirm our findings and optimise urine collection and biomarkers thresholds are required.


Prognostic nutritional index (PNI) is one of the most important factors related to prognosis in many types of cancer. This study aimed to evaluate the PNI on predicting the overall survival (OS) in resectable esophageal squamous cell carcinoma (ESCC).3)]. PNI cutoff value was selected by drawing receiver operating characteristics (ROC) curve, which used OS time as the endpoint. The Kaplan-Meier method and the Cox regression model of multivariate analysis were used to analyze the prognostic relationship between PNI and OS.A total of 165 patients with resectable ESCC were included in our retrospective study. PNI values before surgery were calculated for each patient [PNI = 10 × albumin (gr/dL) + 0.005 × total lymphocyte count (mmP <0.05). The Kaplan-Meier curve showed that the high PNI group has significantly increased OS compared to low PNI group (p = 0.01). Three-year OS rates were 57.5% in the low PNI group while 77.7% in the high PNI group. Univariate analysis showed that advanced pathological stage, large tumor length, and low PNI  were significant risk factors for shorter OS. Multivariate analysis showed that tumor length (P = 0.008) and PNI (P = 0.017) were independent prognostic factors in patients with resectable ESCC.Among the 165 patients, 34 (20.6%) were women and 131 (79.4%) were men. The mean age was 62.67 ± 7.95 years, with the age range from 44 to 85 years. The average PNI was 46.68 ± 8.66. ROC curve showed that the best cutoff value was 43.85. All patients were divided into two groups: 72 patients (43.6%) were in the low PNI group (<43.85), while 93 patients (56.4%) were in the high PNI group (≥ 43.85). Univariate analysis demonstrated that PNI, tumor length, and T-stage and pathological stage were related to the prognosis of patients with ESCC (PNI is a simple and useful predictive marker for the OS time in patients with radical esophagectomy. Esophageal cancer (EC) is one of the most common malignant tumors in the world. This disease has a crude mortality rate of 7.8/100,000 in 2020, which represented 5.5% of all cancer deaths and ranked as the sixth most common cause of cancer death . DespiteDue to dysphagia, swallowing pain, eating obstruction, and tumor consumption, patients with esophageal cancer are prone to malnutrition . Recentlen bloc resection with margins histologically free of disease); (4) have American Society of Anesthesiology (ASA) grade of 1-2. The ASA grade 1 was among 56 patients, which occupied 33.94% of our study group. The ASA grade 2 was among 109 patients, which occupied 66.06% of our study group.From January 2017 to August 2020, a retrospective analysis was conducted in 165 patients with ESCC that underwent radical esophagectomy at the cancer hospital of Shantou University Medical College . All of the patients included in the analysis met the following criteria: (1) have ESCC confirmed by histopathology; (2) had surgery or preceded by adjuvant chemotherapy/radiotherapy before surgery; (3) have curative esophagectomy with R0 resection . The best cut-off value of PNI was selected by drawing the ROC curve used OS time as the endpoint.The PNI was calculated using the following formula: PNI = 10 × albumin (gr/ dL) +0.005 x total lymphocyte count  was calculated. Independent sample P < 0.05 was considered statistically significant.Enumeration data were compared by the chi-square test. The Kaplan-Meier method and the Cox regression model of multivariate analysis were used to analyze the prognostic relationship between PNI and OS. The ROC curve was plotted as shown in p = 0.042), T classification (p = 0.002) and pathological stage (p = 0.014).The relationship between PNI and clinicopathological characteristics of 165 patients enrolled in this study is summarized in p = 0.01). To all patients, three-year OS rates were 57.5% in the low PNI group, while 77.7% in the high PNI group  were alive, while cancer-related death occurred in 32 patients. The Kaplan-Meier analysis and the log-rank test showed that patients with low PNI had a significantly worse prognosis in OS than those with high PNI (NI group .p = 0.045), large tumor length (p < 0.001), and low PNI (p = 0.003) were significant risk factors for shorter OS. Multivariate analysis showed that tumor length (P = 0.008) and PNI (P = 0.017) were independent prognostic factors in patients with resectable ESCC  and Fujian Provincial Department of Science and Technology of China (grant no. 2020Y9070).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The CNN was trained to obtain an average Dice score of 0.94. The interobserver variability comparing two MDs showed mean differences for 2D and 3D respectively of 0.58 to 0.52ml (MD1 vs. 2), −1.33 to −0.17ml (MD1 vs. 3) and −1.89 to −0.70ml (MD2 vs. 3). Paired samples t-tests showed significant differences for 2D  and none for 3D . Intraobsever variability was similar for 2D and 3D ultrasound. Comparison of ultrasound volumes and MRI volumes showed a significant difference for the 2D volumetry of all MDs , and no significant difference for 3D ultrasound . Acquisition time was significantly shorter for 3D ultrasound. Tracked 3D ultrasound combined with a CNN segmentation significantly reduces interobserver variability in thyroid volumetry and increases the accuracy of the measurements with shorter acquisition times.Thyroid volumetry is crucial in the diagnosis, treatment, and monitoring of thyroid diseases. However, conventional thyroid volumetry with 2D ultrasound is highly operator-dependent. This study compares 2D and tracked 3D ultrasound with an automatic thyroid segmentation based on a deep neural network regarding inter- and intraobserver variability, time, and accuracy. Volume reference was MRI. 28 healthy volunteers (24—50 a) were scanned with 2D and 3D ultrasound (and by MRI) by three physicians  with different experience levels . In the 2D scans, the thyroid lobe volumes were calculated with the ellipsoid formula. A convolutional deep neural network (CNN) automatically segmented the 3D thyroid lobes. 