Association For Cancer Research Research Articles

Abstract 716: A novel virus-like particle vaccine presenting HER-2 extracellular domain elicits strong immune responses against mammary carcinoma

Abstract Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in about 20% of invasive breast cancers. Anti-HER-2 monoclonal antibody therapy is effective, but its utility is limited by high costs, side effects and development of resistance, thus underlining the need of new therapeutic approaches. A novel anti-HER-2 vaccine made of virus-like particles (VLPs) displaying the extracellular domain (ECD) of the human oncogene/antigen HER-2 induced protective immune responses against transgenic mouse mammary carcinomas expressing human HER-2. We have developed a versatile antigen display platform that, unlike existing technologies, effectively facilitates directional covalent attachment of large antigens at high density on the surface of VLPs (J. Nanobiotechnology 14: 30, 2016). The system uses a tag/catcher conjugation system that was developed by splitting the CnaB2 domain from the fibronectin-binding protein FbaB of Streptococcus pyogenes into a highly reactive peptide (SpyTag) and a protein (SpyCatcher) binding partner. Interaction between SpyTag and SpyCatcher results in the spontaneous formation of an isopeptide bond, occurring at high efficiency in a wide variety of protein contexts and buffer conditions. Here, we genetically fused with SpyCatcher the full extracellular domain (subdomains I-IV) of human HER-2, and bound the fusion antigen (SpyCatcher-HER2) to the surface of VLPs (derived from the AP205 phage), each presenting 360 SpyTag peptides. The vaccine, referred to as HER2-VLP, effectively overcame immune tolerance and induced Th1 cytokines and high-titer, high affinity, therapeutically potent anti-HER-2 antibodies which inhibited tumor growth in wild-type FVB mice implanted with transgenic mammary carcinomas expressing human HER-2. Furthermore, vaccination with HER2-VLP prevented spontaneous development of human HER2-positive mammary carcinomas in tolerant transgenic mice. Vaccination with a control preparation of untagged VLP and HER-2 ECD did not induce protective immune responses. Polyclonal IgG antibodies elicited by HER2-VLP vaccination had an affinity for human HER-2 comparable to trastuzumab and inhibited the 3D growth in vitro of both trastuzumab-sensitive and trastuzumab-resistant BT-474 human breast cancer cells. In conclusion, the HER2-VLP vaccine has the potential to become a tool in the fight against HER-2-positive human cancer. The results also provide strong proof-of-concept for the use of the versatile VLP platform to develop a variety of vaccines against other tumor antigens. Supported by grants from the Italian Association for Cancer Research (AIRC), the University of Bologna, the Danish Research Council, the Eurostars program and the European Research Council (ERC). Citation Format: Arianna Palladini, Susan Thrane, Christoph M. Janitzek, Jessica Pihl, Stine B. Clemmensen, Wilhelm A. de Jongh, Thomas M. Clausen, Giordano Nicoletti, Lorena Landuzzi, Manuel L. Penichet, Tania Balboni, Marianna L. Ianzano, Veronica Giusti, Thor H. Theander, Morten A. Nielsen, Ali Salanti, Pier-Luigi Lollini, Patrizia Nanni, Adam F. Sander. A novel virus-like particle vaccine presenting HER-2 extracellular domain elicits strong immune responses against mammary carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 716.

Abstract 216: Functional stability, progression and evolution of targeted drug sensitivity of HER-2-positive breast cancer patient-derived xenografts

Abstract Human tumors are dynamic entities that undergo variation, selection and progression within the patient. How well patient-derived xenografts (PDX) recapitulate tumor dynamics? To investigate these aspects we established a collection of breast cancer PDX, representative of the main intrinsic subtypes. From 66 primary breast cancer specimens, we obtained 5 transplantable PDXs (8%). The highest rate of PDX stabilization was obtained for HER2-positive (40%) followed by triple negative (17%) and luminal B (14%) subtypes. In 3/66 cases a human lymphoma developed without any further evidence of breast cancer. Two HER-2-positive and one non-amplified, HER-2++, luminal B PDXs were serially transplanted in mice for 7-25 passages over a period of 2-4 years. Morphological and immunohistochemical (ER, PR, Ki67, p53, HER2, HER1, HER3, IGFR) features were highly stable over serial passages of PDXs, which retained the histology and the expression pattern of the tumor of origin. After the second passage, one HER-2++ amplified PDX, named BO-HAT4, was split in six different sublines, which were then studied separately to analyze random and selective events in long-term evolution. Two sublines progressively acquired a marked acceleration of tumor growth rate, whereas the remaining four did not. Metastatic spread was absent in early passages and appeared sporadically in late passages. In vitro cultures derived from early in vivo passages showed a rapid onset of cell senescence, whereas late in vivo passages gave rise to long-term in vitro cultures. However, we have not yet been able to obtain continuous cell lines from breast cancer PDX. To study the onset of resistance to HER-2 targeted therapies, sequential in vivo passages of BO-HAT4 were treated with trastuzumab, leading to a progressive loss of sensitivity. After one year of treatment BO-HAT4 was completely resistant to trastuzumab. We are currently investigating the molecular changes underlying trastuzumab resistance. Interestingly, both trastuzumab-sensitive and -resistant tumors were highly inhibited by neratinib. In conclusion, the low take rate as PDX prevents the generalized analysis of human breast cancer patients. Individual PDX allow the analysis of target therapy response and onset of resistance, however long-term study of transplantable breast cancer PDX show that tumor progression in these model systems is a late and random event. Supported by grants from the Italian Association for Cancer Research (AIRC) and the University of Bologna, Italy. Citation Format: Lorena Landuzzi, Marianna L. Ianzano, Claudio Ceccarelli, Enrico Di Oto, Giordano Nicoletti, Veronica Giusti, Roberta Laranga, Tania Balboni, Carla De Giovanni, Massimiliano Dall'Ora, Sofia Asioli, Arianna Palladini, Donatella Santini, Maria Pia Foschini, Mario Taffurelli, Pier-Luigi Lollini, Patrizia Nanni. Functional stability, progression and evolution of targeted drug sensitivity of HER-2-positive breast cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 216.

Abstract 2406: Metabolic phenotype and metastasis in patient-derived ovarian cancer xenografts

Abstract Introduction and Aim Metabolic re-programming is an emerging hallmark of cancer. Alterations of the metabolism of glucose (i.e. glycolysis) and oxidative phoshorylation (OxPhos) are frequently observed in cancer cells. Ovarian adenocarcinoma (OC) is the third most common gynecologic malignancy in the developed countries and the most lethal one, which has shown little improvement in overall survival for the last 20 years. OC is a widely metastatic disease disseminating to the organs of the abdomen through ascitic effusion. Understanding the mechanisms facilitating metastatic spread and disease recurrence is needed to develop therapeutic strategies.Our aim is the comprehension of the metabolic perturbations favoring the aggressiveness and metastatic dissemination of OC. Experimental methods We characterized a panel of OC-patient derived xenografts (OC-PDX) for their malignant behavior (ascites production, abdominal metastasis formation and survival time). Their metabolic features were depicted as i) glucose and lactate levels measured in ascites, ii) the monocarboxyate transporters MCT1 and MCT4 of the abdominal tumor masses (by IHC), iii) glycolysis- and OxPhos- related enzymes, as well as glucose and lactate transporters (by gene expression) in abdominal masses and ascites. Results We found a direct correlation between the amount of lactate and the expression of its transporters, thus OC-PDX showing higher expression of MC1/MCT4 and higher amount of lactate were categorized as “high glycolytic” and those with lower MCT1/MCT4 and lactate “low glycolytic”. The low glycolytic OC-PDXs developed more abdominal metastasis than the highly glycolytic ones, indicating an inverse correlation between the glycolytic phenotype and the capability of the OC-PDX to disseminate, invade and grow into the organs of the peritoneal cavity. Interestingly, we found that the abdominal masses express lower levels of the glucose transporters (i.e. GLUT1), glycolysis-related enzymes (i.e. HK, LDHA) and MCT4 compared to cell aggregates from abdominal effusion; suggesting that ovarian cancer cells are able to modulates their metabolism (glycolysis/OxPhos) to best fit the needs favoring metastatic spread. Further studies are needed to fully understand this association. Conclusions Nowadays, metabolism is potentially "targetable”. Our results strongly indicate that perturbing tumor's metabolic re-programming might affect metastatic dissemination. M R B and C G contributed equally. Supported by Italian Association for Cancer Research (AIRC) IG2016 18853 to RG. Citation Format: MariaRosa Bani, Carmen Ghilardi, Alessandra Decio, Alessia Anastasia, Martina Verza, Stefano Indraccolo, Raffaella Giavazzi. Metabolic phenotype and metastasis in patient-derived ovarian cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2406.

