Study of chromatin remodeling genes implicates SMARCA4 as a putative player in oncogenesis in neuroblastoma.

In neuroblastoma (NB), genetic alterations in chromatin remodeling (CRGs) and epigenetic modifier genes (EMGs) have been described. We sought to determine their frequency and clinical impact. Whole exome (WES)/whole genome sequencing (WGS) data and targeted sequencing (TSCA®) of exonic regions of 33 CRGs/EMGs were analyzed in tumor samples from 283 NB patients, with constitutional material available for 55 patients. The frequency of CRG/EMG variations in NB cases was then compared to the Genome Aggregation Database (gnomAD). The sequencing revealed SNVs/small InDels or focal CNAs of CRGs/EMGs in 20% (56/283) of all cases, occurring at a somatic level in 4 (7.2%), at a germline level in 12 (22%) cases, whereas for the remaining cases, only tumor material could be analyzed. The most frequently altered genes were ATRX (5%), SMARCA4 (2.5%), MLL3 (2.5%) and ARID1B (2.5%). Double events (SNVs/small InDels/CNAs associated with LOH) were observed in SMARCA4 (n = 3), ATRX (n = 1) and PBRM1 (n = 1). Among the 60 variations, 24 (8.4%) targeted domains of functional importance for chromatin remodeling or highly conserved domains but of unknown function. Variations in SMARCA4 and ATRX occurred more frequently in the NB as compared to the gnomAD control cohort (OR = 4.49, 95%CI: 1.63-9.97, p = 0.038; OR 3.44, 95%CI: 1.46-6.91, p = 0.043, respectively). Cases with CRG/EMG variations showed a poorer overall survival compared to cases without variations. Genetic variations of CRGs/EMGs with likely functional impact were observed in 8.4% (24/283) of NB. Our case-control approach suggests a role of SMARCA4 as a player of NB oncogenesis.

QuantumClone: clonal assessment of functional mutations in cancer based on a genotype-aware method for clonal reconstruction.

Motivation: In cancer, clonal evolution is assessed based on information coming from single nucleotide variants and copy number alterations. Nonetheless, existing methods often fail to accurately combine information from both sources to truthfully reconstruct clonal populations in a given tumor sample or in a set of tumor samples coming from the same patient. Moreover, previously published methods detect clones from a single set of variants. As a result, compromises have to be done between stringent variant filtering [reducing dispersion in variant allele frequency estimates (VAFs)] and using all biologically relevant variants. Results: We present a framework for defining cancer clones using most reliable variants of high depth of coverage and assigning functional mutations to the detected clones. The key element of our framework is QuantumClone, a method for variant clustering into clones based on VAFs, genotypes of corresponding regions and information about tumor purity. We validated QuantumClone and our framework on simulated data. We then applied our framework to whole genome sequencing data for 19 neuroblastoma trios each including constitutional, diagnosis and relapse samples. We confirmed an enrichment of damaging variants within such pathways as MAPK (mitogen-activated protein kinases), neuritogenesis, epithelial-mesenchymal transition, cell survival and DNA repair. Most pathways had more damaging variants in the expanding clones compared to shrinking ones, which can be explained by the increased total number of variants between these two populations. Functional mutational rate varied for ancestral clones and clones shrinking or expanding upon treatment, suggesting changes in clone selection mechanisms at different time points of tumor evolution. Availability and implementation: Source code and binaries of the QuantumClone R package are freely available for download at https://CRAN.R-project.org/package=QuantumClone. Contact: gudrun.schleiermacher@curie.fr or valentina.boeva@inserm.fr. Supplementary information: Supplementary data are available at Bioinformatics online.

HMCan-diff: a method to detect changes in histone modifications in cells with different genetic characteristics.

Comparing histone modification profiles between cancer and normal states, or across different tumor samples, can provide insights into understanding cancer initiation, progression and response to therapy. ChIP-seq histone modification data of cancer samples are distorted by copy number variation innate to any cancer cell. We present HMCan-diff, the first method designed to analyze ChIP-seq data to detect changes in histone modifications between two cancer samples of different genetic backgrounds, or between a cancer sample and a normal control. HMCan-diff explicitly corrects for copy number bias, and for other biases in the ChIP-seq data, which significantly improves prediction accuracy compared to methods that do not consider such corrections. On in silico simulated ChIP-seq data generated using genomes with differences in copy number profiles, HMCan-diff shows a much better performance compared to other methods that have no correction for copy number bias. Additionally, we benchmarked HMCan-diff on four experimental datasets, characterizing two histone marks in two different scenarios. We correlated changes in histone modifications between a cancer and a normal control sample with changes in gene expression. On all experimental datasets, HMCan-diff demonstrated better performance compared to the other methods.

