Aurélie Dutour

Micro-RNA profiles in osteosarcoma as a predictive tool for ifosfamide response

Micro‐RNAs (miRNA) are currently used as cancer biomarkers for hematological cancers and solid tumors. Osteosarcoma is the first primary malignant bone tumor, characterized by a complex genetic and resistance to conventional treatments. For this latter property, the median survival has not been improved since 1990 despite preoperative administration of chemotherapeutic agents. The prediction of tumor response before chemotherapy treatment would constitute a major progress for this pathology. We assessed in this study if miRNA profiling could surpass the current limitations for osteosarcoma diagnosis. We measured the miRNA expression in different osteosarcoma samples: (i) 27 osteosarcoma paraffin‐embedded tumors from patients, (ii) human osteosarcoma cell lines, and (iii) tumors from a syngeneic rat osteosarcoma model, recapitulating human osteosarcoma. miRNA profiles were determined using microfluidic cards performing high‐throughput TaqMan®‐based PCR assays, called TaqMan® Low Density Arrays. Osteosarcoma of rat and human origins showed a miRNA signature, which could discriminate good from bad responders. In particular, we identified five discriminating miRNAs (miR‐92a, miR‐99b, miR‐132, miR‐193a‐5p and miR‐422a) in patient tumors, which could be easily transferable to diagnosis. These discriminating miRNAs, as well as those identified in rat, targeted the TGFβ, the Wnt and the MAP kinase pathways. These results indicate that our platform constitutes a potent diagnostic tool to predict tumor sensitivity to a drug in attempt to better adapt treatment to tumor biological specificities and also to identify new potential therapeutic strategies.

The in vivo radiosensitizing effect of gold nanoparticles based MRI contrast agents

Owing to the high atomic number (Z) of gold element, the gold nanoparticles appear as very promising radiosensitizing agents. This character can be exploited for improving the selectivity of radiotherapy. However, such an improvement is possible only if irradiation is performed when the gold content is high in the tumor and low in the surrounding healthy tissue. As a result, the beneficial action of irradiation (the eradication of the tumor) should occur while the deleterious side effects of radiotherapy should be limited by sparing the healthy tissue. The location of the radiosensitizers is therefore required to initiate the radiotherapy. Designing gold nanoparticles for monitoring their distribution by magnetic resonance imaging (MRI) is an asset due to the high resolution of MRI which permits the accurate location of particles and therefore the determination of the optimal time for the irradiation. We recently demonstrated that ultrasmall gold nanoparticles coated by gadolinium chelates (Au@DTDTPA-Gd) can be followed up by MRI after intravenous injection. Herein, Au@DTDTPA and Au@DTDTPA-Gd were prepared in order to evaluate their potential for radiosensitization. Comet assays and in vivo experiments suggest that these particles appear well suited for improving the selectivity of the radiotherapy. The dose which is used for inducing similar levels of DNA alteration is divided by two when cells are incubated with the gold nanoparticles prior to the irradiation. Moreover, the increase in the lifespan of tumor bearing rats is more important when the irradiation is performed after the injection of the gold nanoparticles. In the case of treatment of rats with a brain tumor (9L gliosarcoma, a radio-resistant tumor in a radiosensitive organ), the delay between the intravenous injection and the irradiation was determined by MRI.

Inhibition of chondrosarcoma growth by mTOR inhibitor in an in vivo syngeneic rat model.

