A. Italiano

PRIMA-1(MET) induces death in soft-tissue sarcomas cell independent of p53.

BackgroundThe aim of this study was to explore the efficacy and define mechanisms of action of PRIMA-1MET as a TP53 targeted therapy in soft-tissue sarcoma (STS) cells.MethodsWe investigated effects of PRIMA-1MET on apoptosis, cell cycle, and induction of oxidative stress and autophagy in a panel of 6 STS cell lines with different TP53 status.ResultsCell viability reduction by PRIMA-1MET was significantly observed in 5 out of 6 STS cell lines. We found that PRIMA-1MET was capable to induce cell death not only in STS cells harboring mutated TP53 but also in TP53-null STS cells demonstrating that PRIMA-1MET can induce cell death independently of TP53 in STS cells. We identified an important role of reactive oxygen species (ROS), involved in PRIMA-1MET toxicity in STS cells leading to a caspase-independent cell death. ROS toxicity was associated with autophagy induction or JNK pathway activation which represented potential mechanisms of cell death induced by PRIMA-1MET in STS.ConclusionsPRIMA-1MET anti-tumor activity in STS partly results from off-target effects involving ROS toxicity and do not deserve further development as a TP53-targeted therapy in this setting.

Combined targeting of MDM2 and CDK4 is synergistic in dedifferentiated liposarcomas

PurposeMDM2 and CDK4 are frequently co-amplified in well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS). We aimed to determine whether combined MDM2/CDK4 targeting is associated with higher antitumour activity than a single agent in preclinical models of DDLPS.Experimental designDDLPS cells were exposed to RG7388 (MDM2 antagonist) and palbociclib (CDK4 inhibitor), and apoptosis and signalling/survival pathway perturbations were monitored by flow cytometry and Western blotting. Xenograft mouse models were used to assess tumour growth and survival. Treatment efficacy was assessed by Western blotting, histopathology and tumour volume.ResultsRG7388 and palbociclib together exerted a greater antitumour effect than either drug alone, with significant differences in cell viability after a 72-h treatment with RG7388 and/or palbociclib. The combination treatment significantly increased apoptosis compared to the single agents. We then analysed the in vivo antitumour activity of RG7388 and palbociclib in a xenograft model of DDLPS. The combination regimen reduced the tumour growth rate compared with a single agent alone and significantly increased the median progression-free survival.ConclusionsOur results provide a strong rationale for evaluating the therapeutic potential of CDK4 inhibitors as potentiators of MDM2 antagonists in DDLPS and justify clinical trials in this setting.

Bone Marrow Metastases in a Patient with Primary Mediastinal Non-Seminomatous Germ Cell Tumor – an Unusual Pattern of Relapse

Background: Bone marrow is a very unusual site of metastasis for germ cell tumors. Case Report: We report the case of a 21-year-old male patient who was treated with chemotherapy and secondary surgery for a primary mediastinal non-seminomatous germ cell tumor (NSGCT). The patient achieved complete remission. However, 4 months after completion of therapy, he complained of rapidly worsening bone pain. No evidence for disease relapse was found in the computed tomography scan of thorax and abdomen, magnetic resonance imaging of the spine, or bone scan. A blood test revealed pancytopenia and elevated serum tumor markers. A bone marrow aspirate showed infiltration by tumor cells positive for AE1/AE3 and AFP confirming the diagnosis of isolated bone marrow metastatic relapse. Salvage chemotherapy was started and resulted in a rapid decrease of serum tumor markers. However, pancytopenia did not improve and the patient died of severe sepsis 3 weeks later. Conclusion: We report here the first case of isolated bone marrow metastatic relapse of an NSGCT. 2 other cases of bone marrow metastasis in patients with NSGCT have been reported. In these 2 cases, as in our patient, the primary site was not testicular but mediastinal suggesting a non-fortuitous association.

