Semen: The Ultimate Liquid Biopsy For Prostate Cancer?
By Renee Stirling Richards, Ph.D.
Cancer InCytes Magazine - Volume 4, Issue 1, Summer 2015
Published June 30, 2015
This article was originally published as an early publication article on the Cancer InCytes blog on January 30, 2015.
Managing Editor: Arvin Gouw, Ph.D.
Summary For The Public
Current therapies for advanced prostate cancer can extend life but the disease is still largely incurable. Preventative measures and early detection are much more effective at reducing death rates from advanced cancer. Early detection may be achieved with a liquid biopsy - a test that identifies biomarkers of disease in body fluids, particularly blood. However, it is difficult to attract funds for early detection research when exciting new therapeutic possibilities are in the pipeline; possibly even more challenging when semen is proposed as the source of biomarkers.
Prostate cancer is very common, with at least one third of men over the age of 50 harbouring localised disease. Unlike many other cancers, the majority of prostate gland tumours are low risk, slow growing and never progress to life threatening stages. Unfortunately, the established diagnostic schedule, consisting of blood serum prostate specific antigen (PSA) test with digital rectal examination (DRE), is unable to predict the presence of potentially aggressive disease and many men proceed to invasive and risky biopsy unnecessarily. If low risk disease is diagnosed following biopsy, men usually opt for an ‘active surveillance’ routine which monitors disease progress via repeated biopsies. Proceeding to treatment with curative intent is avoided when possible as it is associated with significant side effects, including impotence and urinary incontinence, and is unnecessary in cases of low risk disease. Paradoxically, aggressive disease may still be overlooked because biopsies sample very small sections of the prostate gland.
Advances in medical imaging are providing the first major steps forward in prostate cancer diagnostics in many years. Multi-parametric magnetic resonance imaging (mpMRI) is emerging as an excellent tool to identify and site prostate tumours and guide biopsy, but it misses 10% of significant cancers and is very expensive. High cost diagnostics are not readily available to all and are unsuitable for screening purposes. Cheap, highly predictive biomarker tests to determine which patients should proceed to more expensive and invasive procedures are still desperately needed to reduce prostate cancer morbidity, mortality and financial burden. Early detection of changes associated with tumours that will go on to become aggressive will enable appropriate application of curative treatment in early stage disease, when it is most likely to be successful.
The early detection of localised tumours using a liquid biopsy of blood is challenging. Many biomarkers appear at measurable levels in blood only after blood-tissue barriers are breached and the cancer has begun to spread. If it were possible to obtain an alternative body fluid which contains a much higher concentration of cancer-specific markers, it should be thoroughly examined. Indeed, the prostate gland functions specifically to secrete sperm-nourishing contents into semen, a very accessible body fluid. This provides a rare opportunity to indirectly sample an internal organ without the need for invasive techniques.So, while blood is often the only practical choice for liquid biopsy and the cancer biomarker is the proverbial needle in a haystack, in the case of prostate cancer, the whole sewing kit is readily available!
Semen is produced following compression of the entire prostate gland during ejaculation, resulting in up to 40% of the contents being prostate-derived. Almost 20 years ago it was shown that abnormal prostate cells were present in semen long before diagnosis via traditional biopsy. More recent studies have proven that molecular markers of prostate cancer are detectable in prostate cells obtained from semen. Combining these markers with the serum PSA test significantly improves the ability to identify high risk prostate cancer. The cell-free secretions of semen are yet another source of prostate cancer biomarker candidates.
These promising preliminary findings require follow-up studies and much larger participant numbers. While there is some scepticism regarding the practicality of a semen test for prostate cancer (opponents cite a lack of physiological ability and toleration by potential participants), these initial findings prove that: 1) semen samples for prostate cancer research are obtainable; and 2) semen contains biomarkers of prostate cancer. The mostly age-related limitations to sample collection should diminish as progress is made towards early detection of high risk cancers in younger men, who are more able to provide specimens. The proof-of-principle hurdles are currently being overcome and progress in this area should now begin to accelerate.
