Allelic Loss in a Minimal Region on Chromosome 16Q24 Is Associated with Vitreous Seeding of Retinoblastoma

Research Article

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

Sandrine Gratias,1 Harald Rieder,4 Reinhard Ullmann,5 Ludger Klein-Hitpass,2 Stephanie Schneider,6 Re´ka Bo¨lo¨ni,3 Martin Kappler,7 and Dietmar R. Lohmann1

1Institut fu¨r Humangenetik, 2Institut fu¨r Zellbiologie, and 3Augenklinik, Universita¨tsklinikum Essen, Essen, Germany; 4Institut fu¨r Humangenetik und Anthropologie, Universita¨tsstraße 1, Universita¨tDu¨sseldorf, Du¨sseldorf, Germany; 5Max-Planck Institute for Molecular Genetics, Berlin, Germany; 6Institut fu¨r Klinische Genetik, Universita¨tsklinikum Marburg, Marburg, Germany; and 7Berufsgenossenschaftliches Forschungsinstitut fu¨r Arbeitsmedizin, Ruhr-Universita¨tBochum, Bochum, Germany

Abstract Loss of all or parts of chromosome 16 is observed in 31% of In addition to RB1 gene mutations, retinoblastomas frequently retinoblastomas (51 of 162; summarized in ref. 6). In most of these show gains of 1q and 6p and losses of 16q. To identify tumors, the whole long arm of one homologue is lost (40 of suppressor genes on 16q, we analyzed 22 short tandem repeat 51, 78%). A survey of comparative genomic hybridization (CGH) loci in 58 patients with known RB1 mutations. A subset of analyses has indicated that most partial deletions on chromosome tumors was also investigated by conventional and matrix 16q include chromosome band 16q22 (7 of 11, 64%; ref. 3). To study comparative genomic hybridization. In 40 of 58 (69%) tumors, alterations of this region in further detail, Marchong et al. (6) did LOH analysis of seven microsatellite markers located on 16q21-23.3 we found no loss of heterozygosity (LOH) at any 16q marker. 8 LOH was detected in 18 of 58 (31%) tumors, including five with (Mb 60.9–81.5, Ensembl v36 ) and quantitative multiplex PCR of allelic imbalance at some markers. In one tumor LOH was only five sequence-tagged sites in Mb 61.5 to 75.1 (Ensembl v36) and of observed at 16q24. As the parental origin of allele loss was six exons of the cadherin 11 (CDH11) gene. They found frequent unbiased, an imprinted locus is unlikely to be involved. allelic loss at D16S398 (located at Mb 64.7, observed in 11 of 28 Analysis of gene expression by microarray hybridization and tumors, 39%) and at D16S422 (Mb 81.5, observed in 9 of 23 tumors, quantitative RT real-time PCR did not identify a candidate 39%). Quantitative multiplex PCR showed that sequences located suppressor in 16q24. Cadherin 13 (CDH13), CBFA2T3, and within the CDH11 gene (Mb 63.5–63.7) are most frequently lost (41 WFDC1, which are candidate suppressors in other tumor of 71 tumors, 58%). The long arm of chromosome 16 is a frequent entities with 16q24 loss, did not show loss of expression. In target of deletions in various cancers. Three candidate regions, one in 16q22.1 and two in 16q24.3, have been identified. In some tumors addition, mutation and methylation analysis showed no somatic alteration of CDH13. Results in all tumors with with 16q22.1 loss, E-cadherin (CDH1) is inactivated by mutations or chromosome 16 alterations define a single minimal deleted silenced by epigenetic mechanisms (11, 12). This provides good region of 5.7 Mb in the telomeric part of 16q24 with the evidence for a tumor-suppressor role of CDH1 in these cancers. centromeric boundary defined by retention of heterozygosity The tumor suppressors underlying loss in the telomeric candidate for a single nucleotide variant in exon 10 of CDH13 (Mb 82.7). regions are not defined yet. Here, we used LOH analysis and Interestingly, clinical presentation of tumors with and without microarray expression analysis to identify candidate tumor 16q alterations was distinct. Specifically, almost all retino- suppressors on 16q in retinoblastoma. Moreover, we investigated blastomas with 16q24 loss showed diffuse intraocular seeding. if clinical manifestation is distinct depending on the presence of This suggests that genetic alterations in the minimal deleted alterations on 16q. region are associated with impaired cell-to-cell adhesion. [Cancer Res 2007;67(1):408–16] Materials and Methods Samples and extraction of DNA and RNA. Tumor and blood samples Introduction were obtained from retinoblastoma patients at the time of operative treatment. In addition, blood samples were received from parents of most Retinoblastoma, a rare childhood eye tumor, has served as a patients. Tissues were snap frozen in liquid nitrogen. Informed consent was model to elucidate the genetic events underlying the development obtained from all patients or their parents. Storage and processing of of sporadic and hereditary cancer (reviewed in refs. 1, 2). For samples as well as extraction of nucleic acids was done as previously initiation of this tumor, mutations in both alleles of the RB1 gene, described (10). Material from 58 patients (54unilateral and 4bilateral cases) a tumor suppressor on chromosome 13q14, are required. Retino- with known mutations in the RB1 gene was used for the present study. blastomas often show gains on chromosomes 1q and 6p and losses Microsatellite analysis of markers on chromosome 16. A total of 22 of chromosome 16q (summarized in ref. 3). Therefore, it is reason- short tandem repeat loci with high polymorphic information content were able to assume that mutations in additional genes contribute to analyzed (Fig. 1A; primer sequences are available on request). Intermarker promotion and progression of this tumor (4). The minimum regions distances were even (1–2 Mb along 16q) except a gap of 15 Mb between markers D16S3080 and D16S3050 (at 16q12.1 and 16q21, respectively). PCR of genomic gains on 1q and 6p have been defined and this has led with labeled forward primers (FAM, PET, or NED fluorescent dyes at the to the identification of putative target oncogenes (5–10). 5¶-end, Applied Biosystems, Weiterstadt, Germany) was done in multiplexed assays and analyzed as described (10). If loss of one allele in the tumor was incomplete, the allele ratio was determined as follows: (PI allele tumor / PI Requests for reprints: Dietmar R. Lohmann, Institut fu¨r Humangenetik, 1 Universita¨tsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany. Phone: 49- 201-7234562; Fax: 49-201-7235900; E-mail: [email protected]. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-1317 8 http://www.ensembl.org.

