Spatial sequestration of activated-caspase 3 in aggresomes mediates resistance of neuroblastoma cell to bortezomib treatment.

Neuroblastoma (NB) is the most common pediatric tumor and is currently treated by several types of therapies including chemotherapies, such as bortezomib treatment. However, resistance to bortezomib is frequently observed by mechanisms that remain to be deciphered. Bortezomib treatment leads to caspase activation and aggresome formation. Using models of patients-derived NB cell lines with different levels of sensitivity to bortezomib, we show that the activated form of caspase 3 accumulates within aggresomes of NB resistant cells leading to an impairment of bortezomib-induced apoptosis and increased cell survival. Our findings unveil a new mechanism of resistance to chemotherapy based on an altered subcellular distribution of the executioner caspase 3. This mechanism could explain the resistance developed in NB patients treated with bortezomib, emphasizing the potential of drugs targeting aggresomes.

Cell-Free DNA Extracted from CSF for the Molecular Diagnosis of Pediatric Embryonal Brain Tumors.

BACKGROUND: Liquid biopsies are revolutionary tools used to detect tumor-specific genetic alterations in body fluids, including the use of cell-free DNA (cfDNA) for molecular diagnosis in cancer patients. In brain tumors, cerebrospinal fluid (CSF) cfDNA might be more informative than plasma cfDNA. Here, we assess the use of CSF cfDNA in pediatric embryonal brain tumors (EBT) for molecular diagnosis. METHODS: The CSF cfDNA of pediatric patients with medulloblastoma (n = 18), ATRT (n = 3), ETMR (n = 1), CNS NB FOXR2 (n = 2) and pediatric EBT NOS (n = 1) (mean cfDNA concentration 48 ng/mL; range 4-442 ng/mL) and matched tumor genomic DNA were sequenced by WES and/or a targeted sequencing approach to determine single-nucleotide variations (SNVs) and copy number alterations (CNA). A specific capture covering transcription start sites (TSS) of genes of interest was also used for nucleosome footprinting in CSF cfDNA. RESULTS: 15/25 CSF cfDNA samples yielded informative results, with informative CNA and SNVs in 11 and 15 cases, respectively. For cases with paired tumor and CSF cfDNA WES (n = 15), a mean of 83 (range 1-160) shared SNVs were observed, including SNVs in classical medulloblastoma genes such as SMO and KMT2D. Interestingly, tumor-specific SNVs (mean 18; range 1-62) or CSF-specific SNVs (mean 5; range 0-25) were also observed, suggesting clonal heterogeneity. The TSS panel resulted in differential coverage profiles across all 112 studied genes in 7 cases, indicating distinct promoter accessibility. CONCLUSION: CSF cfDNA sequencing yielded informative results in 60% (15/25) of all cases, with informative results in 83% (15/18) of all cases analyzed by WES. These results pave the way for the implementation of these novel approaches for molecular diagnosis and minimal residual disease monitoring.

Management of a Composite Pheochromocytoma (Pheochromocytoma/Neuroblastoma) in Adult Patient Recurring After Several Years: A Complex Case Report.

Pheochromocytoma/neuroblastoma composite tumors are rare entities for which little is known. We report an atypical case of a 39-year-old man with secondary bone locations of a composite tumor, 7 years after resection of adrenal neuroblastoma, with constitutional alteration of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 whose role is unknown. The diagnosis of a peripheral neuroblastic tumor in adulthood is difficult and even more so when it is a composite tumor. In the absence of a standard of care, management is varied and discussions about treatment modalities for these patients are complex.

Alterations in Homologous Recombination-Related Genes and Distinct Platinum Response in Metastatic Triple-Negative Breast Cancers: A Subgroup Analysis of the ProfiLER-01 Trial.