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There is a critical need for an inexpensive, non-invasive test to determine whether or not prostate cancer is present and if so, whether a lesion is clinically significant. Blood serum is touted as the easiest and most desirable source of novel cancer biomarkers. However, blood markers of cancer appear only when the blood-tissue barrier is disrupted and are subject to a dilution effect in the circulation which often limits utility to cases with high tumour burden. Given that early detection is a key to beating cancer, this is a difficult hurdle to overcome. Attempts to bypass this issue for prostate cancer have relied almost completely on urine as a source of biomarkers. Vigorous prostatic massage or firm digital rectal examination are required to produce prostate cell shedding and ‘milk’ cells with fluid into the urethra for expulsion upon urination. Apart from the obvious discomfort of this process, the method is inherently biased to the posterior aspect of the prostate. On the other hand, ejaculated seminal fluid is the result of global prostatic smooth muscle contraction resulting in ~40% of the ejaculate being prostate-derived. Almost 20 years ago it was discovered that the presence of abnormal prostatic cells in ejaculate correlated with and predated prostate cancer diagnosis via biopsy, sometimes by years. More recent studies have proven that molecular markers of prostate cancer are present in seminal fluid. Why then, are so few groups working in this area? Is prudishness hindering more rapid advancement in the field of prostate cancer diagnostics?
The established two-step prostate cancer diagnostic regimen consisting of a serum Prostate Specific Antigen (PSA) test with Digital Rectal Examination (DRE) followed by biopsy is imprecise and inadequate. While prostate cancer is extremely common, with small localised prostate carcinomas detected at autopsy in >1/3 men aged >50, a large number of cases remain biologically latent and never progress to life-threatening disease [1, 2]. A compromised prostate-blood barrier causes leakage of the normally prostate confined PSA into the circulation in a number of prostate disorders other than cancer, so that most men with elevated serum PSA levels do not have prostate cancer detected. While an elevated level of total serum PSA is commonly used to justify prostate biopsy, there is no PSA concentration threshold which provides a specific and sensitive detection test for prostate cancer, nor the ability to distinguish between indolent and aggressive forms of the disease [3, 4]. Moreover, the commonly used invasive and imprecise transrectal ultrasound (TRUS) guided biopsy accesses only the posterior aspect of the gland and has a false negative rate of up to 25% .
In the presence of low-risk disease based on biopsy grade (Gleason score, clinical stage) and serum PSA, an active surveillance regimen may be implemented whereby further biopsies are performed at set intervals. This puts the patient at a small risk of sepsis, with antibiotic resistant bacteria emerging at increasing rates with, amongst other factors, repeated biopsies [6, 7]. However, proceeding to treatment with curative intent is associated with a risk of more significant side-effects. Adverse effects of radical prostatectomy include erectile dysfunction and urinary incontinence, while radiation therapy is associated with irritative bladder and anorectal symptoms, as well as impotence .
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The use of multiparametric Magnetic Resonance Imaging (mpMRI) to identify and site significant prostate tumours is emerging as a promising new diagnostic tool , but the high cost of mpMRI and the fact that it misses up to 10% of significant tumours, means the need for inexpensive, non-invasive diagnostic and prognostic tests to help inform patient treatment course remains. An accurate early detection test will reduce costs and morbidity associated with unnecessary invasive procedures and promises to improve outcomes for those with high risk disease.
Blood is considered the most desirable sources of cancer biomarkers because of the socially acceptable and routine nature of collection and processing. Both whole cell and molecular approaches are in fact showing promise for detection of disseminated prostate cancer and assessment of treatment response. Tumour cells enter the circulation during the process of metastasis, but these circulating tumour cells (CTCs) are very rare initially, with only one in a billion circulating cells being of tumour origin in patients with known metastatic disease . The number of CTCs increases with tumour burden in these patients, which has enabled development of an FDA approved CTC enumeration test, CellSearch® to assess the presence of metastatic prostate cancer . However, CTC enumeration is of no predictive value for localised prostate cancer and is not useful as an early detection tool . Molecular (i.e. protein, RNA, microRNA, DNA) and metabolic serum markers are expected to provide greater sensitivity and specificity than whole cell approaches. Some of the most exciting, new molecular findings for prostate cancer involve the use of microRNA panels to detect recurrence of advanced disease following radical prostatectomy  and as prognostic markers for late stage disease [14, 15]. While blood markers clearly hold much promise for assessment at this phase, accurate early detection and prognosis is yet to be realised. Higher concentrations of molecular and metabolic biomarkers that provide a more representative portrait of pre-metastatic, localised disease may be obtained from a body fluid proximal to the tumour and into which effluent from the prostate is normally discharged.
