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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.

MAPPYACTS (NCT02613962) is an international prospective precision medicine trial aiming to define tumor molecular profiles in pediatric patients with recurrent/refractory malignancies in order to suggest the most adapted salvage treatment. From February 2016 to July 2020, 787 patients were included in France, Italy, Ireland, and Spain. At least one genetic alteration leading to a targeted treatment suggestion was identified in 436 patients (69%) with successful sequencing; 10% of these alterations were considered “ready for routine use.” Of 356 patients with follow-up beyond 12 months, 107 (30%) received one or more matched targeted therapies—56% of them within early clinical trials—mainly in the AcSé-ESMART platform trial (NCT02813135). Overall, matched treatment resulted in a 17% objective response rate, and of those patients with ready for routine use alterations, it was 38%. In patients with extracerebral tumors, 76% of actionable alterations detected in tumor tissue were also identified in circulating cell-free DNA (cfDNA).Significance:MAPPYACTS underlines the feasibility of molecular profiling at cancer recurrence in children on a multicenter, international level and demonstrates benefit for patients with selected key drivers. The use of cfDNA deserves validation in prospective studies. Our study highlights the need for innovative therapeutic proof-of-concept trials that address the underlying cancer complexity.This article is highlighted in the In This Issue feature, p. 1171

Supplementary Data from The European MAPPYACTS Trial: Precision Medicine Program in Pediatric and Adolescent Patients with Recurrent Malignancies

Patient Information

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

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.

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.

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.

Information on treatment protocols

Abstract MAPPYACTS (NCT02613962) is an international prospective precision medicine trial aiming to define tumor molecular profiles in pediatric patients with recurrent/refractory malignancies in order to suggest the most adapted salvage treatment. From February 2016 to July 2020, 787 patients were included in France, Italy, Ireland, and Spain. At least one genetic alteration leading to a targeted treatment suggestion was identified in 436 patients (69%) with successful sequencing; 10% of these alterations were considered “ready for routine use.” Of 356 patients with follow-up beyond 12 months, 107 (30%) received one or more matched targeted therapies—56% of them within early clinical trials—mainly in the AcSé-ESMART platform trial (NCT02813135). Overall, matched treatment resulted in a 17% objective response rate, and of those patients with ready for routine use alterations, it was 38%. In patients with extracerebral tumors, 76% of actionable alterations detected in tumor tissue were also identified in circulating cell-free DNA (cfDNA). Significance: MAPPYACTS underlines the feasibility of molecular profiling at cancer recurrence in children on a multicenter, international level and demonstrates benefit for patients with selected key drivers. The use of cfDNA deserves validation in prospective studies. Our study highlights the need for innovative therapeutic proof-of-concept trials that address the underlying cancer complexity. This article is highlighted in the In This Issue feature, p. 1171

Background: Paediatric tumours are often characterised by the presence of recurrent DNA copy number alterations (CNAs). These DNA copy number profiles, obtained from a tissue biopsy, can aid in the correct prognostic classification and therapeutic stratification of several paediatric cancer entities (e.g. MYCN amplification in neuroblastoma) and are part of the routine diagnostic practice. Liquid biopsies (LQBs) offer a potentially safer alternative for such invasive tumour tissue biopsies and can provide deeper insight into tumour heterogeneity. Procedure: The robustness and reliability of LQB CNA analyses was evaluated. We performed retrospective CNA profiling using shallow whole-genome sequencing (sWGS) on paired plasma circulating cell-free DNA (cfDNA) and tissue DNA samples from routinely collected samples from paediatric patients (n = 128) representing different tumour entities, including osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, Wilms tumour, brain tumours and neuroblastoma. Results: Overall, we observed a good concordance between CNAs in tissue DNA and cfDNA. The main cause of CNA discordance was found to be low cfDNA sample quality (i.e. the ratio of cfDNA (700 bp)). Furthermore, CNAs were observed that were present in cfDNA and not in tissue DNA, or vice-versa. In neuroblastoma samples, no false-positives or false-negatives were identified for the detection of the prognostic marker MYCN amplification. Conclusion: In future prospective studies, CNA analysis on LQBs that are of sufficient quality can serve as a complementary assay for CNA analysis on tissue biopsies, as either cfDNA or tissue DNA can contain CNAs that cannot be identified in the other biomaterial.

