Beyond PSA: are new prostate cancer biomarkers of potential value to New Zealand doctors?

Prostate cancer is the most commonly registered male cancer in New Zealand making up 25.2% of all registrations, ahead of colorectal cancer and malignant melanoma of the skin, and the third most common cancer registration for both sexes.Prostate cancer was also the third leading cause of male cancer deaths in 20061. Although recent data might be interpreted as suggesting that there has been a decline in the incidence of prostate cancer since the year 20002, this may be an artefact of increased uptake of prostate-specific antigen (PSA) screening at that time. With increased PSA testing comes earlier diagnosis and registration of patients, which in turn will lead to an elevation of diagnosis in younger age groups (giving the pre-2000 increase).The apparent post-2000 decline is thus a result of those patients already being picked up by the test who would have otherwise been diagnosed at that time. The likely result is a paradigm shift in the age distribution of patients with diagnosed prostate cancer, and a return to a steady gradual increase in diagnosed prostate cancer patients, as seen in the pre-PSA years2.PSA testingthe current method of prostate cancer risk and progression assessment a prima facie, falls well short of the performance required of a screen in an age of evidence-based medicine, with sensitivity and specificity of PSA testing being quoted as 74-84% and 90-94% respectively3,4 and a positive predictive value of 21.9% (when using the traditional value of PSA 4.0 ng/mL as a threshold)5.Use of such a test as the basis of clinical decisions for prostate cancer patients renders active surveillance (a programme consisting of regular PSA and DRE (digital rectal examination) testing (in addition to regular biopsy of a patients prostatic tissue) or watchful waiting (where treatment has a stronger palliative element and curative treatments are foregone)116 as the most prudent course of action when a PSA level is shown to be in the grey zone of 2.5 ng/mL-10 ng/mL6.It should be noted, however, that active surveillance and watchful waiting, despite the implication of PSA values, are primarily indicated through key parameters of biopsy results, including Gleason score, clinical grade of disease, number of cores positive upon biopsy and volume of malignant tissue in each positive core. The current dependence on an invasive test for disease prognosis is reflective of the difficulty to differentiate between indolent and aggressive neoplasms with PSA, which is, in essence, a risk-stratification tool.Indeed, this is further underpinned when one observes the high rate of false positive (95 in 1000 men aged 55-69 years who have the PSA test) and a substantial number of false negative results (23 per 1000 men aged 55-69 who have PSA testing and then biopsy)3. As a result, the decisions surrounding treatment become extremely difficult if the sole basis for the decision to treat was a non-invasive test such as PSA (in practice, just as for active surveillance, the decision to treat is primarily indicated through parameters of prostate biopsy).Patients who do not need treatment may opt to be treated and suffer unnecessary side effects. Equally, those who do need treatment may choose not to be treated, and miss the opportunity for an early intervention. It is this dilemma which epitomises the experience of both patient and practitioner in dealing with the inherent uncertainty of PSA testing. Ideally, clinicians would be able to call on an accurate and reliable non-invasive risk-stratification system, whereby patients are empowered with precise knowledge to make more fully informed decisions on their health, and equally have a clearer understanding of the risk of recurrence8.This review discusses novel biomarkers in prostate cancer which have the potential to be incorporated in new risk-stratification systems, and their role in delivering the diagnostic and prognostic precision currently lacking in clinical prostate cancer treatment. We note that this list is not exhaustive, but covers several that would be potentially applicable to the New Zealand clinical situation.PSA testing: the status quoCurrent policy and practiceScreens for genetic susceptibility to breast cancer (BRCA1/2 screening114), or for the presence of early signs of cancer in the cervix (cervical cancer screening113) are both well established in Aotearoa/New Zealand. However, comparable well established methods are not available for screening genetic susceptibility to prostate cancer, despite the similarity in incidences of breast (2572 registrations, 2006) and prostate (2484 registrations, 2006) cancers2.The lack of a well substantiated and non-invasive screening test for early prostate cancer3 (as compared with PAP smear testing