Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience.

INTRODUCTION: Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples. METHODS: Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon 19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon 2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene 1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon 14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples Federico II before shipment. Methodological and molecular data from reference specimens were annotated. RESULTS: Overall, a median DNA concentration of 3.3 ng/µL (range 0.1-10.0 ng/µL) and 13.4 ng/µL (range 2.0-45.8 ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7 ng/µL (range 0.2-11.9 ng/µL) and 9.3 ng/µL (range 0.5-18.0 ng/µL) were also detected. KRAS exon 2 p.G12C, EGFR exon 19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon 14 skipping mutation. CONCLUSIONS: Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice.

Reduced Lipopolysaccharide-Binding Protein (LBP) Levels Are Associated with Non-Alcoholic Fatty Liver Disease (NAFLD) and Adipose Inflammation in Human Obesity.

Lipopolysaccharide (LPS) and its binding protein LBP have emerged as potential contributors to the progression from overweight/obesity to overt metabolic diseases and NAFLD. While LPS is known to activate hepatocyte inflammation, thus contributing toward NAFLD development, the role of LBP is more intricate, and recent data have shown that experimental reduction in hepatic LBP promotes NAFLD progression. In this cross-sectional investigation, we evaluated circulating LBP in relation to obesity, NAFLD, visceral adipose tissue (VAT) inflammation, and type 2 diabetes (T2D). We recruited 186 individuals (M/F: 81/105; age: 47 ± 10.4 years; BMI: 35.5 ± 8.6 kg/m2); a subgroup (n = 81) underwent bariatric surgery with intra-operative VAT and liver biopsies. LBP levels were higher in obese individuals than non-obese individuals but were inversely correlated with the parameters of glucose metabolism. Reduced LBP predicted T2D independent of age, sex, and BMI (p < 0.001). LBP levels decreased across more severe stages of hepatosteatosis and lobular inflammation, and were inversely associated with VAT inflammation signatures. In conclusion, LBP levels are increased in obese individuals and are associated with a more favorable metabolic profile and lower NAFLD/NASH prevalence. A possible explanation for these findings is that hepatic LBP production may be triggered by chronic caloric excess and facilitate LPS degradation in the liver, thus protecting these individuals from the metabolic consequences of obesity.

Metastasis of lung carcinoid in the thyroid gland after 18 years: it is never too late. A case report and review of the literature.

Metastasis to the thyroid gland is a rare event. To date, only 11 cases of metastasis from neuroendocrine tumors (NETs) originating in the lung have been reported. We present a case of a patient in his 40s harboring two nodules in the thyroid gland that were diagnosed as well-differentiated NET (G1). Eighteen years before the patient underwent a lung lobectomy of the right upper lobe for a bronchial typical carcinoid with metastasis in one lymph node. Normal blood levels of calcitonin virtually ruled out the diagnosis of medullary thyroid carcinoma (MTC) and supported the diagnosis of a possible thyroid metastasis of the previous bronchial NET. Mutational analysis performed on both primary and metastasis tumor tissue did not show any mutation in the 409 genes analyzed.

Circulating dipeptidyl peptidase-4 is independently associated with the presence and severity of NAFLD/NASH in individuals with and without obesity and metabolic disease.

INTRODUCTION: Dipeptidyl peptidase 4 (DPP4) levels are associated to metabolic and cardiovascular diseases in humans; initial evidence reported a relationship between DPP4 and chronic liver diseases. Aim of this study was to investigate hepatic and systemic DPP4 levels/activity in relation to NAFLD/NASH in individuals with and without metabolic disease. METHODS: We recruited fifty-two obese individuals undergoing bariatric surgery and intra-operative liver biopsy at Sapienza University, Rome, Italy. The association between DPP4 levels/activity and NAFLD was also evaluated in 126 non-obese individuals recruited in the same setting. RESULTS: NAFLD patients had significantly higher circulating DPP4 activity than no-NAFLD in both the obese and non-obese cohorts; plasma DPP4 activity and levels linearly correlated with steatosis grade and inflammation at the liver biopsy. Hepatic DPP4 mRNA was not associated to either its circulating levels/activity or NAFLD. In the multivariate logistic regression analysis on all the study participants (n = 178), higher circulating DPP4 activity was associated with NAFLD independently of potential confounders with OR (95% CI): 3.5 (1.2-10.21), p = 0.022. CONCLUSIONS: This study demonstrates the coexistence of increased plasma DPP4 levels and activity in NAFLD. Circulating DPP4 measurement may represent a novel cost-effective strategy for NAFLD/NASH risk stratification and a potential tool for monitoring disease's progression in established NAFLD.

