Array comparative genomic hybridization identifies novel potential therapeutic targets in cholangiocarcinoma

GENES, CHROMOSOMES & CANCER 48:544–551 (2009)

Array Comparative Genomic Hybridization Identifies a Distinct DNA Copy Number Profile in Renal Cell Cancer Associated with Hereditary Leiomyomatosis and Renal Cell Cancer Taru A. Koski,1 Heli J. Lehtonen,1 Kowan J. Jee,2 Shinsuke Ninomiya,2 Simon A. Joosse,3 Pia Vahteristo,1 Maija Kiuru,1 Auli Karhu,1 Heli Sammalkorpi,1 Sakari Vanharanta,1 Rainer Lehtonen,1 Henrik Edgren,4,5 Petra M. Nederlof,3 Marja Hietala,6 Kristiina Aittoma¨ki,1,7 Riitta Herva,8 Sakari Knuutila,2 Lauri A. Aaltonen,1 and Virpi Launonen1* 1

Department of Medical Genetics,Biomedicum Helsinki,University of Helsinki,Helsinki,Finland Department of Pathology,Haartman Institute and HUSLAB,University of Helsinki and Helsinki University Central Hospital, Helsinki,Finland 3 Department of Experimental Therapy,The Netherlands Cancer Institute, Amsterdam,The Netherlands 4 Medical Biotechnology Centre,VTT Technical Research Centre of Finland,University of Turku,Turku,Finland 5 Genome-Scale Biology Research Program,Biomedicum Helsinki,University of Helsinki,Helsinki,Finland 6 Department of Medical Genetics,University of Turku and Turku University Hospital,Turku,Finland 7 Department of Clinical Genetics,Helsinki University Central Hospital,Helsinki,Finland 8 Department of Pathology,Oulu University Hospital,Oulu,Finland 2

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a tumor predisposition syndrome with cutaneous and uterine leiomyomatosis as well as renal cell cancer (RCC) as its clinical manifestations. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (fumarase) gene. In this study, we used array comparative genomic hybridization to identify the specific copy number changes characterizing the HLRCC-associated RCCs. The study material comprised formalin-fixed paraffin-embedded renal tumors obtained from Finnish patients with HLRCC. All 11 investigated tumors displayed the papillary type 2 histopathology typical for HLRCC renal tumors. The most frequent copy number changes detected in at least 3/11 (27%) of the tumors were gains in chromosomes 2, 7, and 17, and losses in 13q12.3-q21.1, 14, 18, and X. These findings provide genetic evidence for a distinct copy number profile in HLRCC renal tumors compared with sporadic RCC tumors of the same histopathological subtype, and delineate chromosomal regions that associate with C 2009 Wiley-Liss, Inc. V this very aggressive form of RCC.

INTRODUCTION

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is characterized by leiomyomas of the uterus and skin as well as renal cell cancer (RCC) (Launonen et al., 2001; Tomlinson et al., 2002). The disease-predisposing gene fumarate hydratase (FH) encodes fumarase, an enzyme involved in mitochondrial tricarboxylic acid cycle (TCAC) (Tomlinson et al., 2002). To date, more than 120 FH mutation-positive HLRCC families have been identified worldwide. Benign leiomyomas occur with high penetrance in these families, but malignant RCC tumors are found in about 20% of the families (Tomlinson et al., 2002; Alam et al., 2003, 2005; Toro et al., 2003; Chan et al., 2005; Badeloe et al., 2006; Chuang et al., 2006; Lehtonen et al., 2006, 2007; Wei et al., 2006; Refae et al., 2007). HLRCC renal carcinomas C 2009 Wiley-Liss, Inc. V

typically occur at young age and are solitary, unilateral, and associated with aggressive disease course (Launonen et al., 2001; Grubb et al., 2007). A key feature in HLRCC renal cell carcinomas is their morphology, as they typically display papillary type 2 histology with large cells with abundant eosinophilic cytoplasm, large Supported by: European Commission, Grant number: LSHCCT-2005-518200, Supported by: the Academy of Finland, Grant numbers: 213183, 214268, 212901; Supported by: the Center of Excellence in Translational Genome-Scale Biology, the Sigrid Juselius Foundation, the Cancer Society of Finland. *Correspondence to: Virpi Launonen, PhD, Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, PO Box 63 (Haartmaninkatu 8), FIN-00014, Finland. E-mail: virpi.launonen@stuk.fi Received 16 November 2007; Accepted 27 February 2009 DOI 10.1002/gcc.20663 Published online 16 April 2009 in Wiley InterScience (www.interscience.wiley.com).