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Figure 1. Pattern of chromosome 16 alterations in retinoblastoma samples. A, results of microsatellite analysis (MSA) in 40 tumors with no LOH (black squares) at any informative marker. Allelic imbalance (blue squares) is scored for makers with allele ratios between 1.3 and 2.5. Gray squares, noninformative markers. Headers of columns with microsatellite analysis results give a summary of the results of conventional CGH and matrix CGH (matrix CGH-a, reported in ref. 17; matrix CGH-b, original): n, no copy number changes on chromosome 16; L, losses; gL, gains and losses; —, not done. B, results of microsatellite analysis in 18 tumors with LOH at some or all informative markers. Parental origin of allele losses for markers with LOH on chromosome 16: m, maternal allele retained; p, paternal allele retained. C, synopsis of the results of microsatellite analysis and matrix CGH in five tumors with complex copy number changes. Green bars, gains; orange bars, losses. Scale (left), map position of chromosome 16 markers according to Ensembl v36.

allele2 tumor) / (PI allele1 blood / PI allele2 blood), where PI is the peak as previously described (14, 15). The cDNAs were purified by phenol/ integral. To obtain allele ratio values >1, the allele with the larger peak area chloroform/isoamyl alcohol/phase lock gel (Eppendorf, Hamburg, Germany) in the tumor was defined as allele1. We used the criteria established in extraction, precipitated, and used to generate biotinylated cRNA by in vitro a previous study (13) to categorize the results as follows: LOH for values transcription for 16 h at 37jC (Bioarray High Yield RNA Transcript Labeling of allele ratio >2.5; allelic imbalance for 1.3 V allele ratio V 2.5; normal for kit, Enzo Life Science, Farmingdale, NY). Purification of cRNA was done values of allele ratio < 1.3. using RNeasy mini columns (Qiagen, Hilden, Germany). Fragmentation of Microarray expression analysis. Synthesis of double-stranded cDNA cRNA, hybridization to HG-U133A oligonucleotide arrays (Affymetrix, Inc., was done with f2.5 Ag of total RNA and anchored T7-oligo-d(T)21-V Santa Barbara, CA), washing, staining, and scanning (GeneArray scanner primer [5¶-GCATTA-GCGGCCGCGAAATTAATACGACTCACTATAGGGA- 2500, Agilent, Palo Alto, CA) were done following standard Affymetrix GA(T)21V-3¶, MWG Biotech, Ebersberg, Germany] for first-strand synthesis protocols (Technical Manual). Signal intensities and detection calls for www.aacrjournals.org 409 Cancer Res 2007; 67: (1). January 1, 2007

Table 1. List of genes in 16q24 differentially expressed between tumors with and without LOH at chromosome 16q

c b Gene symbol Position* (bp) Affymetrix accession no Median expression P < Expression in retina

No LOH LOH

MBTPS1 82644869 201620_at 3,383.4 1,727.3 0.003 2,901.9 KIAA1609 83068608 65438_at 188.9 113.6 0.015 115.5 USP10 83291085 209137_s_at 2,746.7 1,731.4 0.02 1,479.5 ZDHHC7 83565573 218606_at 2,156.95 1,276.40.003 2,691.2 KIAA0513 83618911 204546_at 981.2 644.1 0.003 842 COX4NB 84369736 218057_x_at 1,607.65 1,008.3 0.05 1,102 COX4I1 84390697 202698_x_at 10,967.3 8,523.5 0.001 14,302.3 FBXO31 85920443 222352_at 798.7 364.25 0.015 500.7 MAP1LC3B 85983320 208785_s_at 3,293.25 1,507.8 0.02 2,698.1 ZCCHC14 85997364212655_at 3,112.85 1,691.3 0.015 1,829.9 SLC7A5 86421130 201195_s_at 1,238.2 951.3 0.05 2,717.9 APRT 87403378 213892_s_at 509.8 275.8 0.015 475.7 GALNS 87407643 206335_at 764.65 521.9 0.03 Absent HSPC176 87451007 218354_at 1,229.05 920.6 0.015 1,303 ANKRD11 87861536 219437_s_at 1,284.5 868.8 0.02 827.6 SPG7 88102306 202104_s_at 957.6 707.1 0.02 Absent PCOLN3 88238346 201933_at 1,194.45 902.2 0.03 572.8 KIAA1049 88467527 221495_s_at 2,473.65 1,591.3 0.02 2,886.8