Adipose tissue remodelling in obese subjects is a determinant of presence and severity of fatty liver disease.

AIMS: Experimental data suggest that visceral adipose tissue (VAT) dysfunction contributes to non-alcoholic fatty liver disease (NAFLD) development in obesity, however, data on humans are limited. Aims of this study were to investigate the relationship between NAFLD and VAT morphofunctional impairment and to determine whether the extent of VAT remodelling is associated with liver damage and metabolic alterations in obesity. METHODS: We analysed data from 40 obese individuals candidate to bariatric surgery in whom paired intraoperative liver and omental biopsies were performed for diagnosing NAFLD and VAT inflammation by immunohistochemistry and mRNA expression studies. RESULTS: Within our study population, NAFLD was significantly associated with greater VAT CD68+ macrophages infiltration (P = .04), fibrosis (P = .04) and impaired microvascular density (P = .03) as well as increased expression of markers of local hypoxia, apoptosis and inflammation (UNC5B, CASP7, HIF1-α, IL-8, MIP2, WISP-1, all P < .01). The degree of VAT inflammation correlated with the severity of hepatic injury (steatosis, inflammation, fibrosis; all P < .01) and impaired gluco-metabolic profile. CONCLUSIONS: In obese patients, NAFLD is associated in a dose-dependent manner with signs of VAT remodelling, which reflect more severe clinical metabolic impairment. Our study depicts morphological alterations and novel mediators of VAT dysfunction, adding knowledge for future therapeutic approaches to NAFLD and its metabolic complications.

Granzyme B Expression in Visceral Adipose Tissue Associates With Local Inflammation and Glyco-Metabolic Alterations in Obesity.

Granzyme B (GrB) is a serine protease produced by immune and non-immune cells, able to promote multiple processes, like apoptosis, inflammation, extracellular matrix remodeling and fibrosis. GrB expression in visceral adipose tissue (VAT) was associated with tissue damage, local inflammation and insulin resistance in obesity murine model, but there is no data in humans. Aim of this study was to explore the expression of GrB in VAT from obese subjects in relation to adipose tissue injury, inflammation, metabolic alterations and GrB circulating levels. For this purpose, 85 obese individuals undergoing bariatric surgery and 35 healthy subjects (as control) were recruited at Sapienza University, Rome, Italy. Study participants underwent clinical work-up and routine biochemistry. mRNA expression of GrB in VAT and of a panel of VAT inflammatory markers was analyzed by real-time PCR. Serum GrB levels were measured by Elisa Affymetrix EBIO. We observed that 80% of obese patients expressed GrB mRNA in VAT, and GrB VAT expression was associated with the presence of local inflammation and glucose homeostasis alterations. Moreover, GrB serum levels, which were higher in obese subjects compared to non-obese healthy individuals, were associated with GrB expression in VAT and glyco-metabolic impairment. Our data show, for the first time in humans, that obese subjects with "sick" fat and altered glucose tolerance exhibit GrB expression in VAT, and suggest that GrB might contribute to obesity-related VAT inflammatory remodeling and glucose homeostasis dysregulation. Moreover, increased circulating GrB levels might represent a possible peripheral marker of VAT dysfunction in metabolic diseases.

Reduced Biliverdin Reductase-A Expression in Visceral Adipose Tissue is Associated with Adipocyte Dysfunction and NAFLD in Human Obesity.

