Caterina Chiappetta

Brown Fat Expresses Adiponectin in Humans

The presence of brown adipose tissue (BAT) in humans is unclear. Pheochromocytomas (PHEO) are rare tumors of neuroectodermal origin which occur in 0.1-0.2% of patients with hypertension. We sought to evaluate the presence and activity of BAT surrounding adrenal PHEO in a well-studied sample of 11 patients who were diagnosed with PHEO and then underwent adrenalectomy. Areas of white fat (WAT) and BAT surrounding PHEO were obtained by Laser Capture Microdissection for analysis of uncoupling protein (UCP)-1 and adiponectin mRNA expression. Adiponectin and UCP-1 mRNA levels were significantly higher in BAT than in WAT (0.62 versus 0.15 and 362.4 versus 22.1, resp., P < 0.01 for both). Adiponectin mRNA levels significantly correlated with urinary metanephrines (r = 0.76, P < 0.01), vanilly mandelic acid (VMA) (r = 0.95, P < 0.01), and serum adiponectin levels (r = 0.95, P < 0.01). Serum adiponectin levels significantly decreased (24.2 ± 2 μg/mL versus 18 ± 11 μg/mL, P < 0.01) after adrenalectomy in PHEO subjects. This study provides the following findings: (1) BAT surrounding PHEO expresses adiponectin and UCP-1 mRNA, (2) expression of adiponectin mRNA is significantly higher in BAT than in WAT surrounding PHEO, and (3) catecholamines and serum adiponectin levels significantly correlate with BAT UCP-1 and adiponectin mRNA.

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.

Expression of phosphoinositide-specific phospholipase C enzymes in normal endometrium and in endometriosis.

OBJECTIVE To delineate the panel of expression of phosphoinositide-specific phospholipase C (PI-PLC) signaling enzymes in normal endometrium and in endometriosis. DESIGN Clinical/experimental study. SETTING University. PATIENT(S) Healthy donor woman and endometriosis-affected woman. INTERVENTION(S) Normal endometrium and endometriosis surgical biopsies were analyzed using gene expression analyses methodology (reverse transcriptase-polymerase chain reaction [PCR], bioanalyses). MAIN OUTCOME MEASURE(S) Gene expression (messenger RNA concentration) measures of 12 PI-PLC enzymes: PI-PLC β1, PI-PLC β2, PI-PLC β3, PI-PLC β4, PI-PLC γ1, PI-PLC γ2, PI-PLC δ1, PI-PLC δ3, PI-PLC δ4, PI-PLC ε, PI-PLC η1, and PI-PLC η2. RESULT(S) PI-PLC β1, PI-PLC β3, PI-PLC δ1, and PI-PLC δ3 enzymes were detected, although differently expressed in normal and endometriosis tissues. CONCLUSION(S) The involvement of PI-PLC enzymes in inflammation and the consistency of susceptible endometriosis loci with PI-PLC genes mapping corroborate the hypothesis that PI signaling might be involved in the pathogenesis of endometriosis.

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 Cushings 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.

Primary peripheral PNET/Ewing's sarcoma arising in the meninges, confirmed by the presence of the rare translocation t(21;22) (q22;q12)

Peripheral primitive neuroectodermal tumor/Ewings sarcoma (ES) (pPNET/ES) of intracranial origin are very rare. These tumors are characterized by specific translocations involving a gene on chromosome 22q12, the most common being t(11;22) (q24;q12). We report a case of 37‐year‐old man with pPNET/ES arising in the meninges and bearing the rare translocation t(21;22) (q22;q12). The tumor was composed of sheets and nests of monotonous small cells with round to oval nuclei, finely dispersed chromatin, small nucleolus and scant cytoplasm. We discuss the importance of the differential diagnosis with central primitive neuroectodermal tumors (cPNET).