Activity of trabectedin and the PARP inhibitor rucaparib in soft-tissue sarcomas

BackgroundTrabectedin has recently been approved in the USA and in Europe for advanced soft-tissue sarcoma patients who have been treated with anthracycline-based chemotherapy without success. The mechanism of action of trabectedin depends on the status of both the nucleotide excision repair (NER) and homologous recombination (HR) DNA repair pathways. Trabectedin results in DNA double-strand breaks. We hypothesized that PARP-1 inhibition is able to perpetuate trabectedin-induced DNA damage.MethodsWe explored the effects of combining a PARP inhibitor (rucaparib) and trabectedin in a large panel of soft-tissue sarcoma (STS) cell lines and in a mouse model of dedifferentiated liposarcoma.ResultsThe combination of rucaparib and trabectedin in vitro was synergistic, inhibited cell proliferation, induced apoptosis, and accumulated in the G2/M phase of the cell cycle with higher efficacy than either single agent alone. The combination also resulted in enhanced γH2AX intranuclear accumulation as a result of DNA damage induction. In vivo, the combination of trabectedin and rucaparib significantly enhanced progression-free survival with an increased percentage of tumor necrosis.ConclusionThe combination of PARP inhibitor and trabectedin is beneficial in pre-clinical models of soft-tissue sarcoma and deserves further exploration in the clinical setting.

Heterogeneous Mechanisms of Secondary Resistance and Clonal Selection in Sarcoma during Treatment with Nutlin

Nutlin inhibits TP53-MDM2 interaction and is under investigation in soft-tissue sarcomas (STS) and other malignancies. Molecular mechanisms of secondary resistance to nutlin in STS are unknown. We performed whole-transcriptome sequencing (RNA-seq) on three pretreatment and secondary resistant STS cell lines selected based on their high primary sensitivity to nutlin. Our data identified a subset of cancer gene mutations and ploidy variations that were positively selected following treatment, including TP53 mutations in 2 out of 3 resistant cell lines. Further, secondary resistance to nutlin was associated with deregulation of apoptosis-related genes and marked productive autophagy, the inhibition of which resulted in significant restoration of nutlin-induced cell death. Collectively, our findings argue that secondary resistance to nutlin in STS involved heterogeneous mechanisms resulting from clonal evolution and several biological pathways. Alternative dosing regimens and combination with other targeted agents are needed to achieve successful development of nutlin in the clinical setting.

Prospective assessment of the predictive value of the BRCA1 gene status in sarcoma patients treated with trabectedin: an updated analysis of the EORTC 62091 trial

We describe the predictive value of BRCA1 gene status on trabectedin efficacy and found no correlation despite the mechanisms of action of this drug that rely on DNA repair systems.

Activity and safety of crizotinib in patients with alveolar soft part sarcoma with rearrangement of TFE3: European Organization for Research and Treatment of Cancer (EORTC) phase II trial 90101 'CREATE'

BackgroundnAlveolar soft part sarcoma (ASPS) is an orphan malignancy associated with a rearrangement of transcription factor E3 (TFE3), leading to abnormal MET gene expression. We prospectively assessed the efficacy and safety of the MET tyrosine kinase inhibitor crizotinib in patients with advanced or metastatic ASPS.nnnPatients and methodsnEligible patients with reference pathology-confirmed ASPS received oral crizotinib 250u2009mg bd. By assessing the presence or absence of a TFE3 rearrangement, patients were attributed to MET+u2009and MET- sub-cohorts. The primary end point was the objective response rate (ORR) according to local investigator. Secondary end points included duration of response, disease control rate (DCR), progression-free survival (PFS), progression-free rate, overall survival (OS) and safety.nnnResultsnAmong 53 consenting patients, all had a centrally confirmed ASPS and 48 were treated. A total of 45 were eligible, treated and assessable. Among 40 MET+u2009patients, 1 achieved a confirmed partial response (PR) that lasted 215u2009days and 35 had stable disease (SD) as best response (ORR: 2.5%, 95% CI 0.6% to 80.6%). Further efficacy end points in MET+u2009cases were DCR: 90.0% (95% CI 76.3% to 97.2%), 1-year PFS rate: 37.5% (95% CI 22.9% to 52.1%) and 1-year OS rate: 97.4% (95% CI 82.8% to 99.6%). Among 4 MET- patients, 1 achieved a PR that lasted 801u2009days and 3 had SD (ORR: 25.0%, 95% CI 0.6% to 80.6%) for a DCR of 100% (95% CI 39.8% to 100.0%). The 1-year PFS rate in MET- cases was 50% (95% CI 5.8% to 84.5%) and the 1-year OS rate was 75% (95% CI 12.8% to 96.1%). One patient with unknown MET status due to technical failure achieved SD but stopped treatment due to progression after 17 cycles. The most common crizotinib-related adverse events were nausea [34/48 (70.8%)], vomiting [22/48 (45.8%)], blurred vision [22/48 (45.8%)], diarrhoea (20/48 (41.7%)] and fatigue [19/48 (39.6%)].nnnConclusionnAccording to European Organization for Research and Treatment of Cancer (EORTC) efficacy criteria for soft tissue sarcoma, our study demonstrated that crizotinib has activity in TFE3 rearranged ASPS MET+u2009patients.nnnClinical trial numbernEORTC 90101, NCT01524926.