Abstract 4299: Mutual influence of ROS, pH and CLIC1 membrane protein in the regulation of G1/S phase progression in human glioblastoma stem cells

Abstract Glioblastoma (GB) is the most lethal, aggressive and diffuse brain tumor. The main challenge of successful treatment is targeting the cancer stem cell (CSC) sub-population responsible for tumor origin, progression and recurrence. Chloride Intracellular Channel 1 (CLIC1), highly expressed in CSCs, is constitutively present in the plasma membrane where it is associated with chloride ion permeability. In vitro, CLIC1 inhibition leads to a significant arrest of GB CSCs in G1 phase of the cell cycle. Furthermore, CLIC1 knockdown impairs tumor growth in vivo. Here we demonstrate that CLIC1 membrane localization and function is specific for GB CSCs. Mesenchymal stem cells (MSCs) do not show CLIC1-associated chloride permeability and inhibition of CLIC1 protein function has no influence on MSC cell cycle progression. Investigation of the basic functions of GB CSCs reveals a constitutive state of oxidative stress and cytoplasmic alkalinization compared with MSCs. We now report that these three elements are temporally linked during CSC G1/S transition. Impeding CLIC1-mediated chloride current prevents both intracellular ROS accumulation and pH changes. CLIC1 membrane functional impairment results in GB CSCs resetting from an allostatic tumorigenic condition to a homeostatic steady state. In contrast, inhibiting NADPH oxidase and the NHE1 proton pump results in cell death of both GB CSCs and MSCs. Our results show that CLIC1 membrane protein is crucial and specific for GB CSC proliferation, and is a promising pharmacological target for successful brain tumor therapies. This work was supported by grant n.16713, IG 2015 to MM Italian Association for Cancer Research (AIRC). Citation Format: Ivan Verduci, Marta Peretti, Federica Maddalena Raciti, Valentina Carlini, Alessandra Patarozzi, Sara Barozzi, Massimiliano Garrè, Sarah Sertic, Alex Costa, Antonio Daga, Federica Barbieri, Tullio Florio, Michele Mazzanti. Mutual influence of ROS, pH and CLIC1 membrane protein in the regulation of G1/S phase progression in human glioblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4299.

Abstract P4-21-33: HER2 activity regulates the pro-trastuzumab immune tumor microenvironment

Abstract It is well know that trastuzumab activity against HER2-positive breast carcinomas (BCs) depends also on immune cells. Our genomic analysis of 53 HER2-positive BCs indicated that those exquisitely sensitive to treatment are addicted to this oncoprotein and are enriched in immune pathways and T-cells infiltration, raising the hypothesis that HER2 regulates immune cells recruitment. The aim of this study was to investigate how HER2 activity contributes to mold tumor microenvironment. Gene expression profile analysis of 53 HER2-pos BCs showed that trastuzumab-sensitive tumors express significantly higher levels of chemokines involved in immune cells recruitment (IFNγ-related CXCLs and CCLs) and higher levels of immune checkpoint ligands (PD-L1, PD-L2) than tumors that do not benefit from trastuzumab. Immunohistochemistry analysis on FFPE tumor specimens showed a significant positive association between tumor cell positivity for CXCL9, CXCL10 and CCL2 and trastuzumab sensitivity. In vitro analysis in HER2-positive BC cell lines revealed that while IFNγ-related chemokines (CXCL9 and CXCL10) are not directly regulated by HER2 signaling, CCL2 was significantly up-regulated both at mRNA and protein levels by HER2 stimulation with EGF/HRG and down-modulated by HER2 inhibition with trastuzumab. The EGF-induced increase in CCL2 expression was abrogated by the concomitant treatment with PI3K inhibitor but not with MEK inhibitor, indicating that HER2 regulates CCL2 expression via the PI3K pathway. HER2 signals derived from cell stimulation with EGF/HRG caused also the upmodulation of PD-L1/PD-L2 both at mRNA and protein levels while their inhibition with trastuzumab reduced their levels. Overall, our results support the notion that the activated HER2 oncogene concomitantly induces the expression of CCL2 and PD-L1/PD-L2 regulating infiltration of pro-trastuzumab immune cells and their suppression. Thus, it is likely that activity of trastuzumab in therapy-responsive tumors derives from its ability in relieving suppression of recruited antitumor effector immunity. Supported by Italian Association for Cancer Research (AIRC). Citation Format: Tagliabue E, Forte L, Regondi V, Ghirelli C, Aiello P, Triulzi T. HER2 activity regulates the pro-trastuzumab immune tumor microenvironment [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-21-33.

P3.02b-008 Quantification and Monitoring of Treatment Response in EGFR Mutant NSCLC Patients by Digital-PCR in Plasma cftDNA: Topic: EGFR Biomarkers

The identification of activating epidermal growth factor receptor (EGFR) mutations is essential for deciding therapy of non-small cell lung cancer (NSCLC) patients. Circulating cell-free tumor DNA (cftDNA) holds promise as a non-invasive methodology for tumor monitoring in solid malignancies. Among advanced NSCLC patients with an acquired resistance to EGFR-tyrosine kinase inhibitors (TKIs), about 50% carry T790M mutation, but its frequency in EGFR-TKI-naıve patients and dynamic change during therapy remains unclear. We hypothesized that EGFRmutation analysis detection in cftDNA for NSCLC may be feasible for monitoring treatment response to EGFR-TKIs and also predict drug resistance. EGFR sensitive mutations and T790M were analyzed using digital PCR (d-PCR) (Quant studio 3D, life technologies) in longitudinally (at baseline, at 4, 8, 20, 60, 120, 180, 270, 360 days) collected plasma samples (n=50) from 8 tissue-confirmed EGFR-mutant NSCLC patients treated with an EGFR-TKI (Gefitinib N = 4; Erlotinib N = 1; Afatinib N = 3). DNA extracted from plasma of 8 healty blood donors were used to detect the specificity of EGFR mutant assay. Tumor assessment was performed according to RECIST criteria 1.1 every two months. The sensitivity of d-PCR in plasma versus tissue was 71.4%. No EGFR mutation was present in the 8 control cases (specificity of 100%). Of four patients who developed progression disease (PD), in the samples of progression, T790M was detected in 75% of cases. The frequency of T790M in pre-TKI plasma samples was of 37.5%. EGFR sensitive mutations decreased at PD while T790M mutation increased in 75% of patients. Patients with concomitant pre-TKI EGFR 19 deletion and T790M showed a PD before of 12 months compared to those with L858R. T790M was frequently detected when new lesions were developed. Four patients had T790M level decreased to undetectable level with longer PFS than those with detectable T790M in blood. Our results indicated that d-PCR was a highly sensitive and useful method for detecting the T790M mutation. Moreover, dynamically monitoring T790M change might help determining EGFR-TKI resistance. We thank Italian Association for Cancer Research (AIRC) for supporting the study.