The ALK receptor in sympathetic neuron development and neuroblastoma.

The ALK gene encodes a tyrosine kinase receptor characterized by an expression pattern mainly restricted to the developing central and peripheral nervous systems. In 2008, the discovery of ALK activating mutations in neuroblastoma, a tumor of the sympathetic nervous system, represented a breakthrough in the understanding of the pathogenesis of this pediatric cancer and established mutated ALK as a tractable therapeutic target for precision medicine. Subsequent studies addressed the identity of ALK ligands, as well as its physiological function in the sympathoadrenal lineage, its role in neuroblastoma development and the signaling pathways triggered by mutated ALK. This review focuses on these different aspects of the ALK biology and summarizes the various therapeutic strategies relying on ALK inhibition in neuroblastoma, either as monotherapies or combinatory treatments.

SV-Bay: structural variant detection in cancer genomes using a Bayesian approach with correction for GC-content and read mappability.

MOTIVATION: Whole genome sequencing of paired-end reads can be applied to characterize the landscape of large somatic rearrangements of cancer genomes. Several methods for detecting structural variants with whole genome sequencing data have been developed. So far, none of these methods has combined information about abnormally mapped read pairs connecting rearranged regions and associated global copy number changes automatically inferred from the same sequencing data file. Our aim was to create a computational method that could use both types of information, i.e. normal and abnormal reads, and demonstrate that by doing so we can highly improve both sensitivity and specificity rates of structural variant prediction. RESULTS: We developed a computational method, SV-Bay, to detect structural variants from whole genome sequencing mate-pair or paired-end data using a probabilistic Bayesian approach. This approach takes into account depth of coverage by normal reads and abnormalities in read pair mappings. To estimate the model likelihood, SV-Bay considers GC-content and read mappability of the genome, thus making important corrections to the expected read count. For the detection of somatic variants, SV-Bay makes use of a matched normal sample when it is available. We validated SV-Bay on simulated datasets and an experimental mate-pair dataset for the CLB-GA neuroblastoma cell line. The comparison of SV-Bay with several other methods for structural variant detection demonstrated that SV-Bay has better prediction accuracy both in terms of sensitivity and false-positive detection rate. AVAILABILITY AND IMPLEMENTATION: https://github.com/InstitutCurie/SV-Bay CONTACT: valentina.boeva@inserm.fr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Lin28B and Let-7 in the Control of Sympathetic Neurogenesis and Neuroblastoma Development.

The RNA binding protein Lin28B is expressed in developing tissues and sustains stem and progenitor cell identity as a negative regulator of the Let-7 family of microRNAs, which induces differentiation. Lin28B is activated in neuroblastoma (NB), a childhood tumor in sympathetic ganglia and adrenal medulla. Forced expression of Lin28B in embryonic mouse sympathoadrenal neuroblasts elicits postnatal NB formation. However, the normal function of Lin28B in the development of sympathetic neurons and chromaffin cells and the mechanisms involved in Lin28B-induced tumor formation are unclear. Here, we demonstrate a mirror-image expression of Lin28B and Let-7a in developing chick sympathetic ganglia. Lin28B expression is not restricted to undifferentiated progenitor cells but, is observed in proliferating noradrenergic neuroblasts. Lin28 knockdown in cultured sympathetic neuroblasts decreases proliferation, whereas Let-7 inhibition increases the proportion of neuroblasts in the cell cycle. Lin28B overexpression enhances proliferation, but only during a short developmental period, and it does not reduce Let-7a. Effects of in vivo Lin28B overexpression were analyzed in the LSL-Lin28B(DBHiCre) mouse line. Sympathetic ganglion and adrenal medulla volume and the expression level of Let-7a were not altered, although Lin28B expression increased by 12- to 17-fold. In contrast, Let-7a expression was strongly reduced in LSL-Lin28B(DbhiCre) NB tumor tissue. These data demonstrate essential functions for endogenous Lin28 and Let-7 in neuroblast proliferation. However, Lin28B overexpression neither sustains neuroblast proliferation nor affects let-7 expression. Thus, in contrast to other pediatric tumors, Lin28B-induced NB is not due to expansion of proliferating embryonic neuroblasts, and Let-7-independent functions are implicated during initial NB development. SIGNIFICANCE STATEMENT: Lin28A/B proteins are highly expressed in early development and maintain progenitor cells by blocking the biogenesis and differentiation function of Let-7 microRNAs. Lin28B is aberrantly upregulated in the childhood tumor neuroblastoma (NB). NB develops in sympathetic ganglia and adrenal medulla and is elicited by forced Lin28B expression. We demonstrate that Lin28A/B and Let-7 are essential for sympathetic neuroblast proliferation during normal development. Unexpectedly, Lin28B upregulation in a mouse model does not affect neuroblast proliferation, ganglion size, and Let-7 expression during early postnatal development. Lin28B-induced NB, in contrast to other pediatric cancers, does not evolve from neuroblasts that continue to divide and involves Let-7-independent functions during initial development.