*According to Ensembl v36. cMedian of signal values determined by the Microarray Suite 5 statistical algorithm. bWilcoxon one-way test m2 approximation.

further analysis were determined using GeneChip Microarray Suite 5.0 Results of matrix CGH of these samples are available at Gene Expression Software (Affymetrix). Scaling across all probe sets of a given array to an Omnibus (GEO submission no. GSE5359).11 average intensity of 1,000 units compensated for variations in the amount CDH13 methylation and mutation analysis. Bisulfite treatment of DNA and quality of cRNA samples and other experimental variables. Further was done as described (25). Primers were modified from Toyooka et al. (26). processing of the signal values and gene information was done with Primer sequences are available on request. Reactions at 25 AL containing standard spreadsheet software (Excel, Microsoft Corporation). 3 AL bisulfite-treated DNA, 0.2 mmol/L of each deoxynucleotide triphos- Quantitative reverse transcription real-time PCR. Reverse transcrip- phate, 2 Amol/L of each primer, 2.5 ALof10 PCR buffer, and 1 unit Taq tion (RT) and quantitative real-time PCR were done using Assays on Polymerase (AmpliTaq Gold, Applied Biosystems) were exposed to a thermal Demand (CDH13 assay ID Hs00169908_m1, Applied Biosystems) as profile starting with 95jC for 10 min followed by 35 cycles of 95jC/15 s, previously described (8). For relative quantification, the expression of 65jC/30 s, and 72jC/15 s, and ending with 72jC for 7 min. Products were h-actin (Human ACTB Endogenous Control, Part No. 4352935E, Applied separated by agarose gel electrophoresis and visualized with ethidium Biosystems) was analyzed. bromide. Constitutional and M.Sss I in vitro methylated DNAs (NEB, Comparative genomic hybridization. Conventional CGH was done as Frankfurt, Germany) were used as positive controls. Primers for PCR and previously described (16). CGH results of 16 of the tumors analyzed in sequencing of all 14exons of CDH13 (Vega gene ID OTTHUMG- this study had been reported previously (16). Results of matrix CGH from 00000072884) were chosen from intronic and untranslated regions using 16 tumors included in this study had been reported (17). In addition, samples Primer3 software (27). Primer sequences are available on request. Templates M5715 and M5450 were analyzed by matrix CGH using a submegabase for sequencing were generated by PCR in 25 AL reactions with 80 ng DNA; resolution tiling path bacterial artificial chromosome array, comprising 10 pmol of each primer (GoTaq Green Mastermix, Promega, Madison, WI); the human 32k Re-Array set9 [DNA kindly provided by Pieter de Jong and a thermal profile starting with 95jC/2 min followed by 35 cycles of (BACPAC Resources, Children’s Hospital Oakland, Oakland, CA); refs. 18–21], 95jC/30 s, 51jC/30 s, and 72jC/1 min, and ending with 72jC for 5 min. the 1 Mb Sanger set (clones kindly provided by Nigel Carter, Wellcome Trust For sequence reactions, a BigDye Terminator v1.1 kit was used according Sanger Centre, Cambridge, United Kingdom; ref. 18) and a set of 390 to the kit protocol. Products were analyzed on an ABI PRISM 3100 Genetic subtelomeric clones (assembled by members of the European Cooperation in Analyzer (Applied Biosystems). the Field of Scientific and Technical Research B19 initiative: Molecular Statistical analysis of genetic findings and patient data. The cytogenetics of solid tumors). Hybridizations were done as described by Wilcoxon test (normal approximation) and Significance Analysis of Micro- Erdogan et al. (22). Detailed step-by-step protocols are also available array (SAM, Stanford University, Stanford, CA) were used to identify genes online.10 Further analysis and visualization of matrix CGH data was done that were consistently differentially expressed between tumors with and using the software package CGHPRO (23). Data were normalized by subgrid without LOH. Detailed data on clinical presentation, treatment, and follow- Lowess. No background subtraction was done. Circular Binary Segmentation up of patients were obtained. We used data warehouse software (Cognos

(24), in combination with a threshold of F0.2 log2 ratio, was used for the Series 7.1; Cognos, Inc., Ottawa, ON, Canada) to link all clinical and genetic objective determination of presence and size of chromosomal imbalances. data and to set the stage for data mining, which was done using the tools provided by the software environment.

9 http://bacpac.chori.org/pHumanMinSet.htm. 10 http://www.molgen.mpg.de/~abt_rop/molecular_cytogenetics/. 11 http://www.ncbi.nlm.nih.gov/geo/.

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The influence of each aberration on seeding was tested by Fisher’s exact higher probability of diffuse seeding and at least local seeding for the studied test. To study the combined effect of the aberrations on seeding, a aberration. Additionally, age was included in the model as covariate. proportional odds model was done using the cumulative logit as link Calculations were done using SAS version 9.1 (SAS Institute, Inc., Cary, NC), function in a logistic regression. Therefore, this variable was sorted in and tests were conducted two-sided with a significance level of a = 5%. descending order to model the probabilities of diffuse seeding and of at least Statistical evaluation of the findings was also done using JMP 5.1 software local seeding. Effect estimates (expressed as odds ratios, OR) >1 denote a (SAS Institute).

