The behaviour change behind a successful pilot of hypoglycaemia reduction with HYPO-CHEAT.

Background: Children with hypoglycaemia disorders, such as congenital hyperinsulinism (CHI), are at constant risk of hypoglycaemia (low blood sugars) with the attendant risk of brain injury. Current approaches to hypoglycaemia detection and prevention vary from fingerprick glucose testing to the provision of continuous glucose monitoring (CGM) to machine learning (ML) driven glucose forecasting. Recent trends for ML have had limited success in preventing free-living hypoglycaemia, due to a focus on increasingly accurate glucose forecasts and a failure to acknowledge the human in the loop and the essential step of changing behaviour. The wealth of evidence from the fields of behaviour change and persuasive technology (PT) allows for the creation of a theory-informed and technologically considered approach. Objectives: We aimed to create a PT that would overcome the identified barriers to hypoglycaemia prevention for those with CHI to focus on proactive prevention rather than commonly used reactive approaches. Methods: We used the behaviour change technique taxonomy and persuasive systems design models to create HYPO-CHEAT (HYpoglycaemia-Prevention-thrOugh-Cgm-HEatmap-Assisted-Technology): a novel approach that presents aggregated CGM data in simple visualisations. The resultant ease of data interpretation is intended to facilitate behaviour change and subsequently reduce hypoglycaemia. Results: HYPO-CHEAT was piloted in 10 patients with CHI over 12 weeks and successfully identified weekly patterns of hypoglycaemia. These patterns consistently correlated with identifiable behaviours and were translated into both a change in proximal fingerprick behaviour and ultimately, a significant reduction in aggregated hypoglycaemia from 7.1% to 5.4% with four out of five patients showing clinically meaningful reductions in hypoglycaemia. Conclusions: We have provided pilot data of a new approach to hypoglycaemia prevention that focuses on proactive prevention and behaviour change. This approach is personalised for individual patients with CHI and is a first step in changing our approach to hypoglycaemia prevention in this group.

The hypoglycaemia error grid: A UK-wide consensus on CGM accuracy assessment in hyperinsulinism.

Objective: Continuous Glucose Monitoring (CGM) is gaining in popularity for patients with paediatric hypoglycaemia disorders such as Congenital Hyperinsulinism (CHI), but no standard measures of accuracy or associated clinical risk are available. The small number of prior assessments of CGM accuracy in CHI have thus been incomplete. We aimed to develop a novel Hypoglycaemia Error Grid (HEG) for CGM assessment for those with CHI based on expert consensus opinion applied to a large paired (CGM/blood glucose) dataset. Design and methods: Paediatric endocrinology consultants regularly managing CHI in the two UK centres of excellence were asked to complete a questionnaire regarding glucose cutoffs and associated anticipated risks of CGM errors in a hypothetical model. Collated information was utilised to mathematically generate the HEG which was then approved by expert, consensus opinion. Ten patients with CHI underwent 12 weeks of monitoring with a Dexcom G6 CGM and self-monitored blood glucose (SMBG) with a Contour Next One glucometer to test application of the HEG and provide an assessment of accuracy for those with CHI. Results: CGM performance was suboptimal, based on 1441 paired values of CGM and SMBG showing Mean Absolute Relative Difference (MARD) of 19.3% and hypoglycaemia (glucose <3.5mmol/L (63mg/dL)) sensitivity of only 45%. The HEG provided clinical context to CGM errors with 15% classified as moderate risk by expert consensus when data was restricted to that of practical use. This provides a contrasting risk profile from existing diabetes error grids, reinforcing its utility in the clinical assessment of CGM accuracy in hypoglycaemia. Conclusions: The Hypoglycaemia Error Grid, based on UK expert consensus opinion has demonstrated inadequate accuracy of CGM to recommend as a standalone tool for routine clinical use. However, suboptimal accuracy of CGM relative to SMBG does not detract from alternative uses of CGM in this patient group, such as use as a digital phenotyping tool. The HEG is freely available on GitHub for use by other researchers to assess accuracy in their patient populations and validate these findings.

