ADAM33 gene silencing by promoter hypermethylation as a molecular marker in breast invasive lobular carcinoma

<p>Abstract</p> <p>Background</p> <p>ADAM33 protein is a member of the family of transmembrane glycoproteins composed of multidomains. ADAM family members have different activities, such as proteolysis and adhesion, making them good candidates to mediate the extracellular matrix remodelling and changes in cellular adhesion that characterise certain pathologies and cancer development. It was reported that one family member, <it>ADAM23</it>, is down-regulated by promoter hypermethylation. This seems to correlate with tumour progression and metastasis in breast cancer. In this study, we explored the involvement of ADAM33, another ADAM family member, in breast cancer.</p> <p>Methods</p> <p>First, we analysed <it>ADAM33 </it>expression in breast tumour cell lines by RT-PCR and western blotting. We also used 5-aza-2'-deoxycytidine (5azadCR) treatment and DNA bisulphite sequencing to study the promoter methylation of ADAM33 in breast tumour cell lines. We evaluated ADAM33 methylation in primary tumour samples by methylation specific PCR (MSP). Finally, <it>ADAM33 </it>promoter hypermethylation was correlated with clinicopathological data using the chi-square test and Fisher's exact test.</p> <p>Results</p> <p>The expression analysis of <it>ADAM33 </it>in breast tumour cell lines by RT-PCR revealed gene silencing in 65% of tumour cell lines. The corresponding lack of ADAM33 protein was confirmed by western blotting. We also used 5-aza-2'-deoxycytidine (5-aza-dCR) demethylation and bisulphite sequencing methodologies to confirm that gene silencing is due to <it>ADAM33 </it>promoter hypermethylation. Using MSP, we detected <it>ADAM33 </it>promoter hypermethylation in 40% of primary breast tumour samples. The correlation between methylation pattern and patient's clinicopathological data was not significantly associated with histological grade; tumour stage (TNM); tumour size; ER, PR or ERBB2 status; lymph node status; metastasis or recurrence. Methylation frequency in invasive lobular carcinoma (ILC) was 76.2% compared with 25.5% in invasive ductal carcinoma (IDC), and this difference was statistically significant (p = 0.0002).</p> <p>Conclusion</p> <p><it>ADAM33 </it>gene silencing may be related to the discohesive histological appearance of ILCs. We suggest that <it>ADAM33 </it>promoter methylation may be a useful molecular marker for differentiating ILC and IDC.</p

ADAM23 Negatively Modulates alpha(v)beta(3) Integrin Activation during Metastasis

The ADAM23 gene is frequently silenced in different types of tumors, and, in breast tumors, silencing is correlated with tumor progression, suggesting that it might be associated with the acquisition of a metastatic phenotype. ADAM23 exerts its function mainly through the disintegrin domain, because its metalloprotease domain is inactive. Analysis of ADAM23 binding to integrins has revealed a specific interaction with alpha(v)beta(3) integrin mediated by the disintegrin domain. Altered expression of alpha(v)beta(3) integrin has been observed in different types of tumors, and expression of this integrin in the activated form has been shown to promote metastasis formation. Here, we investigated the possibility that interaction between ADAM23 and alpha(v)beta(3) integrin might negatively modulate alpha(v)beta(3) activation during metastatic progression. ADAM23 expression was knocked down using short hairpin RNA in the MDA-MB-435 cell line, which has been