149 - Prostate Cancer Biomarkers

149 Prostate Cancer Biomarkers
Simpa S. Salami, MD, MPH, Ganesh S. Palapattu, MD, Alan W. Partin, MD, PhD, and Todd M. Morgan, MD
T
he development and implementation of prostate cancer biomark- challenging group. After a diagnosis of prostate cancer has been
ers have continued to expand rapidly over the past decade. made, balancing the risks and benefits of active surveillance with
In an era in which early detection based on prostate-specific primary therapy as the initial management strategy is a central
antigen (PSA) has been under fire from the United States Preventative question. Knowledge regarding biologic behavior could drastically
Services Task force, new markers to aid in biopsy decision have improve the decision-making process for a patient contemplating
become available in the clinic. Furthermore, attention has turned from curative intent therapy (e.g., radical prostatectomy or radiation) or
detection of any cancer to a focus on detecting clinically significant active surveillance. Finally, post-treatment biomarkers hold promise
disease, often interpreted as Gleason score ≥7 cancer. In addition, for improving our ability to make decisions regarding adjuvant or
biomarkers that can aid in distinguishing aggressive from indolent salvage therapies in men at high risk of developing metastatic disease.
disease for men who have cancer continue to be sought after, and a Several molecular approaches have been undertaken in pursuit
number of these are now clinically available. However, substantial of finding the optimal prostate cancer biomarker. An overview of
limitations exist for all of the currently available markers, and it is basic cellular processes starts first with a DNA sequence (gene) that
critical to understand the strengths and potential weaknesses for each is transcribed to mRNA (transcript) and then translated to a protein
of these before using them for clinical decision making. that can then carry out specific cellular functions (e.g., catalyze
Prostate cancer is one of the few malignancies with a serum-based, biochemical reactions leading to the formation of products such as
clinically informative biomarker. Since its discovery in 1979 until metabolites). A guiding principle of prostate cancer biomarker
clinical application in the late 1980s through 1990s, the PSA has development is that prostate cancer cells, a priori, are different in
evolved into an invaluable tool for detecting, staging, and monitor- some molecular way than their benign counterparts. Further, another
ing prostate cancer in men. The widespread use of PSA screening important observation is that aggressive prostate cancer cells are
led to a dramatic increase in prostate cancer diagnoses in the mid- similarly different in comparison with their more indolent counter-
1990s, and the subsequent drop in prostate cancer deaths is at least parts. The identification and quantification of these molecular dif-
partially attributable to the adoption of population-based PSA ferences in tissues and bodily fluids form the basis of prostate cancer
screening. Whereas the majority of prostate cancers in the 1980s biomarker discovery.
and early 1990s commonly arose through finding an abnormal Advances in molecular oncology and breakthroughs in laboratory
digital rectal examination (DRE) or elevated PSA, or both, today techniques have exponentially expanded our repertoire of innovative
most prostate cancer arises as clinically nonpalpable (stage T1c) tools for the discovery of novel ways of predicting the future. In this
disease with PSA levels between 2.5 and 10 ng/mL. Widespread chapter, we discuss the process and phases of biomarker development,
application of PSA screening and the long natural history of prostate paying particular attention to scientific rationale and clinical applica-
cancer have also resulted in a stage migration to nonpalpable, clinically tion of blood-, urine-, and tissue-based biomarkers.
localized (stage T1c) disease to go along with the parallel reduction
in mortality (Diamandis et al., 2000; Lilja, 1997; McCormack et al.,
1995; Polascik et al., 1999; Pound et al., 1997, 1999; Rittenhouse BIOMARKER DEVELOPMENT
et al., 1998; Stephenson and Stanford, 1997).
Although PSA screening has improved survival, however, outcomes Although there are many groups now developing methods for early
are not the same for all PSA-detected disease (Diamandis et al., detection of prostate cancer, the early detection research network
2000; Gretzer et al., 2002; Yousef and Diamandis, 2001). Indeed (EDRN) has developed a particularly robust infrastructure for bio-
the most common cause of mortality for all men with prostate cancer marker development. The EDRN is a National Cancer Institute (NCI)
is heart disease. This is not to downplay the influence of prostate funded program set forth with the objective to identify, develop,
cancer on the mortality of the nearly 30,000 men who die from it and validate promising biomarkers and technologies for the early
annually in the United States but rather to highlight the critical need detection of cancer (Clements, 1989; Schedlich et al., 1987; Srivastava,
for reliable tools that allow for identification of harmful cancers. 2014; Srivastava et al., 2001). As suggested by its name, it is actually
Although PSA is the most well-known prostate cancer tumor a network of academic centers working in collaboration with industry,
marker, it is organ specific and not cancer specific. There is significant public health groups, informatics centers, and patient advocates.
overlap in serum PSA levels among men with cancer and those The structure of the network consists of five scientific components.
with benign disease. Thus even substantially elevated serum PSA The first of these are the biomarker development laboratories
levels may be due to inflammation or benign prostatic hyperplasia (BDLs), which is responsible for discovery of novel biomarkers and
(BPH) rather than malignancy (Catalona et al., 1991; Oesterling technologies. They identify biomarkers and participate in the early
et al., 1988; Partin et al., 1990). To this end, application of PSA phases of validation. The biomarker reference laboratories (BRLs)
derivatives such as PSA density, PSA velocity, age-adjusted values, facilitate the development and validation of assays that measure
and, more recently, molecular derivatives may be used to improve these new biomarkers. Without such efforts, assays results may be
clinical decisions compared with using PSA in isolation. difficult to reproduce when deployed at a different site. The BRLs
There are three main domains in which clinically localized also assist in developing a more robust and efficient clinical assay.
prostate cancer biomarkers are needed: (1) early detection, (2) The third scientific component are the clinical epidemiological valida-
newly diagnosed prostate cancer, and (3) post-treatment. By tion centers (CEVCs). These are large clinical practices with infra-
identifying men at greatest risk of harboring harmful disease, improved structure to procure ample clinical samples and to lead clinical trials.
early detection biomarkers could markedly reduce the group of men They are responsible for procuring samples for the BDLs. The fourth
subjected to prostate biopsy. The cohort of men who possess an component of the network is the data management and coordinating
elevated PSA after an initial negative biopsy represents a particularly center (DMCC). This component is the spine of the network in the
3478
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Chapter 149 Prostate Cancer Biomarkers 3479
sense that they provide all the logistical and statistical support for proposed by Pepe et al. (Kumar et al., 1997; Lövgren et al., 1997;
all phases of development. Importantly, this EDRN component is Pepe et al., 2008; Takayama et al., 1997). Use of the PRoBE design
responsible for creating a reliable, secure, and practical means by is intended to overcome spectrum bias, in which there is differential
which data may be collected for network studies. The fifth and final selection of cases and controls. In addition, this approach overcomes
component is the informatics center responsible for developing ascertainment bias, defined as inclination for differential levels of
software for information management. As the EDRN experience grew, the biomarker to undergo diagnostic testing, because all subjects
experts in oncology, clinical trials, biostatistics, and informatics came receive the same testing. A common strategy has been to develop
together and refined what is considered best practice for the develop- prospective registries to be used for validation studies by blinding
ment of new biomarkers (Lilja, 1997; Pepe et al., 2001; Rittenhouse samples, which are stored at the National Cancer Institute. In this
et al., 1998; Young et al., 1995). way, validation studies can be performed using large cohorts for a
Similar to the phases of drug development, biomarker development series of biomarkers as long as sample size is sufficient.
has been broken down into a number of sequential phases. Conceptu- The ultimate objective for new biomarkers is to reduce the burden
ally, progression of a biomarker through each of the five phases of of cancer. This takes place in phase 5, cancer control studies. With
development indicates increasing strength of evidence in favor of deployment over time, biomarkers are expected to efficiently identify
its clinical application. Phase 1 consists of biomarker discovery. cancers earlier in the disease course, leading to greater construct
Although this may occur at any laboratory, the BDLs are intended validity. To achieve a successful outcome in phase 5 requires reduction
to be factories of discovery along the lines of genomics, proteomics, in cancer mortality that can be attributed to the biomarker use without
glycomics, and metabolomics. The objective of this phase is to identify prohibitive costs or overdiagnosis. This lofty goal has been achieved
potential biomarkers and prioritize each for validation. The typical by few biomarkers.
outcome of phase 1 development is a novel biomarker with some Inherently, the cost necessary to take a biomarker from phase 1
measure of test sensitivity and specificity in a subset of cancer and through phase 5 is often in excess of millions of dollars. A common
control subjects. In this way, a preclinical assay is moved forward strategy applied by the EDRN is to partner with multiple institutions
into phase 2. and industry to fund work along the different phases with a common
Phase 2 has the objective of more reliably measuring the sensitivity goal of moving biomarkers into clinical practice that are likely to
and specificity of the new biomarker in its ability to differentiate improve the health of a population. It is worth pointing out that
case status from control. Here the BRL often participates to optimize the EDRN’s approach is not the only means by which biomarkers
the clinical assay and assess the reproducibility of the assay in may be developed but the outline here developed by the EDRN
preparation for a clinical trial. It would not be unusual to see scientists provides a rigorous framework for doing so.
comparisons of biomarkers measured in tissue samples compared
with levels from less invasive approaches such as voided urine or
serum. The clinical samples deployed at this stage tend to be more ASSESSMENT OF BIOMARKER PERFORMANCE
representative of the target population and, as a result, biomarkers
may fail at this phase if they were initially developed on an overly In the previous section, we have discussed study designs as applied
selected set of tissues. by the EDRN in the development and validation of biomarkers. In
In phase 3, investigators examine the ability of new biomarkers to this section, we briefly discuss common ways by which biomarker
detect preclinical disease and evaluate how much lead time is provided performance may be assessed. Typically, this will be conducted using
by the biomarker relative to clinical presentation. The criteria necessary the clinical grade assay in the intended population along the indicated
