Molecular Diagnosis of Prostate Cancer: Are We Up to Age?

Transcription

Molecular Diagnosis of Prostate Cancer: Are We Up to Age?
Tapan Bhavsar, Peter McCue, and Ruth Birbe
Prostate cancer (PCa), a highly heterogeneous disease, is the one of the leading cause of
morbidity and mortality in the developed countries. Historically used biomarkers such as
prostatic acid phosphatase (PAP), serum prostate-specific antigen (PSA), and its precursor have
not stood the challenge of sensitivity and specificity. At present, there is need to re-evaluate the
approach to diagnose and monitor PCa. To this end, molecular markers that can accurately
identify men with PCa at an early stage, and those who would benefit from early therapeutic
intervention, are the need of the hour. There has been unprecedented progress in the
development of new PCa biomarkers through advancements in proteomics, tissue DNA and
protein/RNA microarray, identification of microRNA, isolation of circulating tumor cells, and
tumor immunohistochemistry. This review will examine the current status of prostate cancer
biomarkers with emphasis on emerging biomarkers by evaluating their diagnostic and
prognostic potentials.
Semin Oncol 40:259-275 & 2013 Elsevier Inc. All rights reserved.
P
rostate cancer (PCa) is the one of the leading
causes of morbidity and mortality in developed countries. In the United States and
Europe, PCa claims around 30,350 and 85,200 every
year.1,2 As many as 232,090 men in the United States
and 237,800 men in Europe are newly diagnosed
with PCa annually.3
PCa is a highly heterogeneous disease that
includes genetic aberrations, local invasion of extracellular matrix, micrometastatic cell extravasations to
circulation, and invasion of secondary organ tissues
followed by resistance to hormonal modulation.
Historically, human prostatic acid phosphatase
(PAP) (or serum acid phosphatase [AP]), the first
serum biomarker for PCa, lacked sufficient sensitivity as a reliable biomarker for response to systemic
therapy or recurrence. Later, serum prostate-specific
antigen (PSA) was used for screening men with an
existing diagnosis of PCa4 and was regarded as an
ideal marker for identifying recurring disease subsequent to treatment. However, PCa has been detected
in about 15% of men with normal (upper limit,
4.0 ng/mL) or very low levels of total PSA, thereby
making it difficult to reliably rule out the possibility
Department of Pathology, Thomas Jefferson University, Philadelphia, PA.
Conflicts of interest: none.
Address correspondence to Ruth Birbe, MD, Department of Pathology,
Thomas Jefferson University, 285 Main Bldg, 132 South 10th
St, Philadelphia, PA 19107. E-mail: [email protected]
0270-9295/ - see front matter
& 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1053/j.seminoncol.2013.04.002
Seminars in Oncology, Vol 40, No 3, June 2013, pp 259-275
of PCa at any PSA level.5 ProPSA, the precursor form
of PSA, served as an additional indicator in differentiating PCa from benign processes.6 Unfortunately
neither of these markers has stood the challenge of
sensitivity and specificity. At the present, there is
need to re-evaluate the approach to diagnose and
monitor PCa. New biomarkers are clearly needed.
To this end, in lieu of analyzing the PCa tumor cells
from biopsy specimens, PCa was studied indirectly
by analyzing blood, urine, and prostatic ejaculates
for variations in proteins with the hope of developing a panel of biomarkers that would provide more
diagnostic information than any one modality.
Although the subject of ongoing debate, molecular markers that can accurately identify men with
PCa at an early stage, and those who would benefit
from early therapeutic intervention, are the need of
the hour. There has been unprecedented progress in
the development of new PCa biomarkers through
advancements in proteomics, tissue DNA and protein/RNA microarray, identification of microRNA
(miRNA), isolation of circulating tumor cells, and
tumor immunohistochemistry (IHC). The contribution of Human Genome Sequencing Project and
National Cancer Institute–sponsored PCa Specialized
Program of Research Excellence (SPORE) cancer
centers in the United States has enriched this field.
These biomarkers, after clinical validation, would be
sine qua non to guide clinicians in defining prognosis and monitoring patients, as well as in the
assessment of treatment options of PCa.
The biomarkers can be broadly classified into
protein markers, including those detected on tissue,
259
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urine, and other body fluids, DNA- and RNA-based
markers, and novel markers identified through proteomic studies.
This review will examine the current status of PCa
biomarkers with emphasis on emerging biomarkers by
evaluating their diagnostic and prognostic potentials.
MOLECULAR MARKERS ON TISSUE
Ki-67/MIB-1
Ki-67, a cell-proliferation associated protein marker
with an expression pattern under cell cycle regulation,7 had been described as a promising biomarkers
of PCa. IHC staining for Ki-67 was shown to discriminate latent PCa revealed by the presence of a
decreased proportion of actively proliferating cells
from clinical cancers with poorer prognosis.8–10 However, several studies show that it does not seem to be
an independent prognostic marker for PCa recurrence
after radical prostatectomy, although it may have some
clinical utility in a subset of patients.11–13
p53
Nuclear accumulation of p53, a marker of growth
control and apoptosis, has been associated with
advanced stages of PCa,14 metastases,15 and an overall
worse prognosis.16–22 Studies conducted on localized
PCa have reported frequencies of p53 alteration
ranging from 4%–60% and as high as 94% in more
advanced castrate-resistant PCa (CRPC) Interestingly,
DNA sequencing analysis has suggested a lower rate
of p53 mutations than indicated by overexpression in
IHC analysis.23 More recently, it has been found that
focal p53 expression by IHC in primary PCa is
predictive of cancer recurrence after radical prostatectomy. However, altered p53 occurs in many malignancies and it is not a specific marker for PCa.
