Somatostatin receptor type 2 (SST2) is the main pharmacological target of somatostatin (SS) analogues widely used in patients with pancreatic neuroendocrine tumours (P-NETs), this treatment being ineffective in a subset of patients. Since it has been demonstrated that Filamin A (FLNA) is involved in mediating GPCR expression, membrane anchoring and signalling, we investigated the role of this cytoskeleton protein in SST2 expression and signalling, angiogenesis, cell adhesion and cell migration in human P-NETs and in QGP1 cell line. We demonstrated that FLNA silencing was not able to affect SST2 expression in P-NET cells in basal conditions. Conversely, a significant reduction in SST2 expression (−43±21%, P<0.05 vs untreated cells) was observed in FLNA silenced QGP1 cells after long term SST2 activation with BIM23120. Moreover, the inhibitory effect of BIM23120 on cyclin D1 expression (−46±18%, P<0.05 vs untreated cells), P-ERK1/2 levels (−42±14%; P<0.05 vs untreated cells), cAMP accumulation (−24±3%, P<0.05 vs untreated cells), VEGF expression (−31±5%, P<0.01 vs untreated cells) and in vitro release (−40±24%, P<0.05 vs untreated cells) was completely lost after FLNA silencing. Interestingly, BIM23120 promoted cell adhesion (+86±45%, P<0.05 vs untreated cells) and inhibited cell migration (−24±2%, P<0.00001 vs untreated cells) in P-NETs cells and these effects were abolished in FLNA silenced cells. In conclusion, we demonstrated that FLNA plays a crucial role in SST2 expression and signalling, angiogenesis, cell adhesion and cell migration in P-NETs and in QGP1 cell line, suggesting a possible role of FLNA in determining the different responsiveness to SS analogues observed in P-NET patients.
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- Author: Giovanna Mantovani x
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Eleonora Vitali, Valeria Cambiaghi, Alessandro Zerbi, Carlo Carnaghi, Piergiuseppe Colombo, Erika Peverelli, Anna Spada, Giovanna Mantovani, and Andrea G Lania
Alessandra Mangone, Barbara Altieri, Mario Detomas, Alessandro Prete, Haider Abbas, Miriam Asia, Yasir S Elhassan, Giovanna Mantovani, and Cristina L Ronchi
Treatment for advanced adrenocortical carcinoma (ACC) consists of mitotane alone or combined with etoposide, doxorubicin, and cisplatin (EDP). Although both therapies are widely used, markers of response are still lacking. Since inflammation-based scores have been proposed as prognostic factors in ACC, we aimed to investigate their role in predicting the response to first-line chemotherapy.
We performed a retrospective analysis of patients with advanced ACC treated with mitotane monotherapy or EDP ± mitotane. Clinical parameters (tumour stage at diagnosis, resection status, Ki67, time from diagnosis to treatment start, performance status, plasma mitotane levels, time in mitotane target ≥ 80%, clinically overt cortisol hypersecretion), and pretreatment inflammation-based scores (neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio, derived neutrophil-to-lymphocyte ratio) were investigated. The primary endpoints were overall survival (OS) and time-to-progression (TTP) from treatment initiation, the secondary endpoint was the best objective response to treatment.
We included 90 patients (59% = women, median age = 51 years) treated with mitotane monotherapy (n = 40) or EDP ± mitotane (n = 50). In the mitotane monotherapy cohort, NLR ≥ 5 and PLR ≥ 190 predicted shorter OS (hazard ratio (HR): 145.83, 95% CI: 1.87–11,323.83; HR: 165.50, 95% CI: 1.76–15,538.04, respectively), remaining significant at multivariable analysis including clinical variables. NLR was also associated with shorter TTP (HR: 2.58, 95% CI: 1.28–5.20), but only at univariable analysis. Patients with NLR ≥ 5 showed a worse treatment response than those with NLR < 5 (P = 0.040). In the EDP ± mitotane cohort, NLR ≥ 5 predicted shorter OS (HR: 2.52, 95% CI: 1.30–4.88) and TTP (HR: 1.95, 95% CI: 1.04–3.66) at univariable analysis.
