P 1
ACTIVITY OF A NEW RUTHENIUM COMPLEX ON A MODEL OF BREAST CANCER
Gretta Veronica Badillo Pazmay(1) - Maura Montani (1) - Fabio Marchetti (2)
- Corrado Di Nicola (2) - Riccardo Pettinari (3) - Claudio Pettinari (3) - Alba
Hysi (4) - Stefano Ferraro (5) - Cristina Marchini (6) - Manuela Iezzi (4) - Giu-
lio Lupidi (7) - Augusto Amici (6)
School of Bioscience and Veterinary Medicine, University of Camerino, Camerino (MC), Italy (1) - School of Science and Technology, University of Camerino, Camerino, Italy (2) - School of Pharmacy, University of Camerino, Camerino, Italy (3) - Department of Medicine and Aging Sci-
ences. Aging Research Center (CeSI)Università di Chieti. Via dei Vesti- ni,31 –66100 Chieti (4) - Scuola di Scienze Ambientali- Università di
Camerino. via S.Agostino 1, 62032 Camerino (MC) (5) - School of Biosci-
ence and Veterinary Medicine, Via Gentile III da Varano 62032 Camerino (MC) (6) - School of Pharmacy, Via S. Agostino 1, 62032 Camerino (MC) (7)
Introduction: Ruthenium shows patterns of antitumor activity
and clinical toxicity that are different from those of platinum. In addition, some ruthenium complexes although displaying low cytotoxicity against primary tumors have greater antimetastatic effects mediating the inhibition of metastatic progression[1]. Our efforts are aimed at testing this agent and its derivatives, in a rich group of cell lines representing the complexity of cancer disease and detecting possible mechanism of action [2-4].
Methods: NAMI-A was prepared according to a patented pro-
cedure (Mestroni et al., 1998); Cisplatin was obtained by Sigma Chemical Co. (St. Louis, MO); UNICAM1 complex was prepa- red into our Inorganic Chemistry section of the University of Camerino. We evaluated the effects UNICAM1, NAMI-A and
85
Cisplatin on cell viability and motility through MTT and Wound-healing assays, respectively. We also investigated the effects on Tumor Growth; by i.p. treatment, on the body weight, retention of Ru and Pt in tissues of interest (histological analy- sis) and in serum.
Results: UNICAM1, IC50 value, is about 100 times higher than
Cisplatin one, demonstrating the absence of toxicity. UNICAM1 proved to inhibit cell motility, with an inhibition of 60% and 75% after 24 and 48h, respectively. In vivo experi- ments, showed an inhibitory effect of UNICAM1 on tumor growth (q1x6 and q3X4 treatments); particularly with the q3x4 treatment, that looks like Cisplatin effect. In addition, we reco- vered the presence of Ru (UNICAM 1 and NAMI-A) and Pt (Cisplatin) traces in Serum vs time, and in the kidneys and liver after ~1 month of final dose; demonstrating a high clearance ability for UNICAM1. The body weight decrease was monito- red obtaining a physiological course of growth with UNICAM 1 which looks like the group of animals without chemotherapeutic treatment.
Conclusion: The success of Pt-drugs has prompted research on
other transition metals, which has led to the identification of gold and ruthenium compounds with high antitumor and anti- metastatic activity. Consequently, metallodrugs appear to have the potential to achieve powerful new therapeutic effects, which cannot be induced by organic compounds. It is likely that this field will retain a high level of research interest. New synthesis and studies of metallodrugs are projected to improve the quality of life of patients and play a key role in chemotherapy and ra- diotherapy within this century.
References
1. Marchetti, F.; Pettinari, C.; Pettinari, R.; Cerquetella, A.; Di Nicola, C.; Macchioni, A.; Zuccaccia, D.; Monari, M.; Piccinel- li, F. Inorg. Chem. 2008, 47, 11593.
2. Gagliardi R, Sava G, Pacor S, Mestroni G, Alessio E (1994) Antimetastatic action and toxicity on healthy tissues of Na[trans- RuCl4(DMSO)Im] in the mouse. Clin Exp Metastasis 12:93–100.
3. F. Merigo, M. Montani, F. Boschi, P. Marzola, R. Orru`, P. Farace, A. Sbarbati and A. Amici. Mesenchymal stem cells share molecular signature with mesenchymal tumor cells and favor early tumor growth in syngeneic mice. Oncogene (2008) 27, 2542–2551.
