ß-Catenin-Driven Differentiation Is a Tissue-Specific Epigenetic Vulnerability in Adrenal Cancer.

Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent ß-catenin-activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific ß-catenin-containing complexes, and the epigenome. On chromatin, ß-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, ß-catenin bound histone methyltransferase EZH2. SF1/ß-catenin and EZH2/ß-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/ß-catenin from chromatin and favored EZH2/ß-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities. SIGNIFICANCE: Oncogenic ß-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for ß-catenin-driven cancers.

B cell subsets in thymus from infants with Down syndrome

ABSTRACT: B lymphocytes are still poorly characterized in the human thymus, and due to the immunological peculiarities of Down syndrome, a detailed investigation of thymic B lymphocytes and their subsets was performed in <2-year-old infants. This is a period of intense thymic activity, and here we compared thymuses from infants with Down syndrome (DG) and age-matched control group (CG) with heart disease as the only described malformation, no clinical signs of immunodeficiency and normal blood lymphocyte numbers. METHODS: This study, approved by the institutional ethics committee, included 10 thymic samples from DG infants, and 20 from CG infants. Depending on thymus size and the type of surgical approach, babies were submitted to total thymectomy or only the removal of a small thymic fragment (mainly in the DG), always during cardiac surgery in order to correct congenital defects. Flow cytometry was used for immunophenotyping of cell suspension obtained from thymic tissue; and the B lymphocyte topographic localization was investigated by immunohistochemistry, and a quantitative evaluation of B-cells in each region was carried out using methods of digitized images analysis. RESULTS: Flow cytometry revealed that CD20+ cells represent around 1% of total lymphocytes in both groups, with higher naive and lower unswitched memory B lymphocyte frequencies in DG when compared to the CG, while B1 cells were present in much higher frequency in DG than in CG. All the other B-cell subpopulations (transitional, switched memory, plasmablasts and plasmocytes) were statistically equivalent between groups. Immunohistochemistry showed higher frequencies of CD20+ cells in medullary region compared to cortex, without differences between the groups (DG = 16.8% and CG = 17.1% of total cell number), while the cortex region had few B cells in both groups, with significantly lower frequency in DG group, reaching 0.25%, compared with the CG, which showed 0.6%. CONCLUSION: Flow cytometry revealed a different distribution of B-cell subtypes in DG thymus compared to CG and, thus, data obtained from such rare material could be relevant in the characterization and further analysis and understanding of the immunological mechanisms that involve the participation of B lymphocyte subpopulations in the thymus. FAPESP (grant 2014/50489-9).

Intra-individual Variability of Serum Aldosterone and Implications for Primary Aldosteronism Screening.

CONTEXT: Primary aldosteronism (PA) screening relies on an elevated aldosterone to renin ratio with a minimum aldosterone level, which varies from 10 to 15 ng/dL (277-415.5 pmol/L) using immunoassay. OBJECTIVE: To evaluate intra-individual coefficient of variation (CV) of aldosterone and aldosterone to direct renin concentration ratio (A/DRC) and its impact on PA screening. METHODS: A total of 671 aldosterone and DRC measurements were performed by the same chemiluminescence assays in a large cohort of 216 patients with confirmed PA and at least 2 screenings. RESULTS: The median intra-individual CV of aldosterone and A/DRC was 26.8% and 26.7%. Almost 40% of the patients had at least one aldosterone level <15 ng/dL, 19.9% had at least 2 aldosterone levels <15 ng/dL, and 16.2% had mean aldosterone levels <15 ng/dL. A lower cutoff of 10 ng/dL was associated with false negative rates for PA screening of 14.3% for a single aldosterone measurement, 4.6% for 2 aldosterone measurements, and only 2.3% for mean aldosterone levels. Considering the minimum aldosterone, true positive rate of aldosterone thresholds was 85.7% for 10 ng/dL and 61.6% for 15 ng/dL. An A/DRC >2 ng/dL/µIU/mL had a true positive rate for PA diagnosis of 94.4% and 98.4% when based on 1 or 2 assessments, respectively. CV of aldosterone and A/DRC were not affected by sex, use of interfering antihypertensive medications, PA lateralization, hypokalemia, age, and number of hormone measurements. CONCLUSION: Aldosterone concentrations had a high CV in PA patients, which results in an elevated rate of false negatives in a single screening for PA. Therefore, PA screening should be based on at least 2 screenings with concomitant aldosterone and renin measurements.

