Adverse environmental exposures in pregnancy can significantly alter the development of the fetus resulting in impaired child neurodevelopment. Such exposures can lead to epigenetic alterations like DNA methylation, which may be a marker of poor cognitive, motor and behavioral outcomes in the infant. Here we review studies that have assessed DNA methylation in cord blood following maternal exposures that may impact neurodevelopment of the child. We also highlight some key studies to illustrate the potential for DNA methylation to successfully identify infants at risk for poor outcomes. While the current evidence is limited, in that observations to date are largely correlational, in time and with larger cohorts analyzed and longer term follow-up completed, we may be able to develop epigenetic biomarkers that not only indicate adverse early life exposures but can also be used to identify individuals likely to be at an increased risk of impaired neurodevelopment even in the absence of detailed information regarding prenatal environment. Neurodevelopment begins early in fetal life, with neuronal migration, synaptogenesis and myelination of neurons occurring from the second month of pregnancy . EnvironOur current ability to predict poor motor, cognitive and neurobehavioral outcomes in the neonatal period is limited. Neuroimaging has achieved the most attention as a tool for early prediction and monitoring of development. Cranial magnetic resonance imaging (MRI) allows for qualitative assessment of grey and white matter injury and is superior to ultrasound imaging for the detection of white matter changes . While MGiven the current limitations of accurately assessing future neurodevelopmental outcomes, identifying cord blood biomarkers which aid in the prediction of later cognitive, motor and behavioral function would be a significant step towards targeted, effective interventions implemented at a time-point where their effects are likely to be greatest. DNA methylation in cord blood represents one such potential biomarker that is gaining research momentum. This epigenetic process reflects the biological convergence of perinatal exposures that can have important long-term consequences. Here we review literature assessing the potential for epigenetic biomarkers in cord blood to be used to predict poor neurodevelopmental outcome. While most studies to date have employed targeted gene approaches, the capacity for genome wide arrays to identify novel epigenetic biomarkers will also be discussed.Epigenetic modifications alter gene expression without changes to the DNA sequence and are often reversible. Typically epigenetic modifications encompass DNA methylation, histone modifications and non-coding RNAs. DNA methylation is the most commonly studied epigenetic modification and typically involves the addition of a methyl group to a cytosine adjacent to a guanine, denoted CpG, however cytosine methylation also occurs at CH sites  and this has been repeatedly reported in the brain (reviewed in ). The meDNA methylation is the most studied epigenetic change. It has a critical role in development  and due One of the many advantages of studying DNA methylation is the ease with which it can be assessed in peripheral bodily fluids such as blood, urine, sperm, stool samples and saliva . The use(Nr3c1) expression in the brain due to differences in maternal behavior are associated with DNA methylation of Nr3c1 [Early studies that assessed brain DNA methylation patterns and their association with offspring behavior were conducted in rodents where environmental exposures are amenable to manipulation. For example, alterations in glucocorticoid receptor of Nr3c1 . That stof Nr3c1 . Howeverof Nr3c1 . Howeverof Nr3c1 ,29,30,31As this is a new and emerging research field, only a limited number of studies are available that have specifically assessed cord blood epigenetic state and matched this with offspring neurodevelopmental outcome. These studies are outlined in HES1, a gene that encodes a transcription factor involved in neuronal cell proliferation and differentiation, and in diencephalon development [HES1 methylation and child cognitive development was validated in a further 200 cord blood samples from the Southampton Women’s Survey cohort, where more specific child cognitive functions were assessed at age seven years, including cognitive flexibility, executive function and memory [HES1 methylation and neurodevelopment extends to child behavioral outcomes. In one-year-old children from the Growing Up in Singapore Towards Healthy Outcomes (GUSTO) cohort, lower externalizing behavior scores were also associated with greater cord blood HES1 methylation [Most studies that have assessed epigenetic biomarkers associated with neurodevelopmental outcomes to date have adopted approaches targeting genes in pathways that are known to be susceptible to prenatal programming effects  ,32 and/oelopment . This asd memory . This ashylation . These sA large body of evidence exists linking pre- and early post-natal stress exposures with altered fetal and infant brain development. Examples of such stress effects include those induced following acute and chronic maternal stress exposures , malnutrition (famine) and exposure to toxins . The leading biological model explaining how these early life exposures result in an increased vulnerability to poor neurodevelopment in the offspring is through excess fetal exposure to stress-induced hormones, such as glucocorticoids . Excess glucocorticoid exposure has widespread effects on the developing brain, including delayed neuronal maturation, myelination, vascularization, and synapse formation , and resIncreased cortisol exposure of the developing fetus following maternal stress exposure is facilitated by a decrease in placental cortisol inactivation, through reduced activity of the 11 beta hydroxy-steroid dehydrogenase type 2 (11βHSD2) enzyme. This results in excess cortisol crossing to the fetus, ultimately perturbing fetal growth and development. Decreased 11βHSD2 activity and expression have been observed in pregnancies complicated by various prenatal acute and chronic stress exposures and maternal mental health conditions (such as anxiety and depression) ,56. ExceNR3C1 which encodes the glucocorticoid receptor and HSD11B2, encoding 11βHSD2 [NR3C1) consists of 9 exons, with multiple transcripts arising from alternate exon 1 promoter regions, which all translate various isoforms of the same protein. In rats, studies have typically reported the exon 1F promoter region as the most susceptible to altered methylation state following prenatal stress exposure. Human studies are consistent with those from rodents, with increased methylation of the equivalent homolog of the rat Nr3c1 promoter region observed following early life stress [NR3C1 and HSD11B2 in the placenta has been assessed for associations with neurodevelopmental indices. In one such study, DNA methylation of the HSD11B2 gene in placenta from term deliveries was linked with infant’s neuro-behavior assessed prior to hospital discharge [HSD11B2 DNA methylation was greater in intrauterine growth restricted infants, and was inversely associated with poorer quality of movements measured on the Neonatal Intensive Care Unit Network Neurobehavioral Scales (NNNS). While this study did not assess longer term neurodevelopmental outcomes, previous studies have shown that poor quality of movements measured on the NNNS is predictive of a lower score on the Bayley’s Psychomotor Developmental Index at 18–24 months of age [Promise for cord blood epigenetic status of genes involved in HPA function or cortisol metabolism to predict poor neurodevelopment has arisen from a number of animal and human studies. In this context, the most extensively phenotyped genes include  11βHSD2 . The hume stress ,32. In hischarge . The exts of age ,60 and aNr3c1 DNA immunoprecipitation and chip hybridization performed for those genes that demonstrated differential Nr3c1 DNA binding compared to control (unexposed) offspring. While more than 1000 genes were differentially expressed between control and betamethasone-exposed offspring on Day 52, only 81 genes remained differentially expressed between the two groups by Day 65. Exposure to the synthetic glucocorticoid therefore appeared to result in precocious development of the guinea pig hippocampus. However, Nr3c1 DNA binding and DNA methylation were differentially affected by the betamethasone treatment. Specifically, hypomethylation was observed in promoter regions of genes encoding Eme2 and Cntrob, while hypermethylation of Gnpda1 was observed in hippocampi of betamethasone compared to control offspring [To investigate the direct consequences of excess prenatal glucocorticoid exposure on the epigenetic landscape of brain tissue, Matthews and colleagues exposed pregnant guinea pigs to the synthetic glucocorticoid betamethasone during periods of rapid neurogenesis and brain growth  . DNA andffspring . These fIn humans, longitudinal studies of women and children following exposure to natural disasters in pregnancy have allowed the relationship between both prenatal objective and subjective stress on child developmental outcomes and DNA methylation to be determined. Such studies have been performed in pregnant women exposed to the 1998 Quebec Ice Storm, where both prenatal stress could be measured objectively  and subjectively (through women’s perceived stress rating of this period). This dual assessment has allowed for the impacts of stress on child development and DNA methylation status to be determined. In this cohort, higher maternal subjective stress ratings were associated with subsequent infant temperament , child bNr3c1 gene in the rat hippocampus, accompanied by decreased Nr3c1 expression [NR3C1 methylation have been reported in human neonates that correspond to maternal mental well-being. In an Australian birth cohort (Barwon Infant Study), maternal depression and anxiety at 28 weeks’ gestation were nominally associated with increased DNA methylation of different sites in the NR3C1 1F promoter region in cord blood mononuclear cells [NR3C1 gene promoter regions in cord blood mononuclear cells, and this corresponded to altered stress responses in the three month old infants [IGF2/H19 [Maternal anxiety and depression during pregnancy are associated with poor child behavioral and cognitive outcomes, and an increased incidence of child mental health problems (reviewed in ). Data fpression   [NR3C1 CpG2 site  , maternapG2 site . This hyWhile poor infant outcomes following maternal depression have been documented in the literature, controversy exists over whether pharmacological treatment of maternal depression is beneficial. Guidelines from the USA, UK and Australia all recognize the limited or inadequate evidence around the safe use of anti-depressants in pregnancy, given that risk factors for poor pregnancy and child health outcomes, including smoking, obesity and preterm birth, occur more frequently in women with depression compared to healthy women ,80,81. INR3C1 methylation response to maternal depression. Male infants exposed to maternal depression in second or third trimester showed increased NR3C1 DNA methylation in buccal swabs, while female infants demonstrated hypomethylation in response to the same exposure [Sex of the fetus may also play an important mediating role in determining DNA exposure  (Table 2exposure . BDNF) [BDNF is a neuronal growth factor with multiple roles in neuronal proliferation, differentiation, growth, apoptosis and synaptic plasticity across development and the lifespan. In a prospective study of mother—infant dyads, maternal depression was assessed during either second or third trimester, using the Edinburgh Post-natal Depression Scale, and buccal swabs were collected from infants at age two months. Using a targeted approach to assess methylation at five CpG sites within the BDNF promoter IV region, decreased DNA methylation was observed at CpG3 following prenatal depression [BDNF is involved in many developmental processes in the fetal brain, as well as ongoing synaptic function