26, 6, and 6 random lobe scans were used for training, validation, and testing, respectively. On MRI (T1 VIBE sequence) the thyroid was manually segmented by an experienced MD. MRI thyroid volumes ranged from 2.8 to 16.7 Exact thyroid volumetry plays an essential role in monitoring and treatment of many thyroid diseases, especially for the radioiodine therapy (RIT) in hyperthyroidism: The needed activity, commonly calculated using Marinelli’s formula, is proportional to the thyroid mass . The latIn contrast, we present an analysis on volumetry and variability in a realistic setup using 3D compounded ultrasound volumes acquired with a tracked ultrasound system. Analyzing intra- and interobserver variability in thyroid volumetry and comparing 2D and 3D US in this task is a fairly well-analyzed topic in literature , 15–23. We introduce a fully automatic method for 3D volumetry of thyroid lobes using tracked 3D US.We comprehensively evaluate our method compared to 2D US by analyzing intra- and interobserver variability and accuracy by comparing to MRI.We evaluate the acquisition time performance of our method compared to 2D US.We provide a novel tracked 3D US thyroid dataset as the first bigger and labelled open source dataset that will become publicly available upon acceptance.mm. For the 3D US, electromagnetic tracking, and video acquisition, a PIUR tUS system was used . This device employs two 6D tracking sensors, operating at a frequency of 80 Hz with accuracy in position of 1.40 mm RMS and in angle of 0.50° RMS. Video sequences were acquired with 89 fps. 3D US probes, which consist of matrix arrays, can also be used to obtain US volumes. However, these probes are not widely available, rather bulky, and often do not cover the entire thyroid making a compounding necessary. The image processing (3D US compounding using image data and tracking information) and thyroid segmentation was done using ImFusion Suite Version 2.9.7 . PyTorch was used for the implementation of a deep neural network for automatic 3D US thyroid segmentation and Tensorflow was used for the evaluation of the network. The model was trained on an NVIDIA Titan V 12 GB HBM2 using Polyaxon to schedule different runs (https://polyaxon.com/). The training time was around 10 hours in average. The inference on new volumes however took only few seconds. The segmentation pipeline was implemented and trained by our team and differed from the one offered by piur imaging in their latest release. We did not use piur’s segmentation tool for this study to be able to modify the segmentation pipeline for our project and to be able to provide this as work open-source for the research community.This study was conducted in the scope of an Institutional Review Board Approval from the Ethical Commission of the Technical University of Munich . All volunteers gave written informed consent to the US and MRI scans. Eligible participants had to be anamnestically healthy and at least 18 years old and were recruited from the hospital and research lab environment. Exclusion criteria for possible candidates were subtotal or total thyroidectomy, distinct postoperative or inherent changes in the neck anatomy and MRI contraindications . Twenty-eight healthy volunteers, aged 23 to 50, eighteen males and ten females, participated in the study. One high-resolution T1 VIBE (Volumetric interpolated breath-hold examination) MRI scan of the neck was acquired with a Biograph mMR machine  with a field strength of 3 T. The resolution of the MRI image was 0.625x0.625x1.00 mm3, and the field of view 320x320x80 mm . The accThree 2D and three 3D US scans for each volunteer were acquired by three physicians  with different levels of experience . The ellipsoid formula (correction factor 0.48) was applied to estimate the thyroid volumes from the 2D US scans. No isthmus correction was included. To acquire a volumetric image for a 3D US, the physician slowly sweeps the transducer over both lobes (one sweep per lobe). These sweeps are automatically compounded into a 3D US stack. The 3D US thyroid volumes were calculated based on the automatic segmentation. The resolution of the segmentation mask matched the one of the US image. As a result, volume calculation succeeded by counting all voxels labeled as thyroid. The workflow for the 3D volumetry task can be seen in For the automatic segmentation of the 3D US scans, a fully convolutional deep neural network developed by Roy et al.  named QuSD). The interobserver variability was estimated by applying the method proposed by Choi et al. [α = 0.05 for all tests in both groups. To compare 2D and 3D US volume measurements to MRI, we applied paired samples t-tests between MRI the first scan sequence from each MD with the same significance level resulting in the same critical values per group.From the complete data set, scans from 16 volunteers were used for training and validation. Thus, the network was trained on images that were segmented by the same network in a later step for the volumetry estimation. We, therefore, split the evaluation into two groups, one including scans that were fed to the network during training (15 volunteers) (Group 1) and the other with only unseen data (13 volunteers) (Group 2) to be able to detect a potential bias. To estimate the intraobserver variability of the acquisitions, we used a formula from Lyshchik et al. . We exami et al. . Here, tWe will present all results for Group 1 and Group 2 separately.Intraobserver variability is summarized in The interobserver variability can be seen in For the US volume, the first scan series per MD was chosen and compared to the MRI volume. Time needed for the 2D scans was on average 58.8 sec (SD 11.05 sec) for MD1, 55.0 sec (SD 8.8 sec) for MD2 and for 35.7 sec (SD 5.8 sec) MD3, while the 3D scans were performed on average in 26.0 sec (SD 5.5 sec) by MD1, 21.1 sec (SD 5.9 sec) by MD2 and in 21.6 sec (SD 5.2 sec) by MD3.SD = 6.12%). The interobserver difference was 9.51% (SD = 8.8%) [SD = 10.5%) and 11.3% (SD = 8.5%). The interobserver variability amounted to 15.3% (SD = 16.6%) [cm and those with a diameter bigger than 2cm. In the first group the interobserver variability was 7.0% (SD = 4.9%) and –5.1% (SD = 3.6%) in the second group [Intra- and interobserver variability in thyroid volumetry is a fairly well analysed problem. Vulpoi et al. had three MDs examine 30 children with 2D US. One physician scanned 25 patients twice to gather data on intraobserver variability, yielding 6.29% ( = 8.8%) . Lee et = 16.6%) . In the nd group , 18.Our study focused on the total thyroid volume. In comparison to the results reported by Vulpoi et al., Lee et al. and Choi et al., our results for intra- and interobserver variability in 2D US  are slightly higher.SD = 0.7%) for 2D US and 5.9% (SD = 0.3%) for 3D US. The accuracy for 2D US was 15.9% and 2.8% for 3D US. In the second study the intraobserver variability amounted to 14.4% (SD = 1.9%) in 2D US and to 3.36% (SD = 0.25%) for 3D US. The accuracy amounted to 15.3% for 2D US and 5.2% for 3D US. The second study showed no significant variation between the thyroid weight and both US modalities [Lyshchik et al. conducted two studies on comparing the volume of thyroid nodules in children both with 2D and 3D US. In the first study one physician scanned 102 children with 129 nodules three times, and delineated their thyroids. The delineated thyroids were controlled by a second expert. The second study included 47 children prior to thyroidectomy. Here, the thyroid weights after surgery were used as ground truth. The nodule volumes were calculated with the ellipsoid formula for 2D and multiplanar volume approximation, respectively manual planimetry, for the 3D scans. Both studies showed very similar results. 