545

f

e


(5 yrs) RCC RCC 49 36 71

Alive 42 72

Kiuru et al., 2001. Lehtonen et al., 2006. g No 2nd hit in the FH gene found in the renal tumor.

Papillary type 2 (4) Papillary type 2 (4) Papillary type 2 (4)

Papillary type 2 (NId) Papillary type 2 (3–4) Papillary type 2 (4)

NId 9 Whole Kidney 2 8 Whole Kidney RCC
(24 yrs) RCC 27 Alive 68 26 35 68

Papillary type 2 (4) Papillary type 2 (NId) 5 9 RCC RCC 49 43 48 32

33 39

34 40

RCC RCC

22 13

Papillary type 2 (3) Papillary type 2 (3)

Solitary, unilateral, growth to vena cava, metastases in lungs and mediastinum Solitary, unilateral, metastases in lungs and bones Solitary, unilateral, growth to vena cava, metastases in retroperitoneum Solitary, unilateral, metastases in lymph nodes Solitary, unilateral, metastases in adrenal gland and lymph nodes Solitary, unilateral, metastases in lymph nodes Solitary, unilateral, no metastases Solitary, unilateral, metastases in liver, lung and para-aortal lymph nodes Solitary, unilateral, no metastases Solitary, unilateral, metastases in liver Multiple, unilateral, growth to vena cava, metastasis in lymph node Papillary type 2 (4) 8 RCC 48 42

Histology (Fuhrman grade) RCC tumor size (cm) Cause of death (Follow-Up time) Age at death (yrs) Age at RCC diagnosis (yrs)

TABLE 1. Characteristics of the Finnish HLRCC Patients with RCC

nuclei, and prominent inclusion-like eosinophilic nucleoli. The Fuhrman nuclear grade is high, from 3 to 4. Recently, the histopathological spectrum of RCC in HLRCC expanded as four cases with collecting duct carcinoma and one case with conventional renal cancer were reported (Alam et al., 2003; Toro et al., 2003; Wei et al., 2006; Grubb et al., 2007; Lehtonen et al., 2007). The link between the mitochondrial dysfunction and tumorigenesis is still not completely clarified. Recent evidence suggests that pseudohypoxia caused by accumulation of a TCAC intermediate fumarate in fumarase deficient cells is at least one contributing factor. It has been shown that fumarate is an inhibitor of hypoxia-inducible factor (HIF) prolyl hydroxylases that normally hydroxylate HIFs (Isaacs et al., 2005). The unhydroxylated HIFs are not destroyed by the proteosome, which leads to aberrant stabilization of HIF in HLRCC renal tumors (Isaacs et al., 2005; Pollard et al., 2005). HIFs are transcription factors up-regulating the transcription of genes associated with e.g., angiogenesis and glycolysis (Isaacs et al., 2005). Apart from the FH mutations, genetic alterations in the HLRCC-associated RCCs are poorly understood. Therefore, we used array comparative genomic hybridization (aCGH) to examine genetic aberrations in 11 HLRCC-associated RCCs. We also studied the involvement of the MET gene in tumorigenesis of HLRCC-associated RCC. Germline and somatic MET mutations have been identified in hereditary papillary renal carcinoma (HPRC) and in some cases of sporadic papillary RCC, respectively (Schmidt et al., 1997).

Other features

THE GENOMIC PROFILE OF HLRCC RENAL TUMORS

b

a

Normal and tumor tissues analyzed. Launonen et al., 2001. c Tomlinson et al., 2002. d No information available.

Female Female Male FAM5f FAM5f FAM5f E1a,g E2 E3

H153Rf H153Rf H153Rf

Male Female Male FAM2b FAM3e FAM4f B2a C5a D7

541delAGc R300Xc H153Rf

Female Female FAM1b FAM2b M19 B1

541delAGc 541delAGc

Female Female FAM1 FAM1b M13 M17

M7

541delAG 541delAGc

Female

c b

FAM1

541delAG

c b a

Family Case

Altogether 11 formalin-fixed paraffin-embedded RCC tumors from Finnish patients with HLRCC were included in the study (Table 1). All patients carried a FH germline mutation: six harbored a 541delAG mutation, four had a H153R mutation, and one had a R300X mutation. All studied tumors displayed papillary type 2 histology, and all except one displayed a somatic second hit in the FH gene (Lehtonen et al., 2006). RCCs were diagnosed between 26 and 71 years of age (average 43.5 years, median 39 years). To date, two patients are still alive but all of the remaining nine have died of renal cancer. Eight patients

FH Mutation

Patients

Gender

MATERIALS AND METHODS

Genes, Chromosomes & Cancer DOI 10.1002/gcc

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KOSKI ET AL.

included in the study were females and three were males. We also analyzed formalin-fixed paraffin-embedded normal kidney tissue from four of the studied patients.

quality with the QC Metrics tool of the software. Genomic regions of amplifications and deletions were defined based on ADM algorithm with a moving average of 1 Mb.