Core Needle Biopsy in Lymphoma Diagnosis: The Diagnostic Performance and the Role of the Multidisciplinary Approach in the Optimization of Results.

To compare the diagnostic accuracy of core needle biopsies (CNBs) and surgical excisional biopsies (SEBs), samples of lymphoid proliferation from a single institution from 2013 to 2017 (N=476) were divided into groups of CNB (N=218) and SEB (N=258). The diagnostic accuracy of these samples was evaluated as a percentage of conclusive diagnosis, according to the World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues . The contribution of clinical data, the assessment of sample adequacy by a pathologist during the procedure, the number and size of fragments, the needle gauge, the ancillary tests, and the type of lymphoid proliferation were also examined. The diagnostic accuracy of SEB was 97.3% and CNB 91.3% ( P =0.010). Additional factors considered essential for establishing the final diagnosis in some cases were: clinical information (20.6% CNB, 7.4% SEB; P <0.001); immunohistochemistry (96.3% CNB, 91.5% SEB; P =0.024); flow cytometry (12% CNB, 6.8% SEB; P =0.165); and other complementary tests (8.2% CNB, 17.3% SEB; P =0.058). Factors that did not influence performance were the evaluation of sample adequacy during the procedure, the number and size of fragments, and the needle gauge. Increased percentage of nondiagnostic CNB was observed in T-cell lymphomas (30%), followed by classic Hodgkin lymphoma (10.6%). The main limitation of CNB was the evaluation of morphologically heterogenous diseases. CNB is useful and safe in lymphoma diagnosis provided it is carried out by a team of experienced professionals. Having an interventional radiology team engaged with pathology is an essential component to achieve adequate rates of specific diagnoses in CNB specimens.

Retrospective Analysis of Prognostic Factors in Pediatric Patients with Adrenocortical Tumor from Unique Tertiary Center with Long-Term Follow-Up.

Pediatric adrenocortical tumors (PACTs) represent rare causes of malignancies. However, the south/southeast regions of Brazil are known to have a high incidence of PACTs because of the founder effect associated with a germline pathogenic variant of tumor suppressor gene TP53. We aimed to retrospectively analyze the types of variables among hormone production, radiological imaging, tumor staging, histological and genetic features that were associated with the occurrence of malignancy in 95 patients (71% females) with PACTs from a unique center. The worst prognosis was associated with those aged > 3 years (p < 0.05), high serum levels of 11-desoxicortisol (p < 0.001), tumor weight ≥ 200 g (p < 0.001), tumor size ≥ 5 cm (p < 0.05), Weiss score ≥ 5 (p < 0.05), Wieneke index ≥ 3 (p < 0.001) and Ki67 ≥ 15% (p < 0.05). Furthermore, patients with MacFarlane stage IV had an overall survival rate almost two times shorter than patients with other stages (p < 0.001). Additionally, the subtractions of BUB1B-PINK1 (<6.95) expression (p < 0.05) and IGF-IR overexpression (p = 0.0001) were associated with malignant behavior. These results helped identify patients who are likely to have an aggressive course; further multicenter prospective studies are required to confirm our results. In conclusion, PACTs with these patterns of prognostic factors could be treated using an adjuvant approach that may improve the overall survival in such patients.

Genetic and clinical aspects of paediatric pheochromocytomas and paragangliomas.

