Lara K. Boyd

Distinct Genomic Alterations in Prostate Cancers in Chinese and Western Populations Suggest Alternative Pathways of Prostate Carcinogenesis

Prostate cancer is significantly more common in Western men than in Asian men, but the basis for this difference remains unknown. Because genomic studies of Asian prostate cancer are very limited, we used a genome-wide approach to reveal the genomic alterations in Chinese prostate cancers. We found a significant reduction in the frequency of certain somatic genomic changes that are commonly found in Western prostate cancers, including the 21q22.2-22.3 deletion, which involves the TMPRSS2:ERG fusion gene, and 10q deletion, which causes PTEN inactivation. Array results were confirmed by PCR-based molecular copy-number counting in selected samples. The different frequencies of these genomic changes were further evaluated by fluorescent in situ hybridization and immunohistochemistry analyses of tissue microarray samples. These alterations might be key genetic changes underlying the regional/ethnic difference in clinical incidence and might be induced by specific environmental and/or genetic risk factors that Western men are exposed to. Our findings suggest that tumors arise in Western and Chinese populations by alternative pathogenetic mechanisms.

The complexity of prostate cancer: genomic alterations and heterogeneity

Although prostate cancer is the most common malignancy to affect men in the Western world, the molecular mechanisms underlying its development and progression remain poorly understood. Like all cancers, prostate cancer is a genetic disease that is characterized by multiple genomic alterations, including point mutations, microsatellite variations, and chromosomal alterations such as translocations, insertions, duplications, and deletions. In prostate cancer, but not other carcinomas, these chromosome alterations result in a high frequency of gene fusion events. The development and application of novel high-resolution technologies has significantly accelerated the detection of genomic alterations, revealing the complex nature and heterogeneity of the disease. The clinical heterogeneity of prostate cancer can be partly explained by this underlying genetic heterogeneity, which has been observed between patients from different geographical and ethnic populations, different individuals within these populations, different tumour foci within the same patient, and different cells within the same tumour focus. The highly heterogeneous nature of prostate cancer provides a real challenge for clinical disease management and a detailed understanding of the genetic alterations in all cells, including small subpopulations, would be highly advantageous.

Androgen-Induced TMPRSS2:ERG Fusion in Nonmalignant Prostate Epithelial Cells

Fusion genes play important roles in tumorigenesis. The identification of the high-frequency TMPRSS2 fusion with ERG and other ETS family genes in prostate cancer highlights the importance of fusion genes in solid tumor development and progression. However, the mechanisms leading to these fusions are unclear. We investigated whether androgen, through stimulating its receptor, could promote spatial genome reorganization and contribute to the generation of the TMPRSS2:ERG fusion. We show that treatment with androgen can induce the TMPRSS2:ERG fusion in both malignant and nonmalignant prostate epithelial cells. Although the fusion could be detected in malignant cells following 24-hour treatment, prolonged exposure to androgen was required to detect the fusion transcript in nonmalignant cells. We associated the fusion incidence with genetic factors, including androgen-induced gene proximity, androgen receptor exon1 CAG repeat length and expression of the PIWIL1 gene. This study demonstrates that fusions can be induced prior to malignant transformation and generation of the fusion is associated with both gene proximity and loss of the ability to prevent double-strand breaks.

High-resolution genome-wide copy-number analysis suggests a monoclonal origin of multifocal prostate cancer

