PROSTATE SPECIFIC ANTIGEN (PSA)

INTRODUCTION

Prostate cancer (CaP) is the common malignancy in men globally and ranked second
after lung and bronchus cancer with respect to death (Angelis et al., 2007; Katz and
Katz, 2008). Serum prostate specific antigen (PSA) has been widely regarded as the
most clinically useful marker for the detection of CaP and BPH (Wu, 1994; Brawley
et al., 2009). Tumor markers, measurable either n serum or from tissue specimens are
generally useful in the detection, screening, staging, prognosis and monitoring
therapy (Loeb and Catalona, 2008). PSA was first described in 1971 and purified in
1979 in seminal plasma and the prostate. PSA is a single chain glycoprotein with 240
amino acids having a molecular weight of approximately 34 KDa (Noldus et al.,
1997; Singh, 2009). PSA is synthesized by the epithelial cells of prostatic acini and
ducts and is secreted as a normal constituent of seminal fluid .

PSA, a useful tumor marker is a single chain glycoprotein consisting of 93% amino
acids and 7 % carbohydrates. In healthy males, the prostate epithelium synthesizes
and secretes PSA and efficiently prevents the escape of the protease into the
circulation. However, minor amount of PSA does enter into the blood circulation.
Hence it is worthwhile to determine serum PSA concentrations in patients of benign
and malignant conditions of prostate, which could help in differential diagnosis.

What are the benefits and limitations of the PSA test?

Benefits Limitations
May indicate the presence of cancer in its May lead to unnecessary tests and
earliest stages treatment
Simple blood test (not harmful) Cannot distinguish beween slow growing
and advanced cancer.
Currently only test we have as red flag to The PSA test cannot diagnose prostate
indicate follow - up cancer but can tell you if theres
 aproblem with the prostate.

As a review from a Journal of PSA and beyond: Biomarkers in prostate

cancer by James G. et al., (2014) suggested that prostate-specific antigen (PSA) is
one of the best-known biomarkers in medicine as PSA continues to play a role in the
diagnosis and management of prostate cancer. PSA is a protein marker type using
blood serum as a sample and analyse using immunoassay method to detect in a
patients with normal prostate and benign prostatic hyperplasia (BPH), and in both
primary and metastatic prostate cancer cells. Total PSA is an organ-specific but not
disease-specific determination.

Total PSA production is determined by the number of PSA-producing cells,

the level of PSA gene expression, the rate of PSA protein secretion per cell, and the
degree of PSA leakage from glandular acini into the serum. Serum PSA levels may be
elevated from inspissated secretions, distorted architecture, or disruption of basement
membrane integrity occurring with prostatic infarction, prostatitis, ejaculation, digital
rectal manipulation (DRE), or prostatic instrumentation.

The role of PSA can be divided into three major role which is screening,
predicting outcomes of the long-term risk of a particular prostate cancer, especially
high-grade of a lethal disease and also monitoring of patients on hormone therapy for
advanced/metastatic prostate cancer or those with PSA recurrence following
definitive therapy. Most clinicians who deal with prostate cancer on a regular basis
agree that PSA testing which is screening needs to be used in a smart manner, and
that the main concerns involve is an overtreatment rather than over-diagnosis. A total
lack of screening may result in men with highly aggressive tumours missing their
window of opportunity for a cure from this prostate cancer disease. According to
Antonarakis ES, et al.,(2012), PSA levels can be used before radical prostatectomy to
predict outcomes, including tumour volume, grade of disease, and biochemical
progression. The ability of PSA to indicate biomedical progression also makes it
useful as a surveillance marker for recurrent disease before a tumour is detectable by
DRE or bone scan. Postoperatively, PSA doubling time has been shown to stratify the
risk of clinical progression to metastases and death in those with PSA recurrence.

In a study of 2950 men done by Thompson IM, et al.,(2004) shows that the
relationship of PSA to prevalence of prostate cancer and high-grade prostate cancer
using Gleason score (7) in those with a PSA level lower than 4.0 ng/mL, a level
once considered normal can be proven that low PSA levels do not necessarily
exclude a diagnosis of cancer and the patients PSA level is now seen to reflect a
continuum of a prostate cancer risk. This can be seen in the table 1 below:

PSA level Number of men Men with prostate Men with Gleason score
(ng/ml) cancer, n (%) 7 prostate cancer, n (%)
0.0-0.5 486 32 (6.6%) 4 (0.8%)
0.6-1.0 791 80 (10.1%) 8 (1.0%)
1.1-2.0 998 170 (17.0%) 20 (2.0%)
2.1-3.0 482 115 (23.9%) 22 (4.6%)
3.1-4.0 193 52 (26.9%) 13 (6.7%)
Table 1: the rates of prostate cancer in those with a PSA level lower than 4.0 ng/mL

PSA level is also influenced by age, race, and prostate volume. An age-
specific range is now commonly used as been shown in the table 2. PSA increases
with age and is higher in people of certain races, such as African Americans.