and plasticity, altered DNA methylation of this gene may have quite detrimental consequences for neurodevelopment. Other genes that appear to be susceptible to changes in methylation with prenatal depression include those encoding neurotrophins, such as brain derived neurotrophic factor (BDNF) . BDNF ispression . The conpression , potentiSLC6A4) and DNA methylation of the SLC6A4 promoter region [SLC6A4 promoter region (at CpG sites 6 and 9) in cord blood [SLC6A4 gene promoter, and therefore may contribute to altered SLC6A4 expression in the offspring. Decreased expression of SLC6A4 during development may have long term consequences on child neurodevelopment, via increasing 5-HT reuptake. The consequences of altered 5-HT reuptake may include physical, emotional and/or behavioral disturbances throughout life, given that serotonin plays key functions in regulating mood, sleep and cognitive performance. Future longitudinal studies of these children will increase our understanding of the longer-term impact of maternal depression and child SLC6A4 methylation on neurodevelopment.The epigenetic impact of maternal depression in pregnancy has also been investigated in pathways regulating neurotransmitter uptake, in particular serotonin (5-HT). Serotonin is constitutively expressed in neurons, with its uptake regulated by the serotonin transporter (5-HTT). While best known as a neurotransmitter, serotonin also has critical neurotrophic roles during development, mediating neuronal growth and differentiation . Variatir region . Maternard blood . This asNR3C1 DNA methylation in those exposed to child maltreatment [NR3C1 is observed in blood of patients with borderline personality disorder (BPD) [NR3C1 methylation has been explored more recently in 11–21 year olds. Participants completed a comprehensive suite of psychological health assessments, evaluating behavioral, perceived and standardized symptomology, as well as providing peripheral blood for DNA methylation in lymphocytes to be determined at 41 CpG sites spanning the NR3C1 gene [NR3C1 exon 1F promoter region and childhood maltreatment and poor psychological health. Specifically, early life childhood maltreatment was associated with increased methylation of a CpG site located in the exon 1F promoter region, which was highly correlated with the development and severity of borderline personality symptoms. Methylation of a second CpG site located in the NR3C1 exon 1E promoter region was also associated with depression symptoms, with methylation at both sites inversely associated with health related quality of life. Maltreatment in childhood, including physical, sexual and emotional abuse or neglect, can have lasting effects on a child’s mental health, increasing the risk of multiple psychopathologies, such as depression, anxiety and borderline personality disorder, and increasing the risk for suicide . The conreatment ,89. Simier (BPD) . This as3C1 gene   who took part in the 1958 British Birth Cohort study reported the degree of childhood abuse experienced in the first 16 years of life, and provided peripheral blood for DNA analysis . Over 90SLC6A4 gene in peripheral blood mononuclear cells, and demonstrated the greatest activity during a period of enforced social isolation [SLC6A4 methylation, indicating a gradient response. In humans, poor maternal care provision has also been associated with altered DNA methylation. Increased methylation of the BDNF and oxytocin receptor genes in whole blood has been observed in adults who report low maternal care provision during the first 16 years of life [Quality of parental care provision in early life can also determine child and adult mental health outcomes. Low maternal care increases the risk of poor mental health in later life . The biosolation . In thos of life . Notably of life . This stANGPT2 and PRPF18 were associated with first trimester maternal circulating mercury levels [Numerous heavy metals such as mercury, cadmium and arsenic are known to impact fetal development, and prenatal exposures of these heavy metals are associated with poor child outcomes . The Wory levels . Given tPEG3 differentially methylated region (DMR) [The impact of cadmium on cord blood methylation state has also been investigated. Out of the three studies that have assessed cadmium, two found maternal circulating cadmium levels were significantly associated with cord blood DNA methylation changes. The largest of these studies included 319 infant–mother pairs, assessed nine imprinted genes and showed a significant association between maternal cadmium concentrations and altered cord blood DNA methylation at the on (DMR) . A smallon (DMR) . In the on (DMR) . These cSeveral other studies have investigated whether maternal arsenic levels are associated with DNA methylation changes in cord blood. In the same cohort mentioned above in which mercury was investigated, there were no changes in cord blood DNA methylation associated with maternal arsenic levels . HoweverBDNF) following exposures to prenatal and early life stress and adversity, as potential factors that may contribute to the postnatal phenotype. While postnatal factors can impact child neurodevelopment, the overarching goal of epigenetic biomarker research is to identify an epigenetic signature that: (i) can discriminate children at risk for poor neurodevelopmental health outcomes as early as possible, so that limited health funding resources can be directed to those most at risk; and (ii) identify novel mechanistic pathways that can inform new interventions. Early identification tools and interventions designed to address cognitive and mental health disparities, particularly in low socio-economic areas, are urgently required to alleviate the associated health, social and economic burden. To achieve this, what is needed is an unbiased approach to assess DNA methylation in a genome wide manner without the bias of “candidate genes” or “brain specific genes”. The identification of biomarkers that can predict neurodevelopmental outcome does not require that the genes have some brain related function, despite these genes receiving the most research attention to date. In fact, DNA methylation of CpG sites or regions that are not associated with genes, will still serve as great biomarkers provided they can reliably predict neurodevelopmental outcomes. What is now needed are genome wide DNA methylation approaches in cord blood that can identify children at risk who will require early interventions. These studies will need to take into account different cell compositions and the potential impact this has on DNA methylation profiles. Further, epigenome wide analyses that are unbiased would be most beneficial to increasing our understanding in this area, and should be performed in multiple large cohorts.Epigenetic changes that occur in response to an adverse prenatal environment may constitute a biological mechanism by which early adversity results in long term changes to child health, contributing, or increasing susceptibility, to pathology. Existing data support differences in DNA methylation of specific genes (e.g., the glucocorticoid receptor, serotonin transporter and


Neuroendocrine tumours are a heterogeneous group of diseases with a significant variety of diagnostic tests and treatment modalities. Guidelines were developed by North American and European groups to recommend their best management. However, local particularities and relativisms found worldwide led us to create Brazilian guidelines. Our consensus considered the best feasible strategies in an environment involving more limited resources. We believe that our recommendations may be extended to other countries with similar economic standards. The World Health Organization (WHO) categorises neuroendocrine tumours (NETs) according to the tumour prognosis, considering the histopathology and proliferative index of tumours . HoweverNETs are tumours featuring neuroendocrine differentiation and may develop in various organs. The morphological classification proposed by WHO suggested three distinct prognostic groups: 1) grade 1 (G1) [Ki67 ≤ 2% and <2 mitoses/10 high-power fields (HPF)], (2) grade 2 (G2) (Ki67 3%–20% and 2–20 mitoses/10 HPF) and (3) grade 3 (G3) (Ki67 > 20% and >20 mitoses/10 HPF) [ grade 1 The pathology report should be used as the gold diagnostic standard.Immunohistochemistry (IHC) should be conducted for all suspected cases of NETs with a minimal panel of markers  to confirm the neuroendocrine nature of the tumour [IVA], and reduce the chance of diagnostic errors.2). The average of mitosis should be based on a count using 10 HPF. We recommend that the determined Ki67 should be expressed as a percentage of 500–2000 neoplastic cells counted in the areas of greater nuclear staining, known as hot spots. In a situation where a pathological analysis is performed using aspirated biopsy or a fine needle in which the cell count is below 500 cells, histologic grading is not recommended. Thus, the pathology report should be limited to confirming the neuroendocrine nature of the tumour.The proliferative index should be evaluated by counting mitotic figures using optical microscopy and the percentage of positive cells identified by IHC to determine the Ki67. The degree of determination requires a count of mitotically active cells in several areas, with at least 50 HPF  or poorly differentiated, according to the architectural features  and cytomorphological . We recommend that reporting the histologic degree of differentiation regardless of the proliferative index, i.e. NETs with a high proliferative index (>20%) should be described according to their cell differentiation and not solely as carcinomas , 3 [IVA]Poorly differentiated tumours should be classified in small or large cells [VB].Although there are many different staging systems for NETs, for example, the modified ENETS staging system for pancreatic NETs , for resRoutine testing for other markers is not necessary [VE], except in cases of primary site determination where pancreatic markers  can guide the diagnosis of occult primary NETs [VB].Histological parameters should be described in all cases of primary tumour resection  –3, 5 .The 5-HIAA in the urine (24 h) should be measured in patients with carcinoid syndrome [IIA] and in all patients with metastatic midgut NETs involving the liver, even in the absence of specific symptoms [IVB].Dosage is optional for prognostic purposes  [IIIC].For patients receiving somatostatin analogues (SAs), 5-HIAA should be measured immediately before the next dose, in order to avoid artificially low values. [VA]Measuring serum insulin, proinsulin and C-peptide following prolonged fasting (72 h) during hypoglycemia is considered the gold standard for diagnosis of insulinoma. However, the fasting test has limited application, because it requires hospitalisation to monitor blood glucose. In practice, documentation of clinical and laboratory hypoglycemia in the presence of Whipple’s triad accompanied by hypersinsulinemia is usually sufficient and recommended for diagnosing insulinoma.The absolute values of glucose and insulin are the most relevant data, and any measurable insulin ≥ 3 mcU/ml ≥ 20, 8 pmol/l when blood glucose levels fall to values ≤ 55mg/dl or 3 mmol/l should be considerable abnormal , 11. TheGastrin levels are useful for differentiating various types of stomach NETs . Type I:We do not recommend hormonal screenings for all patients in the absence of clinical indications, except for urinary 5-HIAA in asymptomatic patients with liver metastatic midgut NETs [VA]. In these cases, the identification of subclinical elevations of this marker may be useful for monitoring carcinoid heart disease (CHD).In rare cases of atypical carcinoid syndrome and 5-HIAA concentrations are in the upper normal limits, platelet serotonin measurement may be useful [IVC].The most commonly used conventional imaging tests for NETs are ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI). US tests have extensive variability in the detection of pancreatic NETs 17%–79%), making this method unsuitable for identifying NETs . However%–79%, ma68Ga