Table 2. Summary of genetic findings in DNAfrom tumor samples

ID RB1 gene mutation and Gains on Gains on 16q allele Vitreous Age at diagnosis c b allele loss in DNAfrom tumor* chromosome 1q chromosome 6p loss seeding (d)

Unilateral retinoblastoma M74g.59683C>T, LOH Complete high Partial LOH — 1,135 M1727 g.76430C>T, LOH Complete high No gain LOHx Diffuse 808 M2919 g.64347G>Ak, LOH Intermediate No gain LOH — — M5450 g.59683C>T, LOH Complete high Complete LOHx Diffuse 1,815 M5715 g.153209insT, LOH Complete high Partial LOHx Diffuse 1,002 M6301 g.64348C>Tk, NLOH Complete high Complete LOHx Diffuse 1,099 M6302 Hypermethylation, LOH Complete high Complete LOHx Diffuse 890 M7848 g.156713C>T, LOH Intermediate Complete LOHx Diffuse 1,315 M10288 g.64348C>T, LOH Complete high Complete LOH{ Diffuse 775 M19484 g.39523del4, LOH Complete high Partial LOHx Diffuse 985 M19489 g.150037C>T, LOH No gain Partial LOHx Diffuse 373 M22402 g.162368G>T, LOH Complete high Complete LOH — — M22590 g.161996G>C, LOH Complete high No gain LOHx Diffuse 603 M22641 g.59683C>T, LOH Complete high Partial LOH — 1,051 M22731 g.70330G>A, LOH Intermediate Partial LOHx Diffuse 1,054 M22860 g.59683C>T, g.77051T>C, NLOH No gain No gain LOH No 1,725 M24794 g.78225del7, LOH Complete high Partial LOHx Diffuse 470 M461 g.64348C>T, LOH No gain — NLOH — 181 G1142 g.156713C>Tk, NLOH Complete high Partial NLOH Local 330 G1166 g.39551delAT, LOH No gain Partial NLOH — — M1324g.78238C>T, g.78250C>T, NLOH No gain No gain NLOHx Diffuse 433 M1820 g.156797ins100, LOH Intermediate Partial NLOH — 516 M2087 Hypermethylation, LOH No gain No gain NLOH Local 461 M2968 Hypermethylation, LOH No gain No gain NLOH — 132 M3297 g.78238C>T, g.5470C>T, NLOH No gain Complete NLOHx Diffuse 511 M4042 g.64348C>T, g.77029delAT, NLOH No gain No gain NLOH — 237 M5500 g.64348C>T, LOH Intermediate Partial NLOH No 467 M5701 g.39562G>T, LOH No gain No gain NLOH No 380 M6489 g.64348C>T, LOH No gain No gain NLOH No 245 M6680 g.73752G>Ak, g.162029ins8, NLOH No gain No gain NLOH No 74 M6685 g.76898C>T, LOH No gain Partial NLOH No 300 M6807 g.150037C>T, LOH No gain Partial NLOH No 160 M7378 Hypermethylation, LOH No gain No gain NLOH{ Diffuse 128 M7398 g.41954G>T, LOH Intermediate Complete NLOHx Diffuse 293 M7413 g.64348C>T, LOH No gain No gain NLOH Local 292 M7422 g.59695C>T, LOH No gain Complete NLOH — 2,315 M7899 g.64348C>T, LOH No gain No gain NLOH{ Diffuse 293 M8999 g.78228insAA, LOH Intermediate Partial NLOH Local — M10282 g.64348C>T, LOH No gain No gain NLOH — 61 M22058 Hypermethylation, LOH Intermediate Complete NLOH — 411 M22067** g.150098insAk, NLOH No gain No gain NLOH No 120 M22136 g.78285T>Ck, g.78238C>T, NLOH Intermediate Complete NLOH — 628 M22233 g.70329C>Tk, LOH No gain Complete NLOH — 897 M22455 g.41946delT, NLOH No gain No gain NLOH Local 192 M23215 g.39562delG, LOH Complete high Partial NLOH Local 876 M23449 g.78238C>T, LOH Intermediate No gain NLOH No 579 M23818 g.78238C>T, g.76492T>A, NLOH Intermediate Complete NLOHx Diffuse 942 M23869 g.64348C>Tk, LOH Intermediate Complete NLOHx Diffuse 448 M23978 g.59683C>T, g.70318delT, NLOH No gain No gain NLOH No 326

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Table 2. Summary of genetic findings in DNAfrom tumor samples (Cont’d)

ID RB1 gene mutation and Gains on Gains on 16q allele Vitreous Age at diagnosis c b allele loss in DNAfrom tumor* chromosome 1q chromosome 6p loss seeding (d)

M24307 g.65363G>C, LOH Intermediate No gain NLOH — 241 M24431 g.162237C>T, LOH Intermediate No gain NLOH No 397 M24733 g.73774G>Tk, LOH Intermediate Complete NLOHx Diffuse 746 M24784 g.153241delA, LOH No gain No gain NLOH — 111 M24821 g.150037delC, g.44667G>T, NLOH Complete high Complete NLOH — 540 Bilateral retinoblastoma M20517 g.77000G>Ck, LOH No gain No gain NLOH No 240 M22013 g.76430C>Tk, NLOH Intermediate Partial NLOH No 129 M22507 g.1863delGAinsTTk, LOH No gain No gain LOH No 1,058 M23209 g.64328A>Gk, NLOH No gain No gain NLOH Local 522

Abbreviation: NLOH, no LOH. *Mutation description with reference to the genomic sequence of the RB1 gene (accession no. L11910). cData from ref. 10. bData from ref. 8. x CDH13 with normal coding sequence and unmethylated. kMutation is also present in peripheral blood DNA (germ-line mutation). {CDH13 with normal coding sequence. **Unilateral multifocal retinoblastoma.

