Publications by authors named "Rosa Camerlingo"

21 Publications

  • Page 1 of 1

A Reversible Shift of Driver Dependence from EGFR to Notch1 in Non-Small Cell Lung Cancer as a Cause of Resistance to Tyrosine Kinase Inhibitors.

Cancers (Basel) 2021 Apr 22;13(9). Epub 2021 Apr 22.

Department of Advanced Biomedical Sciences, University "Federico II", 80131 Naples, Italy.

Notch1 plays a key role in epithelial-mesenchymal transition (EMT) and in the maintenance of cancer stem cells. In the present study we tested whether high levels of activated Notch1 in oncogene-driven NSCLC can induce a reversible shift of driver dependence from EGFR to Notch1, and thus causing resistance to EGFR inhibitors. Adherent cells (parental) and tumor spheres (TS) from NSCLC H1975 cells and patient-derived CD133-positive cells were tested for EGFR and Notch1 signaling cascade. The Notch1-dependent modulation of EGFR, NCID, Hes1, p53, and Sp1 were then analyzed in parental cells by binding assays with a Notch1 agonist, DLL4. TS were more resistant than parental cells to EGFR inhibitors. A strong upregulation of Notch1 and a concomitant downregulation of EGFR were observed in TS compared to parental cells. Parental cell exposure to DLL4 showed a dose-dependent decrease of EGFR and a simultaneous increase of NCID, Hes1, p53, and Sp1, along with the dislocation of Sp1 from the promoter. Furthermore, an enhanced interaction between p53 and Sp1 was observed in TS. In NSCLC cells, high levels of active Notch1 can promote a reversible shift of driver dependence from EGFR to Notch1, leading to resistance to EGFR inhibitors.
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http://dx.doi.org/10.3390/cancers13092022DOI Listing
April 2021

Next Generation Sequencing-Based Profiling of Cell Free DNA in Patients with Advanced Non-Small Cell Lung Cancer: Advantages and Pitfalls.

Cancers (Basel) 2020 Dec 17;12(12). Epub 2020 Dec 17.

Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori-IRCCS-Fondazione G. Pascale, 80131 Naples, Italy.

Lung cancer (LC) is the main cause of death for cancer worldwide and non-small cell lung cancer (NSCLC) represents the most common histology. The discovery of genomic alterations in driver genes that offer the possibility of therapeutic intervention has completely changed the approach to the diagnosis and therapy of advanced NSCLC patients, and tumor molecular profiling has become mandatory for the choice of the most appropriate therapeutic strategy. However, in approximately 30% of NSCLC patients tumor tissue is inadequate for biomarker analysis. The development of highly sensitive next generation sequencing (NGS) technologies for the analysis of circulating cell-free DNA (cfDNA) is emerging as a valuable alternative to assess tumor molecular landscape in case of tissue unavailability. Additionally, cfDNA NGS testing can better recapitulate NSCLC heterogeneity as compared with tissue testing. In this review we describe the main advantages and limits of using NGS-based cfDNA analysis to guide the therapeutic decision-making process in advanced NSCLC patients, to monitor the response to therapy and to identify mechanisms of resistance early. Therefore, we provide evidence that the implementation of cfDNA NGS testing in clinical research and in the clinical practice can significantly improve precision medicine approaches in patients with advanced NSCLC.
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http://dx.doi.org/10.3390/cancers12123804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766403PMC
December 2020

Patient-derived organoids as a potential model to predict response to PD-1/PD-L1 checkpoint inhibitors.

Br J Cancer 2019 11 31;121(11):979-982. Epub 2019 Oct 31.

Department of Precision Medicine, University of Campania L. Vanvitelli, Naples, Italy.

Selection of cancer patients for treatment with immune checkpoint inhibitors remains a challenge due to tumour heterogeneity and variable biomarker detection. PD-L1 expression in 24 surgical chordoma specimen was determined immunohistochemically with antibodies 28-8 and E1L3N. The ability of patient-derived organoids to detect treatment effects of nivolumab was explored by quantitative and qualitative immunofluorescence and FACS analysis. The more sensitive antibody, E1L3N (ROC = 0.896, p = 0.001), was associated with greater tumour diameters (p = 0.014) and detected both tumour cells and infiltrating lymphocytes in 54% of patients, but only 1-15% of their cells. Organoids generated from PD-L1-positive patients contained both tumour cells and PD-1/CD8-positive lymphocytes and responded to nivolumab treatment with marked dose-dependent diameter reductions of up to 50% and increased cell death in both PD-L1-positive and negative organoids. Patient-derived organoids may be valuable to predict individual responses to immunotherapy even in patients with low or no immunohistochemical PD-L1 expression.
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http://dx.doi.org/10.1038/s41416-019-0616-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889147PMC
November 2019

Conditioned medium of primary lung cancer cells induces EMT in A549 lung cancer cell line by TGF-ß1 and miRNA21 cooperation.