BACKGROUND: a specific subset of metastatic triple-negative breast cancers (mTNBC) is characterized by homologous recombination deficiency (HRD), leading to enhanced sensitivity to platinum-based chemotherapy. Apart from mutations in BRCA1/2 genes, the evaluation of other HRD-related alterations has been limited to date. As such, we analyzed data from mTNBC patients enrolled in the ProfiLER-01 study to determine the prevalence of alterations in homologous recombination-related (HRR) genes and their association with platinum sensitivity. METHODS: next-generation sequencing and promoter methylation of BRCA1 and RAD51C were performed on tumors from patients with mTNBC, using a panel of 19 HRR genes. Tumors were separated into three groups based on their molecular status: mutations in BRCA1/2, mutations in other HRR genes (BRCA1/2 excluded) or BRCA1/RAD51C promoter methylation and the absence of molecular alterations in HRR genes (groups A, B and C, respectively). Sensitivity to platinum-based chemotherapy was evaluated through the radiological response. RESULTS: mutations in BRCA1/2 were detected in seven (13.5%) patients, while alterations in other HRR genes or hypermethylation in BRCA1 or RAD51C were reported in 16 (30.7%) patients; furthermore, no alteration was found in the majority of patients (n = 29; 55.8%). Among 27 patients who received platinum-based chemotherapy, the disease control rate was 80%, 55% and 18% (groups A, B and C, respectively; p = 0.049). Regarding group B, patients with disease control exhibited mutations in FANCL, FANCA and the RAD51D genes or RAD51C methylation; Conclusion: mutations in HRR genes and epimutations in RAD51C were associated with disease control through platinum-based chemotherapy. As such, apart from well-characterized alterations in BRCA1/2, a more comprehensive evaluation of HRD should be considered in order to enlarge the selection of patients with mTNBC that could benefit from platinum-based chemotherapy.

Suprarenal Masses in Very Young Infants: Is It Safe to Watch and Wait? Report of a SIOPEN Observational Study Results.

Background: To assess whether expectant observation of infants ≤ 90 days old with small suprarenal masses (sSRMs) could avoid unnecessary surgery without impacting outcome. Methods: Infants ≤ 90 days with a ≤ 5 cm mass, without midline extension or lymph node or distant spread were registered (ClinicalTrials.org:NCT01728155). Once staging was completed, they were followed with ultrasound, MRI and urinary catecholamines. Surgical resection was only planned if there was a ≥40% mass volume increase or for a mass persisting after 48 weeks of the planned observation. Results: Over a 5-year period, 128 infants were registered. No infant had detectable MYCN amplification in the peripheral blood. Surgery was performed in 39 (30.5%) patients, in 18 during and in 21 after the planned 48-week observation, and 74% were confirmed to be neuroblastomas. Non-life-threatening surgical complications occurred in two cases. The 3-year overall survival and event-free survival were 100% and 87.1%, respectively. The 16 events observed were volume increase (N = 11) and progression to neuroblastoma stage MS (N = 5). Patients with solid masses or MIBG-positive masses had lower EFS. Conclusions: Expectant observation for infants with sSRMs with clinical follow-up and timely imaging (including MRI scan) is safe and effective, allowing surgery to be avoided in the majority of them.

Environmental cues from neural crest derivatives act as metastatic triggers in an embryonic neuroblastoma model.

Embryonic malignant transformation is concomitant to organogenesis, often affecting multipotent and migratory progenitors. While lineage relationships between malignant cells and their physiological counterparts are extensively investigated, the contribution of exogenous embryonic signals is not fully known. Neuroblastoma (NB) is a childhood malignancy of the peripheral nervous system arising from the embryonic trunk neural crest (NC) and characterized by heterogeneous and interconvertible tumor cell identities. Here, using experimental models mimicking the embryonic context coupled to proteomic and transcriptomic analyses, we show that signals released by embryonic sympathetic ganglia, including Olfactomedin-1, induce NB cells to shift from a noradrenergic to mesenchymal identity, and to activate a gene program promoting NB metastatic onset and dissemination. From this gene program, we extract a core signature specifically shared by metastatic cancers with NC origin. This reveals non-cell autonomous embryonic contributions regulating the plasticity of NB identities and setting pro-dissemination gene programs common to NC-derived cancers.

Frequency and Prognostic Impact of ALK Amplifications and Mutations in the European Neuroblastoma Study Group (SIOPEN) High-Risk Neuroblastoma Trial (HR-NBL1).