DNA Isolation

Mutation Screening of the MET and CDH19 Genes

DNA was extracted using Qiagen DNeasy Kit (Quiagen GmbH, Hilden, Germany). A pool of normal female or male genomic DNA (derived from five normal individuals each) was used as a reference. The concentration and purity of genomic DNA were checked by NanoDrop ND1000 UV–VIS spectrophotometer (NanoDrop Technologies, Wilmington, Delaware) and the integrity was assessed by high-resolution agarose gel electrophoresis (Flesh GelTM MetaPhor, Cambrex, USA).

Mutation screening was performed by direct sequencing of all tumor samples (n ¼ 11). Only exons 14-19 of the MET gene were analyzed because they contain majority of the previously reported mutation sites (Trusolino and Comoglio, 2002). All encoding exons of the CDH19 gene were screened for mutations. The primer sequences and PCR conditions are available from the authors on request.

Array-Based Comparative Genomic Hybridization

For genome-wide array CGH, we used oligonucleotide-based human genome CGH 244K (Agilent Technologies, Palo Alto, CA) with microarray formats containing approximately 240,000 probes (mapped according to NCBI Genome Build 35 by the manufacturer). A total 1.5 lg of each genomic DNA was fragmented for labeling by AluI and RsaI (Promega, Madison, WI), and the digested DNA was purified with QIAprep mini kit (Quiagen, Valencia, CA). The digested length of DNA was checked with a high-resolution agarose gel electrophoresis (Flesh GelTM MetaPhor, Cambrex). Cy3-dUTP and Cy5-dUTP (PerkinElmer, Wellesley, MA) were used for the label, and subsequently the yield and specific activity of the label were analyzed by NanoDrop ND-1000 UV-VIS spectrophotometer (NanoDrop Technologies, Wilmington, DE). In addition, to validate the results, in two cases the ULS-labeling which is recommended for paraffin specimens, was repeated. The protocol followed manufacturer’s instructions (Agilent Genomic DNA ULS labeling kit). Hybridization was performed in a rotating chamber at 65 C for 40 hr. After final washes, slides were scanned using Agilent microarray confocal scanner G2565AA (Agilent Technologies). Images were analyzed using Agilent G2567AA Feature Extraction software (v7.5; Agilent Technologies) with the intensity-dependent linear normalization method. Genomic aberrations were defined using Agilent CGH Analytics (v3.4) software (Agilent Technologies) after checking the hybridization Genes, Chromosomes & Cancer DOI 10.1002/gcc

RESULTS

Genomic aberrations in HLRCC-associated RCCs were studied using Agilent 244k aCGH platform with protocols optimized for formalinfixed paraffin-embedded tumor material. The comparison of genomic profiles between ULSlabeling and normal labeling showed the same result by using the ADM algorithm. Data were derived from all 11 tumors (Table 1) and from four normal kidney samples included in the study. One tumor sample (B2) as well as all normal tissue samples displayed a normal genomic profile. The tumors typically displayed large gains or losses of whole chromosomes or chromosome arms. No high-level amplifications were found in any of the tumors. The most frequent chromosomal aberrations detected in at least 3/11 (27%) of the tumors were gains of the whole chromosomes 2, 7, and 17, whole chromosome losses of 14, 18, and X, and the loss of 13q12.3–q21.1 (Tables 2 and 3). Additionally, one of the three male patients had a loss of chromosome Y. Two tumors (B2 and C5) showed a tendency to gains in telomere regions (data not shown), but the possibility of a methodology related artifact could not be ruled out. Chromosome 19 often shows false-positive changes, especially in the telomeric regions that are typical G-band light areas. However, gain of whole chromosome 19 was not doubtful in case C5. The aCGH data has been placed in a previously published public repository (Scheinin et al., 2008) found at http://www.cangem.org (Experiment ID: CG-EXP-48). In addition to the loss of chromosome 18 in three tumors, a loss of the CDH19 gene at 18q22, was found in two tumors (M7 and M13, Fig. 1).