Results tumor M5715 showed gains in a small region on 16q (Mb 69.44– 69.75; Fig. 1C). In this tumor, the two informative markers next to Identification of a minimal deleted region 16q. Forty of 58 this region have allele loss (D16S3067 at 67.6 Mb and D16S3118 at (69%) tumors showed no LOH at any informative short tandem 12 74.8 Mb), whereas all other informative 16q loci in this tumor had repeat marker (Fig. 1A). Results of conventional CGH (16) and allelic imbalance. The remaining two of the five tumors with LOH matrix CGH (17) were available from 34and 11 of these tumors, and allelic imbalance also revealed complex changes in matrix respectively. In all but three tumors, results of microsatellite CGH that were not detected by conventional CGH (Fig. 1C). For analysis were accordant with those of CGH. Results were example, in tumor M22641, markers from 16q11.2 to 16q22.1 had discordant in tumor M24733, which showed losses at all 16q allelic imbalance, whereas all informative markers toward the probes in matrix CGH but had normal copy number in telomere showed clean loss of one allele. The matrix CGH results of conventional CGH and no allele loss in microsatellite analysis, this tumor showed gains in the region with allelic imbalance and and in tumors M22136 and M23818, which showed loss of 16q in losses in the proximal part of the region with LOH (Fig. 1C). conventional CGH but not in microsatellite analysis. However, for both M22136 and M23818, signal losses observed in conventional CGH were just below the threshold, which was set at 80% of the mean fluorescent signal intensity. Also, it is to be noted that microsatellite analysis of tumor M23818 showed allelic imbalance at one marker D16S398 (Fig. 1A, blue square). Threshold values for classification by microsatellite analysis, conventional CGH, and matrix CGH are similar but not identical. As a consequence, classification of samples with low-level changes may come to different results. The different classification of tumors M24733, M22136, and M23818 suggests that the sensitivity of microsatellite analysis to detect low-level copy number imbalances is lower than that of matrix CGH and conventional CGH. Microsatellite analysis detected LOH in 18 of 58 (31%) tumors (Fig. 1B). In five of these tumors (M6301, M19484, M5715, M22641, and M22731), some markers on 16q had LOH, whereas other markers showed reduced but not missing signals of one of the alleles (allelic imbalance). In conventional CGH, three of the tumors with LOH and allelic imbalance (M6301, M19484, and M5715) showed losses along the entire long arm. In matrix CGH, Figure 2. Age at diagnosis of retinoblastoma (in months) in relationship to chromosome 16 alterations as detected by microsatellite analysis. Retinoblastomas with LOH are diagnosed significantly later than tumors with 12 In preparation. no LOH on 16q (Wilcoxon P < 0.001).