extensively used as a model for alpha(v)beta(3) integrin activation. Ablation of ADAM23 enhanced alpha(v)beta(3) integrin activation by at least 2- to 4-fold and ADAM23 knockdown cells showed enhanced migration and adhesion to classic alpha(v)beta(3) integrin ligands. Ablation of ADAM23 expression also enhanced pulmonary tumor cell arrest in immunodeficient mice. To complement our findings with clinical evidence, we showed that silencing of ADAM23 gene by DNA promoter hypermethylation in a collection of 94 primary breast tumors was significantly associated with lower distant metastases-free and disease-specific survivals and was an independent prognostic factor for poor disease outcome. Our results strongly support a functional role of ADAM23 during metastatic progression by negatively modulating alpha(v)beta(3) integrin activation. [Cancer Res 2009;69(13):5546-52]FAPESP[04/09088-9

Risk factors and action thresholds for the novel coronavirus pandemic. Insights from the Italian Society of Nephrology COVID-19 Survey

365noBackground and aim: Over 80% (365/454) of the nation’s centers participated in the Italian Society of Nephrology COVID-19 Survey. Out of 60,441 surveyed patients, 1368 were infected as of April 23rd, 2020. However, center-specific proportions showed substantial heterogeneity. We therefore undertook new analyses to identify explanatory factors, contextual effects, and decision rules for infection containment. Methods: We investigated fixed factors and contextual effects by multilevel modeling. Classification and Regression Tree (CART) analysis was used to develop decision rules. Results: Increased positivity among hemodialysis patients was predicted by center location [incidence rate ratio (IRR) 1.34, 95% confidence interval (CI) 1.20–1.51], positive healthcare workers (IRR 1.09, 95% CI 1.02–1.17), test-all policy (IRR 5.94, 95% CI 3.36–10.45), and infected proportion in the general population (IRR 1.002, 95% CI 1.001–1.003) (all p < 0.01). Conversely, lockdown duration exerted a protective effect (IRR 0.95, 95% CI 0.94–0.98) (p < 0.01). The province-contextual effects accounted for 10% of the total variability. Predictive factors for peritoneal dialysis and transplant cases were center location and infected proportion in the general population. Using recursive partitioning, we identified decision thresholds at general population incidence ≥ 229 per 100,000 and at ≥ 3 positive healthcare workers. Conclusions: Beyond fixed risk factors, shared with the general population, the increased and heterogeneous proportion of positive patients is related to the center’s testing policy, the number of positive patients and healthcare workers, and to contextual effects at the province level. Nephrology centers may adopt simple decision rules to strengthen containment measures timely.nonenoneNordio M.; Reboldi G.; Di Napoli A.; Quintaliani G.; Alberici F.; Postorino M.; Aucella F.; Messa P.; Brunori G.; Brunori G.; Bosco M.; Malberti F.; Mandreoli M.; Mazzaferro S.; Movilli E.; Ravera M.; Salomone M.; Santoro D.; PostorinoLimido M.A.; Bonomini M.; Stingone A.; Maccarone M.; Di Loreto E.; Stacchiotti L.; Malandra R.; Chiarella S.; D'Agostino F.; Fuiano G.; Nicodemo L.; Bonofiglio R.; Greco S.; Mallamaci F.; Barreca E.; Caserta C.; Bruzzese V.; Galati D.; Tramontana D.; Viscione M.; Chiuchiolo L.; Tuccillo S.; Sepe M.; Vitale F.; Ciriana E.; Santoro D.; Martignetti V.; Caserta D.; Stizzo A.; Romano A.; Iulianiello G.; Cascone E.; Minicone P.; Chiricone D.; Delgado G.; Barbato A.; Celentano S.; Molfino