Belgian Recommendations for Analytical Verification and Validation of Immunohistochemical Tests in Laboratories of Anatomic Pathology.

Analytical verification and validation of immunohistochemical (IHC) tests and their equipment are common practices for today's anatomic pathology laboratories. Few references or guidelines are available on how this should be performed. The study of Sciensano (the Belgian national competent authority regarding licensing of medical laboratories) performed in 2016, demonstrated a significant interlaboratory variation in validation procedures of IHC tests among Belgian laboratories. These results suggest the unavailability of practical information on the approach to the verification and validation of these tests. The existing Belgian Practice Guideline for the implementation of a quality management system in anatomic pathology laboratories has been reviewed to meet this demand and, in addition, to prepare the laboratories for the EU-IVD revised regulations (IVDR). This paper describes Belgian recommendations for the verification and validation of IHC tests before implementation, for ongoing validation, and for revalidation. For each type of test (according to the IVDR classification and the origin) and its intended use (purpose), it addresses how to perform analytical verification/validation by recommending: (1) the number of cases in the validation set, (2) the performance characteristics to be evaluated, (3) the objective acceptance criteria, (4) the evaluation method for the obtained results, and (5) how and when to revalidate. A literature study and a risk analysis taking into account the majority of variables regarding verification/validation of methods have been performed, resulting in an expert consensus recommendation that is a compromise among achievability, affordability, and patient safety. This new consensus recommendation has been incorporated in the aforementioned ISO 15189:2012-based Practice Guideline.

Semi-automated digital quantification of cellular infiltrates for in vivo evaluation of transplanted islets of Langerhans encapsulated with bioactive materials.

BACKGROUND: In the field of xenotransplantation, digital image analysis (DIA) is an asset to quantify heterogeneous cell infiltrates around transplanted encapsulated islets. MATERIALS AND METHODS: RGD-alginate was used to produce empty capsules or to encapsulate neonatal porcine islets (NPI) with different combinations of human pancreatic extracellular matrix (hpECM), porcine mesenchymal stem cells (pMSC) and a chitosan anti-fouling coating. Capsules were transplanted subcutaneously in rats for one month and then processed for immunohistochemistry. Immunostainings for macrophages (CD68) and lymphocytes (CD3) were quantified by DIA in two concentric regions of interest (ROI) around the capsules. DIA replicability and reproducibility were assessed by two blind operators. Repeatability was evaluated by processing the same biopsies at different time points. DIA was also compared with quantification by point counting (PC). RESULTS: Methodology validation: different sizes of ROIs were highly correlated. Intraclass correlation coefficients confirmed replicability and reproducibility. Repeatability showed a very strong correlation with CD3 stains and moderate/strong for CD68 stains. Group comparisons for CD68 IHC at each time point proved internal consistency. Point counting and DIA were strongly correlated with both CD3 and CD68. Capsule biocompatibility: Macrophage infiltration was higher around capsules containing biomaterials than around empty and RGD-alginate-NPI capsules. Lymphocytic infiltration was comparable among groups containing cells and higher than in empty capsules. CONCLUSION: We validated a semi-automated quantification methodology to assess cellular infiltrates and successfully applied it to investigate graft biocompatibility, showing that neonatal porcine islets encapsulated in alginate alone triggered less infiltration than capsules containing islets and bioactive materials.

The Impact of the COVID-19 Pandemic and the Associated Belgian Governmental Measures on Cancer Screening, Surgical Pathology and Cytopathology.

INTRODUCTION: The severe acute respiratory syndrome coronavirus 2 caused a pandemic of coronavirus disease 2019 (COVID-19). Unprecedented public health actions were introduced, including social distancing, travel restrictions and quarantine. The Belgian government announced a national emergency plan, thereby postponing all non-urgent medical consultations and operations. This report analyses the impact of these measures on cancer screening, through assessment of the workload of a laboratory for histopathology and cytopathology. METHODS: Data on monthly numbers of histological and cytological samples, immunohistochemistry and molecular tests were extracted from the laboratory information management system. RESULTS: The global histopathological and cytological workload was substantially reduced. The impact on oncology-related surgical procedures was rather limited. The anti-COVID-19 measures significantly diminished all screening-related samples, such as colon biopsies, breast biopsies and cervical cytology, and strongly reduced the number of samples related to "functional" pathology, such as thyroidectomies and gastric biopsies. CONCLUSIONS: Since many health care interventions are reflected in the workload of a pathology laboratory, this study enabled us to identify areas for "deconfinement" health care actions. Our findings indicate that various areas in medicine were affected, but the impact seemed largest for cancer screening. Health care professionals should assure that consultations related to cancer screening are postponed instead of cancelled.

