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Leiomyosarcomas are rare and aggressive tumors characterized by a complex karyotype. Surgical resection with or without radiotherapy and chemotherapy is the standard curative treatment. Unfortunately, a high percentage of leiomyosarcomas recurs and metastasizes. In these cases, doxorubicin and ifosfamide represent the standard treatment but with low response rates. Here, we evaluated the induction of proteotoxic stress as a possible strategy to kill leiomyosarcoma cells in a therapeutic perspective. We show that aggressive leiomyosarcomas coexist with high levels of proteotoxic stress. As a consequence, we hypothesized that leiomyosarcoma cells are vulnerable to further increases of proteotoxic stress. The small compound 2c is a strong inducer of proteotoxic stress. In leiomyosarcoma cells, it triggers cell death coupled to a profound reorganization of the mitochondrial network. By using stimulated emission depletion microscopy, we have unveiled the existence of DIABLO/SMAC clusters that are modulated by 2c. Finally, we have engineered a new version of 2c linked to polyethylene glycol though a short peptide, named 2cPP. This new prodrug is specifically activated by proteases present in the tumor microenvironment. 2cPP shows a strong antitumor activity in vivo against leiomyosarcomas and no toxicity against normal cells.

Background: The evidence on high-dose ifosfamide (HD-IFO) use in patients with relapsed osteosarcoma is limited. We performed a retrospective study to analyze HD-IFO activity. Methods: Patients with osteosarcoma relapsed after standard treatment [methotrexate, doxorubicin, cisplatin +/&minus, ifosfamide (MAP+/&minus, I)] with measurable disease according to RECIST1.1 were eligible to ifosfamide (3 g/m2/day) continuous infusion (c.i.) days 1&ndash, 5 q21d. RECIST1.1 overall response rate (ORR) (complete response (CR) + partial response (PR)), progression-free survival at 6-month (6m-PFS), duration of response (DOR), and 2-year overall survival (2y-OS) were assessed. PARP1 expression and gene mutations were tested by immunohistochemistry and next-generation sequencing. Results: 51 patients were included. ORR was 20% (1 CR + 9 PR). Median DOR was 5 months (95%CI 2&ndash, 7). Median PFS, 6m-PFS, OS, and 2y-OS were 6 months (95%CI 4&ndash, 9), 51%, 15 months (10&ndash, 19), and 30%, respectively. A second surgical complete remission (CR2) was achieved in 26 (51%) patients. After multivariate analysis, previous use of ifosfamide (HR 2.007, p = 0.034) and CR2 (HR 0.126, p &lt, 0.001) showed a significant correlation with PFS and OS, respectively. No significant correlation was found between outcomes and PARP1 or gene mutations. Conclusions: HD-IFO should be considered as the standard first-line treatment option in relapsed osteosarcoma and control arm of future trial in this setting.

Purpose: No effective therapy is available for unresectable soft-tissue sarcomas (STS). This unmet clinical need prompted us to test whether chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CAR.CIK) are effective in eliminating tumor cells derived from multiple STS histotypes in vitro and in immunodeficient mice. Experimental Design: The experimental platform included patient-derived CAR.CIK and cell lines established from multiple STS histotypes. CAR.CIK were transduced with a retroviral vector encoding second-generation CSPG4-specific CAR (CSPG4-CAR) with 4-1BB costimulation. The functional activity of CSPG4-CAR.CIK was explored in vitro, in two- and three-dimensional STS cultures, and in three in vivo STS xenograft models. Results: CSPG4-CAR.CIK were efficiently generated from patients with STS. CSPG4 was highly expressed in multiple STS histotypes by in silico analysis and on all 16 STS cell lines tested by flow cytometry. CSPG4-CAR.CIK displayed superior in vitro cytolytic activity against multiple STS histotypes as compared with paired unmodified control CIK. CSPG4-CAR.CIK also showed strong antitumor activity against STS spheroids; this effect was associated with tumor recruitment, infiltration, and matrix penetration. CSPG4-CAR.CIK significantly delayed or reversed tumor growth in vivo in three STS xenograft models (leiomyosarcoma, undifferentiated pleomorphic sarcoma, and fibrosarcoma). Tumor growth inhibition persisted for up to 2 weeks following the last administration of CSPG4-CAR.CIK. Conclusions: This study has shown that CSPG4-CAR.CIK effectively targets multiple STS histotypes in vitro and in immunodeficient mice. These results provide a strong rationale to translate the novel strategy we have developed into a clinical setting.

With the ever-expanding therapeutic indications and ongoing clinical trials with Poly(adenosine diphosphate-ribose) Polymerase (PARP) inhibitors, it is of outmost importance to stop and rethink what we know and still do not know concerning one of the major revolutions in target therapies in the last decades. Indeed, many PARP inhibitors (PARPi) are able to bind multiple targets, with a plethora of potential interactions with cancer cell signaling, metabolism and the tumor microenvironment (TME). These interactions can mediate both response and resistance to PARPi, but also represent an opportunity for sequential and/or combinatorial therapies. Here we advocate a "look before you leap" approach in reviewing available clinical and preclinical evidence concerning PARPi, delving into this complex entanglement, trying to unravel the potential for innovative therapeutic strategies revolving on PARP inhibition.

