Genome-wide profiling of chromosomal alterations in renal cell carcinoma using high-density single nucleotide polymorphism arrays

Introduction

Renal cell carcinoma (RCC) accounts for about 3% of all new cancer cases and the incidence rates for all stages have been steadily rising over the last three decades.^1-3 Approximately one-third of patients present with metastatic disease at the initial diagnosis and more than 40% of patients will eventually die from their cancer.^4,5 Despite the introduction of new treatment regimens, surgical resection remains the only curative therapy for RCC.⁶ However, up to 50% of patients undergoing nephrectomy for clinically localized RCC will develop local recurrence or distant metastasis.⁷ These statistics highlight the need for detecting RCC at early stages and identifying markers that can predict prognosis and response to therapy. RCC comprises a heterogeneous group of epithelial tumors with various cytogenetic and molecular abnormalities and different histological features. As is the case for most cancers, clinical variables play a major role in prognosis of localized RCC. Several prognostic algorithms and nomograms for RCC survival or recurrence after nephrectomy have been developed that incorporate multiple clinicopathological prognostic factors such as TNM stage, Fuhrman grade, tumor size, performance status, etc.^5,8 Accumulation of genetic aberrations plays a pivotal role in the initiation and prognosis of RCC and other cancers. Therefore, integration of genetic markers into traditional approaches may allow a more accurate prediction of prognosis.

Clear cell RCC (ccRCC) accounts for 70% to 80% of all RCC and represents the majority of patients who will develop metastatic disease. Loss of chromosome 3p is the most common genetic alteration in ccRCC.^9,10 The von Hippel Lindau (VHL) gene is a tumor suppressor gene located at 3p25.3 region, and mutation of this gene is associated with development of ccRCC.¹¹ Comprehensive genetic profiling of ccRCC tumors may not only provide insights on the mechanisms of tumorigenesis, but also provide potential prognostic biomarkers for the identification of patients at risk for recurrence or metastasis.

Conventional cytogenetic, allelotyping, and comparative genomic hybridization (CGH) studies have identified a number of chromosome aberrations in development ccRCC.^12-19 SNP-CGH array is a powerful new technology to detect both copy number variations and LOH events by applying high-density whole-genome SNP genotyping technology to array CGH.²⁰ High resolution SNP-CGH array may enable us to identify chromosome breakpoints and regions of abnormality which will help us locate genes involved in cancer development and progression. In this study, we applied SNP-CGH array to obtain genome-wide LOH and amplification profile of ccRCC tumors. The Illumina's HumanHap300 SNP chip has a 9 Kb mean SNP spacing, enabling an effective resolution of ∼90 Kb (with a 10 SNP smoothing). We also analyzed correlations between chromosome aberrations and clinicopathological variables, including tumor stage and nuclear grade.

Materials and Methods

Results

Discussion

Illumina's high density SNP arrays have been the choice of platform for genome-wide association study that allowed major advances in identifying germline genetic susceptibility loci to common cancers, including cancers of breast, prostate, colorectum, and lung.^22-25 However, there are only a couple of studies that applied Illumina's high density SNP array to query chromosome aberrations in tumor tissues to date.^26,27 This study is the first such attempt in RCC and provides whole genome LOH profile of ccRCC tumors with the highest resolution to date. Our data showed that LOH are very frequent events in ccRCC tumors, most of which involved large fragments of chromosomes. We also identified focal LOH and amplifications, which may provide clues of new genes involved ccRCC tumorigenesis.

There are four recent studies using Affymetrix's SNP chips, two using 10K chips and two using 100K chips, for copy number aberrations in RCC.^28-31 Our study is the only one using Illumina's 317K SNP chip. We compared the results of these SNP-arrays with ours. There is a complete concordance of the most frequent loss and gain, i.e., the 3p loss and 5q gain. There is also a high concordance of other high frequent regions, for example, chromosome loss at 1p, 4q, 6q, 8p, 9q, 10q, 14q, 19p, and chromosome gain at 7p, 7q, 12q. But we detected more aberrations than other studies and identified some smallest overlapping regions due to the higher density of our SNP chips and larger sample size.

