Selective Loss of Chromosome 11 in Pheochromocytomas Associated with the VHL Syndrome

Selective loss of chromosome 11 in pheochromocytomas associated with the VHL syndrome

Weng Onn Lui1, Jindong Chen2, Sven GlaÈ sker3, Bernhad U Bender3, Casey Madura2, Sok Kean Khoo2, Eric Kort4, Catharina Larsson1, Harmut PH Neumann*,3 and Bin Tean Teh*,1,2

1Department of Molecular Medicine, Karolinska Hospital, Stockholm, Sweden; 2Laboratory of Cancer Genetics, Van Andel Research Institute, Grand Rapids, Michigan, MI 49503, USA; 3Department of Nephrology and Hypertension, Albert-Ludwigs- University, Freiburg, Germany; 4Laboratory of Analytical, Cellular and Molecular Microscopy, Van Andel Research Institute, Grand Rapids, Michigan, MI 49503, USA

By using comparative genomic hybridization (CGH), we tions. These sequential alterations have been well characterized the genetic pro®les of 36 VHL-related characterized in certain types of malignancies such as pheochromocytomas. We then compared the results with colorectal cancer (Fearon and Vogelstein, 1990). those of sporadic and MEN 2-related pheochromocyto- However, in the majority of cancer, this information is mas. In 36 VHL-related tumors, loss of chromosome 3 and still either lacking or incomplete. Furthermore, in some chromosome 11 were found in 34 tumors (94%) and 31 tumors, it is not clear if the subgroups, such as the tumors (86%), respectively. There was signi®cant con- hereditary form and its sporadic counterpart, share the cordance of deletions in chromosomes 3 and 11 (Kap- same genetic routes. In benign parathyroid tumors, for pa=0.64, P=0.0095), suggesting that they are involved in example, we have previously demonstrated the existence two dierent but necessary and complementary genetic of at least two distinct genetic pathways in play, which pathways. The loss of chromosome 11 appeared to be are mutually exclusive (Farnebo et al., 1999). speci®c for VHL-related pheochromocytoma as it was not We have previously studied a large number of present in any of the 10 VHL-related CNS hemangio- patients with VHL (Brauch et al., 1995), an autosomal blastomas studied and was signi®cantly less common when dominant cancer syndrome characterized by pheochro- compared with (a) sporadic pheochromocytomas from mocytoma, retinal angioma, CNS hemangioblastoma previously published results (13%; P=50.0001), and (b) and clear cell renal cell carcinoma. The primary disease MEN 2-related pheochromocytomas from this and pre- gene, VHL, is located on chromosome 3p25 and viously published studies (30%; P=0.0012). In summary, identi®ed in 1993 (Latif et al., 1993). In concurring with this is the ®rst report of a novel consistent genetic Knudson's two-hit mutation theory, these VHL tumors alteration that is selected and speci®c for VHL-related harbor germline inactivating mutations and lose the pheochromocytoma, besides the two hits of the VHL gene. function of the remaining copy through hypermethyla- Oncogene (2002) 21, 1117 ± 1122. DOI: 10.1038/sj/onc/ tion or chromosome 3 deletions. However, besides the 1205149 alterations in the VHL gene represent the ®rst two hits, no other genetic alteration is known in these VHL- Keywords: von Hippel-Lindau; VHL; pheochromocy- related tumors. To this aim, we set out to study 36 toma; comparative genomic hybridization VHL-related pheochromocytoma by comparative genomic hybridization. In addition, we compared their genetic pro®les with those of (1) multiple endocrine Introduction neoplasia type 2 (MEN 2)-related pheochromocytomas; (2) previously reported sporadic pheochromocytomas; Tumorigenesis is a multi-step process during which a and (3) VHL-related CNS hemangioblastomas. Having series of genetic alterations take place. Each genetic identi®ed and con®rmed the loss of chromosome 11 as a alteration in the dividing cells would confer the cells frequent genetic alteration, besides the VHL gene, in the additional transforming advantage propelling them into VHL-related pheochromocytomas, we proceeded to the next step. Consequently, these genetic alterations are perform mutation analysis of the two known pheochro- considered potential targets for therapeutic interven- mocytoma-related tumor suppressor genes on chromosome 11: (1) MEN1 gene in 11q13, a tumor suppressor gene that causes multiple endocrine neoplasia type 1 *Correspondence: BT Teh, Van Andel Research Institute, 333, which has been reported to be associated with Bostwick NE, Grand Rapids, MI 49503, USA; pheochromocytoma (Schussheim et al., 2001); and (2) E-mail: [email protected] or H Neumann, Medizinische SDHD in 11q23, which is responsible for hereditary Universtitatsklinik, Hugstetterstr. 55, 79106 Freiburg, Germany; paraganglioma type 1 (Baysal et al., 2000) and E-mail: [email protected] Received 6 September 2001; revised 25 October 2001; accepted 30 considered to play a role in the pathogenesis of October 2001 pheochromocytoma (Gimm et al., 2000). Genetic profiles of VHL-related pheochromocytoma WO Lui et al 1118 Results ances in all three groups of tumors: VHL-related pheochromocytomas, MEN 2-related pheochromocy- In the total of 51 tumors studied (36 VHL-related tomas and VHL-related hemangioblastomas. Figure 1 pheochromocytomas, 5 MEN 2-related pheochromo- showed the CGH pro®les of VHL-related pheochro- cytomas and 10 VHL-related hemangioblastomas), one mocytomas compared with those of VHL-related or more genetic imbalances were detected in 46 (90%) hemangioblastomas. Loss of chromosome 3p or more of them. Table 1 summarizes frequent genetic imbal- extensively the whole of chromosome 3 is the most