OBJECTIVE: Few and conflicting reports have characterized the genetics of paediatric pheochromocytomas and paragangliomas (PPGLs). This study aimed to investigate the clinical and genetic features of Brazilian children with PPGL. PATIENTS AND METHODS: This study included 25 children (52% girls) with PPGL. The median age at diagnosis was 15 years (4-19). The median time of follow-up was 145 months. The genetic investigation was performed by Sanger DNA sequencing, multiplex ligation-dependent probe amplification and/or target next-generation sequencing panel. RESULTS: Of the 25 children with PPGL, 11 (44%), 4 (16%), 2 (8%), 1 (4%) and 7 (28%) had germline VHL pathogenic variants, SDHB, SDHD, RET and negative genetic investigation, respectively. Children with germline VHL missense pathogenic variants were younger than those with SDHB or SDHD genetic defects [median (range), 12 (4-16) vs. 15.5 (14-19) years; P = .027]. Moreover, 10 of 11 cases with VHL pathogenic variants had bilateral pheochromocytoma (six asynchronous and four synchronous). All children with germline SDHB pathogenic variants presented with abdominal paraganglioma (one of them malignant). The two cases with SDHD pathogenic variants presented with head and neck paraganglioma. Among the cases without a genetic diagnosis, 6 and 2 had pheochromocytoma and paraganglioma, respectively. Furthermore, metastatic PPGL was diagnosed in four (16%) of 25 PPGL. CONCLUSIONS: Most of the paediatric PPGL were hereditary and multifocal. The majority of the affected genes belong to pseudohypoxic cluster 1, with VHL being the most frequently mutated. Therefore, our findings impact surgical management and surveillance of children with PPGL.

Clinical Impact of Pathological Features Including the Ki-67 Labeling Index on Diagnosis and Prognosis of Adult and Pediatric Adrenocortical Tumors.

Adrenocortical tumors (ACT) in the adult and pediatric population are generally considered distinct entities due to differences in molecular events related to tumorigenesis, clinical presentation, and outcome. Furthermore, pathological criteria used for diagnosis and prognostication of ACT in adults are usually inadequate for predicting the biological behavior of ACT in children. Here, we analyzed 146 adult and 44 pediatric (< 15y/o) ACT with long-term clinical follow-up and furthered current evidence on the clinical and pathological differences between pediatric and adult tumors. Predilection for female over male gender was observed in both cohorts, but more so in adults (84% vs. 61%, p = 0.003). Cushing syndrome was more frequent in adults (p < 0.001), whereas virilization, either isolated (p < 0.001) or combined to Cushing (p = 0.047), was more common in children. The Ki67 labelling index (LI) of pediatric adenomas and carcinomas was much higher than their corresponding tumors in adults (p < 0.001). Despite these differences, pathological analyses including the evaluation of Ki67 greatly improved patient prognostication in both age cohorts. Indeed, increased Weiss scores and Ki67 indexes correlated with poor overall- and disease-free survival in adult patients with carcinoma. Among the proliferative indexes tested, Ki67 LI ≥ 10% showed the highest hazard ratio (HR) for recurrence and the Ki67 LI ≥ 3% showed the highest HR for survival. In pediatric tumors, the Wieneke score (p < 0.001) and the Ki67 LI (p < 0.001) showed high accuracy for predicting biological behavior, and increased scores/indexes correlated with worse overall and disease-free survival. In this age cohort, Ki67 LI < 10% was able to rule out malignant behavior, whereas Ki67 LI ≥ 15% may be used to predict the patients with higher risks of recurrence and/or poor outcome.

Phosphodiesterase 2A and 3B variants are associated with primary aldosteronism.

Familial primary aldosteronism (PA) is rare and mostly diagnosed in early-onset hypertension (HT). However, 'sporadic' bilateral adrenal hyperplasia (BAH) is the most frequent cause of PA and remains without genetic etiology in most cases. Our aim was to investigate new genetic defects associated with BAH and PA. We performed whole-exome sequencing (paired blood and adrenal tissue) in six patients with PA caused by BAH that underwent unilateral adrenalectomy. Additionally, we conducted functional studies in adrenal hyperplastic tissue and transfected cells to confirm the pathogenicity of the identified genetic variants. Rare germline variants in phosphodiesterase 2A (PDE2A) and 3B (PDE3B) genes were identified in three patients. The PDE2A heterozygous variant (p.Ile629Val) was identified in a patient with BAH and early-onset HT at 13 years of age. Two PDE3B heterozygous variants (p.Arg217Gln and p.Gly392Val) were identified in patients with BAH and HT diagnosed at 18 and 33 years of age, respectively. A strong PDE2A staining was found in all cases of BAH in zona glomerulosa and/or micronodules (that were also positive for CYP11B2). PKA activity in frozen tissue was significantly higher in BAH from patients harboring PDE2A and PDE3B variants. PDE2A and PDE3B variants significantly reduced protein expression in mutant transfected cells compared to WT. Interestingly, PDE2A and PDE3B variants increased SGK1 and SCNN1G/ENaCg at mRNA or protein levels. In conclusion, PDE2A and PDE3B variants were associated with PA caused by BAH. These novel genetic findings expand the spectrum of genetic etiologies of PA.