Background Chondrosarcomas are the second most frequent primary malignant type of bone tumor. No effective systemic treatment has been identified in advanced or adjuvant phases for chondrosarcoma. The aim of the present study was to determine the antitumor effects of doxorubicin and everolimus, an mTOR inhibitor on chondrosarcoma progression. Methods and Findings Doxorubin and/or everolimus were tested in vivo as single agent or in combination in the rat orthotopic Schwarm chondrosarcoma model, in macroscopic phase, as well as with microscopic residual disease. Response to everolimus and/or doxorubicin was evaluated using chondrosarcoma volume evolution (MRI). Histological response was evaluated with % of tumor necrosis, tumor proliferation index, metabolism quantification analysis between the treated and control groups. Statistical analyses were performed using chi square, Fishers exact test. Doxorubicin single agent has no effect of tumor growth as compared to no treatment; conversely, everolimus single agent significantly inhibited tumor progression in macroscopic tumors with no synergistic additive effect with doxorubicin. Everolimus inhibited chondrosarcoma proliferation as evaluated by Ki67 expression did not induce the apoptosis of tumor cells; everolimus reduced Glut1 and 4EBP1 expression. Importantly when given in rats with microscopic residual diseases, in a pseudo neoadjuvant setting, following R1 resection of the implanted tumor, everolimus significantly delayed or prevented tumor recurrence. Conclusions MTOR inhibitor everolimus blocks cell proliferation, Glut1 expression and HIF1a expression, and prevents in vivo chondrosarcoma tumor progression in both macroscopic and in adjuvant phase post R1 resection. Taken together, our preclinical data indicate that mTOR inhibitor may be effective as a single agent in treating chondrosarcoma patients. A clinical trial evaluating mTOr inhibitor as neo-adjuvant and adjuvant therapy in chondrosarcoma patients is being constructed.

18F-FDG PET SUVmax Correlates with Osteosarcoma Histologic Response to Neoadjuvant Chemotherapy: Preclinical Evaluation in an Orthotopic Rat Model

Assessment of osteosarcoma response to neoadjuvant chemotherapy is performed by histopathologic analysis after surgical resection of the primary tumor. The purpose of this study was to evaluate whether 18F-FDG PET could be a noninvasive surrogate to histopathologic analysis and allow for earlier response evaluation to neoadjuvant chemotherapy in osteosarcoma. Methods: Metabolic response to neoadjuvant chemotherapy was assessed in immunocompetent rats with a preestablished orthotopic osteosarcoma using 18F-FDG PET before and after receiving 2 doses of ifosfamide. Comparison was then made by assessing histologic responses on euthanized animals. Results: Maximum standardized uptake value (SUVmax) measured by 18F-FDG PET after 2 doses of chemotherapy was correlated to histologic classification (P < 0.01). An SUVmax less than 15 corresponded to good responders, whereas an SUVmax greater than 15 but less than 20 and an SUVmax greater than 20 corresponded to partial responders or nonresponders, respectively. A 40% decrease in SUVmax between the first and second 18F-FDG PET scans distinguished between partial and good response to chemotherapy. Conclusion: Determination of SUVmax using semiquantitative 18F-FDG PET predicts response to neoadjuvant chemotherapy earlier than does histologic analysis.

The ENCCA-WP7/EuroSarc/EEC/PROVABES/EURAMOS 3rd European Bone Sarcoma Networking Meeting/Joint Workshop of EU Bone Sarcoma Translational Research Networks; Vienna, Austria, September 24-25, 2015. Workshop Report

This report summarizes the results of the 3rd Joint ENCCA-WP7, EuroSarc, EEC, PROVABES, and EURAMOS European Bone Sarcoma Network Meeting, which was held at the Children’s Cancer Research Institute in Vienna, Austria on September 24–25, 2015. The joint bone sarcoma network meetings bring together European bone sarcoma researchers to present and discuss current knowledge on bone sarcoma biology, genetics, immunology, as well as results from preclinical investigations and clinical trials, to generate novel hypotheses for collaborative biological and clinical investigations. The ultimate goal is to further improve therapy and outcome in patients with bone sarcomas.

Therapeutic pipeline for soft-tissue sarcoma

Introduction: Soft-tissue sarcomas (STS) represent a heterogeneous group of malignant tumors originating from connective tissues. Over recent years, this heterogeneity has led to a molecular breakdown of STS and subsequent use of targeted agents in several molecularly defined subgroups. After the initial success of imatinib in gastrointestinal stromal tumors, several other compounds have shown promising activity in some but not all subgroups of sarcoma. Areas covered: This review discusses the rational and clinical results, when available, that support this subtype-directed approach. In the vast majority of cases, these agents have been tested only in patients with advanced disease; as chemotherapeutic agents are developed as non-histotype-specific therapies, they are not discussed here. The PubMed literature was searched using the terms ‘sarcoma’, ‘angiogenesis’, ‘mTOR’ and ‘targeted agents’. Proceedings of the annual meeting of the American Society of Clinical Oncology as well as those of the Connective Tissue Oncology Society were also searched for relevant information. Expert opinion: Many agents are currently developed in a subtype-specific manner in STS and this represents a significant leap forward. However, much remains to be done to improve our understanding of the molecular biology of this heterogeneous group of diseases.

