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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.

Two cell identities, noradrenergic and mesenchymal, have been characterized in neuroblastoma cell lines according to their epigenetic landscapes relying on specific circuitries of transcription factors. Yet, their relationship and relative contribution in patient tumors remain poorly defined. Here, we demonstrate that the knock-out of GATA3, but not of PHOX2A or PHOX2B, in noradrenergic cells induces a mesenchymal phenotype. Our results document spontaneous plasticity in several models between both identities and show that plasticity relies on epigenetic reprogramming. We demonstrate that an in vivo microenvironment provides a powerful pressure towards a noradrenergic identity for these models. Consistently, tumor cells with a mesenchymal identity are not detected in a series of PDX models. Further study of the intra-tumor noradrenergic heterogeneity reveals two distinct cell populations exhibiting features of chromaffin-like or sympathoblast-like cells. This work emphasizes that both external cues of the environment and intrinsic factors control plasticity and cell identity in neuroblastoma.

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. Key Objective: High risk neuroblastoma (HR-NB) is one of the most difficult childhood cancers to cure. This study examined whether the presence of an ALK alteration (amplification or mutation) was associated with a poor prognosis in a large patient series treated on the prospective European high-risk neuroblastoma trial (HR-NBL1). Knowledge Generated: We found that ALK amplification or clonal mutation was associated with inferior prognosis in patients with HR-NB and both are independent prognostic variables on multivariate analysis. To our knowledge, this is the first study to report the highly prognostic significance of ALK amplification in HR-NB. Relevance: As ALK can be targeted therapeutically, this study convincingly argues for the introduction of ALK inhibitors for upfront management of patients with HR-NB with ALK aberrations. Importantly, the prognostic significance of ALK alterations included a subgroup of trial patients treated with the current standard of care for HR-NB including anti-GD2 immunotherapy. info:eu-repo/semantics/publishedVersion

The majority of neuroblastoma patients have tumors that initially respond to chemotherapy, but a large proportion of patients will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole genome sequencing of 23 paired diagnostic and relapsed neuroblastomas showed clonal evolution from the diagnostic tumor with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK signaling pathway. Seven events were detected only in the relapse tumor while the others showed clonal enrichment. In neuroblastoma cell lines we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18, 61%) and these lesions predicted for sensitivity to MEK inhibition in vitro and in vivo. Our findings provide the rationale for genetic characterization of relapse neuroblastoma and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease.

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 HERC2. Conclusions: 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.

In cancer, clonal evolution is characterized based on single nucleotide variants and copy number alterations. Nonetheless, previous methods failed to combine information from both sources to accurately reconstruct clonal populations in a given tumor sample or in a set of tumor samples coming from the same patient. Moreover, previous methods accepted as input all variants predicted by variant-callers, regardless of differences in dispersion of variant allele frequencies (VAFs) due to uneven depth of coverage and possible presence of strand bias, prohibiting accurate inference of clonal architecture. We present a general framework for assignment of functional mutations to specific cancer clones, which is based on distinction between passenger variants with expected low dispersion of VAF versus putative functional variants, which may not be used for the reconstruction of cancer clonal architecture but can be assigned to inferred clones at the final stage. The key element of our framework is QuantumClone, a method to cluster variants into clones, which we have thoroughly tested on simulated data. QuantumClone takes into account VAFs and genotypes of corresponding regions together with information about normal cell contamination. We applied our framework to whole genome sequencing data for 19 neuroblastoma trios each including constitutional, diagnosis and relapse samples. We discovered specific pathways recurrently altered by deleterious mutations in different clonal populations. Some such pathways were previously reported (e.g., MAPK and neuritogenesis) while some were novel (e.g., epithelial-mesenchymal transition, cell survival and DNA repair). Most pathways and their modules had more mutations at relapse compared to diagnosis.

Purpose: The tumor genomic copy number profile is of prognostic significance in neuroblastoma patients. We have studied the genomic copy number profile of cell-free DNA (cfDNA) and compared this with primary tumor arrayCGH (aCGH) at diagnosis. Experimental Design: In 70 patients, cfDNA genomic copy number profiling was performed using the OncoScan platform. The profiles were classified according to the overall pattern, including numerical chromosome alterations (NCA), segmental chromosome alterations (SCA), and MYCN amplification (MNA). Results: Interpretable and dynamic cfDNA profiles were obtained in 66 of 70 and 52 of 70 cases, respectively. An overall identical genomic profile between tumor aCGH and cfDNA was observed in 47 cases (3 NCAs, 22 SCAs, 22 MNAs). In one case, cfDNA showed an additional SCA not detected by tumor aCGH. In 4 of 8 cases with a silent tumor aCGH profile, cfDNA analysis revealed a dynamic profile (3 SCAs, 1 NCA). In 14 cases, cfDNA analysis did not reveal any copy number changes. A total of 378 breakpoints common to the primary tumor and cfDNA of any given patient were identified, 27 breakpoints were seen by tumor aCGH, and 54 breakpoints were seen in cfDNA only, including two cases with interstitial IGFR1 gains and two alterations targeting TERT. Conclusions: These results demonstrate the feasibility of cfDNA copy number profiling in neuroblastoma patients, with a concordance of the overall genomic profile in aCGH and cfDNA dynamic cases of 97% and a sensitivity of 77%, respectively. Furthermore, neuroblastoma heterogeneity is highlighted, suggesting that cfDNA might reflect genetic alterations of more aggressive cell clones. Clin Cancer Res; 22(22); 5564–73. ©2016 AACR. See related commentary by Janku and Kurzrock, p. 5400

