Screening of prostate cancer suffers from a lack of specificity, identifying many false positives, mainly through the use of routine PSA screening and follow-up biopsy tissue analysis [21
]. Men with elevated PSA-levels but negative biopsies will typically be re-evaluated and may be rebiopsied, although this has been proven unnecessary for the vast majority [22
]. Epigenetics, more specifically DNA methylation, has been postulated to aid the diagnostic process. Because the presence of cancer cells missed by standard histopathology can be detected through DNA methylation [14
], a confirmatory epigenetic assay can reinforce a negative finding or indicate the need for a rebiopsy in men with an initial negative biopsy [2
DNA methylation plays a role in gene silencing and acts as a powerful biomarker by repressing tumor suppressor genes during oncogenesis [24
]. The functional loss of key genes in normal cells is progressive in nature, accumulating deregulating oncogenic hits and eventually disturbing several protective regulatory pathways [25
]. In multiple cancer types, including prostate cancer, this phenomenon is present as a field effect, offering the possibility to detect aberrant methylation at a distance from the actual tumor [14
], and is currently used to develop an assay to improve upon standard histopathology in prostate cancer detection.
Several genes have been described as methylation biomarkers for prostate cancer, including the three genes that are the subject of this analysis. GSTP1
is likely the most studied epigenetic lesion in relation to prostate cancer [26
]. In addition, the gene is also of potential use as a screening marker in non-invasive samples such as urine and blood [29
have also been described as prostate cancer markers with important roles in detecting the epigenetic field effect surrounding cancer foci [14
]. In this paper, a multiplex assay detecting methylation of GSTP1APC
has been developed and evaluated. The transition of singleplex to multiplex should lead to a decreased variance and restrain potential sources of error. In addition, it allows smaller sample quantities to be used as input for the assay, which is critical given that often limited cancer is found in needle biopsy tissue.
Multiplexing the three epigenetic assays and the ACTB control gene into one reaction leads to copy number and methylation ratio alterations relative to individual singleplex assays. The use of different fluorophores for the individual primers is most likely the main reason, although altered thermodynamics and PCR efficiencies cannot be completely excluded. The effect appears to be consistent for each individual assay, but not between different assays (Figure ). Although this changes the interpretation of the calculated methylation ratios, the final outcome, i.e. detection or absence of methylation, should be determined with the same accuracy as with individual singleplex assays. Although only a small test cohort was available for verification, similar correlations between methylation and the presence of cancer foci were obtained. A small decrease in MCC was observed for APC and RASSF1. However, it can be argued that due to the consistency in copy number changes, the ratios should also be altered in a consistent manner. This supports the belief that the observed, minor decreases in correlation are random effects, errors that are not linked to the transition of singleplex to multiplex.
A typical biopsy tissue core using an 18-gauge needle produces a cylindrical mass of approximately 1
mm thick. A variable amount of tissue is cut from the FFPE block for analysis, especially if an atypical focus is noted and recuts are performed. This leaves an uneven amount of tissue remaining within the paraffin block. Thus, the smaller the tissue requirement to do further testing, including analytical, molecular analyses, the better.
The assays were initially developed using prostatectomy samples and 40 microns of biopsy tissue from FFPE blocks. However, such amounts of tissue are not always readily available for confirmatory assays, e.g. confirming a negative histology outcome or detecting occult cancer. Therefore, it was tested whether the multiplex assay could generate the same methylation outcome in smaller biopsies of 20 or even 10
μm. The smallest biopsy volume clearly possessed a suboptimal performance, where methylation was more often missed due to erroneous sample localization (Table ). The minimally required amount of input DNA for reliable, repeatable detection of methylation was not observed for the 10
μm, as opposed to 20 and 40
μm samples (Figure and Table ). The occasional lack of detecting methylation in cancer-positive samples from 20
μm biopsies could relate to tissue sampling errors, older samples or lack of sufficient input. Additionally, not all tissue sections may have methylated promoter regions for any of the 3 genes. In general, the intuitive rule that more recent, larger samples are better for Q-MSP analysis was proven true (Table ). Because 20-micron samples preserved in FFPE for up to 5 or 6
years seem to be borderline, requiring more DNA for testing seems reasonable for older, archived samples. As a general guideline, samples down to 20 micron provide sufficient input DNA up to an age of 6
years. For recently archived tissue, up to one year, 20-micron samples appear as adequate as their 40-micron counterparts.
A 2-fold dilution experiment ranging from 512 to 2 copies of the plasmid vector used for the multiplex assay was set up with 24 repeats per dilution factor (data not shown) to determine the limit of detection and analytical sensitivity of the assay. As little as 16 methylated copies of APC and GSTP1 and 32 copies of RASSF1 could be detected reliably, i.e. in more than 95% of the samples. This illustrates the high analytical sensitive of an MSP-based epigenetic assay.
In conclusion, an epigenetic, confirmatory multiplex assay for detecting the presence of prostate cancer cells missed by histology has been further developed. It performed similar relative to the individual singleplex assays for the most important test trait, biomarker gene methylation status. Epigenetic testing can be executed in an accurate and reliable manner in FFPE core tissue biopsies, with as little as 20
μm of sample volume.