Segmental Chromosomal Aberrations in Localized Neuroblastoma Can be Detected in Formalin-Fixed Paraffin-Embedded Tissue Samples and Are Associated With Recurrence.

BACKGROUND: Array comparative genomic hybridization (CGH) analyses of frozen tumors have shown strong associations between the pattern of chromosomal aberrations and outcome in patients with advanced-stage neuroblastoma. New platforms for analyzing chromosomal aberrations using formalin-fixed paraffin-embedded (FFPE) tissue have recently been developed. We sought to determine whether chromosomal microarray analysis (CMA) using FFPE tumors is feasible and if segmental chromosomal aberrations were prognostic of recurrence in localized neuroblastoma. METHODS: Patients with MYCN nonamplified International Neuroblastoma Staging System stage 1 and 2 disease who recurred were identified. CMA was performed with diagnostic FFPE samples using OncoScan™ FFPE Express 2.0. The prognostic significance of chromosomal pattern was validated in 105 patients with available CGH results. RESULTS: In 26 evaluable patients, 11 recurred locally, nine had metastatic relapse, and six remained progression free >3 years from diagnosis. No chromosomal aberrations were identified in four tumors. Numerical chromosomal aberrations (NCAs) without segmental chromosomal aberration (SCA) were identified in 11 patients: six progressed locally, two had metastatic progression and 3 remained progression-free. Eleven patients had SCAs: four progressed locally, six developed metastatic progression and one remained progression-free. Five or more SCAs were only detected in tumors from patients who developed metastases (P = 0.0004). In the validation cohort, SCAs were associated with inferior event-free survival (EFS) compared to NCA (5-year EFS 68% ± 8.3% vs. 91% ± 3.6%, respectively; P = 0.0083). CONCLUSIONS: It is feasible to evaluate chromosomal aberrations using FFPE neuroblastoma tissue. SCA is associated with inferior EFS in localized neuroblastoma patients, and multiple SCAs may be predictive of metastatic relapse.

SegAnnDB: interactive Web-based genomic segmentation.

MOTIVATION: DNA copy number profiles characterize regions of chromosome gains, losses and breakpoints in tumor genomes. Although many models have been proposed to detect these alterations, it is not clear which model is appropriate before visual inspection the signal, noise and models for a particular profile. RESULTS: We propose SegAnnDB, a Web-based computer vision system for genomic segmentation: first, visually inspect the profiles and manually annotate altered regions, then SegAnnDB determines the precise alteration locations using a mathematical model of the data and annotations. SegAnnDB facilitates collaboration between biologists and bioinformaticians, and uses the University of California, Santa Cruz genome browser to visualize copy number alterations alongside known genes. AVAILABILITY AND IMPLEMENTATION: The breakpoints project on INRIA GForge hosts the source code, an Amazon Machine Image can be launched and a demonstration Web site is http://bioviz.rocq.inria.fr.

Recent insights into the biology of neuroblastoma.

