Yong-Jie Lu

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.

Expression profile of genes from 12p in testicular germ cell tumors of adolescents and adults associated with i(12p) and amplification at 12p11.2–p12.1

Gain of 12p material is invariably associated with testicular germ cell tumors (TGCTs) of adolescents and adults, most usually as an isochromosome 12p. We analyzed TGCTs with i(12p) using a global approach to expression profiling targeting chromosomes (comparative expressed sequence hybridization, CESH). This indicated overexpression of genes from 12p11.2–p12.1 relative to testis tissue and fibroblasts. The nonseminoma subtype showed higher levels of expression than seminomas. Notably, 12p11.2–p12.1 is amplified in about 10% of TGCTs and CESH analysis of such amplicon cases showed high levels of overexpression from this region. Microarray analysis, including cDNA clones representing most UniGene clusters from 12p11.2–p12.1, was applied to DNA and RNA from 5 TGCTs with amplification of 12p11.2–p12.1 and seven TGCTs with gain of the entire short arm of chromosome 12. Expression profiles were consistent with the CESH data and overexpression of EST595078, MRPS35 and LDHB at 12p11.2–p12.1 was detected in most TGCTs. High-level overexpression of BCAT1 was specific to nonseminomas and overexpression of genes such as CMAS, EKI1, KRAS2, SURB7 and various ESTs correlated with their amplification. Genes such as CCND2, GLU3, LRP6 and HPH1 at 12p13 were also overexpressed. The overexpressed sequences identified, particularly those in the region amplified, represent candidate genes for involvement in TGCT development.

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.

Relationship Between MYCN Copy Number and Expression in Rhabdomyosarcomas and Correlation With Adverse Prognosis in the Alveolar Subtype

PURPOSE Amplification of the transcription factor MYCN is an important molecular diagnostic tool in stratifying treatment for neuroblastoma. Increased copy number and overexpression of MYCN in the pediatric cancer rhabdomyosarcoma has been described in a number of small studies with conflicting conclusions about its association with clinicopathologic characteristics. We aimed to study the phenomenon in the largest series to date. PATIENTS AND METHODS Using quantitative polymerase chain reaction, we measured MYCN copy number and expression levels in rhabdomyosarcoma samples from 113 and 92 individuals with a confirmed diagnosis of rhabdomyosarcoma, respectively. RESULTS Increased copy number of MYCN was found to be a feature of both the embryonal and alveolar subtypes. The copy number and expression levels were significantly greater in the alveolar subtype, although the range of expression in both subtypes spanned several orders of magnitude. MYCN copy number showed a significant correlation with expression in the alveolar subtype; this relationship between copy number and expression could be modeled as a logarithmic function. It is notable that relatively high expression frequently occurred in embryonal rhabdomyosarcoma without high copy number and that low expression was found in some cases with high copy number. In patients with alveolar rhabdomyosarcoma, overexpression (greater than median) or gain of genomic copies of MYCN were significantly associated with adverse outcome. CONCLUSION MYCN deregulation is a feature of rhabdomyosarcoma tumorigenesis, defines groups of patients with a poor prognosis, and is a potential target for novel therapies.

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.

Role for Amplification and Expression of Glypican-5 in Rhabdomyosarcoma

Overexpression of genes, through genomic amplification and other mechanisms, can critically affect the behavior of tumor cells. Genomic amplification of the 13q31-32 region is reported in many tumors, including rhabdomyosarcomas that are primarily pediatric sarcomas resembling developing skeletal muscle. The minimum overlapping region of amplification at 13q31-32 in rhabdomyosarcomas was defined as containing two genes: Glypican-5 (GPC5) encoding a cell surface proteoglycan and C13orf25 encompassing the miR-17-92 micro-RNA cluster. Genomic copy number and gene expression analyses of rhabdomyosarcomas indicated that GPC5 was the only gene consistently expressed and up-regulated in all cases with amplification. Constitutive overexpression and knockdown of GPC5 expression in rhabdomyosarcoma cell lines increased and decreased cell proliferation, respectively. A correlation between expression levels of nascent pre-rRNA and GPC5 (P = 0.001), but not a C13orf25 transcript containing miR-17-92, in primary samples supports an association of GPC5 with proliferative capacity in vivo. We show that GPC5 increases proliferation through potentiating the action of the growth factors fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and Wnt1A. GPC5 enhanced the intracellular signaling of FGF2 and HGF and altered the cellular distribution of FGF2. The mesoderm-inducing effect of FGF2 and FGF4 in Xenopus blastocysts was also enhanced. Our data are consistent with a role of GPC5, in the context of sarcomagenesis, in enhancing FGF signaling that leads to mesodermal cell proliferation without induction of myogenic differentiation. Furthermore, the properties of GPC5 make it an attractive target for therapeutic intervention in rhabdomyosarcomas and other tumors that amplify and/or overexpress the gene.

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.