3D US showed a lower user dependency than 2D US, had a more accurate thyroid nodule volumetry, was less dependent on nodule size and performed better on irregular nodule outlines. In the first study the intraobserver variability was 16.1% , between the MRI-ROI and mean 2D US measurements was found. No significant interobserver variability among the MDs could be determined [ml for B-Mode, −0.06 ± 0.09ml for tUS, 0.01 ± 0.18ml for CT and −0.20 ± 0.32ml for MRI. These results align with our results in showing that tracked 3D US shows a smaller difference to MRI than 2D US. Furthermore, it shows the second largest mean volume error in volume estimation for MRI. This aligns with our observation of a probable continuous underestimation of our MRI segmentations.With respect to MRI, Reinartz et al. compared 2D US thyroid volumetry to MRI (T1 FFE). Three different experienced MDs performed US scans on patients prior to RIT. The thyroid volumes were calculated by using the ellipsoid formula for the 2D US measurements and by manual segmentation. A significant difference of 22.7% . Based on this data, the network can handle small changes in the ultrasound image. For more prominent changes in the imaging parameters , resulting in more considerable changes in the ultrasound image, we believe that the CNN would have to be adjusted in its architecture and fine-tuned to increase its generalizability. Compared to the current clinical practice, the improved performance can be based on the nature of the 3D acquisition and the automatic segmentation. 3D US itself makes a difference compared to 2D US as it removes the variability of the probe placement with respect to the anatomy through volume compounding. The automatic segmentation allows for a complete thyroid segmentation, which provides a shape closer to the actual thyroid form than an ellipsoid approximation.Only young and healthy volunteers were enrolled, simplifying the segmentation and volumetry tasks.The CNN was trained with 16 volunteers only, allowing for a good Dice score but leaving space for improvements. These can include cross-validation as well as training on 3D input data instead of 2D images. The latter one might allow for additional spatial information which could improve the network performance; yet, it would require a significantly larger dataset. Visual analysis also showed non-optimal segmentation results in few scans . The netOur study shows that 3D US outperforms 2D US significantly. This was consequently seen in both groups 1 and 2 denoting “training” and “new” data. Regarding the intraobserver variability, 3D US outperforms 2D US with 2 out of 3 MDs. In the interobserver variability 3D US increases the similarity of acquisition scans between two MDs in 2 out of 3 cases. The results also show that the improvement with 3D US is the biggest for the least experienced MD. We show that the 3D US volumetry is more accurate than 2D US using MRI as ground truth. The tracked 3D US acquisition is significantly faster as well. Therefore, the combination of tracked 3D US and automatic segmentation is not only feasible but offers a more accurate alternative to conventional US imaging. The labelled thyroid 3D US dataset will be freely available helping further projects in deep learning for thyroid volumetry within the research community. We suggest conducting similar studies on patients with thyroid morbidities to stratify the clinical benefit of our framework.


This survey study assesses the occurrence of short-term adverse events in adults 60 years or older who received a booster dose of the BNT162B2 mRNA vaccine. This survey study assessed the occurrence of adverse effects (AEs) in adults 60 years or older who received a booster dose.On July 29, 2021, concerns of waning immunity after Pfizer-BioNTech BNT162B2 mRNA vaccination led the Israeli Ministry of Health to start a campaign to administer booster (third) doses to individuals who received their second dose at least 5 months prior.AAPOR reporting guideline for survey studies.This study was conducted among members of Clalit Health Services (CHS), which insures more than half of the Israeli population. Study participants were individuals 60 years or older who received the booster dose of the Pfizer-BioNTech BNT162b2 mRNA COVID-19 vaccine in the first 5 days of the campaign . The CHS institutional review board approved this study, with a waiver of informed consent because deidentified survey data were used. This study followed the Individuals aged 60 to 79 years were sent a text message with a request to complete an online survey  and 20 to 28 days  after the initiation of the campaign. For participants who responded to both surveys, we considered only the latter response. Individuals’ demographic and clinical characteristics were extracted from their electronic health records.Of 82 392 CHS members 60 years or older who received a booster dose during the study period, 66 094 were contacted, with 27 046 (40.9%) responding to the survey. The median age of respondents was 71 years ; 43.6% of respondents were aged 60 to 69 years; 44.5%, 70 to 79 years; and 12.0%, 80 years or older. The proportion of female respondents was 45.3%, and 49.2% had at least 1 risk factor for severe COVID-19 .Of the respondents, 30.0% reported at least 1 AE, 24.8% reported local reactions, and 16.6% reported systemic reactions. The most common AEs included pain at the injection site (23.5%), fatigue (9.7%), and malaise (7.2%) .Most of the respondents (67.8%) reported that their general feeling after the booster was similar to the feeling after the second dose; 18.7% and 11.1% reported a milder or worse response, respectively. Only 1.2% sought medical attention owing to an AE.Females were more likely than males to report AEs (39.0% vs 22.6%). The proportion of females who reported systemic reactions was nearly double that of males (22.9% vs 11.4%). Individuals aged 60 to 69 years were more likely to report systemic AEs than were individuals 70 years or older (20.5% vs 13.6%).4 A study by Menni et al4 found a similar proportion of systemic reactions among older individuals after the second vaccine dose as after the third dose in our study (16.4% vs 16.6%).We found that AEs after the BNT162b2 mRNA vaccine booster dose were generally mild and usually did not require medical care. The proportion of self-reported AEs that occurred in our study was similar or lower than that after the administration of the second vaccine dose in several previous studies.5 A limitation of our study was the different survey methods in different age groups, which might have resulted in differences in reported proportions of AEs.The proportion of female respondents who reported systemic AEs was greater than the proportion of male respondents, with higher proportions among participants in the younger age group (60-69 years) than in the older age groups. Similar results were reported in previous studies after administration of the second vaccine dose.