positron emission tomography (PET/CT68Ga) and CTMD have comparable accuracies in the diagnosis of duodenopancreatic NETs [CT with multislice detector (CTMD) has an average detection capability of 73% (39%–94%) for pancreatic NETs . False ntic NETs . MRI hastic NETs .US for primary assessment of NETs should not be used  [IVD].MRI, CTMD and EUS have a similar sensitivity to detect pancreatic NET , 18. HowThree-phase CT is the preferred method for staging liver metastases from NETs [VA]. MRI can be used to evaluate liver metastases, if available  [IVB].CT enterography is optional and experimental  [IVC].The sensitivity of EUS for diagnosing duodenopancreatic NETs is 87%, and its specificity is 98% . For insEUS can be used for the diagnosis of pancreatic or duodenal NETs  [IIA].18F-Fluorodesoxiglucose (18F/FDG) is a radiopharmaceutical analogue of glucose and PET/CT68Ga is an analogue (agonist) of somatostatin receptor type 2. While the performance of scintigraphy octreoscan is similar to multidetector CT , b68Ga-SA, if available, is indicated and preferable to octreoscan for staging G1 and G2 NETs .PET/CT68Ga-SA is recommended in cases of unknown primary lesions or suspicious lesions [PET/CT lesions  [IIIA].68Ga-SA can be used to evaluate suspected recurrences after surgery [PET/CT surgery  [IIIA].68Ga apparently has superior sensitivity compared with WBMR for detecting WD NETs, specifically in bone and primary lesions .WBMR, if available, is a method with high accuracy for screening patients with NETs without ionising radiation  [IIIB].Patients with unknown primary tumours are characterised as such when conventional tests such as CT, MRI, functioning peptide dosage, endoscopy and colonoscopy cannot determine NET origin. The importance of identifying an occult primary NET relies on the potential to impact patient survival –37, for 68Ga is considered an excellent imaging method for detecting occult primary tumours .PET/CTSurgery can detect primary tumours intraoperatively independently of preoperative location [VC].68Ga is one of the most accurate methods for detecting WD occult NETs .If PET/CTEnterotomography can be considered, particularly in cases of suspected Crohn’s disease  [VC].Endoscopic capsule  [IVC] anIHC in the investigation of occult NETs can be useful, especially in occult pancreatic NETs (see pathology section) [VC].There are very few studies to guide the follow-up of patients with NETs. Most recommendations have been based on expert opinions .NET G3: Clinical assessment without radiological tests should be performed every three months for two years and every six months from the third to fifth year [VB]. Conventional imaging (CT and MRI) of the abdomen, pelvis and chest can be used in cases of suspected recurrence.Gastrointestinal and pancreatic NET G1 or G2 treated with R0/R1 resection: Clinical and imaging tests (CT and/or MRI) should be performed every 4–6 months for two years. After this period, perform annual examinations every 3–5 years or earlier if there is suspicion of relapse [VB]. Tumours <1 cm require monitoring only in the presence of negative factors of prognosis, such as poorly differentiated histology or high mitotic index [IIIB].68Ga are not recommended for follow-up in patients with no evidence of disease as determined by conventional imaging [VE].Functional tests such as octreoscan and PET/CTChromogranin A measurement alone is not recommended [IVE].Conventional imaging tests (CT and/or MRI) should be performed every 3–6 months [VA].In cases of rapid disease progression or dubious examination results, a new liver biopsy may be considered to reassess proliferation [VC].Because there is no level 1 evidence for the follow-up of patients with NETs, there is room for personalisation of frequency of follow-up visits and imaging tests.Type 1: Endoscopic assessment every 12 months in recurrent patients and every 24 months in non-recurrent patients [VB]. There was no consensus on the follow-up period. The follow-up should be based on clinical judgments in individual cases.Type 2: Consider the same recommendations for multiple endocrine neoplasia (see MEN section).Type 3: CT thorax, pelvis and abdomen should be performed every 3–4 months for the first two years and every 6–12 months from the third to fifth year [VB].Colorectal NETs <1 cm G3 and G1–G3 from 1 to 2 cm: Annual monitoring according to the protocol for adenomatous polyps .For cases of NETs >2 cm, we recommend annual colonoscopies for five years.68Ga should not be included in the routine follow-up.Octreoscan or PET/CTWe recommend complete resection with curative intent for all NETs.ForWD pancreatic NETs <2 cm, particularly G1 tumours, we recommend surveillance, with CT scan performed every six months [IVB]. For growing tumours and/or those with higher ki67 index, surgery is recommended –53 [IVB]Midgut NETs should be treated with resection of mesenteric lymph nodes in order to prevent mesenteric fibrosis [VB].The practice of resection of the primary NET in the metastatic setting is based on small retrospective series. In general, patients with good performance status and symptomatic primaries have indication for resection. Patients with liver transplant indications should also have the primary tumour resected. In asymptomatic metastatic patients, surgery for primary G1 or G2 tumours of the small intestine should be considered for the risk of obstruction, mesenteric fibrosis and intestinal ischemia .Resection of G1 and G2 midgut primary symptomatic NETs, with or without involvement of mesenteric and symptomatic primary colon or rectum NETs  [IVA].Resection of the pancreatic or gastric NET primary in the metastatic disease setting is not recommended  [IVE].Resection of asymptomatic primary tumours of the midgut can be considered in select cases, where the tumour presents a significant risk for complications such as obstruction and mesenteric fibrosis  [IVC].Patients with G1 and G2 NETs presenting with metastases limited to the liver may be considered for resection of entire tumour. In order to obtain R0 resection, combined radiofrequency ablation should be considered for local control . In geneThe decision for liver resection of metastatic WD NETs should be discussed by multidisciplinary teams [VA]. R0 resection should be aimed whenever possible  [IVB].Ablative techniques such as radiofrequency can be combined with surgery in order to obtain negative margins. Intraoperative US [IVA] should be performed for staging liver lesions .Liver resection should be performed by experienced teams [VA].A simultaneous approach to primary and liver tumours can be considered  [IVB].Debulking is indicated for functioning NETs to alleviate symptoms not adequately controlled with systemic therapy and/or locoregional therapies of the liver –64. The Recommended for patients with good performance status, functioning WD NETs, with predominant liver disease and poorly controlled clinical syndrome despite systemic and/or locoregional therapies , 64  orWe prefer pure embolisation for midgut G1 NETs, limiting the addition of chemotherapy to cases of G2 pancreatic or G2 gastrointestinal NETs [VB].Both embolisation and chemoembolisation may be repeated, if no significant liver dysfunction is detected [VB].Radiofrequency ablation is associated with symptomatic and/or radiological response in 70%–80% of NET patients, with an overall five-year survival rate of 50% . RadioblPercutaneous radiofrequency ablation can be considered for palliation of WD inoperable and progressive NET patients with a low volume of liver metastasis  [IVB]. TAblation with open radiofrequency can be used as a complement to surgery, in an attempt to achieve free margins and/or manage lesions with difficult resectability  [IVC].90Yttrium as the radiation-emitting radioisotope. 90Yttrium releases beta radiation (β) with an average tissue penetration of 2.5 mm and a maximum penetration of 11 mm. Radioembolisation offers doses at 100–300 Gy, which would not be possible through external radiotherapy due to liver toxicity .Assessments to rule out extrahepatic diseases should include total abdomen CT/MRI, chest CT, octreoscan or preferably, if available, PET/CTEligible patients must have the primary tumour resected prior to transplantation.Liver transplantation may be a potentially curative option for selected patients with WD NET. Eligible cases for transplantation, however, should be very well selected –74.LiveTwo phase III placebo-controlled studies have demonstrated the antitumour effects of SAs (octreotide and lanreotide) in WD gastroenteropancreatic (GEP) NETs with Ki67 < 10% , 76, regSAs are the first-line systemic treatment of choice for WD, inoperable and advanced progressive WD GEP NETs, with Ki67 < 10% , 76 [IB]68Ga prior to SA-based treatment [IIB], although this is not mandatory [We favour the confirmation of SSTR2 expression through octreoscan or PET/CTandatory .We consider octreotide and lanreotide to have similar safety and efficacy for treating WD GEP NETs [VB].Watchful waiting can be recommended in WD NETs, preferably those with G1, non-functioning, low-volume, and oligosymptomatic disease [VC]. WD G1 NETs are indolent and may stabilise for months to years without treatment as observed in the median of progression-free survival (PFS) in the placebo arm of the CLARINET study . FurtherDespite its unfavourable toxicity profile, interferon-α is still used and, in many low-resource countries, it is one of the few available treatments for NET. Several studies have shown that interferon-α provides tumour control, mostly through disease stabilisation and symptomatic response in carcinoid syndrome . HoweverInterferon-α can be used in cases of SA-resistant and progressive WD NETs , 78 [IC]Chemotherapy, although not studied in phase III trials, is considered an effective treatment for G1 and G2 pancreatic NETs, with the response rates of 30%–40% with temozolomide and capecitabine , capecitPatients with radiological and/or symptomatic progressive WD pancreatic NET  [IIB].Patients with WD gastrointestinal NET who have exhausted other treatment options  [IID].Suggested regimens: Capecitabine combined with temozolomide or oxaliplatin, FOLFOX and streptozotocin with or without 5FU.Increased expression of Ki67 can be used to recommend chemotherapy in WD GEP [VB].MGMT expression should not guide the use of temozolomide/dacarbazine  [IVD].Everolimus, an oral inhibitor of mTOR, was effective in WD pancreatic, gastrointestinal or pulmonary NETs. The RADIANT-2 study , a phaseEverolimus monotherapy is recommended for non-functioning and progressive gastrointestinal or pulmonary WD NETs [IA]. Everolimus may be used as a first-line or second-line treatment after SA  [IIB].Everolimus in combination with SA is recommended for patients with functioning tumours [IIA]. Utilisation is preferable after progression on SA monotherapy .Everolimus can be used as a monotherapy in functioning tumours to control hormonal syndromes , 84 .The phase III placebo-controlled RADIANT-3 trial for patients with advanced, progressive pancreatic NET showed significantly prolonged PFS in favour of everolimus, with a median of 11 months versus 4.6 months  . SimilarIn pancreatic NETs, the choice between the two drugs should consider their toxicity profiles. Serious adverse events (G3 and grade 4) most commonly associated with sunitinib were neutropenia (12%), hypertension (10%), and hand and foot syndrome (6%), while everolimus commonly promoted stomatitis (7%), anemia (6%) and hyperglycemia (5%) , 87. In Everolimus and sunitinib monotherapy are recommended for WD functional or non-functional, progressive advanced NETs , 87 [IA]Both drugs can be used in first-line therapy or following SA and/or chemotherapy , 87 .111Indium-labeled octreotide or PET/CT68Ga .177Lutetium has not been established, although improved PFS has been demonstrated in the second-line setting. For limited data on delayed safety analysis of this treatment and perceptions of greater toxicity of chemotherapy and targeted-therapy after 177Lutetium, we prefer to indicate it after other systemic therapies have failed [VB].The best timing to administer 