4. Mirco Galie, Carlo Sorrentino, Maura Montani, Luigi Micos- si, Emma Di Carlo. Mammary carcinoma provides highly tu- mourigenic and invasive reactive stromal cells. Carcinogenesis vol.26 no.11 pp.1868–1878, 2005.
P 2
SLAMF1/CD150 IS A SIGNALING RECEPTOR EXPRESSED BY A SUBSET OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS CHARACTERIZED BY A FAVORABLE PROGNOSIS.
Cinzia Bologna (1) - Roberta Buonincontri (1) - Sara Serra (1) - Tiziana Vaisitti (1) - Valentina Audrito (1) - Davide Brusa (1) - Davide Rossi (2) - Marta Coscia (3) - Gianluca Gaidano (2) - Silvia Deaglio (1)
Department of Medical Sciences, University of Turin and HuGeF, Turin, Italy (1) - Division of Hematology, Department of Translational Medicine,
“Amedeo Avogadro” University of Eastern Piedmont, Novara, Italy (2) -
Division of Hematology, AO Città della Salute e della Scienza, Turin, Italy
(3)
Human SLAMF-1 (signaling lymphocytic activation molecule) is expressed on hematopoietic cells where it acts as a co- stimulatory molecule through self-interactions. Moreover, it performs as a microbial sensor, regulating bacterial phagosome functions through an ubiquitous cellular autophagic machinery. We investigated the role of SLAMF-1 in human B cells, ex- ploiting chronic lymphocytic leukemia (CLL) as a model. CLL is characterized by the expansion of a monoclonal population of mature B lymphocytes, with a highly variable clinical course. We demonstrated that SLAMF-1 is expressed at variable levels by CLL cells and marks the subset characterized by a good prognosis. Silencing of SLAMF1 expression in Mec-1 cell line led to a down-modulation of pathways connected to cell death, intracellular vescicle formation and recirculation, as determined by global gene expression analysis. Moreover, the apoptotic re- sponse to fludarabine treatment observed in control Mec-1 cells (constitutively SLAMF-1+) was completely lost in silenced
cells. Consistent with previous findings, fludarabine activated autophagy in the Mec-1 cells. However, in SLAMF1- cells no
modulation of the autophagic flux was highlighted, suggesting that deletion of the molecule is responsible for this phenome- non. In line with this finding, fludarabine responses were differ- ent in CLL patients divided according to SLAMF-1 expression, as well as in SLAMF-1high vs low cells separated through cell
sorting from the same patient. Functional experiments con- firmed that the engagement of the receptor initiates a signaling cascade that involves the direct interaction with the adaptor molecule Eat-2 and that converges on the activation of the MAP kinases. Prolonged engagement of SLAMF-1 led to the appear- ance of autophagic vesicles and to the increase of LC3B for- mation. The modulation of autophagy was mediated by ROS and by the sequential phosphorylation of Jnk1/2 and Bcl-2: the final result was the release of Beclin-1 from Bcl-2 and the con- sequent assembly of the autophagic complex, including Vps34. Taken together, these results suggest that SLAMF-1 could rep- resent a novel marker for the subset of CLL patients with an indolent clinical course. These results also suggest a link be- tween the activation of the autophagic process and a milder form of the diseae, with a better response to fludarabine treat- ment.
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P 3
ACTIVATED D16HER2 HOMODIMERS AND SRC KINASE SIGNALING AXIS IS A PREDICTOR OF TRASTUZUMAB BENEFIT.