Age (years) Total PSA, normal reference range (ng/ml)

Younger than 50 0.0 2.5
50-59 0.0 3.5
60-69 0.0 4.5
 70 and older 0.0 6.5
Table 2: PSA level by age

To improve PSA specificity, the determination of PSA density, which is equal

to serum PSA divided by prostate gland volume, should be done. This is because PSA
density could help differentiate between BPH and early nonpalpable cancer,
especially at serum levels of 4.0 to 10.0 ng/mL. Other than that, serial PSA
measurements, termed PSA velocity (ng/mL/year) and PSA doubling time (the
relative time for the PSA level to double), can reflect biological changes within the
prostate over time. A PSA velocity greater than 0.75 ng/mL/year has been shown to
increase both the positive predictive value of PSA testing and the likelihood of
diagnosing cancers while they are organ-confined. However as years gone by, this
two method have shown its limitation for a better result as many studies has been
done. While PSA is organ-specific, it is not cancer-specific. This limited specificity
results in a large number of negative prostate biopsies, and their associated
complications. Thus, the ratio of free PSA to total PSA is lower in men with prostate
cancer than in those without, and when the PSA level is between 4.0 and 10.0 ng/mL
with a negative digital rectal examination, the ratio can help determine the need for a
prostate biopsy.

As a conclusion, despite many testing controversies using PSA as a biomarker

in cancer biology, PSA still remains the most useful biomarkers in determining
prostate cancer. To get the better result using PSA method, serum PSA test results
require experienced contextual interpretation and judgment when applied to
individual men. The heterogeneity of prostate cancer means multiplexed models
combining several biomarkers can be expected to improve the accuracy of prostate
cancer diagnosis as we strive to reduce the number of unnecessary prostate biopsies
and improve detection of clinically significant prostate cancers while avoiding over-
diagnosis and overtreatment of biologically irrelevant tumours.
REFERENCE

Angelis GD, Rittenhouse HG, Mikolajczyk SD, Shamel LB, Semjonow A (2007).
Twenty years of PSA: From prostate Antigen to Tumor Marker, Rev. Urol. 9(3): 113-
123.

Antonarakis ES, Feng Z, Trock BJ, et al. The natural history of metastatic
progression in men with prostate-specific antigen recurrence after radical
prostatectomy: Long-term follow-up. BJU Int 2012;109:32-39.

Brawley OW, Ankerst DP, Thompson IM (2009). Screening for Prostate Cancer. CA
Cancer J. Clin., 59(4):264-273. Epub 2009 Jun 29.

James G. Huang, Nicholas Campbell, and Larry Goldenberg. PSA and beyond:
Biomarkers in prostate cancer. BC Medical Journal, Vol. 56, No. 7, September 2014,
page(s) 334-341.

Katz A, Katz A (2008). The top 13: what family physicians should know about
prostate cancer. Can. Fam. Phys. Rev. 54(2): 198-203.

Loeb S, Catalona WJ (2008). What to do with an abnormal PSA Test.Oncologist, 13:

299305.

Noldus J, Chen Z, Stamey T (1997). Isolation and characterization of free form

prostate specific antigen (f-PSA) in sera of men with prostate cancer. J. Urol., 158:
16061609.

Singh JC (2009). Cut-off value for PSA: do we need a change. J. Postgrad. Med.,
55(2): 150-151
Thompson IM, Pauler DK, Goodman PJ, et al. Prevalence of prostate cancer among
men with a prostate-specific antigen level < or = 4.0 ng per milliliter. N Engl J Med
2004;350:2239-2246.