PURPOSE Total cell-free DNA (cfDNA) and tumor-derived cfDNA (ctDNA) can be used to study tumor-derived genetic aberrations. We analyzed the diagnostic and prognostic potential of cfDNA and ctDNA, obtained from pediatric patients with rhabdomyosarcoma. METHODS cfDNA was isolated from diagnostic plasma samples from 57 patients enrolled in the EpSSG RMS2005 study. To study the diagnostic potential, shallow whole genome sequencing (shWGS) and cell-free reduced representation bisulphite sequencing (cfRRBS) were performed in a subset of samples and all samples were tested using droplet digital polymerase chain reaction to detect methylated RASSF1A ( RASSF1A-M). Correlation with outcome was studied by combining cfDNA RASSF1A-M detection with analysis of our rhabdomyosarcoma-specific RNA panel in paired cellular blood and bone marrow fractions and survival analysis in 56 patients. RESULTS At diagnosis, ctDNA was detected in 16 of 30 and 24 of 26 patients using shallow whole genome sequencing and cfRRBS, respectively. Furthermore, 21 of 25 samples were correctly classified as embryonal by cfRRBS. RASSF1A-M was detected in 21 of 57 patients. The presence of RASSF1A-M was significantly correlated with poor outcome (the 5-year event-free survival [EFS] rate was 46.2% for 21 RASSF1A-M ‒positive patients, compared with 84.9% for 36 RASSF1A-M ‒negative patients [ P < .001]). RASSF1A-M positivity had the highest prognostic effect among patients with metastatic disease. Patients both negative for RASSF1A-M and the rhabdomyosarcoma-specific RNA panel (28 of 56 patients) had excellent outcome (5-year EFS 92.9%), while double-positive patients (11/56) had poor outcome (5-year EFS 13.6%, P < .001). CONCLUSION Analyzing ctDNA at diagnosis using various techniques is feasible in pediatric rhabdomyosarcoma and has potential for clinical use. Measuring RASSF1A-M in plasma at initial diagnosis correlated significantly with outcome, particularly when combined with paired analysis of blood and bone marrow using a rhabdomyosarcoma-specific RNA panel.

PURPOSE Rhabdomyosarcomas (RMS) are rare neoplasms affecting children and young adults. Efforts to improve patient survival have been undermined by a lack of suitable disease markers. Plasma circulating tumor DNA (ctDNA) has shown promise as a potential minimally invasive biomarker and monitoring tool in other cancers; however, it remains underexplored in RMS. We aimed to determine the feasibility of identifying and quantifying ctDNA in plasma as a marker of disease burden and/or treatment response using blood samples from RMS mouse models and patients. METHODS We established mouse models of RMS and applied quantitative polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) to detect ctDNA within the mouse plasma. Potential driver mutations, copy-number alterations, and DNA breakpoints associated with PAX3/ 7-FOXO1 gene fusions were identified in the RMS samples collected at diagnosis. Patient-matched plasma samples collected from 28 patients with RMS before, during, and after treatment were analyzed for the presence of ctDNA via ddPCR, panel sequencing, and/or whole-exome sequencing. RESULTS Human tumor-derived DNA was detectable in plasma samples from mouse models of RMS and correlated with tumor burden. In patients, ctDNA was detected in 14/18 pretreatment plasma samples with ddPCR and 7/7 cases assessed by sequencing. Levels of ctDNA at diagnosis were significantly higher in patients with unfavorable tumor sites, positive nodal status, and metastasis. In patients with serial plasma samples (n = 18), fluctuations in ctDNA levels corresponded to treatment response. CONCLUSION Comprehensive ctDNA analysis combining high sensitivity and throughput can identify key molecular drivers in RMS models and patients, suggesting potential as a minimally invasive biomarker. Preclinical assessment of treatments using mouse models and further patient testing through prospective clinical trials are now warranted.

In the clinical management of paediatric solid tumours, histological examination of tumour tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumour is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumour samples. This methylation pattern can be detected in the cell-free DNA. Here, we provide proof-of-concept of histopathologic classification of paediatric tumours using cell-free reduced representation bisulphite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumour type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in paediatric oncology in a cost-effective and minimally invasive manner.

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.