Bcl-2
Overexpression of Bcl-2 protein by IHC has been
commonly detected in advanced CRPC. Bcl-2 protein
expression in primary PCa, along with p53, is a
predictor of cancer recurrence after radical prostatectomy and radiotherapy.24–26 Furthermore, the
combination of p53 and Bcl-2 protein expression
is an independent predictor of recurrence after surgery
in radical prostatectomies. Curiously, this combination
is not predictive of postoperative recurrence when
evaluated in the pretreatment needle biopsy.
Alpha-Methylacyl CoA Racemase
Alpha-methylacyl CoA racemase (AMACR) is an
enzyme involved in fat metabolism and functions as
a growth-promoter independent of androgens in
PCa.27,28 IHC staining using monoclonal antibodies
T. Bhavsar, P. McCue, and R. Birbe
to AMACR is now a common practice to assist in the
diagnosis of PCa; a multi-institutional study has shown
sensitivity and specificity of 97% and 92%, respectively.29 The specificity of AMACR in the detection of
PCa in biopsy specimens is regarded as an improvement over the serum PSA test,30 in as much as one
study has shown 100% specificity and 97% sensitivity.31,32 On the flip side, AMACR has been identified in
prostatic intraepithelial neoplasia (PIN) and occasionally in benign conditions. To err on the side of caution,
a PCa diagnosis should not be based solely on a
positive AMACR stain, especially when the luminal
staining is weak and/or noncircumferential. To circumvent this hurdle, in clinical practice, AMACR is
used in combination with PCa-negative basal cell
stains (p63, 34bE12, and cytokeratins 5 and 6).33,34
AMACR mRNA and protein levels also have been
shown to be overexpressed in PCa, making it a highly
specific tissue biomarker.29,32,35 The detection of
AMACR in prostatic secretions36 and urine37 has
shown promise, although this enzyme is found in
other malignancies such as renal cell carcinoma.
Prostate-Specific Membrane Antigen
Prostate-specific membrane antigen (PSMA) is a
type II transmembrane glycoprotein predominantly
expressed in the prostate epithelium. Significantly
increased expression of PSMA is observed in PCa,
particularly in poorly differentiated, metastatic, and
CRPC.38 Multiple studies have suggested that PSMA
correlates well with Gleason score and/or the stage
of disease. It can be used to monitor post-treatment
disease progression.39–44 PSMA-specific reverse transcriptase–polymerase chain reaction (RT-PCR) has
shown promise as a sensitive biomarker in identifying micrometastases in lymph nodes that are not
detectable by light microscopy.45 PSMA also functions in the radiologic imaging of PCa. The immunoscintigraphic test, ProstaScint (Cytogen, Princeton, NJ)
localizes PCa cells by targeting PSMA with an indium
111–labeled monoclonal antibody,46 and has been
used for imaging soft tissue sites of metastatic PCa
for presurgical staging, especially in high-risk
patients. It may be of utility in the evaluation of
biochemical relapse after local therapy.
Osteopontin Splicing Isoforms
The diagnosis and prediction of prognosis in PCa
has been augmented by a number of alternatively
spliced genes such as osteopontin (OPN), kallikreins, spliced variant of fibroblast growth factor
receptor (FGF-R2), and truncated form of vascular
endothelial growth factor receptor flt-4 (VEGFR-3).47
OPN is a glycophosphoprotein that acts as an
important autocrine and paracrine signal affecting
the growth and behavior of PCa cells.48,49 Its
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Molecular diagnosis of prostate cancer
expression has been correlated with stage, grade,
and early tumor progression in PCa.49,50 Osteopontin splicing isoforms (OPN-SI) have been shown to
be strongly associated with PCa occurrence and
tumor cell differentiation.51–55 The three isoforms,
OPNa, OPNb, and OPNc splice variants, are all
overexpressed in PCa in relation to benign prostatic
hyperplasia (BPH). OPNc has been shown to be the
most upregulated isoform in PCa, outperforming the
remaining isoforms and serum PSA serum levels in
PCa diagnostic accuracy.
BMP-6
Bone morphogenetic proteins (BMP) are involved
in new bone formation and other organ development. The expression of both BMP-6 mRNA and
protein is upregulated in PCa, in as much as BMP-6
mRNA expression has been demonstrated in 95%–
100% of primary PCa with solid organ metastases,
18%–36% with organ-confined disease, and 85% of
bone metastases.56,57 Taking a step forward, CpG
demethylation of the BMP-6 promoter has been
shown to be responsible for the high expression in
primary and secondary sites of advanced PCa.58
Interestingly, co-expression of the proteins BMP-6,
bone sialoprotein, and thymidine phosphorylase was
observed in 90% of the radical prostatectomy specimens with bone metastases and only in 29% of men
with curative treatment,59 making these proteins
useful markers to identify at-risk patients for disease
progression after treatment.
PTEN
PTEN is a tumor-suppressor gene that encodes for a
protein that dephosphorylates phosphatidylinositol-3phosphate. The deletion of the PTEN gene has a
frequency of about 40% in the western population,
and a mere 7.6% in those of Chinese origin.60
Deletions of PTEN have been associated with Gleason
scores 47, biochemical relapse, and nodal metastasis.