Metastatic bone and soft tissue sarcomas often relapse after chemotherapy (CHT) and molecular targeted therapy (mTT), maintaining a severe prognosis. A subset of sarcoma cancer stem cells (sCSC) is hypothesized to resist conventional drugs and sustain disease relapses. We investigated the immunotherapy activity of cytokine induced killer cells (CIK) against autologous sCSC that survived CHT and mTT. The experimental platform included two aggressive bone and soft tissue sarcoma models: osteosarcoma (OS) and undifferentiated-pleomorphic sarcoma (UPS). To visualize putative sCSC we engineered patient-derived sarcoma cultures (2 OS and 3 UPS) with a lentiviral sCSC-detector wherein the promoter of stem-gene Oct4 controls the expression of eGFP. We visualized a fraction of sCSC (mean 24.2 ± 5.2%) and confirmed their tumorigenicity in vivo. sCSC resulted relatively resistant to both CHT and mTT in vitro. Therapeutic doses of doxorubicin significantly enriched viable eGFP+sCSC in both OS (2.6 fold, n = 16) and UPS (2.3 fold, n = 29) compared to untreated controls. Treatment with sorafenib (for OS) and pazopanib (for UPS) also determined enrichment (1.3 fold) of viable eGFP+sCSC, even if less intense than what observed after CHT. Sarcoma cells surviving CHT and mTT were efficiently killed in vitro by autologous CIK even at minimal effector/target ratios (40:1 = 82%, 1:4 = 29%, n = 13). CIK immunotherapy did not spare sCSC that were killed as efficiently as whole sarcoma cell population. The relative chemo-resistance of sCSC and sensitivity to CIK immunotherapy was confirmed in vivo. Our findings support CIK as an innovative, clinically explorable, approach to eradicate chemo-resistant sCSC implicated in tumor relapse.

Summary Background Trabectedin is an alkylating drug with a unique mechanism of action causing single-strand and double-strand DNA breaks that activate DNA damage-response pathways. Based on our preclinical data, we hypothesised that poly(ADP-ribose) polymerase 1 (PARP1) inhibitors might be an ideal partner of trabectedin and aimed to assess the safety, identify the recommended phase 2 dose, and explore preliminary signs of activity of trabectedin and olaparib combination treatment in patients with bone and soft-tissue sarcoma. Methods We did an open-label, multicentre, phase 1b study, recruiting patients from the national Italian sarcoma network aged 18 years and older with histologically confirmed bone and soft-tissue sarcoma progressing after standard treatments with Eastern Cooperative Oncology Group performance status of 1 or less. In a classic 3 + 3 design, patients received a 24 h infusion of trabectedin on day 1 and olaparib orally twice a day in 21-day cycles across six dose levels (trabectedin 0·675–1·3 mg/m2 every 3 weeks; olaparib 100–300 mg twice a day from day 1 to 21). Intermediate dose levels were permitted to improve safety and tolerability. The primary endpoint was determination of the recommended phase 2 dose (the maximum tolerated dose). Safety and antitumour activity were assessed in all patients who received at least one dose of the study drugs. We report the results of the dose-escalation and dose-expansion cohorts. The trial is still active but closed to enrolment, and follow-up for patients who completed treatment is ongoing. This trial is registered with ClinicalTrials.gov, number NCT02398058. Findings Between Nov 17, 2014, and Jan 30, 2017, of 54 patients assessed for eligibility, we enrolled 50 patients: 28 patients in the dose-escalation cohort and 22 patients in the dose-expansion cohort. Patients received a median of four cycles of treatment (IQR 2–6; range 1–17 [the patients who received the highest number of cycles are still on treatment]) with a median follow-up of 10 months (IQR 5–23). Considering all dose levels, the most common grade 3–4 adverse events were lymphopenia (32 [64%] of 50 patients), neutropenia (31 [62%]), thrombocytopenia (14 [28%]), anaemia (13 [26%]), hypophosphataemia (20 [40%]), and alanine aminotransferase concentration increase (9 [18%]). No treatment-related life-threatening adverse events or deaths occurred. One (2%) patient interrupted treatment without progression without reporting any specific toxicity. Observed dose-limiting toxicities were thrombocytopenia, neutropenia for more than 7 days, and febrile neutropenia. We selected intermediate dose level 4b (trabectedin 1·1 mg/m2 every 3 weeks plus olaparib 150 mg twice a day) as the recommended phase 2 dose. Seven (14%; 95% CI 6–27) of 50 patients achieved a partial response according to Response Evaluation Criteria In Solid Tumors 1.1. Interpretation Trabectedin and olaparib in combination showed manageable toxicities at active dose levels for both drugs. Preliminary data on antitumour activity are encouraging. Two dedicated phase 2 studies are planned to assess activity of this combination in both ovarian cancer (EudraCT2018-000230-35) and soft-tissue sarcomas. Funding Italian Association for Cancer Research, Italian Sarcoma Group, Foundation for Research on Musculoskeletal and Rare Tumors, and Italian Ministry of Health.