Expression of Phosphoinositide-specific phospholipase C enzymes in human osteosarcoma cell lines

The definition of the number and nature of signal transduction pathways networking in the pathogenesis of osteosarcoma raised great interest. Intracellular calcium ions are important second messengers implicated in the control of cell death. The calcium concentration is regulated by signal transduction pathways, including the Phosphoinositides (PI) signaling. Phosphatydil inositol (4,5) bisphosphate (PIP2) is critical for many cellular activities. The levels of PIP2 are regulated by means of Phosphoinositide-specific Phospholipase C (PI-PLC) family of enzymes. We delineated the panel of expression of PI-PLC enzymes in four human osteosarcoma cell lines. In MG-63 cell line, PI-PLC β1, β2, β3, β4, γ1, γ2, δ1, δ3 and ε resulted expressed. In 143B cell line, PI-PLC β1, β2, β3, β4, γ1, γ2, δ1, δ3 and ε were expressed. In SaOS-2 cell line, PI-PLC β1, β3, β4, γ1, γ2, δ1, δ3, ε and η1. In Hs888 cell line, PI-PLC β1, β3, β4, γ1, δ1, δ3, δ4, ε and η1 the administration of U-73122 to cultures briefly modifies the levels of PI-PLC transcripts. The obtained complete expression panel of PI-PLC isoforms will be a useful tool for further functional studies about the role of the PI signal transduction pathway in osteosarcoma.

Use of a new generation of capillary electrophoresis to quantify circulating free DNA in non-small cell lung cancer

Circulating free DNA (cfDNA) is present in higher concentration in non-small-cell lung cancer (NSCLC) patients than in controls. This study was designed to assess the sensitivity and specificity of Agilent 2100 Bioanalyzer to identify patients with NSCLC and to compare it with quantitative RealTime-PCR (RT-qPCR) assay. 30 NSCLC patients and 26 controls were analyzed. The amount of cfDNA was determined both through quantitative RT-PCR targeting the human β-actin gene and by Agilent 2100 Bioanalyzer. Performances of the assays were calculated by the receiver operating characteristic (ROC) curves. The mean cfDNA concentration, obtained through the use of Agilent 2100 Bioanalyzer, in NSCLC patients (94.5 ng/mL) was almost twice the concentration detected in controls (42.8 ng/mL) as well as found by RT-qPCR (22.5 ng/mL vs 7.1 ng/mL, p<0.001). The area under curve of Agilent 2100 Bioanalyzer and RT-PCR showed that there are no statistically significant differences between these tests (p<0.92). This study shows that Agilent 2100 Bioanalyzer is an effective diagnostic tool to discriminate NSCLC patients from healthy individuals and suggests a new approach for early detection of NSCLC.

Expression of phosphoinositide-specific phospholipase C enzymes in human skin fibroblasts

Fibroblasts are involved in a number of functions regulated by different signal transduction pathways, including the phosphoinositide (PI) signaling system and related converting enzymes, such as phosphoinositide-specific phospholipase C (PI-PLC). The PI-PLC family comprises crucial effector enzymes in the PI signal transduction pathway. Once activated, PI-PLC cleaves an important membrane PI, the phosphatidylinositol (4,5) bisphosphate into inositol trisphosphate and diacylglycerol—both are crucial molecules in the transduction of signals. The activity of selected PI-PLC enzymes was reported in fibroblasts, although the complete panel of expression was not available. Each cell type expresses a group of selected PI-PLC isoforms, and knowledge of the panel of expression is a necessary and preliminary tool to address further studies. In the present study, we delineated the expression panel of PI-PLC enzymes in human skin fibroblasts. PI-PLC β1, PI-PLC β3, PI-PLC β4, PI-PLC γ1, PI-PLC γ2, PI-PLC δ1, PI-PLC δ3, PI-PLC δ4, and PI-PLC ϵ were expressed. PI-PLC β1 was weakly expressed, PI-PLC δ4 was inconstantly expressed, and PI-PLC γ2 was weakly expressed.

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.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

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.