PLoS One 2019 25;14(7):e0219597. Epub 2019 Jul 25.

Thoracic Service, Department of Surgery, Memorial Sloan-Kettering Cancer Center, NY, United States of America.

The epithelial-mesenchymal transition (EMT) plays a key role in tumor progression, drug resistance and metastasis. Recently, numerous microRNA (miRNA) have been described to regulate EMT in tumor progression. In this study, we found that conditioned medium from the LC212 non-small-cell lung cancer (NSCLC) cell line (LC212-CM) induces morphological changes and overexpression of Vimentin, CD90, SMAD 2/3, SLUG and TWIST in A549 NSCLC cells, consistent with a mesenchymal phenotype. To identify the soluble mediators in LC212-CM involved in this phenomenon, we performed miRNA profiling and TGF-β1 quantification. We found that LC212-CM contains high levels of TGF-β1 as well as different secreted miRNAs. We focused our attention on Homo sapiens-microRNA21 (hsa-miR21), one of most relevant miRNA associated with lung cancer progression, metastasis and EMT. An hsa-miR21 antagomiR was able to prevent the LC212-CM-induced EMT phenotype in A549 cells. Furthermore, we found that TGF-β1 and hsa-miR21 cooperate in the induction of EMT in A549 cells. Intriguingly, TGF-β1 was found to induce hsa-miR21 expression in A549 cell, thus suggesting that the hsa-miR21 mediates at least in part the pro-EMT effects of TGF-β1. In conclusion, hsa-miR21 and TGF-β1 are involved in autocrine and paracrine circuits that regulate the EMT status of lung cancer cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0219597PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657837PMC
February 2020

Mutated Von Hippel-Lindau-renal cell carcinoma (RCC) promotes patients specific natural killer (NK) cytotoxicity.

J Exp Clin Cancer Res 2018 Dec 4;37(1):297. Epub 2018 Dec 4.

Functional Genomics, Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione "G. Pascale"-IRCCS, Via Semmola, 80131, Naples, Italy.

Background: Previous evidence demonstrated that restoration of wild type VHL in human renal cancer cells decreased in vitro NK susceptibility. To investigate on the role of tumoral VHL status versus NK capability in renal cancer patients, 51 RCC patients were characterized for VHL mutational status and NK function.

Methods: VHL mutational status was determined by direct DNA sequencing on tumor tissue. NK cytotoxicity was measured against specific target cells K562, VHL-wild type (CAKI-1) and VHL-mutated (A498) human renal cancer cells through externalization of CD107a and IFN-γ production. Activating NK receptors, NKp30, NKp44, NKp46, NKG2D, DNAM-1, NCAM-1 and FcγRIIIa were evaluated through quantitative RT-PCR. RCC tumoral Tregs were characterized as CD4CD25CD127Foxp3 and Treg function was evaluated as inhibition of T-effector proliferation.

Results: VHL mutations were detected in 26/55 (47%) RCC patients. IL-2 activated whole-blood samples (28 VHL-WT-RCC and 23 VHL-MUT-RCC) were evaluated for NK cytotoxicity toward human renal cancer cells A498, VHL-MUT and CAKI-1, VHL-WT. Efficient NK degranulation and increase in IFN-γ production was detected when IL-2 activated whole-blood from VHL-MUT-RCC patients were tested toward A498 as compared to CAKI-1 cells (CD107aNK: 7 ± 2% vs 1 ± 0.41%, p = 0.015; IFN-γNK: 6.26 ± 3.4% vs 1.78 ± 0.9% respectively). In addition, IL-2 activated NKs induced higher CD107a exposure in the presence of RCC autologous tumor cells or A498 as compared to SN12C (average CD107aNK: 4.7 and 2.7% vs 0.3% respectively at 10E:1 T ratio). VHL-MUT-RCC tumors were NKp46 cells infiltrated and expressed high NKp30 and NKp46 receptors as compared to VHL-WT-RCC tumors. A significant lower number of Tregs was detected in the tumor microenvironment of 13 VHL-MUT-RCC as compared to 13 VHL-WT-RCC tumors (1.84 ± 0.36% vs 3.79 ± 0.74% respectively, p = 0.04). Tregs isolated from VHL-MUT-RCC patients were less suppressive of patients T effector proliferation compared to Tregs from VHL-WT-RCC patients (Teff proliferation: 6.7 ± 3.9% vs 2.8 ± 1.1%).