Biliverdin reductase A (BVR-A) is an enzyme involved in the regulation of insulin signalling. Knockout (KO) mice for hepatic BVR-A, on a high-fat diet, develop more severe glucose impairment and hepato-steatosis than the wild type, whereas loss of adipocyte BVR-A is associated with increased visceral adipose tissue (VAT) inflammation and adipocyte size. However, BVR-A expression in human VAT has not been investigated. We evaluated BVR-A mRNA expression levels by real-time PCR in the intra-operative omental biopsy of 38 obese subjects and investigated the association with metabolic impairment, VAT dysfunction, and biopsy-proven non-alcoholic fatty liver disease (NAFLD). Individuals with lower VAT BVR-A mRNA levels had significantly greater VAT IL-8 and Caspase 3 expression than those with higher BVR-A. Lower VAT BVR-A mRNA levels were associated with an increased adipocytes' size. An association between lower VAT BVR-A expression and higher plasma gamma-glutamyl transpeptidase was also observed. Reduced VAT BVR-A was associated with NAFLD with an odds ratio of 1.38 (95% confidence interval: 1.02-1.9; χ2 test) and with AUROC = 0.89 (p = 0.002, 95% CI = 0.76-1.0). In conclusion, reduced BVR-A expression in omental adipose tissue is associated with VAT dysfunction and NAFLD, suggesting a possible involvement of BVR-A in the regulation of VAT homeostasis in presence of obesity.

Angiopoietin-Like Protein 4 Overexpression in Visceral Adipose Tissue from Obese Subjects with Impaired Glucose Metabolism and Relationship with Lipoprotein Lipase.

Angiopoietin-like protein 4 (ANGPTL4) regulates lipid partitioning by inhibiting circulating and tissue lipoprotein lipase (LPL); ANGPTL4 loss-of-function variants improve insulin sensitivity and reduce type 2 diabetes (T2D) risk with mechanisms partially unknown. This study was designed to explore metabolic implications of differential ANGPTL4 and LPL expression in human adipose tissue (AT). We recruited eighty-eight obese individuals, with and without abnormal glucose metabolism (AGM), undergoing bariatric surgery; visceral AT (VAT) fragments were obtained intra-operatively and analyzed by immunohistochemistry and mRNA by rt-PCR. Data on hepatic ANGPTL4 mRNA were available for 40 participants. VAT ANGPTL4 expression was higher in AGM individuals than in those with normal glucose tolerance (NGT) and associated with VAT inflammation, insulin resistance, and presence of adipocyte size heterogeneity. Increased ANGPTL4 was associated with AGM with OR = 5.1 (95% C.I.: 1.2-23; p = 0.02) and AUROC = 0.76 (95% C.I.: 1.2-23; p < 0.001). High LPL was associated with the detection of homogeneous adipocyte size, reduced microvessel density, and higher HIF-1α levels and inversely correlated to blood transaminases. In conclusion, in obese individuals, VAT ANGPTL4 levels are increased in the presence of local inflammation and AGM. Conversely, higher LPL expression describes a condition of increased lipid storage in adipocytes, which may serve as a protective mechanism against ectopic fat accumulation and related metabolic disease in obesity.