for “a positive test” are defined in this phase. Robust studies examining clinical indications. Perhaps the most common means for assessing
the impact of covariates on biomarker expression and measurement will validity is calculations of sensitivity, specificity, and accuracy. Sensitivity
take place. Given the growing number of biomarkers, it would not be is defined as the proportion of individuals with the disease who
uncommon to see algorithms developed during this phase to support have a positive biomarker test, whereas specificity is defined as the
the use of panels of biomarkers as opposed to a single biomarker. The proportion of individuals without the disease who will have a negative
tissue repositories used for this phase are typically retrospective cohort test. Accuracy is then the sum of true positives and true negatives
studies in which clinical samples have been procured prospectively divided by the total population. Sensitivity and specificity are graphi-
over time with careful annotation of clinical end points and risk cally summarized using the receiver operating characteristics (ROC)
factors. These subjects are usually identified before cancer is diagnosed, curve. This approach plots sensitivity on the vertical axis and 1-
sometimes years in advance of diagnosis. Having completed phase 3, specificity on the horizontal axis. In doing so, the area under the
a new biomarker would possess a validated clinical assay and strong curve (AUC) is a measure of the accuracy of the test. An AUC of 1
evidence of ability to differentiate case from control according to its signifies an ideal test, whereas AUCs approaching 0.5 indicate a
clinical indication, conducted on a fairly robust sample of subjects. An poorly performing test.
understanding of factors that may affect measurement and expression Despite widespread use, sensitivity and specificity are not intuitive
of the biomarker is established, and plans are made for a prospective to interpret in a clinical setting; therefore positive and negative
validation trial, that is, phase 4. predictive values are often used instead. These are calculated based
In phase 4, indications for applying a new biomarker are clearly on sensitivity and specificity as well as the prevalence of the disease.
defined according to the labeling. Potential subjects are identified The definition of positive predictive value is the probability that a
for eligibility, consented, and enrolled with careful annotation of positive test indicates presence of cancer. Similarly, negative predictive
all demographic, disease, risk factors, and cancer end points. The value is the probability that the patient does not have cancer when
goal of phase 4 is to quantify the performance characteristics of the biomarker test is negative. In many ways, these measures are
a biomarker in the target population of interest. This is typically more relevant to clinical practice, and there is an increasing tendency
done by examination of sensitivity, specificity, positive, and nega- to use these as the end point for biomarker validation trials.
tive predictive values (NPVs). Secondarily, this phase also examines In prostate cancer early detection, it is useful to think in terms
characteristics of cancers diagnosed using the biomarker; considers of pretest and post-test probability. When new biomarkers are
implementation feasibility and costs; and considers potential impact introduced, the natural question becomes, “How much does this
on overall mortality. add to what we already know in terms of risk prediction?” The
Unlike drug studies, in which randomized clinical trials are likelihood ratio (LR) is useful in this regard because the test indicates
conducted as validation of treatment effect, biomarkers are rarely how much a new biomarker raises or lowers the pre-biopsy probability
tested with such trials given the large sample sizes, long durations, of disease. A positive LR is calculated as the probability of a positive
and prohibitive costs (Andriole et al., 2009; Lundwall and Lilja, test in those with cancer divided by the probability of that test result
1987; Schröder et al., 2009). Fortunately, robust clinical validation in disease-free individuals. Similarly, a negative LR is calculated as
design specifically for biomarker discovery (prospective randomized the probability of a negative test result among healthy men divided
open label, blinded end point study design, or PRoBE) has been by the probability of the same result among those with prostate
3480 PART XV The Prostate
cancer. Likelihood ratios greater than 1 indicate that the test results leader sequence (Lundwall and Lilja, 1987; Oesterling et al., 1988;
increases the likelihood that patient has cancer, whereas likelihood Partin et al., 1990). Cleavage of pre-pro-PSA results in an inactive
ratios less than 1 decrease the probability of the condition. In the 244-amino acid proenzyme termed proPSA. Finally, cleavage of a
literature, likelihood ratios greater than 5 or less than 0.2 are regarded leader amino acid sequence of proPSA by hK2 produces the active
as meaningful shifts in pretest probability. form of PSA (Kumar et al., 1997; Lövgren et al., 1997; Magklara
Prostate biomarkers today are proliferating at a greater rate thanks et al., 2000; Meng et al., 2002; Takayama et al., 1997).
to the many innovations in high throughput methods (Prensner PSA was first identified and purified in the late 1970s, but wide-
et al., 2012; Takayama et al., 2001). As such, understanding the status spread use in clinical urology did not occur for another decade
of biomarkers with result with regard to the US Food and Drug (Ablin et al., 1970; Christensson et al., 1990; Kuriyama et al., 1980;
Administration (FDA) and other approvals is helpful (Ablin et al., McCormack et al., 1995; Oesterling et al., 1988; Otto et al., 1998;
1970; Füzéry et al., 2013; Kuriyama et al., 1980; Oesterling et al., Seamonds et al., 1986; Sensabaugh, 1978; Stamey et al., 1987).
1988; Seamonds et al., 1986; Sensabaugh, 1978; Stamey et al., 1987). Although ectopic expression of PSA has been reported in smaller
FDA approval is considered to be the final end point in the concentrations in the tissue of malignant breast tumors (Partin et al.,
development process because that indicates governmental approval 2003; Yu et al., 1994a, 1994b) normal breast tissue (Christensson
for a specific indication and provides for biomarker validity and safety. et al., 1990; Monne et al., 1994; Zhang et al., 2000), breast milk
Such processes are not undertaken lightly and often cost millions of (Lilja et al., 1991; Végvári et al., 2010; Yu and Diamandis, 1995),
dollars. Currently FDA-approved biomarkers include PSA, percent-free and adrenal and renal carcinomas (Levesque et al., 1995; McCormack
PSA, PHI, and PCA3. However, the FDA does not actively regulate et al., 1995; Sensabaugh, 1978), PSA is highly organ specific because
so-called laboratory-developed tests (LDTs) that are performed in a it is produced primarily by prostatic luminal epithelial cells.
central Clinical Laboratory Improvement Amendments (CLIA)-certified The function of this androgen-regulated protease is to liquefy
laboratory. CLIA laboratory standards established by the Centers for semen through its action on the gel-forming proteins semenogelin
Medicare and Medicaid Services exist to ensure quality laboratory and fibronectin within the semen after ejaculation (Goldfarb et al.,
testing but not necessarily biomarker validity nor safety. The CLIA 1986; Lilja, 1985; Lilja and Weiber, 1984; McGee and Herr, 1988).
program examines performance characteristics of the tests with regard In serum, PSA circulates in bound (complexed, cPSA) and unbound
to laboratory precision, analytical sensitivity and specificity, reporting forms (free, fPSA) (Fig. 149.2). Three proteins that are known to
ranges, reference ranges, and criteria for the test systems. A CLIA-certified bind to PSA in the blood are ACT, α2-macroglobulin (A2M), and
LDT can be offered commercially for use in the clinic, but rigorous α1-protease inhibitor (API) (Christensson et al., 1990; McCormack
clinical validation may or may not have taken place. et al., 1995; Otto et al., 1998; Vieira et al., 1994). Binding of free
PSA to ACT inactivates the protease, but the complex PSA-ACT remains
immunodetectable (Meng et al., 2002; Partin et al., 2003).
BLOOD-BASED BIOMARKERS The majority (approximately 70%) of PSA in serum is protein
Prostate-Specific Antigen (PSA or hK3) bound, most to ACT in an irreversible fashion. Only 5% to 10% of
PSA is bound to A2M and 1% to 2% to API. Binding of PSA to A2M
Initially developed as a biomarker for monitoring prostate cancer still allows some proteolytic activity but renders the PSA-A2M complex
patients after treatment, PSA continues to be a lightning rod for undetectable by most current assays because all PSA epitope sites
controversy in the setting of prostate cancer screening. Given its become masked (Fig. 149.3) (Carter et al., 1992; Christensson et al.,
unique prominence, a clear understanding of PSA is needed to lay 1990; Oesterling et al., 1993; Zhang et al., 2000). PSA-ACT and
the foundation for other potential prostate cancer biomarkers. Also PSA-API are detected by PSA assays, as is fPSA.
known as hK3 (human kallikrein 3), PSA is a member of the PSA expression is strongly androgen dependent (Henttu et al.,
kallikrein gene family (Fig. 149.1). Originally, only three genes of 1992; Ohwaki et al., 2010). Immunohistochemical detection of PSA
this family of genes were identified: the pancreatic/renal kallikrein within the prostate is characterized by bimodal peaks between birth
(hKLK1), human kallikrein 2 (hKLK2), and PSA (hKLK3) genes and 6 months of age and after 10 years, correlating directly with
(Diamandis et al., 2000; Lilja, 1997; McCormack et al., 1995; Rit- testosterone levels (Goldfarb et al., 1986; Stamey et al., 1987). Serum
tenhouse et al., 1998; Yu et al., 1994; Yu et al., 1994a, 1994b). Since PSA becomes detectable at puberty with increases in luteinizing
the identification of 12 other kallikrein genes, this family of proteases hormone and testosterone (Stamey et al., 1987; Vieira et al., 1994).
now consists of 15 members and is described with a distinct nomen- In the absence of prostate cancer, serum PSA levels vary with
clature (Diamandis et al., 2000; Monne et al., 1994; Yousef and age, race, and prostate volume. Importantly, on a per-cell basis,
Diamandis, 2001). These proteins are all serine proteases and are PSA expression is similar between benign and malignant prostate
located on the long arm of chromosome 19 within the region span- cells (Armitage et al., 1988; Dalton, 1989; Meng et al., 2002; Nadler
ning q13.2-q13.4. These serine proteases have similar amino acid et al., 1995).
sequences, with hK1 expressing 60% and hK2 expressing 78%
homology with PSA (Clements, 1989; Schedlich et al., 1987; Yu and
Diamandis, 1995). hK2 and hK3 (PSA) are released in zymogen
form from the prostatic epithelium and are found in seminal fluid
PSA-ACT
as well as serum. Because they share structural homology, they can
Complexed PSA PSA-A2M
form complexes with endogenous protease inhibitors such as α2-
PSA-API
macroglobulin (A2M) and α1-antichymotrypsin (ACT) (Levesque
et al., 1995; Lilja, 1997; Rittenhouse et al., 1998; Young et al., 1995). PSA
To understand PSA and its related derivative biomarkers, it is –7proPSA
important to understand how PSA is processed. PSA begins as a proPSA –4proPSA
zymogen, termed pre-pro-PSA, that contains a 17–amino acid –2proPSA
Free PSA BPSA
Other free PSA
(Intact PSA, i.e., iPSA)
Gene KLK1 KLK15 KLK3 KLK2 KLK4 KLK5
KLK6-14 Fig. 149.2. Molecular forms of prostate-specific antigen (PSA). Molecular
derivatives of PSA include free PSA, such as proPSA (and the various clipped
Protein PSA hK2 forms), BPSA (benign PSA), and other free PSA forms such as intact, inac-
tivated PSA. Complexed PSA includes free PSA that is bound to proteases
Fig. 149.1. Human kallikrein gene map. Map of the human kallikrein locus such as ACT (α1-antichymotrypsin), API (α1-protease inhibitor), and A2M
and corresponding proteins as described by Yousef and Diamandis (2001). (α2-macroglobulin).
TABLE 149.1 Molecular Derivatives of