Hypercoagulation Screening as Marker of Occult Cancer in a Population of Healthy Blood Donors: The Hypercan Prospective Study

INTRODUCTION and AIM: The HYPERCAN study is an ongoing prospective Italian multicenter trial, designed to test whether the persistence of a laboratory hypercoagulation abnormalities may predict early cancer diagnosis in healthy subjects (Project A), or prognosis and response to therapy in patients with cancer (Project B). The aim of this abstract is to present a preliminary analysis of data collected for project A.MATERIAL AND METHODS: A large healthy population of Italian blood donors from Bergamo and Milan areas (North Italy) is prospectively enrolled after informed written consent and followed-up for 5 years for the occurrence of cancer. As established by a monitoring schedule, blood donors are periodically screened for a series of serological, biochemical and clinical parameters, and tested for viral infections. We plan to enroll 10,000 donors of both gender, age range 35-65, in 5 years. Blood samples from each study subject are collected at enrollment and after 8-10 months, and processed to obtain plasma, buffy coat, and serum subsamples, which are stored in a dedicated biobank until testing for hypercoagulation biomarkers. Demographic and clinical data are collected, including age, gender, body mass index (BMI), current medications, relevant comorbidities, routine hematological and biochemical workup. In addition, subjects are asked to fill in a questionnaire on lifestyle, smoke and dietary habits. An identification of all malignant tumors, according to the categories 140-208 (International Classification of Diseases) is carried out every 6 months.RESULTS: Between April 2012 and June 2016, 6,607 blood donors (70% males; median age 48 years) have been recruited. Routine biochemical and hematological workups are into the normal range values in more than 90% of subjects. The analysis of questionnaires reveals that 57% of the donors were not smokers, 15% regular smokers, 28% ex-smokers; 49% of them were moderate/low alcohol consumer (≥ 2 drink/die). The crude incidence of all malignant cancers in the Bergamo/Milan area is 552 cases/100,000 persons/year in males and 382 cases/100,000 person/year in females. According to the Cancer Registry, 57 cancer cases (38 males and 19 females) are to be expected in the general population in the age range 35-65 years, after a median follow-up of 2.5 years. In our population, at June 2016, after a median follow-up of 2.5 years, we recorded a total of 46 cancer cases (35 M / 11 F). Five cases were excluded because diagnosis occurred within 6 months from enrollment (incident cancer cases); the remaining 41 cases included in the analysis were diagnosed with cancer 6-28 months from enrollment (median time to diagnosis = 24.8 months). Median age at diagnosis of cancer was 53 years. The most frequent tumor type in male donors was prostate cancer (25.8%), followed by colo-rectal (19.3%) and thyroid (12.9%) cancers. In female donors, breast cancer was the most frequent (40%). Currently, the enrollment of healthy donors and follow-up is ongoing, as well as the identification of new cancer cases. Next, according to the original plan, samples from 3 matched cancer-free donors for each cancer-case donor (cancer: non-cancer = 1:3 ratio) will be analyzed in parallel for hypercoagulation markers.CONCLUSIONS: This preliminary analysis reveals that the distribution of tumor types in our population reflects the same as reported for the general population of the same geographic area. Second, in our population of healthy blood donors, the incidence of cancer diagnosis is lower than that predicted from epidemiological data. This message supports the concept that a healthy lifestyle can be effective to prevent cancer.Project funded by AIRC "5xMILLE" n. 12237 grant from the "Italian Association for Cancer Research (AIRC)". DisclosuresFalanga:Pfizer: Speakers Bureau; Aspen: Speakers Bureau; Janssen: Speakers Bureau.

Abstract 1066: Synthetic miR-22 inhibits alternative non homologous end-joining DNA repair and increases sensitivity to PARP-inhibition in multiple myeloma cells

Abstract Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by deep genomic instability. Alternative non-homologous end-joining (A-NHEJ) DNA repair is up-regulated in a significant fraction of cancers and it is more error-prone than the major LIG4-dependent NHEJ repair pathway (classic-NHEJ). Principal components of A-NHEJ (PARP1, POLQ and LIG3) represent therefore a new class of cancer cell-specific therapeutic targets. There is Increasing evidence that microRNAs take actively part in the regulation of the DNA damage/repair network. Based on this rationale, we aimed to elucidate the role of miRNAs in the regulation of A-NHEJ-driven genomic instability in MM. We first examined the prognostic role of DNA Ligases involved in NHEJ pathways interrogating the public MM gene expression dataset GSE9782, which revealed higher expression of LIG3 in patients with shorter survival; conversely LIG4 expression was not associated with worse prognosis, thus supporting the idea that LIG3 has a pivotal role in genomic instability and progression of MM. Next, we aimed to identify LIG3-targeting miRNAs by an integrated approach that predicted miR-22 as negative regulator of LIG3 in MM patients. On this basis we evaluated miR-22 and LIG3 expression in a panel of MM cell lines and primary samples compared to plasma cells from healthy donors. We detected significant down-regulation of miR-22 and up-regulation of LIG3, strictly correlated to disease progression. Subsequently, we induced miR-22 overexpression (by transduction or transfection) in a panel of MM cell lines and we found cell growth inhibition, apoptosis induction and cell cycle arrest. Importantly, transfection of miR-22 mimics reduced the viability of primary MM cells but not of HD-PBMCs. We next examined the effects of enforced overexpression of miR-22 on DNA-damage response and we observed an increase of the DNA damage marker yH2AX, reduction of LIG3 at mRNA and protein level, and decrease of LIG3-3’UTR luciferase activity, indicating specific 3’ UTR targeting. Importantly, we observed inhibition of A-NHEJ repair by a functional assay based on the plasmid EJ2-GFP. Finally, miR-22 mimics potentiated cytotoxic effects of PARP-inhibitor Olaparib (Selleckchem), both in vitro and in vivo, confirming that co-inhibition of LIG3 and PARP-1 have a synergistic effect on A-NHEJ dependent MM cells. Taken together, our preliminary findings indicate that miR-22 inhibits A-NHEJ by specific targeting of LIG-3 and modulates sensitivity to PARP inhibition. Further investigations could shed new light on A-NHEJ in MM pathogenesis and define a role for miR-22 as therapeutic agent in this still incurable disease. Acknowledgement: This work has been supported by Italian Association for Cancer Research (AIRC). PI: PT. “Special Program Molecular Clinical Oncology - 5 per mille” n.9980, 2010/15 Citation Format: Daniele Caracciolo, Eugenio Morelli, Maria Teresa Di Martino, Daniela Talarico, Cirino Botta, Nicola Amodio, Cinzia Federico, Emanuela Altomare, Lavinia Biamonte, Maria Angelica Stamato, Pierosandro Tagliaferri, Pierfrancesco Tassone. Synthetic miR-22 inhibits alternative non homologous end-joining DNA repair and increases sensitivity to PARP-inhibition in multiple myeloma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1066.