in cervical cancer) requires a more aggressive and concerted effort from policymakers, clinicians and researchers to address the uncertainties and errors manifest in the PSA test, which defines the current status of prostate screening and on a more global level, the plight of mens health, in this country.As a reflection of where the New Zealand healthcare system stands with its current prostate screening proceduresout of the eight criteria outlined by the New Zealand National Health Committee (NHC) screening assessment, prostate cancer screening meets only one criterionthat prostate cancer is a condition which is a suitable candidate for screening3. Indeed, PSA and direct rectal examination (DRE) are described as unsuitable tests as:cneither can be described as reliable, accurate, sensitive or specific enough for screening asymptomatic men.d National Health Committee (2004)However, there exists a growing body of evidence which tentatively suggests that screening for prostate cancer is not without its benefits. Specifically, criterion three outlined by the NHCthat there is an effective and accessible treatment or intervention for the condition identified through early detection3would seem to be supported by data presented from the Scandinavian Prostate Cancer Group-4 trial144 demonstrating a reduction in metastatic disease incidence (RR=0.65; p=0.006) and disease-specific death (RR=0.82; p=0.09) for clinically localised prostate cancer specimens after a 12-year follow-up period with radical prostatectomy, as compared to watchful waiting.Additionally, data extracted from a cohort of 7578 men in Sweden, randomised to screening, demonstrated a prostate cancer-specific mortality reduction of almost 50% (RR=0.56; p=0.002) over 14 years compared to non-screened controls145, which would provide randomised controlled trial evidence demanded by the fourth criterion stipulated by the NHCthat a screening programme is effective in reducing morbidity and mortality.Although the inevitable risk of overdiagnosis has been acknowledged by the study authors and elsewhere145,146, these recent developments perhaps signal that it may be pertinent to once again review the current government policy on prostate cancer screening.Strengths and limitationsPSA testing has demonstrable strengths. With 90% of new cases detected early enough for curative treatment115 (where the treatments offer cure rates between 70%-90%) and changes in prostate cancer mortality ranging from 10%-39% in countries in Western Europe, North America and Australia116 we can recognise that, although flawed, PSA is having a positive effect of the clinical treatment of prostate cancer.In addition, when we consider that prostate cancer has a tendency to progress slower than other cancers (and even slower with androgen ablation therapy), the burden associated with the myriad of medical interventions such as radiotherapy, surgery and hospice care will often become more costly than an early, curative intervention administered on the basis of a routine PSA test116.Moreover, the natural course of prostate cancer means that if we were to forego PSA testing and diagnose on the appearance of symptoms, 70% of these cases will already have metastases. It must be acknowledged too, that PSA should only be seen as the initial step in prostate cancer assessmentTRUS (transrectal ultrasound) biopsy remains the gold standard in delivering diagnostic and prognostic data on prostate cancer. Figure 1. Current use of PSA in monitoring progression, diagnosis and prognosis of disease Note: The PSA Grey Zone (2.5ng/uL - 10ng/uL) 6 extends across the whole continuum of prostate cancer progression. These recognised limitations of PSA testing have led to international initiatives towards developing and validating new biomarkers with higher sensitivity and specificity which alone, or in conjunction with current screening methods, are able to deliver more definitive results on the presence and nature of cancer in the prostate, in a fast, cost-effective and non-invasive manner. Through the clinical application of novel biomarkers and effective implementation in the healthcare system, clinicians may aspire to deliver well informed and clear-cut decisions on the course of prostate cancer patients treatments and prognoses, and ultimately deliver better health outcomes for men in Aotearoa/New Zealand. Novel biomarkers: beyond PSA As researchers delve further into the elements underlying sporadic prostate cancer, we begin to unearth increasing evidence of this being a heterogeneous disease18. Unlike the discovery of the Bcr-Abl gene in chronic myeloid leukaemia, it is unlikely that more research will reveal a single specific gene locus that is responsible for prostate cancer. Naturally, such a multifaceted disease demands an equally multifaceted approach to