Introduction: Type 2 diabetes (T2DM), which is more prevalent  and the main driver of the diabetes epidemic, now affects 5.9% of the world’s adult population, with almost 80% of the total in developing countries. At present, 537 million adults (20–79 years) are living with diabetes—1 in 10. This number is predicted to rise to 643 million by 2030 and 783 million by 2045. In India, reports show that 69.2 million people are living with diabetes (8.7%) as per 2015 data. Long-term metformin treatment is a known pharmacological cause of vitamin B12 (Vit B12) deficiency, as was evident within the first 10–12 years after it started to be used. Methods: This was a cross-sectional study conducted in the Postgraduate Department of Medicine in one of the tertiary hospitals in Kashmir. A total of 1600 consecutive patients with T2DM were taken for the study. Out of which 700 patients met the inclusion criteria. These 700 patients were divided into two groups: those taking metformin, and those who were not on metformin. Cumulative metformin doses were recorded in patients taking metformin, using history of dose and duration of treatment. Serum Vit B12 levels were taken for all patients. Based on the results of Vit B12 levels, patients were classified into normal levels (20 pmol/L), possible B12 deficiency (150–220 pmol/l), and definite deficiency (<150 pmol/L). Results: Our results depicted that patients on prolonged metformin therapy showed an increase in Vit B12 deficiency by 11.16%. The prevalence of clinical neuropathy in the metformin-exposed group was 45%, whereas, a prevalence of 31.8% was found in the non-metformin group. The mean age of patients with neuropathy was higher than those without neuropathy (59.01 ± 7.14 vs. 49.95 ± 7.47) . Conclusions: In our study, we found that metformin use is associated with Vit B12 deficiency, which is dependent upon the cumulative dose of metformin. Importantly, prolonged metformin use is also associated with an increase in the prevalence of clinical neuropathy. Diabetes is becoming the fastest epidemic of the 21st century. Type 2 diabetes (T2DM), which occurs in more than 90% of all diabetes cases and is the primary cause of the diabetes epidemic, now affects 5.9% of the world’s adult population, with nearly 80% of the total occurring in developing countries . The IntMetformin is the most commonly prescribed oral anti-diabetic drug in patients with T2DM . MetformThis was a cross-sectional study conducted in the Postgraduate Department of Medicine at Government Medical College Srinagar in North India, from September 2014 to November 2016. A total of 1600 consecutive patients of T2DM were screened for the study. Among them, 187 patients declined to participate in the study and 713 patients did not meet the inclusion criteria. A total of 700 T2DM patients participated, and were divided into two groups: those taking metformin, and those who were not on metformin. Cumulative metformin doses were recorded in patients taking metformin using their dose and duration of treatment. In all the outpatients and admitted diabetes patients, a detailed history was taken and a clinical examination was performed. Blood pressure, body mass index (BMI), HbA1c and baseline investigations were recorded in every patient. Serum Vit B12 levels were taken in all the patients. Serum samples were stored at room temperature (15–30 °C) for no longer than 7 h. All included patients were subjected to Vit B 12 assay, which was performed using the Roche E-170 Vit B12 electrochemiluminescence immunoassay (ECLIA) method [The inclusion criteria in this study were as follows: age group of 31–70 years, patients with T2DM according to American Diabetes Association, 2017 (ADA) criteria and patients withT2DM undergoing treatment for diabetes. The exclusion criteria for the study were as follows: alcoholism, ongoing pregnancy, liver disease, renal disease, thyroid disorders, history suggestive of malabsorption disorders and history of use of proton pump inhibitors & Vit B12 supplements. The baseline demographic variables, such as age, sex, dietary habits (vegetarian or on mixed diet), and HbA1c levels, were measured in both groups, as well as Vit B12 levels and severity of peripheral neuropathy  . The stut-test and ANOVA with post-hoc (Tukey’s Honest Significant Difference) were also employed for the analysis of data. Graphically, the data was presented using scatter diagram. A p-value of <0.05 was taken as statistically significant.Data analysis was conducted using SPSS 20.0 statistical software  Continuous data were summarized as mean and standard deviation. Categorical data were summarised as frequency and percentage. The difference in the prevalence of vitamin B12 deficiency and clinical neuropathy between metformin and non-metformin groups was analysed using the chi-square test. The association between cumulative metformin dose and vitamin B12 levels was analysed using Pearson’s correlation coefficient and regression analysis. Moreover, Student’s Independent Out of 700 T2DM patients in the age group from 30 to 70 years included in this study, 366 patients (52.29%) were males and 334 (47.71%) were females. The minimum age among patients taken in this study was 31 years and the maximum age of 70 years. The mean age of males was 50 years and that of females was 52.56 years. There were 373 (53.29%) patients in our study living in urban and 327 (46.71%) in rural areas. A total of 98% were married and only 2% were unmarried; 320 (45.71%) were literate and 380 (54.29%) were illiterate.2 and for females it was 28.4 ± 2.16 kg/m2 with a minimum value of 19.6 kg/m2 and maximum value of 36.8 kg/m2.Out of 700 T2DM patients, 451 (64.4%) were on metformin and 249 (35.6%) patients were on insulin, sulfonylureas, and gliptins. We divided these patients into two groups: those taking metformin (metformin group) and those not taking metformin (non-metformin group). In the metformin group, 307 patients were on metformin alone, 116 patients were on metformin and sulfonylureas and 28 patients were on metformin as well as insulin. In the non-metformin group, out of 249 patients, 203 were on insulin and 46 patients were on sulfonylureas and/or voglibose. The mean BMI of the study population was 27.15 ± 2.2; for males it was 26 ± 2.31 kg/mBased on Vit B 12 levels, the patients were divided into three groups: vit-B12-sufficient group, borderline-deficient group and B12-deficient group.The patients were divided into two groups, metformin and non-metformin, with a mean treatment duration as given in The relationship between cumulative dose of metformin and vit B12 deficiency is shown in p value < 