PO-44 - Tissue factor pathway inhibitor-2 (TFPI-2) is cleaved by PRSS3: implication for tumor endothelial cells migration

Tissue Factor Pathway Inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor whose expression is up-regulated by VEGF in microvascular and umbilical vein endothelial cells (EC). Despite this, TFPI-2 has been suggested as anti-angiogenic molecule, due to its ability to inhibit the migration/proliferation of EC induced by VEGF. Nothing is known about the precise mechanism of TFPI-2 function tuning in tumor endothelium. Aim of this study was to investigate the role of TFPI-2 in tumor vasculature, where angiogenesis and vascular remodeling are fundamental for cancer progression. Tumor-EC were isolated from ovarian carcinomas and cultured in vitro in presence of factors reproducing the tumor microenvironment (VEGF, FGF-2, EGF). TFPI-2 and PRSS3 silencing was achieved by small interfering RNA (siRNA). Tumor-EC migration was assayed by the wound healing assay. Transcript expression was examined by qRT-PCR. Proteolytic reactions were monitored by western blot. We show that tumor-EC express TFPI-2, the majority of which is released and found anchored in the extracellular matrix. Silencing the expression of TFPI-2 enhances tumor-EC migration, confirming TFPI-2 as an anti-angiogenic molecule. We had previously shown that the cancer vasculature express PRSS3, a trypsin family member able to cleave proteins containing the kunitz-type domains; we reasoned that it could potentially inhibit TFPI-2. Herein, we demonstrate in a cell free system that TFPI-2 directly interacts with and is degraded by active PRSS3. In a more complex biological context, active PRSS3 is able to remove TFPI-2 from the extracellular matrix put down by tumor-EC. Accordingly, silencing PRSS3 causes the extracellular accumulation of TFPI-2 that results in the inhibition of tumor-EC migration. Our results demonstrate for the first time that TFPI-2 is a direct substrate of PRSS3, which hydrolyses TFPI-2 (most likely at the Kunitz-type domains) blocking its anti migratory capability. The proteolytic inactivation of TFPI-2 by PRSS3 might represent a mechanism favoring cancer by increasing angiogenesis and vascular remodeling. Supported by the Italian Association for Cancer Research (AIRC).

Measurement of Thrombin Generation Is a Positive Predictive Biomarker of V enous Thromboembolism (VTE) in Metastatic Cancer Patients Enrolled in the Hypercan Study

Introduction The HYPERCAN study is an ongoing prospective Italian multicenter trial (Thromb.Res. 2014, Suppl 2, 182), designed to evaluate the role of laboratory hypercoagulation screening to predict early diagnosis (in healthy subjects), or prognosis and response to therapy in patients with either limited or metastatic cancers. Four cancer types are included, i.e. non-small cell lung [NSCL], gastric, colorectal, and breast cancers. The occurrence of clinically manifest VTE events, confirmed by objective diagnostic tests, is also recorded. Patients are followed up for 5 years or death.Aim In a group of patients with metastatic cancer enrolled in the HYPERCAN program, we wanted to evaluate the role of thrombin generation assay (TG), fibrinogen, and D-dimer levels in predicting the occurrence of VTE in the follow up.Methods As of June 2015, overall 831 patients with metastatic cancer have been enrolled. According to protocol, blood samples from these patients are collected at enrollment (baseline), after 3 and 6 chemotherapy cycles, and at end of treatment, or earlier if cancer disease progression. We measured the levels of TG, fibrinogen, and D-dimer in the baseline citrated plasma samples from the first 281 patients enrolled into the study (158 M/123 F; median age 64 years, range 32-84; NSCL = 56.2%, gastric = 11.1%, colorectal = 14.6%, breast = 18.1%). TG was measured by the Calibrated automated thrombogram (CAT assay, STAGO, France) at 5pM TF and results expressed as endogenous thrombin potential (ETP), fibrinogen and D-dimer were measured by commercial assays (Q.F.A. Thrombin; D-dimer HS; Werfen, Italy). Cut-off values were established by ROC curves; Kaplan Meier analysis was performed to define the VTE risk.Results Overall the patient baseline ETP values as well as the fibrinogen and D-Dimer levels were significantly greater than those of a control group of healthy subjects (p<0.0001). Among the cancer types, ETP values were highest in patients with NSCL cancer, and lowest in those with gastric cancer (1899±517 nM*min vs 1622±550 nM*min; p=0.024). D-dimer and fibrinogen levels were greatest in gastric (801±186 ng/ml) and NSCL (484±190 mg/dl) cancers, respectively, and were both lowest in breast cancer patients. After a median follow up of 473 days, overall 37 VTE events were recorded in 36 patients: 16 pulmonary embolism (PE), 16 deep vein thrombosis (DVT), 3 superficial vein thrombosis, and 1 PE + DVT. Of these events, 69% occurred in NSCL, 17% in colon, 8.5% in gastric, and 5.5% in breast cancer patients. Median time to VTE was 5 months from enrollment, >80% of VTE developed during chemotherapy. Baseline ETP levels were significantly higher in patients with VTE than in patients without VTE (2020±618 nM*min vs 1799±467 nM*min; p=0.017). Univariate analysis (Kaplan-Meier) showed that patients with ETP>1750 nM*min had about 3-fold higher risk of developing VTE than those with ETP<1750 nM*min (HR:2.841, 95% CI 1.42-5.69 p=0.002). ETP predictive value remained significant by multivariate analysis after correction for age, gender, and tumor site (HR: 2.341, 95% CI 1.15-4.75, p=0.019). Differently, the baseline levels of D-dimer and fibrinogen did not significantly predict for VTE.Conclusions These results reveal that ETP is a valuable marker in predicting VTE in metastatic cancer patients, therefore it can help to optimize the identification of the high risk subjects, which remains an important challenge and may improve the design of interventional studies of efficacy and safety of primary thromboprophylaxis in cancer patients during chemotherapy.Project funded by AIRC "5xMILLE" n. 12237 grant from the "Italian Association for Cancer Research (AIRC)". DisclosuresSantoro:Celgene: Research Funding.

Platelet Adhesion Under Flow Condition in Patients with Essential Thrombocythemia (ET) and Polycythemia Vera (PV): Analysis According to the Mutational Status