risk-stratification, screening and diagnosis. Novel biomarkers for sporadic prostate cancer have been found on many echelons of the central dogma of genetics: genetic (specifically DNA), epigenetic, transcriptomic, proteomic and metabolomic approaches all show promise for use in clinical medicine in the future. Genomics TMPRSS2-ERGThis marker can be detected using RT-PCR methods, applied to urine samples from subjects whose prostate has been massaged. Discovery of this gene fusion is potentially the most significant advance in the last decade in the molecular pathology of prostate cancer.TMPRSS2 is a prostate specific gene19,20 on chromosome 21 that codes for a transmembrane-bound serine protease20. The protease is predicted to react with a number of proteins on the cell surface, as well as extracellular matrix components, soluble proteins and proteins on nearby cells21.ERG is a member of the ETS family of transcription factors which are able to activate or repress expression of genes involved in cellular proliferation, differentiation and apoptosis22. Figure 2. The potential significance of the TMPRSS2-ERG fusion Note: The androgen-sensitive promoter region of the TMPRSS2 gene, through fusion to ETS family genes, could lead to androgen-driven overexpression of ETS family genes such as ERG. These in turn have been shown to cause downstream effects such as a high expression of the histone deacetylase I (HDAC I) gene, upregulation of Wnt pathways and downregulation of tumour necrosis factor and cell death pathways.23 Genes from the ETS family and TMPRSS2 lie nearby on chromosome 21, and hence fusions typically occur via rearrangements including deletion and translocation24. Cross et al22have suggested the possibility of certain sequences in TMPRSS2 and ERG which make some men more prone to these fusions that are seen in 49% of localised prostate cancers22. Furthermore, the timing of the occurrence of these fusions is particularly significant - TMPRSS2-ERG fusions have not been detected in morpohologically benign prostatic tissue but arise at a very specific point in the pathogenesis of prostate cancer, namely the high-grade prostatic intra-epithelial neoplastic stage (HGPIN) (essentially analogous to carcinoma in situ). In addition, in late-stage androgen receptor-negative cancers, TMPRSS2-ERG fusions were still present in the DNA but were not expressed25, which aligns with the current understanding of the bypass mechanisms involved in androgen-independence and the fact that TMPRSS2 contains an androgen-dependent promoter region22. The clinical significance of these novel discoveries in the TMPRSS2-ERG fusion will be delineated more clearly as further studies are published. In terms of prognostication, there have been groups who have looked at TMPRSS2-ERG fusions in comparison to measures such as Gleason Score, survival data and tumour recurrence. In general, TMPRSS2-ERG fusions were shown to be linked with worse prognoses22: 44% of Gleason pattern 5 contained TMPRSS2-ERG fusions compared with 7% of Gleason pattern 2 tumours26 Non-fusion patients had a 90% survival at 8 years compared with 25% survival at 8 years in those identified having a particular pattern of TMPRSS2 fusion known as 2+ Edel (duplication of TMPRSS2-ERG fusion sequences and interstitial deletion of sequences 5 to ERG)27 Tumours with TMPRSS2-ERG fusions had a higher recurrence rate after radical prostatectomy with an odds ratio of 7.1 (95% confidence interval 1.1-45)28. Despite their prostate specificity and their appearance in Prostatic Intra-epithelial Neoplasia (PIN), TMPRSS2-ERG fusions are unlikely to be suitable for screening as they have been found by Hessels et al29 to show low sensitivity (37% in a cohort of 108). However, in the same study, the fusions were detected with a positive predictive value (PPV) of 94%29, which suggests that it could be a useful risk-assessment tool whereby a clinician could request further biopsies in the cases where patients have a negative initial biopsy but persistently elevated PSA and positive test for the gene fusion product. A similar pattern of low sensitivity but a high positive predictive value is seen in TMPRSS2-ERG fusions and their association with five key histological features30: Blue-tinged mucin Cribriform growth pattern Intraductal tumour spread Macronucleoli Signet-ring cell features Ninety-three percent of cases in 253 prostate cancers with three of more of these features were TMPRSS2-ERG fusion positive (high PPV) but equally, 24% of TMPRSS2-ERG fusions did not show any of these features (low sensitivity) 30. Its positive predictive value is comparable to the morphological features of HNPCC and BRCA-associated breast cancers, but the link between genotype and phenotype is not yet fully understood. Tumour morphology and association between TMPRSS2-ERG fusions thus stands as a potentially useful addition to the current armoury of diagnostic and risk-stratification methods, but further research is required in the field before we see collaboration between clinicians and histopathologic and cytogenetic services in New Zealand. Urinary 8-hydroxydeoxyguanosine (8-OHdG)It is widely agreed that reactive oxygen species (ROS) are direct causes of DNA damage. 