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In two tumors, M5450 and M24794, LOH was detected only at quantitative RT real-time PCR to measure CDH13 transcripts markers in telomeric parts of 16q. In conventional CGH, both because this method can detect lower levels of expression tumors had no copy number changes on chromosome 16, whereas compared with array hybridization. We used h-actin (ACTB)asa in matrix CGH, both tumors showed DNA losses (Fig. 1C). control because this gene was expressed at high levels in normal Notably, tumor M24794 had losses only in a part of the region retina and in all retinoblastoma samples tested by microarray with LOH. This suggests that LOH in the other regions is due to analysis (lower, median, and upper quartile of signal values are isodisomy. Results of microsatellite analysis in all tumors with 32,470; 37,880; and 46,601, respectively). We found expression of chromosome 16 alterations are in line with a single minimal CDH13 in 21 of 22 retinoblastomas tested and in RNA from normal deleted region. The boundary of this minimal deleted region retina (Human Retina Total RNA, BD Biosciences Clontech). toward the centromere is defined by retention of heterozygosity Expression levels varied between tumors; however, there was no at D16S422 (Mb 81.5) in tumor M24794. All tumors with LOH correlation with the LOH status on 16q. showed alterations in the region of D16S3026 (Mb 88), which is According to matrix CGH data, which were available from 18 the short tandem repeat polymorphism with the most distal tumors, three tumors had DNA gains (M22641, M5450, and M5715) location known on this chromosome. The parental origin of allele in a minimal region that ranged from Mb 69.44 to 69.75 (Fig. 1C). loss on 16q was determined by genotyping of parental blood DNA This region contains three genes: HYDIN, JGI-931, and HYDNI.1. in 15 patients. Tumors from nine patients showed loss of alleles of Only the first of these genes is represented by a probe set on the paternal origin; in six tumors, the alleles of maternal origin were Affymetrix array (accession no. 220098_at). We found no expression lost (Fig. 1B). of this gene in any retinoblastoma including tumor M22641 that RNA expression data. Microarray expression data were showed gains in matrix CGH. available from 12 retinoblastomas, eight without LOH at any Results of CDH13 methylation and sequencing analysis. The informative marker on 16q and four with LOH, including M22641 methylation status of the CDH13 promoter region was tested in with a mixed pattern of alterations (GEO submission no. GSE5222). 17 tumors. All samples were found to be unmethylated (Table 2). SAM was done to identify genes that, regardless of the absolute Sequencing of the 14exons of CDH13 in 19 tumors and normal RNA expression level, are consistently differentially expressed controls showed a few rare single nucleotide variants [homozy- between tumors with and without LOH. Of the top 20 genes with gous: c335T>A (V112D) tumor M1727, c1038G>A (T346T) tumor differential expression, 13 are located on 16q. These include three M22590, c1953C>G (N651K) tumor M6302; heterozygous: genes that are located in the minimal deleted region: MBTPS1 c1416C>T (N472N) tumor M24794, c1731G>A (D577D) tumor (membrane-bound transcription factor protease, site 1), which M1324]. Sequence analysis of corresponding DNA from blood belongs to the sutiliase (subtilisin-like serine proteases) family, and showed heterozygosity for all variant alleles and, therefore, none two genes that code for zinc finger proteins, ZCCHC14 and of these alterations is a somatic mutation. It is to be noted that ZDHHC7. To the best of our knowledge, these three genes and the retention of heterozygosity in tumor M24794 helps to define the remaining 10 that are located elsewhere on chromosome 16q have centromeric boundary of the minimal deleted region at Mb 82.7 no known role in tumorigenesis. (CDH13, exon 10). According to Ensembl (v36), the minimal deleted region on 16q Correlation of 16q alterations to RB1 mutations and to identified here contains 104genes. Sixty-four of these genes are clinical variables. We found no significant association between represented by probe sets on the Affymetrix chip Hu133A that we the presence of 16q LOH and the origin of the causative mutation used for microarray expression analysis. Of these, 14genes were in the RB1 gene [15 of 44 (34%) and 3 of 13 (23%) patients with not expressed in any tumor analyzed here, and 26 genes showed somatic and germ-line mutations, respectively, P = 0.52, Fisher’s no significant difference in expression between tumors with and exact test two-tailed; Table 2]. Tumors with 16q loss had a higher without LOH on 16q. Specifically, we found no significant frequency of RB1 LOH [16 of 43 (37%) compared with 2 of 15 (13%), difference in expression for CBFA2T3, a candidate suppressor gene without RB1 LOH] but this did not reach statistical significance in breast cancer (28, 29), and for WFDC1, which shows allele loss in (P = 0.11). Interestingly, the distribution of patient’s age at liver cancer (30) and is down-regulated in prostate cancer (31). diagnosis was different depending on the chromosome 16q status Eighteen genes had significant lower expression in tumors with in that tumors with LOH on 16q were diagnosed significantly later LOH (Wilcoxon rank sum test) but none of these genes were (median age at diagnosis 33 months) compared with retinoblas- completely shutdown in any tumor (Table 1). tomas without LOH (median age at diagnosis 11 months, Wilcoxon CDH13, a putative tumor-suppressor gene in several cancer types P < 0.001; Fig. 2). Previously, we found that age at diagnosis is (32–34), is located in the minimal deleted region identified here but distinct depending on the presence of gains on 1q and 6p (8, 10). did not show detectable expression levels on our array. We used Analyzing the age distribution of the tumors studied here, we found

Table 3. Cross-tabulation and Fisher’s exact test of LOH 16q, gain 1q, and gain 6p versus seeding

LOH 16q Gain 1q Gain 6p

Seeding No Yes No Yes No Yes

No 10 1 8 3 8 3 Local 6 0 3 3 3 3 Diffuse 8 12 5 15 5 15 Fisher’s exact test P = 0.002 P = 0.03 P = 0.03

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Table 4. Results of the proportional odds model with seeding as ordinal outcome and LOH 16q, gain 1q, and gain 6p as predictors

Model without age, OR (95% CI) P Model with inclusion of age, OR (95% CI) P

LOH 16q 14.3 (1.60–128) 0.02 23.3 (1.05–519) 0.047 Gain 1q 1.53 (0.27–8.65) 0.63 1.98 (0.31–12.5) 0.47 Gain 6p 3.09 (0.56–17.0) 0.19 3.48 (0.59–20.6) 0.17 Age (y) — — 0.67 (0.23–1.92) 0.45