177Lutetium is recommended for patients with an advanced WD pancreatic or hindgut NET whose disease progressed with SA, locoregional and/or systemic therapy [ therapy  [IIIB].68Ga prior to PRRT .Control of the carcinoid syndrome is important not only for the improvement of symptoms and consequently the patient’s quality of life, but also to prevent or delay the development of complications associated with carcinoid syndrome, such as retroperitoneal fibrosis and carcinoid cardiopathy . SAs havWe recommend the use of SA [IA]. We consider octreotide and lanreotide interchangeable for controlling carcinoid symptoms or other symptoms associated with hormonal syndromes in NET.Physical examination should be performed to discard absorption problems (e.g. fibrosis in application site) [VA].Increased doses of SA can be used  [IIB]. TShorter intervals between injections for patients whose symptoms recur before next injection [VA]Everolimus [IVC] or associated with SA  [IIC].Interferon-α combined with SA  [IIIC].177Lutetium or 90Itrium radioisotopes can be indicated for NETs with proven expression of SSTR2 [IVB] or I123-metaiodobenzylguanine (MIBG) in NET with proven expression of MIBG [ of MIBG  [IVB].Surgical debulking of the liver  [IVB].Locoregional therapies such as embolisation of hepatic artery or radiofrequency ablation in liver predominant disease  [IIIB].Hypoglycemia caused by insulinoma can be fatal. Therefore, glucose control is extremely important, both in the preoperative period (when the vast majority of insulinoma are benign) and unresectable, or metastatic setting. Patients with insulinoma should be hospitalised for management of hypoglycemia.Hospitalisation for control of glycaemia with frequent meals at least every 3 h, continuous infusion of dextrose and glucose monitoring.Diazoxide for glycemic control [VB] can be used. However, this drug is not widely available.SA can be used cautiously for control of glycemia [VB]. This approach should be done carefully as regulatory hormones may be blocked leading to severe hypoglycemia. SA is recommended when SST2 expression is confirmed. Treatment should be initiated with short-acting octreotide for few days to better assess glycemic control. If effective, long-acting SA may be used.Everolimus has antitumour and hyperglycemic effects in metastatic insulinomas  [IVA].Verapamil and glucocorticoids are optional in selected refractory cases [VC].Treatment with targeted therapy or chemotherapy should be performed in combination with glycemic support [VA].CHD is among the most dramatic complications from carcinoid syndrome, as it is associated with poor prognosis . The pepWe recommend screening with echocardiography in patients with elevated 24-h urine 5-HIAA, regardless of carcinoid symptoms [VA].forame ovale by the bubble test. The measurement of NT pro-BNP, a predictor of right-hand-side heart overload is optional [The echocardiogram should be performed by a professional familiar with CHD  [IIIA], optional  [IIIC].In patients without CHD, an echocardiogram should be performed annually or when there at onset of symptoms of cardiac congestive failure [VA].Detailed assessment of the CHD severity can be performed by other diagnostic modalities   [IVC].Patients with CHD should be managed by a multidisciplinary team of oncologists, cardiologists, endocrinologists and surgeons [VA].Pharmacological and non-pharmacological treatment of heart failure, such as water and salt restriction, diuretics and digitalic drugs, can improve symptoms but do not alter clinical outcomes [VB].The definitive treatment of CHD is surgical valve replacement [IIA]. Surgery should be considered if the patient experiences symptoms of heart failure, right ventricular dilatation and decline of right ventricular function , 101. PaValve repair, compared with valve replacement, should be avoided because of the risk of post-repair stenosis and significant valve damage associated with underlying carcinoid syndrome [IVD].Valve replacement, when indicated, should be performed in centres with experience in the treatment of NETs [VB].The use of biological valves (bioprosthesis) is preferred for the lower risk of bleeding compared with metallic valves , 101  with the goal of preventing carcinoid development .Glucagonoma is an aggressive pancreatic NET of glucagon-producing cells that often present with metastatic disease. Clinical manifestations include anemia, weight loss, diabetes and dermatological features of a typical necrolytic migratory erythema. They may also present stomatitis, glossitis, diarrhea, abdominal pain, psychiatric disorders and venous thromboembolism.Surgical treatment, if resectable, and the use of SA as a symptomatic treatment of clinical syndrome of glucagonoma.Consider parenteral nutrition, vitamin supplementation, assessments of the presence of depression and prophylactic anticoagulation in all patients with glucagonoma [VB].Gastrinomas are characterised by ectopic secretion of gastrin from pancreatic or duodenal NET, and resultant gastric ulcers. Severe peptic ulcers with gastroesophageal reflux and diarrhea are characteristic of Zollinger–Ellison syndrome.We recommend the use of high doses of proton pump inhibitors, with or without H2 receptor blockers  [IVA].SA [VA] as an antitumour therapy can be used to control associated diarrhea.Vipoma is a rare pancreatic NET that produces vasoactive intestinal peptide, an important peptide in the neuromodulation of intestinal function. Clinical manifestations include intense watery (choleric) diarrhea, with fluid and electrolytes depletion, risk of acidosis and hypovolemic shock, reductions gastric acid secretion, hyperglycaemia, hypercalcemia and flushing.We recommend SA as the first treatment [IVA]. It is important to manage blood volume, hypocalcemia and acidosis .NECs or G3 are rare and associated with poor prognosis, with a median OS of less than a year . RetrospA retrospective multicentre European study showed higher Ki67 index is associated with better response to platinum-based chemotherapy . Ki67, hResectable G3 NEC should undergo oncological surgery  [IVA].Consider definitive treatment with platinum-based chemotherapy combined with radiotherapy for locally advanced or unresectable tumours  [IVA].Adjuvant chemotherapy with cisplatin (or carboplatin) associated with etoposide or irinotecan for 4–6 cycles can be considered in patients with good performance status after surgical resection  [IVC].Cisplatin (or carboplatin) associated with irinotecan or etoposide for first-line treatment , 106 [IICisplatin (or carboplatin) may be offered again to responders who have received their last dose more than three months before [VB].Temozolomide-based or oxaliplatin-based regimens may be used in WD G3 tumours [VC].Temozolomide or dacarbazine can be used as second-line treatments after platinum regimens  [IIIB].GEP NETs may be associated with hereditary syndromes as MEN-1, von Hippel–Lindau (VHL) and neurofibromatosis-1, among others. In this consensus, we have focused on MEN-1 and VHL as they are the most commonly associated syndrome with NETs.The inheritance pattern of MEN-1 is autosomal dominant with almost complete prevalence by 50 years of age. Patients with MEN-1 have high susceptibility to the development of tumours in the pituitary gland, the parathyroid glands, and duodenal endocrine cells or pancreatic islets . The cliMEN1 germline mutation, whether or not they have any clinic manifestations related to MEN-1. However, it is essential that genetic counselling is carried out and that the DNA collection for mutation research on the MEN1 gene is offered to all patients with the clinical diagnosis of MEN-1. Mutations not identified by Sanger sequencing analysis should be sought by deletion analysis by multiplex ligation-dependent probe amplification (MLPA) .The pituitary adenomas constitute about 15% of all intracranial tumours. The classification is morphological: microadenomas are <1 cm and macroadenomas are >1 cm. Both can be invasive, but pituitary tumours associated with MEN-1 tend to be more aggressive than sporadic forms. Adenomas can be secreting or not.The first choice of therapy for prolactinomas should include dopamine agonists, and cabergoline is the preferred medication , 121 [IASurgical treatment is recommended in cases of resistance or intolerance to dopamine agonists  [IVA].Temozolomide is indicated for rare cases of aggressive and resistant tumours , 125 [IITreatment with radiation therapy may be considered in tumours with local complications  [IVB].Repeated surgical resection can be performed in recurrent tumours [IVB].Acromegaly is a rare disease, but with high morbidity and mortality rates. Surgery is the primary treatment for most cases. SA, in particular, dopaminergic agonists and GH receptor antagonists comprise the treatment for cases that are not controlled surgically or cases with restrictions to surgery. Radiation therapy has been less often used.Surgery is the first treatment option .We recommend the use of SA to treat symptoms and signs [IB]. Both octreotide and lanreotide are clinically equivalent .Pegvisomant can be used in SA-refractory cases , 124 [IBTrans-sphenoidal surgery of the pituitary gland is the first choice of therapy  [IIA].We recommend drug therapy with ACTH modulators or blockers of adrenal synthesis of cortisol and/or radiation therapy for cases uncontrolled by surgery  [IVB].Bilateral adrenalectomy can be considered as an option if other therapies fail  [IIIC].Pituitary surgery, usually by the trans-sphenoidal approach, is the treatment of choice  [IVA]Radiotherapy is indicated for recurrent or partially removed tumours  [IVA]A germline mutation in the short arm of chromosome 3 causes the inhibition of von Hippel–Lindau protein, which results in the increased hypoxia-inducing factor leading to increased production of PDGF, a stimulator of epithelial and vascular cell growth, that promotes hypervascular cystic tumours, carcinomas including clear renal cells and pancreatic NET, with a penetrance of approximately 15%, as well as paragangliomas and pheochromocytomas .All patients with a clinical diagnosis of VHL syndrome, even without a family history, should carry out a molecular test  [IVA].68Ga, if available.We recommend screening VHL patients with usual clinical evaluation and annual imaging tests  beginning at 20 years of age [VB]. To differentiate pancreatic cystadenoma and cysts with septate and thick content, we suggest octreoscans or PET/CTGenetic testing is also recommended for the first-degree and second-degree family members to identify asymptomatic carriers of VHL disease [VA].We recommend radiological monitoring in cases of cysts in the pancreas. Surgery is indicated in exceptional cases for the compression of adjacent organs or development of NET.et al .We prefer sunitinib as the first choice in pancreatic NET associated with VHL syndrome [VC] and everolimus as the first choice in MEN-1-associated pancreatic NET [VC]The management of NETs is challenging and complex because of their vast heterogeneity. Thus, each case should be discussed and handled in a multidisciplinary environment. The development of national guidelines is an important roadmap for assertive diagnostic and therapeutic choices, not only based on scientific evidences but also adaptable to particular scenarios. In this Brazilian NET guideline, we constructed recommendations for diagnosis and treatment that differ from those found in current guidelines. We believe that our considerations are useful and can assist medical decisions for the best management of patients.The incidence of NETs, although still low, has increased and will become more frequent in medical practice. Continuous efforts toward updated knowledge should be made, especially through collaborative groups. Working together can be more effective in standardising procedures, evaluating risks and benefits of available treatments, and optimising resources. Finally, clinical and translational research should be encouraged as it is essential in elucidating the carcinogenesis mechanisms of NETs and a key step in therapeutic advances.