Table 1 Frequent non-random DNA copy number changes in 51 familial pheochromocytomas and hemangioblastomas Germline Losses Patient Sample Mutation Chr3 Chr11 Chr1 Others Gains

Pheochromocytomas VHL-associated I 1 505T4C3 11 II 2 505T4C3 12q III 3 505T4C 3 11p, 11q13-qter 18q IV 4 505T4C 3 11p, 11q13-qter 5 505T4C 6 505T4C 3 11p, 11q13-qetr V 7 505T4C 3 11p, 11q13-qter 9q32-qter, 19 VI 8 505T4C 3 11p 1p11-p31 9p, 13q21-q22 VII 9 505T4C3 11 10 505T4C3 11 11 505T4C 3 11p VIII 12 505T4C3 11 IX 13 505T4C 3p 11p, 11q13-qter 9p 6p, 7 X 14 505T4C 3p 11p 15q15-qter 7p13-q21 15 505T4C 3 11p, 11q13-qter 16 505T4C 3 11p, 11q13-qter 17 505T4C 3 11p, 11q13-qter 18 505T4C 3 11p, 11q13-qter XI 19 505T4C 3 11p, 11q13-qter XII 20 505T4C 3 11p, 11q13-qter XIII 21 505T4C 3 11p 2p, 17 XIV 22 505T4C 3 11p 23 505T4C XV 24 505T4C 3 11p, 11q13-qter 8 25 505T4C3p 26 505T4C 3 11p, 11q13-qter 27 505T4C 3 11p, 11q13-qter 4, 5q14-q22, 6q11-q23 9p, 13q14-q22 XVI 28 775C4G 3 11p, 11q13-qter XVII 29 746T4A 3 11p, 11q13-qter XVII 30 746T4A 3 11p, 11q13-qter 1p11-p31 6q11-q23, 9p, 13q14-q32 31 746T4A 3 11p, 11q13-qter 1p11-p31 4, 9p, 13q14-q32 32 746T4A 3 11p, 11q13-qter 9p, 12p, 12q13-q21 13q14-q32 XIX 33 695G4A3p XX 34 775C4G 3 11p, 11q13-qter 1p11-p31 2q11.2-q24 35 775C4G 3 11p, 11q13-qter 1p11-p31 2q11.2-q24 XXI 36 775C4G 3p 11p 1p11-p31 9p 9q32-qter, 19p Men-2-associated XXII 37 3 11p 1p11-p31 13q, 14q 38 3 11p 1p11-p31 13q, 14q, 21q XXIII 39 3 1p11-p31 2q21-q24, 4q, 9p 19 XXIV 40 3q 1p 6q, 21q 4p 41 3 1p 4q13-q21, 8, 13q, 21q 1q, 4p14-pter, 19, 22q Hemangioblastomas VHL-associated I 1 529insGCC 3 6q, 9p, 18 17, 19, 20q, 22q II 2 446A4G 3 6q, 9p, 18 16, 17, 19, 20q, 22q 3 446A4G3 III 4 443insTCT 3 18 5 443insTCT 3 6 443insTCT IV 7 699C4G 3 1p31-qter 4, 6q, 7p14-p15, 9p V 8 2 kb del 9 2 kb del VI 10 ? kb del 3p21-pter