Clinical impact of dysplastic changes in acquired aplastic anemia: A systematic study of bone marrow biopsies in children and adults.

Aplastic anemia (AA) is a rare disorder characterized by suppression of bone marrow function, which can progress to myelodysplastic syndrome (MDS) or to acute myeloid leukemia (AML). To determine if there are characteristics in bone marrow biopsies in children and adults previously diagnosed with acquired AA, which could predict progression to MDS, we evaluated 118 hypocellular bone marrow biopsies from adults (76 patients) and children (42) diagnosed initially with acquired AA previously to any treatment. Histology was reviewed according to a detailed protocol including Bennett and Orazi criteria for hypocellular myelodysplastic syndrome (h-MDS) and Bauman et al. criteria for refractory cytopenia of childhood (RCC). Twelve patients (10.2%; 6 children and 6 adults) progressed to MDS after a median time of 56 months. Criteria described by Bennett and Orazi suggestive of h-MDS in bone marrow biopsies were detected in 16 cases (13.5%; 8 adults and 8 children), and none in patients that progressed to MDS/AML. Twenty adults' biopsies (26.3%) had the histological criteria used for the diagnosis of pediatric RCC, and none showed MDS/AML evolution. Ten children (23.8%) were reclassified morphologically as RCC, and only one progressed to MDS. In this population with acquired aplastic anemia (AAA), no histological/immunohistochemical (H/IHC) bone marrow findings could discriminate patients with higher risk for myeloid clonal progression, which questions the diagnosis of h-MDS/RCC based only on the finding of dysplasia in the cases without increased blasts and/or the characteristic genetic abnormalities.

New Insights Into Pheochromocytoma Surveillance of Young Patients With VHL Missense Mutations.

CONTEXT: Von Hippel-Lindau (VHL) disease is an autosomal dominant syndrome caused by germline mutations in the VHL gene. Guidelines recommend pheochromocytoma (PHEO) biochemical screening should start at age 5 years. OBJECTIVE: Genotype-phenotype correlations in VHL, focusing on PHEO penetrance in children, were studied. DESIGN: We retrospectively evaluated 31 individuals (median age at diagnosis was 26 years) with diagnosed VHL disease. RESULTS: PHEO was diagnosed in six children with VHL. A large PHEO (5 cm) was detected in a 4-year-old boy with p.Gly114Ser mutation. PHEO penetrance was 55% starting at age 4 years. VHL missense mutations were identified in 11 of 22 families (50%), frameshift mutations in four (18.2%), stop codon in three (13.6%), splicing site in two (9.1%), and large gene deletion in two (9.1%). The codon 167 (n = 10) was a hotspot for VHL mutations and was significantly associated with PHEO (90% vs. 38%; P = 0.007). PHEOs and pancreatic neuroendocrine tumors (PNETs) were strongly associated with VHL missense mutations compared with other mutations (89.5% vs. 0% and 73.7% vs. 16.7%; P = 0.0001 and 0.002, respectively). In contrast, pancreatic cysts (91.7% vs. 26.3%; P = 0.0001), renal cysts (66.7% vs. 26.3%; P = 0.027), and central nervous system hemangioblastomas (91.7% vs. 47.3%; P = 0.012) were more frequent in VHL with nonmissense mutations. CONCLUSION: VHL missense mutations were highly associated with PHEO and PNETs. Our data support that in children with VHL harboring missense mutations, biochemical screening for PHEO should be initiated at diagnosis.

KCNJ5 Somatic Mutation Is a Predictor of Hypertension Remission After Adrenalectomy for Unilateral Primary Aldosteronism.