Purpose The analysis of circulating tumor DNA (ctDNA), a fraction of total cell-free DNA (cfDNA), might be of special interest in retinoblastoma patients. Because the accessibility to tumor tissue is very limited in these patients, either for histopathological diagnosis of suspicious intraocular masses (biopsies are proscribed) or for somatic RB1 studies and genetic counseling (due to current successful conservative approaches), we aim to validate the detection of ctDNA in plasma of non-hereditary retinoblastoma patients by molecular analysis of RB1 gene. Experimental design In a cohort of 19 intraocular unilateral non-hereditary retinoblastoma patients for whom a plasma sample was available at diagnosis, we performed high-deep next-generation sequencing (NGS) of RB1 in cfDNA. Two different bioinformatics/statistics approaches were applied depending on whether the somatic RB1 status was available or not. Results Median plasma sample volume was 600 μL [100–1000]; median cfDNA plasma concentration was 119 [38–1980] and 27 [11–653] ng/mL at diagnosis and after complete remission, respectively. In the subgroup of patients with known somatic RB1 alterations (n = 11), seven of nine somatic mutations were detected (median allele fraction: 6.7%). In patients without identified somatic RB1 alterations (n = 8), six candidate variants were identified for seven patients. Conclusions Despite small tumor size, blood-ocular barrier, poor ctDNA blood release and limited plasma sample volumes, we confirm that it is possible to detect ctDNA with high-deep NGS in plasma from patients with intraocular non-hereditary retinoblastoma. This may aid in diagnosis of suspicious cases, family genetic counseling or follow-up of residual intraocular disease.

Sequencing of cell-free DNA in the blood of cancer patients (liquid biopsy) provides attractive opportunities for early diagnosis, assessment of treatment response, and minimally invasive disease monitoring. To unlock liquid biopsy analysis for pediatric tumors with few genetic aberrations, we introduce an integrated genetic/epigenetic analysis method and demonstrate its utility on 241 deep whole-genome sequencing profiles of 95 patients with Ewing sarcoma and 31 patients with other pediatric sarcomas. Our method achieves sensitive detection and classification of circulating tumor DNA in peripheral blood independent of any genetic alterations. Moreover, we benchmark different metrics for cell-free DNA fragmentation analysis, and we introduce the LIQUORICE algorithm for detecting circulating tumor DNA based on cancer-specific chromatin signatures. Finally, we combine several fragmentation-based metrics into an integrated machine learning classifier for liquid biopsy analysis that exploits widespread epigenetic deregulation and is tailored to cancers with low mutation rates. Clinical associations highlight the potential value of cfDNA fragmentation patterns as prognostic biomarkers in Ewing sarcoma. In summary, our study provides a comprehensive analysis of circulating tumor DNA beyond recurrent genetic aberrations, and it renders the benefits of liquid biopsy more readily accessible for childhood cancers., Liquid biopsies enable minimally invasive applications for diagnosis and treatment monitoring. Here the authors analyse fragmentation patterns of circulating tumour DNA on multiple levels and develop a bioinformatic tool, LIQUORICE, to accurately detect and classify paediatric cancers with low mutational burden.

Circulating tumor DNA (ctDNA) is a powerful tool for the molecular characterization of cancer. The most frequent pediatric kidney tumors (KT) are Wilms' tumors (WT), but other diagnoses may occur. According to the SIOP strategy, in most countries pediatric KT have a presumptive diagnosis of WT if they are clinically and radiologically compatible. The histologic confirmation is established after post-chemotherapy nephrectomy. Thus, there is a risk for a small fraction of patients to receive neoadjuvant chemotherapy that is not adapted to the disease. The aim of this work is to perform molecular diagnosis of pediatric KT by tumor genetic characterization based on the analysis of ctDNA. We analyzed ctDNA extracted from plasma samples of 18 pediatric patients with KT by whole-exome sequencing and compared the results to their matched tumor and germline DNA. Copy number alterations (CNAs) and single nucleotide variations (SNVs) were analyzed. We were able to detect tumor cell specific genetic alterations-CNAs, SNVs or both-in ctDNA in all patients except in one (for whom the plasma sample was obtained long after nephrectomy). These results open the door to new applications for the study of ctDNA with regards to the molecular diagnosis of KT, with a possibility of its usefulness for adapting the treatment early after diagnosis, but also for disease monitoring and follow up.