PURPOSE: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through activating point mutations or genomic amplification. We studied ALK genetic alterations in high-risk (HR) patients on the HR-NBL1/SIOPEN trial to determine their frequency, correlation with clinical parameters, and prognostic impact. MATERIALS AND METHODS: Diagnostic tumor samples were available from 1,092 HR-NBL1/SIOPEN patients to determine ALK amplification status (n = 330), ALK mutational profile (n = 191), or both (n = 571). RESULTS: Genomic ALK amplification (ALKa) was detected in 4.5% of cases (41 out of 901), all except one with MYCN amplification (MNA). ALKa was associated with a significantly poorer overall survival (OS) (5-year OS: ALKa [n = 41] 28% [95% CI, 15 to 42]; no-ALKa [n = 860] 51% [95% CI, 47 to 54], [P < .001]), particularly in cases with metastatic disease. ALK mutations (ALKm) were detected at a clonal level (> 20% mutated allele fraction) in 10% of cases (76 out of 762) and at a subclonal level (mutated allele fraction 0.1%-20%) in 3.9% of patients (30 out of 762), with a strong correlation between the presence of ALKm and MNA (P < .001). Among 571 cases with known ALKa and ALKm status, a statistically significant difference in OS was observed between cases with ALKa or clonal ALKm versus subclonal ALKm or no ALK alterations (5-year OS: ALKa [n = 19], 26% [95% CI, 10 to 47], clonal ALKm [n = 65] 33% [95% CI, 21 to 44], subclonal ALKm (n = 22) 48% [95% CI, 26 to 67], and no alteration [n = 465], 51% [95% CI, 46 to 55], respectively; P = .001). Importantly, in a multivariate model, involvement of more than one metastatic compartment (hazard ratio [HR], 2.87; P < .001), ALKa (HR, 2.38; P = .004), and clonal ALKm (HR, 1.77; P = .001) were independent predictors of poor outcome. CONCLUSION: Genetic alterations of ALK (clonal mutations and amplifications) in HR-NB are independent predictors of poorer survival. These data provide a rationale for integration of ALK inhibitors in upfront treatment of HR-NB with ALK alterations.

Targeting netrin-3 in small cell lung cancer and neuroblastoma.

The navigation cue netrin-1 is well-documented for its key role in cancer development and represents a promising therapeutic target currently under clinical investigation. Phase 1 and 2 clinical trials are ongoing with NP137, a humanized monoclonal antibody against netrin-1. Interestingly, the epitope recognized by NP137 in netrin-1 shares 90% homology with its counterpart in netrin-3, the closest member to netrin-1 in humans, for which little is known in the field of cancer. Here, we unveiled that netrin-3 appears to be expressed specifically in human neuroblastoma (NB) and small cell lung cancer (SCLC), two subtypes of neuroectodermal/neuroendocrine lineages. Netrin-3 and netrin-1 expression are mutually exclusive, and the former is driven by the MYCN oncogene in NB, and the ASCL-1 or NeuroD1 transcription factors in SCLC. Netrin-3 expression is correlated with disease stage, aggressiveness, and overall survival in NB. Mechanistically, we confirmed the high affinity of netrin-3 for netrin-1 receptors and we demonstrated that netrin-3 genetic silencing or interference using NP137, delayed tumor engraftment, and reduced tumor growth in animal models. Altogether, these data support the targeting of netrin-3 in NB and SCLC.

Targeted Therapy of TERT-Rearranged Neuroblastoma with BET Bromodomain Inhibitor and Proteasome Inhibitor Combination Therapy.

PURPOSE: TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. EXPERIMENTAL DESIGN: Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. RESULTS: The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. CONCLUSIONS: OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.

Age Dependency of the Prognostic Impact of Tumor Genomics in Localized Resectable MYCN-Nonamplified Neuroblastomas. Report From the SIOPEN Biology Group on the LNESG Trials and a COG Validation Group.