8-hydroxydeoxyguanosine (8-OHdG), an oxidised nucleoside of DNA, is a frequently detected lesion where mismatch repair plays a key role43.Upon DNA repair, 8-OHdG is excreted in the urine and thus can not only be a measure of DNA repair capacity, but also a biomarker for oxidative stress and potential carcinogenic initiation44, 45. Increased urinary DNA lesions were detected by Chiou et al43 in both prostate and bladder cancer patients (58.5ng/mg creatinine of urinary DNA lesions in prostate cancer patients compared with 36.1ng/mg creatinine of Urinary DNA lesions in healthy patients) with a sensitivity of 31% and a specificity of 100%. Although their study population was small (and the fact that a biomarker of oxidative stress is not prostate-specific), the specificity of the test and the non-invasive nature of it suggests that with further investigation urinary 8-OHdG has potential as a biomarker which can allow for risk-stratification in those who have elevated serum PSA or a strong family history of prostate cancer. 8-OHdG is frequently detected in both non-malignant and malignant tissue. However, in non-malignant tissues extensive oxidative DNA damage drives cells to cell-cycle arrest (metabolic blockage), while in neoplastic prostate cancer cells it activates repair mechanisms favouring the escape from senescence and the expansion of DNA-damaged clones133.The combination of 8-OHdG in urine, measured along with cell-cycle check point evaluators such as CDKN1A, a cyclin-dependent kinase inhibitor and the product of the growth-arrested and DNA damage inducible gene Gadd45, from a parallel blood sample, may provide a greater understanding of the progression towards malignancy134, 135. Transcriptomics HepsinHepsin is a type II membrane associated serine protease whose structure and similarity to other serine proteases suggests that hepsin is involved in tumour growth, and hence hepsin stands as an attractive target in cancer biomarker development. Its prostate-specificity is best demonstrated through evidence of overexpression of hepsin (median 46.1-fold) in cancerous prostate tissue in 90% of prostate cancer samples (n=90)46. These findings have been confirmed through the work of Magee et al47 in an analysis of 4712 genes. In the same analysis, Hepsin was found to be over-expressed in prostatic intra-epithelial neoplasia in comparison to BPH which points to a relationship between Hepsin and neoplastic transformation. In addition, one can propose that such a biomarker can aid in the prognostication of Gleason 4 and 5 tumours with the discovery of a correlation between increased Hepsin expression and higher Gleason score46. The major shortcoming of the use of Hepsin is the fact that it can only be detected in tissue specimens and, despite attempts to use RNA extracted from urine for quantitating hepsin136 is not currently detectable from urine or serum samples48. Thus, the arrival of Hepsin as a prognostic tool for differentiation of indolent from aggressive tumours depends firmly on the discovery of novel methods of detection that will render it more accessible to clinical practice. Prostate cancer antigen 3 (DD3PCA3)DD3PCA3 is a novel, prostate-specific gene found to be up-regulated in cancerous prostate cells and over-expressed in >95% of clinical specimens31,33. PCA3 is more specific for prostate cancer than serum prostate-specific antigen (PSA), which is prostate-specific but not cancer-specific41. The proof of its prostate specificity has been shown through RT-PCR methodologies, in which PCA3 mRNA expression was low but quantifiable in benign prostatic tissue, but undetectable in normal and malignant tissue from other organs32. Equally, proof of over-expression of DD3PCA3in malignant prostate tissue with a median 66-fold up-regulation (compared to expression in benign tissue) has been demonstrated by Northern Blot analyses31. DD3PCA3 has been concluded to express non-coding mRNA (defined through the presence of alternative splicing, polyadenylation, lack of an extended open reading frame and numerous stop codons) for which there is no discrete cytoplasmic protein productdespite overexpression of the mRNA transcript31. The function of the DD3PCA3 gene and its non-coding mRNA transcript are currently undefined; hence, there is equally little known about the role of the DD3PCA3 gene in pathogenesis of prostate cancer. The magnitude of overexpression of the DD3PCA3 gene in malignant