Patterns of care and outcome Radiation-Induced Soft Tissue Sarcomas

PURPOSEnRadiation therapy is used as a radical treatment for many cancers and is delivered in the neoadjuvant or adjuvant setting, frequently combined with other treatment modalities such as chemotherapy and surgery. However, radiation exposure is a well established risk factor for developing secondary malignancies. Soft-tissue sarcomas are the most common types of radiation-induced tumors in the general population.nnnMETHODSnWe have analyzed 510 patients from the French Sarcoma Group database. Prognostic factors for locoregional-free Survival (LRFS), metastases-free survival (MFS), and overall Survival (OS) were identified by univariate and multivariate analyses using a Cox regression model.nnnRESULTSnMedian overall survival for patients M0 with R0/R1 surgery was 65.1 months (95% CI, 50.0-98.4). The 5-year and 10-year OS rates were 52.9% (47.1-58.2) and 41.0% (34.2-47.7), respectively. On multivariate analysis, the predictors of worse OS were age, R1 margin status, grade 3 and size >55 mm.nnnCONCLUSIONSnWe report here the largest series of patients with radiation-induced soft-tissue sarcomas. We demonstrate that a majority of patients can be cured provided they are managed with adequate surgery.

Abstract 2699: ERCC5 (XPG) status and clinical activity of trabectedin in patients with advanced soft-tissue sarcoma

Trabectedin is approved in Europe for the treatment of advanced soft tissue sarcoma (STS) and provides objective response and disease stabilisation rates ranging from 5%-10% and 30-40%, respectively. Although the precise mechanism of action of trabectedin is not fully elucidated, various in vitro studies have shown that its activity depends, at least in part, on the nucleotide excision repair (NER) status of the cells. Indeed, NER-deficient cell lines are less sensitive to trabectedin than their wild-type counterparts. Our aim was to determine whether the status of ERCC5 (XPG), a 3′-endonuclease which plays a crucial role in the excision of the damaged DNA, was associated with the clinical activity of trabectedin in advanced STS patients. We analyzed both, the single nucleotide polymorphism (Asp1104His, G>C, exon 15) and the expression status (real-time quantitative RT-PCR) of ERCC5 in tumors from a cohort of 119 patients with advanced STS (median age at the start of treatment: 49 years old). All patients included in this retrospective analysis were treated between 1999 and 2007 in phase I-II clinical trials or in the context of a compassionate-use program. Trabectedin was given at different doses (0.5-3 mg/m2) with either a 3-h infusion or a 24-h continuous infusion schedule. The two most frequent histological subtypes were leiomyosarcoma (non-uterine: 21, uterine: 11) and liposarcoma (myxoid round cell: 24, other: 15). Overall, tumour control (CR, PR, and SD ≥ 6 months) was achieved in 42 patients (35%, 95% CI 26-44). The median progression-free survival (PFS) and overall survival (OS) of the entire patient group were 3.3 months (95% CI 2.5-4.1) and 12 months (95% CI 7.8-16.1), respectively. Variant allele carriers for ERCC5 were found in 58 cases: G/C (40; 33%), C/C (17; 15%). ERCC5 mRNA was found to be overexpressed in 48% of analyzed cases. In patients with tumors homozygous for the wild-type allele (Asp), ERRC5 mRNA overexpression was associated with significantly higher median PFS: 17.1 months (95% CI 4.7-29.4) versus 1.8 months (95% CI 1.4-2.2), p=0.002. In the group of patients with tumors heterozygous or homozygous for the variant allele (His), median PFS was poor regardless to the expression status of ERRC5 mRNA: 2.8 months (95% CI 1.3-4.2) versus 3.4 months (95% CI 1.4-5.5), p=0.50. We also compared the effect of wild-type and variant ERCC5 expression in human 94RD27 XPG-deficient fibroblasts (deletion of the last 261 aa) on the sensitivity to trabectedin. Preliminary results show that cells stably expressing wild-type ERCC5 are more sensitive to trabectedin than cells expressing the variant allele of ERCC5. Altogether, our data suggest that overexpression of wild-type ERCC5 might be predictive of clinical benefit from trabectedin in advanced STS patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2699.