Non-coding variants disrupting a tissue-specific regulatory element in HK1 cause congenital hyperinsulinism.

Gene expression is tightly regulated, with many genes exhibiting cell-specific silencing when their protein product would disrupt normal cellular function1. This silencing is largely controlled by non-coding elements, and their disruption might cause human disease2. We performed gene-agnostic screening of the non-coding regions to discover new molecular causes of congenital hyperinsulinism. This identified 14 non-coding de novo variants affecting a 42-bp conserved region encompassed by a regulatory element in intron 2 of the hexokinase 1 gene (HK1). HK1 is widely expressed across all tissues except in the liver and pancreatic beta cells and is thus termed a 'disallowed gene' in these specific tissues. We demonstrated that the variants result in a loss of repression of HK1 in pancreatic beta cells, thereby causing insulin secretion and congenital hyperinsulinism. Using epigenomic data accessed from public repositories, we demonstrated that these variants reside within a regulatory region that we determine to be critical for cell-specific silencing. Importantly, this has revealed a disease mechanism for non-coding variants that cause inappropriate expression of a disallowed gene.

Somatostatin receptors in congenital hyperinsulinism: Biology to bedside.

Congenital hyperinsulinism (CHI), although a rare disease, is an important cause of severe hypoglycemia in early infancy and childhood, causing preventable morbidity and mortality. Prompt diagnosis and appropriate treatment is necessary to prevent hypoglycaemia mediated brain damage. At present, the medical treatment of CHI is limited to diazoxide as first line and synthetic somatostatin receptor ligands (SRLs) as second line options; therefore understanding somatostatin biology and treatment perspectives is important. Under healthy conditions, somatostatin secreted from pancreatic islet δ-cells reduces insulin release through somatostatin receptor induced cAMP-mediated downregulation and paracrine inhibition of ß- cells. Several SRLs with extended duration of action are now commercially available and are being used off-label in CHI patients. Efficacy remains variable with the present generation of SRLs, with treatment effect often being compromised by loss of initial response and adverse effects such as bowel ischaemia and hepatobiliary dysfunction. In this review we have addressed the biology of the somatostatin system contexualised to CHI. We have discussed the clinical use, limitations, and complications of somatostatin agonists and new and emerging therapies for CHI.

HYPO-CHEAT's aggregated weekly visualisations of risk reduce real world hypoglycaemia.

Background: Children with congenital hyperinsulinism (CHI) are at constant risk of hypoglycaemia with the attendant risk of brain injury. Current hypoglycaemia prevention methods centre on the prediction of a continuous glucose variable using machine learning (ML) processing of continuous glucose monitoring (CGM). This approach ignores repetitive and predictable behavioural factors and is dependent upon ongoing CGM. Thus, there has been very limited success in reducing real-world hypoglycaemia with a ML approach in any condition. Objectives: We describe the development of HYPO-CHEAT (HYpoglycaemia-Prevention-thrOugh-CGM-HEatmap-Technology), which is designed to overcome these limitations by describing weekly hypoglycaemia risk. We tested HYPO-CHEAT in a real-world setting to evaluate change in hypoglycaemia. Methods: HYPO-CHEAT aggregates individual CGM data to identify weekly hypoglycaemia patterns. These are visualised via a hypoglycaemia heatmap along with actionable interpretations and targets. The algorithm is iterative and reacts to anticipated changing patterns of hypoglycaemia. HYPO-CHEAT was compared with Dexcom Clarity's pattern identification and Facebook Prophet's forecasting algorithm using data from 10 children with CHI using CGM for 12 weeks. HYPO-CHEAT's efficacy was assessed via change in time below range (TBR). Results: HYPO-CHEAT identified hypoglycaemia patterns in all patients. Dexcom Clarity identified no patterns. Predictions from Facebook Prophet were inconsistent and difficult to interpret. Importantly, the patterns identified by HYPO-CHEAT matched the lived experience of all patients, generating new and actionable understanding of the cause of hypos. This facilitated patients to significantly reduce their time in hypoglycaemia from 7.1% to 5.4% even when real-time CGM data was removed. Conclusions: HYPO-CHEAT's personalised hypoglycaemia heatmaps reduced total and targeted TBR even when CGM was reblinded. HYPO-CHEAT offers a highly effective and immediately available personalised approach to prevent hypoglycaemia and empower patients to self-care.

