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Cell line growth curves and PDX data for idasanutlin combination

Supplementary methods, supplementary tables, supplementary figure legends

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Tumour aCGH-specific and cfDNA- specific breakpoints

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

Supplementary Table 2. Primer sequences used for the amplification of ALK regions of interested in the first PCR-step

Table S3: SNVs detected in primary solid Tumor at diagnosis (n=19), Relapse Tumor (n=4 previously published cases), cfDNA at diagnosis (n=19) and cfDNA with relapse (n=9) with WES.

Supplementary Table 1. Clinical and tumor genetic data of studied cohort of 276 NB patients

Purpose: In neuroblastoma, activating ALK receptor tyrosine kinase point mutations play a major role in oncogenesis. We explored the potential occurrence of ALK mutations at a subclonal level using targeted deep sequencing.Experimental Design: In a clinically representative series of 276 diagnostic neuroblastoma samples, exons 23 and 25 of the ALK gene, containing the F1174 and R1275 mutation hotspots, respectively, were resequenced with an extremely high depth of coverage.Results: At the F1174 hotspot (exon 23), mutations were observed in 15 of 277 samples (range of fraction of mutated allele per sample: 0.562%–40.409%). At the R1275 hotspot (exon 25), ALK mutations were detected in 12 of 276 samples (range of fraction of mutated allele: 0.811%–73.001%). Altogether, subclonal events with a mutated allele fraction below 20% were observed in 15/27 ALK-mutated samples. The presence of an ALK mutation was associated with poorer 5-year overall survival (OS: 75% vs. 57%, P = 0.0212 log-rank test), with a strong correlation between F1174 ALK mutations and MYCN amplification being observed.Conclusions: In this series, deep sequencing allows the detection of F1174 and R1275 ALK mutational events at diagnosis in 10% of cases, with subclonal events in more than half of these, which would have gone undetected by Sanger sequencing. These findings are of clinical importance given the potential role of ALK mutations in clonal evolution and relapse. These findings also demonstrate the importance of deep sequencing techniques for the identification of patients especially when considering targeted therapy. Clin Cancer Res; 21(21); 4913–21. ©2015 AACR.See related commentary by George, p. 4747

Detailed Clinical and Sequencing Data of all Cases.

A TERT targeting breakpoint seen in the cfDNA is not seen in the primary NB DNA (case 58)

Correlation of genomic profiles determined by tumor aCGH and cfDNA analysis

Quality control of cfDNA

Table S2 : detailed description of time points for plasma sampling.

Supplementary Figure 2. Comparison of polymorphism rs3738868 allele fractions located within the intronic region between exon 25 and exon 26 (position chr2:29432625) between the first deep sequencing and validation experiment. The 263 neuroblastoma samples with a depth of coverage higher than 1000X at the polymorphism position are reported on the graph. The concordance between the two experiments is highlighted by the R-squared value

Figure S1 : Sequencing plan; Figure S2: Examples of a high resolution electrophoresis profiles of cfDNA; Figure S3; Mean Coverage observed between Tumor, Germline and cfDNA sequencing; Figure S4: Estimation of the sensitivity of WES and targeted sequencing of cfDNA; Figure S5: CtDNA fraction in total cfDNA at diagnosis (n=19 patients) and at the 2nd timepoint (CR/PR, n=8 patients); Figure S6: Scatterplot of MAFs between WES on primary tumor and cfDNA (A) and WES and TargetSeq (B); Figure S7: Clonal Evolution detected with Target Sequencing; Figure S8: Example of resurgence of MAFs (Case 6) which will be followed by a relapse.

Comparison of recurrent SCA seen by aCGH versus cfDNA Oncoscan® analysis

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.

supplemental figure legend. supplemental figure legend

Supplementary Figure 1. Background variability observed by high coverage Hiseq2500 Illumina® sequencing in 24 germline DNA controls.

Information on treatment protocols

BackgroundALKactivating mutations are identified in approximately 10% of newly diagnosed neuroblastomas andALKamplifications in a further 1-2% of cases. Lorlatinib, a third generation 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 has suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway.AimsTo study the preclinical activity of ALK inhibitors alone and in combination with chemotherapy or idasanutlin.MethodsWe 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 vivodosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient derived xenografts (PDX).ResultsLorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in theALK-amplified PDX model with the highest ALK expression. In this PDX lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth.ConclusionOur study suggests that in neuroblastoma, high ALK expression could be associated with response to lorlatinib and either chemotherapy or idasanutlin. The synergy between MDM2 inhibition and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma.STATEMENT OF TRANSLATIONAL RELEVANCENeuroblastoma is a pediatric tumor of the developing sympathetic nervous system. Around 50% of high-risk neuroblastoma patients are curable. Mutations or amplification of Anaplastic Lymphoma Kinase (ALK) have emerged as a marker with which to further risk-stratify patients. The ALK inhibitor lorlatinib will soon be used alongside chemotherapy in upfront treatment of high-risk patients with ALK-aberrant disease. In this preclinical study, we used a panel ofALKaberrant neuroblastoma models to evaluate ALK inhibitors focusing on lorlatinib in combination with conventional chemotherapy and the small molecule MDM2 inhibitor idasanutlin. In both approaches we found synergy in models with high basal ALK expression without MAPK pathway alterations. We conclude that in neuroblastoma the level of ALK expression could be an additional biomarker predictive of clinical response to ALK inhibitors.