The hydrophobic pocket of the epigenetic reader protein BRD4 has been engineered to carry a photosensitive amino acid to identify novel interacting partners, providing mechanistic insights into BRD4’s function in transcription and beyond. l-phenylalanine (pAzF) without compromising its ability to recognize acetylated lysine residues in histone proteins. We establish the binding efficiency, substrate specificity and crosslinking ability of the engineered ‘reader’ module in biochemical assays. Applying IBPP, we uncovered novel acetylated interacting partners of BRD4, such as transcription factors, expanding on its previously unappreciated role in diverse biological processes. By setting up an azide-acetyllysine photoreaction deep inside the bromodomain aromatic cage as a means to detect protein acetylation, our approach provides a potentially general platform for rapid and unbiased profiling of interacting partners of diverse epigenetic readers whose functions in eukaryotic gene regulation remain convoluted.Chemical modifications on DNA, RNA and histones are recognized by an array of ‘reader’ modules to regulate transcriptional programming and cell fate. However, identification of reader-specific interacting partners in a dynamic cellular environment remains a significant challenge. Herein, we report a chemoproteomic approach termed ‘interaction-based protein profiling’ (IBPP) to characterize novel interacting partners of potentially any reader protein. IBPP harnesses a photosensitive amino acid introduced into the hydrophobic pocket of a reader module to crosslink and enrich transient interacting partners that are inaccessible to traditional methods. Using bromodomain-containing protein 4 (BRD4) as a paradigm, we engineer an ‘aromatic cage’ of the bromodomain to introduce 4-azido- Available methods are either based on in vitro binding of modified truncated peptides to recombinant readers or the use of cell lysates as a potential source of ‘effector’ molecules.8 Such peptide-based assays are not suitable for fully recapitulating the highly dynamic interactions between modified proteins and their readers.2 This becomes particularly challenging in the presence of other gene regulatory complexes in the chromatin landscape where both DNA and histone modifications regulate reader functions. Traditional enrichment methods can identify only the strongest and most abundant interactions; these approaches lack the temporal control necessary for profiling the dynamic interactome of bromodomains, whose Kd towards acetylated proteins vary from 10–100 µM.9The biological consequence of reader-mediated recognition of PSMs is highly dynamic and context dependent. For example, binding of H3K4Me10 IBPP represents a substantial departure from the earlier approaches, as it shifts focus from simple peptide-based probes to full-length engineered readers capable of capturing even the transiently interacting, low abundance partners in cellular milieu.We envisioned a novel approach based on protein interface engineering for characterizing the reader-specific interacting partners with high temporal precision . The cen11 An epigenetic reader involved in gene regulation in diverse cellular processes,9 BRD4 is implicated in multiple malignancies due to its oncogenic translocation with the NUT gene and misregulated reading of acetylated non-histone proteins.12 Here we describe the engineering of BRD4 to include 4-azido-l-phenylalanine 13 in its aromatic cage, validate the biochemical integrity of modified proteins, and show the ability of engineered BRD4 to crosslink to interacting proteins in a complex cellular mixture. We further establish the generality of this approach by applying it to other distantly related bromodomains. Finally, by uncovering and validating novel interacting partners identified through IBPP, we identify potential functions of BRD4 beyond its canonical role in transcription.To validate the IBPP approach, we focused on BRD4, a bromodomain and extra terminal (BET) containing protein.14 Some of these residues can be altered without significant loss of binding affinity, particularly those that do not cause significant structural change. For instance, the Y139F mutation in BRD4 and the equivalent Y147F mutation in BPTF-bromodomain cause only a 2-fold loss in binding affinity towards acetylated histone peptide.15 Such observations led us to hypothesize that 1, an analogue of phenylalanine, could be introduced into the BRD4 aromatic cage without significantly affecting the binding to its interacting partners. We focused on employing pAzF because of the minimal structural perturbation, high crosslinking efficiency and availability of suitable methods for introduction into proteins by both bacterial and mammalian expression.17Detailed structural studies on bromodomains have led to the identification of an aromatic cage formed by multiple hydrophobic residues that bind to acetylated lysine .14 Some 1 into BRD4, we employed amber suppressor codon (TAG) mutagenesis using an evolved orthogonal M. jannaschii TyrRS–tRNACUATy pair.13 Following a series of initial optimizations, we were able to express eight mutants  in high yields  assay with tetramethylrhodamine (TAMRA)-labeled tetra-acetylated histone peptide 2  and S2†.ectively  and S2†.19 We observed that binding affinities of both wild type and mutant proteins towards peptide 2 were significantly reduced by the presence of 200 nM JQ1 (3) and tetra-acetylated (4) peptides revealed that only the latter could inhibit the binding of BRD4 and its selected mutants towards 2  with a random sequence , R585AzF BRD1  and W2950AzF BPTF   . Protein2 as described above  and their mutants  using peptide ed above . In the 23 The lysate was incubated with L92AzF followed by crosslinking using UV light. Crosslinked proteins were pulled down with Ni-NTA beads and washed extensively. The eluted proteins were subjected to Western blotting using anti-His antibody. The presence of multiple higher molecular weight bands . Samples that were not exposed to UV light served as negative controls in our experiment. The identified proteins from UV- and non UV-treated  samples were compiled, and the exclusive unique peptides detected for each protein under each condition were averaged. Proteins that were present in both biological replicates and demonstrated a minimum of two unique peptides were selected for further analysis. Applying these criteria, we identified 398 proteins and placed them into three categories: 6 proteins were exclusive to UV-treated sample, 3 were exclusive to non UV-treated sample, and 389 were shared by both the samples  from the UV-treated sample : ILF3, SHMT, HNRNPK and PDIA1   , it’s ce2 To investigate such a complex network and identify interacting components, we have outlined an approach called IBPP involving protein engineering, UV-light mediated crosslinking and subsequent characterization of these components. As a proof-of-concept, we have successfully engineered multiple bromodomains using amber codon suppression to introduce an azide functionality that can be activated by light, an agent known to manipulate biology with high spatiotemporal precision.30 The potential of these engineered readers to interact with and crosslink to novel binding partners in complex cellular milieu was subsequently examined. Employing IBPP, we expanded the potential functions of BRD4 in diverse biochemical processes through the recognition of acetylated non-histone interacting partners such as transcription factors, chromatin regulators and proteins involved in RNA processing. To our knowledge, this current work represents the first example of reader-specific profiling of ‘acetylome’ (a collection of acetylated proteins) by setting up an azide-acetyllysine photoreaction deep inside the aromatic cage of bromodomains. We further biochemically validated a set of non-histone interacting partners to provide evidence that such non-canonical interactions may potentially regulate BRD4-mediated functions in chromatin remodeling, protein folding as well as in cellular metabolism. We anticipate that future experiments with full-length engineered readers expressed in mammalian cells and in-cell crosslinking will allow precise mapping of physiologically relevant interacting partners. Furthermore, a large number of human proteins carry reader domains (>60 bromodomains31 and ∼200 PHD fingers32) to recognize chemical modifications in histone and non-histone proteins. The molecular mechanisms by which these modules participate in diverse physiological processes  are largely unknown. We envision extending the IBPP approach delineated here to shed light on the role of these readers in human biology and disease.Reader-mediated transcriptional programming is manifested through the combinatorial readout of DNA, histone, and non-histone modifications that cause transcriptional machinery to target specific genomic loci.