Oncogene Genetic profiles of VHL-related pheochromocytoma WO Lui et al 1119 common abnormality, which is detected in 34/36 (94%) of VHL-related pheochromocytomas and in 7/10 (70%) of VHL-related hemangioblastoma. The two VHL-related pheochromocytomas and three VHL- related hemangioblastomas without this loss also did not have any other detected genetic imbalances suggesting two possibilities: (1) sample `contamination' from too high percentage of normal tissues in the tumor samples; or (2) the imbalances were too small beyond the detection by CGH. In MEN 2-related pheochromocytoma, four samples showed loss of the whole chromosome 3 and one showed 3q loss only. The second most common genetic imbalance is the loss of chromosome 11, which was found in 31/36 VHL-related pheochromocytomas (86%): 25 of these tumors had loss of both 11p and 11q, whereas six have only 11p loss. The chromosome 11 loss was further con®rmed by microsatellite markers in 10 matched tumor sets (data not shown). In two cases of MEN 2- related pheochromocytomas (40%), loss of 11p was also found. However, most signi®cantly, chromosome 11 loss was not detected in any of the seven cases of VHL-related CNS hemangioblastomas that showed genetic imbalances. The third most common genetic imbalance was loss of 1p11 ± p31, which was present in 6/36 (17%) VHL- related pheochromocytomas and 5/5 (100%) MEN 2- related pheochromocytomas, and 1/10 (10%) VHL- related hemangioblastomas. The results of the loss of chromosomes 3 and 11 in these VHL-related pheochromocytomas were previously reported by a study using microsatellite markers (Bender et al., 2000) therefore further supporting our present results. We then compared our results with previously Figure 1 Summaries of DNA copy number alterations detected reported CGH results on benign pheochromocytomas: by CGH in 36 VHL-related benign pheochromocytomas (a), and 10 VHL-related CNS hemangioblastomas (b). Each line represents sporadic and MEN 2-related (Table 2). To date, two one alteration detected in one tumor with losses located to the left papers had been published on CGH of pheochromo- and gains to the right of the chromosome ideograms cytomas: one on both sporadic and MEN 2-related

Table 2 Recurrent chromosomal losses (by CGH) in benign pheochromocytomas from dierent genetic background in this study and literatures Sporadic (benign) VHL-related EdstroÈm et al., 2000; Dannenberg et al., 2000 This study Total Chromosomal loss (n=36) (n=32) P-value

(a) VHL-related versus sporadic 3p/whole chromosome 3 34 (94%) 9 (28%) 50.0001 11 (p or p and q) 31 (86%) 4 (13%) 50.0001 1p11-p31 6 (17%) 27 (84%) 50.0001

MEN2-related VHL-related This study and EdstroÈm et al., 2000 This Study Total Chromosomal loss (n=36) (n=32) P-value*

(b) VHL-related versus MEN2-associated 3p/whole chromosome 3 34 (94%) 5 (50%) 0.00031 11 (p or p and q) 31 (86%) 3 (30%) 0.0012 1p11-p31 6 (17%) 9 (90%) 50.0001

