Somatic DNA copy number alterations in non-dysplastic Barrett's esophagus.

BACKGROUND: The purpose of this study was to analyze non-dysplastic Barrett's esophagus (NDBE) biopsy tissue and compare the rate of somatic DNA copy number alterations (CNAs) in patients who subsequently progressed to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC) to those patients who did not. METHODS: A retrospectively collected database of Barrett's esophagus (BE) patients spanning a 16-year period was queried. Patients who progressed from NDBE to HGD or EAC were identified and compared to patients who did not. Initial biopsy specimens were microdissected and extracted DNA underwent Multiplex Ligation-dependent Probe Amplification (MLPA) for CNAs. Comparisons between progressors and non-progressors were made with Fisher's exact and two-sample t tests. Logistic regression assessed factors associated with progression. RESULTS: Of the 2459 patients in the BE database, 36 patients progressed from NDBE to either HGD or EAC. There were eight progressors who had biopsy specimens with adequate DNA for analysis. The progressor and non-progressor cohort had similar demographic information and medical history. The progressor group trended towards being older at diagnosis (72 ± 10 vs. 64 ± 13 years, p = 0.097) and fewer progressors reported reflux symptoms (50 vs. 94.7%, p < 0.001). Progressor specimens had more overall CNAs (75% vs. 33.6%, p = 0.026). On univariable analysis, there was an association between progression and absence of GERD symptoms (OR 16.54 [3.42-80.03], p = 0.001), any CNA (OR 5.10 [1.18-23.30], p = 0.035), and CNA in GATA6 or ERBB2 (OR 6.72 [1.18-38.22], p = 0.032). CONCLUSIONS: Patients who progressed from NDBE to HGD or EAC were older at first diagnosis of BE and fewer of the progressors reported symptoms of reflux when compared to non-progressors. Progression was associated with the presence of any CNA and specific CNAs in GATA6 or ERBB2.

Distinct Genomic Alterations in Prostate Tumors Derived from African American Men.

We aim to understand, from acquired genetic alterations in tumors, why African American (AA) men are more likely to develop aggressive prostate cancer. By analyzing somatic mutations in 39 genes using deeper next-generation sequencing with an average depth of 2,522 reads for tumor DNA and genome-wide DNA copy-number alterations (CNA) in prostate cancer in a total of 171 AA/black men and comparing with those in 860 European American (EA)/white men, we here present several novel findings. First, >35% of AA men harbor damaging mutations in APC, ATM, BRCA2, KDM6A, KMT2C, KMT2D, MED12, ZFHX3, and ZMYM3, each with >1% of mutated copies. Second, among genes with >10% of mutated copies in tumor cells, ZMYM3 is the most frequently mutated gene in AA prostate cancer. In a patient's tumor with >96% frameshift mutations of ZMYM3, we find allelic imbalances in 10 chromosomes, including losses of five and gains of another four chromosomes, suggesting its role in maintaining genomic integrity. Third, when compared to prostate cancer in EA/white men, a higher frequency of CNAs of MYC, THADA, NEIL3, LRP1B, BUB1B, MAP3K7, BNIP3L and RB1, and a lower frequency of deletions of RYBP, TP53, and TMPRSS2-ERG are observed in AA/black men. Finally, for the above genes with higher frequency of CNAs in AA than in EA, deletion of MAP3K7, BNIP3L, NEIL3 or RB1, or gain of MYC significantly associates with both higher Gleason grade and advanced pathologic stage in AA/black men. Deletion of THADA associates with advanced pathologic stage only. IMPLICATIONS: A higher frequency of damaging mutation in ZMYM3 causing genomic instability along with higher frequency of altered genomic regions including deletions of MAP3K7, BNIP3L, RB1, and NEIL3, and gain of MYC appear to be distinct somatically acquired genetic alterations that may contribute to more aggressive prostate cancer in AA/black men.

Feasibility and performance of a novel probe panel to detect somatic DNA copy number alterations in clinical specimens for predicting prostate cancer progression.