Predictive markers for pathological complete response after neo-adjuvant chemotherapy in triple-negative breast cancer.

A combination of Sox10 and GATA3 was previously identified as a marker for metastatic triple-negative breast cancer (TNBC), but it is uncertain whether their expression is associated with pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). This study investigates the predictive value of clinicopathological characteristics, as well as protein expression of Sox10, GATA3, p53 and p63, in a consecutive series of TNBC patients treated with NAC. Archived hematoxylin & eosin stained slides of core biopsies and resection specimens from 35 TNBC patients were reviewed. The following clinicopathological characteristics were determined at the biopsy level: age at diagnosis, cancer type, Nottingham grade, lympho-vascular invasion, syncytial growth, necrosis, clear cell differentiation, myxoid peritumor stroma, stromal tumor-infiltrating lymphocytes (sTILs) and presence of an in situ component. The MD Anderson residual cancer burden (RCB) score and corresponding RCB class were determined. Immunohistochemistry for Sox10, p53, GATA3 and p63 was performed at the biopsy level. sTILs, either as a continuous or as a dichotomous variable, were the only parameter that was significantly associated with pCR in univariable and multivariable analyses. Assessment of sTILs showed moderate to good interobserver agreement. High sTILs (≥40%) were significantly associated with increased pCR rates, and this association was observer-independent. This retrospective study of a consecutive community-based cohort of TNBC patients confirms that sTILs are a robust, observer-independent predictor for therapeutic response after NAC. The combination of Sox10, GATA3 and p53 immunoreactivity is unlikely to harbor any predictive value for pCR in TNBC.

Haploid Germ Cells Generated in Organotypic Culture of Testicular Tissue From Prepubertal Boys.

While in mice various studies have described the completion of spermatogenesis in vitro using either organotypic culture of prepubertal testicular tissue or 3D culture of isolated cells, in humans it has not been possible to achieve germ cell differentiation from immature testicular tissue (ITT). In our study, we evaluated the ability of human ITT to differentiate via a long-term organotypic culture of frozen-thawed 1 mm3 testicular fragments from five prepubertal boys in two different culture media. Tissue and supernatants were analyzed at regular intervals up to day 139. Sertoli cell (SC) viability and maturation was evaluated using immunohistochemistry (IHC) for SOX9, GDNF, anti-Mullerian hormone (AMH) and androgen receptor (AR), and AMH concentration in supernatants. Spermatogonia (SG) and proliferating cells were identified by MAGE-A4 (for SG) and Ki67 (for proliferating cells) via immunohistochemistry (IHC). Apoptotic cells were studied by active caspase 3. To evaluate Leydig cell (LC) functionality testosterone was measured in the supernatants and steroidogenic acute regulatory protein (STAR) IHC was performed. Germ cell differentiation was evaluated on Hematoxylin-Eosin histological sections, via IHC for synaptonemal complex 3 (SYCP3) for spermatocytes, Protein boule-like (BOLL) for spermatocytes and round spermatids, angiotensin-converting enzyme (ACE), protamine 2 and transition protein 1 (for elongated spermatids) and via chromogenic in situ hybridization (CISH). We reported the generation of meiotic and postmeiotic cells after 16 days of culture, as shown by the histological analyses, the presence of differentiation markers and the increase of haploid germ cells. We showed SC viability and maturation by a decrease of AMH secretion in the supernatants (p ≤ 0.001) while the number of SOX9 positive cells did not show any variation. A decrease of spermatogonia (p ≤ 0.001) was observed. The number of apoptotic cells did not vary. LC functionality was shown by the increase in STAR expression (p ≤ 0.007) and a peak in testosterone secretion, followed by a reduction (p ≤ 0.001) with stabilization. According to our knowledge, this is the first report of generation of haploid cells in human ITT. Differentiating germ cells have to be further evaluated for their ability to complete differentiation, their fecundability and epigenetic characteristics.

Enhanced Vascular Biocompatibility and Remodeling of Decellularized and Secured Xenogeneic/Allogeneic Matrices in a Porcine Model.