Lorenzo Castagnoli (1) - Manuela Iezzi (2) - Valentina Ciravolo (1) - Gaia
Cristina Ghedini (1) - Tiziana Triulzi (1) - Patrizia Gasparini (1) - Roberta
Zappasodi (3) - Patrizia Nanni (4) - Elda Tagliabue (1) - Serenella Pupa (1)
Fondazione IRCCS - Istituto Nazionale dei Tumori, Dept. Experimental Oncology and Molecular Medicine, Milan, Italy (1) - Aging Research Cen-
tre, G. d’Annunzio University, Chieti, Italy (2) - Fondazione IRCCS - Istituto
Nazionale dei Tumori, “C. Gandini” Medical Oncology, Bone Marrow Transplantation Unit, Milan, Italy (3) - Department of Experimental Pathol-
ogy, University of Bologna, Bologna, Italy (4)
Introduction: Almost 90% of HER2 positive breast cancers
patients (BCs) express the splice variant of HER2 lacking of exon 16 (d16HER2). This deletion promotes the formation of stable activated d16HER2 homodimers. The oncogenic activity and Trastuzumab susceptibility of d16HER2-positive mammary tumors, as well as the relationship of d16HER2 with wild type (WT) HER2-driven pathological and clinical features in human HER2-overexpressing BCs, await clarification. Toward this aim, we compared survival and tumor multiplicity of transgenic (tg) mouse models for the human d16 and WT HER2 isoforms and Trastuzumab activity in mice orthotopically transplanted with derived mammary tumor cell lines transgenically express- ing d16 and WT HER2. We also analyzed functional relation- ship between activated d16HER2 and Src kinase (pSrc) and evaluated in HER2-positive BCs a potential association between the expression of d16HER2 and pSrc and their impact on prog- nosis.
Materials and methods: We evaluated: d16HER2- and
WTHER2-driven tumorigenesis and Trastuzumab activity in tg and parental FVB mice; downstream signaling axes by western blot of spontaneous d16 and WT HER2 tumor lysates; expres- sion of both HER2 isoforms and pSrc in murine and human specimens by immunofluorescence using confocal microscopy. We performed a gene expression analysis of human HER2- positive BCs treated adjuvantly with Trastuzumab on the Illu- mina Whole-Genome DASL® platform and data were analyzed
according to d16HER2 and pSrc expression levels using GSEA bioinformatic tool.
Results: We revealed that d16HER2 significantly accelerates
mammary tumorigenesis (p<0.001) and responds more effi- ciently to Trastuzumab (p<0.001) compared to WTHER2. Analysis of signaling downstream of d16HER2 and WTHER2 revealed that only activated d16HER2 is significantly functional and directly linked to expression of pSrc. In HER2-positive BCs we found a significant correlation between high levels of d16HER2 and pSrc (p=0.0016), suggesting that expression of activated d16HER2 is mirrored in high Src activity, consistent with results in tg mice. HER2-positive BCs who expressed high pSrc-d16HER2 levels exhibited the greatest benefits of Trastuzumab treatment (p=0.022).
Conclusions: Our data provide evidence that d16HER2-
positive tumors are significantly responsive to Trastuzumab and that high expression of activated d16HER2 and Src represents a signaling axis marking Trastuzumab susceptibility.
Supported by AIRC and Minister of Health
P 4
ROLE OF ZNF224 IN CHRONIC LYMPHOCYTIC LEUKEMIA
Elena Cesaro (1) - Giorgia Montano (1) - Michela Ciano (1) - Dario Bruzzese (2) - Paola Costanzo (1)
Department of Molecular Medicine and Medical Biotechnologies, Univer- sity of Naples Federico II, Naples, Italy (1) - Department of Medical Statis-
tics, University of Naples Federico II, Naples, Italy (2)
Introduction: Chronic Lymphocytic Leukemia (CLL) is an in-
dolent non-Hodgkin's lymphoma and the most common type of leukemias in adult. It is characterized by the accumulation of lymphocytes with impaired apoptosis.
ZNF224 is a member of the KRAB-zinc finger family of tran- scription factors, that inhibits the transcription of its target gene through the interaction with KAP1, the universal co-repressor of KRAB-ZFPs. KAP1 in turn recruits activities modifying the chromatin structure to silence gene expression. Also, the tran- scriptional repression activity of ZNF224 required the methyla- tion of H4R3 by PRMT5, a type II protein arginine methyltrans- ferase, implicated in signaling and transcription regulation. Morever, ZNF224 acts as a transcriptional co-regulator of of the zinc finger protein Wilms’tumor 1 (WT1), thus playing a criti- cal role in the regulation of apoptotic events in chronic mye- logenous leukemia. In this study we examine the function of ZNF224 in B cell chronic lymphocytic leukemia. We analyzed the expression levels of ZNF224 and Cyclin D3, a protein with oncogenic potential and a key cell cycle regulatory component, in CLL patients.
