R. Tim D. Oliver

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 Association of CCND1 Overexpression and Cisplatin Resistance in Testicular Germ Cell Tumors and Other Cancers

Development of chemoresistance limits the clinical efficiency of platinum-based therapy. Although many resistance mechanisms have been demonstrated, genetic/molecular alterations responsible for drug resistance in the majority of clinical cases have not been identified. We analyzed three pairs of testicular germ cell tumor cell lines using Affymetrix expression microarrays and revealed a limited number of differentially expressed genes across the cell lines when comparing the parental and resistant cells. Among them, CCND1 was the most significantly differentially expressed gene. Analysis of testicular germ cell tumor clinical samples by quantitative reverse transcription PCR analysis revealed that overall expression of CCND1 was significantly higher in resistant cases compared with sensitive samples (P < 0.0001). We also found that CCND1 was dramatically overexpressed both in induced and intrinsically resistant samples of ovarian and prostate cancer. Finally combined CCND1 knockdown using small-interfering RNA and cisplatin treatment inhibited cell growth in vitro significantly more effectively than any of these single treatments. Therefore, deregulation of CCND1 may be a major cause of cisplatin resistance in testicular germ cell tumors and may also be implicated in ovarian and prostate cancers. CCND1 could be potentially used as a marker for treatment stratification and as a molecular target to improve the treatment of platinum-resistant tumors.

Detection of TMPRSS2:ERG fusion gene in circulating prostate cancer cells.

AIM To investigate the existence of TMPRSS2:ERG fusion gene in circulating tumor cells (CTC) from prostate cancer patients and its potential in monitoring tumor metastasis. METHODS We analyzed the frequency of TMPRSS2:ERG and TMPRSS2:ETV1 transcripts in 27 prostate cancer biopsies from prostatectomies, and TMPRSS2:ERG transcripts in CTC isolated from 15 patients with advanced androgen independent disease using reverse transcription polymerase chain reaction (RT-PCR). Fluorescence in situ hybridization (FISH) was applied to analyze the genomic truncation of ERG, which is the result of TMPRSS2:ERG fusion in 10 of the 15 CTC samples. RESULTS TMPRSS2:ERG transcripts were found in 44% of our samples, but we did not detect expression of TMPRSS2:ETV1. Using FISH analysis we detected chromosomal rearrangements affecting the ERG gene in 6 of 10 CTC samples, including 1 case with associated TMPRSS2:ERG fusion at the primary site. However, TMPRSS2:ERG transcripts were not detected in any of the 15 CTC samples, including the 10 cases analyzed by FISH. CONCLUSION Although further study is required to address the association between TMPRSS2:ERG fusion and prostate cancer metastasis, detection of genomic truncation of the ERG gene by FISH analysis could be useful for monitoring the appearance of CTC and the potential for prostate cancer metastasis.

Hedgehog Signalling in Androgen Independent Prostate Cancer

OBJECTIVES Androgen-deprivation therapy effectively shrinks hormone-naïve prostate cancer, both in the prostate and at sites of distant metastasis. However prolonged androgen deprivation generally results in relapse and androgen-independent tumour growth, which is inevitably fatal. The molecular events that enable prostate cancer cells to proliferate in reduced androgen conditions are poorly understood. Here we investigate the role of Hedgehog signalling in androgen-independent prostate cancer (AIPC). METHODS Activity of the Hedgehog signalling pathway was analysed in cultured prostate cancer cells, and circulating prostate tumour cells were isolated from blood samples of patients with AIPC. RESULTS AIPC cells were derived through prolonged culture in reduced androgen conditions, modelling hormone therapy in patients, and expressed increased levels of Hedgehog signalling proteins. Exposure of cultured AIPC cells to cyclopamine, which inhibits Hedgehog signalling, resulted in inhibition of cancer cell growth. The expression of the Hedgehog receptor PTCH and the highly prostate cancer-specific gene DD3(PCA3) was significantly higher in circulating prostate cancer cells isolated from patients with AIPC compared with samples prepared from normal individuals. There was an association between PTCH and DD3(PCA3) expression and the length of androgen-ablation therapy. CONCLUSIONS Our data are consistent with reports implicating overactivity of Hedgehog signalling in prostate cancer and suggest that Hedgehog signalling contributes to the androgen-independent growth of prostate cancer cells. As systemic anti-Hedgehog medicines are developed, the Hedgehog pathway will become a potential new therapeutic target in advanced prostate cancer.