specimens when compared to the near-negligible amounts of DD3PCA3 expression in benign prostatic tissue confirms that the ultimate cause of the lack of a cytoplasmic protein product from PCA3 mRNA expression lies in the transcription as opposed to translation of other processing steps31. Although conflicting literature does exist on the subject of the DD3PCA3 genes clinical utility, the majority pertaining to the matter confirm that DD3PCA3 has strong diagnostic value, particularly in differentiating early-stage prostate cancer from benign prostatic hyperplasia (BPH)34,35,36. PPV of 52.2% in men with PCA3 \u2265100 is reported by Roobol et al 2010a and Robool et al 2010b. This marker stands as one of the most attractive risk-stratification tools to detect early prostate cancer for a gamut of reasons: The DD3PCA3 test does not require a biopsy- the mRNA is collected from urine after DRE and prostatic massage34. DD3PCA3 levels are directly reflective of tumour burden (as it is mRNA from cancer cells) and are not affected by prostate size, unlike PSA (which is a surrogate serum marker). This reduces the number of false positives detected in BPH cases and hence increases overall specificity32. DD3PCA3 mRNA expression adds the most value to current diagnostic tools at PSA values between 2.5ng/ml and 4.0ng/ml34. The quantitative PCA3 score has been found to correlate to the frequency of prostate cancer-positive biopsythus it can act as a means to stratify patients into categories of prostate cancer risk32. In theory, it has all the hallmarks of a test which can deliver the much sought after specificity that is currently lacking in determining whether to biopsy or not. However, current validation studies have struggled to produce definitive results confirming DD3PCA3 mRNA as a clinically applicable biomarker. Five studies which look the performance of DD3PCA3 which use \u22652.5ng/ml or \u22653.0ng/ml as PSA cut-off values gave the following values (as an average across the five studies)37, 38,39,40,41: PPV: 28.3% Sensitivity: 62.6% Specificity: 74.8% (Sample Size [average]: 303) Values for sensitivity have been quoted as high as 82% at 2.5ng/ml PSA cut-off42 and for specificity. Mearini et al34 claim 100% sensitivity (when PSA and DD3PCA3 are combined) in a tPSA range <4ng/ml. It must also be noted that PCA3 scores and PSA cut-offs can be varied to change the specificity and sensitivity, whereby a higher PCA3/PSA cut-off will produce very high specificity (i.e. very few false positive results) but much compromised sensitivity (high number of false negative results) and vice versa with lowered cut-off values. In addition, the means by which PCA3 is assayed for (i.e. the technology used) can also alter these results. What these values demonstrate is a classic teething issue of a novel biomarker; the lack of consistency in the type of assay used to identify the marker as well as small sample sizes hampers the production of consistent results and ultimately prevents the attainment of a definitive answer on the applicability of DD3PCA3 as a prostate cancer biomarker. This being said, its prostate-specificity and its potential to differentiate between indolent neoplasms and early malignant tumours ensures that further extensive research will be conducted into the utility of DD3PCA3 as a biomarker aiding clinicians in early diagnosis of prostate cancer. Epigenomics Glutathione-S-transferase P1 (GSTP1)From the family of Glutathione-S-transferases, GSTP1 conjugates chemically reactive electrophiles with glutathione, thus preventing DNA damage from reactive oxygen species and carcinogens which release reactive electrophilic metabolites49. Promoter hypermethylation of the region expressing GSTP1 has been directly linked to the loss of GSTP1 expression in prostate cancer50,51,52; indeed, this somatic genomic alteration is manifest in over 90% of prostate cancersmaking it the most frequent epigenetic event reported in prostate cancer51,52,53. With respect to its role in cancer pathogenesis, GSTP1 hypermethylation and the resulting loss of expression is a process presently considered as a promoter of cancer (as opposed to an initiator), with loss of GSTP1 increasing susceptibility of DNA to oxidants and free radicals54. GSTP1 hypermethylation is an attractive target for more intensive investigation into its role as a prostate cancer biomarker for many reasons: Its role in the pathogenesis of prostate cancer has been elucidated and the mechanism is well understood. GSTP1 hypermethylation is not frequently observed in normal prostate tissue50,53(although there have been reports of GSTP1 hypermethylation in high grade prostatic intra-epithelial neoplasia). GSTP1 hypermethylation is less frequent in non-prostate genitourinary malignancies (e.g. renal and bladder cancer) 54. GSTP1 is not limited by the