A2M Prostate-Specific Antigen
PSA TYPE % IN SERUM

A A A
B B B Complexed PSA 60–95
E ACT E PSA-ACT 60–90
PSA PSA PSA
C C C PSA-API 1–5
D D D PSA-A2M 10–20
Free PSA 5–45
ACT, α1-antichymotrypsin; API, α1-protease inhibitor; A2M, α2-macroglobulin;

A B C PSA, prostate-specific antigen.
Fig. 149.3. Prostate-specific antigen (PSA)–binding proteases. (A) Free PSA
with A to E representing immunoreactive epitopes of free PSA. (B) α1-
Antichymotrypsin (ACT) blocks the E epitope during binding. (C) α2-Macroglobulin (Kaplan et al., 2012; Thompson et al., 2006). Interpretation of PSA
(A2M) blocks all immunoreactive sites on PSA, making this derivative difficult values should always take into account the presence of prostate
to measure in serum. disease, previous diagnostic procedures, and prostate-directed
treatments.
In men without BPH, the rate of change in PSA is 0.04 ng/mL
Elevated serum PSA levels are probably a product of disruption per year, compared with 0.07 to 0.27 ng/mL per year in men with
of cellular architecture within the prostate gland (Mejak et al., 2013; BPH who are between the ages of 60 and 85 years (Carter et al.,
Stamey et al., 1987). The loss of the barrier afforded by the basal 1992; Oesterling et al., 1993; Yuan et al., 1992). PSA velocity has
layer and basement membranes within the normal gland is a likely long held interest as a potential predictor of prostate cancer in the
site for the egress of PSA into the circulation. This can occur in the early detection setting and as a marker of higher-risk disease in
setting of prostate disease (BPH, prostatitis, prostate cancer) and patients on active surveillance. There are now substantial data showing
with prostate manipulation (prostate massage, prostate biopsy) (Ercole that PSA velocity is of minimal, if any, use for prostate cancer screen-
et al., 1987; Morote Robles et al., 1988; Stamey et al., 1987; Wang ing, including in patients at higher baseline risk of developing prostate
et al., 1981). Prostatic inflammation (acute and chronic) and urinary cancer (Mikropoulos et al., 2018; Vickers et al., 2014). As such, PSA
retention can cause PSA elevations to variable degrees (Armitage velocity should not be used as a trigger for biopsy. In the active
et al., 1988; Dalton, 1989; Etzioni et al., 2005; Marks et al., 2006; surveillance setting, there are now more recent data that PSA kinetics
Nadler et al., 1995; Thompson et al., 2003). Prostatic trauma, such can improve prediction of reclassification over time, although the
as occurs after prostatic biopsy, can result in a temporary spike clinical utility of this metric still remains to be determined (Cooper-
in serum PSA that persists for 4 or more weeks before returning berg et al., 2018). PSA density, which is total PSA divided by prostate
to baseline values (Kaplan et al., 2012; Thompson et al., 2006; Yuan volume, is another PSA-derived metric and provides substantially
et al., 1992). more predictive information than PSA velocity. Proposed cutoffs for
Most studies of the effect of ejaculation on serum PSA have shown biopsy in the early detection setting have ranged from 0.08 ng/mL2
no significant change in PSA (Kirkali et al., 1995; McCormack et al., to 0.15 ng/mL2 (Aminsharifi et al., 2018; Nordström et al., 2018).
1995; Woodrum et al., 1998), although in men 50 years of age and Nordstrom et al. (2018) reported that PSA density cutoffs of 0.10 ng/
older ejaculation can lead to a very transient increase in PSA (Catalona mL2 and 0.15 ng/mL2 would lead to detection of 77% and 49% of
et al., 1998; Herschman et al., 1997; Partin et al., 1998; Tchetgen Gleason score ≥7 tumors, respectively, in a population-based cohort
et al., 1996). However, PSA appears to return to baseline within 24 with PSA of at least 3 ng/mL. In the multicenter Prostate Cancer
hours (Rajaei et al., 2013). Men with a new PSA elevation should Active Surveillance Study, PSA density is a key predictor of adverse
be asked about sexual activity within 24 hours of PSA testing and, reclassification on surveillance biopsy (Newcomb et al., 2016).
if so, should be asked to abstain before a repeat blood draw. Long-
distance cycling is another potential cause of false PSA elevation, Free Prostate-Specific Antigen
with PSA levels increasing by approximately 10% after bicycle rides
exceeding 55 kilometers (Mejak et al., 2013). Although the majority of serum PSA is found complexed to the
Although these factors can cause small-scale changes in PSA levels, proteases (primarily ACT), 5% to 45% of PSA exists as enzymatically
the presence of prostate disease (prostate cancer, BPH, and inactive fPSA (Table 149.1; McCormack et al., 1995; Woodrum et al.,
prostatitis) is certainly the most important factor affecting serum 1998). PSA produced from malignant cells appears to more fre-
PSA (Ercole et al., 1987; Morote Robles et al., 1988; Wang et al., quently escape proteolytic processing, resulting in a greater fraction
1981), and it is primarily the impact of BPH and prostatitis on of serum PSA complexed to ACT and a lower percentage of total
PSA levels that confounds the accuracy of PSA in the screening PSA that is free compared with men without prostate cancer
setting. Prostate-directed treatment (for BPH and cancer) can lower (Catalona et al., 1997; Christensson et al., 1993; Leinonen et al.,
serum PSA by decreasing the volume of prostatic epithelium available 1993; Lilja, 1993; Stenman et al., 1994). This principle led to the
for PSA production and by decreasing the amount of PSA produced development of fPSA testing as a means to improve the accuracy of
per cell. 5α-reductase inhibitors (5ARI) such as finasteride and PSA as a prostate cancer screening biomarker, and the FDA has
dutasteride have been shown to lower PSA levels by roughly 50% approved its use in men with a serum total PSA level of 4 to 10 ng/
after 12 months of treatment (Guess et al., 1993). This has resulted mL and a negative DRE (Catalona et al., 1998; Partin et al., 1998).
in the so-called “doubling rule,” whereby PSA levels are multiplied A number of studies have evaluated %fPSA cut points to determine
by a factor of 2 in men undergoing 5ARI therapy to guide decisions potential thresholds that optimize the performance of this tool.
regarding prostate cancer risk. However, using a multiple of 2 Christensson et al. (1993) measured free and total PSA fractions in
may overestimate PSA values in the first 6 months of treatment men with and without prostate cancer and found that a free/total
and underestimate PSA levels after several years of treatment PSA cutoff of 0.18 (18% fPSA) significantly improved the ability to
(Etzioni et al., 2005; Marks et al., 2006; Thompson et al., 2003). In distinguish between subjects with and without cancer compared
addition, compelling data from the Prostate Cancer Prevention Trial with use of total PSA alone. As many as 20% to 65% of unnecessary
(PCPT) and other sources suggest that 5ARI therapy substantially biopsies may be avoided when using %fPSA cutoff values ranging
improves the performance characteristics of PSA in the screening between 14% and 28% while maintaining sensitivity rates from
setting with an AUC of 0.76 (finasteride) versus 0.68 (placebo) 70% to 95% within the tPSA range of 4 to 10 ng/mL (Catalona
et al., 1998; Partin et al., 1998; Polascik et al., 1999; Pound et al., In addition to contributing to detection, fPSA may provide
1997; 1999; Stephenson and Stanford, 1997; Veltri and Miller, 1999; prognostic information. Serial measurement of %fPSA within archival
Vessella et al., 2000). In a prospective, multi-institutional study of serum has shown significant differences in aggressive and nonag-
men aged 50 to 75 years with PSA levels between 4 and 10 ng/mL gressive prostate cancers (Carter et al., 1997). This study suggested
and palpably benign prostate glands, a %fPSA cutoff of 25% detected that the longitudinal measurement of %fPSA changes may not only
95% of cancers (sensitivity) while avoiding 20% of unnecessary aid in detection but also contribute information regarding disease
biopsies (specificity) (Catalona et al., 1998; Gretzer et al., 2002). behavior. A number of studies have reported on the correlation
This resulted in an AUC for %fPSA that was significantly higher than between %fPSA and pathological outcomes, showing some correlation
that of total PSA (0.72 vs. 0.53). between low %fPSA and aggressive pathological features (Aus et al.,
Although there remains no single, established %fPSA threshold, 2003; Masieri et al., 2012; Morote et al., 2000; Shariat et al., 2006).
proposed cut points generally range from 15% to 25%. The National In addition, Shariat et al. (2006) reported an independent association
Comprehensive Cancer Network Prostate Cancer Early Detection between lower %fPSA and biochemical recurrence in 402 men who
Guidelines incorporate %fPSA as an option to help with decision underwent radical prostatectomy for clinically localized disease.
making before biopsy and recommend a threshold of 10% (Carroll Perhaps the clinical setting in which %fPSA is most often used
and Mohler, 2018). However, an important recent development has is in patients with an elevated PSA and a prior negative prostate
been the increasing availability and usage of predictive tools that biopsy (Djavan et al., 2000; Hayek et al., 1999; Stephan et al., 1997).
incorporate multiple clinical variables such as total PSA, %fPSA, Stephan et al. (1997) reported a 5% cancer underdiagnosis rate when
and DRE findings (Hernandez et al., 2009; Pepe et al., 2001; Zaytoun using a %fPSA value of 21% to trigger rebiopsy, and Catalona et al.
et al., 2011). In particular, the updated calculator based on results (1997) reported that a threshold of 28% detected 95% of cancers
of the prostate cancer prevention trial (PCPT) provide a calibrated and avoided 12% of repeat biopsies. More recent European data
estimate of prostate cancer risk that can facilitate shared clinical reported an AUC of 0.73 for %fPSA in a repeat biopsy population,
decision making (Fig. 149.4; Andriole et al., 2009; Ankerst et al., exceeding that of PSA and PCA3 (Auprich et al., 2012). At a threshold
2012; Schröder et al., 2009). fPSA decreases in a similar fashion as of 18%, %fPSA demonstrated a sensitivity of 85% and specificity
total PSA in the setting of 5ARI therapy; thus the percentage of fPSA of 41%. However, the AUC for %fPSA was only 0.52 in another
is not altered significantly by these medications (Keetch et al., 1997; European repeat biopsy cohort, suggesting a need for other biomarkers
Pannek et al., 1998). in this setting (Scattoni et al., 2013).
Fig. 149.4. Web-based calculation of prostate-cancer risk using the PCPT risk calculator operationalized
by the University of Texas Health Science Center at San Antonio (http://deb.uthscsa.edu/URORiskCalc/
Pages/calcs.jsp). Distinct nomograms using different patient factors and biomarkers are available, and
the output for the most commonly used nomograms gives the risk of high-risk cancer and any prostate
cancer using a pictorial interface.
Prostate lumen BPSA
Prostate luminal epithelial