In the clinical management of pediatric solid tumors, histological examination of tumor tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumor is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumor samples. This methylation pattern can be detected in the cell-free DNA. Here, we provide proof-of-concept of histopathologic classification of pediatric tumors using cell-free reduced representation bisulfite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumor type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in pediatric oncology in a cost-effective and minimally invasive manner.Translational relevanceObtaining a correct diagnosis in pediatric oncology can be challenging in some tumor types, especially in renal tumors or central nervous system tumors. Furthermore, the diagnostic odyssey can result in anxiety and discomfort for these children. By applying a novel technique, reduced representation bisulfite sequencing on cell-free DNA (cf-RRBS), we show the feasibility of obtaining the histopathological diagnosis with a minimally invasive test on either plasma or cerebrospinal fluid. Furthermore, we were able to derive the copy number profile or tumor subtype from the same assay. Given that primary tumor material might be difficult to obtain, in particular in critically ill children or depending on the tumor location, and might be limited in terms of quantity or quality, our assay could become complementary to the classical tissue biopsy in difficult cases.

BACKGROUND: Liquid biopsies are revolutionary tools to detect tumour-specific genetic alterations in body fluids. Here, we assess whether the circulating tumor DNA (ctDNA) in Cerebral Spinal Fluid (CSF) could be used for tumor genetic profiling of Medulloblastomas (MB). METHODS: We extracted the ctDNA of 4-5 CSF droplets after lumbar puncture from patients with Medulloblastoma (12), or ETMR (1). ctDNA (quantity mean 30ng; range 8-177ng) and matched genomic DNA from primary tumors were sequenced by Whole Exome Sequencing (WES) (Illumina 100PE) using Nimblegen Medexome Capture. SNVs/mutations were called using GATK-UnifiedGenotyper, GATK-HaplotypeCaller and Samtools, and filtered on constitutional DNA from public databases. Copy Number profiles were generated with CNVkits. RESULTS: 10/13 cfDNA WES yielded satisfactory depth (>10x). A mean of 466 (range 93-945) SNVs were detected in the primary tumor and 474 (range 18-922) in the CSF. A mean of 416 (range 18-872) commons SNVs were observed between the cfDNA and the primary tumor, comprising classical Medulloblastoma genes such as SMO or MLL2. Interestingly, several SNVs were observed either in the tumor only (mean 50; range 3-115) or in CSF only (mean 58; range 0-148) suggesting a clonal heterogeneity. For 5 cases, Copy Number profiles were also available, allowing the detection of MYCN amplification in 1 MB or 19q13 miRNA cluster amplification in the ETMR.

Circulating tumor DNA (ctDNA) is a powerful tool for the molecular characterization of cancer. The most frequent pediatric kidney tumors (KT) are Wilms' tumors (WT), but other diagnoses may occur. According to the SIOP strategy, in most countries pediatric KT have a presumptive diagnosis of WT if they are clinically and radiologically compatible. The histologic confirmation is established after post-chemotherapy nephrectomy. Thus, there is a risk for a small fraction of patients to receive neoadjuvant chemotherapy that is not adapted to the disease. The aim of this work is to perform molecular diagnosis of pediatric KT by tumor genetic characterization based on the analysis of ctDNA. We analyzed ctDNA extracted from plasma samples of 18 pediatric patients with KT by whole-exome sequencing and compared the results to their matched tumor and germline DNA. Copy number alterations (CNAs) and single nucleotide variations (SNVs) were analyzed. We were able to detect tumor cell specific genetic alterations-CNAs, SNVs or both-in ctDNA in all patients except in one (for whom the plasma sample was obtained long after nephrectomy). These results open the door to new applications for the study of ctDNA with regards to the molecular diagnosis of KT, with a possibility of its usefulness for adapting the treatment early after diagnosis, but also for disease monitoring and follow up.

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.

