Bone marrows from neuroblastoma patients: an excellent source for tumor genome analyses

Abstract

Neuroblastoma is the most common extra-cranial solid tumor in childhood. Presence of disseminated tumor cells (DTCs) in the bone marrow (BM) at diagnosis and at relapse is a common event in stage M neuroblastomas. Although the clinical heterogeneity of disseminated neuroblastomas is frequently associated with genomic diversity, so far, only little information exists about the genomic status of DTCs. This lack of knowledge is mainly due to the varying amount of BM infiltrating tumor cells, which is usually below 30% even at diagnosis thereby hampering systematic analyses. Thus, a valuable chance to analyze metastatic and relapse clones is, so far, completely unexploited. In this study, we show that the enrichment of tumor cells in fresh or DMSO frozen BM samples with a minimum of 0.05% or 0.1% infiltration rate, respectively, by applying magnetic bead-based technique increased the DTC content to a sufficient level to allow SNP array analyses in 49 out of 69 samples. In addition, we successfully used non-enriched BM samples with ≥30% DTCs including non-stained and immunostained cytospin and BM smear slides for SNP array analyses in 44 cases. We analyzed the genomic profile of DTCs by an ultra-high density SNP array technique with highest performance detecting all segmental chromosomal aberrations, amplified regions, acquired loss of heterozygosity events and minor aberrations affecting single genes or parts thereof.

Keywords: Bone marrow; Disseminated tumor cells; Enrichment; Neuroblastoma; SNP array.

Figure 1

DTC enrichment, DNA extraction and SNP array analysis. After density gradient centrifugation (a), mononuclear fraction containing tumor cells was incubated with FITC‐labeled anti‐GD2 antibodies and subsequently anti‐FITC magnetic beads (b). Selection of GD2+ neuroblastoma cells was performed in a column which was placed in a magnetic field and the negative fraction was collected (c). DTCs were recovered after removing the column from the magnet (d). DNA was extracted from the negative (e) and positive (f) fractions for SNP array analysis (g and h).

Figure 2

Tumor cell content before and after enrichment. The tumor cell content of archival samples before enrichment was classified into four groups: group A: 0.1%–1%, group B: 2%–9%, group C: 10%–30% and group D: 31%–50%. The fresh BM samples were listed in group E. The tumor cell content before and after enrichment in each group is represented by orange and green bars, respectively.

Figure 3

SNP array profiles of BM‐derived DTCs. a. SNP array data from an enriched DMSO frozen BM sample at relapse with 5.5% tumor cell infiltration before and 85% after enrichment and the corresponding tumor free sample (REF). The red box shows a region with copy neutral LOH in chromosome 14q in the DTCs which is not detectable in the reference. In this region the smooth signal is on level 2 representing two copies while the middle track of the allele peaks is missing in this region meaning two identical copies or copy neutral LOH. b. SNP array of an enriched fresh BM sample with 0.05% tumor cell infiltration before and 68% after enrichment shows two deletions at the short arm of chromosome 1, a large deletion at the distal part (tel. – 36.4 Mb) and a small deletion at 50.3–51.6 Mb (arrows represent breakpoints). In these areas the smooth signal does not descend to 1 because of the amount of normal cells. Also the four tracks in the allele peaks are caused by the mixture of normal cells and tumor cells. c. SNP array profile of the DNA extracted from a BM cytospin slide after GD2/CD56 staining with 90% tumor cell infiltration shows two regions with 3 copies in the long arm of chromosome 6. In these regions the copy number state in smooth signal is close to level 3 (due to a mixture of 10% normal cells) and allele peaks show 4 tracks representing gains (i.e. trisomy). Breakpoints are at 74.4 Mb, 155.0 Mb and 157.9 Mb (arrows). d. SNP array profile from a BM smear slide with 50% tumor cell infiltration at diagnosis and the corresponding data from the primary tumor (TU) show a deletion of the distal part of chromosome 8p. The breakpoints at 27.3 Mb are identical in both samples (arrows). The allele peaks of the tumor disclose a loss of heterozygosity (LOH) in the deleted segment and accordingly the copy number state in smooth signal is 1. In the DTC sample the smooth signal is between 1 and 2 and the allele peaks in this region show four tracks due to a mixture of 50% normal cells and 50% tumor cells. AP: allele peaks, SMS: smooth signal.

Figure 4

SNP array of an enriched frozen BM sample of a male patient at diagnosis with 6% tumor cell infiltration before and 40% after enrichment compared to the primary tumor (TU) and tumor free MNCs as reference (REF). The Log2 ratios show a deletion within the ATRX gene on Xq21.1 in the DTCs and in the tumor. The deletion concerns only parts of the ATRX gene and is not present in the reference sample.

Figure 5

Whole genome views (WGVs) of BM‐derived DTCs which were created by the ChAS software. WGVs of DTCs from a. enriched DMSO frozen BM sample with 0.69% DTC infiltration before and 60% after enrichment, b. enriched fresh BM sample with 0.05% DTC infiltration before and 68% after enrichment, c. immunostained cytospin slide with 95% DTC infiltration and d. BM smear slide with 50% DTC infiltration, show different numerical and segmental chromosomal aberrations and amplifications (short arm of chromosome 2 in a and c) in different chromosomes. The left y‐axis in the upper box of each sample shows the weighted log 2 ratio and the right y‐axis the copy number (smooth signal, dark blue line). The lower boxes in a, b, c and d represent the allele peaks. The numbers in the x‐axis indicate the chromosome numbers and X and Y.