Temporal lobe pleomorphic xanthoastrocytoma and acquired BRAF mutation in an adolescent with the constitutional 22q11.2 deletion syndrome.

DiGeorge syndrome, or velocardiofacial syndrome (DGS/VCFS), is a rare and usually sporadic congenital genetic disorder resulting from a constitutional microdeletion at chromosome 22q11.2. While rare cases of malignancy have been described, likely due to underlying immunodeficiency, central nervous system tumors have not yet been reported. We describe an adolescent boy with DGS/VCFS who developed a temporal lobe pleomorphic xanthoastrocytoma. High-resolution single nucleotide polymorphism array studies of the tumor confirmed a constitutional 22q11.21 deletion, and revealed acquired gains, losses and copy number neutral loss of heterozygosity of several chromosomal regions, including a homozygous deletion of the CDKN2A/B locus. The tumor also demonstrated a common V600E mutation in the BRAF oncogene. This is the first reported case of a patient with DiGeorge syndrome developing a CNS tumor of any histology and expands our knowledge about low-grade CNS tumor molecular genetics.

A multicenter, cross-platform clinical validation study of cancer cytogenomic arrays.

Cytogenomic microarray analysis (CMA) offers high resolution, genome-wide copy number information and is widely used in clinical laboratories for diagnosis of constitutional abnormalities. The Cancer Genomics Consortium (CGC) conducted a multiplatform, multicenter clinical validation project to compare the reliability and inter- and intralaboratory reproducibility of this technology for clinical oncology applications. Four specimen types were processed on three different microarray platforms-from Affymetrix, Agilent, and Illumina. Each microarray platform was employed at two independent test sites. The results were compared in a blinded manner with current standard methods, including karyotype, FISH, or morphology. Twenty-nine chronic lymphocytic leukemia blood, 34 myelodysplastic syndrome bone marrow, and 30 fresh frozen renal epithelial tumor samples were assessed by all six laboratories. Thirty formalin fixed paraffin embedded renal tumor samples were analyzed at the Affymetrix and Agilent test sites only. All study samples were initial diagnostic samples. Array data were analyzed at each participating site and were submitted to caArray for central analysis. Laboratory interpretive results were submitted to the central analysis team for comparison with the standard-of-care assays and for calculation of intraplatform reproducibility and cross-platform concordance. The results demonstrated that the three microarray platforms 1) detect clinically actionable genomic changes in cancer compatible to standard-of-care methods; 2) further define cytogenetic aberrations; 3) identify submicroscopic alterations and loss of heterozygosity (LOH); and 4) yield consistent results within and between laboratories. Based on this study, the CGC concludes that CMA is a sensitive and reliable technique for copy number and LOH assessment that may be used for clinical oncology genomic analysis.

Clinical utilization of high-resolution single nucleotide polymorphism based oligonucleotide arrays in diagnostic studies of pediatric patients with solid tumors.

High-resolution single nucleotide polymorphism (SNP) arrays have been effectively implemented as a first tier test in clinical cytogenetics laboratories for the detection of constitutional chromosomal abnormalities in patients with suspected genomic disorders. We recently published our experience utilizing SNP array analysis of bone marrow aspirates as a clinical test for patients with suspected leukemia or lymphoma in the Clinical Cancer Cytogenetics Laboratory at The Children's Hospital of Philadelphia. In the present report we summarize our clinical experience using the Illumina HumanHap610 BeadChip array (Illumina, San Diego, CA) for whole genome analysis of pediatric solid tumors. A total of 168 DNA samples isolated from a variety of solid tumors, including brain tumors, sarcomas, neuroblastomas, and Wilms tumors, as well as benign neoplasms and reactive processes, were analyzed over a 2 1/2 year period. One hundred thirty-seven of 168 (82%) specimens had at least one copy number alteration or region of loss of heterozygosity detected by the SNP array. Thirty-three of 168 (20%) of cases had a normal karyotype or targeted fluorescence in situ hybridization (FISH) study, but had an abnormal finding by the array analysis. Sixty-three of 168 (37%) samples for which cytogenetic studies were unsuccessful or not performed demonstrated an abnormal array result. In 44 of 168 cases (26%) the array and karyotype or FISH were abnormal, but each demonstrated alterations not detected by the other methodology. Based on our experience in the last 2 1/2 years, we suggest that SNP array analysis can be used as a first tier clinical test for the majority of pediatric solid tumors.

Implementation of high resolution single nucleotide polymorphism array analysis as a clinical test for patients with hematologic malignancies.

Single nucleotide polymorphism-based oligonucleotide arrays have been used as a research tool to detect genomic copy number changes and allelic imbalance in a variety of hematologic malignancies and solid tumors. The high resolution, genome-wide coverage, minimal DNA requirements, and relatively short turnaround time are advantageous for use in a clinical setting. We validated the Illumina HumanHap550 BeadChip array for clinical use by analyzing 127 pediatric leukemia and lymphoma samples that had previously been characterized by means of standard cytogenetic analysis and fluorescence in situ hybridization. A higher resolution Illumina HumanHap610 BeadChip array was ultimately used for clinical testing. To date, 180 samples from children with a suspected or confirmed hematologic malignancy have been analyzed. Of the 180 clinical samples, 130 (72%) bone marrow or lymphoma specimens had aberrations revealed by the array that were not seen in the karyotypes. These typically included deletions in genes associated with B- or T-cell malignancies, such as CDKN2A/B, PAX5, and IKZF1. There were also 75 regions of copy number neutral loss of heterozygosity (>5 Mb threshold) detected in 49 samples in this cohort, which could be categorized as constitutional or acquired abnormalities. On the basis of our experience in the last 2 years, we suggest that single nucleotide polymorphism arrays are a valuable addition to, but not a replacement for, standard cytogenetic approaches for hematologic malignancies.

Activating mutations in BRAF characterize a spectrum of pediatric low-grade gliomas.

In the present study, DNA from 27 grade I and grade II pediatric gliomas, including ganglioglioma, desmoplastic infantile ganglioglioma, dysembryoplastic neuroepithelial tumor, and pleomorphic xanthoastrocytoma was analyzed using the Illumina 610K Beadchip SNP-based oligonucleotide array. Several consistent abnormalities, including gain of chromosome 7 and loss of 9p21 were observed. Based on our previous studies, in which we demonstrated BRAF mutations in 3 gangliogliomas, 31 tumors were screened for activating mutations in exons 11 and 15 of the BRAF oncogene or a KIAA1549-BRAF fusion product. There were no cases with a KIAA1549-BRAF fusion. A BRAF V600E mutation was detected in 14 of 31 tumors, which was not correlated with any consistent pattern of aberrations detected by the SNP array analysis. Tumors were also screened for mutations in codon 132 in exon 4 of IDH1, exons 2 and 3 of KRAS, and exons 2-9 of TP53. No mutations in KRAS or TP53 were identified in any of the samples, and there was only 1 IDH1 R132H mutation detected among the sample set. BRAF mutations constitute a major genetic alteration in this histologic group of pediatric brain tumors and may serve as a molecular target for biologically based inhibitors.