Targeted imaging of αvβ3 expressing sarcoma tumor cells in vivo in pre-operative setting using near infrared: A potential tool to reduce incomplete surgical resection

Tumor size and location along with efficacy of pre-operative imaging are limiting factors for optimal surgical excision for osteosarcoma. Our general hypothesis is that targeting αvβ3 integrin-rich osteosarcoma neoangiogenesis should provide improved delivery of diagnostic compounds and assist surgeons intra operatively using near-infrared imaging techniques. We evaluated in an orthotopic metastatic osteosarcoma in rats the potential of AngioStamp™ targeting αvβ3 integrins and detected intra operatively by near infrared (NIR) illumination (Fluobeam™) as a novel, intra operative imaging technique. To determine the potential of this association in improving tumor and metastasis detection, we compared the quality and sensitivity of tumor/metastasis margin delineation and tumor resection using intra-operative NIR imaging to the ones guided by pre-operative imaging (i.e., MRI subsequently confirmed by histopathological analysis). Chemotherapy being essential in osteosarcoma treatment, we evaluated the capacity of AngioStamp™ to specifically localize to the tumor after chemotherapy treatment. We showed a significantly lesser extent of healthy tissue resection after surgical excision when assessing tumor margin intra operatively using AngioStamp™/Fluobeam™ association compared to pre-operative MRI post-operatively confirmed by histopathological analysis (p<0.01). Importantly, intra-operative NIR illumination of lungs revealed more metastases than were detected by CT Scan or under intra-operative white light examination (p<0.01). Importantly, chemotherapy did not alter AngioStamp™ tumor specific targeting nor the sensitivity of tumor detection. Our preclinical data confirm the potential of intra-operative imaging for improved primary tumor and lung metastasis excision. Based on these promising results, we now propose to evaluate this approach as a mean to improve surgical excision while maintaining tumor control in other sarcoma or tumors overexpressing αvβ3 integrins.

Description of the immune microenvironment of chondrosarcoma and contribution to progression

ABSTRACT Chondrosarcoma (CHS) is a rare bone malignancy characterized by its resistance to conventional systemic and radiation therapies. Whether immunotherapy targeting immune checkpoints may be active in these tumors remains unknown. To explore the role of the immune system in this tumor, we analyzed the immune environment of chondrosarcomas both in human sample, and in a syngeneic rat model, and tested the contribution of T lymphocytes and macrophages in chondrosarcoma progression. Immunohistochemical stainings were performed on human chondrosarcoma samples and on Swarm rat chondrosarcoma (SRC) model. Selective immunodepletion assays were performed in SRC to evaluate immune populations involvement in tumor progression. In human and rat chondrosarcoma, immune infiltrates composed of lymphocytes and macrophages were identified in the peritumoral area. Immune infiltrates composition was found correlated with tumors characteristics and evolution (grade, invasiveness and size). In SRC, selective depletion of T lymphocytes resulted in an accelerated growth rates, whereas depletion of CD163+ macrophages slowed down tumor progression. Splenocytes isolated from CHS-bearing SRC showed a specific cytotoxicity directed against chondrosarcoma cells (27%), which significantly decreased in CD3-depleted SRC (11%). The immune environment contributes to CHS progression in both human and animal models, suggesting that immunomodulatory approaches could be tested in bone chondrosarcoma.

Bcl-xl as the most promising Bcl-2 family member in targeted treatment of chondrosarcoma

