Laurent Alberti

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.

IL-6 as an intracrine growth factor for renal carcinoma cell lines.

Interleukin‐6 (IL‐6) is produced at high levels by renal cell carcinoma cell lines. The molecular mechanisms involved in its possible role as an autocrine growth factor were investigated. IL‐6 and IL‐6 receptor expression was investigated in 8 renal cell carcinoma (RCC) cell lines. The modulation of RCC cell line proliferation by an anti‐IL‐6 Ab, an IL‐6 antisense oligonucleotide (ASON) directed against the second exon of IL‐6 and cytokines inhibiting IL‐6 production (IL‐4 and IL‐13) was investigated. All 8 RCC cell lines expressed IL‐6 mRNA, produced IL‐6 and expressed the soluble and membrane‐bound gp130 chain of IL‐6 receptor. The gp80 chain of IL‐6 receptor was undetectable at the surface of the 8 RCC cell lines tested, while the soluble form of gp80 was detectable in the supernatant of one of these cell lines. The addition of a blocking IL‐6 Ab did not inhibit the proliferation of any of the 8 RCC cell lines. In contrast, IL‐6 ASON inhibited specifically IL‐6 production and the proliferation of all RCC cell lines. Exogenous IL‐6 failed to restore RCC cell line proliferation blocked by ASON, indicating that IL‐6 acts through an intracrine loop in RCC cell lines. IL‐13 and IL‐4 inhibited the proliferation of 7 of the 8 cell lines without interfering with IL‐6 or IL‐6 receptor expression. IL‐6 ASON inhibited the proliferation of the 8 RCC cell lines tested additively with IL‐4 or IL‐13. IL‐6 is an intracrine growth factor in renal cell carcinoma cell lines.

CD40L induces multidrug resistance to apoptosis in breast carcinoma and lymphoma cells through caspase independent and dependent pathways.

BackgroundCD40L was found to reduce doxorubicin-induced apoptosis in non Hodgkins lymphoma cell lines through caspase-3 dependent mechanism. Whether this represents a general mechanism for other tumor types is unknown.MethodsThe resistance induced by CD40L against apoptosis induced by a panel of cytotoxic chemotherapeutic drugs in non Hodgkins lymphoma and breast carcinoma cell lines was investigated.ResultsDoxorubicin, cisplatyl, etoposide, vinblastin and paclitaxel increased apoptosis in a dose-dependent manner in breast carcinoma as well as in non Hodgkins lymphoma cell lines. Co-culture with irradiated L cells expressing CD40L significantly reduced the percentage of apoptotic cells in breast carcinoma and non Hodgkins lymphoma cell lines treated with these drugs. In breast carcinoma cell lines, these 5 drugs induced an inconsistent increase of caspase-3/7 activity, while in non Hodgkins lymphoma cell lines all 5 drugs increased caspase-3/7 activity up to 28-fold above baseline. Co-culture with CD40L L cells reduced (-39% to -89%) the activation of caspase-3/7 induced by these agents in all 5 non Hodgkins lymphoma cell lines, but in none of the 2 breast carcinoma cell lines. Co culture with CD40L L cells also blocked the apoptosis induced by exogenous ceramides in breast carcinoma and non Hodgkins lymphoma cell lines through a caspase-3-like, 8-like and 9-like dependent pathways.ConclusionThese results indicate that CD40L expressed on adjacent non tumoral cells induces multidrug resistance to cytotoxic agents and ceramides in both breast carcinoma and non Hodgkins lymphoma cell lines, albeit through a caspase independent and dependent pathway respectively.