I.; Coppola S.; Raiola I.; Abategiovanni M.; Borrelli S.; Margherita C.; Bruno F.; Ida M.; Aliperti E.; Potito D.; Cuomo G.; De Luca M.; Merola M.; Botta C.; Garofalo G.; Alinei P.; Paglionico C.; Roano M.; Vitale S.; Ierardi R.; Fimiani V.; Conte G.; Di Natale G.; Romano M.; Di Marino V.; Scafarto A.; Meccariello S.; Pecoraro C.; Di Stazio E.; Di Meglio E.; Cuomo A.; Maresca B.; Rotaia E.; Capasso G.; Auricchio M.; Pluvio C.; Maddalena L.; De Maio A.; Palladino G.; Buono F.; Gigliotti G.; Mandreoli M.; Mancini E.; La Manna G.; Storari A.; Mosconi G.; Cappelli G.; Scarpioni R.; Gregorini M.; Rigotti A.; Mancini W.; Bianco F.; Boscutti G.; Amici G.; Tosto M.; Fini R.; Pace G.; Cioffi A.; Boccia E.; Di Lullo L.; Di Zazzo G.; Simonelli R.; Bondatti F.; Miglio L.; Rifici N.; Treglia A.; Muci M.; Baldinelli G.; Rizzi E.; Lonzi M.; De Cicco C.; Forte F.; De Paolis P.; Grandaliano G.; Cuzziol C.; Torre V.M.; Sfregola P.; Rossi V.; Fabio G.; Flammini A.; Filippini A.; Onorato L.; Vendola F.; Di Daniela N.; Alfarone C.; Scabbia L.; Ferrazzano M.; Grotta B.D.; Gamberini M.; Fazzari L.; Mene P.; Morgia A.; Catucci A.; Palumbo R.; Puliti M.; Marinelli R.; Polito P.; Marrocco F.; Morabito S.; Rocca R.; Nazzaro L.; Lavini R.; Iamundo V.; Chiappini M.; Casarci M.; Morosetti M.; Hassan S.; Alfarone C.; Ferrazzano M.; Firmi G.; Galliani M.; Serraiocco M.; Feriozzi S.; Valentini W.; Sacco P.; Garibotto G.; Cappelli V.; Saffioti C.; Repetto M.; Rolla D.; Lorenz M.; Pedrini L.; Polonioli D.; Galli E.; Ruggenenti P.; Scolari F.; Bove S.; Costantino E.; Bracchi M.; Mangano S.; Depetri G.; Malberti F.; La Milia V.; Farina M.; Zecchini S.; Savino R.; Melandri M.; Guastoni C.; Paparella M.; Gallieni M.; Minetti E.; Bisegna S.; Messa P.; Righetti M.; Badalamenti S.; Guastoni C.; Alberghini E.; Bertoli S.; Fabbrini P.; Albrizio P.; Rampino T.; Colturi C.; Rombola G.; Lucatello A.; Guerrini E.; Ranghino A.; Lenci F.; Fanciulli E.; Santarelli S.; Damiani C.; Garofalo D.; Sopranzi F.; Santoferrara A.; Di Luca M.; Galiotta P.; Brigante M.; Manganaro M.; Maffei S.; Berto I.; Besso L.; Viglino G.; Besso L.; Cusinato S.; ChiarinottiChiappero D.F.; Tognarelli G.; Gianoglio B.; Salomone M.; Forneris G.; Biancone L.; Savoldi S.; Vitale C.; Boero R.; Filiberti O.; Borzumati M.; Gesualdo L.; Lomonte C.; Gernone G.; Pallotta G.; Di Paolo S.; Vernaglione L.; Specchio A.; Stallone G.; Dell'Aquila R.; Aucella F.; Sandri G.; Russo F.; Napoli M.; Marangi A.; Morrone L.; Di Stratis C.; Fresu A.; Cicu F.; Murtas S.; Manca O.; Pani A.; Pilloni M.; Pistis R.; Cadoni M.; Contu B.; Logias F.; Ivaldi R.; Fancello S.; Cossu M.; Lepori G.; Lepori G.; Vittoria S.; Battiati E.; Arnone M.; Rome M.; Barbera A.; Granata A.; Collura G.; Dico C.L.; Pugliese G.; Di Natale E.; Rizzari G.; Cottone L.; Longo N.; Battaglia G.; Marcantoni C.; Giannetto G.; Tumino G.; Randazzo F.; Bellissimo L.; Faro F.L.; Grippaldi F.; Urso S.; Quattrone G.; Todaro I.; Vincenzo D.; Murgo A.; Masuzzo M.; Pisacane A.; Monardo P.; Santoro D.; Pontorierro M.; Quari C.; Bauro A.; Chimenz R.R.; Alfio D.; Girasole F.; Cascio A.L.; Caviglia A.; Tornese F.; Sirna F.; Altieri C.; Cusumano R.; Saveriano V.; La Corte A.; Locascio G.; Rotolo U.; Rome M.; Musso S.; Risuglia L.; Blanco G.; Minardo G.; Castellino S.; Zappulla Z.; Randone S.; Di Francesca M.; Cassetti C.C.; Oddo G.; Buscaino G.; Mucaria F.; Barraco V.I.; Di Martino A.; Mucaria F.; Rallo D.; Dani L.; Campolo G.; Manescalchi F.; Biagini M.; Agate M.; Panichi V.; Casani A.; Traversari L.; Garosi G.; Brunori G.; Tabbi M.; Selvi