Nuclear Factor-κB
Nuclear factor-κB (NF-κB) activation has been
shown to occur in the transition from a preneoplastic state to PCa. The overexpression of NF-κB
has been shown to have a positive correlation
with advanced PCa tumor stage and independently
predicts a high risk of biochemical relapse of
PCa.61 This is especially relevant in patients
with positive surgical margins and lymph node
metastasis.
Those identified include somatic cytochrome
c (CYCS), intestinal cell kinase (ICK), inhibitor of
NF-κB kinase subunit beta (IKBKB), and glutamate
decarboxylase 1 (GAD1).62,63 In a similar vein, CD10
has proved to be an important biomarker whose loss
or decreased expression is thought to be an early
(and frequent) event in human PCa.64 Syndecan-1
overexpression, as analyzed by tissue microarray,
predicts early recurrence and is significantly associated with tumor-specific survival, high Gleason
score, Ki-67, and Bcl-2 overexpression.65 A significant decrease in protein expression of annexins
1 and 7 in hormone-refractory PCa has been correlated with tumor progression.66
MOLECULAR MARKERS IN SERUM
The clinical utility of serum markers for PCa
diagnosis and disease progression is obvious. Much
effort has been applied to identify efficacious
candidates.
Crisp-3/SGP28
Cysteine-rich secretory protein 3 (CRISP-3) is a
specific granule protein of 28 kd (SGP28), purified
from human neutrophils. The expression of CRISP-3/
SGP28 mRNA is increased 20- to 20,000-fold in
Gleason grade 3 PCa. A quantitative enzyme-linked
immunosorbent assay (ELISA)-based assay is under
development to study the diagnostic potential of
CRISP-3/SGP28 in plasma and body fluids.67
hK2
Using a quantitative RT-PCR, hK2 protein expression is found to be increased in high-grade PIN, PCa,
and lymph node metastases. A highly sensitive hK2specific immunoassay shows promise in diagnostic
applicability over free PSA ratio in the “gray zone” to
distinguish PCa from BPH. Moreover, serum hK2
alone or in combination with total PSA and free PSA
may improve the detection of extraprostatic or
advanced disease.68–71
Osteoprotegerin
Serum levels of osteoprotegerin (OPG) have been
found to be significantly higher in patients with
advanced PCa compared to patients at other stages
of prostatic disease. An increased serum OPG concentration may be embraced as a marker of early
relapse from androgen ablation therapy and as a
monitor in patients with bone metastases.72,73
Novel Tissue Markers
Chromogranin A
The use of prostatic tissue microarrays has
enriched the field with a varitey of new PCa markers.
Chromogranin A (CGA or GRN-A), a member of
the granin family of proteins, is an acidic protein
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identified in all neuroendocrine cell types. CGA has
clinical potential as a biomarker for early, progressive, and recurrent PCa.74 Several studies have
suggested a role for CGA in the initial detection of
PCa, particularly when used in combination with
free-total PSA ratio.75 Conversely, not all PCa exhibit
neuroendocrine differentiation, limiting the overall
efficacy of chromogranin A in diagnosing and monitoring PCa patients.
Vascular Endothelial Growth Factor
Human Glandular Kallikrein 2
Other Serum Markers
Human glandular kallikrein 2 is closely related to
PSA and is expressed at high levels in prostate
tissue.76,77 It has been used in various combinations
with free and total PSA to statistically improve the
sensitivity and specificity of identifying PCa.78,79
The methodology of DNA microarray emerges as an
important innovation in the search of serum markers
with the aim of selecting differentially expressed
genes. Using the approach of antibody microarray, five
proteins have been identified, including von Willebrand factor, immunoglobulin M, alpha-1 chymotrypsin, immunoglobulin G, and vilin, with significantly
higher serum levels in PCa patients.95 In addition,
serum levels of hK2/fPSA, Hepsin, AMACR, neuronspecific enolase, urokinase-type plasminogen activator
system, β-catenin, and hK11 have been shown to be
promising markers in the diagnosis of PCa.
Transforming Growth Factor-β and
Interleukin-6
Elevated serum levels of transforming growth
factor-β (TGF-β1) and interleukin-6 (IL-6), via an
increased p21 expression through Smad and STAT3
signaling, respectively, have been associated with
poor prognosis. Additionally, elevated serum TGF-β1
levels have been associated with an increased risk of
metastatic disease.80–82 Increased serum levels of
IL-6, along with IL-8 and IL-11, have been demonstrated in patients with metastatic and CRPC.83-86
The measurement of preoperative levels of both
TGF-β1 and IL-6 makes an effective contributor in
improving the accuracy of predicting regional lymph
node metastases, disease progression, and biochemical recurrence.82,87
Caveolin-1
Caveolin-1 (Cav-1) is an integral membrane protein
and a main component of caveolae membranes
in vivo. Abundant stromal Cav-1 immunostaining is
present in almost all patients with BPH. A small subset
of samples of primary PCa has been shown to have
significantly decreased levels of stromal Cav-1.88
E-cadherin
E-cadherin, a marker of cell adherence, has been
studied extensively in prostatectomy specimens. In
general, an inverse correlation with tumor grade and
stage is found. Loss of E-cadherin expression is a
promising marker of poor prognosis in localized
PCa.89
EGFR Family, c-erbB-1/c-erbB-2
Both the epithelial growth factor receptor (EGFR)
and the oncogene c-erbB2/HER-2 have been found to
be overexpressed in PCa. There is an increasing level
of expression from localized to metastatic disease.90
Serum levels of the ligand VEGF were found to be
significantly higher in those PCa patients with
metastatic disease.91 Increased plasma VEGF level
is an independent prognostic factor in PCa.92,93
Moreover, it also has been demonstrated that the
related microvessel density (MVD) count was significantly higher in PCa patients with metastatic disease
when compared with those without it.94
MOLECULAR MARKERS IN URINE
Urine biomarkers are promising for PCa detection
as they are readily measurable and can be obtained
noninvasively.