Conclusions: VHL tumoral mutations improve NKs effectiveness in RCC patients and need to be considered in the evaluation of immune response. Moreover therapeutic strategies designed to target NK cells could be beneficial in VHL-mutated-RCCs alone or in association with immune checkpoints inhibitors.
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http://dx.doi.org/10.1186/s13046-018-0952-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6278085PMC
December 2018

Neutrophil Extracellular Traps as an Adhesion Substrate for Different Tumor Cells Expressing RGD-Binding Integrins.

Int J Mol Sci 2018 Aug 9;19(8). Epub 2018 Aug 9.

Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, 80145 Naples, Italy.

Neutrophil extracellular traps (NETs), in addition to their function as a host defense mechanism, play a relevant role in thrombus formation and metastatic dissemination of cancer cells. Here we screened different cancer cell lines endogenously expressing a variety of integrins for their ability to bind to NETs. To this end, we used NETs isolated from neutrophil-like cells as a substrate for adhesion assays of HT1080, U-87 MG, H1975, DU 145, PC-3 and A-431 cells. Levels of α5, αIIb, αv, β1, β3 and β5 chains were determined by western blot analysis in all cell lines and levels of whole integrins on the plasma membrane were assessed by fluorescence-activated cell sorting (FACS) analysis. We found that high levels of α5β1, αvβ3 and αvβ5 enhance cell adhesion to NETs, whereas low expression of α5β1 prevents cell attachment to NETs. Excess of cyclic RGD peptide inhibited cell adhesion to NETs by competing with fibronectin within NETs. The maximal reduction of such adhesion was similar to that obtained by DNase 1 treatment causing DNA degradation. Our findings indicate that NETs from neutrophil-like cells may be used as a substrate for large screening of the adhesion properties of cancer cells expressing a variety of RGD-binding integrins.
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http://dx.doi.org/10.3390/ijms19082350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121671PMC
August 2018

Effect of ABT-888 on the apoptosis, motility and invasiveness of BRAFi-resistant melanoma cells.

Int J Oncol 2018 Sep 27;53(3):1149-1159. Epub 2018 Jun 27.

Istituto Nazionale Tumori -IRCCS- 'Fondazione G. Pascale', 80131 Naples, Italy.

Melanoma is a molecularly heterogeneous disease with many genetic mutations and altered signaling pathways. Activating mutations in the BRAF oncogene are observed in approximately 50% of cutaneous melanomas and the use of BRAF inhibitor (BRAFi) compounds has been reported to improve the outcome of patients with BRAF-mutated metastatic melanoma. However, the majority of these patients develop resistance within 6-8 months following the initiation of BRAFi treatment. In this study, we examined the possible use of the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor, ABT-888 (veliparib), as a novel molecule that may be successfully employed in the treatment of BRAFi-resistant melanoma cells. Sensitive and resistant to BRAFi dabrafenib A375 cells were exposed to increasing concentrations of ABT-888. Cell viability and apoptosis were assessed by MTT assay and Annexin V-FITC analysis, respectively. The cell migratory and invasive ability was investigated using the xCELLigence technology and Boyden chamber assays, respectively. ABT-888 was found to reduce cell viability and exhibited pro-apoptotic activity in melanoma cell lines, independently from the BRAF/NRAS mutation status, in a dose-dependent manner, with the maximal effect being reached in the 25-50 µM concentration range. Moreover, ABT-888 promoted apoptosis in both the sensitive and resistant A375 cells, suggesting that ABT-888 may be useful in the treatment of BRAFi-resistant subsets of melanoma cells. Finally, in accordance with the involvement of PARP1 in actin cytoskeletal machinery, we found that the cytoskeletal organization, motility and invasive capability of both the A375 and A375R cells decreased upon exposure to 5 µM ABT-888 for 24 h. On the whole, the findings of this study highlight the pivotal role of PARP1 in the migration and invasion of melanoma cells, suggesting that ABT-888 may indeed be effective, not only as a pro-apoptotic drug for use in the treatment of BRAFi-resistant melanoma cells, but also in suppressing their migratory and invasive activities.
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http://dx.doi.org/10.3892/ijo.2018.4457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6065454PMC
September 2018

Antioxidant and anti-proliferative properties of extracts from heterotrophic cultures of .