A.; Cencioni L.; Fagugli R.; Timio F.; Leveque A.; Manes M.; Mennella G.; Calo L.; Fiorini F.; Abaterusso C.; Calzavara P.; Nordio M.; Meneghel G.; Bonesso C.; Gambaro G.; Gammaro L.; Rugiu C.; Dell'Aquila R.; Dell'Aquila R.; Ronco C.; Rugiu C.Nordio, M.; Reboldi, G.; Di Napoli, A.; Quintaliani, G.; Alberici, F.; Postorino, M.; Aucella, F.; Messa, P.; Brunori, G.; Brunori, G.; Bosco, M.; Malberti, F.; Mandreoli, M.; Mazzaferro, S.; Movilli, E.; Ravera, M.; Salomone, M.; Santoro, D.; Postorinolimido, M. A.; Bonomini, M.; Stingone, A.; Maccarone, M.; Di Loreto, E.; Stacchiotti, L.; Malandra, R.; Chiarella, S.; D'Agostino, F.; Fuiano, G.; Nicodemo, L.; Bonofiglio, R.; Greco, S.; Mallamaci, F.; Barreca, E.; Caserta, C.; Bruzzese, V.; Galati, D.; Tramontana, D.; Viscione, M.; Chiuchiolo, L.; Tuccillo, S.; Sepe, M.; Vitale, F.; Ciriana, E.; Santoro, D.; Martignetti, V.; Caserta, D.; Stizzo, A.; Romano, A.; Iulianiello, G.; Cascone, E.; Minicone, P.; Chiricone, D.; Delgado, G.; Barbato, A.; Celentano, S.; Molfino, I.; Coppola, S.; Raiola, I.; Abategiovanni, M.; Borrelli, S.; Margherita, C.; Bruno, F.; Ida, M.; Aliperti, E.; Potito, D.; Cuomo, G.; De Luca, M.; Merola, M.; Botta, C.; Garofalo, G.; Alinei, P.; Paglionico, C.; Roano, M.; Vitale, S.; Ierardi, R.; Fimiani, V.; Conte, G.; Di Natale, G.; Romano, M.; Di Marino, V.; Scafarto, A.; Meccariello, S.; Pecoraro, C.; Di Stazio, E.; Di Meglio, E.; Cuomo, A.; Maresca, B.; Rotaia, E.; Capasso, G.; Auricchio, M.; Pluvio, C.; Maddalena, L.; De Maio, A.; Palladino, G.; Buono, F.; Gigliotti, G.; Mandreoli, M.; Mancini, E.; La Manna, G.; Storari, A.; Mosconi, G.; Cappelli, G.; Scarpioni, R.; Gregorini, M.; Rigotti, A.; Mancini, W.; Bianco, F.; Boscutti, G.; Amici, G.; Tosto, M.; Fini, R.; Pace, G.; Cioffi, A.; Boccia, E.; Di Lullo, L.; Di Zazzo, G.; Simonelli, R.; Bondatti, F.; Miglio, L.; Rifici, N.; Treglia, A.; Muci, M.; Baldinelli, G.; Rizzi, E.; Lonzi, M.; De Cicco, C.; Forte, F.; De Paolis, P.; Grandaliano, G.; Cuzziol, C.; Torre, V. M.; Sfregola, P.; Rossi, V.; Fabio, G.; Flammini, A.; Filippini, A.; Onorato, L.; Vendola, F.; Di Daniela, N.; Alfarone, C.; Scabbia, L.; Ferrazzano, M.; Grotta, B. D.; Gamberini, M.; Fazzari, L.; Mene, P.; Morgia, A.; Catucci, A.; Palumbo, R.; Puliti, M.; Marinelli, R.; Polito, P.; Marrocco, F.; Morabito, S.; Rocca, R.; Nazzaro, L.; Lavini, R.; Iamundo, V.; Chiappini, M.; Casarci, M.; Morosetti, M.; Hassan, S.; Alfarone, C.; Ferrazzano, M.; Firmi, G.; Galliani, M.; Serraiocco, M.; Feriozzi, S.; Valentini, W.; Sacco, P.; Garibotto, G.; Cappelli, V.; Saffioti, C.; Repetto, M.; Rolla, D.; Lorenz, M.; Pedrini, L.; Polonioli, D.; Galli, E.; Ruggenenti, P.; Scolari, F.; Bove, S.; Costantino, E.; Bracchi, M.; Mangano, S.; Depetri, G.; Malberti, F.; La Milia, V.; Farina, M.; Zecchini, S.; Savino, R.; Melandri, M.; Guastoni, C.; Paparella, M.; Gallieni, M.; Minetti, E.; Bisegna, S.; Messa, P.; Righetti, M.; Badalamenti, S.; Guastoni, C.; Alberghini, E.; Bertoli, S.; Fabbrini, P.; Albrizio, P.; Rampino, T.; Colturi, C.; Rombola, G.; Lucatello, A.; Guerrini, E.; Ranghino, A.; Lenci, F.; Fanciulli, E.; Santarelli, S.; Damiani, C.; Garofalo, D.; Sopranzi, F.; Santoferrara, A.; Di Luca, M.; Galiotta, P.; Brigante, M.; Manganaro, M.; Maffei, S.; Berto, I.; Besso, L.; Viglino, G.; Besso, L.; Cusinato, S.; Chiarinottichiappero, D. F.; Tognarelli, G.; Gianoglio, B.; Salomone, M.; Forneris, G.; Biancone, L.; Savoldi, S.; Vitale, C.; Boero, R.; Filiberti, O.; Borzumati, M.; Gesualdo, L.; Lomonte, C.; Gernone, G.; Pallotta, G.; Di