In conclusion, inflammation-based scores, calculated from routinely measured parameters, may help predict response to chemotherapy in advanced ACC.
Donatella Treppiedi, Genesio Di Muro, Giusy Marra, Anna Maria Barbieri, Federica Mangili, Rosa Catalano, Andreea Serban, Emanuele Ferrante, Marco Locatelli, Andrea G Lania, Maura Arosio, Anna Spada, Erika Peverelli, and Giovanna Mantovani
Cushing’s disease (CD) is a rare endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor. Pasireotide is the only pituitary-targeted drug approved for adult patients. Nevertheless, many side effects are encountered and curative therapy is still challenging. Ubiquitin-specific peptidase 8 (USP8) plays a crucial role in the modulation of corticotroph cells growth and ACTH secretion. Here, we explored the anticancer potential of the USP8 inhibitor RA-9 in USP8-WT human tumor corticotroph cells and murine AtT-20 cells. Our results showed that RA-9 causes cell proliferation decrease (−24.3 ± 5.2%, P < 0.01) and cell apoptosis increase (207.4 ± 75.3%, P < 0.05) in AtT-20 cells, as observed with pasireotide. Moreover, RA-9 reduced ACTH secretion in AtT-20 cells (−34.1 ± 19.5%, P < 0.01), as well as in AtT-20 cells transfected with USP8 mutants, and in one out of two primary cultures in vitro responsive to pasireotide (−40.3 ± 6%). An RA-9 mediated decrease of pERK1/2 levels was observed in AtT-20 cells (−52.3 ± 13.4%, P < 0.001), comparable to pasireotide, and in primary cultures, regardless of their in vitro responsiveness to pasireotide. Upregulation of p27 was detected upon RA-9 treatment only, both in AtT-20 cells (167.1 ± 36.7%, P < 0.05) and in one primary culture tested (168.4%), whilst pCREB level was similarly halved in AtT-20 cells by both RA-9 and pasireotide. Altogether, our data demonstrate that RA-9 is efficient in exerting cytotoxic effects and inhibitory actions on cell proliferation and hormone secretion by modulating the expression of pERK1/2, pCREB and p27. Inhibition of USP8 might represent a novel strategy to target both USP8-WT and USP8-mutated tumors in CD patients.
Olimpia Alice Manzardo, Miriam Cellini, Rita Indirli, Alessia Dolci, Paolo Colombo, Flaminia Carrone, Elisabetta Lavezzi, Giovanna Mantovani, Gherardo Mazziotti, Maura Arosio, and Andrea Gerardo Antonio Lania
TNM 8th edition introduces changes in the staging of patients with differentiated thyroid carcinoma (DTC). This study aims at assessing the value of TNM 8th edition in predicting response to therapy and structural recurrence of DTC. Four hundred and eighty DTC patients were retrospectively evaluated by 7th and 8th editions of TNM staging system in relationship with risk stratification, response to therapy and recurrence of disease as defined by 2015 ATA guidelines. As compared to the 7th edition, TNM 8th led to downstage 136 patients (28.3%), with 97.5% of patients falling into lower stages (I–II) and only 2.5% remaining in higher stages (III–IV) (P < 0.001). Patients who were downstaged in stages I–II by TNM 8th were classified more frequently at intermediate-high risk (P < 0.001), had more frequently structural incomplete response to therapy (P = 0.009) and had higher risk of structural recurrence (P = 0.002) as compared to patients who were in the same TNM stages but were not downstaged. Specifically, the risk of structural recurrence was significantly higher in patients in whom the downstaging was induced by changes in tumour classification (hazard ratio (HR) 6.18, 95% CI 2.20–17.40; P = 0.001) but not in those who were downstaged for the increase in age cut-off (HR 2.80, 95% CI 0.86–9.19; P = 0.09). In conclusion, TNM 8th edition did not show reliability in predicting aggressiveness of DTC. In fact, the downstaging of DTC patients especially when performed due to changes in tumour classification may overlook patients predisposed to structural recurrence, potentially causing uncertainty in the therapeutic decision-making at the time of disease’s diagnosis.