PURPOSE: For localized, resectable neuroblastoma without MYCN amplification, surgery only is recommended even if incomplete. However, it is not known whether the genomic background of these tumors may influence outcome. PATIENTS AND METHODS: Diagnostic samples were obtained from 317 tumors, International Neuroblastoma Staging System stages 1/2A/2B, from 3 cohorts: Localized Neuroblastoma European Study Group I/II and Children's Oncology Group. Genomic data were analyzed using multi- and pangenomic techniques and fluorescence in-situ hybridization in 2 age groups (cutoff age, 18 months) and were quality controlled by the International Society of Pediatric Oncology European Neuroblastoma (SIOPEN) Biology Group. RESULTS: Patients with stage 1 tumors had an excellent outcome (5-year event-free survival [EFS] ± standard deviation [SD], 95% ± 2%; 5-year overall survival [OS], 99% ± 1%). In contrast, patients with stage 2 tumors had a reduced EFS in both age groups (5-year EFS ± SD, 84% ± 3% in patients < 18 months of age and 75% ± 7% in patients ≥ 18 months of age). However, OS was significantly decreased only in the latter group (5-year OS ± SD in < 18months and ≥ 18months, 96% ± 2% and 81% ± 7%, respectively; P = .001). In < 18months, relapses occurred independent of segmental chromosome aberrations (SCAs); only 1p loss decreased EFS (5-year EFS ± SD in patients 1p loss and no 1p loss, 62% ± 13% and 87% ± 3%, respectively; P = .019) but not OS (5-year OS ± SD, 92% ± 8% and 97% ± 2%, respectively). In patients ≥ 18 months, only SCAs led to relapse and death, with 11q loss as the strongest marker (11q loss and no 11q loss: 5-year EFS ± SD, 48% ± 16% and 85% ± 7%, P = .033; 5-year OS ± SD, 46% ± 22% and 92% ± 6%, P = .038). CONCLUSION: Genomic aberrations of resectable non-MYCN-amplified stage 2 neuroblastomas have a distinct age-dependent prognostic impact. Chromosome 1p loss is a risk factor for relapse but not for diminished OS in patients < 18 months, SCAs (especially 11q loss) are risk factors for reduced EFS and OS in those > 18months. In older patients with SCA, a randomized trial of postoperative chemotherapy compared with observation alone may be indicated.

ALK mutation dynamics and clonal evolution in a neuroblastoma model exhibiting two ALK mutations.

The ALK gene is a major oncogene of neuroblastoma cases exhibiting ALK activating mutations. Here, we characterized two neuroblastoma cell lines established from a stage 4 patient at diagnosis either from the primary tumor (PT) or from the bone marrow (BM). Both cell lines exhibited similar genomic profiles. All cells in the BM-derived cell line exhibited an ALK F1174L mutation, whereas this mutation was present in only 5% of the cells in the earliest passages of the PT-derived cell line. The BM-derived cell line presented with a higher proliferation rate in vitro and injections in Nude mice resulted in tumor formation only for the BM-derived cell line. Next, we observed that the F1174L mutation frequency in the PT-derived cell line increased with successive passages. Further Whole Exome Sequencing revealed a second ALK mutation, L1196M, in this cell line. Digital droplet PCR documented that the allele fractions of both mutations changed upon passages, and that the F1174L mutation reached 50% in late passages, indicating clonal evolution. In vitro treatment of the PT-derived cell line exhibiting the F1174L and L1196M mutations with the alectinib inhibitor resulted in an enrichment of the L1196M mutation. Using xenografts, we documented a better efficacy of alectinib compared to crizotinib on tumor growth and an enrichment of the L1196M mutation at the end of both treatments. Finally, single-cell RNA-seq analysis was consistent with both mutations resulting in ALK activation. Altogether, this study provides novel insights into ALK mutation dynamics in a neuroblastoma model harbouring two ALK mutations.

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.

Circulating microRNA biomarkers for metastatic disease in neuroblastoma patients.

In this study, the circulating miRNome from diagnostic neuroblastoma serum was assessed for identification of noninvasive biomarkers with potential in monitoring metastatic disease. After determining the circulating neuroblastoma miRNome, 743 miRNAs were screened in 2 independent cohorts of 131 and 54 patients. Evaluation of serum miRNA variance in a model testing for tumor stage, MYCN status, age at diagnosis, and overall survival revealed tumor stage as the most significant factor impacting miRNA abundance in neuroblastoma serum. Differential abundance analysis between patients with metastatic and localized disease revealed 9 miRNAs strongly associated with metastatic stage 4 disease in both patient cohorts. Increasing levels of these miRNAs were also observed in serum from xenografted mice bearing human neuroblastoma tumors. Moreover, murine serum miRNA levels were strongly associated with tumor volume. These findings were validated in longitudinal serum samples from metastatic neuroblastoma patients, where the 9 miRNAs were associated with disease burden and treatment response.

The RNA helicase DDX17 controls the transcriptional activity of REST and the expression of proneural microRNAs in neuronal differentiation.