Paolo, S.; Vernaglione, L.; Specchio, A.; Stallone, G.; Dell'Aquila, R.; Aucella, F.; Sandri, G.; Russo, F.; Napoli, M.; Marangi, A.; Morrone, L.; Di Stratis, C.; Fresu, A.; Cicu, F.; Murtas, S.; Manca, O.; Pani, A.; Pilloni, M.; Pistis, R.; Cadoni, M.; Contu, B.; Logias, F.; Ivaldi, R.; Fancello, S.; Cossu, M.; Lepori, G.; Lepori, G.; Vittoria, S.; Battiati, E.; Arnone, M.; Rome, M.; Barbera, A.; Granata, A.; Collura, G.; Dico, C. L.; Pugliese, G.; Di Natale, E.; Rizzari, G.; Cottone, L.; Longo, N.; Battaglia, G.; Marcantoni, C.; Giannetto, G.; Tumino, G.; Randazzo, F.; Bellissimo, L.; Faro, F. L.; Grippaldi, F.; Urso, S.; Quattrone, G.; Todaro, I.; Vincenzo, D.; Murgo, A.; Masuzzo, M.; Pisacane, A.; Monardo, P.; Santoro, D.; Pontorierro, M.; Quari, C.; Bauro, A.; Chimenz, R. R.; Alfio, D.; Girasole, F.; Cascio, A. L.; Caviglia, A.; Tornese, F.; Sirna, F.; Altieri, C.; Cusumano, R.; Saveriano, V.; La Corte, A.; Locascio, G.; Rotolo, U.; Rome, M.; Musso, S.; Risuglia, L.; Blanco, G.; Minardo, G.; Castellino, S.; Zappulla, Z.; Randone, S.; Di Francesca, M.; Cassetti, C. C.; Oddo, G.; Buscaino, G.; Mucaria, F.; Barraco, V. I.; Di Martino, A.; Mucaria, F.; Rallo, D.; Dani, L.; Campolo, G.; Manescalchi, F.; Biagini, M.; Agate, M.; Panichi, V.; Casani, A.; Traversari, L.; Garosi, G.; Brunori, G.; Tabbi, M.; Selvi, A.; Cencioni, L.; Fagugli, R.; Timio, F.; Leveque, A.; Manes, M.; Mennella, G.; Calo, L.; Fiorini, F.; Abaterusso, C.; Calzavara, P.; Nordio, M.; Meneghel, G.; Bonesso, C.; Gambaro, G.; Gammaro, L.; Rugiu, C.; Dell'Aquila, R.; Dell'Aquila, R.; Ronco, C.; Rugiu, C

Risk factors and action thresholds for the novel coronavirus pandemic. Insights from the Italian Society of Nephrology COVID-19 Survey

Background and aim: Over 80% (365/454) of the nation\u2019s centers participated in the Italian Society of Nephrology COVID-19 Survey. Out of 60,441 surveyed patients, 1368 were infected as of April 23rd, 2020. However, center-specific proportions showed substantial heterogeneity. We therefore undertook new analyses to identify explanatory factors, contextual effects, and decision rules for infection containment. Methods: We investigated fixed factors and contextual effects by multilevel modeling. Classification and Regression Tree (CART) analysis was used to develop decision rules. Results: Increased positivity among hemodialysis patients was predicted by center location [incidence rate ratio (IRR) 1.34, 95% confidence interval (CI) 1.20\u20131.51], positive healthcare workers (IRR 1.09, 95% CI 1.02\u20131.17), test-all policy (IRR 5.94, 95% CI 3.36\u201310.45), and infected proportion in the general population (IRR 1.002, 95% CI 1.001\u20131.003) (all p &lt; 0.01). Conversely, lockdown duration exerted a protective effect (IRR 0.95, 95% CI 0.94\u20130.98) (p &lt; 0.01). The province-contextual effects accounted for 10% of the total variability. Predictive factors for peritoneal dialysis and transplant cases were center location and infected proportion in the general population. Using recursive partitioning, we identified decision thresholds at general population incidence 65 229 per 100,000 and at 65 3 positive healthcare workers. Conclusions: Beyond fixed risk factors, shared with the general population, the increased and heterogeneous proportion of positive patients is related to the center\u2019s testing policy, the number of positive patients and healthcare workers, and to contextual effects at the province level. Nephrology centers may adopt simple decision rules to strengthen containment measures timely