LMNA splice-site mutation (c.357-2A>G), LMNA nonsense mutation  in exon 4, LMNA missense mutation  in exon 6, Hutchinson-Gilford Progeria Syndrome, and no LMNA mutations were analyzed. For each cell type, the percentage of dysmorphic nuclei, and other morphological features such as average nuclear area and average eccentricity were obtained. Compared to blind observers, our procedure implemented in Matlab codes possessed similar accuracy to manual counting of dysmorphic nuclei while being significantly more consistent. The automatic quantification of nuclear defects revealed a correlation between in vitro results and age of patients for initial symptom onset. Our results demonstrate the method’s utility in experimental studies of diseases affecting nuclear shape through automated, unbiased, and accurate identification of dysmorphic nuclei.Nuclear shape defects are a distinguishing characteristic in laminopathies, cancers, and other pathologies. Correlating these defects to the symptoms, mechanisms, and progression of disease requires unbiased, quantitative, and high-throughput means of quantifying nuclear morphology. To accomplish this, we developed a method of automatically segmenting fluorescently stained nuclei in 2D microscopy images and then classifying them as normal or dysmorphic based on three geometric features of the nucleus using a package of Matlab codes. As a test case, cultured skin-fibroblast nuclei of individuals possessing  LMNA) gene causes premature aging up to seven times faster than normal, usually culminating in death due to cardiovascular complications [LMNA gene mutations are associated with a range of heart disease symptoms including cardiomyopathy, heart failure, and arrhythmia [LMNA mutations cause heart disease is under investigation, it is known that the LMNA gene expresses both the Lamin A and C proteins via alternative splicing. These proteins form the integral meshes of the nuclear lamina, which provides structural support to the nucleus [LMNA mutations including heterozygous splice site, nonsense, and missense mutation, which cause diverse heart diseases with no other major pathologies [LMNA mutations are widely hypothesized to alter the mechanical properties of cell nuclei through the production of defective structural proteins, thus compromising the functionality and health of certain cell types [Abnormal cellular structures arising from proteins expressed by mutant genes can negatively impact tissue and organ function predisposing individuals inheriting genetic mutations to disease –3. One pications . Other trhythmia ,4,5. Whi nucleus . Consequ nucleus . Indeed, nucleus . This ishologies ,10. Furtll types ,11–13. All types –15. NuclWhile dysmorphic nuclei are a hallmark of many diseases such as laminopathies ,13 and cMany existing methods of automatically detecting irregularities in nuclear morphology calculate a single feature related to nuclear shape, measuring nuclear defect levels as the difference in mean values of the feature between entire cell populations. Examples include quantifying nuclear shape asymmetry averages  or the dLMNA mutations (LMNA-CMs), unaffected family members and unrelated individuals as negative controls (Controls), and a patient with Hutchinson-Gilford Progeria Syndrome as a positive control (HGPS). The developed method identifies dysmorphic nuclei with accuracy comparable to manual observation in a significantly more consistent and unbiased manner and allowed for correlating of subtle differences in nuclear shape of fibroblasts to a phenotype the patients presented in the clinic.In this work, we have developed a method of automatically segmenting nuclei in two-dimensional fluorescent images and classifying each as possessing a normal or dysmorphic morphology based on three nuclear shape features by a package of Matlab codes. As proof of concept, this technique was used to quantify the prevalence of dysmorphic nuclei in skin fibroblasts of individuals with three different A 7.6cm x 8.3cm rectangular glass coverslip  was cleaned via sonication for 30 minutes in 200 proof ethanol solution. The coverslip was then spin-coated with a 10:1 mixture of polydimethylsiloxane (PDMS) and curing agent , and cured at 65°C for at least 12 hours. Finally, the large coverslip was cut into smaller, rectangular 14mm x 12.5mm coverslips using a diamond scriber . Coverslips were placed PDMS side facing downward onto 100 μL drops of 0.05 mg/mL fibronectin solution for 10 minutes. Coverslips were then rinsed 3 times in Phosphate Buffered Saline  and stored in 4°C PBS until seeded with cells.LMNA splice-site mutation (c.357-2A>G) [LMNA nonsense mutation  in exon 4 [LMNA missense mutation  in exon 6 [LMNA G608G point mutation [Human fibroblast cells were collected from three families with different mutations of heterozygous 57-2A>G)   with 10% Fetal Bovine Serum  and 1% Antibiotic-Antimycotic . At 24 hours of incubation, the media was changed to MEM with 2% FBS. After an additional 24 hours of incubation, the cells were fixed.As the division rate depended on patient age, the optimal cell seeding density was determined by seeding cells on isotropic coverslips in amounts varying from 75,000 to 400,000 cells per coverslip, and expanding them as described above. After 48 hours of incubation, these coverslips were examined and the optimal cell seeding density for confluency at 2 days was identified.Staining was performed using standard techniques . BrieflyThe microscope slides containing the stained samples were imaged using an UPLFLN 40x oil immersion objective  and digital CCD camera ORCA-R2 C10600-10B  mounted on an IX-83 inverted motorized microscope . Fluorescence images of ten randomly selected fields of view per slide were taken at 40x magnification (6.22 pixels/μm resolution) for each sample.d) between the area of the convex hull (h) of each nucleus and the area enclosed by its calculated boundary (a) was divided by the convex hull area  rather than algebraically. Areas of negative curvature correspond to both blebs and invaginations in the nucleus due to the inward folding of the nuclear envelope, creating regions where the nuclear boundary becomes concave as opposed to the positive boundary curvature created by the smooth surface of a normal nucleus (NCmax) in the boundary curvature profile of each nucleus was also recorded and used as a feature to assess nuclear shape abnormality  was calculated for each nucleus boundary using the method described in Driscoll, et al. . The ext nucleus . Any nucormality :NCmax=mmax variables on the axes. On the 3D plot, each nucleus in the validation set corresponded to a single point color coded according to agreements , disagreement , and agreement on defective).To better elucidate observer-code and observer-observer differences, three dimensional (3D) plots were generated with the RC, MNC, and NC2 value for the trend and significance for both constants were calculated. P values of 0.05 or less were considered statistically significant. The level of agreement in classification of nuclei among different manual observers and the Matlab analysis codes was assessed using percent agreement, but Cohen’s Kappa was also calculated as it was possible for the manual observers to guess [Analysis of Variance (ANOVA) with Tukey’s method was used for mean comparisons among conditions. The student t-test was used if comparison was made between a single pair of data points. Linear trends were determined using linear regression, and both the Rto guess ,30.A training data set was created by cropping 243 LMNA-CM patient and control nuclei from their images using ImageJ . All nuclei in the training set were manually designated by at least two observers as either dysmorphic or normally shaped . Using tA classification scheme was created using the three limits for each feature. Any nucleus possessing a relative nuclear concavity, mean negative curvature, or maximum negative curvature below the lower limit for that feature was designated as normal Step 1, . Then, aTo demonstrate the method’s versatility, it was used to analyze images of nuclei labeled by different fluorescent stains. When the same nuclei were visualized using both Anti-Lamin A/C Antibody  and DAPIThe nuclei from an HGPS , Neg-CONConsistent with morphological observations of HGPS cell nuclei, the average percentage of dysmorphic nuclei was significantly greater in HGPS patient coverslips than in those of all negative control individuals . SurprisThe average area of all HGPS nuclei was significantly lower than that of dysmorphic HGPS nuclei alone . No signIn 2 = 0.84, P < 0.003) between patient separation from age-matched negative control prediction and the onset of symptoms age. While the Coriell Institute does not provide the information of when this particular patient  developed heart disease symptoms, it is typical for individuals with the HGPS disease to present with heart disease symptoms very early in life [Unlike HGPS patients  between LMNA-CM-A and both the positive and negative controls  spanning four different S1 Text(PDF)Click here for additional data file.S1 CodeThe detailed description of the code is provided in (M)Click here for additional data file.S2 CodeThe detailed description of the code is provided in (M)Click here for additional data file.S3 CodeThe detailed description of the code is provided in (M)Click here for additional data file.S4 CodeThe detailed description of the code is provided in (M)Click here for additional data file.S5 CodeThe detailed description of the code is provided in (M)Click here for additional data file.S6 CodeThe detailed description of the code is provided in (M)Click here for additional data file.S7 CodeThe detailed description of the code is provided in (M)Click here for additional data file.S1 FigStarting with an unprocessed, 2D fluorescent nuclei image and ending with the generation of nuclei boundaries, which are overlaid on the original image. Boxes represent steps in the image analysis process while arrows represent progression to a new or previous step.(TIF)Click here for additional data file.S2 Figa Following automatic segmentation of nuclei, an image is displayed with all detected nuclei highlighted and numbered, including one undetected pair of overlapping nuclei; b User is prompted to choose an action after reviewing the displayed image, entering ‘6’ in order to exclude the overlapping nuclei from further analysis, and then entering the number corresponding to that body in the image; c The overlapping nuclei are excluded from analysis, and highlighted in red.(PDF)Click here for additional data file.S3 Figa Lamin A+C immunofluorescence label and b DAPI, and then segmented by the image analysis Matlab code; c Average area, d average eccentricity, and e average mean negative curvature were then calculated based on nuclear boundaries in both the DAPI and Lamin A+C stain images (n = 48 nuclei). f The percentage of nuclei designated as dysmorphic was also calculated for all images and averaged by stain type (n = 12 images). Error bars represent one standard deviation. Scale bar: 25 μm.Nuclei were visualized with both (TIF)Click here for additional data file.S4 Figa Original grayscale image; b Binary image generated from first intensity threshold; c Regions of original grayscale image isolated based on detected bodies in binarized image; d Region of original image corresponding to a single nucleus; e Binary image generated from second intensity threshold; f Outline of nucleus generated; g Outline smoothed and given sub-pixel resolution using snake algorithm; h Nuclei labeled and outlined with snake algorithm boundaries; i Overlapping nuclei (red outline) excluded from analysis; j Example data of nuclear area and orientation. Scale bar: 25 μm.(PDF)Click here for additional data file.S5 FigPlot shows no correlations for the data.(PDF)Click here for additional data file.