The Repressor Element 1-silencing transcription factor (REST) represses a number of neuronal genes in non-neuronal cells or in undifferentiated neural progenitors. Here, we report that the DEAD box RNA helicase DDX17 controls important REST-related processes that are critical during the early phases of neuronal differentiation. First, DDX17 associates with REST, promotes its binding to the promoter of a subset of REST-targeted genes and co-regulates REST transcriptional repression activity. During neuronal differentiation, we observed a downregulation of DDX17 along with that of the REST complex that contributes to the activation of neuronal genes. Second, DDX17 and its paralog DDX5 regulate the expression of several proneural microRNAs that are known to target the REST complex during neurogenesis, including miR-26a/b that are also direct regulators of DDX17 expression. In this context, we propose a new mechanism by which RNA helicases can control the biogenesis of intronic miRNAs. We show that the processing of the miR-26a2 precursor is dependent on RNA helicases, owing to an intronic regulatory region that negatively impacts on both miRNA processing and splicing of its host intron. Our work places DDX17 in the heart of a pathway involving REST and miRNAs that allows neuronal gene repression.

Heterogeneous MYCN amplification in neuroblastoma: a SIOP Europe Neuroblastoma Study.

BACKGROUND: In neuroblastoma (NB), the most powerful prognostic marker, the MYCN amplification (MNA), occasionally shows intratumoural heterogeneity (ITH), i.e. coexistence of MYCN-amplified and non-MYCN-amplified tumour cell clones, called heterogeneous MNA (hetMNA). Prognostication and therapy allocation are still unsolved issues. METHODS: The SIOPEN Biology group analysed 99 hetMNA NBs focussing on the prognostic significance of MYCN ITH. RESULTS: Patients <18 months (18 m) showed a better outcome in all stages as compared to older patients (5-year OS in localised stages: <18 m: 0.95 ± 0.04, >18 m: 0.67 ± 0.14, p = 0.011; metastatic: <18 m: 0.76 ± 0.15, >18 m: 0.28 ± 0.09, p = 0.084). The genomic 'background', but not MNA clone sizes, correlated significantly with relapse frequency and OS. No relapses occurred in cases of only numerical chromosomal aberrations. Infiltrated bone marrows and relapse tumour cells mostly displayed no MNA. However, one stage 4s tumour with segmental chromosomal aberrations showed a homogeneous MNA in the relapse. CONCLUSIONS: This study provides a rationale for the necessary distinction between heterogeneous and homogeneous MNA. HetMNA tumours have to be evaluated individually, taking age, stage and, most importantly, genomic background into account to avoid unnecessary upgrading of risk/overtreatment, especially in infants, as well as in order to identify tumours prone to developing homogeneous MNA.

Genomic Amplifications and Distal 6q Loss: Novel Markers for Poor Survival in High-risk Neuroblastoma Patients.

Background: Neuroblastoma is characterized by substantial clinical heterogeneity. Despite intensive treatment, the survival rates of high-risk neuroblastoma patients are still disappointingly low. Somatic chromosomal copy number aberrations have been shown to be associated with patient outcome, particularly in low- and intermediate-risk neuroblastoma patients. To improve outcome prediction in high-risk neuroblastoma, we aimed to design a prognostic classification method based on copy number aberrations. Methods: In an international collaboration, normalized high-resolution DNA copy number data (arrayCGH and SNP arrays) from 556 high-risk neuroblastomas obtained at diagnosis were collected from nine collaborative groups and segmented using the same method. We applied logistic and Cox proportional hazard regression to identify genomic aberrations associated with poor outcome. Results: In this study, we identified two types of copy number aberrations that are associated with extremely poor outcome. Distal 6q losses were detected in 5.9% of patients and were associated with a 10-year survival probability of only 3.4% (95% confidence interval [CI] = 0.5% to 23.3%, two-sided P = .002). Amplifications of regions not encompassing the MYCN locus were detected in 18.1% of patients and were associated with a 10-year survival probability of only 5.8% (95% CI = 1.5% to 22.2%, two-sided P < .001). Conclusions: Using a unique large copy number data set of high-risk neuroblastoma cases, we identified a small subset of high-risk neuroblastoma patients with extremely low survival probability that might be eligible for inclusion in clinical trials of new therapeutics. The amplicons may also nominate alternative treatments that target the amplified genes.

Activated ALK signals through the ERK-ETV5-RET pathway to drive neuroblastoma oncogenesis.