Nat Prod Res 2019 Jun 15;33(11):1659-1663. Epub 2018 Jan 15.

a Dipartimento di Biologia , Università di Napoli Federico II , Napoli , Italy.

This study explores the possibility to use the extremophilic microalga (strain 064) as a source of natural biomolecules with beneficial and protective effects on human health. was cultivated in heterotrophy conditions and cells extracts for their antioxidant and anti-proliferative properties were tested. extracts showed high antioxidant power tested through ABTS assay and revealed high glutathione and phycocyanin contents. Based on Annexin-V FITC/propidium iodide and MTT analysis, algae extracts inhibited the proliferation of human adenocarcinoma A549 cells (51.2% inhibition) through the induction of apoptosis without cell cycle arrest. Besides, cytotoxicity and cytometry assays showed a positive pro-apoptotic mechanism. On these bases, we suggest that from heterotrophic culture, for its therapeutic potential, could be considered a good candidate for further studies with the aim to isolate bioactive anti-cancer molecules.
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http://dx.doi.org/10.1080/14786419.2018.1425853DOI Listing
June 2019

Phenotype characterization of human melanoma cells resistant to dabrafenib.

Oncol Rep 2017 Nov 18;38(5):2741-2751. Epub 2017 Sep 18.

Institute of Genetics and Biophysics (IGB), A. Buzzati-Traverso, CNR, I-80131 Naples, Italy.

In the present study, the phenotype of melanoma cells resistant to dabrafenib (a B-RAF inhibitor) was investigated, to shed more light on melanoma resistance to B-RAF inhibition. Melanoma cells resistant to dabrafenib were generated using 3 different cell lines, A375, 397 and 624.38, all carrying B-RAFV600E, and they were characterized by cytofluorometric analysis, Ion Torrent technology, immunofluorescence and biochemistry. All dabrafenib-resistant cells showed, in addition to a re-activation of MAPK signaling, morphological changes compared to their sensitive counterparts, accompanied by an increase in CD90 (mesenchymal marker) expression and a decrease in E-cadherin (epithelial marker) expression, suggesting an epithelial-to-mesenchymal-like phenotypic transition. However, melanoma cells with TGF-β1-induced epithelial-to-mesenchymal transition (EMT) were more sensitive to dabrafenib treatment compared to the sensitivity noted in the non-TGF‑β1‑induced EMT melanoma cells, suggesting that TGF-β1-induced EMT was not associated with dabrafenib resistance. Although dabrafenib-resistant cells exhibited increased cell motility and E-cadherin/vimentin reorganization, as expected in EMT, all of them showed unvaried E-cadherin mRNA and unchanged Snail protein levels, while Twist1 protein expression was decreased with the exception of A375 dabrafenib-resistant melanoma cells, where it was unaffected. These findings suggest a distinct active EMT-like process adopted by melanoma cells under drug exposure. Furthermore, dabrafenib-resistant cells exhibited stem cell-like features, with Oct4 translocation from the cytoplasm to peri-nuclear sites and nuclei, and increased CD20 expression. In conclusion, our data, in addition to confirming that resistance to dabrafenib is dependent on re-activation of MAPK signaling, suggest that this resistance is linked to a distinct active EMT-like process as well as stem-cell features adopted by melanoma cells.
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http://dx.doi.org/10.3892/or.2017.5963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780027PMC
November 2017

New cell block containing agarose for cytopathological diagnosis of tumor samples.

Diagn Cytopathol 2017 Nov 30;45(11):1057-1060. Epub 2017 Jun 30.

Pathology Unit, Istituto Nazionale Tumori "Fondazione G. Pascale" - IRCCS, Naples, Italy.

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http://dx.doi.org/10.1002/dc.23778DOI Listing
November 2017

The Hydrogen Sulfide Releasing Molecule Acetyl Deacylasadisulfide Inhibits Metastatic Melanoma.

Front Pharmacol 2017 27;8:65. Epub 2017 Feb 27.

Department of Pharmacy, University of Naples Federico II Naples, Italy.