Materials and methods: The study was conduced on peripher-
al blood sample obtained from 60 CLL patients. Patients either were untreated or had received no treatment for leatest one years before the study. A ZNF224 and Cyclin D3 mRNA levels were estimated by Real-Time PCR using SYBR Green
Results: We observed that CLL cells obtained from patient
samples exhibited higher level of ZNF224 and Cyclin D3 than lymphocyte from healthy donors (p<0,001). Morever, we ob- served a positive correlation between ZNF224 and Cyclin D3 expression and between ZNF224 and lymphocyte number (ALC) in CLL patients, while, there was no association between ZNF224 and the lymphocyte doubling time (LTD), thus leading us to speculate a survival role for ZNF224 in malignant B-cells. Furthermore, we observed a decrease of ZNF224 by fludarabine treatment, a drug widely used in the treatment of CLL, both in leukemia cells than in CLL patients. This reduction is accompa- nied by a significant increase of apoptosis in leukemia cells.
Conclusions: Our data suggest that high levels of ZNF224 ex-
pression are probably related to B cell malignancies, through the transcriptional regulation of specific genes involved in cell cycle control, as CyclinD3, and/or survival. Further studied are needed to elucidate the role of ZNF224 the pathogenesis of chronic lymphocytic leukaemia, thus paving the way to the de- velopment of new therapeutic approach in leukemia.
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P 5
EXPRESSION AND PROGNOSTIC ROLE OF CXCR4/CXCL12/CXCR7 AND MTOR PATHWAYS IN NEUROENDOCRINE TUMORS (NETS)
Luisa Circelli (1) - Concetta Sciammarella (2) - Antongiulio Faggiano (2) -
Salvatore Tafuto (3) - Luciano Pezzullo (4) - Elsa Guadagno (5) - Maria Luisa
Del Basso De Caro (5) - Fabiana Tatangelo (6) - Nunzia Simona Losito (6) -
Giovanni Botti (1) - Francesco Izzo (3) - Stefania Scala (1) - Annamaria
Colao (2)
Oncological Immunology, National Cancer Institute "Fondazione G.Pascale", Naples, Italy (1) - Department of Clinical Medicine and Sur-
gery, University of Naples,"Federico II", Naples, Italy (2) - Department of
Abdominal Oncology, National Cancer Institute "Fondazione G.Pascale", Naples, Italy (3) - Thyroid and Parathyroid Surgery Unit, National Cancer
Institute "Fondazione G.Pascale", Naples, Italy (4) - Department of Func-
tional and Biomorfological Science, University of Naples,"Federico II", Naples, Italy (5) - Department of Pathology, National Cancer Institute
"Fondazione G.Pascale", Naples, Italy (6)
Background: Neuroendocrine tumors (NETs) are rare and het-
erogeneous neoplasms with variable biological behavior. The incidence of NETs is about 1-5 cases/100,000/year with a pro- gressive increase of the incidence and prevalence. NETs are op- timally treated with surgery and somatosatin analogs (SSA’s) to control symptoms but are relatively insensitive to systemic chemotherapy. As a result,patients with advanced unresectable NETs have a poor prognosis. In 2011, two targeted therapies, sunitinib and everolimus were approved in the subset of pro- gressive pancreatic NETs (pNETs). The chemokine receptor CXCR4 has been shown to signal on mTOR pathway in gastric and renal cancer. CXCR4 interacts with the chemokine CXCL12 to exert proliferative and chemotactic effects. CXCL12 was shown to recognise with high affinity the orphan receptor CXCR7. To identify possible prognostic factors and new therapeutic targets in NET patients, the role of the axis CXCR4-CXCL12-CXCR7 and mTor pathway was evaluated.
Methods: 61 human NET were included into the study: 40 gas-
tro-entero-pancreatic (GEP),21 medullary thyroid cancer (MTC). The mRNA was extracted from fresh /paraffin- embedded tissue and CXCR4, CXCL12 and CXCR7 was de- termined by qRT-PCR.CXCR4,CXCL12 and CXCR7 and mTOR pathway (mTOR,p-mTOR,p-p70S6K,p-4eBP1) was evaluated by immunohistochemistry (IHC) in our cohort. Ex- pression of CXCR4/CXCR7 was also evaluated on NCIH727 (Bronchial-NET), BON (P-NET) and TT (Medullary-Thyroid) cell lines by qRT-PCR and confirmed by Western-Blotting.