Activating mutations of the ALK receptor occur in a subset of neuroblastoma tumors. We previously demonstrated that Alk mutations cooperate with MYCN overexpression to induce neuroblastoma in mice and identified Ret as being strongly upregulated in MYCN/Alkmut tumors. By a genetic approach in vivo, we now document an oncogenic cooperation between activated Ret and MYCN overexpression in neuroblastoma formation. We show that MYCN/RetM919T tumors exhibit histological features and expression profiles close to MYCN/Alkmut tumors. We show that RET transcript levels decrease precedes RET protein levels decrease upon ALK inhibition in neuroblastoma cell lines. Etv5 was identified as a candidate transcription factor regulating Ret expression from murine MYCN/Alkmut tumor transcriptomic data. We demonstrate that ETV5 is regulated both at the protein and mRNA levels upon ALK activation or inhibition in neuroblastoma cell lines and that this regulation precedes RET modulation. We document that ALK activation induces ETV5 protein upregulation through stabilization in a MEK/ERK-dependent manner. We show that RNAi-mediated inhibition of ETV5 decreases RET expression. Reporter assays indicate that ETV5 is able to drive RET gene transcription. ChIP-seq analysis confirmed ETV5 binding on the RET promoter and identified an enhancer upstream of the promoter. Finally, we demonstrate that combining RET and ALK inhibitors reduces tumor growth more efficiently than each single agent in MYCN and AlkF1178L-driven murine neuroblastoma. Altogether, these results define the ERK-ETV5-RET pathway as a critical axis driving neuroblastoma oncogenesis downstream of activated ALK.

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.

Whole-Exome Sequencing of Cell-Free DNA Reveals Temporo-spatial Heterogeneity and Identifies Treatment-Resistant Clones in Neuroblastoma.

Purpose: Neuroblastoma displays important clinical and genetic heterogeneity, with emergence of new mutations at tumor progression.Experimental Design: To study clonal evolution during treatment and follow-up, an innovative method based on circulating cell-free DNA (cfDNA) analysis by whole-exome sequencing (WES) paired with target sequencing was realized in sequential liquid biopsy samples of 19 neuroblastoma patients.Results: WES of the primary tumor and cfDNA at diagnosis showed overlap of single-nucleotide variants (SNV) and copy number alterations, with 41% and 93% of all detected alterations common to the primary neuroblastoma and cfDNA. CfDNA WES at a second time point indicated a mean of 22 new SNVs for patients with progressive disease. Relapse-specific alterations included genes of the MAPK pathway and targeted the protein kinase A signaling pathway. Deep coverage target sequencing of intermediate time points during treatment and follow-up identified distinct subclones. For 17 seemingly relapse-specific SNVs detected by cfDNA WES at relapse but not tumor or cfDNA WES at diagnosis, deep coverage target sequencing detected these alterations in minor subclones, with relapse-emerging SNVs targeting genes of neuritogenesis and cell cycle. Furthermore a persisting, resistant clone with concomitant disappearance of other clones was identified by a mutation in the ubiquitin protein ligase HERC2Conclusions: Modelization of mutated allele fractions in cfDNA indicated distinct patterns of clonal evolution, with either a minor, treatment-resistant clone expanding to a major clone at relapse, or minor clones collaborating toward tumor progression. Identification of treatment-resistant clones will enable development of more efficient treatment strategies. Clin Cancer Res; 24(4); 939-49. ©2017 AACR.

Microenvironment-Driven Shift of Cohesion/Detachment Balance within Tumors Induces a Switch toward Metastasis in Neuroblastoma.

Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. Disseminated forms have high frequency of multiple tumoral foci whose etiology remains unknown; NB embryonic origin limits investigations in patients and current models. We developed an avian embryonic model driving human NB tumorigenesis in tissues homologous to patients. We found that aggressive NBs display a metastatic mode, secondary dissemination via peripheral nerves and aorta. Through tumor transcriptional profiling, we found that NB dissemination is induced by the shutdown of a pro-cohesion autocrine signal, SEMA3C, which constrains the tumoral mass. Lowering SEMA3C levels shifts the balance toward detachment, triggering NB cells to collectively evade the tumor. Together with patient cohort analysis, this identifies a microenvironment-driven pro-metastatic switch for NB.