Variation in Glycemic Outcomes in Focal Forms of Congenital Hyperinsulinism-The UK Perspective.

Context: In focal congenital hyperinsulinism (CHI), localized clonal expansion of pancreatic ß-cells causes excess insulin secretion and severe hypoglycemia. Surgery is curative, but not all lesions are amenable to surgery. Objective: We describe surgical and nonsurgical outcomes of focal CHI in a national cohort. Methods: Patients with focal CHI were retrospectively reviewed at 2 specialist centers, 2003-2018. Results: Of 59 patients with focal CHI, 57 had heterozygous mutations in ABCC8/KCNJ11 (51 paternally inherited, 6 de novo). Fluorine-18 L-3,4 dihydroxyphenylalanine positron emission tomography computed tomography scan identified focal lesions in 51 patients. In 5 patients, imaging was inconclusive; the diagnosis was established by frozen section histopathology in 3 patients, a lesion was not identified in 1 patient, and 1 declined surgery. Most patients (n = 56) were unresponsive to diazoxide, of whom 33 were unresponsive or partially responsive to somatostatin receptor analog (SSRA) therapy. Fifty-five patients underwent surgery: 40 had immediate resolution of CHI, 10 had persistent hypoglycemia and a focus was not identified on biopsy in 5. In the 10 patients with persistent hypoglycemia, 7 underwent further surgery with resolution in 4 and ongoing hypoglycemia requiring SSRA in 3. Nine (15% of cohort) patients (1 complex surgical access; 4 biopsy negative; 4 declined surgery) were managed conservatively; medication was discontinued in 8 children at a median (range) age 2.4 (1.5-7.7) years and 1 remains on SSRA at 16 years with improved fasting tolerance and reduction in SSRA dose. Conclusion: Despite a unifying genetic basis of disease, we report inherent heterogeneity in focal CHI patients impacting outcomes of both surgical and medical management.

Pancreatic ductal adenocarcinoma cells employ integrin α6ß4 to form hemidesmosomes and regulate cell proliferation.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6ß4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin ß4 reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, suggesting a requirement for α6ß4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC. Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.

Clustering of Hypoglycemia Events in Patients With Hyperinsulinism: Extension of the Digital Phenotype Through Retrospective Data Analysis.