Imatinib mesilate for the treatment of gastrointestinal stromal tumour

Background: The molecular hallmark of gastrointestinal stromal tumours (GISTs), the mutation of the KIT gene, was discovered 10 years ago. GISTs have since been recognized as separate pathological entities among sarcomas, and have become a model for targeted treatment of solid tumours. Imatinib mesilate, which was approved in 2002 for the treatment of patients with advanced GIST, has dramatically changed the course of the disease. Objective: This article will focus on the development of imatinib mesilate in the treatment of patients with GIST. Methods: A Pubmed search was performed using the keywords ‘imatinib’, ‘gastrointestinal stromal’, ‘GIST’, ‘KIT’ and ‘PDGFR’. Websites of the American Society of Clinical Oncology and the European Society of Medical Oncology were searched for data reported in abstract form at recent symposiums. Personal communications from opinion leaders were sought for additional information that might be relevant. Results: Imatinib has changed the clinical course of patients with advanced GISTs and further development in the adjuvant setting as well as prospective assessment of predictive factors are the current focus of ongoing research.

Lymphoma and Myeloma Cell Resistance to Cytotoxic Agents and Ionizing Radiations Is Not Affected by Exposure to Anti–IL-6 Antibody

Background Production of high levels of IL-6 is often correlated with resistance to cytotoxics or ionizing radiations, in cancer cell lines as in various cancer patients. We investigated whether monoclonal antibodies directed against IL-6 may enable to reverse resistance of cancer cell lines. Methodology/Principal Findings We exposed ten haematological cancer cells from lymphoma, myeloma, or leukemia origins to cytotoxics or ionizing radiations and assessed the effects of anti–IL-6 antibody addition on cell proliferation, apoptosis, or IL-6 signaling. A strong correlation between IL-6 secretion, measured by ELISA, and resistance to doxorubicin as ionizing radiations was observed in the multiple myeloma U266 and the Burkitts lymphoma Daudi and Namalwa cells. Although an anti–IL-6 antibody combined to both treatments efficiently blocked IL-6 signaling in U266 cells, expressing the IL-6 receptor gp80, it did not increase treatment-induced anti-proliferative and pro-apoptotic effects on these cells, as well as on Daudi and Namalwa cells. This lack of effect could be related to diverse factors: 1) a higher release of the soluble form of IL-6 receptor gp80 in response to doxorubicin and irradiation from all cell lines, 2) an impaired level of the IL-6 pathway inhibitor SOCS3 in Daudi cells, and 3) an increased release of IL-10 and TNFα, two cytokines involved in cell radio- and chemoresistance. Conclusions/Significance These data support the fact that IL-6 is not the preponderant actor of cell resistance to cytotoxics and ionizing radiations, which seems to be regulated by a complex network of proteins.

Impact of KIT exon 10 M541L allelic variant on the response to imatinib in aggressive fibromatosis: analysis of the desminib series by competitive allele specific Taqman PCR technology

BackgroundAggressive fibromatosis (AF) is a rare fibroblastic proliferative disease with a locally aggressive behavior and no distant metastasis, characterized by driver mutations in CTNNB1 or the APC gene. When progressive and/or symptomatic AF is not amenable to local management, a variety of medical treatments may be efficient, including imatinib mesylate. The phase II “Desminib trial” included 40 patients with AF to evaluate the toxicity and efficacy of imatinib resulting in a 65% tumor control rate at 1xa0year. We investigated a potential predictive value of KIT exon 10 M541L variant (KITL541) on this prospective series.MethodsDNA was extracted in sufficient quantity from 33 patients included in the Desminib trial. The detection of KITL541 was performed by Competitive Allele-Specific Taqman® PCR technology. Chi-2 analyses were performed to search for a correlation between KIT status and tumor response. Progression free (PFS) and overall survival (OS) were compared by log-rank test after Kaplan-Meier analysis.ResultsIn 6 out of 33 cases (18%), the technique failed to determine the mutational status; 5 patients (19%) harboured KITL541 and 22 patients (81%) were classified as KIT wild type. Compared with total cohort, KITL541 frequency did not distinguish between different clinical characteristics. In the KITL541 and the KITWT subgroups, the tumor control rate at 1xa0year was 100% and 68%, respectively (pu2009=u20090.316). The median PFS of patients harboring KITL541 or not is 29.9 and 24.5xa0months, respectively (pu2009=u20090.616), and the median OS is not reached, in any of the groups.ConclusionOur results do not support a predictive effect of KITL541 on the efficacy of imatinib for patients with AF.