CONTEXT: Primary aldosteronism (PA) is the most common cause of endocrine hypertension (HT). HT remission (defined as blood pressure <140/90 mm Hg without antihypertensive drugs) has been reported in approximately 50% of patients with unilateral PA after adrenalectomy. HT duration and severity are predictors of blood pressure response, but the prognostic role of somatic KCNJ5 mutations is unclear. OBJECTIVE: To determine clinical and molecular features associated with HT remission after adrenalectomy in patients with unilateral PA. METHODS: We retrospectively evaluated 100 patients with PA (60 women; median age at diagnosis 48 years with a median follow-up of 26 months). Anatomopathological analysis revealed 90 aldosterone-producing adenomas, 1 carcinoma, and 9 unilateral adrenal hyperplasias. All patients had biochemical cure after unilateral adrenalectomy. KCNJ5 gene was sequenced in 76 cases. RESULTS: KCNJ5 mutations were identified in 33 of 76 (43.4%) tumors: p.Gly151Arg (n = 17), p.Leu168Arg (n = 15), and p.Glu145Gln (n = 1). HT remission was reported in 37 of 100 (37%) patients. Among patients with HT remission, 73% were women (P = 0.04), 48.6% used more than three antihypertensive medications (P = 0.0001), and 64.9% had HT duration <10 years (P = 0.0015) compared with those without HT remission. Somatic KCNJ5 mutations were associated with female sex (P = 0.004), larger nodules (P = 0.001), and HT remission (P = 0.0001). In multivariate analysis, only a somatic KCNJ5 mutation was an independent predictor of HT remission after adrenalectomy (P = 0.004). CONCLUSION: The presence of a KCNJ5 somatic mutation is an independent predictor of HT remission after unilateral adrenalectomy in patients with unilateral PA.

Targeted Assessment of G0S2 Methylation Identifies a Rapidly Recurrent, Routinely Fatal Molecular Subtype of Adrenocortical Carcinoma.

PURPOSE: Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy with few therapies; however, patients with locoregional disease have variable outcomes. The Cancer Genome Atlas project on ACC (ACC-TCGA) identified that cancers of patients with homogeneously rapidly recurrent or fatal disease bear a unique CpG island hypermethylation phenotype, "CIMP-high." We sought to identify a biomarker that faithfully captures this subgroup.Experimental Design: We analyzed ACC-TCGA data to characterize differentially regulated biological processes, and identify a biomarker that is methylated and silenced exclusively in CIMP-high ACC. In an independent cohort of 114 adrenocortical tumors (80 treatment-naive primary ACC, 22 adrenocortical adenomas, and 12 non-naive/nonprimary ACC), we evaluated biomarker methylation by a restriction digest/qPCR-based approach, validated by targeted bisulfite sequencing. We evaluated expression of this biomarker and additional prognostic markers by qPCR. RESULTS: We show that CIMP-high ACC is characterized by upregulation of cell cycle and DNA damage response programs, and identify that hypermethylation and silencing of G0S2 distinguishes this subgroup. We confirmed G0S2 hypermethylation and silencing is exclusive to 40% of ACC, and independently predicts shorter disease-free and overall survival (median 14 and 17 months, respectively). Finally, G0S2 methylation combined with validated molecular markers (BUB1B-PINK1) stratifies ACC into three groups, with uniformly favorable, intermediate, and uniformly dismal outcomes. CONCLUSIONS: G0S2 hypermethylation is a hallmark of rapidly recurrent or fatal ACC, amenable to targeted assessment using routine molecular diagnostics. Assessing G0S2 methylation is straightforward, feasible for clinical decision-making, and will enable the direction of efficacious adjuvant therapies for patients with aggressive ACC.

Targeting the polarization of tumor-associated macrophages and modulating mir-155 expression might be a new approach to treat diffuse large B-cell lymphoma of the elderly.