BACKGROUND: Hyperinsulinism (HI) due to excess and dysregulated insulin secretion is the most common cause of severe and recurrent hypoglycemia in childhood. High cerebral glucose use in the early hours results in a high risk of hypoglycemia in people with diabetes and carries a significant risk of brain injury. Prevention of hypoglycemia is the cornerstone of the management of HI, but the risk of hypoglycemia at night or the timing of hypoglycemia in children with HI has not been studied; thus, the digital phenotype remains incomplete and management suboptimal. OBJECTIVE: This study aims to quantify the timing of hypoglycemia in patients with HI to describe glycemic variability and to extend the digital phenotype. This will facilitate future work using computational modeling to enable behavior change and reduce exposure of patients with HI to injurious hypoglycemic events. METHODS: Patients underwent continuous glucose monitoring (CGM) with a Dexcom G4 or G6 CGM device as part of their clinical assessment for either HI (N=23) or idiopathic ketotic hypoglycemia (IKH; N=24). The CGM data were analyzed for temporal trends. Hypoglycemia was defined as glucose levels <3.5 mmol/L. RESULTS: A total of 449 hypoglycemic events totaling 15,610 minutes were captured over 237 days from 47 patients (29 males; mean age 70 months, SD 53). The mean length of hypoglycemic events was 35 minutes. There was a clear tendency for hypoglycemia in the early hours (3-7 AM), particularly for patients with HI older than 10 months who experienced hypoglycemia 7.6% (1480/19,370 minutes) of time in this period compared with 2.6% (2405/92,840 minutes) of time outside this period (P<.001). This tendency was less pronounced in patients with HI who were younger than 10 months, patients with a negative genetic test result, and patients with IKH. Despite real-time CGM, there were 42 hypoglycemic events from 13 separate patients with HI lasting >30 minutes. CONCLUSIONS: This is the first study to have taken the first step in extending the digital phenotype of HI by describing the glycemic trends and identifying the timing of hypoglycemia measured by CGM. We have identified the early hours as a time of high hypoglycemia risk for patients with HI and demonstrated that simple provision of CGM data to patients is not sufficient to eliminate hypoglycemia. Future work in HI should concentrate on the early hours as a period of high risk for hypoglycemia and must target personalized hypoglycemia predictions. Focus must move to the human-computer interaction as an aspect of the digital phenotype that is susceptible to change rather than simple mathematical modeling to produce small improvements in hypoglycemia prediction accuracy.

Corrigendum: Efficacy of Dose-Titrated Glucagon Infusions in the Management of Congenital Hyperinsulinism: A Case Series.

[This corrects the article DOI: 10.3389/fendo.2020.00441.].

Efficacy of Dose-Titrated Glucagon Infusions in the Management of Congenital Hyperinsulinism: A Case Series.

Background: Congenital hyperinsulinism (CHI), a rare disease of excessive and dysregulated insulin secretion, can lead to prolonged and severe hypoglycemia. Dextrose infusions are a mainstay of therapy to restore normal glycemia, but can be associated with volume overload, especially in infants. By releasing intrahepatic glucose stores, glucagon infusions can reduce dependency on dextrose infusions. Recent studies have reported positive outcomes with glucagon infusions in patients with CHI; however, to date, there are no reports describing the clinical utility of titrated doses of infused glucagon to achieve glycemic stability. Objective: To assess the potential clinical utility of dose-titrated glucagon infusions in stabilizing glycemic status in pediatric patients with CHI, who were managed by medical and/or surgical approaches. Methods: Patients with CHI (N = 33), with or without mutations in the ATP-sensitive K+ channel genes, ABCC8, and KCNJ11 requiring glucagon by dose titration in addition to intravenous dextrose and medical therapy with diazoxide/octreotide to achieve glycemic stability were recruited. Following glucagon titration and a 24-h glucose stable period, glucose infusion rate (GIR) was reduced over a 24-h period. Achievement of glycemic stability and decrease in GIR were considered end points of the study. Results: All patients achieved glycemic stability with glucagon infusion, demonstrating clinical benefit. GIR reduced from 15.6 (4.5) to 13.4 (4.6) mg/kg/min mean (SD) (p = 0.00019 for difference; n = 32; paired t-test) over 24 h. By univariate analysis, no individual baseline characteristic was associated with changes in the GIR. However, by baseline-adjusted modeling, mutational status of the patient (p = 0.011) was inversely associated with a reduction in GIR. Adverse events were infrequent with diarrhea possibly attributed to glucagon treatment in 1 patient. With long-term treatment following GIR reduction, necrolytic migratory erythema was observed in another patient. Conclusion: These data suggest that dose-titrated glucagon infusion therapy aids hypoglycemia prevention and reduction in GIR in the clinical management of patients with CHI.

Case report: contradictory genetics and imaging in focal congenital hyperinsulinism reinforces the need for pancreatic biopsy.