Internal nicks
–7 proPSA (inactive) 237 PSA (active) 237
cell
APLILS R
hK2 Proteolysis
Partial cleavage
Intact PSA
(iPSA)
–4 [–4]proPSA 237 –2 [–2]proPSA 237
ILSR SR
Fig. 149.5. Differential cleavage and activation of pro–prostate-specific antigen (PSA). ProPSA is released
from the prostate epithelial cell with a 7–amino acid leader sequence. hK2 cleaves the amino acid leader
to activate PSA. Active PSA undergoes proteolysis to yield inactive PSA (iPSA) and may also undergo
internal degradation to form benign PSA (BPSA). Partial cleavage of the 7–amino acid leader sequence
yields inactive forms of proPSA (i.e., [–2]pPSA or [–4]pPSA).
Free Prostate-Specific Antigen Isoforms

fPSA in serum comprises three isoforms: proPSA, BPH-associated pPSA
Lumen
PSA (BPSA), and intact fPSA (Jansen et al., 2009; Mikolajczyk et al., hK2 PSA
Inactive PSA
2002). These three isoforms exist in approximately equal concentra-
tions in serum, and each has shown promise as a prostate cancer
biomarker. As discussed earlier, PSA originates with a 17–amino
acid chain that is cleaved to yield a precursor inactive form of PSA
termed proPSA (Kumar et al., 1997; Mikolajczyk et al., 1997, 2000,
2001; Peter et al., 2001; Zhang et al., 1995). As depicted in Fig.
149.5, the precursor form of PSA contains a 7–amino acid proleader
peptide, in addition to the 237 constituent amino acids of mature
PSA, and is termed proPSA or [−7]proPSA. This leader amino acid
chain is cleaved by hK2, resulting in the active form of PSA. Incomplete
removal of the 7–amino acid leader chain has led to the identification Basement membrane
of various other truncated or clipped forms of proPSA. These include
proPSAs with 2–, 4–, and 5–leader amino acids ([–2]pPSA, [–4] ACT
Serum
pPSA, and [–5]pPSA), and all are primarily expressed in the peripheral ACT
zone of the prostate. With cellular disruption, these enzymatically Bound PSA Free PSA pPSA
Bound PSA
inactive forms circulate as fPSA and may constitute the majority
of the circulating fPSA in patients with prostate cancer (Mikolajczyk Normal Cancer
et al., 1997; Fig. 149.6).
Reports by Mikolajczyk et al. (2000, 2001) have revealed signifi- Fig. 149.6. Prostate-specific antigen (PSA) synthesis in normal versus cancer
cantly elevated levels of these truncated forms of proPSA in prostate tissue. ProPSA is secreted into the lumen, where the 7–amino acid leader
cancer tissue. In particular, the [−2]proPSA isoform, cleaved between sequence is cleaved by hK2 to yield active PSA. Some of the active PSA
leucine 5 and serine 6 of the propeptide, has shown increasing diffuses into the serum, where it is bound to proteases such as α1-
promise as a serum prostate cancer biomarker (Lazzeri et al., 2012; antichymotrypsin (ACT). The luminal active PSA undergoes proteolysis, and
2013; Le et al., 2010). Although the underlying biology behind the the resulting inactive PSA (iPSA) may also enter the circulation to circulate
increased levels of [−2]proPSA in prostate cancer remains unclear, in the unbound or free state. In prostate cancer, loss of the tissue architecture
it may be that the decreased PSA processing in prostate cancer results may permit a relative increase in bound PSA and proPSA in serum.
in a relative increase in proPSA and its cleaved forms, especially [–2]
proPSA. In addition, Makarov et al. (2009) have proposed that proPSA
production may be driven by benign tissue adjacent to malignant in patients with a serum total PSA of 2 to 10 ng/mL. In addition,
prostatic epithelium. %[−2]proPSA was closely correlated with increasing Gleason score.
From a clinical standpoint, [−2]proPSA has been extensively The levels of [−2]proPSA have been incorporated into a formula
validated in the screening setting, before either initial biopsy or that also includes free and total PSA and is called the Prostate Health
repeat biopsy (Guazzoni et al., 2011; Lazzeri et al., 2012, 2013; Le Index (PHI) (Lazzeri et al., 2013). PHI is FDA approved in men aged
et al., 2010; Loeb et al., 2015, 2017). Lazzeri et al. (2013) reported 50 years and older with total PSA 4 to 10 ng/mL and negative DRE.
on a prospective European cohort of 646 patients with total PSA The accuracy of PHI for predicting clinically significant prostate cancer
between 2 and 10 ng/mL who were undergoing initial prostate biopsy. in the early detection setting is consistently better than PSA or %fPSA,
The %[−2]proPSA was a strong independent predictor of prostate and adding PHI to existing risk calculators such as PCPT significantly
cancer at biopsy in a model that included free and total PSA, and the improves their performance (Loeb et al., 2015, 2017; Tosoian et al.,
AUC for %[−2]proPSA was 0.67. The %[−2]proPSA was also a strong 2017). PHI is also strongly associated with reclassification in men
predictor of Gleason score ≥7 prostate cancer in this study. In the on active surveillance (Hirama et al., 2014; Tosoian et al., 2012).
United States, a prospective National Cancer Institute (NCI) EDRN This test is discussed in more detail in Chapter 152.
validation study demonstrated excellent performance characteristics Another isoform of fPSA, referred to as benign PSA or BPSA, is
for %[−2]proPSA (Sokoll et al., 2010). Including %[−2]proPSA in the identical to mature PSA except for internal cleavages between Lys
base model consisting of free and total PSA significantly improved 182 and Lys 145 (Mikolajczyk et al., 2002). Its expression is generally
the predictive accuracy, and %[−2]PSA outperformed PSA and %fPSA limited to transition zone tissue, and it is highly expressed in the
in predicting the presence of prostate cancer at the time of biopsy setting of BPH (Canto et al., 2004; Mikolajczyk et al., 2000; Wang
et al., 2000). BPSA is closely related to prostate volume and does 1999; Tremblay et al., 1997). In contrast, in cancerous tissue hK2
not appear to have any prognostic capacity in terms of distinguishing is expressed more intensely. Furthermore, tissue expression of hK2
potentially indolent from aggressive disease (de Vries et al., 2005; appears to correlate with more aggressive pathological features, includ-
Naya et al., 2004). Although serum BPSA alone is unlikely to dif- ing Gleason grade (Darson et al., 1997, 1999; Tremblay et al., 1997).
ferentiate between hyperplasia and cancer, in combination with assays A number of studies have shown an association between serum
for [−2]proPSA, it may allow additional discrimination (Rhodes hK2 levels and the presence of prostate cancer, suggesting that it
et al., 2012; Stephan et al., 2009). may be used in conjunction with PSA to improve the selection of
So-called “intact PSA” comprises an additional fPSA subset that patients for prostate needle biopsy (Becker et al., 2000; Nam et al.,
has been identified as a predictive serum biomarker (Hori et al., 2000; Vickers et al., 2007). Furthermore, men with low-grade disease
2013; Nurmikko et al., 2000; Steuber et al., 2007a). Preliminary have lower concentrations of serum hK2 than men with more aggres-
studies have demonstrated that the ratio of intact to free PSA may sive cancer (Darson et al., 1999). Data from a multicenter study
improve the accuracy of prostate cancer detection (Nurmikko et al., demonstrated a statistically significant difference between men with
2000, 2001; Steuber et al., 2002). However, some studies of intact biopsies positive for cancer, looking at hK2 alone and in combination
PSA include proPSA in their intact PSA assay, making it difficult to with fPSA/tPSA (Kwiatkowski et al., 1998). Combining fPSA and
determine the predictive value of intact PSA alone (Peltola et al., 2011). hK2, Partin et al. (1999) demonstrated an increased cancer detection
This parameter has primarily been used as part of a multi-kallikrein rate within the tPSA range of 2 to 10 ng/mL. Serum hK2 may also
panel, which is discussed later in this chapter (Vickers et al., 2008). offer prognostic information: studies show correlations with aggressive
pathological features as well as biochemical recurrence (Haese et al.,
Prostate-Specific Membrane Antigen 2001; Steuber et al., 2007b).
Currently, hK2 used clinically as one of the components of the
The glycoprotein prostate-specific membrane antigen (PSMA) has been 4Kscore, a multi-kallikrein panel reported by Vickers et al. (2008).
evaluated for a number of years as a potential serum, urine, and/or Early reports used a large cohort of men from the Goteborg arm of
tissue biomarker of prostate cancer. A folate hydrolase, PSMA is found the European Randomized Study of Screening for Prostate Cancer
embedded within the cell membrane of all prostatic epithelial cells. It (ERSPC) to combine, within a statistical model, four kallikrein forms
is a type II transmembrane protein with an extracellular C-terminus (total PSA, fPSA, intact PSA, and hK2) to accurately predict the
that exists as a dimer and binds glutamate and glutamate-like structures presence of prostate cancer in men with a PSA of at least 3.0 ng/mL
(Fair et al., 1997; Israeli et al., 1997). The gene for PSMA has been (AUC 0.84). These findings were then validated in a separate ERSPC
cloned, fully sequenced, and localized to the short arm of chromosome cohort (Rotterdam arm) in which it was suggested that use of the
11 (11p11-p12). Although PSMA is predominantly expressed in the multi-kallikrein panel could avoid 513/1000 biopsies with only
secretory acinar epithelium of the prostate gland, it has been isolated 54/177 missed low-grade cancers and 12/100 missed high-grade
in other tissues, including in the central nervous system (astrocytes cancers (Vickers et al., 2010). In addition, of 392 men in this arm
and Schwann cells) and intestine (jejunal brush border). who underwent radical prostatectomy between 1994 and 2004, the
Of interest for diagnosis (Douglas et al., 1997), prognosis (Perner four kallikrein markers were independently associated with pathologi-
et al., 2007), and imaging (Ristau et al., 2013) is the discovery of cally aggressive disease features and improved the accuracy of the
elevated expression of this protein in tissue from prostate cancer base model (AUC 0.81 to 0.84) (Carlsson et al., 2013).
compared with normal prostate tissue (Chang et al., 1999; Elgamal Although the statistical model has not been the same in all studies,
