Sakunthala C. Kudahetti

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.

Ki-67 and outcome in clinically localised prostate cancer: analysis of conservatively treated prostate cancer patients from the Trans-Atlantic Prostate Group study

Treatment decisions after diagnosis of clinically localised prostate cancer are difficult due to variability in tumour behaviour. We therefore examined one of the most promising biomarkers in prostate cancer, Ki-67, in a cohort of 808 patients diagnosed with prostate cancer between 1990 and 1996 and treated conservatively. Ki-67 expression was assessed immunohistochemically, in two laboratories, by two different scoring methods and the results compared with cancer-specific and overall survival. The power of the biomarker was compared with Gleason score and initial serum prostate-specific antigen (PSA). Both methods showed that Ki-67 provided additional prognostic information beyond that available from Gleason score and PSA: for the semi-quantitative method, Δχ2 (1 d.f.)=24.6 (P<0.0001), overall survival χ2=20.5 (P<0.0001), and for the quantitative method, Δχ2 (1 d.f.)=15.1 (P=0.0001), overall survival χ2=10.85 (P=0.001). Ki-67 is a powerful biomarker in localised prostate cancer and adds to a model predicting the need for radical or conservative therapy. As it is already in widespread use in routine pathology, it is confirmed as the most promising biomarker to be applied into routine practice.

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.

HPV infection and immunochemical detection of cell-cycle markers in verrucous carcinoma of the penis

Penile verrucous carcinoma is a rare disease and little is known of its aetiology or pathogenesis. In this study we examined cell-cycle proteins expression and correlation with human papillomavirus infection in a series of 15 pure penile verrucous carcinomas from a single centre. Of 148 penile tumours, 15 (10%) were diagnosed as pure verrucous carcinomas. The expression of the cell-cycle-associated proteins p53, p21, RB, p16INK4A and Ki67 were examined by immunohistochemistry. Human papillomavirus infection was determined by polymerase chain reaction to identify a wide range of virus types. The expression of p16INK4A and Ki67 was significantly lower in verrucous carcinoma than in usual type squamous cell carcinoma, whereas the expression of p53, p21 and RB was not significantly different. p53 showed basal expression in contrast to usual type squamous cell carcinoma. Human papillomavirus infection was present in only 3 out of 13 verrucous carcinomas. Unique low-risk, high-risk and mixed viral infections were observed in each of the three cases. In conclusion, lower levels of p16INK4A and Ki67 expressions differentiate penile verrucous carcinoma from usual type squamous cell carcinoma. The low Ki67 index reflects the slow-growing nature of verrucous tumours. The low level of p16INK4A expression and human papillomavirus detection suggests that penile verrucous carcinoma pathogenesis is unrelated to human papillomavirus infection and the oncogenes and tumour suppressor genes classically altered by virus infection.

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.

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.

The retinoblastoma protein/p16INK4A pathway but not p53 is disrupted by human papillomavirus in penile squamous cell carcinoma

Stankiewicz E, Prowse D M, Ktori E, Cuzick J, Ambroisine L, Zhang X, Kudahetti S, Watkin N, Corbishley C & Berney D M
(2011) Histopathology58, 433–439
The retinoblastoma protein/p16INK4Apathway but not p53 is disrupted by human papillomavirus in penile squamous cell carcinoma

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.

Immunohistochemistry for p16, but not Rb or p21, is an independent predictor of prognosis in conservatively treated, clinically localised prostate cancer

Aims: Treatment decisions are difficult in clinically localised prostate cancer and further biomarkers of aggressive behaviour are required. We investigated the hypothesis that the tissue expression of three cell cycle markers, Rb, p21 and p16, would provide helpful prognostic information in a well characterised series of prostate cancers which were clinically localised and treated conservatively. Methods: The immunohistochemical staining expression of these markers was assessed in tissue microarrays and correlated with 10 year prostate cancer survival and overall survival and then compared with pathological data including contemporary Gleason score, age, measures of tumour extent and initial serum prostate specific antigen (PSA) level. Results: Rb overexpression did not show any significant association with Gleason score or prostate cancer survival. p21 protein expression showed a significant association with prostate cancer survival (p = 0.02) and overall survival (p = 0.01) in a univariate model but not in a multivariate model with pathological and serum PSA data. There was a significant association between p16 cytoplasmic expression and prostate cancer survival (HR = 2.52, 95%CI = 1.79–3.55, p < 0.001) and overall survival (HR = 1.54, 95% CI = 1.20–1.98, p = 0.001) in a univariate model. p16 expression remained an independent prognostic factor for prostate cancer survival (HR = 1.50, 95%CI = 1.05–2.14, p = 0.03). Conclusion: We conclude that p16 cytoplasmic expression can be used as a predictor of outcome in conservatively treated prostate cancer. Rb and p21 show no independent association with outcome and therefore further research is not warranted.