The high density of relatively evenly distributed SNPs enables us to pinpoint the locations of chromosome breakpoints and the sizes of LOH and amplification. The predominant LOH and chromosome gains in ccRCC occur to a large fragment of chromosome arm (from 10 to 100 Mb) and often the whole chromosome arm (Fig.1a & 1b and Tables 1 & 2). Consistent with literature, the most common LOH was 3p and gain was 5q. Other recurrent large LOH regions (>10%) include 8p21-pter (33.3%), 6q23-27 (30.4%), 14q24.1-qter (29.9%), 9q32-qter (20.8%), 9p21-23 (19.2%), 10q22.3-qter (15.4%), 1p36 (15.8%), 17p, 18q and 22q; and other frequent large regions of chromosome amplification include 1q25.1-qter, 7q21.13-qter, and 8q24.12-qter. These recurrent large chromosome aberrations have mostly been reported previously by different studies with varying frequencies;^{9, 10, 12-18, 28-34} however, this present study represents the most complete and detailed account of all the chromosome aberrations.

These large LOH and amplification regions clearly encompass tumor suppressor genes and proto-oncogenes that are involved in ccRCC tumorigenesis, but also include regions that may be caused by general genetic instability, as suggested by the positive associations between LOH events and shorter telomeres. Almost all of the LOH at 3p, except one, encompass VHL gene at 3p25, confirming the critical role of VHL inactivation in ccRCC tumorigenesis. The only exception (not including VHL) is an interstitial LOH, spanning 3p12-14, 3p21-22, 3p24.1-24.2, and 3p24.3. This data is also consistent with a previous observation of the VHL-independent carcinogenic pathway in ccRCC.³⁵ The candidate genes in these regions include FHIT at 3p14.2³⁶ and RASSF1A at 3p21³⁷ The second most frequent LOH region is 8p12-pter. This region is one of the most frequently altered regions in human cancers.³⁸ Chromosome breaks that disrupt the NRG1 gene at 8p12 have been observed in breast and pancreatic cancers and it has been proposed that alteration of the NRG1 gene occurs through breakage at a non-common fragile site^38,39 In this study, one smallest LOH in 8p12 had a size of 0.29 Mb and only contains one gene (NRG1) (Table 3). Previous CGH studies have identified 8p12 as a frequent LOH region in ccRCC and validated by fluorescence in situ hybridization (FISH) and chromosome banding.^19,40 Our study narrowed this region and suggests, for the first time, that 8p12 aberration through breakage of NRG1 at a fragile site is involved in kidney cancer. Chromosome 6q (6q21 and 6q26) and 9q32 contains three established common fragile sites,⁴¹ which match to two other most frequent LOH at 6q23-27 (30.4%) and 9q32-qter (20.8%). These data, together with a substantial number of breakpoints at 3p14, which harbors the best known fragile site gene (FHIT), suggest that many of the observed chromosome aberrations are caused by fragile site instability in tumor cells.

Besides VHL and the other genes in chromosome 3, there are other known tumor suppressors that are located in regions of LOH, for example, p53 at 17p13.1, p16 or other genes at 9p21,⁴⁴ and PTEN at 10q23.3. These genes have been implicated in RCC.^42-45 However, the frequencies of LOH range from 10% to 20%, compared with 86% of VHL, indicating a minor role of these tumor suppressors in ccRCC tumorigenesis. The smallest overlapping regions of LOH may contain potential new genes involved in ccRCC. The number of genes in these regions ranges from 1 to 44, some of which encode hypothetical proteins with unknown function. An interesting gene is RFP2 at 13q14.3, which encodes a transmembrane E3 ubiquitin ligase and is a candidate tumor suppressor.⁴⁶ Future biochemical studies are warranted to measure the expression of these genes and validate their involvement in ccRCC.

Among the four large chromosome amplification regions (Table 2), 5q is the most frequent, including 10 whole 5q amplification, 21 large amplification encompassing 5q32-ter, and 1 focal amplification in 5q35.3 with a length of 0.42 Mb (Fig. 1b). No specific pro-oncogenes have been implicated in ccRCC tumorigenesis in the 5q region. The 0.42 Mb focal amplification region in 5q35.3 contains 15 genes, one of which is SQSTM1, a ubiquitin-binding protein that was shown overexpressed in breast and prostate tumors.^47,48 The LOH of large region encompassing SQSTM1 has been validated by FISH analysis and spectral karyotyping,^19,49 and future functional studies are warranted to evaluate whether SQSTM1 is involved in ccRCC tumorigenesis.