Relationship between hepatic and systemic angiopoietin-like 3, hepatic Vitamin D receptor expression and NAFLD in obesity.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide and an independent risk factor for cardiovascular mortality. Angiopoietin-like proteins (ANGPTLs) are targets for vitamin D receptor (VDR)-mediated gene transcription and this axis may promote NAFLD. ANGPTL3 is a hepatokine which inhibits lipoprotein lipase and its experimentally induced inactivation reduces hepatosteatosis. Little is known on ANGPTL3 in human NAFLD and no data exist on its relationship with hepatic VDR/VD-related genes. The aim of this research was to investigate hepatic ANGPTLs and VDR/VD-related gene expression in human obesity in relation to NAFLD. METHODS: We conducted a cross-sectional investigation on forty obese subjects with/without NAFLD. We evaluated hepatic ANGPTL3, ANGPTL4, ANGPTL8, LPL, VDR, CYP27A1 and CYP2R1 mRNA expression in liver biopsies by RT-PCR; VDR expression was further investigated by immunohistochemistry; circulating ANGPTL3 was measured by Milliplex assay. RESULTS: Compared to non-NAFLD, NAFLD individuals had significantly higher hepatic VDR, ANGPTL3 and LPL expression. ANGPTL3 correlated with steatosis grade, LPL, VDR, CYP27A1 and CYP2R1 expression. Plasma ANGPTL3 concentrations were positively associated with clinical/histological markers of NAFLD/NASH and with hepatic ANGPTL3 expression. Greater hepatic VDR expression was the main determinant of hepatic ANGPTL3 after adjusting for multiple confounders. CONCLUSIONS: Hepatic ANGPTL3 expression correlates with greater VDR expression, presence and severity of NAFLD and translates in increased circulating ANGPTL3, likely as a result of its modulation by up-regulated hepatic VDR in NAFLD. This study provides novel insights to potential mechanisms underlying ANGPTLs-mediated ectopic fat accumulation and NAFLD development in obesity.

KMT2C modulates migration and invasion processes in osteosarcoma cell lines.

In this study we investigated the role of KMT2C (a chromatin-modifying and remodelling protein) in osteosarcoma progression through cell migration and invasion assays in osteosarcoma primary and metastatic cell lines. Wound healing and transwell assays were used to detect changes of cell migration and matrigel assay was used to evaluate changes of cell invasion in primary and metastatic osteosarcoma cell lines after KMT2C siRNA transfection. We found that primary osteosarcoma cell lines showed the highest capacity of migration before mRNA KMT2C silencing and the highest capacity of invasion after mRNA KMT2C silencing; on the contrary, osteosarcoma metastatic cell line showed the highest capacity of migration after mRNA KMT2C silencing and the highest capacity of invasion before mRNA KMT2C silencing. Our study supports data in favour of selective enhancer changes, KMT2C-mediated, in metastatic osteosarcoma probably due to the different microenvironment between primary and metastatic sites.

Elevated plasma copeptin levels identify the presence and severity of non-alcoholic fatty liver disease in obesity.

INTRODUCTION: Copeptin is the stable surrogate marker of vasopressin (VP), which is released in response to elevated plasma osmolality or low blood pressure. Elevated plasma copeptin levels are associated with higher risk of insulin resistance-related disorders, such as type 2 diabetes (T2DM), metabolic syndrome (MS), and cardiovascular disease, and experimental reduction of circulating VP levels is shown to significantly decrease hepatic fat content in obese rats, independently from body adiposity. However, the association between copeptin and non-alcoholic fatty liver disease and steatohepatitis (NAFLD/NASH) in humans has not been explored yet. The aim of this study was to explore the relationship between plasma copeptin and the presence/severity of NAFLD/NASH. METHODS: For this study, we recruited 60 obese patients candidate to bariatric surgery for clinical purposes in which intraoperative liver biopsies were performed for diagnosing NAFLD/NASH. Circulating copeptin levels were also assessed in 60 age- and sex-comparable non-obese individuals without NAFLD at liver ultrasonography. Plasma copeptin was measured by sandwich immunoluminometric assay (Thermo Fisher Scientific). RESULTS: Obese patients with biopsy-proven NAFLD (53%) had significantly higher copeptin levels than both obese individuals without NAFLD and non-obese subjects (ob/NAFLD+ 9.5 ± 4.9; ob/NAFLD- 6.4 ± 2.6; and non-ob/NAFLD- 7.4 ± 5.1 pmol/L; p = 0.004 and p = 0.01 respectively). Plasma copeptin concentration positively correlated with hepatic macro- and micro-vesicular steatosis (r = 0.36, p = 0.026; r = 0.31, p = 0.05), lobular inflammation (r = 0.37, p = 0.024) and significantly increased throughout degrees of NASH severity, as expressed as absence, borderline, and overt NASH at the liver biopsy (r = 0.35, p = 0.01). Greater circulating copeptin predicted the presence of NASH with OR = 1.73 (95% CI = 1.02-2.93) after multivariate adjustment for age, sex, renal function and presence of T2DM and MS components. CONCLUSIONS: Increased plasma copeptin is independently associated with the presence and severity of NAFLD and NASH, pointing to a novel mechanism behind human fatty liver disease potentially modifiable by pharmacological treatment and lifestyle intervention.