BackgroundHigh-risk neuroblastoma is a pediatric cancer with still a dismal prognosis, despite multimodal and intensive therapies. Tumor microenvironment represents a key component of the tumor ecosystem the complexity of which has to be accurately understood to define selective targeting opportunities, including immune-based therapies.MethodsWe combined various approaches including single-cell transcriptomics to dissect the tumor microenvironment of both a transgenic mouse neuroblastoma model and a cohort of 10 biopsies from neuroblastoma patients, either at diagnosis or at relapse. Features of related cells were validated by multicolor flow cytometry and functional assays.ResultsWe show that the immune microenvironment of MYCN-driven mouse neuroblastoma is characterized by a low content of T cells, several phenotypes of macrophages and a population of cells expressing signatures of myeloid-derived suppressor cells (MDSCs) that are molecularly distinct from the various macrophage subsets. We document two cancer-associated fibroblasts (CAFs) subsets, one of which corresponding to CAF-S1, known to have immunosuppressive functions. Our data unravel a complex content in myeloid cells in patient tumors and further document a striking correspondence of the microenvironment populations between both mouse and human tumors. We show that mouse intratumor T cells exhibit increased expression of inhibitory receptors at the protein level. Consistently, T cells from patients are characterized by features of exhaustion, expressing inhibitory receptors and showing low expression of effector cytokines. We further functionally demonstrate that MDSCs isolated from mouse neuroblastoma have immunosuppressive properties, impairing the proliferation of T lymphocytes.ConclusionsOur study demonstrates that neuroblastoma tumors have an immunocompromised microenvironment characterized by dysfunctional T cells and accumulation of immunosuppressive cells. Our work provides a new and precious data resource to better understand the neuroblastoma ecosystem and suggest novel therapeutic strategies, targeting both tumor cells and components of the microenvironment.

In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through activating point mutations or genomic amplification. We studiedDiagnostic tumor samples were available from 1,092 HR-NBL1/SIOPEN patients to determineGenomicGenetic alterations of

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.

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., What's new? Mutations that affect chromatin remodeling can lead to cancer. In this paper, the authors investigated the impact of variations in chromatin remodeling genes and epigenetic modifier genes on neuroblastoma patients. They compared the frequency of these variations in NB cases with data from the Genome Aggregation Database (gnomAD). Neuroblastoma cases had a higher frequency of SMARCA4 and ATRX gene variations than the general population. Furthermore, NB patients with CRG/EMG mutations had poorer overall survival than NB cases without such mutations. These findings highlight the importance of chromatin remodeling in neuroblastoma as an avenue for new therapeutics.

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.

Purpose: In neuroblastoma, activating ALK receptor tyrosine kinase point mutations play a major role in oncogenesis. We explored the potential occurrence of ALK mutations at a subclonal level using targeted deep sequencing. Experimental Design: In a clinically representative series of 276 diagnostic neuroblastoma samples, exons 23 and 25 of the ALK gene, containing the F1174 and R1275 mutation hotspots, respectively, were resequenced with an extremely high depth of coverage. Results: At the F1174 hotspot (exon 23), mutations were observed in 15 of 277 samples (range of fraction of mutated allele per sample: 0.562%–40.409%). At the R1275 hotspot (exon 25), ALK mutations were detected in 12 of 276 samples (range of fraction of mutated allele: 0.811%–73.001%). Altogether, subclonal events with a mutated allele fraction below 20% were observed in 15/27 ALK-mutated samples. The presence of an ALK mutation was associated with poorer 5-year overall survival (OS: 75% vs. 57%, P = 0.0212 log-rank test), with a strong correlation between F1174 ALK mutations and MYCN amplification being observed. Conclusions: In this series, deep sequencing allows the detection of F1174 and R1275 ALK mutational events at diagnosis in 10% of cases, with subclonal events in more than half of these, which would have gone undetected by Sanger sequencing. These findings are of clinical importance given the potential role of ALK mutations in clonal evolution and relapse. These findings also demonstrate the importance of deep sequencing techniques for the identification of patients especially when considering targeted therapy. Clin Cancer Res; 21(21); 4913–21. ©2015 AACR. See related commentary by George, p. 4747