Purpose of our study was to explore a new immunotherapy for high grade soft tissue sarcomas (STS) based on cytokine-induced killer cells (CIK) redirected with a chimeric antigen receptor (CAR) against the tumor-promoting antigen CD44v6. We aimed at generating bipotential killers, combining the CAR specificity with the intrinsic tumor-killing ability of CIK cells (CAR+.CIK). We set a patient-derived experimental platform. CAR+.CIK were generated by transduction of CIK precursors with a lentiviral vector encoding for anti-CD44v6-CAR. CAR+.CIK were characterized and assessed in vitro against multiple histotypes of patient-derived STS. The anti-sarcoma activity of CAR+.CIK was confirmed in a STS xenograft model. CD44v6 was expressed by 40% (11/27) of patient-derived STS. CAR+.CIK were efficiently expanded from patients (n = 12) and killed multiple histotypes of STS (including autologous targets, n = 4). The killing activity was significantly higher compared with unmodified CIK, especially at low effector/target (E/T) ratios: 98% vs 82% (E/T = 10:1) and 68% vs 26% (1:4), (p

Conventional treatments reached an unsatisfactory therapeutic plateau in the treatment of advanced unresectable bone and soft tissue sarcomas that remain an unsolved medical need. Several evidences support the concept that adoptive immunotherapy may effectively integrate within the complex and multidisciplinary treatment of sarcomas.In this work we reviewed adoptive immunotherapy strategies that have been explored in sarcoma settings, with specific focus on issues related to their clinic transferability. We schematically divided approaches based on T lymphocytes specific for MHC-restricted tumor-associated antigens or relying on MHC-independent immune effectors such as natural killer (NK), cytokine-induced killer (CIK) or γδ T cells.Preclinical findings and initial clinical reports showed the potentialities and drawbacks of different adoptive immunotherapy strategies. The expansion of tumor infiltrating lymphocytes is difficult to be reproduced outside melanoma. Genetically redirected T cells appear to be a promising option and initial reports are encouraging against patients with sarcomas. Adoptive immunotherapy with MHC-unrestricted effectors such as NK, CIK or γδ T cells has recently shown great preclinical potential in sarcoma setting and biologic features that may favor clinical transferability. Combination of different immunotherapy approaches and integration with conventional treatments appear to be key issues for successful designing of next clinical trials.

Malignant Pleural Mesothelioma (MPM) is an aggressive tumor characterized by poor prognosis and continuously increasing incidence due to widespread exposure to asbestos. Novel approaches for MPM management could take advantage of the intrapleural administration of drugs. Our group has recently developed a novel nanoplatform using gold nanoparticles (GNPs) aimed at the local treatment of lung diseases [Cova E et al. 2015]. We already proved that engineered GNPs loaded with pemetrexed and specifically decorated with the anti-CD146 expressed by MPM cells, were highly effective in inhibiting cancer cells [Stella GM et al. Eur Respir J 2015 46: OA5003]. The MAP kinase pathway is known to regulate proliferation and survival of tumor cells, including MPM [Myioshy S et al. 2012]. Clinical improvement of selumetinib slowed down due to reported adverse events [Janne PA et al, 2013]. Here, we aimed at investigating the therapeutic efficacy of selumetinib on MPM. Therefore, we preliminary tested selumetinib on two different MPM cell lines, MSTO-211H and H2452, representative of biphasic and epithelioid subtypes respectively (Fig.1). We found that selumetinib was efficacy in inhibiting MSTO-211H line as evidenced by half maximal inhibitory concentration (IC50) of 1.59 uM after 96 h incubation. However, selumetinib was almost ineffective in inhibiting H2452 cells by using the same drug concentrations suggesting a resistance to MEK inhibitors, as already observed for melanoma cell lines (Emery et al., 2009). At least in selected cases, selumetinib might be an useful drug to be loaded by functionalized targeted GNPs

Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause. The most recent hypotheses on IPF pathogenesis suggest a central role of epithelial cell damage, followed by a dysregulated molecular cross talk between epithelial cells and fibroblasts. Thus, IPF progression has often been assimilated to that of cancer, and several signaling patterns appear to be disrupted in both diseases. Here, we analyze the expression in an IPF series of a panel of molecules, which are known to play a role in tumorigenic process. Our findings, although preliminary, reveal that IPF landscape is enriched in neoplastic potential expressed in a context of complex genomic polyclonality and cellular heterogeneity. These results provide a rationale for further investigations aimed to exploit—in a similar fashion to cancer—targeted therapies for a “precision medicine” approach to IPF.

Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem–like cells (sCSC) that are likely responsible for relapses and drug resistance. In this study, we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS, including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs, autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4, a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients, we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas, including putative sCSCs, supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease. Cancer Res; 74(1); 119–29. ©2013 AACR.

Follow-up aims to precociously identify recurrences, metastases or treatment-related adverse events so as to undertake the appropriate therapy. Guidelines admit lack of knowledge on optimal surveillance schedule, but suggest follow-up based on experts' opinion and risk stratification. To identify the impact, if any, of regular follow-up, we interrogated our prospectively collected database whether early detection of recurrences affected both clinical management and, likely, the outcome.We required information to be available on primary surgery and ≥3°years of follow-up for non-recurring patients. We analysed recurrence characteristics (asymptomatic versus symptomatic, low- versus high tumour burden) and computed tomography (CT) scan counts to detect one recurrence. Kaplan-Meier method estimated recurrence-free survival (RFS), post-recurrence progression-free survival (PR-PFS), and disease-specific overall survival (OS). Comparisons used Hazard ratios (HR) with 95% confidence intervals (CIs). Multivariate analyses employed the Cox proportional hazards model. All tests were two-sided.Between 01/2001 and 12/2012 we found 233 study-eligible patients. Estimated 5- and 10-year RFS were 61.8% and 50.4%, respectively. After a 68-month median follow-up, we observed 94 (40.3%) recurrences [73/94 (77.7%) asymptomatic versus 21/94 (22.3%) symptomatic and 45/94 (47.9%) low- versus 49/94 (52.1%) high tumour burden]. Multivariate analysis revealed that symptomatic and high tumour burden recurrences were highly predictive of both worse PR-PFS (HR:3.19, P 0.001; HR:2.80, P = 0.003, respectively) and OS (HR:3.65, P 0.001; HR:2.38, P = 0.026, respectively). Finally, 29 second (primary) cancers were detected during follow-up.Regular follow-up detects recurrences at an earlier stage and may be associated with a better PR-PFS and OS for these patients. In the absence of randomised trials, these evidences support follow-up effort and cost.