0.001). This clearly shows that, with an increase in cumulative dose of metformin, vitamin B12 levels decrease.As we can see from The relationship between neuropathy score ) and metformin use is shown in We defined definite and possible (borderline) deficiency as serum vit B12 levels of <150 and <220 pmol/L, respectively ,18,19,20Out of 700 T2DM patients, 451 (64.4%) and 249 (35.6%) patients belonged to the metformin and non-metformin group, respectively . The mean = 84) compared with the non-metformin-exposed group (n = 52) , but another study conducted by Gupta et al. [p value 0.002). Mean B12 levels were significantly lower in the metformin group: 311 pg/mL (±194.4), p value 0.03. A study by Omar Marar et al. [p < 0.00001}, and Joline WJ Beulens et al. [The prevalence of vit B12 deficiency in the metformin and non-metformin observed groups was 33.26% and 22.1%, respectively. This shows that patients on prolonged metformin therapy showed an 11.16% increase in vit B12 deficiency. A study conducted in Indian population by Singh A K et al.  found tha et al. , found ar et al.  showed ts et al.  also shop-value < 0.001). Pearson’s correlation coefficient for a cumulative dose of metformin and the observed vitamin B12 levels was −0.662, which is statistically significant . This clearly shows that, with an increase in the cumulative dose of metformin, the vitamin B12 levels decrease   (ificant) . These rp-value < 0.001) , showing that HbA1c levels in the clinical neuropathy group were higher, by a mean of 0.78%, than those in patients without neuropathy  . Howeveruropathy . This suThe major limitation of our study is that it was a cross-sectional study, and we could not find any evidence of a temporal relationship between exposure and outcome. We did not include patients with vitamin B12 supplements, which may have helped us more with treatment suggestions.In our study, we found that metformin use is associated with vitamin B12 deficiency, depending on the cumulative metformin dose. Prolonged metformin use is associated with an increase in the prevalence of clinical neuropathy, possibly due to vitamin B12 deficiency caused by metformin use. Poor glycemic control is also associated with an increased prevalence of clinical neuropathy. Therefore, the rationale is to screen patients who are to undergo metformin therapy for vitamin B12 deficiency and monitor for B12 deficiency once the patient has been started on metformin.


Spodoptera frugiperda, the key invasive pest of maize, is a serious concern due to its biology and the current global restriction on applying synthetic pesticides. Entomopathogenic fungi are considered to be a potential biological control strategy. The pathogenicity of 12 isolates of Beauveria bassiana in the immature stages and feeding efficacy of S. frugiperda were evaluated. The B. bassiana isolates QB-3.45, QB-3.46 and QB-3.428 caused the highest egg mortality rates of 87.3, 82.7 and 79.3%, respectively, when applied at a concentration of 1 × 108 conidia/mL and measured at 7 days post-treatment. Neonate mortality rates of 45.6 to 53.6% were observed with the same isolates. The B. bassiana isolates caused significant cumulative mortality rates ranging from 71.3 to 93.3% at 14 days post-treatment and reduced larval feeding efficacy from 69.4 to 77.8% at 48 h post-treatment. This study supports using the effective B. bassiana isolates as a biological control agent against S. frugiperda. The significant mortality of the eggs and neonatal larvae and the reduction in the feeding efficacy of the second instar larvae of the S. frugiperda that were treated with isolates of B. bassiana supports the application of entomopathogenic fungi as a biocontrol agent for the effective control of the S. frugiperda population.The control of  Maize is one of the primary crops producing commercial cereal commodities worldwide, second to rice and wheat . In addiSpodoptera frugiperda, JE Smith, 1797, (Lepidoptera: Noctuid) is one of the most destructive, invasive pests with more than 350 host-plant species due to its polyphagous nature; however, it prefers to feed on maize. FAW is native to the Americas [Fall armyworm (FAW), Americas ,7. MaizeAmericas ,9, whichAmericas . FAW larAmericas ,12.FAW is considered an A1 quarantine invasive pest in Africa . In 2019The fall armyworm was first detected in Africa and it then expanded throughout the world to locations such as Nepal, Indonesia, and Swaziland ,20,21. FMost maize growers in Indonesia prefer using pesticides to control this invasive pest in order to avoid the yield losses of maize to FAW . The conMetarhizium anisopliae, Beauveria bassiana and Bacillus thuringiensis are the most effective biocontrol agents among the active biopesticide products that have been registered by the United States Environmental Protection Agency as of 2016 [B. bassiana, which can be effectively transmitted and produces a variety of toxin-causing infections in the host [B. bassiana was first isolated from silkworm cadavers by Agostino Bassi in the 19th century and it has since infected more than 200 species of insects belong to different orders [Biocontrol agents, such as entomopathogenic fungi, have prominent virulence factors and are rapidly emerging as prime substitutes for synthetic insecticides . Metarhi of 2016 . The mosthe host ,53. B. bt orders .Beauveria bassiana is competitive with synthetic insecticides against forest and farm insect pests in China [B. bassiana were screened in order to evaluate their pathogenicity against the S. frugiperda adult in Nairobi, Kenya [B. bassiana resulted in affecting the larval development of S. frugiperda in Eldoret, Kenya [S. frugiperda in Mexico [B. bassiana proved to be effective for the management of S. frugiperda in Thailand [B. bassiana isolates are considered to be a sound biocontrol agent for the management of FAW [in China . Six isoi, Kenya . Tomato t, Kenya . Entomopn Mexico ,59. The Thailand . B. basst of FAW ,62.Beauveria, from which several bio-insecticides have been derived and used in Indonesia [B. bassiana isolates were used in the present study, originating from different hosts and already tested for their virulence against Solenopsis invicta Buren, one of the most important pests in China [B. bassiana was indicated to be an entomopathogen in many insects’ orders; lepidopteran pests in particular have been effectively controlled by this fungus [Among the identified species of fungi, the most utilized are in the genus ndonesia . Entomopndonesia . Twelve in China . B. basss fungus . Three ds fungus . ConsequEntomopathogenic fungi (EPF) are environmentally friendly and could replace synthetic