that tumors without gains on 1q are diagnosed at a median age of (M22731) at markers located in the minimal deleted region that 10 months (first and third quartiles 4and 14months, respectively) showed no copy number changes (isodisomy). Isodisomy at 16q, and tumors without gains on 6p at an age of 10 months (first and which was also reported in some breast cancer samples (40), third quartiles 4and 15 months, respectively; Table 2). Tumors with suggests a classic two-step mutation inactivation of putative gains on 1q or 6p are diagnosed at a median age of 25 months (first tumor-suppressor genes in this region (41). Taking into account and third quartiles 15 and 33 months and 14and 34months, that 16q was implicated in parent-of-origin effects (42), an respectively). We found no significant association between the alternative explanation might be that loss of the active copy of presence of LOH on 16q and partially or completely differentiated an imprinted gene in this region contributes to tumor progression. histology, tumor necrosis, or tumor calcification (data not shown). However, we could show that the parental origin of 16q losses is Some patients with retinoblastoma show tumor cells floating not limited to only one sex and, therefore, our data do not support within the vitreous cavity (vitreous seeding). The presence of the observation that loss of the active allele of an imprinted locus vitreous seeding was ascertained by indirect ophthalmoscopy at the in this region plays a role in the progression of retinoblastoma. A first presentation of the patient. Eyes with local seeding two-step inactivation of a tumor suppressor may result in loss of show few tumor cells in the vitreous that are confined to the gene expression. By analysis of microarray expression data, we immediate vicinity of the bulk tumor. Diffuse seeding is diagnosed if found that 18 of 64genes located in the retinoblastoma minimal tumor cells are scattered in the vitreous cavity. We analyzed if deleted region and represented on the microarray show lower vitreous seeding is associated with the presence of genetic median expression in tumors with LOH at 16q. However, none of alterations on 16q, 1q, and 6p. For each aberration, there was a these genes showed loss of expression in any sample. shift to the higher categories, i.e., local and diffuse seeding (Table 3). Lung cancer is a second tumor in patients with hereditary In all cases, Fisher’s exact test rejected the hypothesis of retinoblastoma (43). This indicates that this tumor may develop independence between the aberration and seeding. The highest along similar mutational pathways. The CDH13 gene, which is shift was observed for LOH 16q (P = 0.002). A similar result was located in the retinoblastoma minimal deleted region defined here, obtained when applying the proportional odds model (Table 4). is a candidate suppressor in lung cancer (33). Using quantitative RT Again, LOH 16q showed the highest effect on seeding with an OR of real-time PCR, we found that RNA levels of this gene are variable but 14.3 [95% confidence interval (95% CI), 1.60–127.8]. The wide range show no correlation with allele loss in the minimal deleted region. of the 95% CI is due to the small number of patients. In contrast, Moreover, Marchong et al. (6) previously showed that there is no gain 1q and gain 6p showed no influence on seeding in the differential expression of CDH13 between retina and retinoblastoma. combined analysis (OR, 1.53; 95% CI, 0.27–8.65 and OR, 3.09, 95% CI, To find out if a mutant form of CDH13 is expressed in retino- 0.56–17.0, respectively). These results changed only slightly when blastoma, we sequenced the coding region in 19 retinoblastomas age was included in the model as continuous covariate (Table 4). but identified no somatic mutation. Therefore, it is unlikely that The OR of LOH 16q increased to 23.3 with a larger 95% CI (1.05– inactivation of CDH13 is the target of 16q loss in retinoblastoma. 519), whereas the results of gain 1q and 6p did not show major All retinoblastoma samples analyzed in our study are either changes (OR, 1.98; 95% CI, 0.31–12.5 and OR, 3.48; 95% CI, 0.59–20.6). homozygous or compound heterozygous for mutations at the RB1 Age had no significant effect on seeding (OR, 0.67; 95% CI, 0.23– locus (44–46),13 which is to be expected if inactivation of the RB1 1.92). Similar results were obtained with other age transformations gene initiates the development of retinoblastoma. In addition, this like logarithm or classification of age in groups (data not shown). indicates that the tumor cell content of the samples used for DNA preparation is high. Most retinoblastomas with LOH on 16q showed Discussion almost complete loss of one allele at every informative marker on this chromosome arm. Interestingly, 3 of 18 tumors showed allelic Our results define a minimal deleted region on 16q that extends imbalance at several adjacent informative markers on 16q. from Mb 82.7 to the telomere of this chromosome arm. This According to matrix CGH, the genetic changes in these particular particular region has also been identified as a candidate location of tumors are complex (Fig. 1C). Specifically, matrix CGH shows that one or more tumor suppressor genes in other tumor entities, allelic imbalance can correspond to copy number gains (tumor namely gallbladder carcinoma (35), lung cancer (36), prostate M22641), losses (tumor M5715), or may go without detectable cancer (31), ovarian cancer (32), and, most notably, breast cancer changes (M22731). It is understandable that allelic imbalance is (37) and hepatocellular cancer (38). Copy number analyses have observed if there are relative gains of one chromosome homologue. shown that most tumors with 16q alterations have a physical It is less obvious why tumors M5715 and M22731 show allelic deletion of this region (39), and this is also true for most of our samples with allele loss. However, we identified some retinoblastomas with LOH (M22641 and M24794) or allelic imbalance 13 D.R. Lohmann, unpublished data.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. 