e22509 Background: Receptor tyrosine kinases (RTKs) and their signal transducers are suitable targets for the treatment of advanced OS. We evaluated the antitumor activity of the RTK inhibitor P and the MEK inhibitor T and deeply investigated molecular mechanisms behind their activity and potential escape. Methods: Flow cytometry and western blot analyses were carried out in 7 OS cell lines to study the expression of RTK P targets and the activation of their pathways, respectively. Cell viability and colony growth were evaluated after 72h and 7-day treatment respectively, with scalar doses of both single agents and their constant combination. Cell cycle distribution and apoptosis were evaluated by flow cytometry after 72h. In vivo antitumor activity was studied in NOD/SCID mice bearing MNNG-HOS xenografts after 3 weeks of treatment. Cell migration was studied by scratch assays. The involvement of MAPK-PI3K pathway key transducers was explored by Vantage 3D RNA Panel and Nanostring technology, validated by western blot and confirmed by silencing experiments. Results: P targets are expressed on OS cell lines and their pathways are activated. P+T have synergistic antitumor activity (combination index < 1) in OS cell lines by inducing apoptosis (6/7) and inhibiting both ERK1/2(7/7) and AKT (7/7). Furthermore, in vivo antitumor activity was shown in OS bearing mice (tumor volume: P+T/untreated = 0.036, p = 0.002). P+T significantly down-modulated RTK EphaA2 (mean log2 fold change RNA P+T/untreated = -2.02±0.50) and induced Janus kinase MEK6 (mean log2 fold change RNA P+T/untreated = 2.9±0.51). EphA2 silencing reduced cellular proliferation and migration of OS cells. Impeding MEK6-up-regulation in P+T treated cells significantly increased the antitumor effect (51.5±14.3%) of the studied drugs. Conclusions: P+T exert antitumor activity in OS preclinical models through ERK and AKT inhibition and EphA2 downmodulation. MEK6-upregulation after P+T is likely implied in escape mechanism.

11018Background: Given that trabectedin (T) causes single- and double-strand breaks along DNA, we hypothesized to combine trabectedin with the selective PARP1-inhibitor olaparib (O) to induce irrep...

Leiomyosarcoma represents about 24% of all soft tissue sarcomas and can originate from retroperitoneum, uterus, or extremities. Adequate local control may be achieved with surgery and radiotherapy. In the presence of unresectable metastases either doxorubicin- or gemcitabine-based chemotherapy is the standard of treatment. Nevertheless, prognosis remains poor regardless of the selected chemotherapy regimen, and new effective therapeutic agents for patients with advanced leiomyosarcoma are needed. Trabectedin, a promising new DNA-damaging agent with a mechanism of action that is different from that of traditional alkylating agents, is approved in Europe for the treatment of patients with advanced soft tissue sarcoma, after failure of anthracyclines and ifosfamide, or who are unsuited to receive these agents and in combination with pegylated liposomal doxorubicin (PLD) for the treatment of patients with relapsed platinum-sensitive ovarian cancer. We present a case of a 76-year-old patient with progressive metastatic lung lesions from a previously resected primary leiomyosarcoma of the thigh and moderate renal failure, who achieved 17 months of disease stability during third-line treatment with trabectedin. Trabectedin was not associated with any cumulative toxicity and was consistently well tolerated for a total of 22 treatment cycles. Current evidence on trabectedin is also presented.

Cytokine-induced killer cells (CIKs) are ex vivo expanded T-NK lymphocytes capable of HLA-unrestricted antitumor activity. CIKs are promising candidates for adoptive cancer immunotherapies; they can be generated and infused in autologous settings of cancer patients, or from donors, after allogeneic hematopoietic cell transplant. Ex vivo expansion rates of CIKs are greatly variable among patients, with consequent potential clinical limitations for "poor expanders." We compared the standard expansion protocol with a new one, which included the timed addition of irradiated allogeneic peripheral blood mononuclear cells. Our hypothesis is that allogeneic stimulation might provide CIK cells with a proliferative boost and simultaneously decrease their alloreactivity versus third parties, if HLA-mismatched from the allogeneic stimulators. Allo-stimulated CIKs (AS-CIK) reached significantly higher expansion rates compared with standard controls, regardless if generated form healthy donors (131- vs. 32-fold) or cancer patients (117- vs. 14-fold). The expansion of the CD3CD56 subset was 2243-fold for AS-CIKs compared with 362 for standard CIKs. AS-CIKs efficiently killed osteosarcoma targets in vitro, results were comparable with that of standard CIKs. Standard and AS-CIKs did not show differences in phenotype and telomere length. The alloreactivity of AS-CIKs against third party HLA-mismatched peripheral blood mononuclear cells was reduced compared with standard CIKs (37% vs. 23%). In conclusion, alloreactivity of CIK cells may be exploited enhancing their final ex vivo expansion. In clinical perspective these findings may facilitate the extension of CIK-based immunotherapy to larger numbers of patients and, translated into hematopoietic cell transplant settings, contribute to reduce the risk of graft versus host disease in the hypothesis of infusions across HLA barriers.

