Impact of Disseminated Neuroblastoma Cells on the Identification of the Relapse-Seeding Clone

Abstract

Purpose: Tumor relapse is the most frequent cause of death in stage 4 neuroblastomas. Since genomic information on the relapse precursor cells could guide targeted therapy, our aim was to find the most appropriate tissue for identifying relapse-seeding clones.Experimental design: We analyzed 10 geographically and temporally separated samples of a single patient by SNP array and validated the data in 154 stage 4 patients.Results: In the case study, aberrations unique to certain tissues and time points were evident besides concordant aberrations shared by all samples. Diagnostic bone marrow-derived disseminated tumor cells (DTCs) as well as the metastatic tumor and DTCs at relapse displayed a 1q deletion, not detected in any of the seven primary tumor samples. In the validation cohort, the frequency of 1q deletion was 17.8%, 10%, and 27.5% in the diagnostic DTCs, diagnostic tumors, and DTCs at relapse, respectively. This aberration was significantly associated with 19q and ATRX deletions. We observed a significant increased likelihood of an adverse event in the presence of 19q deletion in the diagnostic DTCs.Conclusions: Different frequencies of 1q and 19q deletions in the primary tumors as compared with DTCs, their relatively high frequency at relapse, and their effect on event-free survival (19q deletion) indicate the relevance of analyzing diagnostic DTCs. Our data support the hypothesis of a branched clonal evolution and a parallel progression of primary and metastatic tumor cells. Therefore, searching for biomarkers to identify the relapse-seeding clone should involve diagnostic DTCs alongside the tumor tissue. Clin Cancer Res; 23(15); 4224-32. ©2017 AACR.

Figure 1

Graphical representation of clonal expansion in a stage 4 neuroblastoma patient. Each quadrant represents the clonal architecture of a tissue/time point. Each color represents a group of chromosomal aberrations and the size of each colored area represents the proportion of cells with these aberrations within the analyzed samples (the detail of subclonal frequencies of different aberrations is provided in the supplementary table S3). The primary tumor displayed a branched clonal evolution leading to extensive intra-tumor heterogeneity. A 1q terminal deletion (W) which was present in the diagnostic DTCs and also in the both DTCs and metastatic tumor at relapse was not detected in any of the primary tumor samples. Certain chromosomal aberrations were acquired independently in geographically different tissues at diagnosis and at relapse, indicating parallel tumor progression. A: 18 breakpoints corresponding to 13 aberrations including ATRX deletion (exons: 2-8) present homogenously in all samples/time points; H: Chromosome Y loss and J: 1p33(49,772-49,927)×3 (affecting AGBL4 gene) are present homogenously in the DTCs at diagnosis and relapse samples and heterogeneously in the tumor at diagnosis; W: 1q42.2qter(231,262-249,250)×1 is present heterogeneously in the DTCs at diagnosis and homogenously in the relapse samples; X: 21 breakpoints corresponding to 13 aberrations (including 19q and PTPRD deletions) are homogenously present in the relapse samples; Y: duplication of chromosome 17 is heterogeneously present in the relapse samples; and Z: 5pterp15.2(1-12,709)×1 is heterogeneously present only in the metastatic tumor at relapse. Breakpoints are given in kb. The areas with blue, green and pink spectrums represent different groups of aberrations heterogeneously present in the tumor and DTCs at diagnosis (Supplementary Table S3).

Figure 2

Venn diagram representing the number of breakpoints detected either homogenously or heterogeneously in different samples of one patient. Besides aberrations unique to certain tissues and time points, 20 breakpoints were shared between all samples consist of 18 breakpoints homogeneously present in all samples, representing the genomic profile of the common ancestor clone, and two breakpoints present homogenously in the diagnostic DTCs and relapse samples and heterogeneously in the primary tumor.

Figure 3

A. Frequencies of 1q and 19q terminal deletions and ATRX deletion in DTCs at diagnosis, diagnostic tumors, and DTCs at relapse. B. Frequencies of 19q terminal and ATRX deletions in the DTCs with and without 1q terminal deletions at diagnosis. Samples were restricted to patients ≥18 months without MNA.

Figure 4

Deleted regions of 1q in different patients. For patients with more than one sample with this deletion, only one sample has been represented in the figure since deleted segments are the same for different samples. The deleted region in patient 64 represents the SRO.

Figure 5

Event-free and overall survival curves for patients with and without 1q, 19q, or ATRX deletions in the DTCs at diagnosis. Samples were restricted to patients ≥18 months without MNA.