Xiaoyu Qu

A Three-Marker FISH Panel Detects More Genetic Aberrations of AR, PTEN and TMPRSS2/ERG in Castration-Resistant or Metastatic Prostate Cancers than in Primary Prostate Tumors

TMPRSS2/ERG rearrangement, PTEN gene deletion, and androgen receptor (AR) gene amplification have been observed in various stages of human prostate cancer. We hypothesized that using these markers as a combined panel would allow better differentiation between low-risk and high-risk prostate cancer. We analyzed 110 primary prostate cancer samples, 70 metastatic tumor samples from 11 patients, and 27 xenograft tissues derived from 22 advanced prostate cancer patients using fluorescence in situ hybridization (FISH) analysis with probes targeting the TMPRSS2/ERG, PTEN, and AR gene loci. Heterogeneity of the aberrations detected was evaluated. Genetic patterns were also correlated with transcript levels. Among samples with complete data available, the three-marker FISH panel detected chromosomal abnormalities in 53% of primary prostate cancers and 87% of metastatic (Met) or castration-resistant (CRPC) tumors. The number of markers with abnormal FISH result had a different distribution between the two groups (P<0.001). At the patient level, Met/CRPC tumors are 4.5 times more likely to show abnormalities than primary cancer patients (P<0.05). Heterogeneity among Met/CRPC tumors is mostly inter-patient. Intra-patient heterogeneity is primarily due to differences between the primary prostate tumor and the metastases while multiple metastatic sites show consistent abnormalities. Intra-tumor variability is most prominent with the AR copy number in primary tumors. AR copy number correlated well with the AR mRNA expression (rho = 0.52, P<0.001). Especially among TMPRSS2:ERG fusion-positive CRPC tumors, AR mRNA and ERG mRNA levels are strongly correlated (rho = 0.64, P<0.001). Overall, the three-marker FISH panel may represent a useful tool for risk stratification of prostate cancer patients.

Cells Comprising the Prostate Cancer Microenvironment Lack Recurrent Clonal Somatic Genomic Aberrations

Prostate cancer–associated stroma (CAS) plays an active role in malignant transformation, tumor progression, and metastasis. Molecular analyses of CAS have demonstrated significant changes in gene expression; however, conflicting evidence exists on whether genomic alterations in benign cells comprising the tumor microenvironment (TME) underlie gene expression changes and oncogenic phenotypes. This study evaluates the nuclear and mitochondrial DNA integrity of prostate carcinoma cells, CAS, matched benign epithelium and benign epithelium–associated stroma by whole-genome copy-number analyses, targeted sequencing of TP53, and FISH. Array comparative genomic hybridization (aCGH) of CAS revealed a copy-neutral diploid genome with only rare and small somatic copy-number aberrations (SCNA). In contrast, several expected recurrent SCNAs were evident in the adjacent prostate carcinoma cells, including gains at 3q, 7p, and 8q, and losses at 8p and 10q. No somatic TP53 mutations were observed in CAS. Mitochondrial DNA (mtDNA) extracted from carcinoma cells and stroma identified 23 somatic mtDNA mutations in neoplastic epithelial cells, but only one mutation in stroma. Finally, genomic analyses identified no SCNAs, LOH, or copy-neutral LOH in cultured cancer-associated fibroblasts, which are known to promote prostate cancer progression in vivo. Implications: The gene expression changes observed in prostate cancer–adjacent stroma and the attendant contribution of the stroma to the development and progression of prostate cancer are not due to frequent or recurrent genomic alterations in the TME. Mol Cancer Res; 14(4); 374–84. ©2016 AACR.