BACKGROUND: To assess the feasibility of a novel DNA-based probe panel to detect copy number alterations (CNAs) in prostate tumor DNA and its performance for predicting clinical progression. METHODS: A probe panel was developed and optimized to measure CNAs in trace amounts of tumor DNA (2 ng) isolated from formalin-fixed paraffin-embedded tissues. Ten genes previously associated with aggressive disease were targeted. The panel's feasibility and performance were assessed in 175 prostate cancer (PCa) patients who underwent radical prostatectomy with a median 10-year follow-up, including 42 men who developed disease progression (either metastasis and/or PCa-specific death). Association with disease progression was tested using univariable and multivariable analyses. RESULTS: The probe panel detected CNAs in all 10 genes in tumor DNA isolated from either diagnostic biopsies or surgical specimens. A four-gene model (PTEN/MYC/BRCA2/CDKN1B) had the strongest association with disease progression; 64.3% of progressors and 22.5% of non-progressors had at least one CNA in these four genes, odds ratio (OR) (95% confidence interval) = 6.21 (2.77-13.87), P = 8.48E-06. The association with disease progression remained significant after adjusting for known clinicopathological variables. Among the seven progressors of the 65 patients with clinically low-risk disease, three (42.9%) had at least one CNA in these four genes. CONCLUSIONS: The probe panel can detect CNAs in trace amounts of tumor DNA from biopsies or surgical tissues at the time of diagnosis or surgery. CNAs independently predict metastatic/lethal cancer, particularly among men with clinically low-risk disease at diagnosis. If validated, this may improve current abilities to assess tumor aggressiveness.

The study on copy number alteration of clear cell renal cancer in Chinese population.

Objectives: Copy number alteration (CNA) is one of the important genetic variations. Although there are many studies on renal cancer CNA, few studies are based on the Chinese population. In our study, our objective is to acquire the whole-genome CNA landscape in Chinese population and explore the tumor risk-associated functional genes in the CNA regions, by detecting whole-genome in the clear cell renal cancer (ccRCC) tissues. Methods: We enrolled 35 formalin fixed paraffin embedded samples, which were processed by Oncoscan assay, and then acquired the data of whole-genome CNA. Then genes annotation and enrichment analyzing were processed. Furthermore, the gene burden and the affected bp (base pair) per Mbp (million bp) regions in whole-genome were analyzed by comparison of different T stage affected by CNA. Results: We acquired the whole-genome CNA landscape by Oncoscan detection, and found out the high-frequency CNA regions which were not reported in previous studies, for example, 11P11, 22q11.23, 20q11.3 (PDRG1), and Xp22.33 so on. During the analyzing of genes annotation and enrichment, we found out some ccRCC functional genes in the CNA regions which might play a role in the biological process, for example, the copy number loss of DNA repair genes (TTC5、PARP2, etc.) and tumor suppressor genes (TADA3, VHL, BAP1, ERC2-IT1, etc.), the copy number gain of oncogenes (ABL2, MET, HUWE1, etc.) and Notch signal pathway genes (MDK, etc.). Besides, gene fusion (GSTTP and GSTTP2) was noticed at 22q11.23 which copy number loss occurred, and the frequency is 46%. And between the different T stage patients affected by CNA, the T2+T3 group carried more high-frequency CNA regions (P-value was 0.012). Conclusions: In this study, the whole-genome ccRCC CNA landscape in Chinese population was acquired, a few functional genes and fusion genes were found out. However, a larger scale of samples is still needed to validate our results.

Fatty acid synthase is a primary target of MiR-15a and MiR-16-1 in breast cancer.

Fatty acid synthase (FASN) is upregulated in breast cancer and correlates with poor prognosis. FASN contributes to mammary oncogenesis and serves as a bona fide target in cancer therapies. MicroRNAs inhibit gene expression through blocking mRNA translation or promoting mRNA degradation by targeting their 3'-UTRs. We identified four microRNAs in two microRNA clusters miR-15a-16-1 and miR-497-195 that share a common seed sequence to target the 3'-UTR of the FASN mRNA. In reporter assays, both of these microRNA clusters inhibited the expression of a reporter construct containing the FASN 3'-UTR. However, only ectopic miR-15a-16-1, but not miR-497-195, markedly reduced the levels of endogenous FASN in breast cancer cells. Both miR-15a and miR-16-1 contributes to inhibiting FASN expression and breast cancer cell proliferation. Consistently, a sponge construct consisting of eight repeats of the FASN 3'-UTR region targeted by these microRNAs could markedly increase endogenous FASN levels in mammary cells. When FASN expression was restored by ectopic expression in breast cancer cells, retarded cell proliferation caused by miR-15a-16-1 was partially rescued. In conclusion, we demonstrated that FASN expression is primarily downregulated by miR-15a and miR-16-1 in mammary cells and FASN is one of the major targets of these two tumor suppressive microRNAs.