BACKGROUND/PURPOSE: Calcifications and absence of growth potential are the major drawbacks of glutaraldehyde-treated prosthesis. Decellularized and secured xeno-/allogeneic matrices were assessed in a preclinical porcine model for biocompatibility and vascular remodeling in comparison to glutaraldehyde-fixed bovine pericardium (GBP; control). METHODS: Native human (fascia lata, pericardium) and porcine tissues (peritoneum) were used and treated. In vitro, biopsies were performed before and after treatment to assess decellularization (hematoxylin and eosin/DAPI). In vivo, each decellularized and control tissue sample was implanted subcutaneously in 4 mini-pigs. In addition, 9 mini-pigs received a patch or a tubularized prosthesis interposition on the carotid artery or abdominal aorta of decellularized (D) human fascia lata (DHFL; n = 4), human pericardium (DHP; n = 9), porcine peritoneum (DPPt; n = 7), and control tissue (GBP: n = 3). Arteries were harvested after 1 month and subcutaneous samples after 15-30 days. Tissues were processed for hematoxylin and eosin/von Kossa staining and immunohistochemistry for CD31, alpha-smooth muscle actin, CD3, and CD68. Histomorphometry was achieved by point counting. RESULTS: A 95% decellularization was confirmed for DHP and DPPt, and to a lower degree for DHFL. In the subcutaneous protocol, CD3 infiltration was significantly higher at day 30 in GBP and DHFL, and CD68 infiltration was significantly higher for GBP (p < 0.05). In intravascular study, no deaths, aneurysms, or pseudoaneurysms were observed. Inflammatory reaction was significantly higher for DHFL and GBP (p < 0.05), while it was lower and comparable for DHP/DPPt. DHP and DPPt showed deeper recellularization, and a new arterial wall was characterized. CONCLUSIONS: In a preclinical model, DPPt and DHP offered better results than conventional commercialized GBP for biocompatibility and vascular remodeling.

Porcine pulmonary valve decellularization with NaOH-based vs detergent process: preliminary in vitro and in vivo assessments.

BACKGROUND: Glutaraldehyde fixed xenogeneic heart valve prosthesis are hindered by calcification and lack of growth potential. The aim of tissue decellularization is to remove tissue antigenicity, avoiding the use of glutaraldehyde and improve valve integration with low inflammation and host cell recolonization. In this preliminary study, we investigated the efficacy of a NaOH-based process for decellularization and biocompatibility improvement of porcine pulmonary heart valves in comparison to a detergent-based process (SDS-SDC0, 5%). METHODS: Native cryopreserved porcine pulmonary heart valves were treated with detergent and NaOH-based processes. Decellularization was assessed by Hematoxylin and eosin/DAPI/alpha-gal/SLA-I staining and DNA quantification of native and processed leaflets, walls and muscles. Elongation stress test investigated mechanical integrity of leaflets and walls (n = 3 tests/valve component) of valves in the native and treated groups (n = 4/group). Biochemical integrity (collagen/elastin/glycosaminoglycans content) of leaflet-wall and muscle of the valves (n = 4/group) was assessed and compared between groups with trichrome staining (Sirius Red/Miller/Alcian blue). Secondly, a preliminary in vivo study assessed biocompatibility (CD3 and CD68 immunostaining) and remodeling (Hematoxylin and eosin/CD31 and ASMA immunofluorescent staining) of NaOH processed valves implanted in orthotopic position in young Landrace pigs, at 1 (n = 1) and 3 months (n = 2). RESULTS: Decellularization was better achieved with the NaOH-based process (92% vs 69% DNA reduction in the wall). Both treatments did not significantly alter mechanical properties. The detergent-based process induced a significant loss of glycosaminoglycans (p < 0,05). In vivo, explanted valves exhibited normal morphology without any sign of graft dilatation, degeneration or rejection. Low inflammation was noticed at one and three months follow-up (1,8 +/- 3,03 and 0,9836 +/- 1,3605 CD3 cells/0,12 mm2 in the leaflets). In one animal, at three months we documented minimal calcification in the area of sinus leaflet and in one, microthrombi formation on the leaflet surface at 1 month. The endoluminal side of the valves showed partial reendothelialization. CONCLUSIONS: NaOH-based process offers better porcine pulmonary valve decellularization than the detergent process. In vivo, the NaOH processed valves showed low inflammatory response at 3 months and partial recellularization. Regarding additional property of securing, this treatment should be considered for the new generation of heart valves prosthesis. Graphical abstract of the study.