Background : Pediatric cancers are characterized by few recurrent genetic alterations, but genetic heterogeneity and clonal evolution can play a role in tumor progression. Liquid biopsies now enable monitoring of tumor-specific genetic alterations in sequential samples by analysis of cell free DNA (cfDNA). Methods : We have enrolled 28 consecutive patients with newly diagnosed high risk pediatric cancer (neuroblastoma n=8; rhabdomyosarcoma n=7; Ewing sarcoma n=3; other cerebral or extracerebral high risk cancers n=10) in a prospective clinical study NGSkids (clingov trial : NCT02546453) with an aim to study clonal evolution based on sequential cfDNA analysis. With a median follow up of 22 months, 7 patients have experienced relapse. Molecular analysis consisted of standardized Illumina© 100PE 100x whole exome sequencing (WES) of tumor tissue and paired germline material , and WES of cfDNA extracted from plasma samples at diagnosis, during treatment and follow up, with 2-9 sequential samples available per patient. cfDNA analysis was performed using an in-house procedure, with 20-200 ng of cfDNA subjected to WES following modified library construction and capture approaches to account for cfDNA molecule size (target depth 100x). Following filtering on germline to focus on tumor cell specific alterations, SNVs were called using GATK-UnifiedGenotyper, GATK-HaplotypeCaller, Samtools and Mutect. Copy-number profiles were generated using Varscan and DNAcopy. Results:: At diagnosis, cfDNA quantities were higher in advanced stages of disease (localized stages - mean 64 ng/ml of plasma (range 24-172); metastatic stages - mean 505 ng/ml of plasma (range 20-2,782)). CfDNA WES analysis yielded satisfactory depth in all cases. At diagnosis, a mean of 9 tumor cell specific SNVs common to both primary tumor and corresponding cfDNA was observed (range 1 - 57), with a mean of 11 and 6 specific to the primary and cfDNA respectively, indicating spatial heterogeneity. Whereas cfDNA samples obtained at follow-up in patients without evidence of disease revealed no or few tumor cell specific SNVs, interestingly, cfDNA samples obtained at relapse harbored additional, new relapse-specific SNVs (mean 10; range 2-42) in all cases with relapse, targeting genes such as MAPK and MLL4. Deep sequencing (10,000X) capture techniques with a panel encompassing all identified SNVs is currently being applied to all cfDNA samples, including 1-8 intermediate samples per patient, with an aim to develop models of clonal evolution. Discussion and Conclusion: CfDNA WES proves to be an extremely powerful tool to study spatial and temporal heterogeneity in pediatric high risk cancers, providing further proof of the importance of clonal evolution in cancer progression. Full characterization of cfDNA at relapse, which might represent more aggressive clones, might orient towards targeted treatment approaches. Citation Format: Mathieu Chicard, Adrien Danzon, Nathalie Clémént, Irene Jimenez, Eve Lapouble, Gaelle Pierron, Angela Bellini, Nada Leprovost, Sylvain Baulande, Paul Fréneaux, François Doz, Daniel Orbach, Isabelle Aerts, Hélène Pacquement, Jean Michon, Franck Bourdeaut, Olivier Delattre, Gudrun Schleiermacher. Whole-exome sequencing cell free DNA analysis documents new tumor specific alterations at relapse of high-risk pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2592.

International audience; New protocols based on ALK-targeted therapy by crizotinib or other ALK-targeting molecules have opened for the treatment of patients with neuroblastoma (NB) if their tumors showed mutation and/or amplification of the ALK gene. However, tumor samples are not always available for analysis of ALK mutational status in particular at relapse. Here, we evaluated the ALK mutational status of NB samples by analysis of circulating DNA, using the droplet digital PCR (ddPCR) system. ddPCR assays was developed for the detection of ALK mutations at F1174 and R1275 hotspots found in NB tumors and was applied for the analysis of circulating DNA obtained from 200L of serum or plasma samples collected from 114 patients with NB. The mutations F1174L (exon 23 position 3520, T>C and position 3522, C>A) and the mutation R1275Q (exon 25 position 3824, G>A) were detected in circulating DNA. The sensitivity of our test was 100%, 85%, and 92%, respectively, and the specificity was 100%, 91%, and 98%, respectively. In conclusion, the assay that we have developed offers a reliable, noninvasive blood test to assess ALK mutational status at F1174 and R1275 hotspots and should help clinicians to identify patients showing an ALK mutation in particular when no tumor tissue is available.