*n.s.=not signi®cant

Oncogene Genetic profiles of VHL-related pheochromocytoma WO Lui et al 1120 tumors (EdstroÈ m et al., 2000), while the other solely on selected and involved in two dierent but necessary sporadic tumors (Dannenberg et al., 2000). In compar- and complementary genetic pathways. It is likely that ing with sporadic pheochromocytomas, our study loss of chromosome 11 confers further growth showed that loss of chromosomes 3p and 11 in VHL- advantage to the tumors besides the inactivation of related pheochromocytomas, 94 and 86% respectively, the VHL gene. To date such genetic events have been were very signi®cantly more common than their demonstrated in familial cancer. For example, in sporadic counterparts (P=50.0001). The kappa familial breast cancer, BRCA1- and BRCA2-related statistic for the correlation of chromosomes 3 and 11 breast cancer and ovarian cancer, a high incidence of deletions among pheochromocytoma tumors was 0.63 p53 mutation has been identi®ed (Ramus et al., 1999; (P=0.0095), indicating good correlation of these two Smith et al., 1999) indicating the selection of p53 markers. The loss of chromosome 3 is not surprising mutants and its role in the tumors progression of since according to Knudson's two-hit mutation theory, BRCA1- and BRCA2-related tumors. the VHL-related tumors are supposed to have loss of The VHL protein is involved in the ubiquination of chromosome 3 (second-hit) where the mutated VHL hypoxia-induced factor (HIF)-1 (Kamura et al., 1999; tumor suppressor gene (®rst-hit) is located. However, Maxwell et al., 1999) and in the presence of mutated our ®nding of the loss of chromosome 11 is novel pVHL, HIF-1 accumulates and increases its transcrip- pointing to its selection and role(s) in VHL-related tional activity resulting in overexpression of angio- pheochromocytomas. Furthermore, there was a sig- geneic peptides (e.g., vascular endothelial growth ni®cant lack of chromosome 1p loss in the former factor) (Iliopoulos et al., 1996) and the hypervascular- group when comparing with sporadic or MEN 2- ized state of the majority of VHL-related tumors. related tumors (P=50.0001), further suggesting that However, VHL disease has been distinctly subdivided the VHL-related tumors most probably run a dierent into two clinical subtypes based on the presence or pathway of tumorigenesis. We also showed that the absence of pheochromocytomas, which are also loss chromosome 11 was pheochromocytoma-speci®c correlated with distinct group of mutations (Chen et since it is not present in any of the CNS hemangio- al., 1995). Interestingly, unlike other VHL-related blastomas studied. The other genetic imbalances are tumors (e.g., renal cell carcinoma, retinal angioma summarized in Table 1. Finally, within the VHL- and CNS hemangioblastoma), pheochromocytoma is related pheochromocytoma group, we found that there not characterized by hypervascularization. It is are more non-chromosome 3, non-chromosome 11 possible that following VHL inactivation, pheochro- losses in the non-505 than the 505 mutation group mocyotmas may diverge to follow a distinct genetic (P=0.036). pathway in which loss of chromosome 11 is involved. As far as we know, there are only two known The exact molecular mechanism underlying the pheochromocytoma-related tumor suppressor genes, distinction between the two clinical subgroups and i.e., SDHD and MEN1, but we did not ®nd any how chromosome 11 loss is involved need further mutation in the tumors that showed LOH of studies. In this study we attempted to explore if any chromosome 11 although we could not rule out other of the two known pheochcromocytoma-related tumor genetic aberrations like large deletion or hypermethyla- suppressor genes in chromosome 11 was involved. No tion. mutation was found which suggested a few possibilities: (1) other tumor suppressor gene(s) located in chromosome 11 may be involved; (2) the retained Discussion allele may be inactivated by other inactivating mechanisms such as methylation or promoter muta- Pheochromocytoma is a neuroendocrine tumor arising tion and (3) haploinsuciency of gene(s) on chromo- from the paraganglia system found in the adrenal some 11 is by itself sucient to promote tumor medulla, whereas those found along the para-vertebral progression (Kwabi-Addo et al., 2001). and para-aortic axis, but outside the adrenals, are Finally, we also found that sporadic or MEN 2- called paraganglioma. The majority of pheochromocy- related pheochromocytomas appeared to have a tomas are sporadic cases but approximately 10% are distinct genetic pathway when compared with the familial cases, which are mainly associated with von VHL-related cases. In particular, loss of chromosome Hippel-Lindau disease (VHL), multiple endocrine 1p is important for both MEN 2-related and sporadic neoplasia type 2 (MEN 2) and neuro®bromatosis type tumors but not the VHL-related pheochromocytomas. 1 (NF 1) (EdstroÈ m et al., 2000). As the genes These ®ndings are consistent with a recent report by responsible for these hereditary cancer syndromes were Benn et al. (2000), who found loss of 1p in 61% of identi®ed (i.e., ®rst and second hit), attention has been sporadic pheochromocytomas and 80% of MEN2- shifted to identi®cation of additional genetic altera- related pheochromocytomas but none of the two tions, which represent the subsequent `hits' in their VHL-related pheochromocyotmas. It will be interest- tumor progression. In the present study, we demon- ing to conduct a large study to correlate the clinical strated the selection of chromosome 11 loss in VHL- parameters of these various types of pheochromocy- related pheochromocytoma which appears to be highly tomas with these genetic alterations. This information speci®c. Furthermore, the signi®cant concordance of may in¯uence our management of the disease based chromosomes 3 and 11 deletions suggests both are on its genetic pro®les.