Melanoma is the most common form of skin cancer. Given its high mortality, the interest in the search of preventive measures, such as dietary factors, is growing significantly. In this study we tested, and , the potential anti-cancer effect of the acetyl deacylasadisulfide (ADA), a vinyl disulfide compound, isolated and purified from asafoetida a foul-smelling oleo gum-resin of dietary and medicinal relevance. ADA markedly suppressed proliferation of human melanoma cell lines by inducing apoptosis. Moreover, treatment of melanoma cells with ADA reduced nuclear translocation and activation of NF-κB, decreased the expression of the anti-apoptotic proteins c-FLIP, XIAP, and Bcl-2 and inhibited the phosphorylation and activation of both AKT and ERK proteins, two of the most frequently deregulated pathways in melanoma. Finally, the results obtained were substantiated by the findings that ADA significantly and dose-dependently reduced lung metastatic foci formation in C57BL/6 mice. In conclusion, our findings suggest that ADA significantly inhibits melanoma progression and could represent an important lead compound for the development of new anti-metastatic agents.
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http://dx.doi.org/10.3389/fphar.2017.00065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5326790PMC
February 2017

Integrin-dependent cell adhesion to neutrophil extracellular traps through engagement of fibronectin in neutrophil-like cells.

PLoS One 2017 6;12(2):e0171362. Epub 2017 Feb 6.

Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, Naples, Italy.

Neutrophil extracellular traps (NETs), originally recognized as a host defense mechanism, were reported to promote thrombosis and metastatic dissemination of cancer cells. Here we tested the role of integrins α5β1 and ανβ3 in the adhesion of cancer cells to NETs. Neutrophil-like cells stimulated with calcium ionophore (A23187) were used as a stable source of cell-free NETs-enriched suspensions. Using NETs as an adhesion substrate, two human K562 cell lines, differentially expressing α5β1 and ανβ3 integrins, were subjected to adhesion assays in the presence or absence of DNAse 1, blocking antibodies against α5β1 or ανβ3, alone or in combination with DNAse 1, and Proteinase K. As expected DNAse 1 treatment strongly inhibited adhesion of both cell lines to NETs. An equivalent significant reduction of cell adhesion to NETs was obtained after treatment of cells with blocking antibodies against α5β1 or ανβ3 indicating that both integrins were able to mediate cell adhesion to NETs. Furthermore, the combination of DNAse 1 and anti-integrin antibody treatment almost completely blocked cell adhesion. Western blot analysis and immunoprecipitation experiments showed a dose-dependent increase of fibronectin levels in samples from stimulated neutrophil-like cells and a direct or indirect interaction of fibronectin with histone H3. Finally, co-immunolocalization studies with confocal microscopy showed that fibronectin and citrullinated histone H3 co-localize inside the web-structure of NETs. In conclusion, our study showed that α5β1 and ανβ3 integrins mediate cell adhesion to NETs by binding to their common substrate fibronectin. Therefore, in addition to mechanical trapping and aspecific adsorption of different cell types driven by DNA/histone complexes, NETs may provide specific binding sites for integrin-mediated cell adhesion of neutrophils, platelets, endothelial and cancer cells thus promoting intimate interactions among these cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171362PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293257PMC
August 2017

Combination of antibodies directed against different ErbB3 surface epitopes prevents the establishment of resistance to BRAF/MEK inhibitors in melanoma.

Oncotarget 2015 Sep;6(28):24823-41

Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione G. Pascale", Naples, Italy.

Patients with metastatic melanoma bearing V600 mutations in BRAF oncogene clinically benefit from the treatment with BRAF inhibitors alone or in combination with MEK inhibitors. However, a limitation to such treatment is the occurrence of resistance. Tackling the adaptive changes helping cells survive from drug treatment may offer new therapeutic opportunities. Very recently the ErbB3 receptor has been shown to act as a central node promoting survival of BRAF mutated melanoma. In this paper we first demonstrate that ErbB3/AKT hyperphosphorylation occurs in BRAF mutated melanoma cell lines following exposure to BRAF and/or MEK inhibitors. This strongly correlates with increased transcriptional activation of its ligand neuregulin. Anti-ErbB3 antibodies impair the establishment of de novo cell resistance to BRAF inhibition in vitro. In order to more potently ablate ErbB3 activity we used a combination of two anti-ErbB3 antibodies directed against distinct epitopes of its extracellular domain. These two antibodies in combo with BRAF/MEK inhibitors potently inhibit in vitro cell growth and tumor regrowth after drug withdrawal in an in vivo xenograft model. Importantly, residual tumor masses from mice treated by the antibodies and BRAF/ERK inhibitors combo are characterized almost exclusively by large necrotic areas with limited residual areas of tumor growth. Taken together, our findings support the concept that triple therapy directed against BRAF/MEK/ErbB3 may be able to provide durable control of BRAF mutated metastatic melanoma.
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http://dx.doi.org/10.18632/oncotarget.4485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694796PMC
September 2015

Assessing a novel immuno-oncology-based combination therapy: Ipilimumab plus electrochemotherapy.