Recent influenza surveillance data from Africa suggest an important burden of influenza-associated morbidity and mortality. In tropical countries where influenza virus transmission may not be confined to a single season alternative strategies for vaccine distribution via antenatal care (ANC) or semiannual campaigns should be considered.Using data on monthly influenza disease burden in women of child-bearing age and infants aged 0–5 months in Kenya from 2010–2014, we estimated the number of outcomes  that occurred and that may have been averted through influenza vaccination of pregnant women using: 1) a year-round immunization strategy through ANC, 2) annual vaccination campaigns, and 3) semiannual vaccination campaigns.During 2010–2014, influenza resulted in an estimated 279,047 illnesses, 36,276 medical visits, 1612 hospitalizations and 243 deaths in pregnant women and 157,053 illnesses, 65,177 medical visits, 4197 hospitalizations, and 755 deaths in infants aged 0–5 months in Kenya. Depending on the mode of distribution and the vaccine coverage achieved, 12.8–31.4% of influenza-associated disease in pregnant women and 11.6–22.1% in infants aged 0–5 months might have been prevented through maternal influenza immunization. In this model, point estimates for influenza-associated disease averted through maternal vaccination delivered year-round in ANC or semiannually in campaigns were higher than vaccination delivered in a single annual campaign, but confidence intervals overlapped.Vaccinating pregnant women against influenza can reduce the burden of influenza-associated illness, hospitalization and death in both pregnant women and their young infants. Alternative immunization strategies may avert more influenza-associated disease in countries where influenza virus transmission occurs throughout the year. Influenza remains a major cause of morbidity and mortality worldwide. While influenza immunization programs have been effective tools in reducing the disease burden in high-income countries , 2, few Although data are limited, the impact of influenza on adverse outcomes in pregnant women and children is likely to be as great, or greater in developing world settings as compared to more developed countries. A recent systematic review estimated that 99% of influenza-related deaths among children aged less than five years occur in resource-limited settings, and the incidence of severe acute lower respiratory tract infections among infants aged 0–11 months appears to be greater in low-resource settings . In KenyA model was constructed using estimates of influenza disease burden, vaccine coverage, and vaccine effectiveness to estimate influenza outcomes that could have been averted through influenza vaccination of pregnant women in Kenya. Model inputs include data from studies that are human subjects research. These protocols require informed consent and are reviewed and approved by the Kenya Medical Research Institute and the U.S. Centers for Disease Control and Prevention. We compared the following strategies for vaccinating pregnant women: 1) a year-round immunization strategy in which vaccines are provided through antenatal clinics (ANC), 2) annual vaccination campaigns using Southern Hemisphere formulations of influenza vaccine (delivered in March and April), or 3) semi-annual vaccination (every 6 months) campaigns using both Northern and Southern Hemisphere vaccine formulations. Outcomes assessed included averted influenza cases, medical visits, hospitalizations, and deaths in pregnant women and their infants.During the period of analysis, influenza occurred year-round in Kenya, but seasonal peaks in disease were observed. Cases of influenza associated with the 2009 influenza A(H1N1) pandemic virus were first detected in Kenya in July 2009 , and sprWe used the 5-year average mortality rate in hospitalized women aged 15–49 years with a respiratory diagnosis from an HDS site in western Kenya [6.8% (95% CI 1.1–12.5%)], the respiratory admission rate among women aged 15–49 years, and the proportion of respiratory hospitalizations that tested positive for influenza viruses to estimate the influenza-associated mortality rate in hospitalized pregnant women . We usedWe used data on the rate of influenza-associated acute lower respiratory tract infection hospitalizations in women aged 15–49 years and infants aged 0–5 months from a HDS site in western Kenya and applied this rate to the number of pregnant women and infants aged 0–5 months in the HDS site catchment area. Because limited data were available on disease burden among pregnant women in Kenya, we used hospitalization rates among women of childbearing age as a proxy. There are limited data on the risk of influenza-associated hospitalization among pregnant compared to non-pregnant women in Africa. We did not make an adjustment for the sensitivity of influenza testing by rRT-PCR or influenza-associated non-respiratory illnesses in pregnant women or infants. The average annual hospitalization rate over the 5-year study period was 39.9 (95% CI 25.3–62.9) per 100,000 women aged 15–49 years and 129.4 (95% CI 45.8–365.7) per 100,000 infants aged 0–5 months.We estimated the number of medically attended influenza illnesses in Kenyan pregnant women and their infants by applying a ratio of medically attended cases to hospitalizations determined by dividing the total number of medically attended (MA) ILI and SARI attributable to influenza in women aged 15–49 years or infants aged 0–5 months from population-based surveillance by the hospitalization rate in women aged 15–49 years or infants aged 0–5 months. In this study our medically attended case-hospitalization ratio was 22.5 for women aged 15–49 years and 15.5 for infants aged 0–5 months.We estimated the number of influenza illnesses in Kenyan pregnant women and their infants by dividing the medically attended illness rates by the proportion of ILI and SARI that were medically attended. From population-based surveillance, the proportion of all ILI and SARI that were medically attended was 13.0% for women aged 15–49 years and 41.5% for infants aged 0–5 months. We assumed that protection against repeat infection conferred through influenza illness was complete and would last throughout the pregnancy  or the first 6 months of life (against infant infection).The most recent estimate of vaccination coverage of pregnant women in Kenya with two doses of tetanus toxoid is 55.0% , which iAfter comparing the three vaccination strategies assuming all achieved 55% coverage of all women who become pregnant in a year, we made additional comparisons of hospitalizations averted by the different strategies with the following assumptions: 1. If 86% of women have 2 or more antenatal care visits  and 80% Vaccine effectiveness (VE) was estimated using 1-risk ratio (RR)  where thIn September 2009, influenza A(H1N1)pdm09 virus began circulating in Kenya. Monovalent pandemic vaccine was not available in Kenya until April 2010. During this 8 month period, 85% of influenza viruses identified in routine and population-based surveillance in Kenya were influenza A(H1N1)pdm09. To reflect the reduced vaccine effectiveness of the available seasonal vaccine against pandemic influenza A(H1N1) we assumed that VE was 0% from January to April 2010. This was done to ensure that we did not overestimate the potential impact of the available seasonal vaccination during the initial stages of the pandemic. In 2010 the annual campaign was delayed by 2 months to May-June to more accurately reflect pandemic vaccine availability.We estimated the averted burden of influenza-related outcomes that could be achieved with prenatal influenza vaccination in several steps, similar to the methodology used in the United States. First wThe number of outcomes averted each year not only depends directly on vaccination coverage and effectiveness, but also depends on the influenza attack rate–i.e., years with high attack rates will produce a higher number of averted outcomes assuming the same coverage rate and VE. Therefore, we present averted influenza burden for each vaccination strategy in absolute numbers and as the prevented fraction. The prevented fraction is defined in this analysis as the proportion of outcomes averted through vaccination out of potential outcomes in the absence of vaccination. Each year, the prevented fraction is the same for all outcomes  because the number of cases and MA cases were calculated as proportions of the estimated number of hospitalizations each year using data on reported ILI and SARI, and medically-attended ILI and SARI from population-based surveillance.We further calculated the number of pregnant women that would need to be vaccinated in order to prevent one maternal or infant hospitalization, and one maternal or infant death by dividing the total number of women vaccinated per year by the number of averted episodes .In order to assess the impact of higher rates of influenza-associated hospitalization in pregnant women, we used published and data from 3 countries  to estimDuring 2010–2014, we estimate that influenza virus infections among pregnant women caused 279,047 illnesses, 36,276 medical visits, 1612 hospitalizations, and 243 maternal deaths . We estiDelivery of influenza vaccine through routine antenatal care with 55% coverage might have averted 69,780 influenza-associated illnesses, 9071 medical visits, 403 hospitalizations and 58 deaths in pregnant women over the 5-year study period , Fig 2. Delivery of influenza vaccine through routine antenatal care with 55% coverage might have averted 27,575 influenza-associated illnesses, 11,444 medical visits, 737 hospitalizations, and 98 deaths in infants aged 0–5 months over the 5-year study period , Fig 2. The number needed to vaccinate to prevent one maternal or infant hospitalization was similar for all three strategies . Likewise, the number needed to vaccinate to prevent one maternal or infant death was similar for all three strategies .Delivery of influenza vaccine through routine antenatal care with 69% coverage might have averted 87,690 illnesses, 11,400 medical visits, 507 influenza-associated hospitalizations and 73 deaths in pregnant women over the 5-year study period , Fig 3. Delivery of influenza vaccine through routine antenatal care with 69% coverage might have averted 34,692 illnesses, 14,397 medical visits, 927 influenza-associated hospitalizations, and 123 deaths in infants aged 0–5 months over the 5-year study period , Fig 3. The number needed to vaccinate to prevent one maternal or infant hospitalization was similar for all three strategies . Likewise, the number needed to vaccinate to prevent one maternal or infant death was similar for all three strategies .The scenarios described above estimate influenza-associated hospitalization rates from women aged 15–49 years and assume no increased risk of influenza-associated hospitalization in pregnant women. Combining data from 3 surveillance systems in Kenya, Malawi and South Africa, and weighting according to the number of influenza-positive pregnant women enrolled, we found that pregnant women in sub-Saharan Africa have increased risk of influenza-associated hospitalization, odds ratio (OR) = 2.9 (95% CI 1.0–12.3) . BecauseOver a five year period, we estimated that influenza caused thousands of respiratory hospitalizations among pregnant women and their young infants. Young infants had particularly high rates of influenza-associated hospitalization. Regardless of the immunization strategy, if 55% of pregnant women received influenza vaccination during pregnancy approximately 20% of influenza-associated illness in pregnant women and 17% in young infants could be averted. In all scenarios, the averted fraction of influenza-associated illness was lower in young infants than for pregnant women; however, due to the high rates of severe disease in young infants more hospitalizations and deaths are averted in infants than pregnant women through maternal influenza vaccination.In this model, there were no significant differences in the proportion of maternal or infant illnesses averted through different influenza vaccine delivery mechanisms when vaccine coverage was held constant (55%). With different assumptions about vaccine coverage, a higher proportion of maternal and infant influenza-associated illness may have been prevented through delivery of influenza vaccine in antenatal care or semiannual campaigns than in annual campaigns, although confidence limits overlap. The only study to examine the feasibility of vaccinating year-round in antenatal care found that the vaccine prevented influenza-like illness in pregnant women and laboratory-confirmed influenza-associated illness in infants aged 0–5 months .Policy makers in resource limited countries may find annual campaigns the most effective means of protecting pregnant women and young infants if there are insufficient doses of influenza vaccine to achieve >30% coverage. A single annual campaign will likewise reduce the administrative burden of distributing vaccine throughout the year via ANC or distribution twice annually for semiannual campaigns. Because of the relatively short shelf-life of influenza