Oncogene Genetic profiles of VHL-related pheochromocytoma WO Lui et al 1121 Materials and methods described (Canzian et al., 1996). Ten VHL-related pheochromocytomas (eight with chromosome 11 LOH and two We studied 36 VHL-related pheochromocytomas (from 21 without LOH) were studied by using six highly poly- patients), ®ve MEN 2-related pheochromocytomas (from morphic microsatellite markers from chromosome 11: three patients) and 10 VHL-related CNS hemangioblastomas D11S4046, D11S904, D11S905, D11S937, D11S925 and (from six patients). The study was approved by the Ethical D11S968 from Applied Biosystem (ABI). PCR ampli®ca- review board of the University of Freiburg, Germany and the tions were performed according to the manufacturer's Internal Review Board of the Van Andel Research Institute, recommended protocols and set up in the Gene Amp USA. The majority of tumors came from the large, multi- 9700 termocycler (ABI). Amplicons were denatured and generationed, Black Forest VHL family, which is associated size-fractioned on a 4.2% polyacrylamide gel on an ABI with the c505T4C mutation (Brauch et al., 1995) (Table 1). 377 sequencer (ABI). Genotypes were determined using Some of the pheochromocytomas represent what appear to GENESCAN version 3.1 and GENOTYPER version 2.5 be clinically distinct tumors, based on both spatial and (ABI). temporal dierences arising from the same patients. We also included previously reported CGH results on benign Statistical analysis pheochromocytomas for comparison and statistical analysis: 32 sporadic cases (Dannenberg et al., 2000; EdstroÈ m et al., CGH genetic pro®les of these tumors were divided into 2000) and ®ve MEN 2-related (EdstroÈ m et al., 2000). three sub-groups: VHL-related pheochromocytomas, MEN 2-related pheochromocytomas, and VHL-related hemangioblastomas. We then compared the most frequent genetic Comparative genomic hybdrization (CGH) imbalances of VHL-related pheochromocytomas with their High molecular weight DNA was extracted from these fresh sporadic and MEN 2-related counterparts. The sporadic frozen tumors using standard methods. CGH was performed tumors were taken from previously reported series whereas essentially as previously described (Kallioniemi et al., 1992). the MEN 2-related tumors were derived from the present Brie¯y, tumor DNA samples were labeled with FITC-12- series plus those previously reported cases. A Fisher's dUTP (DuPont, Boston, MA, USA) by nick translation and exact test with the Stat View 5.0.1 software was then normal reference DNA was labeled with Spectrum Red (Vysis performed in comparing the VHL-related cases against the Inc., Downers Grove, IL, USA). Tumor and reference DNAs sporadic cases (Table 2a) as well as the MEN 2-related were mixed with unlabeled Cot-1 DNA (Gibco, BRL), cases (Table 2b). The same analysis was also performed to denatured, and applied onto denatured metaphase slides of compare the non-chromosome 3, non-chromosome 1 losses normal lymphocytes (Vysis Inc.). After hybridization at 378C in the tumors with 505 mutations and those with non-505 for 48 h, the slides were washed in 0.46SSC/0.3% NP-40 at cases. There are 6/27 cases with these losses in the former 748C for 2 min and in 26SSC/0.1% NP-40 at room group versus 6/9 in the latter group. To