Reduced biliverdin reductase-A levels are associated with early alterations of insulin signaling in obesity.

Biliverdin reductase-A (BVR-A) is a serine/threonine/tyrosine kinase involved in the regulation of insulin signaling. In vitro studies have demonstrated that BVR-A is a substrate of the insulin receptor and regulates IRS1 by avoiding its aberrant activation, and in animal model of obesity the loss of hepatic BVR-A has been associated with glucose/insulin alterations and fatty liver disease. However, no studies exist in humans. Here, we evaluated BVR-A expression levels and activation in peripheral blood mononuclear cells (PBMC) from obese subjects and matched lean controls and we investigated the related molecular alterations of the insulin along with clinical correlates. We showed that BVR-A levels are significantly reduced in obese subjects and associated with a hyper-activation of the IR/IRS1/Akt/GSK-3ß/AS160/GLUT4 pathway. Low BVR-A levels also associate with the presence of obesity, metabolic syndrome, NASH and visceral adipose tissue inflammation. These data suggest that the reduction of BVR-A may be responsible for early alterations of the insulin signaling pathway in obesity and in this context may represent a novel molecular target to be investigated for the comprehension of the process of insulin resistance development in obesity.

Correction: The nuclear-cytoplasmic trafficking of a chromatin-modifying and remodelling protein (KMT2C), in osteosarcoma.

[This corrects the article DOI: 10.18632/oncotarget.25755.].

The nuclear-cytoplasmic trafficking of a chromatin-modifying and remodelling protein (KMT2C), in osteosarcoma.

Osteosarcoma is the most common paediatric primary non-hematopoietic bone tumor; the survival is related to the response to chemotherapy and development of metastases. KMT2C is a chromatin-modifying and remodelling protein and its expression has never been studied in osteosarcoma. The aim of this study was to understand the role of KMT2C in the osteosarcoma carcinogenesis and metastatic progression to identify a new molecular target and to provide new therapeutic approach. We performed the immunohistochemical and gene expression analysis of KMT2C in 32 samples of patients with diagnosis of osteosarcoma with known clinic-pathological data and we analysed the expression of genes involved in the metastatic pathway in four osteosarcoma cell lines by blocking the KMT2C expression using siRNA. We found a nuclear-cytoplamic trafficking of KMT2C and the cytoplasmic localization was higher than the nuclear localization (p < 0.0001). Moreover, the percentage of cells with cytoplasmic positivity increased from low grade primary tissue to metastatic tissues. The cytoplasmic localization of KMT2C could lead to a change in its function supporting osteosarcoma carcinogenesis and progression. Our hypothesis is that KMT2C could affect the enhancer activity of genes influencing the invasive properties and metastatic potential of osteosarcoma.

Neurotensin Is a Lipid-Induced Gastrointestinal Peptide Associated with Visceral Adipose Tissue Inflammation in Obesity.