Chondrosarcomas are malignant cartilage tumors showing relative resistance to conventional chemo- and radiotherapy. Previous studies showed that chondrosarcoma cells could be sensitized to chemotherapy by inhibiting the Bcl-2 family members Bcl-2, Bcl-xl and Bcl-w using ABT-737. In this study we explored the specific role of Bcl-2 family members to identify the most important player in chondrosarcoma cell survival and chemo resistance. Immunohistochemistry was performed on tissue microarrays containing 137 conventional chondrosarcomas of different grades. Selective inhibition of Bcl-2 (S55746) or Bcl-xl (WEHI-539 or A-1155463) and the combination with doxorubicin or cisplatin was investigated in a panel of 8 chondrosarcoma cell lines using presto blue viability assays and caspase 3/7 glo apoptosis assays. In addition Bcl-2 and Bcl-xl inhibition was investigated in an orthotopic Swarm Rat Chondrosarcoma (SRC) model. Bcl-2 and Bcl-xl were most abundantly expressed in the primary tumors, and expression increased with increasing histological grade. A subset of chondrosarcoma cell lines was sensitive to selective inhibition of Bcl-xl, and synergy was observed with doxorubicin or cisplatin in 3 out of 8 chondrosarcoma cell lines resulting in apoptosis. Conversely, selective inhibition of Bcl-2 was not effective in chondrosarcoma cell lines and could not sensitize to chemotherapy. In vivo, selective inhibition of Bcl-xl, but not Bcl-2 resulted in a decrease in tumor growth rate, even though no sensitization to doxorubicin was observed. These results suggest that among the Bcl-2 family members, Bcl-xl is most important for chondrosarcoma survival. Further research is needed to validate whether single or combination treatment with chemotherapy will be beneficial for chondrosarcoma patients.

Abstract C260: Dual targeting of the PI3K/Akt/mTOR pathway significantly delays chondrosarcoma progression and relapse.

Chondrosarcoma is a rare and usually slow growing sarcoma. This tumor is notoriously resistant to chemotherapy and radiation treatment. To improve survival rates in patients with chondrosarcoma exploring targeted therapies approaches seems promising. Thus we performed a preclinical study evaluating the efficacy of mTOR inhibitor everlimus in an orthotopic rat chondrosarcoma model and showed the potential of this mTORC 1 inhibitor to significantly delay tumor progression and relapse. Based on these data, we initiated preclinical studies in the same chondrosarcoma model to investigate how mTOR inhibition can be optimized. We first evaluated the antitumor effect of a PI3K (GDC-0941), an Akt (G-594) and a dual PI3K/mTOR inhibitor (GDC-0980). In a second step and in order to overcome forms of resistance we combined inhibition of PI3K to Akt or PI3K to the one of mTOR. GDC-0941 based drug combinations (GDC-0941+G-594; GDC-0941+RAD001) potency to inhibit tumor progression was compared to the one of each agent alone. For each setting, the effect of each treatment on mTOR and ERK pathways, apoptosis and cell proliferation was also tested. Results: As monotherapy, PI3K inhibitor (GDC-0941) was found to be the most efficient inducing an inhibition of tumor progression of 58 % whereas RAD001 induced a 49 % inhibition. The comparison of therapeutic efficiency of GDC-0941 based combinations showed an additive effect of G-594 or RAD001 to GDC-0941. Each of these combination caused an inhibition >60% in chondrosarcoma progression, whereas each agent alone induced a 22% to 45 % inhibition of tumor progression. GDC-0941 exposure inhibited chondrosarcoma proliferation as evaluated by Ki67 staining and induced Caspase 3/7 mediated apoptosis when used as single agent or in combined therapies. Our data reveal that all combination treatments blocked Akt activation, and inhibited phosphorylation of both 4EBP1 and S6K1 which resulted in. The tested treatments induced a similar inhibition of mTOR mediated signaling. Chondrosarcoma exposure to GDC-0941 or G-594 caused a 20% inhibition of p70S6K and 4EBP activation similar to the one induced by the rapalog RAD001. The PI3K inhibitor GDC-0941 caused an activation of Erk Pathway as noted by the strong increase in bRaf phosphorylation. Taken together these data confirm that targeting the PI3K/mTOR pathway is an efficient treatment for chondrosarcoma and combined therapy best way to cause inhibition of tumor progression. The effect noted on Erk pathway suggests that new combination approaches targeting the mTOR and Erk pathway could be of interest. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C260. Citation Format: Delphine Jury, Anne Valerie Decouvelaere, Amandine Pasquier, Marcia Belvin, Lorie Friedman, Jean Baptiste Langlois, Jean Philippe Michot, Jean Yves Blay, Aurelie Dutour. Dual targeting of the PI3K/Akt/mTOR pathway significantly delays chondrosarcoma progression and relapse. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C260.