BACKGROUND: Congenital Hyperinsulinism (CHI) is an important cause of severe hypoglycaemia in infancy due to excessive, dysregulated insulin secretion. In focal CHI, a localised lesion within the pancreas hypersecretes insulin and, importantly, hypoglycaemia resolution is possible through limited surgical resection of the lesion. Diagnosis of focal CHI is based on a crucial combination of compatible genetics and specialised imaging. Specifically, a focal lesion arises due to a paternal mutation in one of the ATP-sensitive potassium channel genes, KCNJ11 or ABCC8, in combination with post-zygotic loss of maternal heterozygosity within the affected pancreatic tissue. 6-[18F]Fluoro-L-3,4-dihydroxyphenylalanine (18F-DOPA) positron emission tomography (PET)/computed tomography (CT) imaging is used to detect and localise the lesion prior to surgery. However, its accuracy is imperfect and needs recognition in individual case management. CASE PRESENTATION: We report the case of an infant with hypoglycaemia due to CHI and a paternally inherited KCNJ11 mutation, c.286G > A (p.Ala96Thr), leading to a high probability of focal CHI. However,18F-DOPA PET/CT scanning demonstrated diffuse uptake and failed to conclusively identify a focal lesion. Due to unresponsiveness to medical therapy and ongoing significant hypoglycaemia, surgery was undertaken and a small 4.9 × 1.7 mm focal lesion was discovered at the pancreatic neck. This is the second case where this particular KCNJ11 mutation has been incorrectly associated with diffuse 18F-DOPA uptake, in contrast to the correct diagnosis of focal CHI confirmed by pancreatic biopsy. CONCLUSIONS: Identifying discrepancies between genetic and imaging investigations is crucial as this may negatively impact upon the diagnosis and surgical treatment of focal CHI. This case highlights the need for pancreatic biopsy when a strong suspicion of focal CHI is present even if 18F-DOPA imaging fails to demonstrate a discrete lesion.

Longitudinal Auxological recovery in a cohort of children with Hyperinsulinaemic Hypoglycaemia.

BACKGROUND: Hypoglycaemia due to hyperinsulinism (HI) is the commonest cause of severe, recurrent hypoglycaemia in childhood. Cohort outcomes of HI remain to be described and whilst previous follow up studies have focused on neurodevelopmental outcomes, there is no information available on feeding and auxology. AIM: We aimed to describe HI outcomes for auxology, medications, feeding and neurodevelopmental in a cohort up to age 5 years. METHOD: We reviewed medical records for all patients with confirmed HI over a three-year period in a single centre to derive a longitudinal dataset. RESULTS: Seventy patients were recruited to the study. Mean weight at birth was - 1.0 standard deviation scores (SDS) for age and sex, while mean height at 3 months was - 1.5 SDS. Both weight and height trended to the population median over the follow up period. Feeding difficulties were noted in 17% of patients at 3 months and this reduced to 3% by 5 years. At age 5 years, 11 patients (15%) had neurodevelopmental delay and of these only one was severe. Resolution of disease was predicted by lower maximum early diazoxide dose (p = 0.007) and being born SGA (p = 0.009). CONCLUSION: In a three-year cohort of HI patients followed up for 5 years, in spite of feeding difficulties and carbohydrate loading in early life, auxology parameters are normal in follow up. A lower than expected rate of neurodevelopmental delay could be attributed to prompt early treatment.

Unravelling the genetic causes of mosaic islet morphology in congenital hyperinsulinism.

Congenital hyperinsulinism (CHI) causes dysregulated insulin secretion which can lead to life-threatening hypoglycaemia if not effectively managed. CHI can be sub-classified into three distinct groups: diffuse, focal and mosaic pancreatic disease. Whilst the underlying causes of diffuse and focal disease have been widely characterised, the genetic basis of mosaic pancreatic disease is not known. To gain new insights into the underlying disease processes of mosaic-CHI we studied the islet tissue histopathology derived from limited surgical resection from the tail of the pancreas in a patient with CHI. The underlying genetic aetiology was investigated using a combination of high depth next-generation sequencing, microsatellite analysis and p57kip2 immunostaining. Histopathology of the pancreatic tissue confirmed the presence of a defined area associated with marked islet hypertrophy and a cytoarchitecture distinct from focal CHI but compatible with mosaic CHI localised to a discrete region within the pancreas. Analysis of DNA extracted from the lesion identified a de novo mosaic ABCC8 mutation and mosaic paternal uniparental disomy which were not present in leukocyte DNA or the surrounding unaffected pancreatic tissue. This study provides the first description of two independent disease-causing somatic genetic events occurring within the pancreas of an individual with localised mosaic CHI. Our findings increase knowledge of the genetic causes of islet disease and provide further insights into the underlying developmental changes associated with ß-cell expansion in CHI.