Background: ET and PV are two myeloproliferative neoplasms characterized by a high incidence of both arterial and venous thrombosis, and microcirculatory disturbances. So far, no studies have been performed to characterize ET and PV patient platelet (PLT) adhesion properties in flow conditions, an index of thrombus formation capacity in vitro (Platelets 2012; 23(3): 229-42).Aim: In this study, in a cohort of patients with ET or PV, we wanted to evaluate the PLT adhesiveness in vitro by a dynamic method and to estimate whether this property is influenced by any of the three somatic mutations, i.e. JAK2 V617F, Calreticulin (CalR) and MPL, typical of these diseases, of which, the JAK2 V617F mutation has a significant association with thrombosis occurrence, while CalR and MPL mutations have not. Further, the relations of PLT adhesion to main hematological parameters and to cytoreductive hydroxyurea (HU) therapy were also considered.Patients and Methods: Sixty-nine patients, i.e. 47 ET (20 M/27 F; mean age 60 years, range 33-86) and 22 PV (13 M/9 F; mean age 65 years, range 46-80), and 26 healthy controls (13 M/13 F; 44 years, range 27-61) were included into the study. For the adhesion assay, peripheral venous whole blood was drawn in sodium citrate, recalcified in the presence of heparin, and perfused over a collagen-coated surface for 4 min at a shear rate of 1,000 s-1. PLTs were then stained with an anti-CD62P (P-selectin)-FITC antibody as a PLT activation index, and annexin V-AlexaFluor647 to detect PLT procoagulant phosphatidylserine expression. After staining, images of adherent platelets in random fields were taken using phase contrast and fluorescence imaging with the EVOS® fluorescence microscope (Life Technologies). Results are expressed as the mean±SEM of the percentage of area covered by all PLTs (% coverage), or as the % of adherent PLTs positive for either P-selectin or phosphatidylserine. Main hematological parameters (i.e. PLT count, WBC count, RBC count, Hb, Hct, MCV, MPV), therapeutic regimens, and mutational status (JAK2 V617F, CalR, or MPL) were also recorded. Statistical analysis was performed by SPSS® (IBM) software package.Results: PLT adhesion was significantly (p<0.01) greater in both ET (44.6±2% coverage) and PV patients (45.6±2.7%) compared to controls (36±2.1%). The analysis of PLT adhesion according to the mutational status shows that, in ET patients, PLT adhesion was greatest in JAK2 V617F mutation carriers (n=22), followed by triple negative (n=7), and CalR-positive (n=15). The only 3 MPL-positive ET patients had the lowest PLT adhesion values. In PV patients, those with >50% JAK2 V617F allele burden carriers (n=8; 52.5±2% coverage) presented a significantly higher PLT adhesion than those with <50% allele burden (n=14; 42.4±3.9, p<0.05). Notably, in both ET and PV patients, while the overall PLT adhesion was significantly increased, the percentage expression of P-selectin by adherent PLTs (~ 75%) was not statistically different from that of control subjects. Differently, the expression of procoagulant phosphatidylserine on adherent PLTs was reduced in both ET (p<0.001) and PV patients (p=n.s.) compared to controls. Among hematological parameters, in ET patients, a significant (p<0.001) correlation was found between PLT adhesion with PLT count, but not with WBC count, RBC count, Hb, Hct, MCV, or MPV. As regard to treatments, a significant reduction in PLT adhesion was associated with cytoreductive treatment with HU (p<0.05) in patients with PV, but not with ET.Conclusions: ET and PV platelets show a greater thrombus formation capacity in vitro in a dynamic flow condition, as measured by an increase in adhesion to collagen. This capacity is significantly augmented according to the JAK2 V617F mutational allele burden and decreased by HU therapy. A prospective study of the PLT thrombus formation potential in a dynamic model is ongoing to evaluate the predictive value of this parameter on the thrombotic risk of ET and PV patients.Project funded by "AIRC IG2013" grant Nr. 14505 from the "Italian Association for Cancer Research (A.I.R.C.)". DisclosuresNo relevant conflicts of interest to declare.

Abstract 4169: Tumor endothelium regulates microenvironment-mediated migration via the proteolysis of extracellular TFPI-2 by trypsinogen 4

Abstract Proteases secreted from endothelial cells (EC) play an important part in angiogenesis, tumor metastasis and blood coagulation processes. In particular, perivascular proteases influence tumor vascularization in many different ways: regulating migration and invasion of endothelial and mural cells, activating chemokines, growth factors, growth factor receptors, break down extracellular matrix and modifying cellular adhesion molecules. Understanding the complexity of protease activities and their regulation by endothelium in response to different microenvironment stimuli might help identify new targets for the inhibition of vascularization in cancer and angiogenesis related diseases. The expression of serine protease 3 (PRSS3) a member of the trypsinogen family, in vivo is associated to the tumors vasculature and in vitro characterizes EC isolated from cancer specimens [Ghilardi, BMC Genomics 2008]. Herein, we used EC isolated from cancer specimens (tumor-derived EC) to elucidate a mechanism linking micro-environment cues to endothelium migration. We show that trypsinogen 4 isoform of PRSS3 is the trypsin family member expressed by human endothelium, and its expression is up-regulated by a tumor pro-angiogenic milieu (i.e. the combined action of VEGF, FGF-2 and EGF; particularly rich in the cancer environment). Trypsinogen 4 depletion profoundly impaired the migratory capability of tumor-derived EC initiated and driven by the tumor pro-angiogenic milieu. We identified Tissue Factor Pathway Inhibitor-2 (TFPI-2) as the functional proteolitic target. TFPI-2 negatively regulated the migration of tumor-derived EC; trypsinogen 4 was capable to cleave TFPI-2 and also to displace it from the extracellular matrix (ECM) deposited by tumor-derived EC. Accordingly, we observed an increased accumulation of TFPI-2 in the extracellular matrix of tumor-derived EC silenced for trypsinogen IV. Altogether, the results indicate that the tumor microenvironment triggers and sustains the expression of trypsinogen 4 to impair TFPI-2 function and permits the migration of endothelium; essential for angiogenesis and blood vessel remodeling. Our findings contribute to advance the understanding of tumor vascular biology, and the comprehension of an active role for trypsinogen 4 and TFPI-2 in microenvironment-mediated tumor neo-vascularization. The identification of the specific protease and its functional target may be of aid for the design of inhibitor(s) exploitable as anti-cancer therapeutics. Supported by the Italian Association for Cancer Research (AIRC) and the CARIPLO foundation. Citation Format: Carmen Ghilardi, Figini Sara, Monica Lupi, Alessia Anastasia, Raffaella Giavazzi, Mariarosa Bani. Tumor endothelium regulates microenvironment-mediated migration via the proteolysis of extracellular TFPI-2 by trypsinogen 4. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4169. doi:10.1158/1538-7445.AM2015-4169

Hypercoagulation Screening As a Marker of Thrombosis and Poor Disease Prognosis in Cancer Patients: The Hypercan Prospective Study

Introduction Malignant tumor progression and poor prognosis have been repeatedly associated with the occurrence of hypercoagulation and overt thrombotic events. The HYPERCAN is an ongoing prospective Italian multicentre study, structured in two sub-projects that involve respectively non-cancer and cancer subjects. The HYPERCAN sub-project on cancer patients aims to evaluate whether thrombotic markers may predict for malignant disease recurrence and/or for venous thromboembolic events (VTE) in these patients.Methods Consecutive adult patients with limited resected or metastatic non-small cell-lung (NSCLC), gastric, colorectal, or breast cancers are enrolled, after informed written consent, and followed up for 5 years or death. Enrollment will be continued for up to 3 years (total 4,000 patients planned). Blood sample collection is planned according to tumor stage. Particularly, for metastatic disease, patient citrated whole blood is collected: 1. at enrollment, 2. after the 3rd cycle of anticancer therapy, 3. after the 6thcycle of anticancer therapy, 4. at the end of treatment, or earlier if disease progression. For patients with limited disease, whole blood samples are collected at enrollment (i.e. after surgery, before starting any medical anticancer treatment), and thereafter once a year for 4 years, or at disease recurrence. At each time point, information on treatment, clinical response, and disease outcome, are collected. Plasma samples and clinical information are centralized at the Division of Immunohematology and Transfusion Medicine - Bergamo Hospital (coordinating center). Patient plasma is tested for a preliminary panel of hemostatic markers (D-dimer, Fibrinogen) and thrombin generation (TG).Results Between April 2012 and February 2014, 1,113 cancer patients were enrolled into the study. Follow-up data were available for 1,045 patients (359 metastatic/686 limited-resected) with NSCLC (n=252), gastric (n=74), colorectal (n=163) and breast (n=556) cancers, with an observation median time of 340 days. Forty-eight patients (4.6%) developed VTE with a median time of 123 days from enrollment. VTE was more frequent in metastatic compared to limited resected cancer patients (9.2% vs 2.2%); colorectal and lung cancer patients showed the highest VTE rate, 8.5% and 7.9%, respectively, independently of tumor stage. Currently plasma from 474 patients (196 metastatic/278 limited resected) were tested. Increased D-dimer and fibrinogen levels at enrollment showed to be significant risk factors for VTE by Kaplan-Meier analysis (HR: 5.4, 95% CI 2.2-13.2 p=0.000 for D-dimer; HR: 4.3, 95% CI 1.8-10.4 p=0.004 for fibrinogen) as well as the endogenous thrombin potential (HR: 4.5, 95% CI 1.8-11.4 p=0.000) and the TG peak (HR: 4.0, 95% CI 1.5-10.2 p=0.009). In the group with limited resected disease, the cumulative incidence of recurrence was 3.2% with a median time of 186 days. Increased D-dimer and fibrinogen levels at enrollment were predictive for disease recurrence by Kaplan-Meier analysis (HR: 9.9, 95% CI 2.4-40.1 p=0.001 for D-dimer; HR: 5.9, 95% CI 1.5-23.6 p=0.012 for fibrinogen).Conclusions These early results reveal that tumor stage and site, as well as elevated levels of D-dimer, fibrinogen and TG at enrollment, are associated with an increased risk of VTE in cancer patients. In subjects with limited resected cancer, elevated D-dimer and fibrinogen levels also predict for disease recurrence. These results support the hypothesis that testing for relatively easy plasma markers of thrombosis may be worth for monitoring the cancer response to therapy beyond the thrombotic risk.Project funded by AIRC “5xMILLE” n. 12237 grant from the “Italian Association for Cancer Research (AIRC)”. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1820: Dynamics of HER-2 loss in mammary carcinoma of human HER-2 trangenic mice