Wu JT (1994). Assay for prostate specific antigen (PSA). Problems and possible
solutions. J. Clin. Lab. Anal. 8: 51-62
REVIEW FROM JOURNAL TITLE: MicroRNA in prostate cancer: functional
importance and potential as circulating biomarker by Benjamin L Jackson,
Anna Grabowska and Hari L Ratan

NAME OF MARKER

microRNA

DESCRIPTIONS

Prostate cancer (PCa) is the development of cancer in the prostate associated with a
substantial morbidity and mortality. It has reported that 233,000 new cases and
29,480 deaths occurred in the year 2014. Early diagnosis and treatment before tumor
metastasizes is crucial for improving the patient survival. The 5 year survival ratio for
men diagnosed while the PCa is localized is nearly 100 %. And only 28 % of the men
who diagnosed with metastatic PCa survive beyond 5 years. There are still to be
challenges for the early diagnosis of PCa due to its variable presentation.

Diagnostic testing for PCa has been widely studied and searches from biomarkers,
such as the prostate-specific antigen (PSA) assay, to radiologic imaging, such as
magnetic resonance imaging (MRI), and Biopsy. Although widely used as a diagnosis
tool, serum PSA assays are sensitive but not specific enough to detecting PCa. Novel
biomarkers with enhanced detective accuracy would greatly assist the diagnosis of
PCa.

The miRNAs are important regulators of biologic processes in PCa progression. A

common expression profile characterizing each tumor subtype and stage has still not
been identified for PCa, probably due to molecular heterogeneity as well as
differences in study design and patient selection. Large-scale studies that should
provide additional important information are still missing. Further studies, based on
common clinical parameters and guidelines, are necessary to validate the translational
potential of miRNAs in PCa clinical management. Such common signatures are
promising in the field and emerge as potential biomarkers.

MiRNAs present an appealing target for biomarker discovery. Each miRNA possesses
the ability to interact with a number of cellular pathways, and consequently changes
in the expression of a small number of miRNAs may reflect dysregulation of a broad
range of cellular processes, in keeping with the complexity of neoplasia. Furthermore,
miRNAs are relatively resistant to RNase degradation due to their short sequence
length which improves their longevity in both serum and tissue samples. A number of
techniques for miRNA profiling have been developed, and the principle that increased
tissue expression of miRNAs may lead to elevated serum levels has been
demonstrated experimentally.

It may eventually be possible to identify a panel of miRNAs that can be measured in

serum or tissue and used to better establish which patients with localised prostate
cancer will ultimately progress and should therefore be treated aggressively, or indeed
to establish which patients with more advanced disease are most likely to benefit
from particular therapies, including novel agents acting on the androgen pathway.
Serum miRNA profiling is especially exciting given that it is accessible in a non-
invasive manner, and it may be possible to establish a diagnostic serum miRNA panel
that would reduce the need for prostate biopsies in patients with elevated PSA levels,
some of whom ultimately will not have prostate cancer.

None of these aims have yet been met, but this review aims to summarise the
progress made both with understanding the role of miRNA dysregulation in prostate
cancer tumourigenesis, and with establishing which miRNAs may be useful serum
biomarkers.

INDICATIONS/APPLICATIONS

microRNA specific markers for detection, profiling, diagnosis, prognosis and

therapeutic targets for prostate cancer progressing, esp early stage of prostate
cancer.

MicroRNAs (miRNAs) are made up of -22 endogenous nucleotides and are small,
noncoding RNAs that are important regulators of gene expression at the
posttranscriptional level by degrading or repressing target miRNAs. miRNA
expression profiles can be used for the detection of diagnostic and prognostic markers
for various cancers. Also, alterations of miRNAs in cancer tissues have been
associated with clinicopathological parameters. Along with circulating miRNAs,
tissue miRNAs have shown promise as markers that can predict cancer recurrence
and/or the potential for survival of cancer patients. Additionally, some miRNAs have
therapeutic potential. In this review, we discuss and assess the usefulness of tissue-
derived and circulating miRNAs for the diagnosis and prognosis of prostate cancer.
miR-29b

play a tumour suppressive role by inhibiting EMT. Expression levels were

reduced in prostate cancer tissue compared to patient-matched non-tumour
tissue, and investigators found increased E-cadherin expression and reduced
N-Cadherin, Twist, and Snail expression in cells transfected with miR-29b,
suggesting that miR-29b inhibits EMT.

miR-145

been suggested to lead to enhanced cell proliferation, migration and invasion

in prostate cancer. Fuse and colleagues demonstrated that the capacity of PC3
and DU145 prostate cancer cells to proliferate, migrate, and invade was
impaired by transfection with miR-145.
identified as potential regulators of EMT.
had a reduced propensity to bony invasion in vivo in a murine intra-tibial
injection model, and that in a cohort of 22 patients, reduced expression levels
of miR-143 and miR-145 correlated with higher Gleason grade, PSA, and
bony metastasis .

