The used technique enabled high resolution detection and mapping of all numerical and structural genomic changes in the tumor material. We could also pinpoint several previously undetected rearrangements and map them in detail. These include five new cases of homozygous deletion, which is only infrequently reported in primary neuroblastoma tumors (se discussion below). The technique proved to be fast, robust, reproducible and reliable and it is likely to be a valuable tool in future studies of neuroblastoma tumors, both in research and in the clinical setting.
Regions of deletions
Thirty percent of the tumors had 1p deletion and those were significantly more often MYCN amplified. Tumors with MYCN amplification had generally larger 1p deletions than tumors without MYCN amplification and the five smallest deletions including the p-terminal were found in tumors without MYCN amplification, which confirms an earlier study . So, when identifying the SRO in 1p deletions in NB, this will be delineated by the tumors without MYCN amplification showing the most distal breakpoints. It is possible that different sets of 1p-deleted genes are important for the biological behavior of the MYCN amplified and the non-MYCN amplified cases, respectively. The SRO of deletions in tumors without MYCN amplification was located from 0 to 10.4 Mb. For the non-amplified tumors, an interstitial SRO located between 17.2 Mb to 37.0 Mb (covering 19.8 Mb) was defined. It has previously been reported that tumors with MYCN amplification have 1p deletions extending proximal to 1p36 whereas non-amplified tumors more often have small terminal deletions of 1p36 . Many groups have previously tried to narrow down the shortest region of overlap of deletions on chromosome 1p [20–30]. In the largest study , the smallest region of consistent deletion (SRD) in all but one NB tumor was located between 5.3 Mb and 6.1 Mb which resides inside our SRO for tumors without MYCN amplification.
There was a significant inverse correlation between 11q loss and the amplification of MYCN. Only 2 of the 20 tumors with a loss of chromosome 11 had MYCN amplification. The smallest region on chromosome 11q that was lost was detected in the two tumors that also had MYCN amplification (Figure 2B), the smallest being 24.4 Mb, from 110.1 Mb to 134.5/qter. The SRO in the tumors without MYCN amplification was defined as being 50 Mb, from 84.5 Mb to 134.5/qter. The fact that 11q deletions occurs predominantly in tumors without MYCN amplification is in agreement with previous studies [15, 32].
Homozygous deletions are rare events in primary NB tumors. Only a few have been reported in single cases; the deletion or homozygous gene inactivation of NF1 [26, 33], the deletion of CDKN2A , PTEN and DMBDT1 . In addition, homozygous deletions in chromosome regions 1p36 , 3p22.3  and 2q33 (CASP8)  have been detected in NB cell lines. In our material, we detected homozygous deletions in the CDKN2A gene in chromosome 9p21 in four tumors. This region is frequently deleted in a wide range of malignancies . CDKN2A encodes the transcripts p16INK4a and p14ARF in alternative reading frames. p16INK4a is an inhibitor to the cell cycle activators CDK4 and CDK6, which inactivate the tumor suppressor protein pRB, and p14ARF binds and inactivates MDM2, which is responsible for the degradation of TP53, thereby leading to the stabilization of TP53 (for a review, see Sharpless et al. ). A second region of homozygous deletion was discovered in one NB tumor, located in chromosome region 3p24.1, harboring the gene RBMS3. The protein encoded by this gene is a member of a protein family which binds single-stranded DNA/RNA. We also detected two homozygous deletions in the NB cell line Kelly, one in chromosome 3p, covering the gene LSAMP, and one in the gene PTPRD in chromosome 9p. LSAMP is a neuronal surface glycoprotein that has been identified as a putative tumor suppressor gene in ovarian and renal carcinomas [40, 41], also reported to be diminished in Kelly and SK-N-AS by Stallings et al. . PTPRD is a candidate tumor suppressor gene that encodes a receptor type protein tyrosine phosphatase. This confirms the finding by Stallings et al. who have previously reported that this gene is heterozygously deleted in some NB tumors and cell lines, as well as being homozygously deleted in Kelly . We identified two regions of SRO on chromosome 3p in our material; SRO 1 from 0–5.5 Mb and SRO 2 from 46.9–51.0. Our SRO 2 region overlaps one of the three SROs previously defined by Hoebeeck et al . This region contains among others RASSF1A and ZMYND10; two candidate tumor suppressor genes that are epigenetically silenced in a proportion of NB tumors [44, 45].
Although differences in dosage between the different alleles is very common in NB tumors, given that several tumors are in the triploid range, it is noteworthy that in the present investigation, only three of the 92 analyzed NBs showed a CN-LOH.
Regions of gain and amplification
The most common chromosomal abnormality found in our material was the gain of chromosome 17q, found in 45% of the tumors. The SRO of gains was located from 54.5 Mb to the terminal of the long arm, including the gene PPM1D located at 56.0 Mb. PPM1D has been reported to be the most likely target of the 17q23 gain in NB tumors ; this gene was included in the gained region in all our tumors with 17q gain. Recently, Vandesompele and coworkers proposed that 17q gains may target two segments with one large from 44.3 Mb, in cases with a single region of gain, and one region of superimposed gains located more distally around 60 Mb .
The amplification of chromosome 12 was detected in two NB tumors. The gene MDM2 is located in the amplified region. The overexpression of MDM2 can result in the excessive inactivation of TP53, thereby diminishing its tumor suppressor function. Another gene is YEATS4 (GAS41; glioma amplified sequence), which has high expression in the human brain and is frequently amplified in gliomas . Genes in this region have also previously been found to be amplified in single NB tumor samples or cell lines [15, 49, 50].
TP53 is inactivated by mutations in approximately half of all human tumors, and is believed to be abrogated in most tumors , although TP53 mutations are rare in neuroblastoma tumors [52–55]. However, mechanisms other than TP53 mutations could prevent TP53 activation. Previous studies have shown that silencing CDKN2A by methylation or the deletion or amplification of MDM2 are mechanisms that are responsible for inactivating TP53 in human tumors [56–59]. PPM1D has also been reported as a candidate proto-oncogene that may be involved in tumorigenesis through the inactivation of TP53 . In our study, we detected homozygous and heterozygous deletions of CDKN2A and the amplification of MDM2 and copy number gain of PPM1D, which shows that these genes can be involved in the initiation/progression of neuroblastoma through the inactivation of TP53.
The fact that 17% of the NB tumors presented with no rearrangements was probably due to the tumors not having any rearrangements that could be visualized with the arrays. However, the possibility that some of these tumor samples contained regions of normal stoma cells, in spite of our efforts to obtain pure tumor material for the studies, cannot be ruled out.