Purpose: After standard multimodal therapy, the prognosis of relapsed and unresectable high-grade osteosarcoma is dismal and unchanged over the last decades. Recently, mitogen-activated protein kinases were shown to be activated in osteosarcoma specimens, suggesting, therefore, they are suitable targets for the multikinase inhibitor sorafenib. Thus, we explored sorafenib activity in patients with relapsed and unresectable osteosarcoma. Experimental design: Patients >14 years, progressing after standard treatment, were eligible to receive 400 mg of sorafenib twice daily until progression or unacceptable toxicity. The primary end point was progression-free survival (PFS) at 4 months. Secondary objectives were PFS, overall survival (OS), clinical benefit rate (CBR), defined as no progression at 6 months and safety. This nonrandomized phase II study used a Simon two-stage design. PFS and OS at 95% confidence intervals (95% CIs) were calculated by the Kaplan-Meier method. All tests were two sided. Results: Thirty-five patients were enrolled. PFS at 4 months was 46% (95% CI 28% to 63%). Median PFS and OS were 4 (95% CI 2-5) and 7 (95% CI 7-8) months, respectively. The CBR was 29% (95% CI 13% to 44%). We observed 3 (8%) partial responses (PRs), 2 (6%) minor responses (

The recombinant monoclonal antibody trastuzumab has antiproliferative effect on breast cancer (BC) cells with ErbB2 overexpression. We postulated that a mechanism able to modify ErbB2 expression enhances the antitumor effect of trastuzumab. We analyzed whether granulocyte-colony stimulating factor (G-CSF), widely used in adjuvant cancer therapy to alleviate chemotherapy-induced myelotoxicity, could influence ErbB2 expression in BC cells and patients. The expression of ErbB2 (Herceptest) was analyzed in four BC cell lines (BT474, SKBR3, ZR75.1, and T47D) treated with G-CSF and in five samples biopsies from BC patients subjected to G-CSF rescue after chemotherapy. The effects of G-CSF and trastuzumab alone or their combination on cell growth and apoptosis were investigated. G-CSF receptor was detected on all cell lines and BC patients. G-CSF induced upregulation of ErbB2 in SKBR3, ZR75, and T47D cells. This modulation was not associated with an increase in tumor cell growth in vitro. Trastuzumab alone inhibited colony formation in soft agar but did not induce apoptosis on BC cells with no or low ErbB2 genomic amplification. The combination of trastuzumab and G-CSF enhanced the inhibition of tumor colony formation and induced apoptosis on these cells. This effect was further increased by G-CSF pretreatment. Five of nine BC patients showed an increase of Herceptest score after G-CSF administration. G-CSF treatment increases ErbB2 expression in vitro and in vivo enhancing the activity of trastuzumab on BC cell lines inducing apoptosis of BC cells with low or no ErbB2 genomic amplification.

Tumor cell contamination might induce relapse after autologous transplantation in breast cancer patients. We used an ex vivo purging strategy to decrease the number of contaminating breast tumor cells in leukaphereses without altering the engraftment potential of the hemopoietic progenitor cells. This method is based on immunoselection of CD34+ cells derived from mobilized peripheral blood of patients with metastatic breast cancer and expansion in the presence of flt3 ligand, stem cell factor, interleukin 6, and thrombopoietin. Tumor contamination before and after culture was monitored by mammaglobin messenger RNA amplification by quantitative polymerase chain reaction. We analyzed both adherent and suspended cells obtained after 2 weeks of culture. Hemopoietic progenitors were increased among suspended cells. In this fraction, tumor cell contamination was decreased, whereas it increased within the adherent cell fraction. Experimental models using CD34+ cells from healthy donors spiked with breast cancer cells were also constructed to investigate whether treatment with anti–ErbB-receptor drugs could further reduce the tumor load without affecting the clonogenic potential of hemopoietic cells. For this purpose, we successfully assayed trastuzumab, a monoclonal antibody against ErbB-2, and gefitinib, an epidermal growth factor receptor tyrosine kinase receptor inhibitor. These results suggest that positively selected CD34+ cells from cancer patients contain tumor cells and that ex vivo expansion can reduce the tumor load of the suspended fraction. Target-based agents against ErbB-2, epidermal growth factor receptor, or both—such as trastuzumab or gefitinib—might increase the efficiency of purging.

10569 Background: TR is a DNA damaging agent as 2nd line-therapy in STS. We speculated that its antitumor activity could be potentiated by a “synthetic lethality” approach targeting key molecules i...