Oncoimmunology 2015 Jun 22;4(6):e1008842. Epub 2015 May 22.

Melanoma, Cancer Immunotherapy, and Innovative Therapy Unit; Istituto Nazionale Tumori Fondazione "G. Pascale" ; Naples, Italy.

Melanoma is responsible for most skin cancer-related deaths and is one of the most common cancers diagnosed in young adults. In melanoma, tumors can become established by activation of the negative regulator of cytotoxic T lymphocytes (CTLs), CTL antigen-4 (CTLA-4). Ipilimumab blocks the interaction of CTLA-4 with CD80/CD86 and augments T-cell activation and proliferation. In electrochemotherapy (ECT), local application of short high-voltage pulses renders cell membranes transiently permeable to chemotherapeutic drugs. The combination of ipilimumab and ECT may be beneficial for the treatment of metastatic melanoma; however, no prospective data are available to date. Here, we report the retrospective analysis of patients treated with ipilimumab in an expanded access program (EAP) who also received ECT. Fifteen patients with previously treated metastatic melanoma who received ipilimumab 3 mg/kg every three weeks for four cycles and underwent ECT for local disease control and/or palliation of cutaneous lesions with bleomycin 15 mg/m after the first ipilimumab infusion were included in the analysis. Over the study period, a local objective response was observed in 67% of patients (27% complete response [CR] and 40% partial response [PR]). According to immune-related response criteria, a systemic response was observed in nine patients (five PR and four stable disease [SD]), resulting in a disease control rate of 60%. Evaluation of circulating T-regulatory (T-reg) cells demonstrated significant differences between responders and non-responders. Overall, treatment was well-tolerated and without notable toxicity. In conclusion, the combination of ipilimumab and ECT appears to be beneficial to patients with advanced melanoma, warranting further investigation in prospective trials.
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http://dx.doi.org/10.1080/2162402X.2015.1008842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485758PMC
June 2015

Isolation and assessment of the in vitro anti-tumor activity of smenothiazole A and B, chlorinated thiazole-containing peptide/polyketides from the Caribbean sponge, Smenospongia aurea.

Mar Drugs 2015 Jan 16;13(1):444-59. Epub 2015 Jan 16.

The NeaNat Group, Dipartimento di Farmacia, Università degli Studi di Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy.

The study of the secondary metabolites contained in the organic extract of Caribbean sponge Smenospongia aurea led to the isolation of smenothiazole A (3) and B (4), hybrid peptide/polyketide compounds. Assays performed using four solid tumor cell lines showed that smenothiazoles exert a potent cytotoxic activity at nanomolar levels, with selectivity over ovarian cancer cells and a pro-apoptotic mechanism.
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http://dx.doi.org/10.3390/md13010444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306946PMC
January 2015

CD90 expression in atypical meningiomas and meningioma metastasis.

Am J Clin Pathol 2014 Jun;141(6):841-9

From the Pathology Department,

Objectives: Meningiomas are slow-growing intracranial/intraspinal tumors, with a wide range of histopathologic variants. The more aggressive atypical and malignant types can disseminate via the venous system, lymphatic, system, or cerebrospinal fluid, with the lung and pleura being the most common sites of extracranial metastases. A case of metastatic meningioma with high expression of CD90 was spotted during a review of flow cytometry data for lung malignancies. Therefore, we have analyzed CD90 expression in a series of meningioma metastases with their corresponding primary tumors and in a series of 92 primary meningioma tumors.

Methods: In addition to flow cytometry and immunohistochemical analysis of the case, a series of meningiomas and relative metastases has been evaluated for CD90 immunohistochemical expression. Furthermore, an immunohistochemical analysis has been conducted in a tissue microarray, including typical and atypical meningiomas.

Results: CD90 had high expression in three of four cases of metastases and in their corresponding primary atypical meningioma. In addition, CD90 was significantly expressed in atypical rather than in typical meningiomas (P = .003). However, the correlation of CD90 with patient survival reveals only a trend of statistical association with extracranial metastases.

Conclusions: CD90 is a biomarker overexpressed in atypical meningioma, with a potential role in metastatic switch of this tumor.
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http://dx.doi.org/10.1309/AJCP8Y0EBQMKWQEIDOI Listing
June 2014

The role of CD44+/CD24-/low biomarker for screening, diagnosis and monitoring of breast cancer.

