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Novel amplifications in pediatric medulloblastoma identified by genome-wide copy number profiling

  • Laboratory Investigation - Human/Animal Tissue
  • Published:
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Abstract

Medulloblastoma (MB) is a WHO grade IV, invasive embryonal CNS tumor that mainly affects children. The aggressiveness and response to therapy can vary considerably between cases, and despite treatment, ~30% of patients die within 2 years from diagnosis. Furthermore, the majority of survivors suffer long-term side-effects due to severe management modalities. Several distinct morphological features have been associated with differences in biological behavior, but improved molecular-based criteria that better reflect the underlying tumor biology are in great demand. In this study, we profiled a series of 25 MB with a 32K BAC array covering 99% of the current assembly of the human genome for the identification of genetic copy number alterations possibly important in MB. Previously known aberrations as well as several novel focally amplified loci could be identified. As expected, the most frequently observed alteration was the combination of 17p loss and 17q gain, which was detected in both high- and standard-risk patients. We also defined minimal overlapping regions of aberrations, including 16 regions of gain and 18 regions of loss in various chromosomes. A few noteworthy narrow amplified loci were identified on autosomes 1 (38.89–41.97 and 84.89–90.76 Mb), 3 (27.64–28.20 and 35.80–43.50 Mb), and 8 (119.66–139.79 Mb), aberrations that were verified with an alternative platform (Illumina 610Q chips). Gene expression levels were also established for these samples using Affymetrix U133Plus2.0 arrays. Several interesting genes encompassed within the amplified regions and presenting with transcript upregulation were identified. These data contribute to the characterization of this malignant childhood brain tumor and confirm its genetic heterogeneity.

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Abbreviations

Array-CGH:

Array-comparative genomic hybridization

BAC:

Bacterial artificial chromosome

CNS:

Central nervous system

CNV:

Copy number variation

i(17)(q10):

Isochromosome 17q

MB:

Medulloblastoma

MOR:

Minimal overlapping region

SMAP:

Segmental maximum a posteriori

WHO:

World Health Organization

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Acknowledgments

The authors wish to thank Prof. Jan Dumanski for providing us with 32K arrays, Robin Andersson, at the Linnaeus Centre for Bioinformatics, for developing SMAP within the LCB Data Warehouse, the late Dr. Magdalena Hartman for initiating the CNS tumor biobank at UAS, and Dr. Inga Hansson for immunohistochemical staining of FFPE tissue sections. This work was supported by the Swedish Childhood Cancer Foundation, the Swedish Cancer Society, and Uppsala University. Illumina genotyping was performed at the SNP Technology Platform, Uppsala, Sweden (http://www.genotyping.se), supported by Uppsala University and the Knut and Alice Wallenberg Foundation. Affymetrix expression analysis was performed at the Uppsala Array Platform (http://www.medsci.uu.se/klinfarm/arrayplatform/).

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Authors and Affiliations

  1. Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-75185, Uppsala, Sweden

    Helena Nord, Svetlana Popova & Irina Alafuzoff

  2. Department of Women’s and Children’s Health, Uppsala University, Uppsala University Hospital, SE-751-85, Uppsala, Sweden

    Susan Pfeifer & Bo Strömberg

  3. Section of Neurosurgery Department of Neuroscience, Uppsala University, Uppsala University Hospital, SE-751 85, Uppsala, Sweden

    Pelle Nilsson

  4. Department of Oncology-Pathology, Karolinska Institutet, Cancer Center Karolinska, Karolinska University Hospital in Solna, SE-171 76, Stockholm, Sweden

    Johanna Sandgren, Monica Nistér & Teresita Díaz de Ståhl

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Table S1

Association in KEGG pathways of upregulated genes, in medulloblastoma samples (1253 and 5594) presenting with novel focal amplifications. Enriched KEGG pathways, according to DAVID Functional Annotation Tool (http://david.abcc.ncifcrf.gov), associated with overexpressed transcripts in medulloblastoma samples 1253 and 5594, relative to the average expression level of the corresponding probes in cerebellum. The genes and percentage of involved genes (involved genes/total genes) in the pathway are shown. To avoid overcounting of duplicated genes, P values were calculated using the Fisher exact statistic based on corresponding DAVID gene IDs, by which all redundancy in original IDs is removed. Adjusted P values according to Benjamini correction are also shown (DOC 39 kb)

Table S2

Minimal overlapping regions of aberrations encompassing at least two tumors were determined. Gene expression data for medulloblastoma grade IV, from series GSE10327 [38], were downloaded from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and compared with gene expression data from normal cerebellum from series GSE3526, both using platform GPL570: Affymetrix U133Plus2.0 (see Supplementary Fig. 4 for sample details). Analysis (3′ Expression Arrays-RMA) was run in Expression Console v1.1 (Affymetrix). The B statistic was calculated using the ‘limma’ package [58] of the R language (http://www.r-project.org/), in the framework of an empirical Bayes method [59] and used to select differentially expressed genes in replicated complementary DNA (cDNA) microarray experiments. The top genes significantly up- or downregulated within minimal overlapping regions of aberrations (maximum difference in mean average value) are listed. The log2 fold change ratio is indicated in parenthesis after the gene name. aRegions of completely or partially overlapping loci identified in previous medulloblastoma studies are indicated. bDel: minimal overlapping regions of deletion; gain: minimal overlapping regions of gain. Cancer genes, according to Sanger Cancer Gene Census (http://www.sanger.ac.uk/genetics/CGP/Census/) and genes known to be involved in medulloblastoma formation are indicated in red (DOC 220 kb)