Neuroblastoma (NB) is an embryonal tumor of the sympathetic nervous system which accounts for 8-10% of pediatric cancers. It is characterized by a broad spectrum of clinical behaviors from spontaneous regression to fatal outcome despite aggressive therapies. Considerable progress has been made recently in the germline and somatic genetic characterization of patients and tumors. Indeed, predisposition genes that account for a significant proportion of familial and syndromic cases have been identified and genome-wide association studies have retrieved a number of susceptibility loci. In addition, genome-wide sequencing, copy-number and expression studies have been conducted on tumors and have detected important gene modifications, profiles and signatures that have strong implications for the therapeutic stratification of patients. The identification of major players in NB oncogenesis, including MYCN, ALK, PHOX2B and LIN28B, has enabled the development of new animal models. Our review focuses on these recent advances, on the insights they provide on the mechanisms involved in NB development and their applications for the clinical management of patients.

Midkine and Alk signaling in sympathetic neuron proliferation and neuroblastoma predisposition.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and arises from cells of the developing sympathoadrenergic lineage. Activating mutations in the gene encoding the ALK tyrosine kinase receptor predispose for NB. Here, we focus on the normal function of Alk signaling in the control of sympathetic neuron proliferation, as well as on the effects of mutant ALK. Forced expression of wild-type ALK and NB-related constitutively active ALK mutants in cultures of proliferating immature sympathetic neurons results in a strong proliferation increase, whereas Alk knockdown and pharmacological inhibition of Alk activity decrease proliferation. Alk activation upregulates NMyc and trkB and maintains Alk expression by an autoregulatory mechanism involving Hand2. The Alk-ligand Midkine (Mk) is expressed in immature sympathetic neurons and in vivo inhibition of Alk signaling by virus-mediated shRNA knockdown of Alk and Mk leads to strongly reduced sympathetic neuron proliferation. Taken together, these results demonstrate that the extent and timing of sympathetic neurogenesis is controlled by Mk/Alk signaling. The predisposition for NB caused by activating ALK mutations may thus be explained by aberrations of normal neurogenesis, i.e. elevated and sustained Alk signaling and increased NMyc expression.

Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma.

Neuroblastoma, a tumour derived from the peripheral sympathetic nervous system, is one of the most frequent solid tumours in childhood. It usually occurs sporadically but familial cases are observed, with a subset of cases occurring in association with congenital malformations of the neural crest being linked to germline mutations of the PHOX2B gene. Here we conducted genome-wide comparative genomic hybridization analysis on a large series of neuroblastomas. Copy number increase at the locus encoding the anaplastic lymphoma kinase (ALK) tyrosine kinase receptor was observed recurrently. One particularly informative case presented a high-level gene amplification that was strictly limited to ALK, indicating that this gene may contribute on its own to neuroblastoma development. Through subsequent direct sequencing of cell lines and primary tumour DNAs we identified somatic mutations of the ALK kinase domain that mainly clustered in two hotspots. Germline mutations were observed in two neuroblastoma families, indicating that ALK is a neuroblastoma predisposition gene. Mutated ALK proteins were overexpressed, hyperphosphorylated and showed constitutive kinase activity. The knockdown of ALK expression in ALK-mutated cells, but also in cell lines overexpressing a wild-type ALK, led to a marked decrease of cell proliferation. Altogether, these data identify ALK as a critical player in neuroblastoma development that may hence represent a very attractive therapeutic target in this disease that is still frequently fatal with current treatments.

The neuroblastoma tumor microenvironment: From an in-depth characterization towards novel therapies.

Neuroblastoma is a cancer of the sympathetic nervous system that develops in young children, either as low-risk or high-risk disease. The tumor microenvironment (TME) is now recognized as an important player of the tumor ecosystem that may promote drug resistance and immune escape. Targeting the TME in combination with therapies directly targeting tumor cells therefore represents an interesting strategy to prevent the emergence of resistance in cancer and improve patient's outcome. The development of such strategies however requires an in-depth understanding of the TME landscape, due to its high complexity and intra and inter-tumoral heterogeneity. Various approaches have been used in the last years to characterize the immune and non-immune cell populations present in tumors of neuroblastoma patients, both quantitatively and qualitatively, in particular with the use of single-cell transcriptomics. It is anticipated that in the near future, both genomic and TME information in tumors will contribute to a precise approach to therapy in neuroblastoma. Deciphering the mechanisms of interaction between neuroblastoma cells and stromal or immune cells in the TME is key to identify novel therapeutic combinations. Over the last decade, numerous in vitro studies and in vivo pre-clinical experiments in immune-competent and immune-deficient models have identified therapeutic approaches to circumvent drug resistance and immune escape. Some of these studies have formed the basis for early phase I and II clinical trials in children with recurrent and refractory high-risk neuroblastoma. This review summarizes recently published data on the characterization of the TME landscape in neuroblastoma and novel strategies targeting various TME cellular components, molecules and pathways activated as a result of the tumor-host interactions.