insecticides. However, one negative impact of the EPFs is that they kill or infect the insect much more slowly than a synthetic insecticide due to their different modes of action depending on which insect continuously feeds on the crop. The different life stages of any insect do not respond to any pathogenic stress in the same way. So, it is very normal to test EPF on various life stages in order to understand which life stage is most susceptible or to find the stage that is the most resistant to the said fungal isolate. This method is able to inform the users of the most appropriate stage of the insect to target for effective biological control programs. Our previous study has concluded that the eggs are the most susceptible stage for targeting as compared to the other life stages of FAW ,62. It hB. bassiana were proved to be pathogenic against S. invicta in 2018 [B. bassiana against the immature stages  and the feeding efficacy of FAW. Therefore, the present study aims to investigate the bioassay of isolates of B. bassiana for the management of FAW.These isolates of  in 2018  but afteThe FAW eggs were established using a collection of larvae that were found on maize crops from Guangdong province, China, during the maize growing season. The collected larvae were cultured on an artificial diet  and the B. bassiana was evaluated against the immature stages and the feeding efficacy of FAW. B. bassiana isolates that were obtained from the IZ-GDAS’s laboratory were cultured on potato dextrose agar (PDA) and maintained at 25 ± 2 °C in the dark. The tested isolates of B. bassiana were previously identified by Li et al. [B. bassiana. The information regarding the B. bassiana isolates is available in  were obtained from the adult cylindrical cages. A batch of 50 eggs was separated with a light microscope using a camel hairbrush. Then, 10 mL of the spore suspension at each concentration was sprayed on a set of 50 eggs using manual atomized spray bottles (20 mL). A sterilized paper towel was used at the bottom of the Petri plate for the absorption of the extra spore suspension. The control was treated with distilled water containing 0.05% Tween-80. The batch of treated eggs was air-dried for 1 h under a laminar hood. The eggs (hatched and non-hatched) were recorded at 7 days post-treatment.B. bassiana were fed fresh maize leaves in a rectangular plastic box (28 × 17 × 18 cm3) that was lined with moist filter paper, incubated at 25 ± 2 °C and monitored daily. The maize was grown under lab conditions in order to ensure that the maize leaves were fresh and insecticide-free.The FAW neonatal larvae that emerged from the eggs that were treated with the isolates of The mortality of the neonatal larvae that were treated with the isolates was further recorded daily for 7 days post-emergence. The bioassays were laid out according to a completely randomized design with three replications for each treatment. Mycosis was assessed for the dead cadavers by following the approach of Aktuse et al. . The dea3). A group of 30 second instar larvae of FAW was shifted into the box containing the maize leaves. Each group of 30 larvae represented one replicate and three replications were used for each treatment with a completely randomized design. Each group of second instar larvae in each plastic box was provided with fresh maize leaves that had been treated with 10 mL of 1 × 106, 107 or 108 conidia/mL using a spray tower. Each concentration was sprayed on every second instar larva to ensure that none escaped from the fungal spore suspension by hiding beneath the leaves’ surfaces. The control larvae were treated with distilled water (0.05% Tween-80). The treated larvae were incubated at 25 ± 2 °C and fresh maize leaves were provided daily. The larval mortality rate was recorded daily for 7 days. A mycosis test was conducted in order to confirm that the mortality of the larvae was due to a fungal infection, as described above.Fresh maize leaves were placed individually in a perforated rectangular plastic box  [3). The control was treated with distilled water containing 0.05% Tween-80. The maize leaves that were provided to the group of second instar larvae as an insect diet were weighed before and after being treated. The feeding efficacy percentage was measured by calculating the total weight of the fresh maize leaves, in grams, that were provided to the larvae divided by the total weight of the leaves that were not fed on by the larvae, multiplied by 100 [A group of 15 second instar stages of FAW was treated with 10 mL of 1 × 10 (20 mL) . Fresh md by 100 . The feeB. bassiana were screened for their ability to cause the mortality of the pupae of FAW. The pupae of the FAW were treated with 10 mL of 1 × 106, 1 × 107, or 1 × 108 conidia/mL using manual atomizer sprayer bottles (20 mL). The FAW pupae were placed in a perforated rectangular plastic box (28 × 17 × 18 cm3). Each group of 10 pupae represented one replicate and three replications were used for each treatment. The control was treated with a solution of distilled water containing 0.05% Tween-80. The pupal mortality rate was recorded for 15 days [The isolates of  15 days . A pupa ® version 8.1  were used to perform for the data analysis and to calculate the homogeneous letters, respectively.The mortality data for all of the stages were analyzed using Abbott’s formula  and the B. bassiana induced egg mortality rates of 30.0 and 28.7%, respectively, followed by the QB-3.46, LNSE-22 and QB-3.436 isolates, which caused 25.3, 24.0 and 20.7% FAW egg mortality when applied at a concentration of 1 × 106 conidia/mL and measured at 7 days post-treatment . . However, the QB-3.436 and LNSE-22 isolates caused egg mortality rates of 40.0 and 35.3% . However, the XJWLMQ-32 and SPLE-24 isolates of B. bassiana caused 39.3 and 30.7% egg mortality, respectively, 7 days post-treatment . . QB-3.45nd 35.3% . The QB-reatment .B. bassiana were the least significant and induced additional mortality rates of 14.9 and 13.4% when applied at a concentration of 1 × 106 conidia/mL  against the neonatal larvae of FAW and measured at 7 days post-treatment   (The results indicated that the effects of the QB-3.428 and QB-3.46 isolates of reatment . The QB-< 0.000) . The QB-< 0.000) . FurtherB. bassiana induced cumulative mortality rates of 39.3, 36.7 and 35.3% against eggs and neonates, respectively, followed by the QB-3.436 and LNSE-22 isolates’ mortality rate of 31.3% at a concentration of 1 × 106 conidia/mL and measured at 14 days post-treatment . However, ≤16.7% cumulative mortality was observed for the other tested isolates . Furthermore, the rest of the other isolates