Urine is widely accepted as a more specific, prostate representative body fluid than serum. However, only 15% of freely-voided urine samples contain PSA , meaning that prostate cells and secretions do not shed into the urethra at a rate high enough to facilitate clinical urine analyses. Vigorous DRE and prostatic massage is required to dislodge prostate cells along with prostatic secretions into the urethra for expulsion during urination . From here, tumour-derived epithelial cells and prostatic secretions can be harvested for various molecular analyses. A non-coding RNA, Prostate Cancer Antigen 3 (PCA3, initially designated DD3), became a target for such analyses following the discovery that it is strongly and specifically up-regulated in over 90% of prostate cancer tissue samples . Detection of PCA3 in the epithelial cell fraction of DRE urine is the principle behind the FDA-approved Progensa® test for prostate cancer [18, 19]. This test has a higher specificity (i.e. does not produce as many false positives) but lower sensitivity (i.e. produces more false negatives) compared with serum PSA and as such, may be beneficial as an adjunct to serum PSA but never as a stand-alone diagnostic test . More recently, a panel of four microRNAs obtained from DRE/prostatic massage secretions was shown to have significantly higher predictive power for prostate cancer than serum PSA . While DRE urine is certainly a source of useful biomarkers, the collection method is inherently biased to the posterior aspect of the prostate so that anterior tumours are likely to remain undetectable using this body fluid.
In contrast to DRE urine, seminal fluid (SF) is released following vigorous and global prostatic smooth muscle contractions during ejaculation. The potential utility of SF for early diagnosis of prostate cancer was first highlighted by the finding that abnormal prostate cells are detectable in this body fluid well before pathological change is evident on biopsy . Recently, RNA sequencing of the non-sperm epithelial cell fraction of SF uncovered microRNA markers that exhibit higher predictive value for prostate cancer than serum PSA . A follow up study has shown that combining a number of RNA markers from SF with serum PSA further improves predictive value . Reports have also shown that SF is a source of peptide , protein  and metabolite markers  of prostate cancer. This should not be surprising given that prostate-derived material constitutes ~40% of total SF , making it the obvious body fluid for isolation and purification of prostate-associated proteins [29, 30]. SF is also a rich source of prostate-derived extracellular vesicles or ‘prostasomes’ containing cell-free RNA and proteins [31, 32]: a treasure trove of potential prostate cancer biomarkers.
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SF is the most likely body fluid to provide a representative snapshot of the prostate and the ultimate liquid biopsy for prostate cancer. The use of SF as source material for a clinical test is dismissed far too readily, citing a lack of physiological ability and toleration in potential participants (in the face of vigorous prostatic massage as alternative!) . Some challenges are posed in obtaining donations from older men (inability, value systems), but the joint efforts of pioneering researchers and enlightened participants has allowed the generation of very promising preliminary findings. Moreover, these age-related limitations on recruitment should diminish as progress is made towards detection of high risk cancers in younger people. Discomfiture with the collection method for SF may have hindered advancement in this area to some degree, but the encouraging results beginning to emerge, in combination with a more tolerant and physiologically able younger generation, may provide the fuel that is needed to accelerate the research. Watch this space!
About The Author
Renee Stirling Richards received her Ph.D. in cell and molecular biology at Griffith University in Australia. She is currently a post-doctoral scientist at the University of Queensland Centre for Clinical Research, studying the utility of seminal fluid for prostate cancer biomarker discovery. http://www.uqccr.uq.edu.au/research/research-profiles/dr-renee-stirling-richards.aspx
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