Oncol Rep 2014 Mar 23;31(3):1127-32. Epub 2013 Dec 23.

Department of Experimental Oncology, National Cancer Institute, G. Pascale, Naples, Italy.

Cancer stem cells (CSCs) have been defined as 'a cell within a tumor that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor'. The CSC hypothesis postulates that a small subpopulation of cancer cells drives tumor initiation, growth and metastasis. CSCs have been isolated from breast cancer using CD44+/CD24-/low phenotype. The purpose of the present study was to evaluate the expression of CD44+/CD24-/low in two diverse breast carcinomas (ductal and lobular), and to determine the correlation between expression of CD44+/CD24-/low, and clinicopathological characteristics starting from human fresh breast cancer specimens. We analyzed specimens from 57 patients using CD44 and CD24 markers by flow cytometry and immunohistochemistry and correlated the CD44+/CD24-/low phenotype with clinicopathological characteristics. Moreover, mammosphere formation was tested. In all specimens tested, CD44+/CD24-/low phenotype was detectable with mean percentage of 4.73% as confirmed also by immunohistochemical analyses. A significant statistical association was found among these phenotypic groups and age, grade G3, estrogen and progesterone receptor, Ki-67 as well as lymph node metastasis. No correlation was found for histological type. In conclusion, our data showed that CD44+/CD24-/low phenotype was found at a high frequency in tumors pT2, G3, pN3, positive for Ki-67, and negative for estrogen and progesterone receptors highlighting the hypothesis that CD44+/CD24-/low profile correlates with the more aggressive clinical-pathological features of the disease.
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http://dx.doi.org/10.3892/or.2013.2943DOI Listing
March 2014

Epithelial to mesenchymal transition by TGFβ-1 induction increases stemness characteristics in primary non small cell lung cancer cell line.

PLoS One 2011 30;6(6):e21548. Epub 2011 Jun 30.

Department of Experimental Oncology, National Cancer Institute, Naples, Italy.

Background: Cancer Stem Cells (CSCs) hypothesis asserts that only a small subset of cells within a tumour is capable of both tumour initiation and sustainment. The Epithelial-Mesenchymal Transition (EMT) is an embryonic developmental program that is often activated during cancer invasion and metastasis. The aim of this study is to shed light on the relationship between EMT and CSCs by using LC31 lung cancer primary cell line.

Materials And Methods: A549 and LC31 cell lines were treated with 2 ng/ml TGFβ-1 for 30 days, and 80 days, respectively. To evaluate EMT, morphological changes were assessed by light microscopy, immunofluorescence and cytometry for following markers: cytokeratins, e-cadherin, CD326 (epithelial markers) and CD90, and vimentin (mesenchymal markers). Moreover, RT-PCR for Slug, Twist and β-catenin genes were performed. On TGFβ-1 treated and untreated LC31 cell lines, we performed stemness tests such as pneumospheres growth and stem markers expression such as Oct4, Nanog, Sox2, c-kit and CD133. Western Blot for CD133 and tumorigenicity assays using NOD/SCID mice were performed.

Results: TGFβ-1 treated LC31 cell line lost its epithelial morphology assuming a fibroblast-like appearance. The same results were obtained for the A549 cell line (as control). Immunofluorescence and cytometry showed up-regulation of vimentin and CD90 and down-regulation of cytocheratin, e-cadherin and CD326 in TGFβ-1 treated LC31 and A549 cell lines. Slug, Twist and β-catenin m-RNA transcripts were up-regulated in TGFβ-1 treated LC31 cell line confirming EMT. This cell line showed also over-expression of Oct4, Nanog, Sox2 and CD133, all genes of stemness. In addition, in TGFβ-1 treated LC31 cell line, an increased pneumosphere-forming capacity and tumours-forming ability in NOD/SCID mice were detectable.

Conclusions: The induction of EMT by TGFβ-1 exposure, in primary lung cancer cell line results in the acquisition of mesenchymal profile and in the expression of stem cell markers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021548PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128060PMC
December 2011

Establishment and phenotypic characterization of the first human pulmonary blastoma cell line.

Lung Cancer 2011 Apr 17;72(1):23-31. Epub 2010 Aug 17.

Department of Experimental Oncology, National Cancer Institute, Via Mariano Semmola, 80131 Naples, Italy.

Background: Sarcomatoid carcinomas are a group of poorly differentiated carcinomas containing a sarcomatoid component with the presence of epithelial-mesenchymal transition.