Amplifications of 1q25.1-qter, 7q21.13–qter, and 8q24.12-qter are common events in a few other cancers. Top candidate genes in these 3 large amplification regions and whole 7p arm include MDM4 (1q32), which inhibits the tumor suppressor function of p53 and promotes tumorigenesis;^50,51 CDK6 (7q21.2), a cyclin-dependent kinase which is amplified and overexpressed in several cancers;^52-54 MYC oncogene (8q24.12); and EGFR (7p12). In the three smallest overlapping regions of amplification (Table 3), 1q32.1 contains MDM4 and 17 other genes, 5p15.33 harbors hTERT (human telomerase reverse transcriptase) and 15 other genes, 5q35.3 contains SQSTM1 and 14 other genes. These genes may play important roles in ccRCC tumorigenesis.

In analyses of these chromosome aberrations with clinicopathological features, we found the LOH at 14q was significantly associated with both higher tumor grade and stage. LOH at 14q is one of the most frequent events in ccRCC. Many previous studies have consistently found that this event is associated with higher grade and stage and worse prognosis and survival in ccRCC.^12,55-58 This region is also frequently deleted in other cancer types and associated with advanced tumors and poor prognosis.^59-62 It appears that 14q may harbor a tumor suppressor gene that controls cell proliferation and loss of its function leads to a growth advantage and transformation of low-grade to high-grade tumors. The identity of this gene or genes remains unknown. One interesting note is that HIF-1α is located on 14q21-q24 and is lost in a substantial number of ccRCC tumors. It is well established that VHL-HIF-1α pathway is the major driving force for ccRCC tumorigenesis. VHL proteins functions as part of an E3 ubiquitin ligase complex that binds to HIF-1α for ubiquitin-mediated proteasomal degradation. Inactivation of VHL by mutation, deletion, or hypermethylation results in the intracellular accumulation of HIF-1α, which in turn increases the expression of many hypoxia-inducible genes essential for cancer cell functions under hypoxic conditions, such as glucose transport, angiogenesis, cell growth, and inflammation.⁶³ It is surprising that HIF-1α as a mediator of VHL function is lost in up to 30% of ccRCC tumors. It was also reported that a large panel of VHL-defective RCC cell lines lacked functional HIF-1α.⁶⁴ The tumorigenesis of these cases may occur through a VHL-independent pathway or an alternative hypoxia inducible factor (e.g., HIF-2α).

We showed that high genetic instability, as evidenced by short telomeres contributes to LOH. It is a general phenomenon the effect of alteration of specific chromosomes and genes, since tumors with LOH at any specific chromosomes had shorter telomeres, 13 chromosomes reached statistical significance or borderline significance, and the other 6 also showed shorter telomeres in tumors with LOH (Table 4). It has been well established that telomere dysfunction leads to genomic instability.^65,66 Telomere dysfunction via either telomere shortening or disruption of telomere structure causes end-to-end fusion of unprotected chromosomes. The propagation of breakage-fusion-bridge cycles will lead to genomic instability, including aneuploidy, LOH, and chromosome loss or amplification. Several studies showed telomere shortening in RCC tumors compared to normal tissues.^67-71 In addition, Gisselsson et al. ⁷⁰ showed that telomere shortening generated cytogenetic heterogeneity in a subgroup of RCC. Furthermore, Izumi et al. ⁷¹ measured telomerase activity, telomere length, chromosome aberrations, and DNA ploidy in ccRCC tumors and divided tumors into high telomerase and low telomerase activity groups. They found that the low telomerase group had shorter telomere length, more chromosome aberrations, and fewer DNA diploid cells, supporting that shorter telomere length is associated with chromosome instability. Our data is consistent with these findings.

In conclusion, we performed a high density, whole genome SNP array analysis to profile chromosome aberrations in ccRCC. This study provides a close estimate of the size and frequency of chromosome LOH and amplifications of ccRCC and identifies several smallest overlapping regions that harbor potential tumor suppressor genes and oncogenes involved in the development of ccRCC. These regions and genes may become prognostic and therapeutic biomarkers as well as potential target of therapy.

Supplementary Material