Allelic Loss in a Minimal Region on Chromosome 16q24 Is Associated with Vitreous Seeding of Retinoblastoma

Abstract

In addition to RB1 gene mutations, retinoblastomas frequently show gains of 1q and 6p and losses of 16q. To identify suppressor genes on 16q, we analyzed 22 short tandem repeat loci in 58 patients with known RB1 mutations. A subset of tumors was also investigated by conventional and matrix comparative genomic hybridization. In 40 of 58 (69%) tumors, we found no loss of heterozygosity (LOH) at any 16q marker. LOH was detected in 18 of 58 (31%) tumors, including five with allelic imbalance at some markers. In one tumor LOH was only observed at 16q24. As the parental origin of allele loss was unbiased, an imprinted locus is unlikely to be involved. Analysis of gene expression by microarray hybridization and quantitative RT real-time PCR did not identify a candidate suppressor in 16q24. Cadherin 13 (CDH13), CBFA2T3, and WFDC1, which are candidate suppressors in other tumor entities with 16q24 loss, did not show loss of expression. In addition, mutation and methylation analysis showed no somatic alteration of CDH13. Results in all tumors with chromosome 16 alterations define a single minimal deleted region of 5.7 Mb in the telomeric part of 16q24 with the centromeric boundary defined by retention of heterozygosity for a single nucleotide variant in exon 10 of CDH13 (Mb 82.7). Interestingly, clinical presentation of tumors with and without 16q alterations was distinct. Specifically, almost all retinoblastomas with 16q24 loss showed diffuse intraocular seeding. This suggests that genetic alterations in the minimal deleted region are associated with impaired cell-to-cell adhesion.

Loss at chromosome arm 16q in retinoblastoma: Confirmation of the association with diffuse vitreous seeding and refinement of the recurrently deleted region

“…Data on vitreous seeding were obtained using criteria described previously (Gratias et al, 2007). Statistical analysis was performed using JMP software (Version 7, SAS Institute, Heidelberg, Germany) and with Stata 11.1 (StataCorp.…”

“…Assuming that small regions with LOH were missed in some of the 186 tumors that had shown no LOH in the first round STR we analyzed a second set of STR markers (D16S3098, D16S2641, D16S3061, D16S486, and D16S3074 with intermarker distances < 2 Mb) that were chosen based on the results of SNP array analysis (see below). MSA was performed in multiplexed assays and results were analyzed and classified as described previously (Gratias et al, , 2007.…”

“…Sample collection of retinoblastomas, RNA isolation and microarray analysis were done as described previously (Gratias et al, , 2007. Signal intensity values for further analysis were determined using ''mas5'' function of the affy package of the Bioconductor software (BioC release 2.3, Available at: http://bioconductor.org/).…”

“…In a recent study, Gratias et al (2007) identified a single minimal deleted region of 5.7 Mb in the telomeric part of 16q with the centromeric boundary defined by retention of heterozygosity for a single nucleotide variant in exon 10 of CDH13 (Mb 83.7). However, as the CDH13 gene did not show loss of expression or somatic mutations or epimutations in tumors with loss of heterozygosity (LOH) on chromosome 16 there was no evidence for two-step inactivation of this gene.…”

“…From a clinical point of view, diffuse vitreous seeding is important because its presence in a patient affects therapeutic decisions and, therefore, the strong association to loss of a region on 16q might give a clue as to the mechanisms underlying this adverse tumor growth pattern. However, as the study by Gratias et al (2007) tested a range of clinical parameters for possible association to 16q loss, a confirmatory study is warranted.…”