Materials And Methods: To obtain a stabilized cell line, three mediums were used: IMDM, BEBM and IMDM/BEBM. CD44, CD29, CD90, CD133, CD326 antigens, cytokeratins and vimentin were tested by flow cytometry and immunofluorescence assay. Side population, spheres formation, tumorigenicity in vitro and in vivo and cell cycle analysis were analyzed.

Results: At day of surgery, cytometric analysis revealed that CD133, CD90 and CD326 levels were very low, CD29 and CD44 were about 80%. After 30 days of culture, CD133 levels increased up to 30%, all cells expressed CD90 and vimentin markers and CD326 was lost. The cells grew only in IMDM/BEBM medium. The cell population was heterogeneous with epithelial and mesenchymal cells. After about 15-30 days, only fibroblast-like cells were observed. LC114 cells were not able to grow as spheres and they did not show a side-population phenotype. The cell cycle analysis confirmed an aneuploid population in the tissue and normal diploid population in cell line.

Conclusions: We selected, characterized and stabilized a primary cell line of human pulmonary blastoma by the expression both of stemness and differentiation markers and confirmed the presence of the marker CD133.
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http://dx.doi.org/10.1016/j.lungcan.2010.07.009DOI Listing
April 2011

The role of CD133 in the identification and characterisation of tumour-initiating cells in non-small-cell lung cancer.

Eur J Cardiothorac Surg 2009 Sep 22;36(3):446-53. Epub 2009 May 22.

Department of Experimental Oncology, National Cancer Institute, Naples, Italy.

Objective: Emerging evidence suggests that specific sub-populations of cancer cells with stem cell characteristics within the bulk of tumours are implicated in the pathogenesis of heterogeneous malignant tumours. The cells that drive tumour growth have been denoted cancer-initiating cells or cancer stem cells (hereafter CSCs). CSCs have been isolated initially from leukaemias and subsequently from several solid tumours including brain, breast, prostate, colon and lung cancer. This study aimed at isolating and characterising the population of tumour-initiating cells in non-small-cell lung cancer (NSCLC).

Methods: Specimens of NSCLC obtained from 89 patients undergoing tumour resection at the Cancer National Institute of Naples were analysed. Three methods to isolate the tumour-initiating cells were used: (1) flow cytometry analysis for identification of positive cells for surface markers such as CD24, CD29, CD31, CD34, CD44, CD133 and CD326; (2) Hoechst 33342 dye exclusion test for the identification of a side-population characteristic for the presence of stem cells; (3) non-adherent culture condition able to form spheres with stem cell-like characteristics. Definition of the tumourigenic potential of the cells through soft agar assay and injection into NOD/SCID mice were used to functionally define (in vitro and in vivo) putative CSCs isolated from NSCLC samples.

Results: Upon flow cytometry analysis of NSCLC samples, CD133-positive cells were found in 72% of 89 fresh specimens analysed and, on average, represented 6% of the total cells. Moreover, the number of CD133-positive cells increased markedly when the cells, isolated from NSCLC specimens, were grown as spheres in non-adherent culture conditions. Cells from NSCLC, grown as spheres, when assayed in soft agar, give rise to a 3.8-fold larger number of colonies in culture and are more tumourigenic in non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice compared with the corresponding adherent cells.

Conclusions: We have isolated and characterised a population of CD133-positive cells from NSCLC that is able to give rise to spheres and can act as tumour-initiating cells.
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http://dx.doi.org/10.1016/j.ejcts.2009.03.063DOI Listing
September 2009

Paroxysmal nocturnal hemoglobinuria: significant association with specific HLA-A, -B, -C, and -DR alleles in an Italian population.

Hum Immunol 2008 Mar 10;69(3):202-6. Epub 2008 Mar 10.

Dipartimento di Oncologia Sperimentale, Istituto Nazionale Tumori di Napoli, Napoli, Italy.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the expansion of a PIG-A mutated hematopoietic stem cell. An immune-mediated origin has been suggested for this disease. Because HLA genes represent a susceptibility factor for autoimmunity, we investigated HLA genotype in 42 Italian PNH patients compared with 301 control subjects of the same ethnic origin. A significantly increased frequency of the HLA class I alleles A*0201 (p < 0.05), B*1402 (p < 0.001), and Cw*0802 (p < 0.005), and of the HLA class II DRB1*1501 (p < 0.01) with the linked DQB1*0602 (p
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http://dx.doi.org/10.1016/j.humimm.2008.02.001DOI Listing
March 2008