Results: CXCR4, CXCR7 and CXCL12 mRNA was signifi-
cantly overexpressed in tumour as compared to normal tissue, p<0.001, p<0.009 and p<0.0013, respectively. The IHC score of CXCR4(p<0.001), p-mTOR(p<0.05), p-4EBP1(p<0.01), p- S6K1 (p<0.05) was significantly higher in G1/G2 tumours, while CXCR7 and CXCL12 score was higher but not signifi- cantly in G3 tumour. Preliminary prognostic evaluation suggest that CXCR4 (p<0.001), CXCR7 (p<0.01), CXCL12 (p<0.01), mTOR (p<0.01), p-mTOR (p<0.05) and p-S6K1 (p<0.01) corre- late with unfavorable prognosis. CXCR4/CXCR7 expression levels on NET cells are comparable to positive controls.
Conclusions: CXCR4/CXCL12/CXCR7 and mTOR pathways
are expressed in NETs and might represent a prognostic factor
in these tumors. Concomitant inhibition of CXCR4 and mTOR pathways may improve effectiveness and overcome resistance
P 6
SERUM BIOMARKERS IDENTIFICATION BY NANOPARTICLE TECHNOLOGY IN METASTATIC SOFT TISSUE SARCOMA
Amalia Conti (1) - Claudia Fredolini (2) - Maria Serena Benassi (1) - Davide
Tamburro (2) - Giovanna Magagnoli (1) - Weidong Zhou (2) - Lance Liotta (2)
- Piero Picci (1) - Alessandra Luchini (2)
Laboratory of Experimental Oncology, Istituto Ortopedico Rizzoli, Bolo- gna, Italy (1) - Center for Applied Proteomics and Molecular Medicine,
George Mason University, Manassas, Virginia, United States (2)
Introduction: Soft Tissue Sarcoma (STS) are extremely rare
tumors. They are less than 1% of all adult cancers, and 7-10% of pediatric ones. They are also very complex and varied as there are approximately 50 different benign and 50 malignant histotypes.
The identification of circulating cancer biomarkers advances the possibility for an early detection, it is important in the patients' stratification into distinct risk subgroups, to better monitor tu- mor progression or response to therapy, guiding physicians in choosing the best treatment. However, at present there are no laboratory tests that permit a reliable early detection of STS or to indicate their dissemination. Discovery of new serum protein biomarkers is especially needed for those Soft Tissue Sarcomas which are associated with advanced stage at presentation and poor survival.
Materials and methods: Proteomic technologies are used for
global profiling and identification of disease-associated markers in biological fluids, such as serum. The low-molecular-weight proteome (<30 kDa) is considered as a rich source of new po- tential biomarkers, that often can escape the detection because of the presence of thousands of very abundant proteins in se- rum. In order to determine whether low-abundant serum pro- teins (<30 kDa) can be measured and useful for prognostic strat- ification of STS patients, we analyzed a discovery set of sera from non-metastatic and metastatic STS patients using poly(N- isopropylacrylamide-co-vinylsulfonic acid) hydrogel core-shell nanoparticles with incorporated Cibacron Blue F3G-A. These nanoparticles selectively entrap low molecular weight proteins on the bases of size exclusion and affinity chromatography, pro- tecting them from enzymatic degradation and amplifying the analyte concentration for mass spectrometry (MS) detection.
Results: Differentially abundant candidate peptidome bi-
omarkers that appear to be specific for detection of metastatic compared to non-metastatic STS have been identified revealing the potential utility for this new methodology. Subsequently, the biomarkers identified by MS have been validated on a second set of sera of metastatic and non-metastatic STS patients.
Conclusions: Our data suggest that new proteomic technology
in combination with careful statistical analysis appears to be useful to identify novel biomarkers for Soft Tissue Sarcoma. However, subsequent experiments will be crucial for their clini- cal utility and functional studies.
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P 7
SERUM MASS-SPECTROMETRY TEST IN FIRST LINE ADVANCED NSCLC PATIENTS TREATED WITH STANDARD CHEMOTHERAPY REGIMES.