Adrian F Daly, Bo Yuan, Frederic Fina, Jean-Hubert Caberg, Giampaolo Trivellin, Liliya Rostomyan, Wouter W de Herder, Luciana A Naves, Daniel Metzger, Thomas Cuny, Wolfgang Rabl, Nalini Shah, Marie-Lise Jaffrain-Rea, Maria Chiara Zatelli, Fabio R Faucz, Emilie Castermans, Isabelle Nanni-Metellus, Maya Lodish, Ammar Muhammad, Leonor Palmeira, Iulia Potorac, Giovanna Mantovani, Sebastian J Neggers, Marc Klein, Anne Barlier, Pengfei Liu, L’Houcine Ouafik, Vincent Bours, James R Lupski, Constantine A Stratakis, and Albert Beckers
Somatic mosaicism has been implicated as a causative mechanism in a number of genetic and genomic disorders. X-linked acrogigantism (XLAG) syndrome is a recently characterized genomic form of pediatric gigantism due to aggressive pituitary tumors that is caused by submicroscopic chromosome Xq26.3 duplications that include GPR101. We studied XLAG syndrome patients (n = 18) to determine if somatic mosaicism contributed to the genomic pathophysiology. Eighteen subjects with XLAG syndrome caused by Xq26.3 duplications were identified using high-definition array comparative genomic hybridization (HD-aCGH). We noted that males with XLAG had a decreased log2 ratio (LR) compared with expected values, suggesting potential mosaicism, whereas females showed no such decrease. Compared with familial male XLAG cases, sporadic males had more marked evidence for mosaicism, with levels of Xq26.3 duplication between 16.1 and 53.8%. These characteristics were replicated using a novel, personalized breakpoint junction-specific quantification droplet digital polymerase chain reaction (ddPCR) technique. Using a separate ddPCR technique, we studied the feasibility of identifying XLAG syndrome cases in a distinct patient population of 64 unrelated subjects with acromegaly/gigantism, and identified one female gigantism patient who had had increased copy number variation (CNV) threshold for GPR101 that was subsequently diagnosed as having XLAG syndrome on HD-aCGH. Employing a combination of HD-aCGH and novel ddPCR approaches, we have demonstrated, for the first time, that XLAG syndrome can be caused by variable degrees of somatic mosaicism for duplications at chromosome Xq26.3. Somatic mosaicism was shown to occur in sporadic males but not in females with XLAG syndrome, although the clinical characteristics of the disease were similarly severe in both sexes.
Albert Beckers, Maya Beth Lodish, Giampaolo Trivellin, Liliya Rostomyan, Misu Lee, Fabio R Faucz, Bo Yuan, Catherine S Choong, Jean-Hubert Caberg, Elisa Verrua, Luciana Ansaneli Naves, Tim D Cheetham, Jacques Young, Philippe A Lysy, Patrick Petrossians, Andrew Cotterill, Nalini Samir Shah, Daniel Metzger, Emilie Castermans, Maria Rosaria Ambrosio, Chiara Villa, Natalia Strebkova, Nadia Mazerkina, Stéphan Gaillard, Gustavo Barcelos Barra, Luis Augusto Casulari, Sebastian J Neggers, Roberto Salvatori, Marie-Lise Jaffrain-Rea, Margaret Zacharin, Beatriz Lecumberri Santamaria, Sabina Zacharieva, Ee Mun Lim, Giovanna Mantovani, Maria Chaira Zatelli, Michael T Collins, Jean-François Bonneville, Martha Quezado, Prashant Chittiboina, Edward H Oldfield, Vincent Bours, Pengfei Liu, Wouter W de Herder, Natalia Pellegata, James R Lupski, Adrian F Daly, and Constantine A Stratakis
X-linked acrogigantism (X-LAG) is a new syndrome of pituitary gigantism, caused by microduplications on chromosome Xq26.3, encompassing the gene GPR101, which is highly upregulated in pituitary tumors. We conducted this study to explore the clinical, radiological, and hormonal phenotype and responses to therapy in patients with X-LAG syndrome. The study included 18 patients (13 sporadic) with X-LAG and microduplication of chromosome Xq26.3. All sporadic cases had unique duplications and the inheritance pattern in two families was dominant, with all Xq26.3 duplication carriers being affected. Patients began to grow rapidly as early as 2–3 months of age (median 12 months). At diagnosis (median delay 27 months), patients had a median height and weight standard deviation scores (SDS) of >+3.9 SDS. Apart from the increased overall body size, the children had acromegalic symptoms including acral enlargement and facial coarsening. More than a third of cases had increased appetite. Patients had marked hypersecretion of GH/IGF1 and usually prolactin, due to a pituitary macroadenoma or hyperplasia. Primary neurosurgical control was achieved with extensive anterior pituitary resection, but postoperative hypopituitarism was frequent. Control with somatostatin analogs was not readily achieved despite moderate to high levels of expression of somatostatin receptor subtype-2 in tumor tissue. Postoperative use of adjuvant pegvisomant resulted in control of IGF1 in all five cases where it was employed. X-LAG is a new infant-onset gigantism syndrome that has a severe clinical phenotype leading to challenging disease management.