test the temperature for 1 min. After air-drying, the slides were hypothesis that deletions in chromosomes 3 and 11 were counterstained with 4,6-diamidino-2-phenylindole (DAPI) correlated to each other as components of dierent (Sigma Inc.), 0.1 mg/ml, in an antifade solution (Vectashield, pathways, the kappa statistic was calculated for these Vector Inc.). A control hybridization was also performed two markers. In this context, the kappa statistic could be using normal female DNA against normal male DNA. thought of as a measure of inter-gene reliability (i.e., they Ten three-color digital images (DAPI, FITC, and Spectrum were consistent and inter-related markers for the same Red ¯uorescence) were collected from each hybridization pathway) and analogous to inter-rater reliability. Because using a Zeiss Axioplan 2 (Carl Zeiss Jena GmbH, Jena, of the small number of discordant pairs, the exact method Germany) epi¯uorescence microscope and a Sensys (Photo- was used. metrics, Tuscon, AZ, USA) charge-coupled-device camera interfaced to an IPLab Spectrum 10 workstation (Signal Mutation analysis of the MEN1 and SDHD gene Analytics Corporation, Vienna, VA, USA). Relative DNA sequence copy number changes were detected by analysing Four VHL-associated benign pheochromocytomas, which the ¯uorescence intensities of tumor and normal DNAs along demonstrated loss of both 11p and 11q, were selected for the length of all chromosomes in each metaphase spread. The mutation screening of the MEN1 (10 exons) and SDHD absolute ¯uorescence intensities were normalized so that the (four exons) genes. The primers will be provided on average green-to-red ratio of all chromosomes in each request. PCR was performed in a 50 ml reaction volume metaphase was 1.0. The ®nal results were plotted as a series containing 50 ng DNA, 20 mM Tris-HCl (pH 8.4), 50 mM of green-to-red ratio pro®les and corresponding standard KCl, 1.5 mM MgCl2, 0.2 mM each primer 0.2 mM dATP, deviations (s.d.s) for each human chromosome from p- dGTP, dCTP, dTTP each and two units of Taq DNA telomere to q-telomere. At least 12 ratio pro®les were polymerase (GIBCO BRL, Life Technologies). Ampli®ca- averaged for each chromosome to reduce noise. Green-to- tion was carried out using GeneAmp PCR System 9700 red ratios 41.20 were considered as gains of genetic material, thermal cycler (ABI) at the following settings: after a and ratios 50.80 as losses. Heterochromatic regions, the denaturation at 948C for 5 min, samples were ampli®ed short arm of the acrocentric chromosomes and chromosome for 35 cycles at 948C, 30 s; 55 ± 588C, 30 s; 728C, 45 s, Y were not included in the evaluation. with a ®nal extension at 728C for 10 min. After ampli®cation, all the PCR products were subjected to puri®cation using Microcon YM-100 column (Amicon, Confirmation of loss of chromosome 11 by microsatellite Millipore) following the manufacturer's manual and markers subsequently direct DNA sequencing using ABI PRISM We con®rmed the chromosome 11 LOH by genotyping the BigDye Terminator cycle sequencing ready reaction kit tumor samples that had matched DNA as previously (ABI).

Oncogene Genetic profiles of VHL-related pheochromocytoma WO Lui et al 1122 References

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Oncogene