Maria Giovanna Dal Belllo (1) - Carlo Genova (1) - Erika Rijavec (1) - Anna
Truini (1) - Angela Alama (1) - Simona Coco (1) - Irene Vanni (1) - Giulia Bar-
letta (1) - Federica Biello (1) - Julia Grigorieva (2) - Krista Meyer (2) - Heinrich
Roder (2) - Francesco Grossi (1)
U.O.S. Tumori Polmonari, IRCCS A.O.U. San Martino-IST-Istituto Na- zionale per la Ricerca sul Cancro, Genova, Italy (1) - Biodesix, Biodesix,
Boulder, United States (2)
Introduction: The mass-spectrometry based serum test
VeriStrat® (VS)was shown to be prognostic in various thera- pies and tumor types and predictive of differential overall sur- vival (OS) benefit for erlotinib vs. chemotherapy (CT) in se- cond line NSCLC setting. The mechanism of action is yet un- known, but thought to be related to the host response of an or- ganism to a tumor. Performance of the test in CT is of clinical interest. We investigated the role of VS in 1st line advanced
NSCLC patients (pts) treated with Cisplatin (Cis) or Car- boplatin (Carbo) plus Pemetrexed (P).
Materials and methods: VS classification was available for 55
eligible stage IV, pts with non-squamous histology; pts were classified as VS Good (VSG) or VS Poor (VSP), VS testing of pretreatment serum samples was done blinded to clinical data. OS and progression-free survival (PFS) were analyzed by Kaplan-Meier method and compared using log-rank p-values; Cox models were used in multivariate analysis. Association with categorical variables was analyzed by Fisher’s exact test.
Results: 36 (65%) pts were classified as VSG and 19 (35%) as
VSP. In the overall population, median PFS was 5.7 months (mo) for VSG vs.1.4 mo for VSP (hazard ratio (HR) 0.37 [0.19- 0.72], p=0.002 ); adjusted HR (AHR) 0.38 [0.17-0.86], p=0.021). Median OS was 10.8 mo for VSG vs. 3.4 mo for VSP (HR 0.23 [0.11-0.51], p < 0.001; AHR 0.12 [0.04-0.37], p<0.001). A similar relationship was found in both treatments: In CarboP median PFS in VSG and VSP was 3.8 mo and 2.0 mo respectively (HR 0.38 [0.15-0.94], p=0.030); median OS was 10.8 mo in VSG and 3.4 mo in VSP (HR 0.26 [0.09-0.72], p=0.006). In CisP median PFS was 6.1 mo in VSG and 1.2 mo in VSP (HR 0.40 [0.14-1.12], p=0.070), median OS was 12.3 mo in VSG, 3.8 mo in VSP (HR 0.17 [0.04-0.69], p=0.005).When compared within VS groups, no statistically significant differences between CarboP and CisP was found ei- ther for PFS (VSG: p=0.508, VSP: p=0.718) or OS (VSG: p=0.466 , VSP: p=0.522). VS was significantly associated with disease control rate (p=0.003) and trended towards significance for objective response (p=0.085).
Conclusions: In Platinum-based doublet CT, VSP pts had much
shorter PFS and OS than VSG . The behavior was similar in CisP and CarboP arms. Further research is needed to find alter- native treatments to improve outcomes for VSP pts.
ClinicalTrials.gov Identifier NCT02055144.
P 8
CXCR4/CXCL12/CXCR7 AND TLR2-4 MEDIATED INFLAMMATION IN COLORECTAL LIVER METASTASES
Crescenzo D'alterio (1) - Guglielmo Nasti (2) - Marianeve Polimeno (1) -
Alessandro Ottaiano (2) - Luisa Circelli (1) - Fabiana Tatangelo (3) - Fran-
cesco Izzo (4) - Rosario Vincenzo Iaffaioli (2) - Stefania Scala (5)
Department of Oncological Immunology, National Cancer Institute, “G. Pascale”, Naples, Italy, Napoli, - (1) - Department of Gastrointestinal
Medical Oncology, National Cancer Institute, “G. Pascale”, Naples, Italy, Napoli, Italy (2) - Department of Pathology, National Cancer Institute, “G.
Pascale”, Naples, Italy, Napoli, Italy (3) - Hepatobiliary Surgery Unit, Na-
tional Cancer Institute, “G. Pascale”, Naples, Italy, Napoli, Italy (4) - De-