of B. bassiana caused ≤26.0% cumulative mortality rates . Furthermore, 50.0 and 44.7% cumulative mortality rates were observed with the ZGNKY-01 and SPLE-24 isolates of B. bassiana . The QB-3.428, QB-3.46 and QB-3.436 isolates of B. bassiana caused 13.4% to 14.9% mortality rates of larvae of FAW that were treated with 1 × 107 conidia/mL . The QB-3.45 and QB-3.45 isolates of B. bassiana caused larval mortality of 25.6 and 20.0% and the QB-3.428 and QB-3.436 isolates caused 17.8 and 14.4% larval mortality of FAW when applied at a concentration of 1 × 108 conidia/mL   .B. bassiana significantly reduced the feeding efficacies of the second instar larvae of FAW by 56.9 and 54.2%, respectively, followed by QB-3.428 and QB-3.436, which reduced the feeding efficacy to 48.6 and 43.1% when applied at a concentration of 1 × 106 conidia/mL and measured at 48 h post-treatment . The QB-3.45 and QB-3.46 isolates of B. bassiana reduced the feeding efficacy to 65.3 and 61.1%, respectively, followed by QB-3.428 (58.3%), in second instar larvae when applied at a concentration of 1 × 107 conidia/mL . The QB-3.45 and QB-3.46 isolates of B. bassiana reduced the feeding efficacy rates to 77.8 and 72.2%, respectively, followed by QB-3.428 (69.4%), against the second instar FAW larvae . The QB-3.436 and LNSE-22 isolates of B. bassiana moderately reduced the feeding efficacy to 54.2 and 51.4%, respectively, against the second instar larva of FAW  (The results showed that the isolates of > 0.000) .2, 286 = 280.34; p > 0.000), the treatments , the life stages , and the interaction  on the mortality of FAW  of FAW. These isolates also showed effectiveness in reducing the feeding efficacy of the treated larvae. In the present study, the QB-3.45, QB-3.46 and QB-3.428 isolates of B. bassiana proved to be the most effective for the management of the FAW population. Therefore, these isolates perhaps provide bases for the development of commercial biological insecticides. Further research is needed to find the main toxins that affect the virulence factors of the entomopathogenic fungal isolates.The


Pediatric adrenocortical tumors have a poor prognosis. The EXPeRT consortium recently published recommendations for their management. In this study, we report on 161 pediatric ACT patients registered with the German Malignant Endocrine Tumor studies and explore those recommendations. The median age at the diagnosis was 4.3 years, and the mean follow-up was 4.5 years. The 3-year overall (OS) and event-free survival (EFS) estimates were 65.5% and 50.6%. The clinical presentation and prognosis were defined by age. The OS was impaired for patients aged ≥ 4 years, following the initial biopsy, tumor spillage, and incomplete tumor resection, with unfavorable histology, according to the five-item microscopic score and COG stages III and IV. COG stages III and IV and unfavorable histology were impacted as negative prognostic factors upon the EFS and OS.n = 51, ACx n = 19, and ACC n = 91) had been reported (the median age at the diagnosis was 4.3 years with a range of 0.1–17.8), with lymph node and distant metastases in 10.7% and 18.9% of the patients with ACC/ACx. The mean follow-up was 4.5 years (with a range of 0–16.7). The three-year overall (OS) and event-free survival (EFS) rates were 65.5% and 50.6%. In the univariate analyses, the OS was impaired for patients aged ≥ 4 years (p = 0.001) with the initial biopsy (p = 0.016), tumor spillage (p = 0.028), incomplete tumor resection (p < 0.001), unfavorable histology (p = 0.047), and COG stages III/IV (p = 0.002). Multivariate analysis revealed COG stages III/IV and an unfavorable five-item score as independent negative prognostic factors for the EFS and OS. Conclusions: Age defines the clinical presentation and prognosis in pediatric ACTs. The outcome is best predicted by the COG stage and five-item score.Background: Adrenocortical tumors (ACTs) encompassing the adrenocortical adenoma (ACA), carcinoma (ACC), and tumors of undetermined malignant potential (ACx) are rare endocrine neoplasms with a poor prognosis. We report on pediatric ACT patients registered with the Malignant Endocrine Tumor studies and explore the EXPeRT recommendations for management. Patients: Data from the ACT patients (<18 years) were analyzed. For the risk prediction, the patients were retrospectively assigned to the COG stages and the five-item score. Results: By December 2021, 161 patients with ACT (ACA  TP53 p.R337H founder variant  with a median age at diagnosis of 4.3 years (a range of 0.1–17.8) and a female to male ratio of 2.4:1. Retroperitoneal lymph node involvement and distant metastases were present in 11  and 20  of patients with ACCs/ACx. The patients aged ≥ 4 years more frequently presented with distant metastases compared to patients < 4 years . No difference was observed for retroperitoneal lymph node involvement in these age groups . The patient characteristics are detailed in The MET database registered 161 patients with ACTs , with or without mitotane, were administered. In 16  patients, tumor resection was performed after a median of four cycles of chemotherapy. The response evaluation after two to four cycles of chemotherapy demonstrated a partial response in 7 , stable disease in 4 (21.1%), and progressive disease in 8 (42.1%). In 4  patients, neoadjuvant chemotherapy failed to achieve operability, and they received chemotherapy only. In 20  patients, one to six cycles of neoadjuvant chemotherapy [NN-1/NN-2, p = 0.435). In 21  patients, a biopsy was performed prior to resection. Biopsies were just as frequent in patients ≥ 4 years compared to patients < 4 years  tumor resection was achieved in 124  patients, whereas microscopic (R1) and macroscopic (R2) residues were left in 15 (10.3%) and 7 (4.8%) patients, respectively. The details of the histopathological evaluation are given in 0 resection was achieved less frequently in patients ≥ 4 years compared to patients < 4 years . Intraoperative tumor spillage occurred in 21  patients. Spillage was more frequent in patients ≥ 4 years . A microscopically complete (R3 (with a range of 0.5–3645.0), with significantly larger tumors in children ≥ 4 than <4 years of age (p < 0.001). The tumor volume increased linearly by 14.5 mL per year of age at diagnosis . On the histopathology, the Ki-67 index was >15% in 52  patients, without differences between patients aged < 4 years and ≥4 years . The Wieneke index classified 56  tumors as benign and 23 (26.1%) as malignant, while 9 (10.2%) tumors remained undetermined. The malignant histology was more frequent in patients aged ≥ 4 years compared to those <4 years . According to