Correction: Increased proliferation and altered cell cycle regulation in pancreatic stem cells derived from patients with congenital hyperinsulinism.

[This corrects the article DOI: 10.1371/journal.pone.0222350.].

Increased proliferation and altered cell cycle regulation in pancreatic stem cells derived from patients with congenital hyperinsulinism.

Congenital hyperinsulinism (CHI) is characterised by inappropriate insulin secretion causing profound hypoglycaemia and brain damage if inadequately controlled. Pancreatic tissue isolated from patients with diffuse CHI shows abnormal proliferation rates, the mechanisms of which are not fully resolved. Understanding cell proliferation in CHI may lead to new therapeutic options, alongside opportunities to manipulate ß-cell mass in patients with diabetes. We aimed to generate cell-lines from CHI pancreatic tissue to provide in vitro model systems for research. Three pancreatic mesenchymal stem cell-lines (CHIpMSC1-3) were derived from patients with CHI disease variants: focal, atypical and diffuse. All CHIpMSC lines demonstrated increased proliferation compared with control adult-derived pMSCs. Cell cycle alterations including increased CDK1 levels and decreased p27Kip1 nuclear localisation were observed in CHIpMSCs when compared to control pMSCs. In conclusion, CHIpMSCs are a useful in vitro model to further understand the cell cycle alterations leading to increased islet cell proliferation in CHI.

Focal Congenital Hyperinsulinism as a Cause for Sudden Infant Death.

Congenital hyperinsulinism (CHI) is the commonest cause of persistent and severe hypoglycemia in infancy due to unregulated insulin secretion from pancreatic ß-cells. Prompt early diagnosis is important, as insulin reduces glucose supply to the brain, resulting in significant brain injury and risk of death. Histologically, CHI has focal and diffuse forms; in focal CHI, an inappropriate level of insulin is secreted from localized ß-cell hyperplasia. We report a 4-month-old male infant, who presented with sudden illness and collapse without a recognized cause and died. Postmortem examination revealed pancreatic histopathology compatible with focal CHI. Immunofluoresence staining showed limited expression of p57kip2 ß-cells reinforcing the diagnosis. Mutation testing for genes associated with CHI from DNA from the focal lesion was negative. This case highlights the recognition of focal CHI as a possible cause for sudden infant death. In children dying suddenly and unexpectedly, postmortem pancreatic sections should be carefully examined for focal CHI.

Clinical Diversity in Focal Congenital Hyperinsulinism in Infancy Correlates With Histological Heterogeneity of Islet Cell Lesions.

Background: Congenital Hyperinsulinism (CHI) is an important cause of severe and persistent hypoglycaemia in infancy and childhood. The focal form (CHI-F) of CHI can be potentially cured by pancreatic lesionectomy. While diagnostic characteristics of CHI-F pancreatic histopathology are well-recognized, correlation with clinical phenotype has not been established. Aims: We aimed to correlate the diversity in clinical profiles of patients with islet cell organization in CHI-F pancreatic tissue. Methods: Clinical datasets were obtained from 25 patients with CHI-F due to ABCC8/KCNJ11 mutations. 18F-DOPA PET-CT was used to localize focal lesions prior to surgery. Immunohistochemistry was used to support protein expression studies. Results: In 28% (n = 7) of patient tissues focal lesions were amorphous and projected into adjoining normal pancreatic tissue without clear delineation from normal tissue. In these cases, severe hypoglycaemia was detected within, on average, 2.8 ± 0.8 (range 1-7) days following birth. By contrast, in 72% (n = 18) of tissues focal lesions were encapsulated within a defined matrix capsule. In this group, the onset of severe hypoglycaemia was generally delayed; on average 46.6 ± 14.3 (range 1-180) days following birth. For patients with encapsulated lesions and later-onset hypoglycaemia, we found that surgical procedures were curative and less complex. Conclusion: CHI-F is associated with heterogeneity in the organization of focal lesions, which correlates well with clinical presentation and surgical outcomes.