TMPRSS2-ERG Gene Fusion Rearrangement
Transmembrane protease serine 2 (TMPRSS2) is
an androgen-regulated, type II transmembranebound serine protease overexpressed in PCa. It plays
a role in tumor metastasis through activation of
protease-activated receptor-2. Using Cancer Outlier
Profile Analysis (COPA), an open source program
that is designed to evaluate variance in a data using
the median instead of the mean to maintain the
peaks of outliers, it was found that the overexpression of two genes ERG and ETV1 was highly
correlated with TMPRSS2.
A fusion of TMPRSS2 (21q22.3) with the transcription factor genes ERG (21q22.2) and ETV1
(7p21.1) has been shown96,97 to be one of the
earliest events in PCa. TMPRSS2-ERG is the most
frequent oncogenic gene fusion rearrangement in
PCa.87 It is detected in almost half of PCa patients
and in about a quarter of patients with PIN.98 Several
studies suggest that TMPRSS2:ERG fusion contributes to a more aggressive cancer phenotype that is
associated with a higher tumor stage and PCaspecific death. Parenthetically, while the frequency
of TMPRSS2-ERG rearrangement constitutes approximately 50% of the cases in Westerners, it is a mere
2.5% in the Chinese population.
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The TMPRSS2-ERG fusion can be detected in the
urine after digital rectal examination (DRE) with 93%
specificity99 and 94% positive predictive value.100
The addition of TMPRSS2–ERG detection can
increase the sensitivity of the urine PCA3 test (see
below) from 62% to 73%99 and will increase the
prediction of tumors with a higher Gleason score.101
The combination of TMPRSS2-ERG detection with
PCA3 may become the preferred screening methodology for PCa.102,103 Recently, a multiplex urine
based assay combining PCA3, TMPRSS2:ERG,
annexin A3, sarcosine, and PSA was shown to maximize PCa detection compared with that of any
single biomarker.104 The presence of PCA3 and
TMPRSS2-ERG fusion has been detected in urine
exosomes from PCa patients.105,106
PCA3/DD3
The prostate cancer antigen 3 (PCA3) gene
encodes a prostate-specific mRNA that is overexpressed in PCa tissue.107,108 DD3 (differential display
code 3)/PCA3 is the most PCa-specific gene (along
with aforementioned TMPRSS2:ERG gene fusion)
that has been described to date,109 showing overexpression in 495% of primary PCa specimens and
in metastatic deposits. In biopsy specimens, PCA3/
DD3 expression has shown 72% sensitivity, 83%
specificity, and 88% negative predictive value for
the detection of PCa.110–112
A DD3/PCA3-based RT-PCR assay has been
developed to quantify PCA3 mRNA levels in urine
collected following DRE. This assay is advantageous
as the specimen processing is relatively simple and
uses clean catch urine. The fraction of specimens
yielding sufficient RNA for analysis is 495% and the
assay is readily adaptable to a clinical laboratory
setting using available instrumentation.113 The
assay also could be applicable in the detection of
malignant prostate cells in blood or ejaculates.
Following normalization to the total amount of
prostate RNA, the resultant ratio, known as PCA3
score has shown 67%–69% sensitivity and 79%–83%
specificity.110,113 This PCA3 score is independent
of prostate volume, the number of prior biopsies,
patient’s age, pharmacotherapy (including type I
and II 5 alpha-reductase inhibitors), and inflammatory conditions. The PCA3 score is useful in
detecting PCa in men with commonly observed
alternative causes of increased PSA such as prostatitis and in patients with normal or low PSA. Along
the same lines, in the detection of high-grade PIN,
PCA3 score has been demonstrated to be 16%
higher than in the tissue of men without this
lesion.114 Moreover, the PCA3 scores are higher
in patients with larger, more invasive tumors,
thereby assisting in determining if patients would
require aggressive treatment or active surveillance.
In a semi-quantitative method, transcripts of five
different biomarkers, including PCA3, AR, SRD5A2,
KLK2, and PSMA, were analyzed by multiplex
RT-PCR assay in tissue samples from patients with
PCa and BPH. It was shown that PCA3 detection,
along with associated serum PSA levels, had a 28-fold
higher chance for PCa detection with 92% specificity
and 94% positive predictive value.
uPM3 Test
The uPM3 test is a nucleic acid amplification assay
detecting the relative expression of PSA mRNA and
PCA3/DD3 RNA in the urine using specific beacons
as probes in a thermostatted spectrofluorometer.
This test has demonstrated a sensitivity range of
46.9%–82.3%, specificity range of 56.3%–89%, and
positive predictive range of 59.4%–97.4%. It has
a negative predictive value range of 87.8%–98%115
in PCa detection, especially in the low PSA ranges.