Abstract Progression of HER-2+ breast cancer can result in the emergence of HER-2-negative tumor variants that activate alternative mitogenic pathways, either spontaneously or after therapy. We found that HER-2 loss occurs even in transgenic mouse models in which the oncogene is driven by viral promoters, thus mammary carcinoma of human HER-2 transgenic mice (huHER-2 mice) can be used to study not only the early phases of HER-2-driven mammary carcinogenesis, but also tumor progression beyond HER-2 addiction. Primary mammary carcinomas of huHER-2 mice express high levels of the oncogene, with a marked intratumoral heterogeneity. Cell lines grown from HER-2+ mammary carcinomas frequently undergo a progressive loss of expression. We have established a model system consisting of cell lines, clones and variants that exhibit one of three phenotypes: a) high and stable HER-2 expression in vitro and in vivo, b) high but labile HER-2 expression which is lost either during in vitro culture, after tumor growth in mice or after in vitro treatment with trastuzumab, and c) complete loss of HER-2 expression After HER-2 loss, most variants displayed a transition to an elongated, motile phenotype (epithelial to mesenchymal transition), an increased ability to generate mammospheres, a reduced expression of CD24 and an increased expression of CD44 (denoting mammary cancer stem cells). Tumorigenic and metastatic ability of HER-2-negative cells was increased in comparison to HER-2+ cells. As expected, HER-2 loss was accompanied by resistance to HER-2 targeted monoclonal antibodies and small molecule inhibitors. The study of therapeutic agents directed against downstream targets showed that HER-2-loss was accompanied by a loss of sensitvity to a Src inhibitor, whereas a PI3K inhibitor was highly effective regardless of HER-2 expression. Our results indicate that human HER-2 transgenic mice are a useful model to study the dynamics of HER-2 loss in advanced HER-2+ mammary carcinoma, and to analyze alternative therapeutic strategies. Supported by grants from the Italian Association for Cancer Research (AIRC). Citation Format: Patrizia Nanni, Arianna Palladini, Lorena Landuzzi, Massimiliano Dall'Ora, Marianna Ianzano, Valentina Grosso, Dario Ranieri, Giordano Nicoletti, Roberta Laranga, Carla De Giovanni, Manuela Iezzi, Pier-Luigi Lollini. Dynamics of HER-2 loss in mammary carcinoma of human HER-2 trangenic mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1820. doi:10.1158/1538-7445.AM2014-1820

Abstract 2774: Coexpression of Delta16 isoform and full-length HER-2 in F1 hybrid transgenic mice: effects on tumor growth and malignancy

Abstract HER-2 gene products found in human breast cancer include the full-length p185 oncoprotein and various shorter isoforms that lack C-terminal, N-terminal or internal portions. Delta16 isoform lacks exon 16 and displays the properties of an activated oncogene. Transgenic mice expressing Delta16 in the mammary gland develop more mammary carcinomas, and at a younger age than mice transgenic for full-length HER-2. Human breast cancers, unlike transgenic mice, co-express Delta16 and full-length HER-2. To study mammary carcinogenesis in a mouse model that mimics the human situation, we obtained hybrid mice bearing heterozygous copies of both human transgenes (Delta16/HER-2 mice), and we compared them to parental mice (referred to as Delta16 and HER-2 transgenic mice, respectively). In Delta16/HER2 hybrid mice, mammary carcinogenesis was similar to that of Delta16 mice, with a median latency time of 17 weeks and a mean of 7 tumors per mouse, thus indicating a dominant expression of Delta16 over HER-2. However, after tumor onset, tumor growth rate and metastatic spread were similar in the three mouse lines, thus suggesting that Delta16 was mainly involved in neoplastic transformation and in the early phases of mammary carcinogenesis, rather than in advanced tumor progression. This could result from cancer cell intrinsic activity of the oncogene or from interactions with the microenvironment, in particular with vasculogenesis. Using isoform-specific PCR analysis we found that most tumors expressed only Delta16, some expressed both Delta16 and HER-2, and some expressed only HER-2. The level of Delta16 surface protein expression, as detected by FACS analysis with cross-reactive antibodies, was generally lower than that of HER-2. We established representative cell lines for in vitro studies. Exposure of these cells to trastuzumab, lapatinib, or their combination showed that Delta16 did not confer resistance to HER-2-targeted drugs in comparison to full-length HER-2. In conclusion, the study of Delta16/HER-2 double transgenic mice suggests that the activated isoform Delta16 plays a dominant role in the early phases of mammary carcinogenesis. Supported by grants from the Italian Association for Cancer Research (AIRC) Citation Format: Pier-Luigi Lollini, Valentina Grosso, Dario Ranieri, Arianna Palladini, Marianna Ianzano, Massimiliano Dall'Ora, Lorena Landuzzi, Giordano Nicoletti, Tania Balboni, Roberta Laranga, Carla De Giovanni, Augusto Amici, Serenella M. Pupa, Manuela Iezzi, Patrizia Nanni. Coexpression of Delta16 isoform and full-length HER-2 in F1 hybrid transgenic mice: effects on tumor growth and malignancy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2774. doi:10.1158/1538-7445.AM2014-2774