accessibility of sample collection; it can be identified in a range of body fluids: urine, serum, and ejaculate 54. Although non-invasive procedures including collection of urine and ejaculate are held as the ideal means of attaining diagnostic information, there are key shortcomings with the use of these tissues. It has been shown that GSTP1 methylation levels are higher in plasma compared to urine, suggesting that prostate cancer is preferentially disseminated into the bloodstream rather than the prostatic ductal system54. With ejaculate, the inherent nature of such a collection procedure, particularly with older men, renders this avenue as one unlikely to see significant clinical exposure. Solutions such as prostatic massage to release cancer cells into the prostatic urethra before collection have so far delivered mixed results48,58,59. The difficulties faced in attaining clinically applicable detection rates through non-invasive methods remains a barrier yet to be surmounted. Currently, the most promising results portraying GSTP1 hypermethylation have been produced from tissue samples. The use of quantitative methylation specific PCR (QMSP) in screening for GSTP1 methylation has been reported to deliver 85.5% sensitivity and 96.8% specificity (n=128)56. When further tests were conducted on the same set of tissue specimens to assess the capacity for differentiation between non-cancerous tissue and histologically-proven adenocarcinoma (n=21), the QMSP assay correctly diagnosed the specimens with 90.9% sensitivity and 100% specificity and 100% positive predictive value. In addition, Harden et al57 demonstrate a 15% increase in specificity of the gold-standard of prostate diagnosishistopathologic assessmentthrough combining histopathologic assessment with QMSP for GSTP1. Furthermore, there is evidence that this method may be complemented with a measure of ENT SCTR methylation137. These results highlight the potential for GSTP1 hypermethylation as a means of complementing histopathological diagnosis of prostate samples and furthermore, a means of differentiating indolent and malignant neoplasms in cases where PSA levels alone are unable to discriminate56. Wnt signalling and methylationWnt signalling and its subsequent pathways are known to be crucial in mammalian and embryonic development60, 61. Its role in the pathogenesis of cancer can be summarised by the following diagram (modified from van der Poel HG60): Figure 3. Potential involvement of the Wnt pathway in the development of malignancy. The steps portrayed are: Binding of Wnt ligand to the frizzled transmembrane receptor. Decreased phosphorylation of B-catenin by GSK3-B. Therefore, stabilised B-catenin now accumulates in the nucleus. Nuclear B-catenin converts the TCF/LEF DNA binding complex from a transcriptional repressor into a transcriptional activator. Transcriptional activation of many cancer-related genes61. In the case of prostate cancer, there are a handful of epigenetic changes which are thought to alter the Wnt signalling pathway: Hypermethylation of the APC (adenomatous polyposis coli) gene is increased 8-fold in prostate tumours relative to samples of benign prostatic hypertrophy61. It has been proposed that DNA hypermethylation of the APC gene, an important component of the B-catenin degradation complex, may lead to the nuclear accumulation of B-catenin and hence the activation of the Wnt signalling pathway activating various oncogenes61. Equally, E-cadherin is a cell-membrane protein, which is known to both interact with B-catenin and be involved in the process of epithelial-to-mesenchymal transmission (EMT), a key step in the development of malignancy63. When the promoter for the E-cadherin gene is silenced by methylation, it not only promotes EMT but also the release of B-catenin away from the cell membrane and into the cytoplasmic and nuclear compartments. The presence of B-catenin in the nucleus will hence activate Wnt signalling61,62. Secreted-frizzled related proteins (SFRPs) and Wnt inhibitory factor-1 (Wif-1) are antagonists for Wnt signalling. Thus, silencing of genes which express SFRPs and Wif-1 through hypermethylation will lead to aberrant Wnt signalling and cancer progression. Although silencing of genes encoding SFRPs and Wif-1 has been identified in many cancers, including colorectal, lung, and bladder cancers and lymphocytic leukaemia64, there is insufficient evidence to definitively claim that Wnt antagonist genes play a key role in prostate cancer development. Despite the extensive elucidation of the Wnt signalling pathway, there remain questions over its relevance to prostate cancer and whether assays for hypermethylation of any of the aforementioned genes will aid the delineation of a diagnostic landscape. However, the role of potential cancer promoters, exemplified by Wnt signalling