Duplex Assay for Urinary AMACR and PCA3
As single marker assays, the urinary AMACR and
PCA3 tests have shown similar sensitivities, while
the AMACR assay had a higher specificity. A duplex
assay based on the use of the AMACR and PCA3
scores greatly improves the accuracy and performance of each methodology. Duplex testing may serve
as an adjunct assay to the serum PSA test for
monitoring elderly patients with repeat negative
biopsies.
Survivin
Survivin is a member of the inhibitor of apoptosis
(IAP) family that functions to prevent apoptosis.
In locally advanced PCa, cytoplasmic overexpression
of survivin predicts local progression. Interestingly, a
nuclear expression is associated with improved
survival.116 The evaluation of the expression of
survivin mRNA in voided urine from patients with
bladder, prostate, and renal cancers has shown 100%
specificity but no sensitivity.117
Telomerase
The use of telomerase activity for the detection of
PCa cells obtained after prostatic massage has shown
58% sensitivity and 100% specificity.118 A high
telomerase activity has been found in 90% of PCa
tissue and in 100% of epithelial cells recovered from
urine by immuno-captivation.119
Thymosin b15
Thymosin beta 15 (Tb15), an actin-binding protein, was found to be upregulated at both the mRNA
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and protein level in high-grade PCa. Using a quantitative high-throughput ELISA, elevated concentrations in urine samples were found in patients
preceding PCa recurrence.120,121
Hepsin
Hepsin is a trypsin-like transmembrane serine
protease. It has been shown to be overexpressed
in up to 90% of PCa,122–127 particularly in advanced
stage tumors and in tumors with Gleason scores 47.
Although there are currently no known serum or
urine tests for hepsin detection, the consistent overexpression of hepsin suggests the possibility of a
potentially useful biomarker.
Bradeion
The detection of bradeion using monoclonal
antibody-based immunochromatographic membrane
strip tests on urine samples has been shown to
detect 70% of prostate, renal, and bladder cancers
with no false-positive results.128
Mini-Chromosome Maintenance-5
Urinary mini-chromosome maintenance-5 (MCM5) detection is a simple, accurate, and noninvasive
method for identifying patients with PCa with
sensitivity of 82% and specificity of 73%–93%.129
An increased urinary concentration of 8-OHdG, a
biomarker of generalized cellular oxidative stress,
was detected by ELISA in the urine of patients with
prostate and bladder cancer.130 However, some
studies cast doubt on its potential as a biomarker.131
Urinary 5a-reductase type 2 has shown same utility
as urinary markers in the diagnosis of PCa, but it is
not well studied.132
MOLECULAR MARKERS IN BLOOD
Early Prostate Cancer Antigen
Early prostate cancer antigen (EPCA) is a PCaassociated nuclear structural protein. An increased
expression has been shown in PCa and adjacent areas
but is absent in normal prostate tissue obtained from
healthy donors and BPH samples.133 EPCA has shown
promising results as a clinically useful PCa biomarker
as shown by studies demonstrating 84%–94% sensitivity and 85%–100% specificity for PCa by IHC and
blood ELISA and in precursor PIN and inflammatory
prostatic lesions.134–136 Moreover, what makes it an
intriguing biomarker is that positive EPCA staining in
an initial PCa negative biopsy may precede the
diagnosis of PCa by as much as 5 years.
Prostate Stem Cell Antigen
Prostate stem cell antigen (PSCA) is a prostatespecific glycosyl
phosphatidylinositol-anchored
glycoprotein. PSCA is shown to have strong expression in approximately 85% of PCa, and this expression has been correlated with Gleason score, stage,
progression, and the presence of bone, liver, and
lymph node metastases.137–141 This glycoprotein is
expressed in approximately 73% of high-grade PIN
and 22% of low-grade PIN.139 The prognostic value
of PSCA has been shown in patients with extraprostatic disease; the presence of PSCA mRNA via
peripheral blood RT-PCR is associated with a lower
progression-free survival.141
EPIGENETIC BIOMARKERS IN PROSTATE
CANCER
Glutathione-S-Transferase P1
Glutathione-S-transferases (GSTs) are a ubiquitous
family of multifunctional enzymes that conjugate
reactive substrates with reduced glutathione (GSH)
and are involved in detoxification. Epigenetic alterations are frequent in PCa and are thought to contribute to disease initiation and progression.142,143
Promoter hypermethylation of the GSTP1 gene is a
highly specific but insensitive marker of PCa. The
methylation of the 5V region of the GSTP1 gene is
the most common epigenetic alteration in PCa.144–146
Hypermethylation of the GSTP1 promoter occurs in
more than 90% of PCa and has been significantly
associated with Gleason score 47 and the presence
of a single Gleason 4 and/or 5 grade.144,147 Promoter
hypermethylation is associated with high-grade PIN
and can be used to distinguish PCa and PIN from
BPH.148–151 GSTP1 promoter hypermethylation has
been found in 72% of plasma or serum samples, 50%
of ejaculates, and 36% of urine samples. GSTP1
methylation in cells derived from serum and urine,
if successfully validated, may possess a significant
clinical potential for early detection of PCa and posttreatment monitoring.
CpG island hypermethylation of GSTP1 promoter
also may serve as a useful molecular biomarker for
the detection and diagnosis of PCa.149,152 Peripheral
blood, urine, ejaculate, and prostatic secretions can
be tested for CpG island hypermethylation and may
become a useful adjunct to serum screening tests
and digital rectal examination.