DNA alterations in the tumor genome and their associations with clinical outcome in prostate cancer.

Although most prostate cancer (PCa) cases are not life-threatening, approximately 293 000 men worldwide die annually due to PCa. These lethal cases are thought to be caused by coordinated genomic alterations that accumulate over time. Recent genome-wide analyses of DNA from subjects with PCa have revealed most, if not all, genetic changes in both germline and PCa tumor genomes. In this article, I first review the major, somatically acquired genomic characteristics of various subtypes of PCa. I then recap key findings on the relationships between genomic alterations and clinical parameters, such as biochemical recurrence or clinical relapse, metastasis and cancer-specific mortality. Finally, I outline the need for, and challenges with, validation of recent findings in prospective studies for clinical utility. It is clearer now than ever before that the landscape of somatically acquired aberrations in PCa is highlighted by DNA copy number alterations (CNAs) and TMPRSS2-ERG fusion derived from complex rearrangements, numerous single nucleotide variations or mutations, tremendous heterogeneity, and continuously punctuated evolution. Genome-wide CNAs, PTEN loss, MYC gain in primary tumors, and TP53 loss/mutation and AR amplification/mutation in advanced metastatic PCa have consistently been associated with worse cancer prognosis. With this recently gained knowledge, it is now an opportune time to develop DNA-based tests that provide more accurate patient stratification for prediction of clinical outcome, which will ultimately lead to more personalized cancer care than is possible at present.

Genomic predictors of patterns of progression in glioblastoma and possible influences on radiation field design.

We present a retrospective investigation of the role of genomics in the prediction of central versus marginal disease progression patterns for glioblastoma (GBM). Between August 2000 and May 2010, 41 patients with GBM and gene expression and methylation data available were treated with radiotherapy with or without concurrent temozolomide. Location of disease progression was categorized as within the high dose (60 Gy) or low dose (46 Gy) volume. Samples were grouped into previously described TCGA genomic groupings: Mesenchymal (m), classical (c), proneural (pn), and neural (n); and were also classified by MGMT-Methylation status and G-Cimp methylation phenotype. Genomic groupings and methylation status were investigated as a possible predictor of disease progression in the high dose region, progression in the low dose region, and time to progression. Based on TCGA category there was no difference in OS (p = 0.26), 60 Gy progression (PN: 71 %, N: 60 %, M: 89 %, C: 83 %, p = 0.19), 46 Gy progression (PN: 57 %, N: 40 %, M: 61 %,C: 50 %, p = 0.8) or time to progression (PN: 9 months, N:15 months, M: 9 months, C: 7 months, p = 0.58). MGMT methylation predicted for improved OS (median 25 vs. 13 months, p = 0.01), improved DFS (median 13 vs. 8 months, p = 0.007) and decreased 60 Gy (p = 0.003) and 46 Gy (p = 0.006) progression. There was a cohort of MGMT methylated patients with late marginal disease progression (4/22 patients, 18 %). TCGA groups demonstrated no difference in survival or progression patterns. MGMT methylation predicted for a statistically significant decrease in in-field and marginal disease progression. There was a cohort of MGMT methylated patients with late marginal progression. Validations of these findings would have implications that could affect radiation field size.

Systematic enrichment analysis of potentially functional regions for 103 prostate cancer risk-associated loci.