Decellularized and Secured Porcine Arteries with NaOH-based Process: Proof of Concept.

BACKGROUND: There is a need for small caliber vascular prosthesis. Synthetic grafts are hindered by thrombogenicity and rapid occlusion. Decellularized matrices could be an alternative. We assessed in vitro and in vivo the biocompatibility of porcine artery treated with a chemical/physical process for decellularization and graft securitization with non/conventional pathogens inactivation. METHODS: Porcine carotid arteries (PCA) were treated. First, biopsies (n = 4/tissue) were performed before/after treatment to assess decellularization (hematoxylin and eosin/-4',6-diamidino-2-phenylindole/DNA/Miller). Second, 5 rats received an abdominal aortic patch of decellularized PCA (DPCA). Four pigs received subcutaneous DPCA implants (n = 2/pig). Half were explanted at day 15 and half at day 30. Finally, 2 pigs received DPCA (n = 2) and polytetrafluoroethylene prosthesis (n = 1), respectively, as carotid interposition. Implants were removed at day 30. Inflammation (CD3 and CD68 immunostaining) calcifications (von Kossa staining), remodeling (hematoxylin and eosin), and vascular characterization (CD31 and alpha-smooth muscle actin immunofluorescent staining) were investigated. RESULTS: Ninety-five percentage of decellularization was obtained without structural deterioration. No death occurred. Low inflammatory reaction was found in the 2 models for DPCA. Acquisition of vascular identity was confirmed in the rodent and porcine models. Similarity between native PCA and DPCA was observed after 30 days. In contrast, polytetrafluoroethylene graft showed severe calcifications, higher CD3 reaction, and higher intimal hyperplasia (P < 0.05). CONCLUSIONS: The physical and chemical process ensures decellularization of carotid porcine arteries and their in vivo remodeling with the presence of an endothelium and smooth-muscle-like cells as well as a low level of inflammatory cells.

Enhanced vascular regeneration with chemically/physically treated bovine/human pericardium in rodents.

BACKGROUND: Glutaraldehyde-treated pericardia for cardiovascular applications have poor long-term clinical results. The efficacy of a combined physical/chemical treatment to improve pericardium biocompatibility and vascular regeneration was assessed and compared with detergent treatment and two commercial bovine pericardia: PeriGuard (DGBP) and Edwards pericardium (nDGBP). The physical and chemical process was applied to bovine and human pericardia (DBP-DHP), and the detergent process was applied to bovine (DDBP). MATERIAL AND METHODS: Native (NBP) and treated bovine tissues were assessed for decellularization (HE/DAPI/DNA/α-Gal and MHC-1 staining) and mechanical integrity ex vivo. Twenty Wistar rats received subcutaneous patches of each bovine tissue to assess immunogenic response up to 4 months (flow cytometry). Ten additional rats received four subcutaneous bovine-treated patches (one/condition) to evaluate the inflammatory reaction (CD3/CD68 immunostaining), calcification (von Kossa staining/calcium quantification), and integration assessment (Hematoxylin and eosin staining). Finally, 15 rodents received a patch on the aorta (DBP n = 5, DHP n = 5, and DGBP n = 5), and vascular biocompatibility and arterial wall regeneration were assessed after 4 months (CD3/CD68/CD31/ASMA and Miller staining). RESULTS: DBP reached the higher level of decellularization, no immunogenic response whereas maintaining mechanical properties. DBP induced the lowest level grade of inflammation after 2 months (P < 0.05) concomitantly for better remodeling. No complications occurred with DBP and DHP where vascular regeneration was confirmed. Moreover, they induced a low level of CD3/CD68 infiltrations. CONCLUSIONS: This process significantly reduces immunogenicity and improves biocompatibility of bovine and human pericardia for better vascular regeneration.

Human Insulinomas Show Distinct Patterns of Insulin Secretion In Vitro.