The combinations of GSTP1 along with APC,
RASSF1a, PTGS2, and MDR1 CpG island hypermethylation can distinguish primary PCa from
benign prostate tissue with a sensitivity of 97.3%–
100% and a specificity of 92%–100%.153 The methylation profile of a panel of 10 hypermethylated
genes including GSTP1, RASSF1A, CDH1, APC,
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DAPK, MGMT, p14, p16INK4a, RARb2, and TIMP3
was assessed, in which all genes were significantly hypermethylated in the urine sediments of
PCa except p14 and p16INK4a.154 Similarly, the
methylation profile of a four-gene panel, GSTP1,
RASSF1A, RARb2, and APC, was able to discriminate cancers from controls at 86% sensitivity and
89% specificity.
Two clinical trials are investigating a panel of
hypermethylated genes in urine and serum as an
early detection marker (NCT00340717 and
NCT01441687) and there is a single trial investigating the association of a miRNA expression profile as
a prognostic biomarker (NCT01220427).
Various other hypermethylated genes have been
proposed as prognostic markers for PCa. The gene
encoding for COX2, PTGS2, has been found to be
methylated in 88% of cases and has been correlated with an increased risk of PSA recurrence
independently of Gleason score or pathological
stage.154 A correlation between hypermethylation
at APC, RASSF1a, and RUNX3 with PSA value and
Gleason score has been shown to have a poor
prognosis.155
Interestingly, studies have demonstrated global
hypomethylation associated with advanced metastatic PCa.156–159 An IHC study on human PCa
showed a significant decrease in the global levels
of 5-methylcytosine in patients with recurrent
PCa.157 Retrotransposon elements such as LINE-1
and Alu repeats also have been found to be hypomethylated in PCa.158,159 For various reasons, not the
least of which is complexity, the diagnostic or
prognostic value of global DNA methylation levels
needs further evaluation.
HISTONE MODIFICATIONS AS BIOMARKERS
IN PROSTATE CANCER
The global levels of specific histone modifications,
H3K9Ac, H3K18Ac, H4K12Ac, H3K4Me2, and
H4R3Me2, using IHC have been analyzed in primary
PCa. It has been evident that an overexpression of
H3K27Me3 global levels in metastatic PCa is associated with biochemical recurrence.161–164 The combination of specific histone modifications, H3K4Me2
and H3K18Ac, has been shown to be indicative of
PCa recurrence in low-grade PCa.165 A significant
decrease in H3K27Me3 has been found in metastatic
disease compared to localized disease using ELISA on
serum samples.166 A CHIP-sequencing strategy may
be used to profile concurrent histone modifications
to investigate potential epigenetic biomarkers
for PCa.
miRNAs AS BIOMARKERS IN PROSTATE
CANCER
miRNAs are attractive as potential diagnostic and
prognostic biomarkers for PCa due to easy detection
in body fluids like blood and serum. These moieties
are highly stable and tend to be tissue- and tumorspecific.167,168 A total of 15 differentially expressed
miRNAs have been shown to aid in the discrimination between PCa and benign tissue with 82%
accuracy using miRNA microarray analysis and RTPCR validation.169 In particular, miR-375, miR-141,
and miR-200b are significantly associated with pathological stage and Gleason score using Taqman
miRNA microarray analysis.170
GENETIC POLYMORPHISMS AND
MUTATIONS
MOLECULAR GENE-BASED TESTS
DNA-based tests can be used for the detection of
microsatellite alterations in PCa. However, these
tests are prone to artifact. They require at least
20% of the analyzed genomic DNA from isolated
PCa tumor cells. Methylation-specific PCR (MSP) has
been shown to be useful for the detection of
epigenetic modifications (eg, promoter hypermethylation) as discussed above. The latter has an advantage as it requires a tumor-to-normal ratio of only
0.1%–0.001%.31 RNA-based gene tests to quantify the
mRNA of interest is advantageous over DNA due to
commercially available US Food and Drug Administration (FDA)-approved technologies such as RTPCR, nucleic acid sequence-based amplification
(NASBA), and transcription-mediated amplification
(TMA). Of course, the robustness of the sample
collection, storage and transport are crucial to maintain the sensitivity of RNA-based tests.160
The genetic polymorphisms examined to date for
PCa have exhibited a relatively low penetrance and
have not been validated in subsequent studies. A
number of loci have been shown to have a positive
correlation with PCa risk, including HPC1, HPC2,
HPCX, HPC20, CAPB, PCAP, and an unnamed locus
at 8p22–23.171 A large-scale association study
of 425,000 SNPs within 16,000 genes using 368
matched cases and controls has provided evidence that
genetic variants of ICAM5 are associated with a positive
risk of PCa. Likewise, the BRCA1 mutation also has
been shown to correlate with a positive risk of PCa.