BACKGROUND: More than 100 prostate cancer (PCa) risk-associated single nucleotide polymorphisms (SNPs) have been identified by genome wide association studies (GWAS). However, the molecular mechanisms are unclear for most of these SNPs. METHODS: All reported PCa risk-associated SNPs reaching the genome-wide significance level of P < 1 × 10(-7) (index SNPs), as well as SNPs in linkage disequilibrium (LD, r(2) ≥ 0.5) with them were cataloged. Genomic regions with potentially functional impact were also identified, including UCSC annotated coding regions (exon and snoRNA/miRNA) and regulatory regions, as well as binding regions for transcription factors (TFs), histone modifications (HMs), DNase I hypersensitivity (DHSs), and RNA Polymerase IIA (POLR2A) defined by ChIP-Seq in prostate cell lines and tissues. Enrichment analysis was performed to test whether PCa risk-associated SNPs are located in these functional regions more than expected. RESULTS: A total of 103 PCa risk-associated index SNPs and 7,244 SNPs in LD with these index SNPs were cataloged. Genomic regions with potentially functional impact, grouped in 30 different categories of functionalities, were identified. Enrichment analysis indicated that genomic regions in the following 15 categories were enriched for the PCa risk-associated SNPs: exons, CpG regions, 6 TFs (AR, ERG, FOXA1, HOXB13, CTCF, and NR3C1), 5 HMs (H3K4me1, H3K4me2, H3K4me3, H3K27AC, and H3T11P), DHSs and POLR2A. In contrast, significantly fewer PCa risk SNPs were mapped to binding regions for H3K27me3, a repressive chromatin marker. CONCLUSIONS: The PCa risk-associated SNPs discovered to date may affect PCa risk through multiple different mechanisms, especially by affecting binding regions of TFs/HMs.

Coordinate loss of MAP3K7 and CHD1 promotes aggressive prostate cancer.

Prostate cancer subtypes are poorly defined and functional validation of drivers of ETS rearrangement-negative prostate cancer has not been conducted. Here, we identified an ETS(-) subtype of aggressive prostate cancer (ERG(-)MAP3K7(del)CHD1(del)) and used a novel developmental model and a cell line xenograft model to show that cosuppression of MAP3K7 and CHD1 expression promotes aggressive disease. Analyses of publicly available prostate cancer datasets revealed that MAP3K7 and CHD1 were significantly codeleted in 10% to 20% of localized tumors and combined loss correlated with poor disease-free survival. To evaluate the functional impact of dual MAP3K7-CHD1 loss, we suppressed Map3k7 and/or Chd1 expression in mouse prostate epithelial progenitor/stem cells (PrP/SC) and performed tissue recombination experiments in vivo. Dual shMap3k7-shChd1 PrP/SC recombinants displayed massive glandular atypia with regions of prostatic intraepithelial neoplasia and carcinoma apparent. Combined Map3k7-Chd1 suppression greatly disrupted normal prostatic lineage differentiation; dual recombinants displayed significant androgen receptor loss, increased neuroendocrine differentiation, and increased neural differentiation. Clinical samples with dual MAP3K7-CHD1 loss also displayed neuroendocrine and neural characteristics. In addition, dual Map3k7-Chd1 suppression promoted E-cadherin loss and mucin production in recombinants. MAP3K7 and CHD1 protein loss also correlated with Gleason grade and E-cadherin loss in clinical samples. To further validate the phenotype observed in the PrP/SC model, we suppressed MAP3K7 and/or CHD1 expression in LNCaP prostate cancer cells. Dual shMAP3K7-shCHD1 LNCaP xenografts displayed increased tumor growth and decreased survival compared with shControl, shMAP3K7, and shCHD1 xenografts. Collectively, these data identify coordinate loss of MAP3K7 and CHD1 as a unique driver of aggressive prostate cancer development.

Comparison of clinical outcomes and genomic characteristics of single focus and multifocal glioblastoma.

We investigate the differences in molecular signature and clinical outcomes between multiple lesion glioblastoma (GBM) and single focus GBM in the modern treatment era. Between August 2000 and May 2010, 161 patients with GBM were treated with modern radiotherapy techniques. Of this group, 33 were considered to have multiple lesion GBM (25 multifocal and 8 multicentric). Patterns of failure, time to progression and overall survival were compared based on whether the tumor was considered a single focus or multiple lesion GBM. Genomic groupings and methylation status were also investigated as a possible predictor of multifocality in a cohort of 41 patients with available tissue for analysis. There was no statistically significant difference in overall survival (p < 0.3) between the multiple lesion tumors (8.2 months) and single focus GBM (11 months). Progression free survival was superior in the single focus tumors (7.1 months) as compared to multi-focal (5.6 months, p = 0.02). For patients with single focus, multifocal and multicentric GBM, 81, 76 and 88 % of treatment failures occurred in the 60 Gy volume (p < 0.5), while 54, 72, and 38 % of treatment failures occurred in the 46 Gy volume (p < 0.4). Out of field failures were rare in both single focus and multiple foci GBM (7 vs 3 %). Genomic groupings and methylation status were not found to predict for multifocality. Patterns of failure, survival and genomic signatures for multiple lesion GBM do not appreciably differ when compared to single focus tumors.