et al., 2000; Minner et al., 2011; Silver et al., 1997). There is also there have been multiple studies validating the performance of this
evidence that increased tissue expression of PSMA may confer a assay. In a US-based study of 1012 men undergoing prostate biopsy,
worse prognosis in patients undergoing radical prostatectomy (Minner the 4Kscore would decrease biopsy rates by 30% to 58% while missing
et al., 2011; Perner et al., 2007). 1.3% to 4.7% of Gleason score ≥7 tumors, depending on the threshold
Currently, PSMA has its greatest use in targeted imaging and ther- used (Parekh et al., 2013). A meta-analysis using individual patient
anostics (Barrett et al., 2013; Milowsky et al., 2007; Osborne et al., data has demonstrated the value of all four components of the
2013; Tagawa et al., 2013). In particular, 68Gallium prostate-specific kallikrein panel, and this same data analyzed across multiple different
membrane antigen positron emission tomography (68Ga-PSMA PET) patient subgroups showed a mean increase in AUC of 0.11 when the
has become an increasingly used diagnostic tool in the setting of 4Kscore was added to a base clinical model (Vickers et al., 2018a,b).
biochemical recurrence after primary therapy. A meta-analysis of
15 studies found 69% of patients with detectable disease, leading Circulating Tumor Cells and Circulating Tumor DNA
to increases in use of radiotherapy and salvage lymphadenectomy
along with concomitant decreased use of systemic therapy (Han et al., Circulating tumor cells (CTCs) have long been touted as potential
2018). Another meta-analysis reported 40% of patients with positive prognostic biomarkers and treatment response indicators. The excite-
68
Ga-PSMA PET scans at the time of primary staging and 76% positive ment in this area of research goes back more than 20 years, when
at the time of biochemical recurrence (Perera et al., 2016). Specificity investigators demonstrated the ability to detect PSA mRNA in the blood
of this modality approaches 97%, and even at PSA levels as low as 0 to of men with advanced prostate cancer (Katz et al., 1994). Subsequent
0.2, 42% of patients in this report were found to have positive scans. CTC research in prostate cancer has used a wide range of methods,
capitalizing on features such as size, surface marker expression, and
Human Kallikrein 2 cellular plasticity that differentiate CTCs from circulating mononuclear
cells in the blood (Danila et al., 2011; Pantel and Alix-Panabières, 2010;
Human kallikrein peptidase 2 (hK2) shares many important properties Yu et al., 2011). Typically, CTCs are defined as being CD45− and positive
with PSA and has demonstrated potential as another prostate cancer for an epithelial marker such as epithelial cell adhesion molecule
tumor marker (Becker et al., 2000; Darson et al., 1997; Kumar et al., (EpCAM) and/or cytokeratin. Although the development of CTCs as
1997; Lövgren et al., 1999; Rittenhouse et al., 1998; Young et al., a prostate cancer biomarker has been relatively slow to evolve, there
1992). Among many similarities, hK2 and PSA share 80% amino has been substantial recent progress in this field with a movement
acid homology (see Fig. 149.1), exhibit similar specificity for toward an increasing number of clinical assays.
prostate tissue, and are hormonally regulated by androgens. As Currently, there is only one FDA-approved methodology for identify-
discussed earlier, one of the key functions of hK2 is to activate the ing CTCs: CellSearch (Veridex, Warren, NJ). The CellSearch system
zymogen (proPSA) to the active PSA through cleavage of the amino uses antibodies against EpCAM for CTC capture and then stains with
acid presequence (see Fig. 149.6). antibodies against CD45 (negative) and cytokeratins 8, 18, and 19
Critical to its utility as a biomarker, hK2 expression varies indepen- (positive) to identify individual CTCs. With this system, a CTC count
dent of PSA expression, in tissue and serum (Tremblay et al., 1997). of 5 or more cells per 7.5 mL of blood at any time during the course
For example, in benign epithelium, PSA is intensely expressed of the disease has been associated with a poor prognosis in prostate,
compared with minimal immunoreactivity of hK2 (Darson et al., breast, and colorectal cancers (Cohen et al., 2008; Cristofanilli et al.,
EpCAM antigen
Cytokeratin
CD45 antigen
Anti-EpCAM
ferrofluid Digital rectal exam Urine specimen PCA-3 and PSA mRNA
concentrations measured
in separate tubes
Anti-CK Quantitative ratio of
PCA-3/PSA mRNA
Anti-CD45 Tumor cell Leukocyte = PCA-3 score
Fig. 149.7. CellSearch CTC: Circulating Tumor Cell (Veridex) cell enumeration PCA-3 score PCA-3 score
< cutoff cutoff
system. CD45, CD stands for “cluster of differentiation,” which was originally
called leukocyte common antigen; CK, cytokeratin; EpCAM, epithelial cell Lower risk of Higher risk of
adhesion molecule. The anti-EpCAM ferrofluid captures the cells, and they positive biopsy positive biopsy
are then validated with cytokeratin-positive and CD45-negative staining.
Fig. 149.8. PCA3 assay protocol. After “attentive” digital rectal exam, urine
is collected. RT-PCR determines the mRNA levels for PCA3 and PSA. The
2004; de Bono et al., 2008; Shaffer et al., 2007). In a study of 422 ratio between PCA3/PSA determines the PCA3 score. Prostate cancer risk
patients with metastatic prostate cancer, using the CellSearch platform level suggests need for biopsy. (From Groskopf J, Aubin SM, Deras IL, et al.:
the investigators demonstrated that there was a difference between APTIMA PCA3 molecular urine test: development of a method to aid in the
baseline numbers of cells and those seen 2 to 5 weeks post-treatment diagnosis of prostate cancer. Clin Chem 52:1089–1095, 2006.)
(Fig. 149.7). For example, 57% of the patients had greater than 5 cells
per 7.5 mL of blood before treatment, and 39% had greater than 5
cells per 7.5 mL after treatment (Shaffer et al., 2007). Similar studies URINE-BASED BIOMARKERS
have lent further support to the potential use of CTCs as a response PCA3
indicator, with conversion from unfavorable to favorable CTC counts
(<5 CTCs) frequently occurring in men with advanced prostate cancer Given the ease of collecting urine specimens and the known shedding
receiving hormonal agents or chemotherapy (Danila et al., 2010; de of prostate cells, urine has long held promise as a potential biomarker
Bono et al., 2008; Reid et al., 2010). Further data demonstrating that source in prostate cancer (Truong et al., 2013). It was not until rela-
CTC response independently predicts survival in these settings are tively recently, however, that urinary biomarkers of prostate cancer
needed before CTCs can be used as an efficacy-response surrogate have come into clinical use. The first of these, prostate cancer antigen
marker (Scher et al., 2009, 2013). 3 (PCA3), was initially described by Bussemakers et al. (1999), who
A number of other platforms for CTC detection have been developed, used differential display and Northern blot analysis to compare
and this continues to be an area of rapid growth in the biomarker normal and prostate cancer tissue. They were able to identify a prostate
space. Investigators at Harvard Medical School developed a microfluidic cancer–specific gene on chromosome 9q21-22 that, although it does
system known as the “CTC-Chip,” which has garnered a great deal not encode a protein, is one of the most sensitive and specific prostate
of attention (Nagrath et al., 2007). This system has a high level of cancer biomarkers. Although its function remains unknown, multiple
sensitivity, able to detect a single EpCAM-positive cell among 1 billion studies have demonstrated that PCA3 is a long noncoding RNA
blood cells, and the capture efficiency has been further improved with (lncRNA) that is not expressed outside of the prostate, and PCA3
an updated device (Nagrath et al., 2007; Stott et al., 2010). levels in malignant tissue generally far exceed levels in benign tissue
Going forward, the use of CTCs as a biomarker will likely revolve (de Kok et al., 2002; Popa et al., 2007). Early studies used reverse
less around simple enumeration and more around the specific transcription polymerase chain reaction (RT-PCR) assays to detect
molecular alterations that can be identified—either within the cells PCA3 in urine and showed improved performance characteristics
or from cell-free nucleic acids—providing a real-time “liquid biopsy” for PCA3 over PSA in diagnosing prostate cancer (Hessels et al.,
in men with prostate cancer (Danila et al., 2011, 2014; Dawson et al., 2003; van Gils et al., 2007).
2013). In 2014 a seminal study published in the New England More recently, a transcription-mediated amplification assay was
Journal of Medicine demonstrated that detection of the androgen- developed, offering improved sensitivity and quantitation relative
receptor splice variant 7 (AR-V7) in CTCs predicts resistance to to standard RT-PCR (Groskopf et al., 2006). This has become the
abiraterone and enzalutamide in metastatic castration-resistant commercial assay (Progensa, Hologic Inc.), which is CE marked
prostate cancer (Antonarakis et al., 2014). These results were sub- and FDA approved (2012) to assist with decisions in the setting
sequently validated in a larger study, which also demonstrated that of a prior negative prostate needle biopsy. To enhance the sensitivity
outcomes were best for patients without CTCs, intermediate for of PCA3 detection, urine samples are collected after an “attentive”
patients with AR-V7–negative CTCs, and worst for patients with digital rectal examination (Fig. 149.8). This involves three firm strokes
AR-V7–positive CTCs (Antonarakis et al., 2017). Similar findings on each lobe of the prostate toward the median sulcus, and the first
were demonstrated using another commercially available platform 20 to 30 mL of voided urine should be collected within 1 hour
based on digital imaging, which found that patients with nuclear (Sokoll et al., 2008). The commercial PCA3 score is reported as the
expression of AR-V7 in CTCs responded better to taxane therapy ratio of urine PCA3 mRNA/urine PSA mRNA × 1,000, thus normal-
than abiraterone or enzalutamide (Scher et al., 2016). izing PCA3 expression to PSA expression.
Instead of capturing cells in circulation to evaluate gene expression, Numerous clinical studies have now been performed to evaluate