GENE EXPRESSION MARKERS IN PROSTATE
CANCER
Some of the important susceptibility genes for
developing PCa include androgen receptor (AR) CAG
repeats, MSR1 (macrophage scavenger receptor 1),
266
ELAC2 mutation carriers, BRCA2, ETV6, and
AMACR/P504S. Expression levels of the KLK2
and DD3 genes in prostate biopsies may be used
for diagnostic discrimination of PCa and benign
hyperplasia, especially in patients with low serum
levels of PSA. B7-H3, a member of the B7 family, is
the first immune molecule that possibly participates
in the development of PCa and in predicting
recurrence and progression. Hypermethylation of
the PDLIM4 gene has been shown to be a sensitive
molecular tool in detecting prostate tumorigenesis.172 Increased expression of Golgi phosphoprotein
2 (GOLPH2), a gene that codes for type II Golgi
membrane antigen GOLPH2/GP73, has been shown
in PCa tissue in a comparative study of GOLPH2
protein, basal cell marker p63, and AMACR in benign
and malignant prostate lesions. The abnormal methylation in the promoter region of the DAB2IP gene
has been reported to be responsible for transcriptional silencing and a significant role in PCa progression.173 A link between a genetic variation in
DAB2IP and the risk of aggressive PCa also has been
shown.174 PAR-2, a gene that encodes for a G
protein-coupled receptor activated by specific serine
proteases, is associated with PCa metastasis175 and is
overexpressed in approximately 40% of PCa.176 A
markedly increased expression level of EZH2, a
member of the polycomb group of proteins, has
been demonstrated by gene expression profiling in
patients who succumbed to metastatic disease.
Interestingly, EZH2 has been found to be a better
predictor for clinical progression than surgical margin status, maximum tumor dimension, Gleason
score, or preoperative PSA.162 The expression of
SPINK-1 a protein that inhibits pancreatic secretion
of trypsin, has been detected in urine samples and
correlates with the biochemical progression-free
interval in PCa. Other genes, such as HEPSIN
(TMPRSS1) and Pim-1, have been associated with
PCa. Overexpression of CD44 variant 9 is associated
with invasion177 and decreased fractions of the v3
variant predict biochemical recurrence.178
BIOMARKERS OF CELL CYCLE PROGRESSION
AND SIGNALING PATHWAYS
The use of advanced diagnostic techniques to
evaluate tumors according to the molecular alterations they carry may lead to the discovery of new
markers and targeted treatments of individual tumors.
Cyclin D1 overexpression has been associated
with poor prognosis after prostatectomy.179,180 Specific inactivation of p14 (ARF) in PCa occurs either
through mutations or promoter hypermethylation153,181 and is a rare event. Amplification of the
c-myc gene on 8q24 is found in more than 11%–21% of
advanced PCa182,183 and is associated with hormone
independence184 and poor prognosis.185 Overexpression of c-myc seems to be involved in tumor progression and hormone resistance rather than tumor
development. High levels of p16 in prostatectomy
specimens predict a rapid treatment failure,186
whereas decreased expression after radiotherapy
predicts poor prognosis.187 Low p16 expression is
often found in metastatic PCa lesions.188 The inhibitory role of retinoblastoma (Rb) in the E2F transcription factor complex has been suggested to play a
role in PCa. Inactivation of Rb in mouse models has
shown a phenotype of PIN.188,189 Loss of hMSH2
expression, a member of the MMR protein family, has
been associated with lower recurrence rates after
prostatectomy.190,191 Decreased IHC expression of
p27 is associated with a high Gleason grade, seminal
vesicle invasion, and positive surgical margin. In highgrade PIN, there is an inverse relation between PTEN
and p27 expression.192 For markers of alternative
growth signaling pathways, growth factors associated
with PCa such as IGF-1,193,194 scatter factor/hepatocyte growth factor (HGF),195 and platelet-derived
growth factor (PDGF)196 have been found to activate
the PI3kinase/AKT/mTOR signaling cascade. NF-κB
and subunits are involved in transcriptional regulation of gene expression of multiple growth factors
including Bcl-2, cyclin D1, MMP-9, VEGF,197 and
PSA.198 These are all found to be elevated in PCa.199
NEW TARGETS AND BIOMARKERS FOR
PROSTATE CANCER THERAPY
The F77 antigen, serine peptidase inhibitor Kazal
type 1 (SPINK1) and survivin (see urine molecular
markers above) are some of the new biomarkers for
PCa. The F77 antigen, termed prostate cancer lipid
antigen (PCLA), holds significant value as a biomarker
due to its glycolipid properties. It is expressed on
both androgen-dependent and androgen-independent
PCa, making it a target for early diagnosis and treatment of advanced diseases. In tissue microarray
studies, F77 was shown to stain 112/166 primary
and 29/34 metastatic PCa specimens.200 Another
biomarker, SPINK1, is specific to a subset of aggressive PCa that does not carry genetic rearrangement of
the ETS transcription factors into the TMPRSS2
promoter region, and promotes prostate tumor
growth through EGFR.201
NOVEL PROTEOMIC BIOMARKERS FOR
PROSTATE CANCER
Proteomic and Metabolic Profiling in PCa
Proteomic profiling analyzes the subtle changes in
proteins in tissue, serum, or urine, and can be used
267
to differentiate PCa from benign conditions. Using
mass spectrometry protein profiling with bioinformatics, microRNA (miR-128) has been implicated for
PCa progression.202 The use of metabolite profiling
has identified the expression of sarcosine during
progression from benign prostate tissue to metastatic PCa.203 Using proteomics expression profiles
of androgen-stimulated PCa cells generated by twodimensional electrophoresis (2-DE) and spectrometric analysis, a metastasis-suppressor gene NDKA/
nm23 was identified that may explain a marked
reduction in metastatic potential when these cells
retain a functional androgen receptor pathway.204
Another proteomic study, which mapped the differences in protein expression profiles between normal
and malignant prostate tissues, identified 20 proteins
that were lost in malignant transformation, including
the novel finding of NEDD8, calponin, and the
follistatin-related proteins.205
Proteomic Profiling With Mass Spectrometry
Mass spectrometry (MS) has been used to identify
new individual biomarkers, such as PCa-24 in the
serum206 and calgranulin B/MRP-14 in voided urine
after prostatic massage.207 High-resolution 2-DE have
shown absence of proteins A and B in urine from
PCa and BPH patients, while protein F was highly
expressed in BPH but not in PCa.208 The use of
proteomic patterns from MS has shown 95% sensitivity and 78% specificity in identifying PCa.209
Enhanced results have been demonstrated using
a boosted decision tree algorithm to analyze the
surface-enhanced laser desorption/ionization (SELDI)/
time of flight (TOF) data, showing a sensitivity and
specificity of 97%.210 Moreover, SELDI has been
coupled with an artificial intelligence learning algorithm to identify biomarkers for early detection of PCa
with a high sensitivity (83%), specificity (97%), and a
positive predictive value (96%). The results of these
study show the potential of SELDI proteomics in the
early detection and diagnosis of PCa.