Insulinomas are ß-cell tumors that cause hypoglycemia through inappropriate secretion of insulin. Characterization of the in vitro dynamics of insulin secretion by perifused fragments of 10 human insulinomas permitted their subdivision into three functional groups with similar insulin content. Group A (four patients with fasting and/or postprandial hypoglycemic episodes) showed qualitatively normal responses to glucose, leucine, diazoxide, tolbutamide, and extracellular CaCl2 omission or excess. The effect of glucose was concentration dependent, but, compared with normal islets, insulin secretion was excessive in both low- and high-glucose conditions. Group B (three patients with fasting hypoglycemic episodes) was mainly characterized by large insulin responses to 1 mmol/L glucose, resulting in very high basal secretion rates that were inhibited by diazoxide and restored by tolbutamide but were not further augmented by other agents except for high levels of CaCl2. Group C (three patients with fasting hypoglycemic episodes) displayed very low rates of insulin secretion and virtually no response to stimuli (including high CaCl2 concentration) and inhibitors (CaCl2 omission being paradoxically stimulatory). In group B, the presence of low-Km hexokinase-I in insulinoma ß-cells (not in adjacent islets) was revealed by immunohistochemistry. Human insulinomas thus show distinct, though not completely heterogeneous, defects in insulin secretion that are attributed to the undue expression of hexokinase-I in 3 of 10 patients.

Unambiguous detection of multiple TP53 gene mutations in AAN-associated urothelial cancer in Belgium using laser capture microdissection.

In the Balkan and Taiwan, the relationship between exposure to aristolochic acid and risk of urothelial neoplasms was inferred from the A>T genetic hallmark in TP53 gene from malignant cells. This study aimed to characterize the TP53 mutational spectrum in urothelial cancers consecutive to Aristolochic Acid Nephropathy in Belgium. Serial frozen tumor sections from female patients (n=5) exposed to aristolochic acid during weight-loss regimen were alternatively used either for p53 immunostaining or laser microdissection. Tissue areas with at least 60% p53-positive nuclei were selected for microdissecting sections according to p53-positive matching areas. All areas appeared to be carcinoma in situ. After DNA extraction, mutations in the TP53 hot spot region (exons 5-8) were identified using nested-PCR and sequencing. False-negative controls consisted in microdissecting fresh-frozen tumor tissues both from a patient with a Li-Fraumeni syndrome who carried a p53 constitutional mutation, and from KRas mutated adenocarcinomas. To rule out false-positive results potentially generated by microdissection and nested-PCR, a phenacetin-associated urothelial carcinoma and normal fresh ureteral tissues (n=4) were processed with high laser power. No unexpected results being identified, molecular analysis was pursued on malignant tissues, showing at least one mutation in all (six different mutations in two) patients, with 13/16 exonic (nonsense, 2; missense, 11) and 3/16 intronic (one splice site) mutations. They were distributed as transitions (n=7) or transversions (n=9), with an equal prevalence of A>T and G>T (3/16 each). While current results are in line with A>T prevalence previously reported in Balkan and Taiwan studies, they also demonstrate that multiple mutations in the TP53 hot spot region and a high frequency of G>T transversion appear as a complementary signature reflecting the toxicity of a cumulative dose of aristolochic acid ingested over a short period of time.

Mobilisation of lipophilic pollutants from blubber in northern elephant seal pups (Mirounga angustirostris) during the post-weaning fast.

Northern elephant seals (NES) (Mirounga angustirostris) from the Año Nuevo State Reserve (CA, USA) were longitudinally sampled during the post-weaning fast in order to study the mobilisation and redistribution of various classes of persistent organic pollutants (POPs), such as polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), dichlorodiphenyldichloroethylene (p,p'-DDE) and hexachlorobenzene (HCB) between blubber and blood. Inner and outer blubber layers were analysed separately. Organohalogenated compounds were detected in all blubber samples in the decreasing order of their concentrations: p,p'-DDE > PCBs ⪢ HCB > PBDEs. The concentrations of all studied compounds were homogeneously distributed in the blubber layer at early fast, since the concentrations of POPs were statistically not different in the inner and outer layers. With the progression of the fast, the concentrations of PBDEs, PCBs and p,p'-DDE increased more sharply in inner blubber than in outer blubber. As a result, their levels became significantly higher in inner blubber as compared to outer blubber at late fast. The rise of pollutant concentrations in blubber might result from a less efficient mobilisation than triglycerides and/or a reuptake by adipocytes of some of the pollutants released into the circulation. The mobilisation of pollutants from blubber was higher at late fast. An increase of pollutant concentrations was observed in serum between early and late fast. Lower halogenated congeners (i.e. tetra-CBs) were present in higher proportions in serum, whereas the higher halogenated congeners (i.e. hepta-CBs) were mainly found in the inner and outer blubber layers. The transfer ratios of both PBDEs and PCBs from inner blubber to serum decreased with the number of chlorine and bromine atoms. In addition, the distribution of both types of compounds between serum and blubber was strongly influenced by their lipophilic character (logKow values), with more lipophilic compounds being less efficiently released from blubber to serum.