Aging immune deterioration and Epstein-Barr (EBV) intrinsic mechanisms play an essential role in EBV-positive diffuse large B-cell lymphoma (DLBCL) of the elderly (EBV + DLBCLe) pathogenesis, through the expression of viral proteins, interaction with host molecules and epigenetic regulation, such as miR-155, required for induction of M1 phenotype of macrophages. This study aims to evaluate the relationship between macrophage polarization pattern in the tumor microenvironment and relative expression of miR-155 in EBV + DLBCLe and EBV-negative DLBCL patients. We studied 28 EBV + DLBCLe and 65 EBV-negative DLBCL patients. Tumor-associated macrophages (TAM) were evaluated by expression of CD68, CD163 and CD163/CD68 ratio (degree of M2 polarization), using tissue microarray. RNA was extracted from paraffin-embedded tumor samples for miR-155 relative expression study. We found a significantly higher CD163/CD68 ratio in EBV + DLBCLe compared to EBV-negative DLBCL. In EBV-negative DLBCL, CD163/CD68 ratio was higher among advanced-staged/high-tumor burden disease and overexpression of miR-155 was associated with decreased polarization to the M2 phenotype of macrophages. The opposite was observed in EBV + DLBCLe patients: we found a positive association between miR-155 relative expression and CD163/CD68 ratio, which was not significant after outlier exclusion. We believe that the higher CD163/CD68 ratio in this group is probably due to the presence of the EBV since it directly affects macrophage polarization towards M2 phenotype through cytokine secretion in the tumor microenvironment. Therapeutic strategies modulating miR-155 expression or preventing immuno-regulatory and pro-tumor macrophage polarization could be adjuvants in EBV + DLBCLe therapy since this entity has a rich infiltration of M2 macrophages in its tumor microenvironment.

The role of ARMC5 in human cell cultures from nodules of primary macronodular adrenocortical hyperplasia (PMAH).

The participation of aberrant receptors and intra-adrenal ACTH in hyperplastic tissue are considered mechanisms that regulate hypercortisolism in PMAH. Additionally, germline ARMC5 mutations have been described as the most frequent genetic abnormality found in patients diagnosed with PMAH. Previous functional studies analyzed ARMC5 role using H295R cells. Therefore, we investigated the role of ARMC5 in cell cultures obtained from PMAH nodules containing steroidogenic cells, aberrant receptors and intra-adrenal ACTH. ARMC5 silencing in non-mutated PMAH cell cultures decreased steroidogenesis-related genes and increased CCNE1 mRNA expression and proliferative capacity without affecting cell viability. Additionally, ARMC5 overexpression induced cell death in PMAH mutated cell cultures, thereby decreasing cell viability. We confirmed the role of ARMC5 as an important pro-apoptotic protein involved in PMAH-related steroidogenesis. We also report for the first time the involvement of ARMC5 in controlling proliferation and regulating cell cycle in PMAH cell cultures; these effects need to be explored further.

Low DICER1 expression is associated with poor clinical outcome in adrenocortical carcinoma.

Low DICER1 expression was associated with poor outcome in several cancers. Recently, hot-spot DICER1 mutations were found in ovarian tumors, and TARBP2 truncating mutations in tumor cell lines with microsatellite instability. In this study, we assessed DICER1 e TRBP protein expression in 154 adult adrenocortical tumors (75 adenomas and 79 carcinomas). Expression of DICER1 and TARBP2 gene was assessed in a subgroup of 61 tumors. Additionally, we investigated mutations in metal biding sites located at the RNase IIIb domain of DICER1 and in the exon 5 of TARBP2 in 61 tumors. A strong DICER1 expression was demonstrated in 32% of adenomas and in 51% of carcinomas (p = 0.028). Similarly, DICER1 gene overexpression was more frequent in carcinomas (60%) than in adenomas (23%, p = 0.006). But, among adrenocortical carcinomas, a weak DICER1 expression was significantly more frequent in metastatic than in non-metastatic adrenocortical carcinomas (66% vs. 31%; p = 0.002). Additionally, a weak DICER1 expression was significantly correlated with a reduced overall (p = 0.004) and disease-free (p = 0.005) survival. In the multivariate analysis, a weak DICER1 expression (p = 0.048) remained as independent predictor of recurrence. Regarding TARBP2 gene, its protein and gene expression did not correlate with histopathological and clinical parameters. No variant was identified in hot spot areas of DICER1 and TARBP2. In conclusion, a weak DICER1 protein expression was associated with reduced disease-free and overall survival and was a predictor of recurrence in adrenocortical carcinomas.

Expression of LIN28 and its regulatory microRNAs in adult adrenocortical cancer.