Liliya Rostomyan, Adrian F Daly, Patrick Petrossians, Emil Nachev, Anurag R Lila, Anne-Lise Lecoq, Beatriz Lecumberri, Giampaolo Trivellin, Roberto Salvatori, Andreas G Moraitis, Ian Holdaway, Dianne J Kranenburg - van Klaveren, Maria Chiara Zatelli, Nuria Palacios, Cecile Nozieres, Margaret Zacharin, Tapani Ebeling, Marja Ojaniemi, Liudmila Rozhinskaya, Elisa Verrua, Marie-Lise Jaffrain-Rea, Silvia Filipponi, Daria Gusakova, Vyacheslav Pronin, Jerome Bertherat, Zhanna Belaya, Irena Ilovayskaya, Mona Sahnoun-Fathallah, Caroline Sievers, Gunter K Stalla, Emilie Castermans, Jean-Hubert Caberg, Ekaterina Sorkina, Renata Simona Auriemma, Sachin Mittal, Maria Kareva, Philippe A Lysy, Philippe Emy, Ernesto De Menis, Catherine S Choong, Giovanna Mantovani, Vincent Bours, Wouter De Herder, Thierry Brue, Anne Barlier, Sebastian J C M M Neggers, Sabina Zacharieva, Philippe Chanson, Nalini Samir Shah, Constantine A Stratakis, Luciana A Naves, and Albert Beckers
Despite being a classical growth disorder, pituitary gigantism has not been studied previously in a standardized way. We performed a retrospective, multicenter, international study to characterize a large series of pituitary gigantism patients. We included 208 patients (163 males; 78.4%) with growth hormone excess and a current/previous abnormal growth velocity for age or final height >2 s.d. above country normal means. The median onset of rapid growth was 13 years and occurred significantly earlier in females than in males; pituitary adenomas were diagnosed earlier in females than males (15.8 vs 21.5 years respectively). Adenomas were ≥10 mm (i.e., macroadenomas) in 84%, of which extrasellar extension occurred in 77% and invasion in 54%. GH/IGF1 control was achieved in 39% during long-term follow-up. Final height was greater in younger onset patients, with larger tumors and higher GH levels. Later disease control was associated with a greater difference from mid-parental height (r=0.23, P=0.02). AIP mutations occurred in 29%; microduplication at Xq26.3 – X-linked acrogigantism (X-LAG) – occurred in two familial isolated pituitary adenoma kindreds and in ten sporadic patients. Tumor size was not different in X-LAG, AIP mutated and genetically negative patient groups. AIP-mutated and X-LAG patients were significantly younger at onset and diagnosis, but disease control was worse in genetically negative cases. Pituitary gigantism patients are characterized by male predominance and large tumors that are difficult to control. Treatment delay increases final height and symptom burden. AIP mutations and X-LAG explain many cases, but no genetic etiology is seen in >50% of cases.