Loss of CDH19 gene only.

a

Minimal region of  2 tumors % of tumors with aberration in minimal region

E1 E2 E3

B2 C5 D7

M19 B1

M17

M13

M7

Sample

1p36.31p33 18.2

1p

1p36.31p33

M7 M13 M17 M19 B1 B2 C5 D7 E1 E2 E3 Minimal region of  2 tumors % of tumors with aberration in minimal region

Sample 1q

1p21.3p31.1 18.2

1p

1p21.3p31.1

3p

7 27.3

2 36.4

3q

7 7

2 2 8q24.3 18.2

8

8q24.3

9q34.11-q34.3 18.2

9

9q34.11-q34.3

11p15.4-pter 18.2

11

11p15.4-pter

11q12.2-q13.2 18.2

11

11q12.2-q13.2

18.2

5

5

5

6p21.2p21.31 18.2

6

6p21.2p21.31

18.2

9p

9p

9

10q23.1qter

11p

13q12.3q21.1 27.3

13

13q11q21.1

13q12.3q31.2

36.4

14

14

14

14

14

15

15q26.1q26.3 15q26.1q26.3 18.2

Loci with losses

16q23.1q24.3

17p

12p12.1-q21.1

TABLE 3. DNA Copy Number Losses Detected in HLRCC-Associated RCCs

7

3q

2

2

Loci with gains

TABLE 2. DNA Copy Number Gains Detected in HLRCC-Associated RCCs

45.5

18q22.1a

18 18

18

18q22.1

a

20p

17q 45.5

17 17

18q22.1a

16 18.2

16 16

17

17

17q

20

21q21.1qter

19

22q13.31q13.33 18.2

22

22q13.31q13.33

27.3

X

X

X

X

21q11.2-q21.1

Y

X

THE GENOMIC PROFILE OF HLRCC RENAL TUMORS

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Genes, Chromosomes & Cancer DOI 10.1002/gcc

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KOSKI ET AL.

Figure 1. Array CGH profiles of chromosome 18 generated from two tumor samples M7 and M13 (a). A magnification of the region for 18q22 (61778833-62878833 bp) shows loss of the CDH19 gene in both tumors (b). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

The deletion comprised approximately 1 Mb (from 62322304 bp to 62422219 bp). No pathogenic mutations of the CDH19 gene (NM_021153) were found after direct sequencing of all of the tumor samples (n ¼ 11). The success rate was 85%. The tumor of one patient (C5) was found to carry a heterozygous missense change c.1171G > A Genes, Chromosomes & Cancer DOI 10.1002/gcc

(p.Val391Met). The same change was also found in the corresponding normal tissue of the patient indicating a germline change. The implication of the MET gene in the FH-deficient RCC tumorigenesis was studied by direct sequencing. The MET gene was analyzed with 97% success rate. No mutations were found.

THE GENOMIC PROFILE OF HLRCC RENAL TUMORS

DISCUSSION

The unique set of HLRCC-associated RCCs gave us an opportunity to study genetic alterations contributing to HLRCC tumorigenesis. All of the tumors were from Finnish FH mutation-positive HLRCC patients. Of these tumors, all but one have been shown to carry a second hit in FH in addition to the germline mutation (Lehtonen et al., 2006). All RCC cases displayed papillary type 2 histology, which is the most common histopathological subtype associated with the syndrome. A distinct DNA copy number profile in the HLRCC-associated RCCs was identified using Agilent 244k aCGH platform optimized for formalin-fixed paraffin-embedded tumor material. The most frequent chromosomal aberrations were gains of chromosomes 2, 7, and 17 and losses of 13q12.3–q21.1, 14, 18, and X. We also studied a subset of the tumors using another aCGH method, BAC/PAC clone based aCGH. We were able to confirm a gain of chromosome arm 17q and losses of 13q, 14q, 18p, and X at a frequency of 38% (3/ 8) or higher by this method (unpublished data). Previous cytogenetic analyses of papillary RCC have demonstrated frequent trisomy of 3q, 7, 8, 12, 16, 17, and 20, and loss of the Y chromosome (Kovacs et al., 1991). Especially gains of the chromosomes 3, 12, 16, and 20 have been suggested to associate with a more aggressive behavior of the tumor (Kovacs, 1993a,b, 1999). Subsequently, allelic imbalance and CGH studies have shown, in addition to cytogenetic findings, gains in 5q and losses of 1p, 4q, 6q, 9p, 11p, 13q, 14q, 18, 21q, and X (Sanders et al., 2002; Prat et al., 2006 and references therein). As originally proposed by Delahunt and Eble (1997), papillary renal cell carcinomas can be further divided into type 1 and 2 based on their morphological features. Gunawan et al. (2003) studied cytogenetically the differences in genetic changes between these two groups and found that gain of 1q and losses of 8p, 11, and 18 were more common in papillary type 2 tumors. Using CGH, Jiang et al. (1998) found no differences between the two groups with the exception of chromosome 7 and 17 gains. Gains of 7p and 17p were detected in all type 1 tumors but only in 31% and 38% of the type 2 tumors, respectively. The most typical changes for type 2 tumors (detected in more than 30% of the tumors) were gains of 7, 12q, and 17, and losses of 4q, 6q, 9q, and X. Yang et al. (2005) have proposed to divide the papillary RCCs into two subclasses: The first