MAPPYACTS (NCT02613962) is an international prospective precision medicine study recruiting children and adolescents with recurrent/refractory malignancies. Design: Patients < 18 years at diagnosis underwent on-purpose tumor biopsy/surgery of recurrent/refractory malignancies for molecular characterization by WES and whole RNA sequencing. Results were reviewed in a dedicated weekly molecular tumor board (MTB), followed by discussion with the treating physician in a clinical MTB (CMTB). Whenever possible, patients were enrolled subsequently in clinical trials based on CMTB recommendations. In parallel, plasma was collected for cell free DNA (cfDNA) analysis by WES (100x) and capture sequencing (> 2000x) targeting alterations identified by tumor WES. Results: From February 2016 to October 2018, 500 patients have been included in 17 centers in France, Italy and Ireland. Median age at inclusion was 13 years (range, 1-33). 38% had sarcomas, 28% brain tumors, 22% other solid tumors, 12% hematological malignancies with a median of one prior relapse/progression. Eleven patients were excluded (10 screening failures, 1 consent withdrawal). 489 patients underwent 525 interventions: biopsy (57%), surgery (34%), blood/bone marrow (9%), in 55% done to metastatic sites. Molecular profiling was performed in 433 patients (88%) and was contributive for 390 patients. Known pathogenic germline variants were identified in 33 patients (8%). For 271/390 patients (70%), there were at least one genetic alteration that could represent a potential therapeutic target. A total of 548 ”actionable” alterations were identified (311 SNV, 208 focal CNA, 29 gene fusions). Overall 448 treatment recommendations were given, 1 to 5 per patient (median 2) and 8% had alterations considered as “ready for use” for treatment at relapse. Recommendations involved mainly cell cycle checkpoint (n=97), PI3K/AKT/mTOR (n=83) and RAF/MAPK (n=68), cell cycle (n=67), growth factor receptor signaling (n=62) and DNA damage repair (n=33). Follow-up information is available for 197 patients discussed in the CMTB before January 2018. Fifty-six patients (28%) were treated with a matched targeted agent, 48 of them in a clinical trial, mostly in our proof-of-concept AcSé-ESMART trial. CfDNA WES of the first 40 samples revealed at least 1 tumor specific SNV in all cases, with a mean of 38% of SNVs seen in the tumor also displayed in cfDNA (range, 2-89%) and correlation between cfDNA quantity and percentage of tumor SNVs in the cfDNA (p=0.02). Conclusion: MAPPYACTS underlines the role of precision medicine approaches to identify actionable molecular alterations in recurrent pediatric cancer enabling access to innovative treatment through early clinical trials. The impact of surrogate approach based on cfDNA testing to identify targetable genetic alterations is currently under evaluation. Citation Format: Pablo Berlanga, Gudrun Schleiermacher, Ludovic Lacroix, Gaelle Pierron, Tiphaine Adam de Beaumais, Mathieu Chicard, Yasmine Iddir, Jean Yves Scoazec, Paul Freneaux, Mohamed Amine Bayar, Sophie Cotteret, Samuel Abbou, Michela Casanova, Cormac Owens, Stefan Michiels, Olivier Delattre, Gilles Vassal, Birgit Geoerger. Pediatric precision medicine program in recurrent tumors: Results of the first 500 patients included in the European MAPPYACTS molecular profiling trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT081.

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.

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

Objet Dans le noyau interphasique, la chromatine est organisee en territoires chromosomiques (TC) [1] . Le lien entre cette organisation et la regulation de l’expression du genome reste meconnu. Afin de preciser cette organisation, nous avons etudie un modele de desorganisation : les aneuploidies en supposant que la presence d’un chromosome surnumeraire pouvait entrainer une perturbation de la distribution des TC, elle-meme conduisant a une modification de l’activite transcriptionnelle. Methodes Etude de la position des TC 18 et 21 par FISH 3D dans les noyaux interphasiques de fœtus aneuploides et temoins. Comparaison des volumes des noyaux et des TC. Parallelement, une analyse transcriptomique comparative pangenomique des cellules a ete realisee afin d’objectiver des deregulations d’expression. Resultats Nous n’avons pas observe de difference de positions des TC 18 et 21 entre les fœtus atteints et les temoins. Si les volumes nucleaires n’etaient pas modifies, ceux des TC l’etaient. Nous avons mis en evidence des modifications d’expression d’une partie des genes portes par les chromosomes d’interet ; mais egalement de genes sur les autres chromosomes. Discussion En cas d’aneuploidie, l’organisation des TC aneuploides dans le noyau semble globalement respectee. Il existerait cependant une diminution du volume de ces TC sans modification du volume des noyaux, interrogeant au detriment de quel compartiment, se fait cette compaction. L’analyse transcriptomique renforce l’idee d’une combinaison d’un effet de dosage genique et d’une rupture de l’homeostasie : aux genes deregules portes par les chromosomes trisomiques, s’ajoutent des modifications d’expression du reste du genome. Ces deregulations concernent de multiples voies impliquees dans le developpement mais aussi des genes au potentiel role dans l’apparition de traits phenotypiques. Conclusion Il semblerait exister un lien entre le desequilibre genique des chromosomes aneuploides et les deregulations de l’ensemble du genome. La participation de la reorganisation des TC reste a affiner.

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