cell-free DNA can be analyzed to detect and characterize circulating the utility of urine PCA3 to serve as a prostate cancer biomarker,
tumor DNA (ctDNA). The goal of these approaches is generally to and all have shown that PCA3 scores are closely correlated with the
profile a tumor’s somatic alterations, which can then potentially be likelihood of a positive biopsy (Alshalalfa et al., 2017; Deras et al.,
used for early detection or to inform prognosis and/or treatment 2008; Hessels et al., 2003; Roobol et al., 2010b; van Gils et al., 2008).
in patients with known cancer. For example, the recently described Deras et al. (2008) reported that men with a PCA3 below 5 had a
CancerSEEK test combines ctDNA analysis with additional circulating 14% positive biopsy rate, compared with a 70% positive biopsy rate
markers in a single early detection assay covering multiple cancer in men with PCA3 above 100. However, in another study, patients
types (Cohen et al., 2018). In advanced disease, ctDNA can inform the with PCA3 above 100 had only a 52% positive biopsy rate (Roobol
timing and mechanism of therapeutic resistance and potentially be et al., 2010a). Unlike PSA, PCA3 levels are independent of prostate
used to guide subsequent treatment decisions (Goodall et al., 2017). size (Haese et al., 2008).
One of the critical challenges in using PCA3—or any biomarker Although tissue-based detection of the fusion using either chro-
reported as a continuous score rather than just “positive/negative”—is mosomal analysis (i.e., fluorescence in situ hybridization) or
establishing appropriate cutoffs. Given that PCA3 scores represent immunohistochemical staining for ERG has shown some utility, this
a continuum of risk, different thresholds will lead to differences in biomarker appears to have its greatest potential as a urine-based
sensitivity and specificity of the assay. Multiple cutoffs have been assay (Hessels et al., 2007; Laxman et al., 2006, 2008). A feasibility
proposed, most commonly 10, 25, and 35 (Crawford et al., 2012; study that examined urine samples post-DRE confirmed that the
Haese et al., 2008). Haese et al. (2008) reported on a cutoff of 35, TMPRSS2:ERG fusion can indeed be detected in the urine and may
with positive biopsies in 39% of those above versus 22% in those aid in the decision to proceed with a prostate biopsy (Laxman et al.,
below the threshold. In a comparative effectiveness review commis- 2008). Like PCA3, TMPRSSS2:ERG levels are normalized to urine
sioned by the US Agency for Healthcare Quality and Research, Bradley PSA mRNA expression. Tomlins et al. (2011) reported on the develop-
et al. (2013) showed that a threshold of 25 results in a sensitivity ment and use of a clinical grade assay based on transcription-mediated
of 74% and specificity of 57% (false-positive rate of 43%). It is this amplification (similar to the PCA3 assay) in 1312 men before prostate
lower threshold of 25 that was used in the FDA approval. However, biopsy. TMPRSS2:ERG outperformed serum PSA for predicting the
different thresholds may be required for different patient populations presence of prostate cancer with an AUC of 0.65 to 0.71 versus 0.59
of clinical scenarios. For example, using a PCA3 score of less than to 0.61. In addition, in a subset of men who underwent prostatectomy,
20 in men with repeat prostate biopsy, Wei et al. (2014) reported a higher urine TMPRSS2:ERG score was associated with increased tumor
negative predictive value of 88%, and a sensitivity and specificity of volume, higher-grade disease, and non–organ-confined cancer.
76% and 52%, respectively. However, in men being seen for an Moving toward the concept that single biomarkers will probably
initial prostate biopsy using a PCA3 score of more than 60, Wei not answer the important clinical questions in prostate cancer alone,
et al. reported a positive predictive value of 80% and a sensitivity many investigators have begun to multiplex markers, resulting in
and specificity of 42% and 91%, respectively (Wei et al., 2014). additive value. Specifically, given that TMPRSS2:ERG is found in
There are increasing reports on the use of PCA3 in the initial only half of all prostate cancer foci (approximately 75% of men
screening setting of patients without a prior biopsy. Roobol et al. with cancer), its greatest utility will be in combination with other
(2010b) reported on a cohort of 721 men, the majority of whom biomarkers. It has been evaluated closely in concert with PCA3, and
had not previously undergone a biopsy and showed that PCA3 (AUC the two biomarkers together appear to provide improved performance
0.64) outperformed PSA (AUC 0.58) in predicting cancer on sub- over either one alone (Hessels et al., 2007; Laxman et al., 2008;
sequent biopsy. In a prospective, community-based evaluation of Salami et al., 2013; Tomlins et al., 2016). In the 1312 patient cohort
PCA3 in 1962 men before any prostate biopsy, prostate cancer was reported by Tomlins et al. (2011), combining TMPRSS2:ERG and
detected in 61% of men with a PCA3 of at least 35 and 32% with PCA3 scores with the PCPT nomogram resulted in an AUC for predict-
a PCA3 below 35 (Crawford et al., 2012). The AUC for PCA3 was ing cancer of 075 to 0.79. TMPRSS2:ERG + PCA3 score groups were
0.71 compared with an AUC of 0.57 for PSA. Another study of 3073 also shown to correlate with features of disease aggressiveness such
men undergoing initial biopsy demonstrated that PCA3 was an as Gleason grade, although data in this area continue to be mixed
independent predictor of any prostate cancer and high-grade prostate (Gopalan et al., 2009; Leyten et al., 2014).
cancer after controlling for additional clinical variables (Chevli et al., Three other large studies have reported on the combination of
2014). Although PCA3 performed better than PSA for the detection TMPRSS2:ERG and PCA3 for predicting the presence of prostate
of any cancer (AUC 0.70 vs. 0.60), they performed similarly for the cancer (Sanda et al., 2017). Cornu et al. (2013) showed that PCA3
detection of high-grade cancer (AUC 0.68 vs. 0.68). and TMPRSS2:ERG were independent predictors of prostate cancer in a
To improve the predictive accuracy of PCA3, a number of nomo- multivariable model that also included other clinical parameters. Leyten
grams have been developed that incorporate PCA3 along with other et al. (2014) reported on the prospective use of the combined assay
known clinical predictors to identify men most likely to have a positive in 497 patients at 6 centers in Europe. The combination of PCA3 and
prostate biopsy. For example, Chun et al. (2009) showed that PCA3 TMPRSS2:ERG increased the performance of the European Randomized
improves the accuracy of a base clinical model (AUC 0.73 vs. 0.68) Study of Screening for Prostate Cancer (ERSPC) risk calculator from
and proposed a final model that incorporated a PCA3 cutoff of 17. an AUC of 0.80 to 0.84. In addition, the sensitivity of PCA3 increased
Similarly, Ankerst et al. (2008) reported on an updated version of from 68% to 76% when TMPRSS2:ERG was added. More recently,
the Prostate Cancer Prevention Trial (PCPT) nomogram that includes Sanda et al. (2017) reported that combined testing of urinary T2:ERG
PCA3 as a continuous variable. The final model outperformed the and PCA3 at thresholds that preserved 95% sensitivity for detecting
base PCPT model (0.70 vs. 0.65) and has been operationalized on the aggressive prostate cancer improved specificity from 18% to 39% in
web (as in Fig. 149.4). Another nomogram incorporating PCA3 and the training cohort and from 17% to 33% in the validation cohort.
clinical variables was developed by Elshafei et al. (2015) for predicting Further, 42% of unnecessary prostate biopsies would have been avoided
prostate cancer and high-grade prostate cancer in an initial biopsy using the combined urine assay, with the greatest potential benefit in
setting with C-indices of 0.74 and 0.77, respectively, compared with younger men. Currently, the combined test is commercially available as
0.70 and 0.75, respectively, without PCA3. Given the available data, The Mi-Prostate Score (MiPS, University of Michigan Health System),
the comparative effectiveness review by Bradley et al. (2013) concluded combining serum PSA, urine PCA3, and urine TMPRSS2:ERG to provide
that PCA3 appears to be superior to total PSA in diagnosing prostate a quantitative risk estimate of the likelihood of detecting prostate cancer
cancer, but that further evidence is needed and there is not yet evidence on prostate biopsy as well as the probability of detecting high-grade
that use of PCA3 leads to better health outcomes. cancer (Tomlins et al., 2016). This test may also have a role in the
management of patients on active surveillance for prostate cancer,
Gene Fusions because it has been correlated with features of tumor aggressiveness
in a prospective active surveillance cohort (Lin et al., 2013).
With the landmark discovery of the presence of gene fusions in prostate
cancer by Tomlins et al. (2005), considerable work has been performed Other Urine Biomarkers
to determine their potential utility as prostate cancer biomarkers. In
particular, fusions of the 5′untranslated region of the androgen- Recently, Van Neste et al. (2016) reported that HOXC6 and DLX1
regulated gene transmembrane protease, serine 2 (TMPRSS2) with mRNA levels in urine in combination with clinical factors improved
v-ets erythroblastosis virus E26 oncogene homolog (ERG) or ets the detection of high-grade prostate cancer with overall AUC of 0.86
variant 1 (ETV1) were found to be nearly 100% specific for pros- and 0.90 in the training and validation cohorts, respectively. The
tate cancer and present in at least 50% of PSA-screened prostate authors performed a decision curve analysis, which revealed a strong
cancers. ERG and ETV1 are members of the erythroblastosis virus net benefit with the best reduction in unnecessary biopsies compared
E26 transformation-specific (ETS) transcription factor family, and with other clinical decision-making tools, such as the PCPT risk
TMPRSS2:ERG fusions represent about 90% of all ETS gene fusions calculator and the PCA3 assay. This assay is now commercially
(Tomlins et al., 2009; Young et al., 2012). available as the SelectMDx test.
Annexin A3 in DNA methylation and histone acetylation status. Segments within