10552 Background: FU care poses a burden on both pts and health system. FU aims to precociously identify recurrences, metastases or treatment-related adverse events so to undertake the appropriate therapy as soon as possible. Guidelines (NCCN, ESMO) admit lack of knowledge on optimal surveillance, but suggest FU based on experts’ opinion and risk stratification. Moreover, tumor burden (TB) is a well known negative prognostic factor (Van Glabbeke 2005). Therefore, low-TB at R might affect final outcome. To identify the impact, if any, of regular FU, we examined our prospectively collected database looking for Rs in which the early detection affected both clinical management and, likely, the outcome. Methods: 140 pts were stratified (AFIP classification). High risk (HR) pts had complete history + physical examination (H&P) and CT every 3 mos for 2 years, 4 mos in 3rd year, 6 mos in the 4th and 5th yrs, yearly thereafter; intermediate (IR), low (LR) and very low risk (VLR) pts had H&P + CT every 4 mos for 2 years, every 6 mos up to the 5th year, then yearly. Rs were divided in: low-TB + completely resectable R (Group 1) and high-TB + disseminated R (Group 2). The number of CT needed to detect (NND) one R and the incidence of early (

10583 Background: Regardless optimal surgery LR is still a major challenge in RPS affecting 60% of the patients (pts). To improve these results pre-operative radiotherapy +/- chemotherapy are under investigation. This strategy requires to perform a POB. The surgical removal of the whole needle track is mandatory for limb sarcomas, but it can hardly ever be achieved in RPS. We explored whether POB did increase the risk of LR in terms of tumor cell seeding (along the needle track) or in the tumor mass area. Methods: We scanned our prospectively (01/2000 to 12/2010) collected RPS database including 176 pts. Complete information (records, CT scans, biopsy-track, follow up data) were available and retrospectively examined in 100 pts. Pts were divided into: group A (POB), group B (direct surgery). The 7 year probability of disease free survival (DFS) was estimated for each pt (ASCO 2012 abs 10000). T student test and Fisher's exact test were used to compare the estimated DFS and the incidence of LR in the 2 groups, respectively. Kaplan-Meier method was used to estimate DFS and overall survival (OS). Results: In 51 male, 49 female, median age 58 years (22-81) RPS histotype distribution was: lipo 43, leio 28, pleomorphic 9 and other 20. RPS grades were: G1 29%, G2 32%, G3 39%. POB was performed in 25 pts (25%) without major complications. The baseline probability of 7 year DFS was 0.43 and 0.51 (p= .24) for group A and B, respectively. After a median follow up of 71 mos, LR was 8% and 23% (p> .05) for group A and B, respectively. No relapse along the needle track was detected. As expected, liposarcoma was associated with a higher rate of LR (Odds Ratio 5.6 CI 95% 2-13). Median DFS, local DFS and OS were 24, 88 and 71 months, respectively. Conclusions: With the limit of the retrospective nature of our data, we didn’t find any increased risk of LR for POB in RPS. Although surgery is still the standard therapy in RPS, knowledge of histotype and differential diagnosis might encourage to consider inclusion of pts into pre-operative clinical trials and to avoid surgery for other retroperitoneal tumors (e.g. lymphomas, germinal cell tumors, IgG4-related sclerosing disease).

Background. TR is a DNA-binding alkaloid approved in Europe for the treatment STS as second/third line therapy. TR interferes with gene transcription and nucleotide excision repair, inducing DNA double strand breaks (DSBs). There is a strong clinical interest to increase TR activity by combination with other anti-cancer drugs. PARP-1 inhibitors (PARPi) which disable DNA base-excision repair mechanism causing the accumulation of DSBs, look like a worth to explore TR therapeutic partners. Our preclinical studies were focused on the effects of the combination of TR with the PARPi (OL and veliparib). Methods: We explored DNA damage by comet assay and P-H2AX determination, PARP-1 activity by polyADPribosylation assay in a panel of 18 sarcoma cell lines, evaluating cell viability after 72h treatment with escalating doses of TR, PARPi, and their constant combination. Colony growth, cell cycle, apoptosis and DNA damage were evaluated. Antitumoral activity was checked after 4 weeks of treatment with 3 endovenous injection of TR once a week and OL intraperitoneally 5 days/week, and compared to control and single agents in bone (SJSA-1) and soft tissue sarcoma (DMR) xenografted NOD/SCID mice. TUNEL assay, P-H2AX, PCNA and CD31 immunohistochemistry was done on explanted xenografts. Results: We showed TR-induced DNA damage and PARP-1 activation in sarcoma sensitive cell line. The addition of PARPi completely blocked basal and TR-induced PARP-1 activation. DNA damage response and checkpoints (ATM, ATR, BRCA1, CHK1, CHK2, p53) were activated after single agents and combination treatment inducing cell cycle arrest in S/G2 phase. This block was released after 48h in single agent-treated cells but not in combination-treated cells leading to apoptotic cell death. Colony growth assay and viability tests revealed a strong synergism of the two drugs (Combination Index < 1, based on Chou-Talalay method). Different sarcoma histotypes displayed differences in sensitivity to both drugs and combination (Ewing's sarcoma the most sensitive> myxoid liposarcoma > leiomyosarcoma > osteosarcoma ≥ undifferentiated pleomorphic > fibrosarcoma). The antitumor effect of combined TR and OL was shown after in vivo treatment of SJSA-1 and DMR xenografted mice. A significant tumor volume reduction was observed if compared to both control and single agent and was attributed to increased DNA damage, apoptosis and reduced proliferation, but not to anti-angiogenic potentiation. In fact, a direct effect on tumor vasculature was shown in TR and combination treated group vs control. Conclusions: Our results validate the biological rationale to combine TR and PARPi in sarcoma and suggest exploring this pharmacological approach in the clinical setting. Citation Format: Ymera Pignochino, Federica Capozzi, Carmine Dell'aglio, Marco Basiricò, Lorenzo D'ambrosio, Danilo Galizia, Erica Palesandro, Maria Serena Benassi, Massimo Aglietta, Giovanni Grignani. The antitumor effect of trabectedin (TR) is potentiated by olaparib (OL) in preclinical models of bone and soft tissue sarcomas (STS). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-213. doi:10.1158/1538-7445.AM2013-LB-213