A novel four-color fluorescence in situ hybridization assay for the detection of TMPRSS2 and ERG rearrangements in prostate cancer

Since the identification of the TMPRSS2-ERG rearrangement as the most common fusion event in prostate cancer, various methods have been developed to detect this rearrangement and to study its prognostic significance. We report a novel four-color fluorescence in situ hybridization (FISH) assay that detects not only the typical TMPRSS2-ERG fusion but also alternative rearrangements of the TMPRSS2 or ERG gene. We validated this assay on fresh, frozen, or formalin-fixed paraffin-embedded prostate cancer specimens, including cell lines, primary prostate cancer tissues, xenograft tissues derived from metastatic prostate cancer, and metastatic tissues from castration-resistant prostate cancer (CRPC) patients. When compared with either reverse transcription-polymerase chain reaction or the Gen-Probe method as the technical reference, analysis using the four-color FISH assay demonstrated an analytical sensitivity of 94.5% (95% confidence interval [CI] 0.80-0.99) and specificity of 100% (95% CI 0.89-1.00) for detecting the TMPRSS2-ERG fusion. The TMPRSS2-ERG fusion was detected in 41% and 43% of primary prostate cancer (n = 59) and CRPC tumors (n = 82), respectively. Rearrangements other than the typical TMPRSS2-ERG fusion were confirmed by karyotype analysis and found in 7% of primary cancer and 13% of CRPC tumors. Successful karyotype analyses are reported for the first time on four of the xenograft samples, complementing the FISH results. Analysis using the four-color FISH assay provides sensitive detection of TMPRSS2 and ERG gene rearrangements in prostate cancer.

Methylation profiling identified novel differentially methylated markers including OPCML and FLRT2 in prostate cancer

ABSTRACT To develop new methods to distinguish indolent from aggressive prostate cancers (PCa), we utilized comprehensive high-throughput array-based relative methylation (CHARM) assay to identify differentially methylated regions (DMRs) throughout the genome, including both CpG island (CGI) and non-CGI regions in PCa patients based on Gleason grade. Initially, 26 samples, including 8 each of low [Gleason score (GS) 6] and high (GS ≥7) grade PCa samples and 10 matched normal prostate tissues, were analyzed as a discovery cohort. We identified 3,567 DMRs between normal and cancer tissues, and 913 DMRs distinguishing low from high-grade cancers. Most of these DMRs were located at CGI shores. The top 5 candidate DMRs from the low vs. high Gleason comparison, including OPCML, ELAVL2, EXT1, IRX5, and FLRT2, were validated by pyrosequencing using the discovery cohort. OPCML and FLRT2 were further validated in an independent cohort consisting of 20 low-Gleason and 33 high-Gleason tissues. We then compared patients with biochemical recurrence (n=70) vs. those without (n=86) in a third cohort, and they showed no difference in methylation at these DMR loci. When GS 3+4 cases and GS 4+3 cases were compared, OPCML-DMR methylation showed a trend of lower methylation in the recurrence group (n=30) than in the no-recurrence (n=52) group. We conclude that whole-genome methylation profiling with CHARM revealed distinct patterns of differential DNA methylation between normal prostate and PCa tissues, as well as between different risk groups of PCa as defined by Gleason scores. A panel of selected DMRs may serve as novel surrogate biomarkers for Gleason score in PCa.

Identification of differentially methylated markers among cytogenetic risk groups of acute myeloid leukemia

Aberrant DNA methylation is known to occur in cancer, including hematological malignancies such as acute myeloid leukemia (AML). However, less is known about whether specific methylation profiles characterize specific subcategories of AML. We examined this issue by using comprehensive high-throughput array-based relative methylation analysis (CHARM) to compare methylation profiles among patients in different AML cytogenetic risk groups. We found distinct profiles in each group, with the high-risk group showing overall increased methylation compared with low- and mid-risk groups. The differentially methylated regions (DMRs) distinguishing cytogenetic risk groups of AML were enriched in the CpG island shores. Specific risk-group associated DMRs were located near genes previously known to play a role in AML or other malignancies, such as MN1, UHRF1, HOXB3, and HOXB4, as well as TRIM71, the function of which in cancer is not well characterized. These findings were verified by quantitative bisulfite pyrosequencing and by comparison with results available at the TCGA cancer genome browser. To explore the potential biological significance of the observed methylation changes, we correlated our findings with gene expression data available through the TCGA database. The results showed that decreased methylation at HOXB3 and HOXB4 was associated with increased gene expression of both HOXB genes specific to the mid-risk AML, while increased DNA methylation at DCC distinctive to the high-risk AML was associated with increased gene expression. Our results suggest that the differential impact of cytogenetic changes on AML prognosis may, in part, be mediated by changes in methylation.