Time course of pathogenic and adaptation mechanisms in cystinotic mouse kidneys.

Cystinosis, a main cause of Fanconi syndrome, is reproduced in congenic C57BL/6 cystinosin knockout (KO) mice. To identify the sequence of pathogenic and adaptation mechanisms of nephropathic cystinosis, we defined the onset of Fanconi syndrome in KO mice between 3 and 6 months of age and analyzed the correlation with structural and functional changes in proximal tubular cells (PTCs), with focus on endocytosis of ultrafiltrated disulfide-rich proteins as a key source of cystine. Despite considerable variation between mice at the same age, typical event sequences were delineated. At the cellular level, amorphous lysosomal inclusions preceded cystine crystals and eventual atrophy without crystals. At the nephron level, lesions started at the glomerulotubular junction and then extended distally. In situ hybridization and immunofluorescence revealed progressive loss of expression of megalin, cubilin, sodium-glucose cotransporter 2, and type IIa sodium-dependent phosphate cotransporter, suggesting apical dedifferentiation accounting for Fanconi syndrome before atrophy. Injection of labeled proteins revealed that defective endocytosis in S1 PTCs led to partial compensatory uptake by S3 PTCs, suggesting displacement of endocytic load and injury by disulfide-rich cargo. Increased PTC apoptosis allowed luminal shedding of cystine crystals and was partially compensated for by tubular proliferation. We conclude that lysosomal storage triggered by soluble cystine accumulation induces apical PTC dedifferentiation, which causes transfer of the harmful load of disulfide-rich proteins to more distal cells, possibly explaining longitudinal progression of swan-neck lesions. Furthermore, our results suggest that subsequent adaptation mechanisms include lysosomal clearance of free and crystalline cystine into urine and ongoing tissue repair.

Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity.

Obesity and type 2 diabetes are characterized by altered gut microbiota, inflammation, and gut barrier disruption. Microbial composition and the mechanisms of interaction with the host that affect gut barrier function during obesity and type 2 diabetes have not been elucidated. We recently isolated Akkermansia muciniphila, which is a mucin-degrading bacterium that resides in the mucus layer. The presence of this bacterium inversely correlates with body weight in rodents and humans. However, the precise physiological roles played by this bacterium during obesity and metabolic disorders are unknown. This study demonstrated that the abundance of A. muciniphila decreased in obese and type 2 diabetic mice. We also observed that prebiotic feeding normalized A. muciniphila abundance, which correlated with an improved metabolic profile. In addition, we demonstrated that A. muciniphila treatment reversed high-fat diet-induced metabolic disorders, including fat-mass gain, metabolic endotoxemia, adipose tissue inflammation, and insulin resistance. A. muciniphila administration increased the intestinal levels of endocannabinoids that control inflammation, the gut barrier, and gut peptide secretion. Finally, we demonstrated that all these effects required viable A. muciniphila because treatment with heat-killed cells did not improve the metabolic profile or the mucus layer thickness. In summary, this study provides substantial insight into the intricate mechanisms of bacterial (i.e., A. muciniphila) regulation of the cross-talk between the host and gut microbiota. These results also provide a rationale for the development of a treatment that uses this human mucus colonizer for the prevention or treatment of obesity and its associated metabolic disorders.

Congenital hyperinsulinism caused by hexokinase I expression or glucokinase-activating mutation in a subset of ß-cells.

