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Supplemental figure 1: Comparison of the copy number profiles of 4 cases generated with sWGS on cfDNA (at 100 kb binsize), sWGS on tumor DNA (at 100 kb binsize), sWGS on tumor DNA (at 15 kb binsize) and arrayCGH on tumor DNA (at 180k resolution). Supplemental figure 2 : Heatmap describing the concordance between arrayCGH data on tumor DNA vs sWGS of cfDNA. Numbers on the right reflect the case numbers, followed by a 1 for aCGH data on the tumor, and 2 for sWGS data on the cfDNA; the numbers on the top and bottom indicate the chromosome numbers. Red=copy number gain, blue=copy number loss. Supplemental figure 3: Validation of LIN28B amplification by Q-PCR.Y-axis represents the haploid copy number, X-axis represents the samples. NGP is a NB cell line that was used as a positive control, since this cell line showed gain of LIN28B in arrayCGH, gDNA:genomic DNA, NTC: no template control

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Purpose: Neuroblastoma (NB) is a heterogeneous disease characterized by distinct clinical features and by the presence of typical copy-number alterations (CNAs). Given the strong association of these CNA profiles with prognosis, analysis of the CNA profile at diagnosis is mandatory. Therefore, we tested whether the analysis of circulating cell-free DNA (cfDNA) present in plasma samples of patients with NB could offer a valuable alternative to primary tumor DNA for CNA profiling.Experimental Design: In 37 patients with NB, cfDNA analysis using shallow whole genome sequencing (sWGS) was compared with arrayCGH analysis of primary tumor tissue.Results: Comparison of CNA profiles on cfDNA showed highly concordant patterns, particularly in high-stage patients. Numerical chromosome imbalances as well as large and focal structural aberrations including MYCN and LIN28B amplification and ATRX deletion could be readily detected with sWGS using a low input of cfDNA.Conclusions: In conclusion, sWGS analysis on cfDNA offers a cost-effective, noninvasive, rapid, robust and sensitive alternative for tumor DNA copy-number profiling in most patients with NB. Clin Cancer Res; 23(20); 6305–14. ©2017 AACR.

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.

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.

Objective The enrichment of circulating tumor cells (CTCs) from blood provides a minimally invasive method for biomarker discovery in cancer. Longitudinal interrogation allows monitoring or prediction of therapy response, detection of minimal residual disease or progression, and determination of prognosis. Despite inherent phenotypic heterogeneity and differences in cell surface marker expression, most CTC isolation technologies typically use positive selection. This necessitates the optimization of marker-independent CTC methods, enabling the capture of heterogenous CTCs. The aim of this report is to compare a size-dependent and a marker-dependent CTC-isolation method, using spiked esophageal cells in healthy donor blood and blood from patients diagnosed with esophageal adenocarcinoma. Methods Using esophageal cancer cell lines (OE19 and OE33) spiked into blood of a healthy donor, we investigated tumor cell isolation by Parsortix post cell fixation, immunostaining and transfer to a glass slide, and benchmarked its performance against the CellSearch system. Additionally, we performed DEPArray cell sorting to infer the feasibility to select and isolate cells of interest, aiming towards downstream single-cell molecular characterization in future studies. Finally, we measured CTC prevalence by Parsortix in venous blood samples from patients with various esophageal adenocarcinoma tumor stages. Results OE19 and OE33 cells were spiked in healthy donor blood and subsequently processed using CellSearch (n = 16) or Parsortix (n = 16). Upon tumor cell enrichment and enumeration, the recovery rate ranged from 76.3 ± 23.2% to 21.3 ± 9.2% for CellSearch and Parsortix, respectively. Parsortix-enriched and stained cell fractions were successfully transferred to the DEPArray instrument with preservation of cell morphology, allowing isolation of cells of interest. Finally, despite low CTC prevalence and abundance, Parsortix detected traditional CTCs (i.e. cytokeratin+/CD45-) in 8/29 (27.6%) of patients with esophageal adenocarcinoma, of whom 50% had early stage (I-II) disease. Conclusions We refined an epitope-independent isolation workflow to study CTCs in patients with esophageal adenocarcinoma. CTC recovery using Parsortix was substantially lower compared to CellSearch when focusing on the traditional CTC phenotype with CD45-negative and cytokeratin-positive staining characteristics. Future research could determine if this method allows downstream molecular interrogation of CTCs to infer new prognostic and predictive biomarkers on a single-cell level.

Purpose: Neuroblastoma (NB) is a heterogeneous disease characterized by distinct clinical features and by the presence of typical copy-number alterations (CNAs). Given the strong association of these CNA profiles with prognosis, analysis of the CNA profile at diagnosis is mandatory. Therefore, we tested whether the analysis of circulating cell-free DNA (cfDNA) present in plasma samples of patients with NB could offer a valuable alternative to primary tumor DNA for CNA profiling. Experimental Design: In 37 patients with NB, cfDNA analysis using shallow whole genome sequencing (sWGS) was compared with arrayCGH analysis of primary tumor tissue. Results: Comparison of CNA profiles on cfDNA showed highly concordant patterns, particularly in high-stage patients. Numerical chromosome imbalances as well as large and focal structural aberrations including MYCN and LIN28B amplification and ATRX deletion could be readily detected with sWGS using a low input of cfDNA. Conclusions: In conclusion, sWGS analysis on cfDNA offers a cost-effective, noninvasive, rapid, robust and sensitive alternative for tumor DNA copy-number profiling in most patients with NB. Clin Cancer Res; 23(20); 6305–14. ©2017 AACR.

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.

In the clinical management of pediatric solid tumors, histologic examination of tumor tissue obtained by an invasive biopsy remains the gold standard to establish a conclusive pathologic diagnosis. However, correct diagnosis in pediatric oncology can be challenging in some tumor types, especially in renal tumors or central nervous system tumors. The DNA methylation pattern of a tumor is known to correlate with the histopathologic diagnosis across cancer types and is showing promise in the diagnostic workup of tumor samples. It has previously been shown that this methylation pattern can be detected in the cell-free circulating DNA isolated from blood plasma of cancer patients. 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. Using computational deconvolution of the DNA methylation profiles, we were able to obtain the correct tumor type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free circulating DNA. We could demonstrate that the majority of misclassifications were associated with bad sample quality, evidenced by an increase of the test accuracy to 94.3% when only including good-quality samples. Misclassifications were not associated with the extent of the disease. Furthermore, we were able to derive the copy number profile or tumor subtype from the same assay. Our findings show the potential of the approach to tackle some of the remaining diagnostic challenges in pediatric oncology in a cost-effective and minimally invasive manner. Given the fact 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 these difficult cases. A preprint of the study manuscript is already available at bioRxiv: https://doi.org/10.1101/795047. Citation Format: Ruben Van Paemel, Andries De Koker, Charlotte Vandeputte, Lieke Van Zogchel, Tim Lammens, Geneviève Laureys, Jo Vandesompele, Gudrun Schleiermacher, Matthieu Chicard, Nadine Van Roy, Ales Vicha, Godelieve A. M. Tytgat, Nico Callewaert, Bram De Wilde, Katleen De Preter. Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B48.