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.

Learning smoothing models of copy number profiles using breakpoint annotations.

BACKGROUND: Many models have been proposed to detect copy number alterations in chromosomal copy number profiles, but it is usually not obvious to decide which is most effective for a given data set. Furthermore, most methods have a smoothing parameter that determines the number of breakpoints and must be chosen using various heuristics. RESULTS: We present three contributions for copy number profile smoothing model selection. First, we propose to select the model and degree of smoothness that maximizes agreement with visual breakpoint region annotations. Second, we develop cross-validation procedures to estimate the error of the trained models. Third, we apply these methods to compare 17 smoothing models on a new database of 575 annotated neuroblastoma copy number profiles, which we make available as a public benchmark for testing new algorithms. CONCLUSIONS: Whereas previous studies have been qualitative or limited to simulated data, our annotation-guided approach is quantitative and suggests which algorithms are fastest and most accurate in practice on real data. In the neuroblastoma data, the equivalent pelt.n and cghseg.k methods were the best breakpoint detectors, and exhibited reasonable computation times.

Control-FREEC: a tool for assessing copy number and allelic content using next-generation sequencing data.

SUMMARY: More and more cancer studies use next-generation sequencing (NGS) data to detect various types of genomic variation. However, even when researchers have such data at hand, single-nucleotide polymorphism arrays have been considered necessary to assess copy number alterations and especially loss of heterozygosity (LOH). Here, we present the tool Control-FREEC that enables automatic calculation of copy number and allelic content profiles from NGS data, and consequently predicts regions of genomic alteration such as gains, losses and LOH. Taking as input aligned reads, Control-FREEC constructs copy number and B-allele frequency profiles. The profiles are then normalized, segmented and analyzed in order to assign genotype status (copy number and allelic content) to each genomic region. When a matched normal sample is provided, Control-FREEC discriminates somatic from germline events. Control-FREEC is able to analyze overdiploid tumor samples and samples contaminated by normal cells. Low mappability regions can be excluded from the analysis using provided mappability tracks. AVAILABILITY: C++ source code is available at: http://bioinfo.curie.fr/projects/freec/ CONTACT: freec@curie.fr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Two cases of localized neuroblastoma with multiple segmental chromosomal alterations and metastatic progression.

Surgery alone is curative for most children with localized MYCN-non-amplified neuroblastoma. However, 10-15% will develop recurrent loco-regional disease, and very rarely, patients will relapse metastatically. Currently, it is not possible to predict which child with localized, MYCN-non-amplified neuroblastoma will develop disseminated disease. We report two children who presented with favorable biology, localized neuroblastoma and subsequently relapsed with metastatic disease after treatment with surgery. Whole-genome DNA copy number analyses performed on the diagnostic tumors identified 15 (case 1) and 8 (case 2) segmental chromosomal alterations. Further analysis of the prognostic value of whole-genome analysis in children with localized neuroblastoma is warranted.

Combination Therapies Targeting ALK-aberrant Neuroblastoma in Preclinical Models.

PURPOSE: ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. EXPERIMENTAL DESIGN: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX). RESULTS: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. CONCLUSIONS: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma.

Reversible transitions between noradrenergic and mesenchymal tumor identities define cell plasticity in neuroblastoma.

Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity.

Control-free calling of copy number alterations in deep-sequencing data using GC-content normalization.

SUMMARY: We present a tool for control-free copy number alteration (CNA) detection using deep-sequencing data, particularly useful for cancer studies. The tool deals with two frequent problems in the analysis of cancer deep-sequencing data: absence of control sample and possible polyploidy of cancer cells. FREEC (control-FREE Copy number caller) automatically normalizes and segments copy number profiles (CNPs) and calls CNAs. If ploidy is known, FREEC assigns absolute copy number to each predicted CNA. To normalize raw CNPs, the user can provide a control dataset if available; otherwise GC content is used. We demonstrate that for Illumina single-end, mate-pair or paired-end sequencing, GC-contentr normalization provides smooth profiles that can be further segmented and analyzed in order to predict CNAs. AVAILABILITY: Source code and sample data are available at http://bioinfo-out.curie.fr/projects/freec/.