10066 Background: TR is an alkylating agent approved in Europe for the treatment of advanced STS as second/third line therapy. TR binds to the minor groove of DNA and interferes with gene transcription and nucleotide excision repair mechanism, inducing DNA double strand breaks (DSBs), and S/G2 cell cycle arrest. There is a strong clinical interest to increase TR activity combining it with other anti-cancer drugs. PARP-1 inhibitors disable DNA base-excision repair mechanism causing the accumulation of DSBs and look like a reasonable TR partner to be explored. We focused our in vitro studies on the effects of the combination of TR with the PARP-1 inhibitor Olaparib (OL). Methods: We explored the activity of TR-OL combination against a panel of different histotypes of STS cell lines, evaluating cell viability after 72h treatment with escalating doses of TR (0-2 nM), OL (0-20 µM), and their constant combination. Following colony formation, cell cycle, apoptosis (annexin V+/PI+) and DNA damage (phospho-histone H2AX - Ser139) were checked. Results: The TR-OL combination strongly affects STS cell viability, showing synergism (Combination Index < 1, based on Chou–Talalay method) on 8 out of 13 cell lines tested. We observed a strong synergism, as a massive reduction of colony growth (402.91, MES-SA, and DMR-SN-8.4.98 lines) induced by the combination if compared with each single agent (78% vs. ~27% : p

OS is the most frequent primary bone tumor in children and young adults.The mammalian Target of Rapamycin (mTOR) and the Extracellular Regulated Kinases (ERK)1/2 pathways have been shown to cooperate in survival advantage of OS. Inhibition of this signaling can be a rational pharmacological strategy to impinge on OS progression. This study pursued the dual blockage of ERK1/2 and mTOR pathways by combining the multikinase inhibitor SOR with the rapamycin analog EV. The synergistic anti-profilerative effect was found against 5 out 7 OS cell lines (combination index Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-366. doi:1538-7445.AM2012-LB-366

10058 Background: Targeting pathways involved in progression and metastatization might be a suitable way to overcome chemoresistance in OS. Monoclonal antibodies (anti-IGF1R) andMK-I (SOR, dasatini...

Osteosarcoma (OS) is the most common primary bone tumor in children and young adults. Despite improved prognosis, metastatic or relapsed OS remains largely incurable and no significant improvement has been observed in the last 20 years. Therefore, the search for alternative agents in OS is mandatory. We explored phospho-ERK 1/2, MCL-1, phospho-Ezrin/Radixin/Moesin (P-ERM) as potential therapeutic targets. The activation of these pathways was shown by immunohistochemistry in about 70% of cases and in all OS cell lines analyzed. Mutational analysis revealed no activating mutations in KRAS. While, BRAF gene was found mutated in 4/30 OS samples from patients. Based on these results we tested the multikinases inhibitor sorafenib (BAY 43-9006) in preclinical models of OS. Sorafenib inhibited OS cell lines proliferation, induced apoptosis and downregulated P-ERK1/2, MCL-1, P-ERM in a dose dependent manner. ERM dephosphorylation is not due to ERK inhibition. The downregulation of MCL-1 increased apoptosis in OS cell lines. In chick embryo chorioallantoic membrane the angiogenesis induced by OS supernatants was blocked by sorafenib treatments. Indeed, sorafenib reduced VEGF and MMP2 production. Sorafenib treatment dramatically reduced tumor volume of OS xenografts and lung metastasis in SCID mice. In conclusion, ERK1/2, MCL-1 and ERM pathways are active in OS. Sorafenib is able to interrupt their routes in vitro and in vivo displaying antitumoral activity, antiangiogenic effect and reducing metastasis colonies formation in lungs. These data support the testing of sorafenib as a potential therapeutic option in metastatic or relapsed OS patients failing standard treatments. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C213.

10540 Background: Osteosarcoma is the most common primary bone tumor in young adults. Despite improved prognosis, resistance to chemotherapy remains responsible for failure of OS treatment. The search for alternative agents focused on specific molecular mechanisms in OS is mandatory. Sorafenib (BAY 43–9006) is a multitarget antitumoral drug (RAF, VEGFR-2/3, FLT-3, KIT, FGFR-1, RET, MCL- 1; PDGFR-β). We explored the expression of main sorafenib targets in OS and its activity in preclinical models. Methods: In 30 samples from OS patients and in 7 OS cell lines we performed immunohistochemistry for MAPK-P and Mcl-1 expression as well as mutational analysis of B-RAF and K-RAS. We assessed the cell proliferation inhibition both in vitro (Cell Titer-Glo) and in vivo (xenografts in SCID mice) caused by Sorafenib treatment. The sorafenib molecular mechanism was investigated by Western blot (MAPK-P, MCL-1) and ELISA tests (VEGF, MMP2/9). Results: Expression of MAPK-P and MCL-1 was shown in 20/30 and 24/30 specimen...