OBJECTIVE: LIN28 control cells reprogramming and pluripotency mainly through miRNA regulation and has been overexpressed in many advanced cancers. In this study, we evaluated the prognostic role of LIN28 and its regulatory miRNAs in a large cohort of adrenocortical tumours (ACTs). PATIENTS AND METHODS: LIN28 protein expression was assessed in 266 adults ACTs (78 adenomas and 188 carcinomas) from Brazil and Germany. LIN28A and LIN28B gene expression was analysed in 59 ACTs (31 adenomas and 28 carcinomas) and copy number variation in 39 ACTs. In addition, we determined the expression of let-7 family, mir-9, mir-30 and mir-125 in 28 carcinomas. RESULTS: LIN28A gene was overexpressed in aggressive ACCs when compared with adenomas and nonaggressive ACCs, but no LIN28A copy number variation was found in ACTs. Unexpectedly, weak LIN28 protein expression was significantly associated with reduced disease-free survival in ACC patients (P = 0·01), but for overall survival only a trend was detectable (P = 0·117). In the multivariate analysis, only Ki67 index ≥10% (HR 4·6, P = 0·000) and weak LIN28 protein expression (HR 2·0, P = 0·03) were independent predictors of recurrence in ACC patients. Interestingly, mir-9 expression, a negative LIN28A/B regulator, was significantly higher in aggressive than in nonaggressive ACCs [2076 (from 36 to 9307) vs 133·4 (from 2·4 to 5193); P = 0·011] and was highly associated with reduced overall (P = 0·01) and disease-free survival (P = 0·01). However, mir-9 prognostic role should be further evaluated in a larger cohort. CONCLUSION: Weak LIN28 protein expression was associated with recurrence in ACCs. Additionally, overexpression of mir-9, a negative LIN28A regulator, was associated with poor outcome.

Congenital hyperinsulinism in Brazilian neonates: a study of histology, KATP channel genes, and proliferation of ß cells.

Congenital hyperinsulinism (CHI) is a rare pancreatic ß-cell disease of neonates, characterized by inappropriate insulin secretion with severe persistent hypoglycemia, with regard to which many questions remain to be answered, despite the important acquisition of its molecular mechanisms in the last decade. The aim of this study was to examine pancreatic histology, ß-cell proliferation (immunohistochemistry with double staining for Ki-67/insulin), and ß-cell adenosine triphosphate-sensitive potassium channels genes from 11 Brazilian patients with severe medically unresponsive CHI who underwent pancreatectomy. Pancreatic histology and ß-cell proliferation in CHI patients were compared to pancreatic samples from 19 age-matched controls. Ten cases were classified as diffuse form (D-CHI) and 1 as focal form (F-CHI). ß-cell nucleomegaly and abundant cytoplasm were absent in controls and were observed only in D-CHI patients. The Ki-67 labeling index (Ki-67-LI) was used to differentiate the adenomatous areas of the F-CHI case (10.15%) from the "loose cluster of islets" found in 2 D-CHI samples (2.29% and 2.43%) and 1 control (1.54%) sample. The Ki-67-LI was higher in the F-CHI adenomatous areas, but D-CHI patients also had significantly greater Ki-67-LI (mean value  =  2.41%) than age-matched controls (mean value  =  1.87%) (P  =  0.009). In this 1st genetic study of CHI patients in Brazil, no mutations or new polymorphisms were found in the 33-37 exons of the ABCC8 gene (SUR1) or in the entire exon of the KCNJ11 gene (Kir 6.2) in 4 of 4 patients evaluated. On the other hand, enhanced ß-cell proliferation seems to be a constant feature in CHI patients, both in diffuse and focal forms.

Estudos imunohistoquímicos dos macrófagos na mucosa colônica em crianças na doença de Crohn e na retocolite ulcerativa inespecífica / Immunohistochemical study of macrophages in the colonic mucosa of children with Crohn's disease and ulcerative colitis

