Circos plot of sample NB19 demonstrating chromothripsis.

<p>The tracks are from outside inwards: chromosome numbers, chromosomal positions in Mb, copy number and allele patterns, respectively. Copy number gains and losses are colored blue and red, respectively. The enlarged segment marked in pink shows a highly rearranged chromosome 12 with more than 200 breakpoints indicating chromothripsis. Additionally, a gain of the distal part of the q-arm of chromosome 12 is present. SCAs were identified on chromosomes 1, 2, (both SCAs only in a subpopulation of cells) 7, 8, 9, 11 (three SCAs) and 17.</p

Influence of slide age on array data quality (MAPD values).

<p>The linear trend line is shown in blue. MAPD values below 0.25 (red line) are considered to represent a good quality.</p

Tumor Touch Imprints as Source for Whole Genome Analysis of Neuroblastoma Tumors

<div><p>Introduction</p><p>Tumor touch imprints (TTIs) are routinely used for the molecular diagnosis of neuroblastomas by interphase fluorescence in-situ hybridization (I-FISH). However, in order to facilitate a comprehensive, up-to-date molecular diagnosis of neuroblastomas and to identify new markers to refine risk and therapy stratification methods, whole genome approaches are needed. We examined the applicability of an ultra-high density SNP array platform that identifies copy number changes of varying sizes down to a few exons for the detection of genomic changes in tumor DNA extracted from TTIs.</p><p>Material and Methods</p><p>DNAs were extracted from TTIs of 46 neuroblastoma and 4 other pediatric tumors. The DNAs were analyzed on the Cytoscan HD SNP array platform to evaluate numerical and structural genomic aberrations. The quality of the data obtained from TTIs was compared to that from randomly chosen fresh or fresh frozen solid tumors (n = 212) and I-FISH validation was performed.</p><p>Results</p><p>SNP array profiles were obtained from 48 (out of 50) TTI DNAs of which 47 showed genomic aberrations. The high marker density allowed for single gene analysis, e.g. loss of nine exons in the <i>ATRX</i> gene and the visualization of chromothripsis. Data quality was comparable to fresh or fresh frozen tumor SNP profiles. SNP array results were confirmed by I-FISH.</p><p>Conclusion</p><p>TTIs are an excellent source for SNP array processing with the advantage of simple handling, distribution and storage of tumor tissue on glass slides. The minimal amount of tumor tissue needed to analyze whole genomes makes TTIs an economic surrogate source in the molecular diagnostic work up of tumor samples.</p></div

Circos Plot of copy number and SNP probe data for sample NB05.

<p>The tracks are from outside inwards: chromosome numbers, chromosomal positions in Mb, copy number and allele patterns. Copy number gains and losses are shown in blue and red, respectively. SCAs resulting in deletion of the concerned region where seen at 1p, 2q, 11q, 12p/q and 15q. Gains were found at: 14q, 17q and 20q. Chromosomes 12 and 15 show partial UPDs in addition to the before mentioned deletions. Chromosome 16 discloses a monosomy with a typical two band allele pattern (allele configurations A0 and B0). Most autosomes (3, 4, 5, 6, 7, 8, 9, 10, 13, 18, 19, 21 and 22) show a continuous disomic copy number profile with a normal three band allele pattern (allele configurations AA, AB and BB). A <i>MYCN</i> amplification is present but hardly visible at the short arm of chromosome 2 (small blue dot). The sex chromosomes show monosomies (except for the pseudoautosomal regions on the X chromosome) indicating that this profile belongs to a male patient.</p

Partial <i>ATRX</i> nullisomy in NB samples NB06 and NB36.

<p>Exons 2–10 of the <i>ATRX</i> gene (transcript variant 1; NCBI RefSeq accession number NM_000489) are deleted in both NB samples (NB06 upper panel, NB36 middle panel). The proximal breakpoints are located at the same chromosomal position in both samples (76,925 kb), however, the lengths of the deletion differed in both samples (~ 80 kb for NB06 and ~ 100 kb for NB36). The lower track shows the spatial assembly of the <i>ATRX</i> gene, the exons are indicated by vertical lines or blocks.</p

Number and type of chromosomal aberrations found in the cohort of 44 NB TTIs.