Rb loss is characteristic of prostatic small cell neuroendocrine carcinoma.

PURPOSE: Small cell neuroendocrine carcinoma of the prostate is likely to become increasingly common with recent advances in pharmacologic androgen suppression. Thus, developing molecular markers of small cell differentiation in prostate cancer will be important to guide the diagnosis and therapy of this aggressive tumor. EXPERIMENTAL DESIGN: We examined the status of RB1, TP53, and PTEN in prostatic small cell and acinar carcinomas via immunohistochemistry (IHC), copy-number alteration analysis, and sequencing of formalin-fixed paraffin-embedded specimens. RESULTS: We found retinoblastoma (Rb) protein loss in 90% of small cell carcinoma cases (26 of 29) with RB1 allelic loss in 85% of cases (11 of 13). Of acinar tumors occurring concurrently with prostatic small cell carcinoma, 43% (3 of 7) showed Rb protein loss. In contrast, only 7% of primary high-grade acinar carcinomas (10 of 150), 11% of primary acinar carcinomas with neuroendocrine differentiation (4 of 35), and 15% of metastatic castrate-resistant acinar carcinomas (2 of 13) showed Rb protein loss. Loss of PTEN protein was seen in 63% of small cell carcinomas (17 of 27), with 38% (5 of 13) showing allelic loss. By IHC, accumulation of p53 was observed in 56% of small cell carcinomas (14 of 25), with 60% of cases (6 of 10) showing TP53 mutation. CONCLUSIONS: Loss of RB1 by deletion is a common event in prostatic small cell carcinoma and can be detected by a validated IHC assay. As Rb protein loss rarely occurs in high-grade acinar tumors, these data suggest that Rb loss is a critical event in the development of small cell carcinomas and may be a useful diagnostic and potential therapeutic target.

The regulation of SOX7 and its tumor suppressive role in breast cancer.

Both epigenetic silencing and genetic deletion of tumor suppressors contribute to the development and progression of breast cancer. SOX7 is a transcription factor important to development, and its down-regulation has been reported in tumor tissues and cell lines of prostate, colon, and lung cancers. However, the regulation of SOX7 expression and its functional role in breast cancer have not been reported. The current study demonstrates that SOX7 mRNA and protein expression are down-regulated in breast cancer tissues and cell lines compared with adjacent normal tissues and nontumorigenic cells, respectively. The SOX7 promoter is hypermethylated in breast cancer cell lines compared with nontumorigenic cells, and the inhibition of DNA methylation increases SOX7 mRNA levels. With shRNA-mediated SOX7 silencing, nontumorigenic immortal breast cells display increased proliferation, migration, and invasion and form structures that resemble that of breast cancer cells in a three-dimensional culture system. Conversely, ectopic SOX7 expression inhibits proliferation, migration, and invasion of breast cancer cells in vitro and tumor growth in vivo. Importantly, we discovered that SOX7 transcript levels positively correlated with clinical outcome of 674 breast cancer patients. Overall, our data suggest that SOX7 acts as a tumor suppressor in breast cancer. SOX7 expression is likely regulated by multiple mechanisms and potentially serves as a prognostic marker for breast cancer patients.

Genetic markers associated with early cancer-specific mortality following prostatectomy.