the gene promoter that are composed of GC-rich regions are termed
Another potential urine-based prostate cancer biomarker is annexin CpG islands. Alterations in the methylation status of these regions
A3, which is inversely related to the presence of prostate cancer. This may affect gene expression and have been shown to play a role
protein is part of a family of calcium and phospholipid binding in carcinogenesis (Jones and Baylin, 2002). Furthermore, cumulative
proteins that have been shown to be altered in cancer (Köllermann effects of environmental exposures, such as diet and stress throughout
et al., 2008; Wozny et al., 2007). Evaluating annexin A3 concentrations life, may affect DNA methylation status and thus contribute to the
in post-DRE urine before prostate needle biopsy, Schostak et al. risk of cancer development (Li et al., 2004). The products of a
(2009) evaluated the potential clinical utility of urine-based annexin number of hypermethylated genes have been implicated in prostate
A3, either as a stand-alone biomarker or together with PSA. In a cancer development, including glutathione-S-transferase π (GSTP1),
blinded study that consisted of training and evaluation sets of 243 adenomatous polyposis coli (APC), retinoic acid receptors beta
and 264 men, respectively, these investigators showed that annexin 2 (RARβ2), and RAS association domain family protein isoform
A3 added to the ability of PSA to predict a positive needle biopsy A (RASSF1A) (Mahapatra et al., 2012).
with a combined AUC of 0.81. Using a multiplex approach, Cao GSTP1 belongs to a family of detoxifying enzymes that are involved
et al. (2011) reported on the combination of urine annexin A3, in metabolic reduction of electrophilic carcinogens and was the first
PCA3, TMPRSS2:ERG, and sarcosine to help identify patients with tissue methylation-biomarker to be discovered. Li et al. (2004) noted
prostate cancer. The multi-marker model demonstrated a high level that the GSTP1 gene was unmethylated in all normal human tissues
of predictive accuracy with an AUC of 0.84 in patients with PSA 4 and BPH but was hypermethylated in all 20 prostate cancer specimens
to 10 ng/mL and an AUC of 0.86 across all patients in their cohort. analyzed. Harden et al. (2003a,b) used PCR to detect hypermethylated
GSTP1 in prostate biopsy specimens, and GSTP1 methylation was
miRNA detected in 11 of 15 (73% sensitivity) prostate cancer cases and in
none of 14 (100% specificity) benign controls. Quantitation of GSTP1
Based upon the realization that a majority of the DNA in the genome hypermethylation accurately detected CaP even in small, limited tissue
does not encode protein sequences, the utility of these sequences in samples. Elevated levels of GSTP1 CpG hypermethylation have been
the regulation of gene expression has been under intense investiga- detected in tissues from precancerous lesions (atypia and prostatic
tion. Among the most important components of this regulation intraepithelial neoplasia [PIN]) and within ejaculates, urine, and
are microRNAs (miRNAs). These are small (approximately 19–22 plasma from men with prostate cancer (Bastian et al., 2005; Nakayama
nucleotide) noncoding, single-strand RNAs involved in the regulation et al., 2003). Cairns et al. (2001) have demonstrated the presence of
of mRNA. They have been detected in a wide range of biologic fluid elevated GSTP1 hypermethylation in up to 79% of prostate cancer
and are being explored as potential biomarkers in across a variety specimens. GSTP1 methylation appears to occur early in prostate
of malignancies. Given their potential mechanistic role, they may carcinogenesis, and methylation levels may correlate with features
provide diagnostic and prognostic information. In prostate cancer, of tumor aggressiveness (Jerónimo et al., 2001; Zhou et al., 2004). In
miR-141 has shown promise in a number of studies in terms of its a recent meta-analysis, GSTP1 promoter hypermethylation was shown
association not only with prostate cancer but potentially also with to have a sensitivity of 82% and specificity of 95% for distinguishing
disease aggressiveness (Bryant et al., 2012; Mitchell et al., 2008). In malignant from normal prostate tissue (Van Neste et al., 2012).
addition, Casanovas-Salas et al. (2014) recently reported that urine In addition to GSTP1, hypermethylation of other genes has also
miR-187 is an independent predictor of prostate cancer at needle been noted to occur in close to 100% of prostate cancers (Mahapatra
biopsy. Although these findings must be expanded to larger populations et al., 2012). APC is a tumor suppressor involved in apoptosis and
of individuals, this remains a promising family of potential markers. cell migration, and mutation of APC is often an early event in the
colon carcinogenesis. In a retrospective analysis assessing patients
with newly diagnosed prostate cancer, APC methylation was associated
TISSUE-BASED BIOMARKERS with an independent 50% increase in the likelihood of death from
α-Methylacyl Coenzyme A Racemase prostate cancer (Richiardi et al., 2009). RASSF1, a gene involved in
cell cycle regulation, and RARβ2, a nuclear transcriptional regulator,
α-methylacyl coenzyme A racemase (AMACR) gene, located on are other key genes methylated in a large proportion of prostate
chromosome 5, is upregulated in prostate cancer tissues (Luo et al., cancers and shown to distinguish benign from malignant prostate
2002; Rubin et al., 2002). AMACR functions as an enzyme responsible in a number of studies (Van Neste et al., 2012; Zon et al., 2009).
for the beta-oxidation of branched-chain fatty acids obtained in diets Perhaps the most clinically relevant aspect of these methylation
consisting of beef and dairy products. Luo et al. (2002) demonstrated changes is the discovery that they are frequently detectable in normal
that 88% of prostate cancer cases and untreated metastasis and tissue adjacent to tumors but not in normal tissue well away from
hormone-refractory prostate cancers were strongly positive for AMACR. the primary tumor (Richiardi et al., 2013). Thus these changes may
Immunohistochemical studies by Rubin et al. (2002) have shown be useful indicators of the field effect that has long been known to
that AMACR expression in biopsy tissue may provide 97% sensitivity exist: the presence of molecular changes in histologically normal
and 100% specificity for prostate cancer detection. Furthermore, in tissue bordering a tumor. In patients with a prior negative biopsy,
combination with other markers, such as TP63, that aid in identifying the identification of these molecular changes in the original biopsy
basal cells absent in prostate cancer, measurement of AMACR has tissue may suggest an increased likelihood of occult prostate cancer.
potential for the development of molecular probes to aid in the Trock et al. (2012) showed that methylation of APC in histologically
detection of prostate cancer. As a result, AMACR immunostaining negative prostate biopsies predicted the presence of prostate cancer
is sometimes performed on prostate needle biopsies to confirm the in 86 men undergoing repeat biopsy, with a negative predictive value
presence of cancer. AMACR may also have some prognostic utility: (NPV) of 0.96 and sensitivity of 0.95. Using a quantitative commercial
decreased staining levels are associated with an increased risk of disease methylation assay (ConfirmMDx, MDxHealth, Inc.) that assesses
progression (Rubin et al., 2005). Although other roles for AMACR as methylation of GSTP1, APC, and RASSF1, Stewart et al. (2013) showed
a biomarker have shown some promise—for example, as a urinary an NPV of 0.90 and sensitivity of 0.68. In this retrospective study,
assay for prostate cancer detection—the performance of serum and methylation status was an independent predictor of the presence of
urine tests has not been sufficient to warrant clinical use to date. prostate cancer on repeat biopsy. Similarly, in a multi-institutional
analysis of 350 subjects, Partin et al. (2014) demonstrated that the
Epigenetic Modifications epigenetic assay assessing methylation of GSTP1, APC, and RASSF1
resulted in an NPV of 88% (95% CI 85–91) in patients undergoing
Changes in gene expression may occur as a result of alterations in repeat prostate biopsy. In a recent analysis of 211 African-American
DNA, and epigenetic changes are those not caused by alterations men undergoing repeat prostate biopsy, ConfirmMDx was shown
in DNA sequence. These epigenetic modifications include changes to have a sensitivity and specificity of 74.1% and 60.0%, respectively,
for prostate cancer detection and 78% and 53%, respectively, for Although these tissue-based prognostic biomarker tests are increas-
Gleason score ≥ 7 disease (Waterhouse et al., 2018). The NPVs for ingly used in the evaluation of patients with early stage prostate cancer
detection of all prostate cancer and Gleason score ≥ 7 disease were to assess tumor aggressiveness, recent studies suggest that these assays
78.8% and 94.2%, respectively. These findings suggest accurate may not be robust to tumor multifocality and heterogeneity. For
identification of at-risk patients based on the “halo effect” of aberrant example, Wei et al. (2017) observed discordant RNA expression profiles
methylation. across distinct foci of prostate cancer obtained from the same patient.
A more recent analysis of grade-discordant multifocal prostate cancer
Genomic Expression Profiles demonstrated that derived Prolaris, Oncotype Dx, and Decipher scores
of low-grade prostate cancers do not predict the concomitant presence
For more than a decade, a great deal of attention has been devoted of an unsampled high-grade cancer from the same patient (Hovelson
to discovering large sets of genes that, when evaluated together as et al., 2018). Although there is likely a role for these tissue-based
a single biomarker assay, perform better than any individual gene prognostic biomarkers in the management of prostate cancer, almost
on its own. Relatively recent advances in genomics have facilitated all of the published data have been from retrospective studies. The
the evaluation of gene expression in formalin-fixed paraffin-embedded Genomics in Michigan ImpactiNg Observation or Radiation study
(FFPE) tissue, meaning prostate-archived prostate specimens can be is an ongoing prospective randomized trial assessing the impact of
interrogated for expression of gene profiles that are associated with Decipher on decision making and outcomes after prostatectomy for
aggressive disease features. Gene expression profiling has been men at high risk of local recurrence (NCT02783950).
explored in numerous settings, resulting in genomic classifiers for
response to chemotherapy in breast and colorectal cancers (Del Rio Inherited Genetic Markers
et al., 2007; Iwao-Koizumi et al., 2005; Sparano et al., 2018).
A number of LDTs using genomic expression profiling for risk Up to 40% to 50% of prostate cancer risk may be related to familial
stratification in men with prostate cancer are now being developed and and hereditary factors (Lichtenstein et al., 2000). Familial prostate
commercialized. This includes Prolaris (Myriad Genetics, Inc.), Onco- cancer is a broad term that encompasses 15% to 20% of cases and can
type Dx Prostate (Genomic Health, Inc.), and Decipher (GenomeDx include those patients with a strong family history of prostate cancer
Biosciences, Inc). The Prolaris test assesses 31 cell cycle progression but no detectable genetic mutations (Giri and Beebe-Dimmer, 2016).
(CCP) genes and 15 housekeeping genes, and a number of studies Having a relative with prostate cancer increases risk of developing the
have demonstrated an association between this gene signature and the disease by two- to fourfold (Goldgar et al., 1994; Liss et al., 2015).
risk of progression and death from prostate cancer (Cuzick et al., 2011, Single nucleotide polymorphisms (SNPs) are common genetic variants
2012). Cuzick et al. (2012) reported that, in a conservatively managed that may or may not have a functional role but are associated with an
cohort, Prolaris score was an independent predictor of prostate cancer increased risk of prostate cancer and likely factor into many of these
specific death. In men who underwent subsequent prostatectomy, familial cases. Men possessing more than four prostate cancer–related
Bishoff et al. (2014) retrospectively assessed the performance of this SNPs have a 4.5-fold increase in risk of developing prostate cancer, and
assay on prostate needle biopsies and demonstrated that Prolaris this risk increases to 10-fold for men with a family history of prostate
score was an independent predictor of biochemical recurrence and cancer (Zheng et al., 2008). An example of SNPs being brought into