In Vitro and Vivo Activity Against Multiple Myeloma Cells Of a Novel Locked Nucleic Acid (LNA)-Mir-221 Inhibitor

miR-221/222 are two highly homologous microRNAs (miRNAs), encoded in tandem on the chromosome X, whose up-regulation has been found in several malignancies and are thought to promote cell proliferation via down-regulation of p27 and/or p57, two negative regulators of G1 to S phase cell cycle progression. We demonstrated up-regulation of both miRNAs in malignant plasma cells (PCs) from multiple myeloma (MM) patients belonging to distinct TC (translocation/Cyclin) groups, including TC2 and TC4. A rising body of evidence suggests that silencing miRNAs with oncogenic potential could represent a novel approach for human cancer therapy. We previously demonstrated that silencing miR-221/222 exerts significant anti-MM activity and triggers canonical targets in vitro and in vivo. Here, in the aim to progress to clinical translation of our proof-of-principle findings, we investigated the anti-tumor activity and the appropriateness for systemic delivery of a novel and originally designed LNA-miR-221, a 13-mer antisense miR-221 inhibitor, which took advantage of locked nucleic acid (LNA) technology and phosphorothioate backbone chemistry for increasing affinity for miR-221 and nuclease resistance. We found that enforced ectopic expression of LNA-miR-221 in t(4;14) MM cells significantly inhibited growth and survival of MM cells in vitro. In treated cells, we detected knock down of miR-221/222 together and increased levels of both p27Kip1 mRNA and protein. Specific activity of this LNA-miR-221 inhibitor was confirmed by the use of a 3'UTR reporter (luciferase renilla/firefly) constructs containing, miR-221 target site. This construct was co-transfected either with miR-221/222 mimics or LNA-miR-221 inhibitor into MM cells. As predicted, a reduced luciferase activity was detected in miR-221/222 mimics co-transfected cells with each 3'UTR reporter plasmid, while increase luciferase activity was measured in MM cells co-transfected LNA-miR-221 inhibitors indicating an efficient and stable binding to the miRNA target sequence. Importantly, we evaluated the systemic delivery of the LNA-miR-221 inhibitor with saline solution vehicle alone by intraperitoneal or intravenous injection route against MM xenografts in SCID/NOD mice. Significant anti-tumor activity was achieved after 2 weeks of treatment at similar extent by both injection routes. Retrieved tumors from treated animals showed efficient inhibition of miR-221/222, as demonstrated by increased levels of p27Kip1 protein in vivo. H&E staining and immunohistochemical analysis showed wide necrosis areas, reduced Ki67 and a significant increase of p27Kip1 cytoplasmic expression in retrieved tumors from LNA-miR-221 inhibitor-treated mice. No changes in mice behavior or organ toxicity were observed in treated mice. Taken together these findings support the rationale for development of this novel and highly efficient LNA-miR-221 inhibitor as a promising anti-MM drug in subsequent primate toxicology studies. Supported by the Italian Association for Cancer Research (AIRC), PI: PT. “Special Program Molecular Clinical Oncology - 5 per mille” n. 9980, 2010/15. Disclosures:No relevant conflicts of interest to declare.

Inhibition Of Mir-21 In HS-5 Bone Marrow Stromal Cells In The Presence Of Multiple Myeloma cells Restores RANKL/OPG Ratio: A Potential Therapeutic Approach For Myeloma-Related Bone Disease

Multiple Myeloma (MM)-related osteolytic lesions of the skeleton result from asynchronous bone turnover wherein increased bone resorption by osteoclasts (OCLs) is associated to suppression of bone formation due to inhibition of osteoblasts (OBLs). In this context, the balance between osteoprotegerin (OPG) and the receptor activator for nuclear factor-κB ligand (RANKL) plays a relevant role, because RANKL signaling induces differentiation, activation and survival of OCLs, whereas OPG acts as a decoy receptor of RANKL, thus blocking bone resorption. In the MM milieu, the interaction of malignant plasma cells (PCs) and bone marrow stromal cells (BMSCs) favors the production of RANKL and reduces OPG secretion by BMSCs. The resulting severe imbalance in RANKL/OPG ratio is the main trigger of MM-related bone disease (BD). Emerging evidence indicates that microRNAs (miRNAs) play a crucial role in bone remodelling as they can act on bone effectors (OCLs, OBLs, BMSCs) and PCs. Indeed, several findings suggest that miR-29 family support OBL differentiation, while we have previously demonstrated that miR-29b is down-regulated during osteoclastogenesis and its enforced expression functionally inhibits terminally differentiated OCLs. Moreover, also miR-21 plays an important role in OCL differentiation and is up-regulated by IL-6 via STAT3 that, in turn, promotes survival and proliferation of PCs, favoring bone damage. On these bases, we investigated whether modulation of miR-21 expression in BMSCs may have an impact on MM microenviroment, focusing our attention on OPG production, taking into account that different bioinformatics resources indicate OPG mRNA as a predicted target of miR-21. To this end, we established a co-culture system where primary BMSCs and HS-5, a human BMSC cell line, were exposed to either RPMI 8226 MM cells or primary CD138+ PCs isolated from MM patients. Then, we isolated BMSCs and evaluated miR-21 and OPG expression. We observed strong up-regulation of miR-21 in BMSCs and HS-5 together with a significant reduction of OPG levels in the presence of MM cells. These preliminary data suggested that antagonizing miR-21 in BMSCs exposed to MM cells could restore RANKL/OPG ratio through the up-regulation of OPG. To address this hypothesis, we transduced HS-5 cell line with a lentiviral vector carrying anti-miR-21 sequence (a21 HS-5) or with an empty lentiviral vector carrying only the GFP gene (GFP CNT HS-5). OPG expression was determined by RT-PCR, Western Blotting and ELISA assays after 48h and 72h of exposure to RPMI 8226 cells or primary PCs. We observed a significant increase in OPG production in a21 HS-5 cells compared to controls (Fig. 1, panel A and panel B). We also evaluated RANKL expression in the same samples. Interestingly, constitutive inhibition of miR-21 in HS-5 cell line upon exposure to MM cells induced a significant RANKL down-regulation, thus restoring a physiological RANKL/OPG ratio.We hypothesized that this surprising result may be due to the activity of the protein inhibitor of activated STAT3 (PIAS3). Indeed, PIAS3 binds to STAT3 and blocks its DNA binding ability, thereby inhibiting STAT3-mediated gene activation. It is already known that PIAS3 acts as a negative regulator of the RANKL expression and it is a direct and validated target of miR-21. Therefore, the inhibition of miR-21 induces PIAS3 up-regulation and a decreased RANKL transcription. To confirm the involvement of PIAS3 in RANKL suppression in our system, we performed a WB assay, showing that in a21 HS-5 cells, PIAS3 is indeed over-expressed compared to controls. Taken together, these findings provide the first evidence that inhibition of miR-21 in MM microenviroment restores RANKL/OPG balance, which might result in prevention of skeletal- related events. These findings support the design of innovative miR-21 inhibition-based approaches against MM-related BD.Supported by the Italian Association for Cancer Research (AIRC) “Special Program Molecular Clinical Oncology - 5 per mille”, PI:P.T., n. 9980, 2010/15.” [Display omitted] Disclosures:No relevant conflicts of interest to declare.

1185P - Prognostic Role of ERBB Family Receptors, MYC and Mitogen-Activated Protein Kinase (MAPK) in Patients with Early-Stage non Small-Cell Lung Cancer

ABSTRACT Background EGFR deregulation has been extensively studied in non small-cell lung cancer (NSCLC), but the expression and the role of other ErbB receptors and their downstream signal transductions remains still unclear. MYC and MAPK are key downstream components of the EGFR pathway and have significant roles in cell survival, proliferation, and growth. This study evaluates the prognostic role of EGFR, ErbB2, ErbB3, ErbB4, MYC and MAPK by immunohistochemistry (IHC) in early stage NSCLC. Methods One-hundred nine NSCLC patients were evaluated: median age was 67 years (range 40–84); Male/Female: 93/16; squamous (SCC)/adenocarcinoma (ADC)/BAC/other: 52/36/3/18; smoker/never smoker:100/9, and stage I/II/III:67/17/25. IHC results were evaluated by two independent observers and the tumors with ≥10% positive cells were classified positive, further confirmed by Receiver Operating Characteristic (ROC) analysis. Kaplan-Meier estimates of survival and time to recurrence were calculated for clinical and biologic variables using Cox model for multivariate analysis. Results EGFR was expressed in 55.9%, ErbB2 in 24.7% ErbB3 in 33.9%, ErbB4 in 27.5%, Myc in 23.8% and MAPK in 27.5 % of patients, respectively. EGFR and ErbB3 were associated with SCC (p Conclusions Our results suggest that in early stage NSCLC the co-expression of EGFR, ErbB2 and MAPK predicted a worse prognosis. Such features may have important implications for future targeted therapies. We thank Italian Association for Cancer Research (AIRC) for supporting the study. Disclosure All authors have declared no conflicts of interest.