BACKGROUND: This study sought to identify novel effectors and markers of localized but potentially life-threatening prostate cancer (PCa), by evaluating chromosomal copy number alterations (CNAs) in tumors from patients who underwent prostatectomy and correlating these with clinicopathologic features and outcome. METHODS: CNAs in tumor DNA samples from 125 patients in the discovery cohort who underwent prostatectomy were assayed with high-resolution Affymetrix 6.0 single-nucleotide polymorphism microarrays and then analyzed using the Genomic Identification of Significant Targets in Cancer (GISTIC) algorithm. RESULTS: The assays revealed 20 significant regions of CNAs, 4 of them novel, and identified the target genes of 4 of the alterations. By univariate analysis, 7 CNAs were significantly associated with early PCa-specific mortality. These included gains of chromosomal regions that contain the genes MYC, ADAR, or TPD52 and losses of sequences that incorporate SERPINB5, USP10, PTEN, or TP53. On multivariate analysis, only the CNAs of PTEN (phosphatase and tensin homolog) and MYC (v-myc myelocytomatosis viral oncogene homolog) contributed additional prognostic information independent of that provided by pathologic stage, Gleason score, and initial prostate-specific antigen level. Patients whose tumors had alterations of both genes had a markedly elevated risk of PCa-specific mortality (odds ratio = 53; 95% CI = 6.92-405, P = 1 × 10(-4)). Analyses of 333 tumors from 3 additional distinct patient cohorts confirmed the relationship between CNAs of PTEN and MYC and lethal PCa. CONCLUSIONS: This study identified new CNAs and genes that likely contribute to the pathogenesis of localized PCa and suggests that patients whose tumors have acquired CNAs of PTEN, MYC, or both have an increased risk of early PCa-specific mortality.

DNA methylation alterations exhibit intraindividual stability and interindividual heterogeneity in prostate cancer metastases.

Human cancers almost ubiquitously harbor epigenetic alterations. Although such alterations in epigenetic marks, including DNA methylation, are potentially heritable, they can also be dynamically altered. Given this potential for plasticity, the degree to which epigenetic changes can be subject to selection and act as drivers of neoplasia has been questioned. We carried out genome-scale analyses of DNA methylation alterations in lethal metastatic prostate cancer and created DNA methylation "cityscape" plots to visualize these complex data. We show that somatic DNA methylation alterations, despite showing marked interindividual heterogeneity among men with lethal metastatic prostate cancer, were maintained across all metastases within the same individual. The overall extent of maintenance in DNA methylation changes was comparable to that of genetic copy number alterations. Regions that were frequently hypermethylated across individuals were markedly enriched for cancer- and development/differentiation-related genes. Additionally, regions exhibiting high consistency of hypermethylation across metastases within individuals, even if variably hypermethylated across individuals, showed enrichment for cancer-related genes. Whereas some regions showed intraindividual metastatic tumor heterogeneity in promoter methylation, such methylation alterations were generally not correlated with gene expression. This was despite a general tendency for promoter methylation patterns to be strongly correlated with gene expression, particularly at regions that were variably methylated across individuals. These findings suggest that DNA methylation alterations have the potential for producing selectable driver events in carcinogenesis and disease progression and highlight the possibility of targeting such epigenome alterations for development of longitudinal markers and therapeutic strategies.

The mitochondrial and autosomal mutation landscapes of prostate cancer.

BACKGROUND: Prostate cancer (PCa) is the most common cancer in men. PCa is strongly age associated; low death rates in surveillance cohorts call into question the widespread use of surgery, which leads to overtreatment and a reduction in quality of life. There is a great need to increase the understanding of tumor characteristics in the context of disease progression. OBJECTIVE: To perform the first multigenome investigation of PCa through analysis of both autosomal and mitochondrial DNA, and to integrate exome sequencing data, and RNA sequencing and copy-number alteration (CNA) data to investigate how various different tumor characteristics, commonly analyzed separately, are interconnected. DESIGN, SETTING, AND PARTICIPANTS: Exome sequencing was applied to 64 tumor samples from 55 PCa patients with varying stage and grade. Integrated analysis was performed on a core set of 50 tumors from which exome sequencing, CNA, and RNA sequencing data were available. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Genes, mutated at a significantly higher rate relative to a genomic background, were identified. In addition, mitochondrial and autosomal mutation rates were correlated to CNAs and proliferation, assessed as a cell cycle gene expression signature. RESULTS AND LIMITATIONS: Genes not previously reported to be significantly mutated in PCa, such as cell division cycle 27 homolog (Saccharomyces cerevisiae) (CDC27), myeloid/lymphoid or mixed-lineage leukemia 3 (MLL3), lysine (K)-specific demethylase 6A (KDM6A), and kinesin family member 5A (KIF5A) were identified. The mutation rate in the mitochondrial genome was 55 times higher than that of the autosomes. Multilevel analysis demonstrated a tight correlation between high reactive-oxygen exposure, chromosomal damage, high proliferation, and in parallel, a transition from multiclonal indolent primary PCa to monoclonal aggressive disease. As we only performed targeted sequence analysis; copy-number neutral rearrangements recently described for PCa were not accounted for. CONCLUSIONS: The mitochondrial genome displays an elevated mutation rate compared to the autosomal chromosomes. By integrated analysis, we demonstrated that different tumor characteristics are interconnected, providing an increased understanding of PCa etiology.