Sao quantificadas e analisadas as características histológicas dos macrófagos na mucosa colônica na doença de Crohn e na retocolite ulcerativa inespecífica. Foram estudados 12 pacientes com doença de Crohn 19 com retocolite ulcerativa inespecífica e 10 espécimes de mucosa retal representando o grupo controle, de acordo com o seguinte modelo: período I (PI) = pré-tratamento; período II (PII)= até dois anos de evoluçao e período III (PIII)= mais de dois anos de evoluçao. Os macrófagos foram identificados em mucosa colônica pelo monoclonal CD68 através do método de imunoperoxidase. A quantificaçao dos macrófagos foi feita através de análise de imagem computadorizada cromática, que expressa em porcentagem a área (mm2) ocupada pelas células CD68 positivas. A porcentagem da área ocupada pelos macrófagos se apresentava aumentada em ambas as doenças, em todos os períodos estudados, quando comparada com o grupo controle, porém sem diferença estatisticamente significante. A distribuiçao dos macrófagos dentro da mucosa do grupo controle foi subepitelial, enquanto na dos doentes atingiu toda a altura da mucosa, concentrando-se nas bases das úlceras e ao longo das fissuras. Na doença de Crohn as células CD68 positivas facilitaram a identificaçao dos microgranulomas, eventualmente despercebidos na coloraçao de hematoxilina-eosina. Embora nao houvesse diferença entre pacientes e controles quanto à área ocupada pelos macrófagos, a distribuiçao diferente pode sugerir a participaçao dos macrófagos na lesao destas duas doenças, embora nao permitam diagnóstico diferencial, possivelmente pela variabilidade dos valores. O CD68 nao identificou os diferentes estados funcionais dos macrófagos, mas a sua posiçao na mucosa sugere que com as fissuras e úlceras, sua funçao principal seria a fagocitose, e nos demais sítios a de célula apresentadora de antígenos e recrutadora de outras células inflamatórias.

Estudo imunohistológico do linfócito T (total e auxiliador) e do linfócito B da mucosa colónica de crianças portadoras de doença de Crohn e retocolite ulcerativa inespecífica / Immunohistological study of the T-lymphocyte (total and helper) and the B-lymphocyte of the colonic mucous in children with Crohn's disease and unspecific ulcerative colitis

Para melhor compreensao dos mecanismos imunológicos envolvidos na mucosa colônica da retocolite ulcerativa inespecífica e da doença de Crohn, foram estudadas as mucosas colônicas de 19 crianças com retocolite ulcerativa inespecífica, 12 crianças com doença de Crohn e um grupo controle representado por 10 espécimes de mucosa retal de pacientes com obstipaçao intestinal crônica inespecífica. As mucosas foram coradas com a técnica de imunoperoxidase, e os linfócitos T (total e auxiliador) e B foram identificados com marcadores específicos - para os linfócitos T total, CD3, linfócitos T auxiliador, CD4 e para o B, CD20 - e quantificadas na lâmina própria através de análise de imagem computadorizada cromática em diferentes períodos evolutivos da doença: PI (antes do tratamento), PH (até dois anos de evoluçao) e PIII (com mais de dois anos de evoluçao). No epitélio superficial foi quantificado apenas o linfócito T total, através de contagem linear. O marcador CD3, do linfócito T total, esteve aumentado no epitélio (superficial e glandular) e na lâmina própria das mucosas doentes em relaçao às do controle. Na mucosa dos pacientes, esteve distribuído difusamente, concentrando-se ao redor dos microgranulomas, no interior dos nódulos linfóides e nas áreas lesadas em proximidade aos macrófagos. Na doença de Crohn, PI, sua média na lâmina própria diferiu significantememte em relaçao ao controle. Comportamento semelhante ocorreu com o marcador CD20 do linfócito B nas mucosas doentes, cujas médias foram superiores aos do controle, embora apenas no PI da doença de Crohn essa diferença tenha sido significativa. O marcador CD4 do linfócito T auxiliador também apresentou nas mucosas doentes médias elevadas em relaçao à mucosa controle, mas sem significaçao estatística. As células CD4+ distribuíram-se difusamente por toda altura da mucosa doente, concentrando-se ao redor dos nódulos linfóides e dos microgranulomas. De modo geral, todos os marcadores estudados neste trabalho apresentaram médias superiores aos do grupo controle com diferentes graus de significância nos diferentes períodos estudados. Esses resultados confirmam a participaçao desses elementos na patogênese da retocolite ulcerativa inespecífica e da doença de Crohn. Os achados de correlaçao positiva entre os marcadores CD4 e CD20 na retocolite ulcerativa inespecífica e CD3 e CD20...