Identification of Combinatorial Genomic Abnormalities Associated with Prostate Cancer Early Recurrence.

Multiple biomarkers are needed to distinguish aggressive from indolent prostate cancer. We tested the prognostic utility of a three-marker fluorescent in situ hybridization (FISH) panel (TMPRSS2/ERG rearrangements, AR gain, and PTEN deletion) in a retrospective cohort (n = 210; median follow-up, 5.7 years). PTEN deletion was associated with an increased risk of biochemical recurrence (BcR; hazard ratio, 3.58; 95% CI, 1.39-9.22; P < 0.01) by multivariable Cox regression analyses and earlier BcR (P < 0.02) by Kaplan-Meier analysis. AR gain coexisted with X-chromosome gain and was associated with advanced tumor stage. When this panel was applied, two categories of combinatorial abnormalities proved clinically important. First, PTEN deletion without TMPRSS2/ERG rearrangement was enriched in pT3/4 tumors (70% versus 48%) and tumors with Gleason grades of 8 to 9 (60% versus 17%) compared with the entire cohort. These patients had earlier BcR than patients with normal FISH panel results (P < 0.01). In contrast, patients with PTEN deletion and ERG rearrangement had a BcR rate similar to patients who tested normal for all three markers (P > 0.1). Second, AR gain and concurrent trisomy 10 without TMPRSS2/ERG rearrangement were enriched in pT3/4 tumors and tumors with Gleason grades of 8 to 9. The three-marker FISH panel demonstrated prognostic utility and identified genomic aberrations associated with advanced disease state and early BcR in prostate cancer.

High-Resolution Genomic Profiling of Disseminated Tumor Cells in Prostate Cancer

Circulating tumor cells and disseminated tumor cells (DTCs) are of great interest because they provide a minimally invasive window for assessing aspects of cancer biology, including tumor heterogeneity, a means to discover biomarkers of disease behavior, and a way to identify and prioritize therapeutic targets in the emerging era of precision oncology. However, the rarity of circulating tumor cells and DTCs poses a substantial challenge to the consistent success in analyzing their molecular features, including genomic aberrations. Herein, we describe optimized and robust methods to reproducibly detect genomic copy number alterations in samples of 2 to 40 cells after whole-genome amplification with the use of a high-resolution single-nuclear polymorphism-array platform and refined computational algorithms. We have determined the limit of detection for heterogeneity within a sample as 50% and also demonstrated success in analyzing single cells. We validated the genes in genomic regions that are frequently amplified or deleted by real-time quantitative PCR and nCounter copy number quantification. We further applied these methods to DTCs isolated from individuals with advanced prostate cancer to confirm their highly aberrant nature. We compared copy number alterations of DTCs with matched metastatic tumors isolated from the same individual to gain biological insight. These developments provide high-resolution genomic profiling of single and rare cell populations and should be applicable to a wide-range of sample sources.

PTEN loss is associated with prostate cancer recurrence and alterations in tumor DNA methylation profiles

Background Prostate cancer (PCa) with loss of the tumor suppressor gene PTEN has an unfavorable prognosis. DNA methylation profiles associated with PTEN loss may provide further insights into the mechanisms underlying these more aggressive, clinically relevant tumors. Methods The cohort included patients with clinically localized PCa. Samples taken from the primary tumor were used to determine PTEN genomic deletions using FISH, and to analyze epigenome-wide DNA methylation profiles. Patients were followed for PCa recurrence on average for 8 years after diagnosis. Results The study included 471 patients with data on PTEN loss, and the frequency of hemi- and homozygous PTEN loss was 10.0% and 4.5%, respectively. Loss of PTEN was associated with a significantly higher risk of recurrence (any vs. no PTEN loss; HR = 1.74; 95% CI: 1.03–2.93). Hazard ratios for hemi- and homozygous loss were 1.39 (95% CI: 0.73–2.64) and 2.84 (95% CI: 1.30–6.19), respectively. Epigenome-wide methylation profiling identified 4,208 differentially methylated CpGs (FDR Q-value < 0.01) in tumors with any versus no PTEN loss. There were no genome-wide significant differentially methylated CpGs in homo- versus hemizygous deleted tumors. Tumor methylation data were used to build a methylation signature of PTEN loss in our cohort, which was confirmed in TCGA, and included CpGs in ATP11A, GDNF, JAK1, JAM3, and VAPA. Conclusion Loss of PTEN was positively associated with PCa recurrence. Prostate tumors with PTEN loss harbor a distinct methylation signature, and these aberrantly methylated CpG sites may mediate tumor progression when PTEN is deleted.