Deubiquitinating Enzyme USP9X Suppresses Tumor Growth via LATS Kinase and Core Components of the Hippo Pathway.

The core LATS kinases of the Hippo tumor suppressor pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Recent studies have identified various E3 ubiquitin ligases that negatively regulate the Hippo pathway via ubiquitination, yet few deubiquitinating enzymes (DUB) have been implicated. In this study, we report the DUB USP9X is an important regulator of the core kinases of this pathway. USP9X interacted strongly with LATS kinase and to a lesser extent with WW45, KIBRA, and Angiomotin, and LATS co-migrated exclusively with USP9X during gel filtration chromatography analysis. Knockdown of USP9X significantly downregulated and destabilized LATS and resulted in enhanced nuclear translocation of YAP and TAZ, accompanied with activation of their target genes. In the absence of USP9X, cells exhibited an epithelial-to-mesenchymal transition phenotype, acquired anchorage-independent growth in soft agar, and led to enlarged, disorganized, three-dimensional acini. YAP/TAZ target gene activation in response to USP9X knockdown was suppressed by knockdown of YAP, TAZ, and TEAD2. Deletion of USP9X in mouse embryonic fibroblasts resulted in significant downregulation of LATS. Furthermore, USP9X protein expression correlated positively with LATS but negatively with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. Overall, these results strongly indicate that USP9X potentiates LATS kinase to suppress tumor growth. Cancer Res; 77(18); 4921-33. ©2017 AACR.

Atypical Forms of Congenital Hyperinsulinism in Infancy Are Associated With Mosaic Patterns of Immature Islet Cells.

Objectives: We aimed to characterize mosaic populations of pancreatic islet cells from patients with atypical congenital hyperinsulinism in infancy (CHI-A) and the expression profile of NKX2.2, a key transcription factor expressed in ß-cells but suppressed in δ-cells in the mature pancreas. Patients/Methods: Tissue was isolated from three patients with CHI-A following subtotal pancreatectomy. CHI-A was diagnosed on the basis of islet mosaicism and the absence of histopathological hallmarks of focal and diffuse CHI (CHI-D). Immunohistochemistry was used to identify and quantify the proportions of insulin-secreting ß-cells and somatostatin-secreting δ-cells in atypical islets, and results were compared with CHI-D (n = 3) and age-matched control tissues (n = 3). Results: In CHI-A tissue, islets had a heterogeneous profile. In resting/quiescent islets, identified by a condensed cytoplasm and nuclear crowding, ß-cells were reduced to <50% of the total cell numbers in n = 65/70 islets, whereas δ-cell numbers were increased with 85% of islets (n = 49/57) containing >20% δ-cells. In comparison, all islets in control tissue (n = 72) and 99% of CHI-D islets (n = 72) were composed of >50% ß-cells, and >20% δ-cells were found only in 12% of CHI-D (n = 8/66) and 5% of control islets (n = 3/60). Active islets in CHI-A tissue contained proportions of ß-cells and δ-cells similar to those of control and CHI-D islets. Finally, when compared with active islets, quiescent islets had a twofold higher prevalence of somatostatin/NKX2.2+ coexpressed cells. Conclusions: Marked increases in NKX2.2 expression combined with increased numbers of δ-cells strongly imply that an immature δ-cell profile contributed to the pathobiology of CHI-A.