4582 Background: Biliary tract carcinomas express EGFR and are potential candidates to EGFR target therapies. We recently described somatic mutations of EGFR that can enhance MAPK or Akt activation (Clin Cancer Res, 2006). Some of them are identical to those previously reported to confer sensitivity to some tyrosine kinase inhibitors (TKIs) like erlotinib or gefitinib in lung cancer. Here we report a molecular analysis of EGFR transducers potentially involved in TKI response. Methods: In 49 samples of biliary tract carcinoma we performed mutational analysis of exons from 18 to 21 of EGFR, exons 9 and 20 of phosphatidylinositol 3’-kinase (PI3K), exon 2 of K-Ras, exon 15 of B-Raf and exons from 5 to 8 of PTEN. Nuclear PTEN expression was analyzed by immunohistochemistry and the expression in cancer cells was compared to that of normal cholangiocites. Results: Mutations of EGFR have been detected in 7 out of 49 samples (14.3%). One of them was a new stop-codon mutation. Five hotspot mutations of PI3K (codon 545, 546, 1048 and 1059) were found in 4 cases (8.2%); 3 cases (6.1%) had single mutations in K-Ras and 4 (8.2%) had the V599E mutation in B-Raf. In some samples, mutations of multiple trasducers were present simultaneously. PI3K mutations were significantly more frequent in EGFR mutated samples compared to wild type (28% vs. 4.7% respectively, p No significant financial relationships to disclose.

TO THE EDITOR: A study by Philip et al published in the July 1, 2006, issue of the Journal of Clinical Oncology shows the therapeutic benefit of epidermal growth factor receptor (EGFR) blockade with erlotinib in patients with biliary tract carcinoma. The results are interesting because in a cohort composed of 24 chemotherapy-refractory patients and 18 chemotherapy-naive patients, erlotinib as a single agent can achieve disease control in 50% of patients with a median duration of disease control of 5.1 months. However, the authors conclude that the activity of erlotinib is modest because only 17% of the patients achieved the primary end point of the study, which was progression-free survival at 24 weeks. Although gefitinib and erlotinib are active in unselected patients with non–small-cell lung cancer (NSCLC), clinical characteristics and tumor molecular markers associated with enhanced benefit have been identified. Notably, mutations in the tyrosine kinase domain of EGFR were subsequently found to be associated with sensitivity to small molecule inhibitors in NSCLC. Emerging data also suggest that increased EGFR copy number is a predictor of response to gefitinib therapy, regardless of EGFR kinase domain mutational status. Furthermore, in contrast to wild-type EGFR, the EGFR mutations are mutually exclusive of K-Ras mutations, which are found in biliary tract carcinomas with a particularly high incidence in some geographical areas. Mutations of the EGFR kinase domain have recently been described by our group in 15% of biliary tract carcinoma specimens. One of these mutations was a T790M substitution, which is known to correlate with an acquired resistance to small molecule inhibitor treatment in NSCLC. Whereas 28% of the EGFR-positive patients were progression free at 24 weeks, the study by Philip et al, based on the lack of correlation between EGFR expression level and treatment outcome in other tumor types, includes six undetermined and seven EGFRnegative patients. This patient number is too small to provide evidence of any correlation. Thus, it would be of interest to know if other molecular analyses, such as sequencing of EGFR tyrosine kinase or of downstream members of the signaling pathway, like K-Ras, B-Raf or PTEN, or EGFR gene amplification, have been planned in this group of biliary tract cancer patients treated with erlotinib. This finding will have extraordinary implications and will serve as a critical step toward individualized patient-specific treatment plans based on the molecular constitution of the tumor of each individual.

10533^ Background: After standard therapy OST relapse is an ominous event almost uniformly fatal in non resectable cases. Our group explored the activity of the multikinase inhibitor S demonstratin...

Background Malignant Pleural Mesothelioma (MPM) is an aggressive tumor arising from mesothelial cells lining the pleural cavities characterized by resistance to standard therapies. Most of the molecular steps responsible for pleural transformation remain unclear; however, several growth factor signaling cascades are known to be altered during MPM onset and progression. Transducers of these pathways, such as PIK3CA-mTOR-AKT, MAPK, and ezrin/radixin/moesin (ERM) could therefore be exploited as possible targets for pharmacological intervention. This study aimed to identify ‘druggable’ pathways in MPM and to formulate a targeted approach based on the use of commercially available molecules, such as the multikinase inhibitor sorafenib and the mTOR inhibitor everolimus. Methods We planned a triple approach based on: i) analysis of immunophenotypes and mutational profiles in a cohort of thoracoscopic MPM samples, ii) in vitro pharmacological assays, ii) in vivo therapeutic approaches on MPM xenografts. No mutations were found in ‘hot spot’ regions of the mTOR upstream genes (e.g. EGFR, KRAS and PIK3CA). Results Phosphorylated mTOR and ERM were specifically overexpressed in the analyzed MPM samples. Sorafenib and everolimus combination was effective in mTOR and ERM blockade; exerted synergistic effects on the inhibition of MPM cell proliferation; triggered ROS production and consequent AMPK-p38 mediated-apoptosis. The antitumor activity was displayed when orally administered to MPM-bearing NOD/SCID mice. Conclusions ERM and mTOR pathways are activated in MPM and ‘druggable’ by a combination of sorafenib and everolimus. Combination therapy is a promising therapeutic strategy against MPM. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1363-1) contains supplementary material, which is available to authorized users.