MYD88 somatic mutations in MALT lymphomas

Extranodal marginal zone lymphoma (MZL) of mucosa-associated lymphoid tissue type (MALT lymphoma) is an indolent lymphoma that is found at a number of different extranodal anatomical sites and in total accounts for 7–8% of all non-Hodgkin (Bertoni et al, 2011). Recurrent chromosomal translocations and unbalanced genomic aberrations have been reported in MALT, many of which lead to activation of the nuclear factor (NF)-κB pathway (Bertoni et al, 2011). MYD88 is a central molecule in the cascade that activates NF-κB signalling after stimulation of either Toll-like, interleukin (IL) 1 and IL18 receptors (Ngo et al, 2011). The MYD88 gene is located at chromosome 3p22, a region commonly gained in MALT lymphomas (Rinaldi et al, 2011). MYD88 mutations were first described to occur almost exclusively in the activated B-cell like subtype (39%) of diffuse large B-cell lymphomas (Ngo et al, 2011), which are also characterized by NF-κB activation. Recurrent mutations were also described in 9% of gastric MALT lymphomas (Ngo et al, 2011). The vast majority of mutations, and all those observed in MALT lymphomas, determined a non-synonymous change at amino acid position 265 with a change from leucine to proline (L265P) (Ngo et al, 2011). Very recently, Xu et al (2011) identified the same MYD88 L265P change in 46/51 (90%) lymphoplasmacytic lymphomas (LPL), 87% with an IgM monoclonal protein (Waldenstrom Macroglobulinemia, WM), contrasting with 3/46 (6%) in MZLs, including 1/20 (5%) MALT lymphomas (Xu et al, 2011). These authors suggested that a L265P mutation could be used to differentiate LPL/WM from MZLs (Xu et al, 2011). With the aim of assessing the prevalence of MYD88 L265P mutations in MALT lymphomas, we studied 53 MALT lymphoma cases, previously characterized by genome-wide DNA profiling (Rinaldi et al, 2011). Informed consent was obtained in accordance with the Declaration of Helsinki following the procedures approved by the local ethical committees and institutional review boards of each participating institution. Whole genome-amplification (WGA) was performed as previously described (Mensah et al, 2012). MYD88 exon 5 underwent polymerase chain reaction (PCR) amplification using 5′-AGG GAG GACT GTG GAT GCA GTA CCA A -3′ and 5′-CTG CAG ACG TGT CTG TGA AGT T-3′, as forward and reverse primers (Morin et al, 2011), respectively. PCR products were sequenced as previously described (Mensah et al, 2012), and the sequence chromatograms were viewed with ChromasLite 2.01 (Technelysium Pty Ltd, Brisbane, Qld, Australia) and compared against GenBank NM_002468. Three out of the 53 (6%) of the MALT lymphoma cases revealed non-synonymous MYD88 mutations, all of which were verified using non-WGA DNA (Fig 1). Two cases harboured a T978C mutation causing the reported L265P substitution. Both of these cases presented with orbital adnexa involvement and no additional genomic lesions; one case had bone marrow involvement. One case of salivary gland MALT lymphoma, with trisomies of chromosomes 3 and 18, showed a novel interstitial 27bp deletion (c.1039_1065del) resulting in the loss of amino acids VCDYTNPCT (p.V286_T294del) that precede an internal inhibitory domain (Li et al, 2005; Ngo et al, 2011), which could become disrupted and allow an increased binding of MYD88 to IRAK1/IRAK4. Somatic mutations of MYD88 have been shown to activate down-stream signalling leading to the activation of both NF-κB but also JAK/STAT3 signalling (Ngo et al, 2011), providing a rationale for molecule-targeted pharmaceutical approaches in these patients. In conclusion, our data indicate that MYD88 is deregulated in a small subset of patients with MALT lymphoma. These MYD88 mutation-bearing patients, as well patients affected by LPL/WM (Xu et al, 2011) or by other indolent lymphoid tumours carrying similar mutations (Fabbri et al, 2011; Wang et al, 2011; Yan et al, 2012) will benefit from the development of new therapeutic strategies that target specific pathways irrespective of the lymphoma subtype. This work was supported by Oncosuisse grant OCS-02034-02-2007, Nelia and Amedeo Barletta Foundation (Lausanne, Switzerland) and Italian Association for Cancer Research (AIRC). FB conceived and designed the study. ZML, AR, AC and AAM performed analyses. MP, RDG, GB and EZ collected and characterized MZL samples. ZML, AC and FB interpreted the data and co-wrote the manuscript. All authors read and approved the final manuscript. All authors declare no conflict of interest.

Clinical significances of ErbB family receptors, Myc, and mitogen-activated protein kinase (MAPK) in patients with early-stage non-small cell lung cancer.

e21094 Background: EGFR deregulation has been extensively studied in non small-cell lung cancer (NSCLC), but less is known about the expression and role of other ErbB receptors and their downstream signal transductions. Myc and MAPK are key downstream components of the EGFR pathway and have significant roles in cell survival, proliferation, and growth. The purpose of this study is to determine the prognostic value of EGFR, ErbB2, ErbB3, ErbB4, Myc and MAPK by immunohistochemistry (IHC) in early stage NSCLC. Methods: 109 NSCLC were evaluated: median age was 67 years (range 40–84); Male/Female:93/16; squamous (SCC)/adenocarcinoma (ADC)/BAC/other: 52/36/3/18; smoker/never smoker:100/9, and stage I/II/III:67/17/25. The tumors with ≥10% positive cells were classified positive, further confirmed by Receiver Operating Characteristic (ROC) analysis. Kaplan-Meier estimates of survival and time to recurrence were calculated for clinical and biologic variables using Cox model for multivariate analysis Results: EGFR and EbrB3 were associated with SCC (p&lt;0.0001 and p=0.004, respectively) whereas ErbB2 and Myc with ADC (p=0.004 and p&lt;0.0001, respectively) . EGFR and ErbB3 were significantly associated (p=0.003), as well as MAPK and ErbB4 (p=0.02). At a median follow-up of 75 months the contemporary overexpression of EGFR, ErbB2 and MAPK was associated with shorter disease free survival (DFS) (HR=5.4, p=0.002) and overall survival (OS) (HR=8.9, p&lt;0.0001). At multivariate analysis adjusting for stage, the co-expression of EGFR, ErbB2 and MAPK was an independent predictor for worse DFS and OS (HR=5.7, p=0.004; HR=8.67, p&lt;0.001, respectively). Conclusions: Our results suggest that in early stage NSCLC, the co-expression of EGFR, ErbB2 and MAPK predicted a worse prognosis. ErbB3, ErbB4 and Myc were not prognostic factors. Such features may have important implications for future targeted therapies. We thank Italian Association for Cancer Research (AIRC) for supporting the study.