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.

Assessing the size and stage of testicular germ cell tumours: 1984-2003.

To assess the size and stage of testicular tumours on presentation in the period 1984–2002.

Identification of ZDHHC14 as a novel human tumour suppressor gene

Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase‐dependent apoptotic pathway and heterozygous knockout of ZDHHC14 decreased cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down‐regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer.

Identification of genomic changes associated with cisplatin resistance in testicular germ cell tumor cell lines

Since the introduction of cisplatin into the clinic, the treatment of patients with a variety of solid tumors including testicular germ cell tumors, ovarian and lung cancers, has dramatically improved. One of the main causes for therapeutic failure in these malignancies is the development of drug resistance. Testicular germ cell tumors (TGCTs), the most common malignancy in young men, exhibit extreme sensitivity to cisplatin‐based chemotherapy, making them an ideal model for investigating the mechanisms of cisplatin chemo‐sensitivity and resistance. TGCT development and pathogenesis have been well studied but little is known about the genetic background in chemo‐resistant cases. We investigated genomic differences between three TGCT parental cell lines and their cisplatin resistant derivatives. Using 10K single nucleotide polymorphism (SNP) microarray analysis, we identified two small chromosomal regions with consistent copy number changes across all three pairs of resistant cell lines. These were an 8.7 Mb region at 6q26‐27, which displayed consistent copy number gain and a 0.3 Mb deletion involving 4 SNPs at 10p14. Both the chromosomal gain and loss were confirmed by fluorescence in situ hybridization. The significance of these regions should be further investigated as they may contain key genes involved in the development of chemo‐ resistance to cisplatin‐based treatment in TGCTs and other cancers.

Rapid high‐resolution karyotyping with precise identification of chromosome breakpoints

Many techniques have been developed in recent years for genome‐wide analysis of genetic alterations, but no current approach is capable of rapidly identifying all chromosome rearrangements with precise definition of breakpoints. Combining multiple color fluorescent in situ hybridization and high‐density single nucleotide polymorphism array analyses, we present here an approach for high resolution karyotyping and fast identification of chromosome breakpoints. We characterized all of the chromosome amplifications and deletions, and most of the chromosome translocation breakpoints of three prostate cancer cell lines at a resolution which can be further analyzed by sequence‐based techniques. Genes at the breakpoints were readily determined and potentially fused genes identified. Using high‐density exon arrays we simultaneously confirmed altered exon expression patterns in many of these breakpoint genes.

The novel association of circulating tumor cells and circulating megakaryocytes with prostate cancer prognosis.

Purpose: To develop an approach for the investigation of different subtypes of circulating tumor cells (CTC) and other cells to evaluate their potential prognostic value of prostate cancer. Experimental Design: Malignancy of CTCs undergoing epithelial-to-mesenchymal transition (EMT) was confirmed by repeated FISH. Subgroups of CTCs in 81 patients with prostate cancer (43 castration resistant and 38 untreated localized) were correlated to disease aggressiveness parameters. AUC analysis was applied to compare the performance for metastasis prediction between serum PSA level alone and a combined risk score using both PSA and EMTing CTC count. Circulating megakaryocytes and cancer patient survival association was performed using Cox model. Results: The majority of vimentin (VIM)+/CD45− cells were malignant, with genomic alterations in several genomic regions. The number of cytokeratin (CK)−/VIM+/CD45− CTCs correlated with disease burden, tumor aggressiveness, and poorer survival. Meanwhile, CK+/VIM+/CD45− CTCs were associated with metastases better than other subtypes of CTCs in these limited samples. Combination of PSA level and the number of CK+/VIM+/CD45− CTCs enhanced the prediction of cancer metastases [AUC, 0.921; 95% confidence interval (CI), 0.858–0.985]. The number of circulating megakaryocytes was potentially associated with good patient survival in advanced prostate cancer (HR, 0.849; 95% CI, 0.628–1.146, per cell increase), and the difference between the number of mesenchymal CTCs and megakaryocytes strongly correlated to poor survival (HR, 10.17; 95% CI, 2.164–47.789, if score ≥2.0). Conclusions: This CTC analysis approach and the potential association of megakaryocytes with cancer prognosis may greatly enhance our ability to investigate the cancer metastasis process and to predict/monitor cancer progression. Clin Cancer Res; 23(17); 5112–22. ©2017 AACR.