Many human cancers present as multifocal lesions. Understanding the clonal origin of multifocal cancers is of both etiological and clinical importance. The molecular basis of multifocal prostate cancer has previously been explored using a limited number of isolated markers and, although independent origin is widely believed, the clonal origin of multifocal prostate cancer is still debatable. We attempted to address clonal origin using a genome‐wide copy‐number analysis of individual cancer and high‐grade prostatic intraepithelial neoplasia (HGPIN) lesions. Using Affymetrix array 6.0 copy‐number analysis, we compared the genomic changes detected in 48 individual cancer and HGPIN lesions, isolated from 18 clinically localized prostate cancer cases. Identical genomic copy‐number changes, shared by all same‐case cancer foci, were detected in all 13 informative cases displaying multiple tumor foci. In addition, individual HGPIN lesions in the two multifocal‐HGPIN cases available shared identical genomic changes. Commonly known genomic alterations, including losses at 6q15, 8p21.3‐8p21.2, 10q23.2‐10q23.31, 16q22.3, 16q23.2‐16q23.3 and 21q22.2‐21q22.3 regions and gain of 8q24.3 were the most frequently detected changes in this study and each was detected in all same‐case foci in at least one case. Microarray data were confirmed by fluorescence in situ hybridization in selected foci. Our high‐resolution genome‐wide copy‐number data suggest that many multifocal cases derive from a single prostate cancer precursor clone and that this precursor may give rise to separate HGPIN foci and may further progress to multifocal invasive prostate cancer. These findings, which demonstrate the monoclonal origin of multifocal prostate cancer, should significantly enhance our understanding of prostate carcinogenesis.

Use of SNPs in cancer predisposition analysis, diagnosis and prognosis: tools and prospects

BACKGROUND The development of cancer is accompanied by several genetic alterations. Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation found in the human population. SNP arrays offer a high-resolution, high-throughput technology for genome-wide analysis, allowing the simultaneous detection of genotype and copy number changes. The power of SNP arrays as a research tool has accelerated our understanding of the genetic alterations in cancer, providing potential clinical applications. OBJECTIVE This manuscript reviews the use of SNPs in cancer research and discusses the potential clinical application of analysing SNPs for cancer predisposition analysis, diagnosis and prognosis. We also discuss potential future applications for the analysis of SNPs. METHODS In writing this review, we have reflected on our own extensive experience in the field of cancer genomics and have surveyed peer-reviewed articles focussing on the application of SNPs in cancer research. In addition, we have referred to product websites. CONCLUSION Since its development, SNP array technology has been extensively applied in cancer research. Information generated from SNP array analysis has been providing valuable information. With the full understanding of the rich resources of SNPs and their effects on influencing cellular function, SNP arrays will revolutionise the clinical practice in cancer risk assessment, diagnosis and prognosis making the concept of personalised medicine a reality.

Abstract 3048: Enhanced androgen signalling pathway increases the risk of prostate cancer through induction of TMPRSS2:ERG fusion

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Clinical incidence and mortality rates of prostate cancer vary dramatically among different countries. In a recent study, we associated the difference in clinical incidence with certain somatic genomic alterations, including PTEN deletion and TMPRSS2:ERG fusion, which are found more commonly in Western but rarely in Chinese cancer samples (Mao et al, Cancer Res, 2010; 70:5207-12). TMPRSS2:ERG fusion, which is transcriptionally controlled by androgen receptor (AR), has been reported in more than 50% of prostate cancers. We investigated whether androgen level and AR activity, which varies between Chinese and Western men, could induce this fusion gene and consequently prostate cancer development. Since AR activity is inversely associated with its exon1 CAG repeats length, we determined the differences in repeat length between TMPRSS2:ERG fusion positive and negative UK samples and also between UK and China prostate cancer patients. We found significantly shorter CAG repeat lengths in the UK compared to Chinese cases (P<0.05). We also observed short CAG repeat lengths in the fusion positive (average n=20.3) compared to negative (average n=21.3) cases, although it is not statistically significant (p=0.14) and potentially due to the limited number of samples. We then investigated whether AR stimulation by androgen can induce TMPRSS2:ERG fusion and spatial genome reorganization. We induced the fusion not only in prostate cancer cells, but also in non-malignant prostate epithelial cells following long-term exposure to dihydrotestosterone (DHT). The induced fusion is associated with androgen-stimulated TMPRSS2 and ERG gene proximity. During our study, androgen-induced TMPRSS2 and ERG proximity and fusion were reported, but only in cancer cells (Mani et al, Science, 2009; 326:1230; Lin et al, Cell, 2009; 139:1069-83; and Haffner et al, Nat Genet, 2010; 42:668-75). We also observed that androgen-stimulated gene proximity is associated with AR transcriptional activity, which can be blocked by either RNA polymerase or AR inhibitors. Finally, we investigated the role of double strand breaks in the induction of TMPRSS2:ERG fusion. We associated high frequency of fusion induction with low expression of PIWIL1, which acts as a double strand breaks protector. Overexpression of PIWIL1 prevents DHT induced TMPRSS2:ERG fusion. Interestingly, long term DHT exposure tends to reduce expression of PIWIL1. This study demonstrates that enhanced AR activity can induce genetic alterations such as TMPRSS2:ERG fusion and consequently result in prostate carcinogenesis. The difference in physiological androgen level and AR activity may account for the variation in prostate cancer risk between Western and Chinese men and suggests that control of androgen level or AR activity can potentially prevent prostate carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3048. doi:10.1158/1538-7445.AM2011-3048

