Gene Methylation and Early Detection of Genitourinary Cancer: The Road Ahead

Abstract and Introduction
Abstract

DNA methylation is a common mechanism of inactivation of tumour-suppressor and other cancer genes in neoplastic cells. The advantages of gene methylation as a target for the detection and diagnosis of cancer in biopsy specimens and non-invasive body fluids such as urine or blood has led to many studies of application in genitourinary cancer. Here, we consider the background, promise and status, challenges and future directions of gene methylation and its clinical utility for the early detection of genitourinary cancer. The challenges of, and strategies for, advancing gene-methylation-based detection are relevant to all types of cancer.
Introduction

Genitourinary cancer includes prostate, bladder, renal and testicular cancer. Prostate cancer is the most commonly detected male cancer and the second leading cause of male cancer deaths in the developed world. Bladder and renal cancer are, respectively, the fourth and sixth most common male cancers, being 1.5–2.5-fold less common in women. Testicular cancer is rare but remains the most frequent cancer in males under 35 years of age.[1,2] Population ageing worldwide[2] in regard to prostate cancer, increasing tobacco use in the developing world[3] as a known risk factor for bladder cancer, and the rising incidence of renal[4] and testicular[5] cancer in the western world all indicate that genitourinary cancer will continue to be a significant health burden in the future.

If detected early ( Box 1 ), prostate, bladder, renal or testicular cancer are curable by surgical resection and management. Moreover, populations that are at increased risk for developing genitourinary cancers are known, so the development of new diagnostic, and non-invasive approaches for the early detection of these cancers is extremely important. One source of new targets for the detection of genitourinary tumours is represented by the gene alterations that are known to underlie the molecular basis of these diseases. Here, I focus on DNA methylation as a target for the early detection of genitourinary cancer, and discuss the current challenges to the clinical implementation of methylation-based screenings. DNA methylation, cancer and early detection

Cancer is a disease initiated and driven by the clonal evolution of cells transformed by genetic and epigenetic alterations, which can occur as either inherited (germline) mutations or acquired (somatic) mutations of key genes.[6] These alterations can be used as targets for the detection of tumour cells in clinical specimens such as tissue biopsies or body fluids[7] ( Box 2 ; Fig. 1). A molecular test that targets gene alterations at the DNA level has several conceptual advantages for the successful early detection of cancer. The alteration may be present, and therefore potentially detectable, before the cancer can be found by imaging or traditional pathology, because either the alteration precedes obvious cancer or the abnormal cells represent a tiny fraction of the cell population in the biopsy. DNA is an ideal substrate for molecular detection because it is robust and survives the adverse conditions that many clinical specimens undergo, and can be readily amplified by powerful PCR-based techniques.[8] It is possible to work with minuscule amounts of DNA from early pre-neoplastic lesions or small cancers and achieve the sensitive detection of one cancer cell (or genome copy) in a background of hundreds or thousands of normal cells. Importantly, DNA alterations can be qualitative, as well as quantitative, so it is possible to screen for an absolute change, which is either present or absent in a clonal cell population, and therefore potentially very specific for cancer. Finally, gene alterations can provide simultaneous diagnostic and prognostic information,[9] including insight into the biology of the individual tumour.[10] Therefore, DNA alterations can form the basis of a sensitive and specific, robust and informative test for the detection of cancer.[8,11]

Figure 1. (click image to zoom)
Screening for genitourinary cancer. Tumour cells and free tumour DNA from dead cancer cells can access urine through secretion from the prostate into the urethra, and the proximity of urine to the transitional cell lining of the bladder and the renal system. Tumour cells that lack the capacity to metastasize and free tumour DNA can also access the circulatory system. Direct, but invasive, access by needle biopsy is performed for prostate cancer and, less commonly, renal cancer. DNA is isolated from the clinical specimen urine, blood or biopsy, and analysed for the presence of gene methylation by quantitative real-time methylation-specific PCR (qMSP).


Alterations in DNA methylation, an epigenetic process present in mammalian cells, are a hallmark of human cancer. Around half of the known human genes contain a CpG island within their promoter region. With the exception of genes on the inactive X chromosome in females and imprinted genes, CpG islands are generally protected from methylation in normal cells. This protection is crucial, as methylation of the CpG island is associated with the loss of expression of the particular gene. It has been shown that the transcriptional silencing of CDKN2A (which encodes INK4a), VHL (the gene that encodes the von Hippel–Lindau protein), MLH1 and other tumour-suppressor genes through promoter hypermethylation is a common feature in human cancer, and results in the loss of gene function, similar to other mechanisms such as deletions or point mutations.[12,13] For several reasons, these hypermethylated regions represent excellent targets for new cancer diagnostic approaches based on PCR technology.

There is accumulating evidence that gene methylation is frequent and can occur early in human tumorigenesis.[14] Several genes are known to be hypermethylated in each adult epithelial tumour type at a sufficiently high frequency that a panel of genes would probably provide a target for detection (diagnostic coverage) in most, or even all, cases.[14,15] Diagnostic coverage is one of the two major determinants of sensitivity of a molecular detection test, the other being the detection limits of the technology to be used. Importantly, sensitive technology for the detection of gene methylation exists ( Box 3 ) (Fig. 2).

Figure 2. (click image to zoom)
Analysis and scoring of gene methylation. a | DNA methylation is a chemical modification that does not change the DNA base sequence. Regular PCR cannot distinguish between unmethylated or methylated cytosines. Modification of DNA with sodium bisulphite converts all unmethylated, but not methylated, cytosine to uracil recognized as thymine. Primers and probes are designed to analyse the DNA specimen for the presence or absence of methylation of a gene. b | Conventional gel-based methylation-specific PCR (cMSP). The unmethylated (U) MSP reaction serves as a quality control for template DNA. The presence of a product from the methylated (M) reaction is a marker for the presence of cancer. Positive and negative controls are also analysed. The unmethylated product is designed to be different in size to identify loading error. c | Quantitative real time MSP (qMSP). The DNA specimen is analysed for the presence of methylated target gene and for amplification of either unmethylated gene sequence or a standard unmethylated gene; for example, β-actin. The point at which the curve crosses the cycle threshold is compared with standard dilutions to obtain a quantitative result. d | Low-density arrays of a panel of methylated genes and controls may be the next step in the analysis of gene methylation in clinical specimens. ND, not determined; NTC, no template control.

An important consideration of a biomarker for early cancer detection is its specificity for tumour cells compared with normal or non-neoplastic cells. In patients with sporadic (that is, non-familial) cancer, the functional inactivation of classical tumour-suppressor genes is only found in neoplastic cells. If the hypermethylation of a tumour-suppressor gene is associated with transcriptional inactivation, then conceptually such methylation would be highly specific for neoplastic cells, and is probably the basis for the high specificity observed in many exploratory studies of methylation-based detection of cancer.^[15] That the methylation of classical tumour-suppressor genes is restricted to neoplastic cells means, in theory, that methylation could provide a 'yes or no' answer for the presence of cancer.^[16]