Fig. S3

Identification of a chromosome 8 amplicon in medulloblastoma sample 0013 encompassing MYC. a Whole-genome Illumina 610Q chip profile for sample 0013. Log R ratio and B-allele frequency values are shown. Detailed view of the amplicon on chromosome 8, for both 32K array (b) and Illumina chip (c). Two green lines above the ideogram indicate an amplified region, and a red bar below it indicates a deletion. 32K array and Illumina 610Q chip profiles show the same pattern of aberrations (EPS 5333 kb)

Fig. S4

Identification of two novel amplicons on chromosome 3 in medulloblastoma sample 5594. a Whole-genome Illumina 610Q chip profile for sample 5594. Log R ratio and B-allele frequency values are shown. Detailed view of the amplicons on chromosome 3, for both 32K array (b) and Illumina chip (c). Two green lines above the ideogram indicate an amplified region, and a red bar below it indicates a deletion. 32K array and Illumina 610Q chip profiles show concordant pattern of aberrations (EPS 5529 kb)

Fig. S5

Identification of novel amplicons on chromosome 1 in medulloblastoma sample 1253. a Whole-genome Illumina 610Q chip profile for sample 1253. Log R ratio and B-allele frequency values are shown. Detailed view of the amplicons on chromosome 1, for both 32K array (b) and Illumina chip (c). An additional amplicon was detected in the second biopsy profiled on Illumina chip, indicating the presence of intratumoral heterogeneity. Two green lines above the ideogram indicate an amplified region, and a red bar below it indicates a deletion (EPS 5248 kb)

Fig. S6

Unsupervised two-way hierarchical cluster analysis of 64 medulloblastoma and 9 cerebellum samples. Gene expression data for two of our tumor samples (1253 and 5594) and from medulloblastoma series GSE10327 (samples GSM260959, GSM260960, GSM260961, GSM260962, GSM260963, GSM260964, GSM260965, GSM260966, GSM260967, GSM260968, GSM260969, GSM260970, GSM260971, GSM260972, GSM260973, GSM260974, GSM260975, GSM260976, GSM260977, GSM260978, GSM260979, GSM260980, GSM260981, GSM260982, GSM260983, GSM260984, GSM260985, GSM260986, GSM260987, GSM260988, GSM260989, GSM260990, GSM260991, GSM260992, GSM260993, GSM260994, GSM260995, GSM260996, GSM260997, GSM260998, GSM260999, GSM261000, GSM261001, GSM261002, GSM261003, GSM261004, GSM261005, GSM261006, GSM261007, GSM261008, GSM261009, GSM261010, GSM261011, GSM261012, GSM261013, GSM261014, GSM261015, GSM261016, GSM261017, GSM261018, GSM261019, and GSM261020), as well as data from normal cerebellum from series GSE3526 (samples GSM80616, GSM80617, GSM80618, GSM80619, GSM80626, GSM80636, GSM80637, GSM80638, and GSM80639), downloaded from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), were used, all performed on platform GPL570: Affymetrix U133Plus2.0. The group to which each medulloblastoma sample was classified according to the original publication (A–E) [38] is indicated after the name of the sample. Unsupervised two-way hierarchical clustering was performed with the TMEV program using Pearson correlation [29]. A low-intensity cutoff filter (value 5) was applied. Furthermore, we selected for clustering the smallest set of genes with the greatest variance in expression (variance filter in TMEV program, value 5). Expression data of 492 most differentially expressed genes identified distinct clusters. Sample 1253 clustered together with samples from group B: SHH group, and sample 5594 with group C [38]. As expected, samples from group A and B clustered in separated groups and tumors from group C, D, and E appear closely related [38]. Cerebellum samples clustered separately (EPS 8580 kb)

Fig. S7

Medulloblastoma samples displaying aberrant chromosome 17 profiles. Fifteen samples were detected with aberrations on chromosome 17. Ten cases displayed loss of 17p combined with gain of 17q, three samples (3237, 0499, and 7171) presented with gain of 17q, and two tumors (5335 and 0013) with loss of 17p. Sample 0013 displayed also an interstitial gain of 17q. Among samples with 17p loss combined with 17q gain, five different breakpoints were identified, indicated as A–E, see Supplementary Fig. 6. Breakpoints A 15.84–16.20 Mb, B 16.29–16.65 Mb, C 18.75–19.12 Mb (the most common), D 21.33–21.77 Mb, and E 22.06–22.60 Mb. Each individual clone was assigned as: balanced (CNC = 0, indicated in blue); gained, (CNC = 1 in green); or deleted (CNC = −1 red), see “Materials and methods.” The X-axis shows the clone positions, and the Y-axis depicts fluorescence ratios. Clone mapping information was obtained from Ensemble (http://ensembl.org/biomart), and coordinates from human genome assembly of March 2006 (NCBI Build 36, hg18) were used (EPS 4934 kb)

Fig. S8

Chromosome 17 breakpoints identified in medulloblastoma samples. A–E Five different breakpoint regions were identified, indicated in grey color. The most common one C was present in six tumors, and the others (A, B, D, E) were detected in only one sample each, see Supplementary Fig. 5. The genes encompassed in the different regions are drawn. Four of the breakpoint regions involved copy number polymorphic regions. Coding exons are represented by vertical lines, and connected by horizontal lines representing introns. Arrowheads on the connecting intron lines indicate the direction of transcription. Provisional genes (not reviewed) are indicated in light blue (EPS 937 kb)

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Nord, H., Pfeifer, S., Nilsson, P. et al. Novel amplifications in pediatric medulloblastoma identified by genome-wide copy number profiling. J Neurooncol 107, 37–49 (2012). https://doi.org/10.1007/s11060-011-0716-0

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