Proteomic Techniques for PCa Biomarkers
Proteomic approaches have been used in tissues
and cancer cell lines to identify PCa biomarkers.
Potential biomarkers for high grade PCa identification include nucleoside diphosphate kinase 1, chromobox protein, 39S ribosomal protein L12, cytosol
aminopeptidase, endopeptidase C1p, inorganic
pyrophosphatase, metaxin 2, GST-pi, acyl-CpA
dehydrogenase,
lysophospholipase,
NADHubiquinone oxidoreductase, 60- and 70-kd GRP-78,
b-actin, cytokeratins 7, 8, and 18, and stomatin-like
protein 2, a-actin, and mutant desmin using 2DGE
coupled with MS.211 Another four novel potential
biomarkers—follistatin, chemokine (C-X-C motif)
ligand 16, pentraxin 3, and spondin 2—have been
identified by examining conditioned media from
PCa cell lines using strong cation exchange highperformance liquid chromatography (HPLC) and
HPLC-tandem MS.212 Unfortunately, there have
been only a few PCa biomarkers on biological fluids
using proteomic studies as the concentration of
potential biomarkers varies in different human body
fluids. Technically, it is difficult to identify lowabundance proteins.213,214
Proteomic PCa Biomarkers in Sera
MS-based mass profiling (MP) combined with
multivariate analysis has detected a protein at m/z
7771, identified as platelet factor 4 in PCa
patients.215 It was significantly decreased in the sera
of all the patients with metastatic PCa but not in
those with localized PCa or healthy individuals.
Similarly, another potential PCa biomarker protein
at m/z at 8946, Apolipoprotein A-II, was identified
using HPLC, reverse-phase chromatography, sosium
dodecyl sulfate–polyacrylamide gel electropheresis
(SDS-PAGE), HPLC–MS/MS, and SELDI/TOF-MS.216
Other potential PCa serum protein biomarkers that
have been identified are collective variant forms of
serum amyloid A (SAAs) using SELDI/TOF217 and
squamous cell carcinoma antigen 1 (SCCA1), calgranulin B, and haptoglobin-related protein using anion
displacement liquid chromate-focusing chromatography for serum fractionation, two-dimensional differential in-gel electrophoresis (2D-DIGE).218
FUTURE DIRECTIONS AND CONCLUSIONS
Since PCa is a heterogeneous disease, it is clear
that a defined set of markers will be important in the
early diagnosis, monitoring, and prognoses of PCa. A
wide array of molecular markers discussed here may
be used in the near future as adjuncts to currently
established prognostic parameters. That said, the
practicality of the use of many molecular biomarkers
is questionable, and it will take time for these
markers to find their way into the clinical arena.
The field of fusion genes is promising at the
clinical and translational levels. The high incidence
and the connection with the androgenic signaling
pathway of TMPRSS2-ERG make it a biomarker with
much potential at the diagnostic and prognostic
levels. Microarray studies using exon-specific target
elements can identify specific splice variants that
may be more correlative with PCa phenotypes.
PCA3 has shown improved positive predictive value
and sensitivity for detection of PCa in the low-PSA
ranges after previous negative biopsies. Further
studies may lead to the application of GSTP1 hypermethylation in augmenting standard histopathology
268
for the diagnosis of PCa. Fluorescence-based quantitative RT-PCR assay for the determination of DD3/
PCA3 transcripts shows great promise as a noninvasive diagnostic tool and will have great impact
for the reduction of the number of unnecessary
biopsies. A limited number of molecular markers in
PCa tissue are of clinical use in predicting outcome
or response to therapy. They include p53, Bcl-2,
p16INK4A, p27Kip1, c-Myc, AR, E-cadherin, and
VEGF. With the advancement of new technologies
such as next-generation sequencing and with the
development of platforms for global epigenome
analyses, epigenetic biomarkers may be used as
powerful tools for determining diagnosis, prognosis,
and therapy response in PCa. Modern advances in
the field of proteomic techniques such as 2D-DIGE
and SELDI/TOF-MS hold the promise of identifying
novel PCa biomarkers for early diagnosis and
prognosis.
We close this truncated summary on a practical
note that while extensive clinical validation of these
novel PCa biomarkers remains one of the most
significant challenges, overcoming this impediment
will by no means eliminate the difficulty in their
identification and development.
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Molecular Diagnosis of Prostate Cancer: Are We Up to Age?

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