Absolute Quantitation of DNA Methylation of 28 Candidate Genes in Prostate Cancer Using Pyrosequencing

Aberrant DNA methylation plays a pivotal role in carcinogenesis and its mapping is likely to provide biomarkers for improved diagnostic and risk assessment in prostate cancer (PCa). We quantified and compared absolute methylation levels among 28 candidate genes in 48 PCa and 29 benign prostate hyperplasia (BPH) samples using the pyrosequencing (PSQ) method to identify genes with diagnostic and prognostic potential. RARB, HIN1, BCL2, GSTP1, CCND2, EGFR5, APC, RASSF1A, MDR1, NKX2-5, CDH13, DPYS, PTGS2, EDNRB, MAL, PDLIM4, HLAa, ESR1 and TIG1 were highly methylated in PCa compared to BPH (p < 0.001), while SERPINB5, CDH1, TWIST1, DAPK1, THRB, MCAM, SLIT2, CDKN2a and SFN were not. RARB methylation above 21% completely distinguished PCa from BPH. Separation based on methylation level of SFN, SLIT2 and SERPINB5 distinguished low and high Gleason score cancers, e.g. SFN and SERPINB5 together correctly classified 81% and 77% of high and low Gleason score cancers respectively. Several genes including CDH1 previously reported as methylation markers in PCa were not confirmed in our study. Increasing age was positively associated with gene methylation (p < 0.0001). Accurate quantitative measurement of gene methylation in PCa appears promising and further validation of genes like RARB, HIN1, BCL2, APC and GSTP1 is warranted for diagnostic potential and SFN, SLIT2 and SERPINB5 for prognostic potential.

The Effect of VEGF-Targeted Therapy on Biomarker Expression in Sequential Tissue from Patients with Metastatic Clear Cell Renal Cancer

Purpose: To investigate how biologically relevant markers change in response to antiangiogenic therapy in metastatic clear cell renal cancer (mRCC) and correlate these changes with outcome. Experimental Design: The study used sequential tumor tissue and functional imaging (taken at baseline and 12–16 weeks) obtained from three similar phase II studies. All three studies investigated the role of VEGF tyrosine kinase inhibitors (TKI) before planned nephrectomy in untreated mRCC (n = 85). The effect of targeted therapy on ten biomarkers was measured from sequential tissue. Comparative genomic hybridization (CGH) array and DNA methylation profiling (MethylCap-seq) was performed in matched frozen pairs. Biomarker expression was correlated with early progression (progression as best response) and delayed progression (between 12–16 weeks). Results: VEGF TKI treatment caused a significant reduction in vessel density (CD31), phospho-S6K expression, PDL-1 expression, and FOXP3 expression (P < 0.05 for each). It also caused a significant increase in cytoplasmic FGF-2, MET receptor expression in vessels, Fuhrman tumor grade, and Ki-67 (P < 0.05 for each). Higher levels of Ki-67 and CD31 were associated with delayed progression (P < 0.05). Multiple samples (n = 5) from the same tumor showed marked heterogeneity of tumor grade, which increased significantly with treatment. Array CGH showed extensive intrapatient variability, which did not occur in DNA methylation analysis. Conclusion: TKI treatment is associated with dynamic changes in relevant biomarkers, despite significant heterogeneity in chromosomal and protein, but not epigenetic expression. Changes to Ki-67 expression and tumor grade indicate that treatment is associated with an increase in the aggressive phenotype of the tumor. Clin Cancer Res; 19(24); 6924–34. ©2013 AACR.

Dual colour fluorescence in situ hybridization to paraffin-embedded samples to deduce the presence of the der(X)t(X;18)(p11.2;q11.2) and involvement of either the SSX1 or SSX2 gene: a diagnostic and prognostic aid for synovial sarcoma

Identification of the t(X;18)(p11.2;q11.2) and the fusion gene products, SYT–SSX1 and SYT–SSX2, associated with a high proportion of synovial sarcomas, has been shown to be a useful diagnostic aid. This study demonstrates the application of dual colour fluorescence in situ hybridization to paraffin‐embedded samples to deduce the presence of the derivative X chromosome and also the position of the breakpoint on chromosome X at either the SSX1 or the SSX2 gene. This used region specific markers from chromosomes X and 18 and an optimized protocol involving microwave exposure. Novel and rapid scoring criteria were validated which circumvented potential problems of nuclear truncation and defining cell boundaries. This involved blind analysis of two negative sarcoma samples and three synovial sarcomas in which corresponding frozen material had been previously shown to have the translocation involving different SSX genes. Six new cases diagnosed as synovial sarcoma were also analysed; two monophasic and two biphasic case were deduced to have a breakpoint in the SSX1 gene, one monophasic case an SSX2 breakpoint, and one case did not show rearrangement of the region. The ability to analyse formalin‐fixed, paraffin‐embedded samples in this way has practical implications for aiding the diagnosis of difficult cases, recently ascribed prognostic relevance, and allows further retrospective studies to be carried out. The methodology is also applicable to the identification of other tumour specific translocations in paraffin‐embedded material. Copyright