Congenital hyperinsulinism causes persistent hypoglycemia in neonates and infants. Most often, uncontrolled insulin secretion (IS) results from a lack of functional K(ATP) channels in all ß-cells or only in ß-cells within a resectable focal lesion. In more rare cases, without K(ATP) channel mutations, hyperfunctional islets are confined within few lobules, whereas hypofunctional islets are present throughout the pancreas. They also can be cured by selective partial pancreatectomy; however, unlike those with a K(ATP) focal lesion, they show clinical sensitivity to diazoxide. Here, we characterized in vitro IS by fragments of pathological and adjacent normal pancreas from six such cases. Responses of normal pancreas were unremarkable. In pathological region, IS was elevated at 1 mmol/L and was further increased by 15 mmol/L glucose. Diazoxide suppressed IS and tolbutamide antagonized the inhibition. The most conspicuous anomaly was a large stimulation of IS by 1 mmol/L glucose. In five of six cases, immunohistochemistry revealed undue presence of low-K(m) hexokinase-I in ß-cells of hyperfunctional islets only. In one case, an activating mutation of glucokinase (I211F) was found in pathological islets only. Both abnormalities, attributed to somatic genetic events, may account for inappropriate IS at low glucose levels by a subset of ß-cells. They represent a novel cause of focal congenital hyperinsulinism.

Lanreotide treatment of metastatic hepatocellular carcinoma resulting in partial regression and more than 3 years of progression-free survival.

We describe the case of a 54 years old woman, with hepatitis B, in whom the diagnosis of a 6 cm hepatocellular carcinoma (HCC) in the left liver was made in 2001. Alpha-foeto-protein (AFP) was 63 ng/mL (NI < 10 ng/mL). After work-up including liver and tumor biopsy confirming HCC and only fibrosis in the nontumoral liver, left hepatectomy was performed. Final pathology showed a well differentiated HCC with tumoral portal vein thrombosis. Unfortunately, lung and mediastinal adenopathies were detected by CT scan 17 months later. Mediastinal nodes were punctured by endosonographic ultrasound, confirming HCC. The patient started treatment with Lanreotide 30 mg twice a month (Somatuline PR, Ipsen). Three months later, CT showed decrease in size of the mediastinal nodes and complete disappearance of the lung nodes. This objective response lasted for 42 months. The treatment was without any significant side effect. Retrospectively, immunohistochemistry was performed to detect somatostatine receptors (sstr) 2. Both the primary tumor and the node showed intense membranous and cytoplasmic staining for sstr2. In 2006, AFP rose and CT showed the appearance of a new mediastinal node. At that time, octreoscan was performed and showed uptake in the new node, although insufficient for metabolic radiotherapy. This case suggests that, although a number of randomized controlled trials did not show a benefit of somatostatin analogues in the treatment of advanced HCC, a subset of patients could benefit from treatment provided their tumor expresses sstr2, on which the existing drugs are efficient.

Glucose-induced O2 consumption activates hypoxia inducible factors 1 and 2 in rat insulin-secreting pancreatic beta-cells.

BACKGROUND: Glucose increases the expression of glycolytic enzymes and other hypoxia-response genes in pancreatic beta-cells. Here, we tested whether this effect results from the activation of Hypoxia-Inducible-factors (HIF) 1 and 2 in a hypoxia-dependent manner. METHODOLOGY/PRINCIPAL FINDINGS: Isolated rat islets and insulin-secreting INS-1E cells were stimulated with nutrients at various pO2 values or treated with the HIF activator CoCl2. HIF-target gene mRNA levels and HIF subunit protein levels were measured by real-time RT-PCR, Western Blot and immunohistochemistry. The formation of pimonidazole-protein adducts was used as an indicator of hypoxia. In INS-1E and islet beta-cells, glucose concentration-dependently stimulated formation of pimonidazole-protein adducts, HIF1 and HIF2 nuclear expression and HIF-target gene mRNA levels to a lesser extent than CoCl2 or a four-fold reduction in pO2. Islets also showed signs of HIF activation in diabetic Lepr(db/db) but not non-diabetic Lepr(db/+) mice. In vitro, these glucose effects were reproduced by nutrient secretagogues that bypass glycolysis, and were inhibited by a three-fold increase in pO2 or by inhibitors of Ca²âº influx and insulin secretion. In INS-1E cells, small interfering RNA-mediated knockdown of Hif1α and Hif2α, alone or in combination, indicated that the stimulation of glycolytic enzyme mRNA levels depended on both HIF isoforms while the vasodilating peptide adrenomedullin was a HIF2-specific target gene. CONCLUSIONS/SIGNIFICANCE: Glucose-induced O2 consumption creates an intracellular hypoxia that activates HIF1 and HIF2 in rat beta-cells, and this glucose effect contributes, together with the activation of other transcription factors, to the glucose stimulation of expression of some glycolytic enzymes and other hypoxia response genes.