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.

Emerging drugs for the treatment of soft tissue sarcomas

Soft tissue sarcomas are rare cancers of mesenchymal origin. Recent progress in the understanding of the biology of these rare tumours has enabled the identification of distinct molecular and pathological entities within this heterogenous group of neoplasms, and has paved the way for the development of targeted therapeutics directed against activated kinases. One of the most clear examples is the identification of KIT and platelet-derived growth factor receptor-α kinase mutations in gastrointestinal stromal tumours, a subset of sarcomas arising from precursors of the interstitial cells of Cajal in the digestive tract, which led to the development of imatinib, sunitinib and other tyrosine kinase inhibitors for the treatment of solid tumours. This model has become the paradigm of a targeted treatment of solid tumours designed to inhibit the causal alteration in the oncogenesis of these tumours. This review summarises treatment strategies in the context of advanced disease and discusses new compounds being developed for patients with soft tissue sarcomas.

Prediction of desmoid tumor progression using miRNA expression profiling

Desmoid tumor is a rare connective tissue tumor with locoregional aggressiveness but unpredictable behavior. The miRNA profile was ascertained for 26 patients included in the Desminib phase II trial and an independent validation cohort of 15 patients. Predictive and prognostic supervised analysis on the Desminib cohort failed to identify miRNAs differentially expressed between progressive and non‐progressive patients under imatinib treatment or between progressive and non‐progressive patients after discontinuation of imatinib. However, an unsupervised hierarchical clustering of the Desminib cohort identified two groups (A and B) of 13 patients each, where only the number of previous lines of treatment before inclusion in the study differed significantly between the two groups. Time to progression after discontinuation of imatinib was longer in group B than in group A. Fifteen miRNAs were highly statistically differentially expressed between groups A and B, targeting more than 3000 genes, including AGO1, BCL2, CDK6, SMAD4, PTEN, CCND1, VEGFA, and RB1. These results were confirmed in the independent validation cohort: hierarchical clustering of these 15 miRNAs identified two groups, in which time to recurrence was statistically different (28.8 months vs 68.8 months). These results provide the first indication of the prognostic value of miRNA expression profiling with a possible direct impact on patient management. A more precise miRNA signature must now be determined to select patients who would not benefit from surgical resection of their tumor and who ought to be monitored without treatment.

KIT exon 10 variant (c.1621 A > C) single nucleotide polymorphism as predictor of GIST patient outcome

BackgroundTumor genotype plays a crucial role in clinical management of GIST. Whether genetic polymorphism of KIT may influence GIST patient outcome is unclear.MethodsWe investigated the biological and clinical significance of the presence of KIT exon 10 variant (c.1621 Au2009>u2009C), KITL541, in a transfected cell line (3xa0T3xa0L541) and in two retrospectively collected series of 109 GIST patients in total. The control group consisted of 60 healthy donors collected at the French department of blood transfusion.ResultsIn the 3xa0T3xa0L541 cell line, KITL541 protein exhibited a spontaneous phosphorylation status comparable to that of wild-type KIT but displayed a phosphorylation pattern of AKT and ERK1/2 that was found similar to that of the classical mutated forms of the KIT receptor. Of 109 patients enrolled in this retrospective translational research study, 24 (22xa0%) harboured KITL541, similarly to the control group of healthy donors (nu2009=u200910 of 60, 17xa0%). A higher prevalence of the variant KITL541 was observed in patients with metastatic status at diagnosis (KITL541 correlated nine of 22 versus 15 of 87, pu2009=u20090.02). In addition, patients with KITL541 and localized GIST had a higher rate of relapse at 5xa0years and lower relapse free survival at 5xa0years in univariate, as well as in multivariate analysis. Response rate and duration of response to imatinib was similar in KITL541 and KITM541 patients.ConclusionKITL541 genotype is associated with a higher risk of metastasis at diagnosis and a higher risk of relapse in GIST patients.