Exome sequencing of prostate cancer supports the hypothesis of independent tumour origins.

BACKGROUND: Prostate cancer (PCa) is a clinically and pathologically heterogeneous disease. The rapid development of sequencing technology has the potential to deliver new biomarkers with emphasis on aggressive disease and to revolutionise personalised cancer treatment. However, a prostate harbouring cancer commonly contains multiple separate tumour foci, with the potential to aggravate tumour sampling. The level of intraprostatic tumour heterogeneity remains to be determined. OBJECTIVE: To determine the level of intraprostatic tumour heterogeneity through genome-wide, high-resolution profiling of multiple tumour samples from the same individual. DESIGN, SETTINGS, AND PARTICIPANTS: Multiple tumour samples were obtained from four individuals following radical prostatectomy. One individual (SWE-1) contained >70% cancer cells in all tumour samples, whereas the other three (SWE-2 to SWE-4) required the use of laser capture microdissection for tumour cell enrichment. Subsequently, DNA was extracted from all tissue samples, and exome sequencing was performed. All tumour foci of SWE-1 were also profiled using a high-resolution array for the identification of copy number alterations (CNA). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Shared somatic high-frequency single nucleotide variants (SNV) and CNAs were used to infer the level of intraprostatic tumour heterogeneity. RESULTS AND LIMITATIONS: No high-frequency mutations, common for the three tumour samples of SWE-1, were identified. Ten randomly chosen positions were validated with Sanger sequencing in all foci, which verified the exome data. The high level of intraprostatic heterogeneity was consistent in all individuals. In total, three out of four individuals harboured tumours without an apparent common somatic denominator. Although we cannot exclude the presence of common structural rearrangements, a high-density array was used for the detection of deletions and amplifications in SWE-1, which agreed with the exome data. CONCLUSIONS: We present evidence for the presence of somatically independent tumours within the same prostate. This finding will have implications for personalised cancer treatment and biomarker discovery.

Identification of new differentially methylated genes that have potential functional consequences in prostate cancer.

Many differentially methylated genes have been identified in prostate cancer (PCa), primarily using candidate gene-based assays. Recently, several global DNA methylation profiles have been reported in PCa, however, each of these has weaknesses in terms of ability to observe global DNA methylation alterations in PCa. We hypothesize that there remains unidentified aberrant DNA methylation in PCa, which may be identified using higher resolution assay methods. We used the newly developed Illumina HumanMethylation450 BeadChip in PCa (n = 19) and adjacent normal tissues (n = 4) and combined these with gene expression data for identifying new DNA methylation that may have functional consequences in PCa development and progression. We also confirmed our methylation results in an independent data set. Two aberrant DNA methylation genes were validated among an additional 56 PCa samples and 55 adjacent normal tissues. A total 28,735 CpG sites showed significant differences in DNA methylation (FDR adjusted P<0.05), defined as a mean methylation difference of at least 20% between PCa and normal samples. Furthermore, a total of 122 genes had more than one differentially methylated CpG site in their promoter region and a gene expression pattern that was inverse to the direction of change in DNA methylation (e.g. decreased expression with increased methylation, and vice-versa). Aberrant DNA methylation of two genes, AOX1 and SPON2, were confirmed via bisulfate sequencing, with most of the respective CpG sites showing significant differences between tumor samples and normal tissues. The AOX1 promoter region showed hypermethylation in 92.6% of 54 tested PCa samples in contrast to only three out of 53 tested normal tissues. This study used a new BeadChip combined with gene expression data in PCa to identify novel differentially methylated CpG sites located within genes. The newly identified differentially methylated genes may be used as biomarkers for PCa diagnosis.