Genomic Copy Number Profiling Using Circulating Free Tumor DNA Highlights Heterogeneity in Neuroblastoma

Abstract

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

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

“…In this study, the ctDNA fraction of the overall cfDNA could be calculated by Sequenza (32), confirming a high proportion of ctDNA in the cfDNA fraction (mean, 60%; range, 3%-99%; Supplementary Table S1), whereas for healthy adults, in general, cfDNA quantities of approximately 10 ng/mL of plasma are observed; in high-risk neuroblastoma patients at diagnosis, high amounts of cfDNA (mean, 1,034 ng/mL of plasma; range, 26-13,533) were documented, further confirming the observation of an important shredding of ctDNA into the bloodstream by high-risk neuroblastoma at diagnosis (10,16). Further studies on larger series will enable to stablish whether the amount of ctDNA is linked to the overall disease burden, the degree of necrosis, or both.…”

“…Alterations seen only in cfDNA but not in the primary were 1p deletion and 17q gain in 1 case each. No discrepancies were observed between CNA determined in cfDNA by WES as compared with an analysis using OncoScan for 11 cases reported previously (10).…”

“…Its quality was defined by bioanalyzer agilent 2100 (Agilent) analysis using the High Sensitivity DNA chip with cfDNA quality expressed as the 200-bp fragment fraction. For 11 plasma samples obtained at diagnosis, cfDNA copy number analysis using Oncoscan has been reported previously (10).…”

“…Spatial heterogeneity has been described within a tumor, and between a tumor and its metastasis, for genetic features such as MYCN, or other CNAs (10). Temporal heterogeneity has also been observed with an accumulation of new CNAs, and mutations including ALK and RAS-MAPK mutations at the time of progression (11)(12)(13).…”

“…The overall copy number profile can also be determined in cfDNA using different technologies such as commercially available platforms or low coverage whole-genome sequencing (WGS; refs. 10,17,18).…”