NKAIN2 functions as a novel tumor suppressor in prostate cancer

Recurrent chromosome breakpoints at 6q22.31, leading to truncation and potential loss-of-function of the NKAIN2 gene, in Chinese prostate cancer patients were previously identified. In this study we investigated genomic, methylation and expression changes of NKAIN2 in a large number of prostate cancer samples and determined its functional role in prostate cancer cells. Fluorescence in situ hybridization analysis confirmed that NKAIN2 truncation is specific to Chinese while deletion of the gene is frequent in both Chinese and UK prostate cancers. Significantly reduced expression of NKAIN2 was also detected at both RNA and protein levels. Somatic mutations of NKAIN2 in prostate cancer samples exist but at very low frequency, suggesting that it is a putative tumor suppressor gene (TSG) with haploid insufficiency. Our functional studies showed that overexpression of NKAIN2 in prostate cancer cells inhibits cellular growth by promoting cell apoptosis, and decreasing cell migration and invasion. Conversely, knockdown of NKAIN2 promotes prostate cancer cell growth by inhibiting cell apoptosis, and increasing cell migration and invasion. These data imply that NKAIN2 is a novel TSG whose activity is commonly reduced in prostate cancer. It may restrain the disease development and progression by inducing apoptosis and suppressing cancer cell growth, migration and invasion. This study provides new insights into prostate carcinogenesis and opportunities for development of novel therapies for prostate cancer.

The different genetic alterations between Western and Chinese prostate cancers and the underlying mechanisms.

184 Background: Prostate cancer shows a wide variation in the clinical incidence and mortality rates of different geographical regions. While it is the most common male cancer in Western countries, it is much less frequent in Asian countries. We investigated genomic changes in prostate cancers from UK and China using microarrays to determine the genetic similarities and differences as well as the underlying mechanisms. METHODS We determined genome-wide genomic alterations using Affymetrix SNP array 6.0, and evaluated data using fluorescence in situ hybridisation (FISH) and immunohistochemistry. In addition, we assess androgen induced TMPRSS2 and ERG co-localization and fusion. Microsatellite analysis was used for AR CAG repeat polymorphism in UK and Chinese population. RESULTS Genome-wide analysis of 32 UK and 39 Chinese samples revealed that losses of 21q22 (leading to TMPRSS2:ERG fusion) and 10q23.3 (PTEN) were at much higher frequency in Western than Chinese prostate cancers. Using FISH analysis of 160 UK and 143 Chinese samples, we showed that PTEN deletion and ERG rearrangements were at a significantly higher frequency in samples from UK than China (p<0.001 for both). We found that PTEN and ERG protein were also differentially expressed (p<0.001) in the two populations. Investigating this further, we induced TMPRSS2 and ERG gene proximity and TMPRSS2:ERG fusion in two immortalised prostate epithelial cell lines by exposure to high dose of androgen. This androgen treatment did not cause increased global DNA damage but was associated with low expression of PIWIL1, which is involved in repairing double-strand breaks. Overexpression of PIWIL1 by transfection inhibited androgen induced TMPRSS2:ERG fusion. We found that AR CAG repeat lengths, which associated with AR activity, are significantly shorter in the UK than Chinese patients (p<0.05). CONCLUSIONS We revealed genomic differences in prostate cancer comparing the high-risk (Western) and low-risk (Chinese) populations. We further demonstrated that TMPRSS2:ERG fusion can be induced by androgen. The difference of CAG repeat length between the two populations are potentially associated with TMPRSS2:ERG fusion positive prostate cancers.