vaccines and antigenic drift in circulating influenza viruses, it is essential to use the most up-to-date vaccine available to confer protection to mother and infant. Offering influenza vaccine throughout the year in ANC or via semiannual campaigns would require coordinated distribution of the new season’s vaccine along with timely removal of any remaining doses of the prior season’s vaccine. Policy makers will also need to consider facility-level cold chain capacity and local differences in antenatal care-seeking behaviors to determine the optimal distribution strategy in their country. With limited cold chain capacity it might be necessary to offer influenza vaccination over a longer period of time than a single 2 month campaign. Likewise if women receive few antenatal care visits and/or present late for antenatal care it may be useful to offer influenza vaccination over a longer period of time than a single 2 month campaign in order to improve vaccine coverage. In some cultures, influenza vaccination may also serve as an inducement to seek antenatal care if offered as part of a focused antenatal care package.There were several limitations to this static model for assessing the impact of different modes of influenza vaccine delivery in antenatal care. First, the model did not adjust for vaccine effectiveness or disease severity by circulating strain type, but used the same estimates across all years due to limited available data. The pandemic strain was particularly severe among pregnant women compared to the previous seasonal influenza A(H1N1) virus and years where influenza A(H3N2) is the predominant circulating virus typically result in more influenza-associated hospitalizations and deaths. Likewise, vaccine effectiveness against influenza A(H3N2) may not be as high as effectiveness against influenza A(H1N1) or influenza B viruses. Virus-specific vaccine effectiveness may alter the overall impact of influenza vaccine on health outcomes for pregnant women and their infants; however, it would not impact the relative outcomes by scenario. Second, we assumed that vaccine effectiveness against laboratory-confirmed influenza hospitalization and death were the same as vaccine effectiveness against laboratory-confirmed influenza illness. It is possible that vaccine effectiveness is higher or lower against severe disease and death than against mild disease; however, studies of vaccine effectiveness are generally not powered to measure effectiveness against severe disease. Third, we assumed protection conferred through immunization or natural infection would be complete and last for the first 6 months of an infant’s life. More recent data suggest that this protection may be limited to the first 4 months of life. Fourth, our surveillance only assessed acute respiratory illness in infants. Young infants with influenza infection may be less likely to present with respiratory symptoms than older children so this may have led to an underestimation of the burden of influenza-associated illness in infants aged 0–5 months. A study in Finland found that nearly half of all influenza-positive infants aged 0–5 months were admitted with a non-respiratory diagnosis . FurtherThis approach only estimates the direct effects of vaccination to vaccinated women and their live-born infants, and does not reflect any potential indirect impact of vaccination  or the potential impact of vaccination on disease severity, birth weight, preterm birth, or fetal loss. Another limitation is that many of the assumptions in this model are generated from surveillance of relatively small populations. Assumptions from these small populations about the proportion of outpatient vs. inpatient illnesses, timing of outpatient vs. inpatient illnesses, healthcare seeking for respiratory illness, and the proportion of deaths occurring in the community may not be representative of respiratory illnesses and deaths throughout Kenya. And finally, we assumed that semiannual campaigns delivered over 4 months annually could achieve the same vaccine coverage as year-round antenatal care distribution. It is likely that distribution throughout the year might achieve higher coverage than intermittent campaigns but without recent data on maternal vaccination campaigns in Kenya it is difficult to predict the anticipated coverage from these different strategies.Over the 5-year period of study, depending on the mode of distribution and the vaccine coverage achieved, 12.8–31.4% of influenza-associated illnesses, medical visits, hospitalizations and deaths among pregnant women and 11.6–22.1% of influenza-associated illnesses, medical visits, hospitalizations and deaths among infants aged 0–5 months might have been prevented through maternal influenza immunization. Each of the proposed strategies for delivery of influenza vaccine to pregnant women has advantages and disadvantages in terms of infrastructure requirements  and associated costs. Governments considering introduction of influenza vaccination for pregnant women will have to assess existing infrastructure for vaccine delivery and determine the best vaccine distribution method for their country. Additional analysis of the cost and cost-effectiveness of different methods of vaccine delivery to pregnant women will be important to inform policy decisions in Kenya and other tropical countries where influenza circulation is not confined to a single seasonal peak.


We report on interns’ clinical competence and experiences of an objective structured clinical examination (OSCE) training program over 3 years. We aimed to determine whether repeated formative OSCEs allow teaching hospitals to improve the effectiveness of clinical training and help interns to achieve high scores in the national summative OSCE.This study included 207 participants, among whom 82 were interns who had completed four mock OSCEs and a national OSCE at the clinical center of Cathay General Hospital (CGH). The other 125 participants were final-year medical students from Fu-Jen University who had completed the national OSCE between 2013 and 2015 at one of four teaching hospitals (including CGH). CGH interns were categorized into three groups according to the medical school attended and Fu-Jen University students were grouped according to their training hospitals. CGH held four mock OSCEs (30 stations), whereas each of the four training hospitals for Fu-Jen students each held one or two OSCEs (6–12 stations) annually. Differences in the mean OSCE scores among groups were analyzed. The medical school attended, pre-internship OSCE experience and the frequency of mock OSCEs held by training hospitals were independent factors in this study.The cumulative mean scores for five OSCEs among three groups of students trained at CGH tended to increase from the first OSCE (OSCE1) to the fifth (OSCE5). The mean score of the students who attended Fu-Jen Medical School was higher than that of students who graduated from foreign medical schools in all five OSCEs; however, the differences were significant only for OSCE2 (P = 0.022) and OSCE3 (P = 0.027). The mean national OSCE scores of FJU students showed no statistically significant differences among the four training hospitals for 2013; however, students training at CGH had significantly higher mean scores in the 2014 (P = 0.001) and 2015 (P = 0.005) OSCEs compared with students training at the other three hospitals.Serial administration of formative OSCEs by teaching hospitals enhances the performance of students on the sequential summative OSCE. Such programs provide multiple opportunities for students to practice their clinical skills, and for faculty to develop their teaching, assessment and consensus building skills. Administration of formative and summative objective structured clinical examinations (OSCEs) in teaching programs has been shown to improve final-year medical school student’s examination performance . AlthougOSCEs are well-established as effective assessment tools for clinical competence. As such, they have become an important part of the medical licensing process in many countries , 5. In TOSCE-related policies also influence various aspects of the medical education systems that apply OSCEs as a formative educational mode . All stuThis retrospective study was approved by the Institutional Review Board (IRB) of Cathay General Hospital (CGHIRB No: CGH-P104084). The IRB granted a waiver of informed consent from the participants in this study; their records were anonymized and de-identified prior to analysis. Eighty-two students who received clinical training at CGH between 2013 and 2015 were categorized according to the medical school attended into three groups labelled A, B and C. Group A comprised students from FJU (n = 31); group B contained students from medical school B of Taiwan (n = 30); and group C comprised graduates from foreign medical schools (n = 21). The students in all the groups had completed a set of five OSCEs within 1 academic year between 2013 and 2015; the first four were formative OSCEs in which station raters provided students with direct feedback, and the final examination was the national OSCE. Individual student scores on each OSCE were recorded and the differences in the mean scores among groups A, B and C were analyzed. Regarding previous  experience of OSCEs, participants in group A completed OSCEs six times on average, whereas those in Group B had completed them once and those in Group C had no experience of OSCEs. The first to fourth mock OSCEs (OSCE1–OSCE4) were held during the months of October, December, February, and April, respectively, and the final national OSCE (OSCE5) was held in the last week of April. Three OSCEs  contained six stations (four SP stations and two procedure operation stations) and two OSCEs (OSCE3 and OSCE5) comprised 12 stations (eight SP stations and four procedure operation stations). The allocated fields for each OSCE station covered five departments: internal medicine, surgery, gynecology, pediatrics, and emergency. These fields correspond to five categories of clinical skills: history taking, physical examination, explanation and management, communication and counseling, and procedure skills. Students were required to achieve not only the passing score at a given minimum number of stations (7 stations) but also the overall passing score. The passing score for each station was calculated using borderline-group and the borderline regression methods. Each station in OSCE1–OSCE4 involved 8 min of observation and 2 min of immediate verbal feedback from station raters. Students received formal individualized performance analysis reports, and participated in a 90-min discussion meeting 1 week after each mock OSCE to review the correct procedures for each station with the aim of improving performance at the next examination. The last national OSCE (OSCE5) was a summative examination; students were given 8 min at each station. All raters were clinical physicians with actual scoring experience as certified by the OSCE office. All SPs were certified as having performance experience. Discrimination, difficulty and passing score of each station were calculated and compared according to guidelines set out by the OSCE office.The 125 final-year FJU medical students received clinical training at four teaching hospitals: F hospital (FH), S hospital (SH), K hospital (KH) and CGH. Each student in this group had participated in the national OSCE (the fifth OSCE of CGH) between 2013 and 2015. The mean scores of these students in each teaching hospital were compared. CGH held four mock OSCEs (30 stations), whereas the remaining teaching hospitals held one or two mock OSCEs (6–12 stations) annually.An analysis of variance (ANOVA) was performed to compare the differences among the study groups. A post hoc analysis was subsequently conducted for the groups showing significant differences; given that the sizes of the groups differed, the Scheffe method was used to prevent type-one error.For the 82 students trained at CGH from 2013 to 2015, the cumulative mean scores for OSCE1 through OSCE5 showed positive curve   than did students at the other hospitals. The stations in these mock examinations featured SPs in well-designed clinical scenarios, and raters, who provided immediate feedback. Such a training style is highly beneficial for faculty development and student training . The mocA limitation of this study was the small sample size. However, OSCE training programs are easier to manage when conducted on small groups comprising approximately 30 students. The 2015 CGH OSCE training program, for example, required two workshops (one for raters and one for SPs), 122 raters, 75 SPs and 54 OSCE stations to train only 33 students. All methods for the training of raters and SPs, development of OSCE stations, and administration of OSCEs were qualified and followed the standards set by the OSCE office. Fine tuning of this training program is essential for application in teaching hospitals of different sizes. The administration of mock OSCEs by teaching hospitals enhances the performance students on the summative OSCE, as well as the teaching and assessment abilities of teachers and program directors.