metastasis. Recently, Cooperberg et al. (2013) incorporated the Prolaris practice is the STHLM3 biomarker test. This assay incorporates the four
score with the CAPRA score and reported improved performance kallikreins that are part of the 4Kscore along with 232 risk SNPs and
for predicting biochemical recurrence after radical prostatectomy. relevant clinical variables to form a composite prostate cancer early
Similarly, Prolaris score has been shown to predict outcomes in detection assay with an AUC of 0.74 for detecting Gleason score ≥7
men undergoing external beam radiation therapy for prostate cancer, prostate cancers (Eklund et al., 2018; Grönberg et al., 2015).
including prostate cancer–specific mortality (Freedland et al., 2013). A number of genes have been implicated in heritable prostate
The Oncotype Dx Prostate classifier uses 12 cancer-related genes in cancer, most of which have important roles in the DNA damage
4 biologic pathways and 5 housekeeping genes to predict the likeli- repair machinery. These include BRCA1, BRCA2, CHEK2, ATM, and
hood of aggressive pathological features at the time of prostatectomy PALB2, along with mismatch repair mutations responsible for Lynch
(Knezevic et al., 2013). This is defined as primary Gleason pattern syndrome (MLH1, MSH2, MSH6, and PMS2). BRCA1 and BRCA2
4 or pT3 disease at prostatectomy. Like Prolaris, Oncotype Dx is are critical proteins in the process of homologous recombination,
primarily intended to help with management decisions in patients and pathogenic mutations in these genes have long been known to
with newly diagnosed prostate cancer regarding active surveillance or increase the risk of breast and ovarian cancers in women (Yoshida
primary treatment and has been extensively validated for its clinical and Miki, 2004). More recently, BRCA1 and BRCA2 mutations in
outcome (Cullen et al., 2015; Eure et al., 2017; Klein et al., 2014). men have been associated with a significant increase in the risk of
Last, the Decipher assay is a 22-marker genomic classifier initially prostate cancer, and men with pathogenic BRCA2 mutations are
developed for use in the post-prostatectomy setting (Erho et al., diagnosed at a younger age, have higher Gleason grade tumors,
2013). In men with high-risk disease and those with biochemical and have a shorter median survival time than men with sporadic
recurrence, the genomic classifier was demonstrated to be indepen- prostate cancers (Mitra et al., 2008; Tryggvadóttir et al., 2007). For
dently associated with the development of metastatic disease and men under age 65 years, BRCA1 carriers who have a 1.8-fold increased
prostate cancer–specific mortality (Karnes et al., 2013, 2018; Ross risk of prostate cancer and BRCA2 carriers a 4.5-fold increased risk
et al., 2014; Spratt et al., 2018). In a recent meta-analysis, Spratt of prostate cancer compared with noncarriers (Thompson et al.,
et al. (2017) classified patients by Decipher as low-, intermediate-, 2002). Across all ages, Kote-Jarai et al. (2011) report an estimated
and high-risk categories with 10-year cumulative incidence of 8.6-fold increased risk of prostate cancer in BRCA2-positive men.
metastases after radical prostatectomy of 5.5%, 15.0%, and 26.7% Although Lynch syndrome is well known to be associated with
(P < .001), respectively. In this post-prostatectomy setting, Decipher colorectal, liver, skin, and upper urinary tract cancers, men with
may be used to guide decision making regarding adjuvant or salvage Lynch syndrome are also at an increased risk of developing prostate
radiation therapy and retrospective data that patients with higher cancer. A study of 821 male Lynch syndrome carriers reported a
Decipher scores (≥0.4) may benefit from postoperative radiation 10-fold increase in relative risk of prostate cancer for MSH2 carriers
(Dalela et al., 2017; Den et al., 2015; Gore et al., 2017). Building (Barrow et al., 2013).
on these findings and using the GenomeDx Decipher database, Zhao Other key genes include ATM and CHEK2, involved in double-
et al. developed a 24-gene biomarker signature, PORTOS (Post- strand DNA break repair, as well as the homeobox protein HOXB13,
Operative Radiation Therapy Outcomes Score) to predict response and all of these may be used as a potential marker of prostate cancer
to adjuvant radiation therapy. Decipher is now also available for risk. The Hale et al. (2014) pooled analysis of five studies shows
clinical use in patients with newly diagnosed prostate cancer to that CHEK2 carriers have a prostate cancer odds ratio of 1.98 and
potentially assist with initial management decisions, based on more 3.39 in unselected and familial cases, respectively. The HOXB13
recent biopsy data (Nguyen et al., 2017). G84E variant was identified as a key prostate cancer risk gene through
familial linkage studies, and the relative risk for prostate cancer SUMMARY
increases in mutation carriers by 4.5-fold (Ewing et al., 2012; Huang
and Cai, 2014). There is no evidence that HOXB13 increases the risk Early detection when cancer remains confined to the prostate not only
of aggressive cancers, however. improves cure rates but also decreases mortality from this disease.
Next generation sequencing (NGS)–based testing is clinically Although the discovery and application of PSA have revolutionized
available for single genes such as for BRCA1 or BRCA2, but multi- current prostate cancer detection and management, stage migration
gene panel testing has become more common in the absence of and changes in the natural history of this cancer have outrun the
a known familial mutation. For prostate cancer, these panels typically currently maximized application of this tumor marker. Application of
include BRCA1, BRCA2, ATM, CHEK2, MLH1, MSH2, TP53, and various PSA derivatives, although improving sensitivity, risks impairing
EPCAM, among others specific to the individual commercial platform. specificity. The discovery of molecular derivatives of PSA, PCA3, new
Importantly, although many of the genes included in these panels kallikrein markers, and gene rearrangements are leading to significant
have a clear association with prostate cancer risk, others carry a still improvements in the efficiency of prostate cancer detection.
unknown clinical significance with poorly defined cancer risk. National Innovations and new understanding in the field of molecular
Comprehensive Cancer Network (NCCN) recommendations focus oncology have provided a host of potential prostate cancer tumor
on BRCA testing at this point (Hall et al., 2014). Identification of markers. Because prostate cancer has been shown to be a heteroge-
BRCA mutations—or potentially these other mutations causing neous disease, application of a panel of markers will probably
increased prostate cancer risk—could have important implications ultimately provide added sensitivity and specificity for detection of
for early detection and management of patients at high risk of the disease. Identification of hypermethylated regions, such as for
developing prostate cancer (Bancroft et al., 2014). GSTP1, as well as tissue-based genomic classifiers may substantially
improve the diagnostic and prognostic potential of prostate needle
biopsies. Development of these markers from research into clinically
applicable tools will require clear demonstration of clinical validity
and clinical utility, showing that these new assays have a real impact
KEY POINTS in the clinical care of men with prostate cancer.
• PSMA has been identified in the central nervous system,
intestine, and prostate and now serves as an important
target for advanced molecular imaging. SUGGESTED READINGS
• PSA is a member of the human kallikrein gene family. PSA Bancroft EK, Page EC, Castro E, et al: Targeted prostate cancer screening in
and human kallikrein-2 have been used in prostate cancer BRCA1 and BRCA2 mutation carriers: results from the initial screening
round of the IMPACT Study, Eur Urol 66:489–499, 2014.
detection.
Byrne JC, Downes MR, O’Donoghue N, et al: 2D-DIGE as a strategy to identify
• Ectopic expression of PSA occurs in breast tissue, adrenal, serum markers for the progression of prostate cancer, J Proteome Res
and renal carcinomas. 8(2):942–957, 2009.
• PSA is organ specific, not disease specific; its half-life is 2 de Bono JS, Scher HI, Montgomery RB, et al: Circulating tumor cells predict
to 3 days. survival benefit from treatment in metastatic castration-resistant prostate
• 5α-reductase inhibitors reduce serum PSA by 50%. cancer, Clin Cancer Res 14(19):6302–6309, 2008. Erratum in: Clin Cancer
• Prostate cancer cells make less PSA than normal prostate Res. 2009;15(4):1506.
tissue, gram for gram. PSA expression is strongly Chevli KK, Duff M, Walter P, et al: Urinary PCA3 as a predictor of prostate
influenced by androgens. Ejaculation can lead to a false cancer in a cohort of 3,073 men undergoing initial prostate biopsy, J Urol
191:1743–1748, 2014.
increase in PSA.
Cucchiara V, Cooperberg MR, Dall’Era M, et al: Genomic markers in prostate
• Seventy percent of serum PSA is bound to three proteins: cancer decision making, Eur Urol 73:572–582, 2018.
α2-macroglobulin, α1-protease inhibitor, and α1- Ewing CM, Ray AM, Lange EM, et al: Germline mutations in HOXB13 and
antichymotrypsin. Patients with prostate cancer have a prostate-cancer risk, N Engl J Med 366(2):141–149, 2012.
higher fraction of circulating PSA bound to these proteins, Hu R, Dunn TA, Wei S, et al: Ligand-independent androgen receptor variants
that is, they have a lower free PSA. derived from splicing of cryptic exons signify hormone-refractory prostate
• When PSA is released from the cell, a portion of an cancer, Cancer Res 69(1):16–22, 2009.
attached amino acid chain is cleaved, leaving a smaller Karnes RJ, Bergstralh EJ, Davicioni E, et al: Validation of a genomic classifier
amino acid chain attached, which inactivates its biologic that predicts metastasis following radical prostatectomy in an at risk patient
population, J Urol 190(6):2047–2053, 2013.
activity. This molecule is termed proPSA. When this amino
Lazzeri M, Haese A, de la Taille A, et al: Serum isoform [-2]proPSA derivatives
acid chain is cleaved from proPSA, PSA becomes active as significantly improve prediction of prostate cancer at initial biopsy in a
a serum protease. ProPSA may be used to diagnose total PSA range of 2-10 ng/ml: a multicentric European study, Eur Urol
prostate cancer. 63(6):986–994, 2013.
• PCA3 is a urine-based marker used in the diagnosis of Polascik TJ, Oesterling JE, Partin AW: Prostate specific antigen: a decade
prostate cancer. It can be combined with detection of the of discovery—what we have learned and where we are going, J Urol
TMPRSS2:ERG gene fusion to improve diagnostic accuracy. 162:293–306, 1999.
• Circulating tumor cells and circulating tumor DNA are Roobol MJ, Schröder FH, van Leeuwen P, et al: Performance of the prostate
promising sources of biomarkers, particularly in advanced cancer antigen 3 (PCA3) gene and prostate-specific antigen in prescreened
men: exploring the value of PCA3 for a first-line diagnostic test, Eur Urol
prostate cancer.
58(4):475–481, 2010.
• Tissue-based gene expression signatures are commercially Sokoll LJ, Wang Y, Feng Z, et al: [-2]Proenzyme prostate specific antigen for
available and can be performed on small amounts of prostate cancer detection: a National Cancer Institute early detection research
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additional prognostic information beyond Gleason score Tomlins SA, Rhodes DR, Perner S, et al: Recurrent fusion of TMPRSS2 and ETS
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• Prostate cancer susceptibility genes have been located on a Vickers A, Cronin A, Roobol M, et al: Reducing unnecessary biopsy during
number of chromosomes increase the risk of developing prostate cancer screening using a four-kallikrein panel: an independent
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• Key inherited genes with moderate penetrance that
increase the risk of aggressive prostate cancer are BRCA1,
BRCA2, and ATM. These are all DNA damage repair genes.
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149 - Prostate Cancer Biomarkers

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149 Prostate Cancer Biomarkers

TABLE 149.1 Molecular Derivatives of

PSA TYPE % IN SERUM

ACT, α1-antichymotrypsin; API, α1-protease inhibitor; A2M, α2-macroglobulin;

Prostate lumen BPSA

Prostate luminal epithelial

Free Prostate-Specific Antigen Isoforms

Annexin A3 in DNA methylation and histone acetylation status. Segments within

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