Neurotensin (NT) is a 13-amino acid peptide localized in the neuroendocrine cells of the small intestine, which promotes fat absorption and fatty acids translocation in response to lipid ingestion. NT-knock-out mice fed with a high-fat diet are protected from obesity, fatty liver, and the development of insulin-resistance. In humans, higher plasma levels of pro-NT, which is the stable circulating precursor of NT, predict obesity, type 2 diabetes (T2D), and cardiovascular disease. In obesity, the presence of visceral adipose tissue (VAT) inflammation leads to unfavorable metabolic outcomes and is associated with the development of T2D and non-alcoholic fatty liver disease (NAFLD). In this study, we investigated the relationship between plasma pro-NT levels and the presence of VAT inflammation in biopsies from 40 morbidly obese subjects undergoing bariatric surgery. We demonstrated that higher proNT levels are significantly associated with greater macrophages infiltration, HIF-1α, WISP-1, and UNC5B expression in VAT (all p < 0.01) due to the diagnosis of T2D and NAFLD. The overall results show that, in obesity, pro-NT is a biomarker of VAT inflammation and insulin-resistance. Additionally, NT may be involved in the development of dysmetabolic conditions likely mediated by increased gut fat absorption and the presence of a proinflammatory milieu in the adipose tissue.

Whole-exome analysis in osteosarcoma to identify a personalized therapy.

Osteosarcoma is the most common pediatric primary non-hematopoietic bone tumor. Survival of these young patients is related to the response to chemotherapy and development of metastases. Despite many advances in cancer research, chemotherapy regimens for osteosarcoma are still based on non-selective cytotoxic drugs. It is essential to investigate new specific molecular therapies for osteosarcoma to increase the survival rate of these patients. We performed exomic sequence analyses of 8 diagnostic biopsies of patients with conventional high grade osteosarcoma to advance our understanding of their genetic underpinnings and to correlate the genetic alteration with the clinical and pathological features of each patient to identify a personalized therapy. We identified 18,275 somatic variations in 8,247 genes and we found three mutated genes in 7/8 (87%) samples (KIF1B, NEB and KMT2C). KMT2C showed the highest number of variations; it is an important component of a histone H3 lysine 4 methyltransferase complex and it is one of the histone modifiers previously implicated in carcinogenesis, never studied in osteosarcoma. Moreover, we found a group of 15 genes that showed variations only in patients that did not respond to therapy and developed metastasis and some of these genes are involved in carcinogenesis and tumor progression in other tumors. These data could offer the opportunity to get a key molecular target to identify possible new strategies for early diagnosis and new therapeutic approaches for osteosarcoma and to provide a tailored treatment for each patient based on their genetic profile.

Clinical significance of epithelial-to-mesenchymal transition in laryngeal carcinoma: Its role in the different subsites.

BACKGROUND: During epithelial-to-mesenchymal transition, cancer cells lose adhesion capacity gaining migratory properties. The role of the process on prognosis has been evaluated in 50 cases of laryngeal carcinoma. METHODS: E-cadherin, N-cadherin, ß-catenin, α-catenin, γ-catenin, caveolin-1, and vimentin immunohistochemical expression were evaluated using a double score based on staining intensity and cellular localization. RESULTS: Cytoplasmic E-cadherin and α/γ catenin staining were associated with a decrease in survival, cytoplasmic ß-catenin was associated with advanced stage, and N-cadherin and vimentin expression were associated with poor differentiation and tumor relapse. On the basis of cancer cells, epithelial or mesenchymal morphological and immunophenotypic similarity we identified 4 main subgroups correlated with a transition to a more undifferentiated phenotype, which have a different pattern of relapse and survival. CONCLUSION: The negative prognostic role of epithelial-to-mesenchymal transition has been confirmed and a predictive role in glottic tumors has been suggested, leading us to propose epithelial-to-mesenchymal transition as an additional adverse feature in laryngeal carcinoma.

Involvement of pro-inflammatory cytokines and growth factors in the pathogenesis of Dupuytren's contracture: a novel target for a possible future therapeutic strategy?