Background : Liquid biopsies are revolutionary tools to monitor tumour-specific genetic alterations in sequential samples. In neuroblastoma (NB), significant levels of circulating tumor DNA (ctDNA) in the bloodstream enable the detection of tumour cell-specific markers including MYCN amplification or activating ALK mutations. As clonal evolution plays a role in NB progression, analysis of a single genetic marker will be insufficient for ctDNA-based disease follow-up. Methods : To gain further insights into mechanisms of clonal evolution in NB, we isolated ctDNA from plasma at diagnosis (n=19) and during follow-up (final time-point: partial or complete remission (PR/CR), n=7; progressive disease (PD), n=9) for 19 NB patients for whom primary NB and matched germline DNA whole exome/whole genome sequencing data (WES/WGS) was available. CtDNA (7-100ng) was subjected to Illumina 100PE WES following modified library construction and capture approaches to account for small ctDNA molecules (target depth 100x). SNVs/mutations were called using GATK-UnifiedGenotyper, GATK-HaplotypeCaller and Samtools. Copy-number profiles were generated using Varscan and DNAcopy. Results : CtDNA WES yielded satisfactory depth in all cases. At diagnosis, a majority of observed SNVs were common to the primary NB and corresponding diagnostic ctDNA of a given patient (mean number of SNVs: 19; range 9-69) with MAF (mutated allele fractions) corresponding to the estimated ctDNA content in the total cell free DNA. At diagnosis, few SNVs specific to the NB (mean: 6; range 0-18) or specific to ctDNA (mean:22; range 9-69) were observed, suggesting spatial heterogeneity with different ctDNA amounts released by different clones. In PR or CR ctDNA samples, lower numbers of SNVs with lower MAFs were detected (mean: 11, range 0-12). Interestingly, PD ctDNA samples harboured an increase in MAFs and a higher numbers of SNVs, with additional relapse-specific SNVs (mean: 22; range 0-55) targeting, amongst others, the protein kinase A signaling pathway. Analysis of additional ctDNA samples obtained between diagnosis and relapse (2-6 samples/patient) using deep sequencing techniques demonstrated a disappearance of SNVs coinciding with response to therapy, and re-appearance of SNVs at the time of tumor progression. Discussion and Conclusion: CtDNA WES proves to be an extremely powerful tool to study spatial and temporal heterogeneity in NB, providing further proof of the importance of clonal evolution in NB progression. Full characterization of ctDNA, which might represent more aggressive clones, might orient targeted treatment approaches. Citation Format: Mathieu Chicard, Leo Colmet Daage, Nathalie Clement, Angela Bellini, Sylvain Baulande, Virginie Bernard, Gaelle Pierron, Eve Lapouble, Isabelle Janoueix-Lerosey, Jean Michon, Valérie Combaret, Olivier Delattre, Gudrun Schleiermacher. Whole exome sequencing of circulating tumor DNA highlights spatial and temporal tumor heterogeneity in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4952. doi:10.1158/1538-7445.AM2017-4952

International audience; Rad52 is a key player in homologous recombination (HR), a DNA repair pathway that is dedicated to double strand breaks repair and recovery of perturbed replication forks. Here we show that fission yeast Rad52 homologue is phosphorylated when S phase cells are exposed to ROS inducers such as ultraviolet A radiation or hydrogen peroxide, but not to ultraviolet C or camptothecin. Phosphorylation does not depend on kinases Chk1, Rad3, Tel1 or Cdc2, but depends on a functional stress activated protein kinase (SAPK) pathway and can be partially prevented by anti-oxidant treatment. Indeed, cells lacking Sty1, the major fission yeast MAP kinase of the SAPK pathway, do not display Rad52 phosphorylation and have UVA induced Rad52 foci that persist longer if compared to wild type cells. In addition, spontaneous intrachromosomal HR is diminished in cells lacking Sty1 and, more precisely, gene conversion is affected. Moreover, HR induced by site-specific arrest of replication forks is twice less efficient in cells that do not express Sty1. Importantly, impairing HR by deletion of the gene encoding the recombinase Rhp51 leads to Sty1 dependent Rad52 phosphorylation. Thus, SAPK pathway impinges on early step of HR through phosphorylation of Rad52 in cells challenged by oxidative stress or lacking Rhp51 and is required to promote spontaneous gene conversion and recovery from blocked replication forks.