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 protein in homologous recombination (HR), a DNA repair pathway dedicated to double strand breaks and recovery of blocked or collapsed replication forks. Rad52 allows Rad51 loading on single strand DNA, an event required for strand invasion and D-loop formation. In addition, Rad52 functions also in Rad51 independent pathways because of its ability to promote single strand annealing (SSA) that leads to loss of genetic material and to promote D-loops formation that are cleaved by Mus81 endonuclease. We have previously reported that fission yeast Rad52 is phosphorylated in a Sty1 dependent manner upon oxidative stress and in cells where the early step of HR is impaired because of lack of Rad51. Here we show that Rad52 is also constitutively phosphorylated in mus81 null cells and that Sty1 partially impinges on such phosphorylation. As upon oxidative stress, the Rad52 phosphorylation in rad51 and mus81 null cells appears to be independent of Tel1, Rad3 and Cdc2. Most importantly, we show that mutating serine 365 to glycine (S365G) in Rad52 leads to loss of the constitutive Rad52 phosphorylation observed in cells lacking Rad51 and to partial loss of Rad52 phosphorylation in cells lacking Mus81. Contrariwise, phosphorylation of Rad52-S365G protein is not affected upon oxidative stress. These results indicate that different Rad52 residues are phosphorylated in a Sty1 dependent manner in response to these distinct situations. Analysis of spontaneous HR at direct repeats shows that mutating serine 365 leads to an increase in spontaneous deletion-type recombinants issued from mitotic recombination that are Mus81 dependent. In addition, the recombination rate in the rad52-S365G mutant is further increased by hydroxyurea, a drug to which mutant cells are sensitive.

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.

Background The majority of high-risk neuroblastomas initially respond to chemotherapy, but over half of patients experience therapy-resistant relapses. The molecular defects driving relapse and drug resistance are unknown. Methods We performed Illumina or Complete Genomics whole genome sequencing of 23 paired diagnostic and relapsed neuroblastomas, and corresponding normal lymphocyte DNA, to define genetic alterations associated with relapse. A panel of 18 neuroblastoma cell lines was analyzed for the presence of RAS-MAPK mutations and sensitivity to small molecule inhibitors of this pathway. Results Neuroblastomas that relapsed after chemotherapy showed dramatic clonal evolution, with only 33% of primary tumor mutations also detected at relapse. In 21 out of 23 patients, more somatic coding mutations were observed at relapse (median: 29 unique to relapse, range: 4-129). Unbiased pathway analysis of the somatic mutations detected in the relapse tissues identified a strong enrichment in genes associated with RAS-MAPK signaling (p = 6.1×10−7). 18 of the 23 cases (78%) showed somatic mutations (N = 15) or structural alterations (N = 3) predicted to activate the MAPK pathway, and these were mutually exclusive: ALK (N = 10), NRAS (N = 1), KRAS (N = 1), HRAS (N = 1), BRAF (N = 1), PTPN11 (N = 1), FGRF1 (N = 1) and NF1 (N = 2). These RAS-MAPK mutations were clonally enriched at relapse and exist within clonal or major subclonal tumor populations. Seven of these RAS-MAPK mutations were detected only in the relapse tumor by whole genome sequencing (∼50X coverage), and only 2 of these 7 mutations were detectable in the primary tumor with targeted detection methods (104-105X coverage). Similar MAPK pathway mutations were detected in 11 of 18 human neuroblastoma-derived cell lines, and these lesions are predicted to be sensitive to small molecule inhibition of MEK in vitro (p Conclusions In this study of 23 neuroblastoma cases selected based solely on having diagnostic-relapse specimens available for analysis, MAPK pathway mutations were highly enriched in the relapsed genomes, providing a potential biomarker for new therapeutic approaches to chemotherapy refractory disease. The fact that several ALK-RAS-MAPK mutations were found in the relapse but not in the corresponding primary tumors favors a model in which rare subclones with secondary driver mutations expand over time. However, it remains to be determined whether these mutations occurred de novo after treatment, were present in rare subclones below detection limits, or were undetectable due to spatial heterogeneity of the primary tumor, which will impact the clinical utility of targeted sequencing at diagnosis. Our study provides strong rationale for performing biopsies on relapse neuroblastoma tumors in order to comprehensively characterize the molecular lesions that underlie treatment-refractory disease, determine their prognostic relevance, and guide treatment decisions for patients. Citation Format: Derek A. Oldridge, Thomas F. Eleveld, Virginie Bernard, Jan Koster, Leo C. Daage, Sharon J. Diskin, Linda Schild, Nadia B. Bentahar, Angela Bellini, Mathieu Chicard, Eve Lapouble, Valérie Combaret, Patricia Legoix-Né, Jean Michon, Trevor J. Pugh, Lori S. Hart, JulieAnn Rader, Edward F. Attiyeh, Jun S. Wei, Shile Zhang, Arlene Naranjo, Julie M. Gastier-Foster, Michael D. Hogarty, Malcolm A. Smith, Jaime G. Auvil, Thomas B. K. Watkins, Danny A. Zwijnenburg, Marli E. Ebus, Peter van Sluis, Anne Hakkert, Esther van Wezel, C. Ellen van der Schoot, Ellen M. Westerhout, Johannes H. Schulte, Godelieve A. Tytgat, M. Emmy M. Dolman, Isabelle Janoueix-Lerosey, Daniela S. Gerhard, Huib N. Caron, Olivier Delattre, Javed Khan, Rogier Versteeg, Gudrun Schleiermacher, John M. Maris, Jan J. Molenaar. Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2980. doi:10.1158/1538-7445.AM2015-2980