Dupuytren's contracture (DC) is a benign fibro-proliferative disease of the hand causing fibrotic nodules and fascial cords which determine debilitating contracture and deformities of fingers and hands. The present study was designed to characterize pro-inflammatory cytokines and growth factors involved in the pathogenesis, progression and recurrence of this disease, in order to find novel targets for alternative therapies and strategies in controlling DC. The expression of pro-inflammatory cytokines and of growth factors was detected by immunohistochemistry in fibrotic nodules and normal palmar fascia resected respectively from patients affected by DC and carpal tunnel syndrome (CTS; as negative controls). Reverse transcription (RT)-PCR analysis and immunofluorescence were performed to quantify the expression of transforming growth factor (TGF)-ß1, interleukin (IL)-1ß and vascular endothelial growth factor (VEGF) by primary cultures of myofibroblasts and fibroblasts isolated from Dupuytren's nodules. Histological analysis showed high cellularity and high proliferation rate in Dupuytren's tissue, together with the presence of myofibroblastic isotypes; immunohistochemical staining for macrophages was completely negative. In addition, a strong expression of TGF-ß1, IL-1ß and VEGF was evident in the extracellular matrix and in the cytoplasm of fibroblasts and myofibroblasts in Dupuytren's nodular tissues, as compared with control tissues. These results were confirmed by RT-PCR and by immunofluorescence in pathological and normal primary cell cultures. These preliminary observations suggest that TGF-ß1, IL-1ß and VEGF may be considered potential therapeutic targets in the treatment of Dupuytren's disease (DD).

BRAF and NRAS mutations are heterogeneous and not mutually exclusive in nodular melanoma.

Inhibitors of RAF inhibit the MAPK pathway that plays an important role in the development and progression of those melanoma carrying the V600E BRAF mutation, but there's a subset of such patients who do not respond to the therapy. Various mechanisms of drug resistance have been proposed which include the clonal heterogeneity of the tumor. We have studied a population of nodular melanoma to investigate the intratumor and intertumor heterogeneity by Laser Capture Microdissection (LCM) analysis. Our results showed that BRAF and NRAS mutations were detected in 47% and 33% of nodular melanoma, respectively, and that there is a discrepancy in mutational pattern of tumoral sample because in the 36% of patients a different mutation, in at least 1 area of the tumor, was found by LCM analysis, giving evidence of the presence of different clonal cells populations. Moreover, we found that mutations in BRAF and NRAS are not mutually exclusive because they were simultaneously present in the same tumor specimens and we observed that when the 2 different mutations were present one is a high-frequency mutation and the other is a low-frequency mutation. This was more evident in lymphonodal metastasis that resulted from wild type to mutational analysis, but showed different mutations following LCM analysis. Therefore, we believed that, when primary tumoral sample results negative to mutational analysis, if it is possible, metastases should be investigated to verify the presence of mutations. Generally, it should be searched for other mutations, in addition to BRAF V600E, so as to better understand the mechanism of drug resistance.

Leptin and adiponectin mRNA expression from the adipose tissue surrounding the adrenal neoplasia.

CONTEXT: Interplay between adipose tissue and adrenal glands has been recently suggested, without well-founded actions of locally adipose tissue surrounding the adrenal glands. OBJECTIVE: We hypothesized that the local expression of leptin and adiponectin can be associated with pathological changes of the adrenal glands. PATIENTS AND MAIN OUTCOME MEASURES: We evaluated RT-PCR of leptin and adiponectin mRNA expression from the adipose tissue surrounding adrenal glands in 30 patients, collecting adipose tissue surrounding the adrenal neoplasms, peri-renal and subcutaneous depots. RESULTS: Leptin mRNA levels from adrenal neoplasia and peri-renal fat were significantly higher in aldosterone-producing adenoma than in nonfunctioning adenomas (P < 0.001 and P < 0.02, respectively). In patients with Cushing's syndrome leptin mRNA levels were significantly higher in adrenal fat than in peri-renal (P < 0.05) and subcutaneous adipose tissue (P < 0.001). Adiponectin mRNA expression from adrenal neoplasia was significantly lower than that from peri-renal and subcutaneous fat depots (P < 0.05). Leptin and adiponectin plasma levels significantly correlated with their mRNA expression from the fat depot surrounding the adrenal neoplasia. CONCLUSIONS: Our findings suggest an active role of the fat depot surrounding the adrenal neoplasia, with local secretion of leptin and adiponectin.