In vitro insulin secretion by pancreatic tissue from infants with diazoxide-resistant congenital hyperinsulinism deviates from model predictions.

Congenital hyperinsulinism (CHI) is the major cause of persistent neonatal hypoglycemia. CHI most often occurs due to mutations in the ABCC8 (which encodes sulfonylurea receptor 1) or KCNJ11 (which encodes the potassium channel Kir6.2) gene, which result in a lack of functional KATP channels in pancreatic ß cells. Diffuse forms of CHI (DiCHI), in which all ß cells are abnormal, often require subtotal pancreatectomy, whereas focal forms (FoCHI), which are characterized by localized hyperplasia of abnormal ß cells, can be cured by resection of the lesion. Here, we characterized the in vitro kinetics of insulin secretion by pancreatic fragments from 6 DiCHI patients and by focal lesion and normal adjacent pancreas from 18 FoCHI patients. Responses of normal pancreas were similar to those reported for islets from adult organ donors. Compared with normal pancreas, basal insulin secretion was elevated in both FoCHI and DiCHI tissue. Affected tissues were heterogeneous in their secretory responses, with increased glucose levels often producing a rapid increase in insulin secretion that could be followed by a paradoxical decrease below prestimulatory levels. The KATP channel blocker tolbutamide was consistently ineffective in stimulating insulin secretion; conversely, the KATP channel activator diazoxide often caused an unanticipated increase in insulin secretion. These observed alterations in secretory behavior were similar in focal lesion and DiCHI tissue, and independent of the specific mutation in ABCC8 or KCNJ11. They cannot be explained by classic models of ß cell function. Our results provide insight into the excessive and sometimes paradoxical changes in insulin secretion observed in CHI patients with inactivating mutations of KATP channels.

Morphologic analysis of focal and diffuse forms of congenital hyperinsulinism.

Congenital hyperinsulinism is clinically characterized by an inappropriate insulin secretion resulting in recurrent severe hypoglycemia. Nesidioblastosis, the proliferation of islet cells budding off from ducts, has been considered for years as the histologic lesion responsible for the syndrome. In our morphologic studies, we demonstrate that nesidioblastosis is not specific of the disease, which is actually not a single entity. Indeed, we recognize the existence of 2 different forms-a diffuse form and a focal form-and demonstrate that they can be differentiated by morphologic criteria, even on frozen sections during surgery. This histologic distinction directs the therapeutic approach because the patients experiencing the focal form of the syndrome can be completely cured by a very limited pancreatectomy. Molecular findings confirmed the reliability of this histologic distinction, showing a specific background for each form.

Endoplasmic reticulum accumulation of Kir6.2 without activation of ER stress response in islet cells from adult Sur1 knockout mice.

Trafficking of pancreatic K(ATP) channels to the plasma membrane critically depends on masking the endoplasmic reticulum (ER) retention signals of the SUR1 and Kir6.2 subunits upon their proper assembly into functional hetero-octamers. When expressed in the absence of the partner protein, each subunit might accumulate in the ER and trigger beta-cell ER stress and oxidative stress. To test this hypothesis, Kir6.2 localisation, ER ultra-structure and ER-stress- and oxidative-stress-response gene mRNA levels were evaluated in pancreatic endocrine cells from adult wild-type (WT) and Sur1 knockout (Sur1 ( -/- )) mice. As previously reported, Kir6.2 was mainly expressed on secretory granules and at the plasma membrane of WT islet cells. In contrast, like the ER chaperone calreticulin, Kir6.2 was primarily localised in the rough endoplasmic reticulum (RER) of Sur1 ( -/- ) islet cells. ER retention of Kir6.2 was demonstrated (electron microscopy) by a significant increase in the length and Kir6.2 density of RER in Sur1 ( -/- ) vs WT islet cells. Despite Kir6.2 retention in RER, Xbp1 mRNA splicing and mRNA levels of preproinsulin and ER-stress-response genes Bip, Edem and Gadd153 were similar in WT and Sur1 ( -/- ) islets. However, mRNA levels of the antioxidant enzymes Sod1, Sod2, Gpx2 and catalase were significantly up-regulated in Sur1 ( -/- ) islets. Sequestration of Kir6.2 in RER of Sur1 ( -/- ) islet cells is thus associated with an increase in RER length and mild oxidative stress without activation of the classical ER stress response.