Genomic Imbalances Detected By Oligo Microarray CGH In Clear Cell Renal Cell Carcinoma (ccRCC)

Renal cell carcinoma (RCC) accounts for about3%of all new cancer cases andthe incidence rates for all stages have been steadily rising over the last3decades.[1–3]Approximately one-third of patients present with metastatic disease at the initial di-agnosis and more than40%of patients will eventually die from their cancer.[4,5]De-spite the introduction of new treatment regimens, surgical resection remains the onlycurative therapy for RCC.[6]However, up to50%of patients undergoing nephrectomyfor clinically localized RCC will develop local recurrence or distant metastasis.[7-8]These statistics highlight the need for detecting RCC at early stages and identifyingmarkers that can predict prognosis and response to therapy. RCC comprises a hetero-geneous group of epithelial tumors with various cytogenetic and molecular abnormal-ities and different histological features. Accumulation of genetic aberrations plays apivotal role in the initiation and prognosis of RCC and other cancers.Clear cell RCC (ccRCC) accounts for70–80%of all RCC and represents the ma-jority of patients who will develop metastatic disease. Loss of chromosome3p is themost common genetic alteration in ccRCC.[9,10]The von Hippel Lindau (VHL) geneis a tumor suppressor gene located at3p25.3region, and mutation of this gene is asso-ciated with development of ccRCC.[11]Comprehensive genetic profiling of ccRCCtumors may not only provide insights on the mechanisms of tumorigenesis, but alsoprovide potential prognostic biomarkers for the identification of patients at risk forrecurrence or metastasis.Conventional cytogenetic and comparative genomic hybridization (CGH) studieshave identified a number of chromosome aberrations in development ccRCC.[12–19]Array CGH is a powerful new technology to detect both copy number variations andLOH events.[20]High resolution CGH array may enable us to identify chromosomebreakpoints and regions of abnormality which will help us locate genes involved incancer development and progression.In our study, we obtain genome-wide LOH and amplification profile of ccRCCtumors. The Nimblegen720k CGH array chip is a probe length50-75mer, enabling an effective resolution of5Kb. We also analyzed correlations between chromosome ab-errations and clinic pathological variables, including tumor stage and nuclear grade.The identification of genetic aberrations may help understand the mechanisms oftumorigenesis and has important implications in diagnosis, prognosis and treatment.We applied Nimblegen’s720K high density comparative genomic hybridization（CGH） arrays to profile chromosomal aberrations in clear cell renal cell carcinoma(ccRCC) from50patients and analyzed the association of LOH/amplification eventswith clinic pathological characteristics and telomere length.Objective: Application of array CGH to identify chromosomal imbalances ofccRCC tumor specimens may help to differentiate relevant subsets of tumors, whichwould allow better prognostic and therapeutic decision.Experimental Designs&Methods: After obtaining the patients’ signed consentforms, total genomic DNA was isolated from50fresh ccRCC tumor tissues by usinga commercial kit. Eleven out of50samples were Fuhrman stage I,24were stage IIand the rests were stage III. Oligoarray CGH was performed on the720k oligonucleo-tide chip purchased from Roche NimbleGen System Inc. and experimental procedureswere performed according to the manufacturer’s protocols with minor modifications.Results: Our data showed that all the samples had genomic imbalances, eitherlosses or gains. The most common loss of heterozygosity (LOH) were3p (45cases)including38whole3p arm losses,7large fragment LOH (spanning3p21-36), and1interstitial LOH (spanning3p12-14,3p21-22,3p24.1-24.2and3p24.3), followed bychromosome losses at8p12-pter,6q23.3-27,14q24.2-qter,9q32.1-qter,9p21-23,10q22.3-qter,1p3617p，18q and22q. We also found several smallest overlappingregions of LOH that contained tumor suppressor genes. One smallest LOH in8p12had a size of0.29Mb and only contained one gene (NRG1). The most frequent chro-mosome gains were at5q (30cases), including24whole5q amplification,5largeamplifications encompassing5q32-ter and1focal amplification in5q35.3(0.42Mb).The other common chromosome gains were1q25.1-qter,7q21.13-qter,8q24.12-qterand whole7p arm. Significant associations of LOH at9p,9q,14q and18q were ob-served with higher nuclear grade. Significant associations with tumor stage were ob- served for LOH at14q,18p and21q. Finally, we found that tumors with LOH at2q,6p,6q,9p,9q and17p had significantly shorter telomere length than those with-outLOH. Interestingly, no case with stage I had a loss or gain of chromosome1, but17out of39cases with stage II and III had either losses or gains involving chromosome1.Conclusions: This is the first study to use Nimblegen’s CGH array that provides aclose estimate of the size and frequency of chromosome LOH and amplifications ofccRCC. The identified regions and genes may become diagnostic and prognostic bi-omarkers as well as potential targets of therapy.The result Array CGH is a reliable and fast technics to obtain the imbalance trans-lations of the chromosome of ccRCC. It shows that the3p lose and5q gain were themost significant imbalances detected in our50ccRCC samples, which were likely dueto the fact that the unbalanced chromosome rearrangements involving the two chro-mosomes. We find the higher nuclear grade and stage is significantly associated withLOH/amplification events at each chromosome at9p,9q,14q,18p and20p. LOH at14q,18p and21q significantly associated with tumor stage. The findings warrant fur-ther investigation of the genes obtained from this study will help to make sure wheth-er the refined genes related with the tumorigenesis.