Abstract 3878: The different genetic alterations between Western and Chinese samples indicate alternate pathways of prostate cancer development

Prostate cancer is the most common male cancer in Western countries, but much less frequent in Asian countries. We systematically investigated genomic changes in prostate cancers from UK and China to determine genetic similarity and difference and potential alternative mechanisms of prostate carcinogenesis in these high and low cancer incidence populations. Using Affymetrix array 6.0 microarrays, we analyzed genome-wide genomic alterations in 32 UK and 39 Chinese prostate cancer samples. Distinct difference in genomic alterations between Western and Chinese prostate cancers were found, including loss of 21q22 and PTEN deletion, which are the most common genomic changes in Western prostate cancer but rarely detected in the Chinese samples. To further evaluate the difference between Western and Chinese samples, FISH analysis for PTEN deletion and ERG rearrangements and immunohistochemistry analysis for PTEN and ERG expressions were applied to UK (n=160) and Chinese (n=143) prostate cancer tissue microarrays (TMAs). PTEN deletion and ERG rearrangements were found at a significantly higher frequency in samples from UK (42.3% and 37.4%) than China (14.3% and 7.5% respectively) (P In conclusion, we compared genetic alterations in prostate cancer samples from UK and China and found there are significant differences between the two groups, commencing at pre-invasive, HGPIN, stage. Our findings suggest that tumours arise in Western and Chinese populations by alternative pathogenetic mechanisms. These alterations, differentially presented in these two populations may be key genetic changes underlying the regional/ethnic difference in clinical incidence and may be induced by specific environmental and/or genetic risk factors that Western men are exposed to. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3878. doi:10.1158/1538-7445.AM2011-3878

Abstract 4831: Monoclonal origin of multifocal cancer and the step-wise genetic events of prostate carcinogenesis

Prostate cancer is commonly detected as multiple foci in a single prostate gland and it is of etiological and clinical importance to identify the origin of these multifocal cancer lesions. Due to the limited number of markers analyzed, previous studies to address this issue are inconclusive. A high-resolution genome-wide study should address this issue better. Therefore, we have performed a genome-wide analysis using Affymetrix Array 6.0 of individually laser-capture microdissected foci to compare the genetic similarities and/or differences of these foci within the same prostate gland. Fluorescence in situ hybridization was used to confirm SNP array results. A total of 43 cancer and high-grade prostatic intraepithelial neoplasia (PIN) foci from 16 prostate cancer cases were analyzed for genomic copy number changes. Copy number changes from same-case foci were compared to determine the genetic relationship between the foci. In many cases, we found extensive genetic differences between different foci isolated from the same case. However, omniclonal copy number gains and losses were detected in 11/11 informative case-matched multifocal tumor cases and 8/8 informative case-matched PIN and tumor cases, suggesting that multiple cancer foci arise from a common prostate cancer precursor clone. In addition, a higher degree of similarity was observed between PIN and adjacent cancer foci, as compared to distant lesions. The strong genetic similarities between PIN and prostate cancer foci from the same region suggest clonal expansion of separate PIN lesions to invasive cancer. Based on the monoclonal model, we examined the step-wise accumulation of these genomic copy number changes. Loss of 10q23.2-10q23.31 and 16q were commonly shared in same-case tumor foci and, in many cases, also matched PIN foci, suggesting that they are early events in prostate carcinogenesis. Other common genomic changes, such as loss of 6q, 8p and 21q22.2-21q22.3, were found less frequently in all same-case foci and rarely in PIN lesions, suggesting these genetic events occur at a relatively later stage. In conclusion, using a high-density SNP array, we reveal a monoclonal origin for the majority of multifocal prostate cancer samples analyzed. From these SNP array data, we have also begun to elucidate the step-wise genetic events in prostate carcinogenesis, including early events such as loss of 10q23.2-10q23.31 and 16q Taken together, these findings should significantly enhance our understanding of prostate carcinogenesis and potentially affect the clinical management of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4831. doi:10.1158/1538-7445.AM2011-4831