<p>Each bar represents one tumor. The number of NB typical segmental chromosomal aberrations (SCAs) is shown in red. SCAs with so far unknown prognostic relevance are shown in orange and numerical chromosomal aberrations (NCAs) are shown in blue. Whole chromosome copy neutral losses of heterozygosity are shown in purple. Presence of <i>MYCN</i> gene amplifications is shown in green, intragenic <i>ATRX</i> gene deletions in pink, hyper-rearrangements in dark blue and chromothripsis in yellow.</p

Effects of magnetic bead-based enrichment of NB cells on their gene expression.

<p>qPCR arrays were used to analyze the effects of magnetic bead-based enrichment on the expression of 71 genes in NB cells. In (a) the altered gene expression is shown for cells that have been enriched only once after density gradient separation, whereas in (b) the effect of two following magnetic bead-based enrichment steps is shown. Red dots represent genes that are significantly changed (p<0.05, |log₂FC|>1) at given conditions compared to the baseline (LAN-1 cells before spiking into PB). The expression of genes with |log₂FC|>1 but p>0.05 are not considered as significant, as their expression was not coherently changed in the different biological replicates. The log₂ fold change is indicated on the y-axis and the mean Ct values in the x-axis.</p

Effects of magnetic bead-based enrichment of NB cells on their gene expression.

<p>qPCR arrays were used to analyze the effects of magnetic bead-based enrichment on the expression of 71 genes in NB cells. In (a) the altered gene expression is shown for cells that have been enriched only once after density gradient separation, whereas in (b) the effect of two following magnetic bead-based enrichment steps is shown. Red dots represent genes that are significantly changed (p<0.05, |log₂FC|>1) at given conditions compared to the baseline (LAN-1 cells before spiking into PB). The expression of genes with |log₂FC|>1 but p>0.05 are not considered as significant, as their expression was not coherently changed in the different biological replicates. The log₂ fold change is indicated on the y-axis and the mean Ct values in the x-axis.</p

Experimental design.

<p><b>(a)</b> We spiked LAN-1 NB cells into fresh PB and kept the samples for 0, 24, 48 and 72h at room temperature and, for the same time periods, at 4°C prior to density gradient separation. The LAN-1 cells were enriched from the MNC fraction with magnetic beads to a 99% purity of the tumor cell fractions prior to homogenization in TRIzol. RNA was isolated from all seven samples simultaneously and used for the qPCR array. (<b>b)</b> LAN-1 cells were spiked into PB and tumor-free BM, and density gradient separation was immediately performed. The MNCs were frozen in 20% DMSO for seven days at -80°C. After thawing, the LAN-1 cells were either directly enriched by magnetic bead-based separation, or an additional density gradient separation (*) was performed prior to magnetic bead-based separation. The samples were homogenized in TRIzol and the isolated RNA was used for qPCR (in case of PB) and microarrays (in case of BM). <b>(c)</b> LAN-1 cells were spiked into PB and density gradient separation of MNCs was performed without delay, following two enrichment steps in a row. The >99% LAN-1 cell fractions were homogenized in TRIzol and RNA was isolated from all samples simultaneously. qPCR arrays were performed in order to analyze the effect of enrichment on selected genes.</p

Unsupervised clustering of NB samples after freezing, storage and thawing procedure.

<p>Microarray analysis of three biological replicates (A-C) and the three different pretreatment conditions: fresh (no freezing/thawing), frozen 1 (thawing > magnetic bead-based separation of LAN-1 cells) and frozen 2 (thawing > density gradient separation > magnetic bead-based separation of LAN-1 cells). In the unsupervised clustering of the expression of the analyzed genes, the fresh and the two differently frozen samples (1, 2) did not cluster. The correlation coefficient (R) is illustrated by the color key: white (0) = no correlation and red (1) = high correlation.</p