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class includes type 1, low-grade type 2, and mixed type 1/low grade type 2 tumors, and the second class includes type 2 tumors with highgrade and poor survival. Both types had gains in chromosomes 7, 12, 16, 17, and 20. Class 2 tumors exhibited more frequent gains of 1q, 2, and 8q and losses at 3p, 6q, and 14q. They also displayed fewer gains of chromosomes 3, 7, and 16. The same group has recently noticed similar changes when detecting copy number changes from gene expression data (Furge et al., 2007). The copy number gains seen in HLRCC-associated RCCs seem to overlap partly with aberrations seen previously in sporadic papillary RCCs. However, HLRCC-associated RCCs lack some changes seen in sporadic papillary RCCs, namely gains of 3, 5q, 8, 12, 16, and 20, of which 12q has been found in 31% of the sporadic papillary type 2 RCCs. (Kovacs, 1993a,b; Jiang et al., 1998; Kovacs, 1999; Sanders et al., 2002; Prat et al., 2006) The gain of chromosome 2 that Yang et al. found more often in sporadic high grade papillary type 2 RCCs was present in 4/11 of our tumors that all display the same histological type. On the other hand, the gains of 1q and 8q that were reported to be more frequent in class 2 samples were not seen in our data (Yang et al., 2005). The copy number losses of our HLRCC-associated RCC tumors are also partly overlapping with results of previous studies investigating copy number changes of papillary RCC tumors. Losses of 3p, 6q, 8p, and 11 previously reported in sporadic papillary type 2 RCCs were not found in our tumors (Gunawan et al., 2003; Yang et al., 2005). Our study also highlighted a putative gene, the CDH19 gene, which might be involved in tumorigenesis of the HLRCC-associated RCCs. A specific loss of the CDH19 gene at 18q22.1 was detected in two tumors, and in addition, three other tumors had a loss of whole chromosome 18. Mutation screening for somatic CDH19 mutations was performed in all tumors without identifying any pathogenic changes. However, further studies are required to assess the role of the CDH19 gene in HLRCC-associated tumors. The CDH19 gene was previously found to be down-regulated in head and neck cancer cell lines (Blons et al., 2002) On the other hand, knockdown of CDH19 expression was found to inhibit MCP-1-induced protein-induced angiogenesis (Niu et al., 2008). The MYC locus showed gain in only one of our tumors. Thus, MYC alterations, recently shown to be associated with high-grade papillary type 2 Genes, Chromosomes & Cancer DOI 10.1002/gcc

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tumors, do not seem to be as common in HLRCC-associated papillary type 2 RCCs (Furge et al., 2007). Involvement of the MET gene through somatic mutations was excluded suggesting the existence of distinct pathways for papillary RCCs associated with HLRCC. However, the whole chromosome 7 gain including MET at 7q31.2 was detected in three tumors but the significance of this finding in context with MET pathway is unclear. Patients with HLRCC-associated RCCs have a poor prognosis. Jiang et al. (1998) found that Xp loss was significantly associated with poor clinical outcome but could not confirm the previous association of gains in 12, 16, and 20 with poor prognosis (Kovacs, 1993a,b, 1999; Jiang et al., 1998). Of the patients included in this study, two (C5 and E1) are still alive. However, a specific genetic pattern of genetic alterations associated with favorable prognosis could not be derived from the limited sample set. Our data provide evidence for a distinct DNA copy number profile in HLRCC-associated papillary type 2 renal tumors and a divergent molecular pathway compared with the sporadic papillary type tumors. The results also highlight the chromosomal regions that associate with a very aggressive form of RCC. ACKNOWLEDGMENTS

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Genes, Chromosomes & Cancer DOI 10.1002/gcc