16q Loss in Retinoblastoma and Vitreous Seeding imbalance in regions without gains. To explain similar findings on only four combinations of genetic alterations: Fifteen tumors have chromosome 8q in bladder cancer, it was proposed that allelic no alteration; 6 tumors show 6p gains only; 12 tumors have gains at imbalance can reflect the presence of more than one evolving both 6p and 1q; and 12 tumors show alterations at all three subclone with allele loss (47). In fact, a pattern with LOH in some genomic regions. Second, median age at diagnosis is increasing regions, and allelic imbalance in other regions, on 16q may result if with the number of genetic alterations: 8, 16, 17, and 35 months for clonal selection favors loss of the entire long arm of chromosome tumors with no alterations, 6p gains only, gains at 6p and 1q, and 16 in a retinoblastoma that primarily had LOH in only some regions alterations at all three regions, respectively. This also suggests an of 16q, such as tumors M5450 and M24794. order of genetic events with 6p gains occurring first, followed by 1q Several tumor entities show allele loss on 16q22.1 in addition to gains and, finally, 16q LOH. However, data from more retinoblas- alterations on 16q24.3 (37, 48). Therefore, it is plausible that growth tomas are needed to test this model. of retinoblastoma is enhanced by loss of a second region on 16q. We found that the presence of 16q alterations is strongly Marchong et al. identified genomic loss of chromosome 16q22 in associated with diffuse vitreous seeding, which reflects the ability retinoblastoma with the highest frequency of genomic loss (22 of 41 of retinoblastoma cells to detach from the bulk tumor and samples, 54%) for a sequence-tagged site located in the CDH11 gene proliferate into small cell clusters. Loss of cell-to-cell contacts and (Mb 63.5). Interestingly, using immunoblot analyzes, Marchong et al. single cell invasion of the surround has been seen in tumors such have shown loss or decrease of intact CDH11 and expression of a as diffuse-type gastric cancer, which is associated with mutations variant form in many retinoblastomas. In our study, the proportion in E-cadherin, CDH1 (12, 49). It is understandable that tumors with of tumors with allelic imbalance or loss at markers in the region of E-cadherin loss show a diffuse growth pattern because cadherin the CDH11 gene is also high [D16S3080 and D16S3050 with LOH in genes code for cell adhesion molecules that mediate cell-to-cell 15 of 29 (52%) and 14of 31 (45%)samples, respectively]. At the RNA adhesion. CDH1 and several other cadherin genes are located on level, we found lower expression of CDH11 in tumors compared 16q. The presence of diffuse vitreous seeding in tumor M24794, with normal retina but no tumor showed loss of expression. Further which has no copy number changes on 16q outside of the minimal studies are needed to determine if mutation of CDH11 contributes deleted region, suggests that alteration of this part of 16q only is to progression of retinoblastoma (6). associated with diffuse vitreous seeding. A cadherin gene, CDH13, Marchong et al. (6) found a second hotspot of alteration at is located in this region but shows neither differential expression D16S422 (16q23), the most distal marker that was analyzed in this (6) nor mutations (this study) in retinoblastoma. Further studies study. Nine of 23 (39%) samples showed LOH, a proportion that are needed to determine if other protein coding genes in the compares well with our findings for the identical marker (11 of 35, minimal deleted region have acquired mutations and to find out if 31%). In our study, all tumors with LOH at D16S422 also had LOH at these alterations explain association with vitreous seeding. further distal markers (16q24). Moreover, one tumor without LOH Alternatively, loss in 16q24may not target a protein coding gene at D16S422 showed LOH in 16q24 (M24794). It would be interesting but a micro-RNA (miRNA). Progression of some cancers is to test the tumor samples investigated by Marchong et al. for LOH at accompanied by alterations in miRNA genes, including gene loss 16q24markers. (reviewed in ref. 50). However, as of now, no miRNA gene is In previous analyses, we found that distribution of age at mapped to 16q24(Ensembl v40).From a clinical point of view, diagnosis of unilateral retinoblastoma varies with the presence of diffuse vitreous seeding is important because its presence has a genomic alterations (8, 10). Here, we found that tumors with LOH grave effect on therapeutic decisions in patients with retinoblas- on 16q are diagnosed later than tumors without LOH. To account toma. The strong association to loss of a region on 16q24identified for the correlation of genomic alterations and age at diagnosis, it here will help to identify the mechanisms that underlie this adverse has been suggested that distinct mutational pathways can result in tumor growth pattern. the development of a retinoblastoma after mutational inactivation of the RB1 locus (8, 10, 16). Probably, depending on the mutational Acknowledgments path taken, different time periods pass until a tumor focus with mutations in both RB1 alleles reaches the size that allows a clinical Received 4/12/2006; revised 10/16/2006; accepted 11/3/2006. Grant support: Deutsche Forschungsgemeinschaft grants Lo 530/6-1, Ri 1123/1-1, diagnosis to be made. It is remarkable that the age distribution is and KFO 109 (Klinische Forschergruppe Ophthalmologische Onkologie und Genetik). not distinct between tumors with LOH only at some 16q loci The Kulturstiftung Essen has supported the medical data warehouse. (M5715, M22641, M22731, M5450, and M24794) and tumors with The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance LOH at all informative 16q loci. This suggests that the minimal with 18 U.S.C. Section 1734solely to indicate this fact. deleted region on 16q24contains the target that is critical for the We thank Susanne Weber and Thomas Lehnert of Kulturstiftung Essen; Pieter de Jong and the BACPAC Resources Centre (http://bacpac.chori.org) for providing DNA difference in biological behavior. An alternative explanation is that of the human 32k BAC Re-Array Set; Nigel Carter and the Mapping Core and Map progression of retinoblastoma might be characterized by a Finishing groups of the Wellcome Trust Sanger Institute for initial clone supply and stepwise accumulation of genetic changes (4). Our data support verification of the 1Mb array; the COST B19 Action ‘‘Molecular Cytogenetics of Solid Tumours’’ for the assembly of the subtelomeric array; and Inga Nowak, Saskia Seeland, such a model in two ways. First, co-occurrence of genetic and Michael Zeschnigk for their assistance in mutation and methylation analysis of alterations is not random. Forty-five of 53 tumors show one of the CDH13 gene.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Allelic Loss in a Minimal Region on Chromosome 16q24 Is Associated with Vitreous Seeding of Retinoblastoma

Sandrine Gratias, Harald Rieder, Reinhard Ullmann, et al.

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