Abstract A25: Genome-wide methylation analyses in advanced-stage prostate cancer models

Introduction: Aberrant DNA methylation is a frequent event in prostate cancer (PCa). Investigation of genome-wide methylation changes in PCa promises to provide novel biomarkers that discriminate between indolent and aggressive diseases and to facilitate the development of novel treatment options. Most methylation studies to date have focused on CpG islands (CGI) in the promoter region of known genes. The current study investigated genome-wide methylation changes with the aim to identify recurrent regions differentially methylated in advanced PCa, including metastases and castration-resistance diseases. Methods: Using comprehensive high-throughput array-based relative methylation analysis (CHARM), we assessed genome-wide methylation status in metastatic PCa represented by two PCa cell lines (LNCap and VCap) as well as castration-resistant cancer represented by PCa xenografts cultivated in castrated mice (LuCaP23.1v, 35v, and 96v) and APIPC cells derived from AR-null LNCap maintained in hormone depleted media. To identify differentially methylated regions (DMR), each metastatic PCa cell line was compared with benign prostate epithelial RWPE1 cells. The castration-resistant xenografts were compared with the corresponding castration-sensitive xenografts, LuCaP23.1, 35, and 96, respectively. APIPC was compared with the parent line maintained in regular growth media. Unlike most methylation analyses that only target promoter CGI, CHARM analyses target CpG enriched regions of the entire genome, including genomic sequences up to 2kb outside of promoter CGIs, “CpG island shores”. The shores have been recently demonstrated to harbor more methylation alterations than the islands in tissue differentiation and cancer development. Results: Methylation changes were frequent in PCa metastases and castration-resistant diseases. Using RWPE1 as the reference, 306 DMRs were identified in LNCap (114 hyper- and 192 hypo-methylated), while 128 DMRs (32 hyper- and 96 hypo-methylated) were discovered in VCap cells. Compared with the parent LNCap line, APIPC showed 284 DMRs (80 hyper- and 204 hypomethylated). Using each corresponding castration-sensitive xenograft as the reference, castration-resistant LuCaP23.1v, LuCaP35v, and LuCaP96v demonstrated 587 (378 hyper- and 209 hypo-methylated), 150 (17 hyper- and 133 hypo-methylated), and 761 (353 hyper- and 408 hypo-methylated) DMRs, respectively. Distribution analyses showed that DMRs associated with these advanced PCa are enriched in regions 500bp outside CpG islands, i.e., CpG island shores, rather than in the islands. As expected, recurrent DMRs were evident. LNCap and VCap, representing PCa metastases, demonstrated 20 DMRs in common. Two of these common DMRs were of special interest: one is located at the 3′ UTR of STAT6 , the expression of which was previously shown to correlate with high histological grades of prostate cancer and with tumor size; another DMR is located at intron 1 of HOXA9 within the CGI shore. Aberrant methylation of HOXA9 promoter CGI was reported in multiple cancers such as bladder and non-small cell lung cancer. Among those representing castration-resistant PCa, 11 DMRs were shared between APIPC and LuCaP23.1v, while 2 DMRs were in common between APIPC and LuCaP96v. Conclusion: CHARM studies in advanced prostate cancer enabled us to better understand methylation changes at the whole genome level. Our data confirmed the importance of CpG enriched regions outside the promoter CGI during the development of cancer, specifically, PCa metastases and castration-resistant diseases. Multiple novel recurrent DMRs were identified in the study, which call for further investigation of their role in primary PCa as well as the related changes in gene expression and histone modification, and the prognostic significance of those changes. Citation Format: Xiaoyu Qu, Jerry Davison, Eric Bluemn, Peter Nelson, Robert Vessella, Min Fang. Genome-wide methylation analyses in advanced-stage prostate cancer models [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A25.