the five-item microscopic score, 62  tumors were allocated to favorable and 50 (44.6%) to unfavorable histology. Unfavorable histology was just as frequent in patients ≥ 4 years compared to patients < 4 years . The median tumor volume was 160.8 cmp < 0.001).The post-surgical COG staging system in patients with ACCs and ACx allocated 20  patients to stage I, 18 (18.9%) to stage II, 37 (38.9%) to stage III, and 20 (21.1%) to stage IV. Stage IV patients were older when compared to those with stages I, II, and III  patients and chemotherapy alone to two (5.7%). No adjuvant treatment was administered in 16 (45.7%) patients. In metastatic disease (stage IV), adjuvant treatment encompassed chemotherapy and mitotane in 9  patients, chemotherapy alone in 2 (13.3%), and mitotane alone in 3 (20.0%). No adjuvant treatment was given to one (6.7%) stage IV patient. In 3 stage II cases with T4 tumors, mitotane was given postoperatively. Mitotane was administered over a median duration of 10.8 months (with a range of 0.5–21.6) in stage III and 4.2 months (with a range of 0.2–30) in stage IV cases. Adjuvant therapy was terminated due to side effects in 5  patients.The response evaluation after two to four cycles of adjuvant chemotherapy in 20 patients with residual tumor lesions demonstrated CR in one (5.0%), a partial response in 8 (40.0%), stable disease in 2 (10.0%), and progressive disease in 9 (45.5%). In 19 patients, adjuvant chemotherapy was given without a residual tumor.The median duration of the follow-up was 2.6 years (with a range of 0–16.7) and a mean duration of 4.5 years. The three-year OS and EFS estimates were 65.5% and 50.6% . p < 0.001). Progression or relapse occurred at a median of 7.2 months (with a range of 0–15.4) and 8.2 months (with a range of 1.0–37.9) after diagnosis in 47 (42.7%) patients. Thirty  patients died due to the ACC/ACx after a median time of 17.2 months (with a range of 0.5–44.4) after diagnosis, including those 4 patients with inoperable tumors following neoadjuvant chemotherapy. Patients succumbing to the disease were significantly older than the surviving patients  were locoregional recurrences in 8  patients, distant metastases in 18 (16.4%), and combined relapses in 6 (5.5%).The events were locoregional recurrences in 8  patients, distant metastases in 18 (16.4%), combined relapses in 6 (5.5%), and progression in 15 (13.6%). The events following complete tumor resection  patients suffered from a subsequent event, which was a locoregional relapse in 6  patients, a distant relapse in 3 (13.6%), a combined relapse in 2 (9.1%), and progression in 11 (50.0%). Of 34 patients with Rp = 0.016) but not of the EFS .In the univariate analysis, age at diagnosis ≥ 4 years, an unfavorable five-item microscopic score, tumor spillage, incomplete tumor resection, a tumor volume of ≥ 200 mL, a Ki67 index of >15%, and COG stages III/IV significantly impacted the negative prognostic factors upon the OS and EFS , Table 3p < 0.001). The overall survival of patients with COG stage III decreased linearly by 1.5% per year of age at diagnosis  and in stage IV patients by 2.9%  (The Cox regression determined that the risk of death was 8-fold higher for a tumor volume of ≥200 mL compared to that of <200 mL (< 0.001) .The age at diagnosis, five-item score, and COG III/IV stages were considered in a multivariate Cox regression model. An unfavorable five-item score and COG stage IV significantly impacted the prognostic factors upon the EFS and OS in the final model .Herein, we have reported the outcome of children and adolescents with ACTs registered in the MET studies. Our data confirm that ACCs/ACx in children and adolescents are associated with unfavorable outcomes  ,18. An aAge defines the clinical presentation and prognosis in children and adolescents with ACTs ,20,25,26Tumor spillage has previously been associated with a worse prognosis and almost fatal outcomes ,27. In lDifferent series have shown that complete tumor resection is a major prognostic factor and may cure the patient ,13,29. IThis nicely illustrates the extraordinary challenge in children and adolescents with ACTs: the pathological stratification of pediatric ACTs is difficult, which hinders therapeutic stratification. In the ARAR0332 study, the pathology review was based on the Wieneke index stratification . However1/2 resection and/or spillage, as well as in stage III and IV patients, chemotherapy plus mitotane is recommended. In contrast, the MET studies advised systemic therapy only for metastatic patients . The MET studies were not designed and powered to prove the efficacy of chemotherapy plus mitotane. In particular, the type and length of therapy were virtually decided on an individual basis by treating physicians, as the GPOH-MET 97 study was not an interventional study according to modern standards. We previously reported on children and adolescents with ACCs and mitotane therapy [p = 0.002; EFS HR 1.9, a 95% CI 1.0–3.5, and p = 0.037). These data need to be interpreted carefully; only patients in good clinical condition received chemotherapy plus mitotane. In the ARAR0332 protocol, with the combination of chemotherapy plus mitotane, the 5-year overall survival in stage III patients was excellent (94.7%) but poor (7.1%) in stage IV patients receiving the same regimen [In the EXPeRT/PARTNER guideline, therapeutic recommendations allow for a resection status and COG stage and, thus, distinguish between resected and advanced-stage ACTs . Startin therapy . Especia regimen . This clTo better define the biology of the disease is of crucial importance for the best prognostic stratification and ultimately for improving the outcome in children and adolescents with ACTs. Thus, progress in our understanding of pathogenesis, as well as additional therapeutic avenues, are needed. Recent findings suggest that somatic mutations and methylation markers may predict prognosis ,26,31. CIn summary, international prospective studies are needed to explore important, open questions in children and adolescents with ACTs, such as the role of adjuvant/neo-adjuvant chemotherapy, the best chemotherapy regimen, the best indications, and treatment of poor-responding, metastatic, and relapsed ACT patients. Age defines the clinical presentation and prognosis in children and adolescents with ACTs. The outcome in patients with advanced and metastatic ACTs was poor and was best predicted by the COG stage and five-item microscopic score. International efforts are needed to improve our understanding of the pathogenesis, introduce new therapeutic avenues, and ultimately increase survival in children and adolescents with ACTs.