MiR-143

also downregulated in prostate cancer, and has been shown in vitro to inhibit
proliferation and migration of prostate cancer cells by suppressing V-Ki-ras2
Kirsten rat sarcoma viral oncogene homolog (KRAS) expression, thereby
inhibiting the EGFR/RAS/mitogen-activated protein kinase (MAPK) pathway.
identified as potential regulators of EMT.
had a reduced propensity to bony invasion in vivo in a murine intra-tibial
injection model, and that in a cohort of 22 patients, reduced expression levels
of miR-143 and miR-145 correlated with higher Gleason grade, PSA, and
bony metastasis .

MiR-133 and miR-146a

have also been shown to target EGFR and to produce similar anti-
proliferative, anti-migratory effects in androgen-insensitive prostate cancer
cell lines in vitro.
MiR-205

is downregulated in prostate cancer cells, and this has been implicated in the
stimulation of EMT. Prostate cancer cells transfected with miR-205
demonstrated enhanced expression of epithelial adhesion molecules such as E-
cadherin, and displayed other features of a more epithelial phenotype,
suggesting that miR-205 downregulation may be partly responsible for
stimulating EMT.

MiR-125b

has an androgen-responsive element within the promoter region of its gene.

Mi-R125b upregulation has been demonstrated to result in androgen-
independent growth in prostate tumour xenografts in castrate mice perhaps
through its anti-apoptotic effects (see MiRNAs and avoidance of apoptosis).

MiR-21
In vitro, transfection of LnCaP cells with a miR-21 expressing retrovirus has
been shown not only to stimulate androgen-dependent cell growth, but also to
rescue cells from androgen-deficient growth arrest. This indicates that miR-21
may also mediate castrate-resistant prostate cancer (CRPC) development. Li
and colleagues recently established that tissue miR-21 expression levels may
have clinical importance. They evaluated miR-21 expression levels in a cohort
of 169 radical prostatectomy tissue samples and found increased miR-21
expression was associated with pathological stage, lymph node status,
extracapsular extension and biochemical recurrence. They went on to
demonstrate in vivo tumour growth repression in a mouse model treated with a
miR-21 inhibitor.

MiR-32

is a further example of an androgen-regulated miRNA that is upregulated in

CRPC. MiR-32 upregulation is associated with downregulation of the protein
BTG2, and reduced BTG2 staining in radical prostatectomy specimens has
been shown to predict tumour progression.
MiR-27a
androgen-stimulated upregulation of miR-27a results in reduced prohibitin
expression. Prohibitin is a known tumour-suppressor gene and co-repressor of
the androgen receptor, and miR-27a mediated downregulation of its protein
product results in increased expression of androgen receptor target genes and
increased prostate cancer cell growth.

MiR-141
attempted to profile differential miRNA expression in androgen treated and
untreated LnCaP cells in vitro and in prostate cancer xenografts in intact and
castrated mice. MiR-141 demonstrated the greatest androgen-dependent
expression in that study. MiR-141 is overexpressed in various forms of human
epithelial malignancy including prostate cancer. Separate analysis of prostate
epithelial and stromal cells has demonstrated miR-141 expression to be
epithelium-restricted.

RESULTS

A number of miRNAs have been shown to influence key cellular processes involved
in prostate tumourigenesis, including apoptosis-avoidance, cell proliferation and
migration and the androgen signalling pathway. An overlapping group of miRNAs
have shown differential expression in the serum of patients with prostate cancer of
varying stages compared with unaffected individuals. The majority of studies thus far
however, involve small numbers of patients and have shown variable and
occasionally conflicting results.

Besides that, microRNA also can used for screening chronic diseases includes viral
infections, nervous system disorders, cardiovascular disorders, and diabetes.

REVIEWS

There is evidence from in vitro and in vivo studies that alteration in miRNA function
plays a role in prostatic carcinogenesis. miRNA dysregulation influences a number of
critical cellular processes involved in this process, including but not limited to:
stimulation of the cell cycle, avoidance of apoptosis, epithelial-mesenchymal
transition and modulation of AR-mediated signalling. Further understanding of the
functional importance of miRNA dysregulation may allow the development of novel
therapeutic strategies involving miRNA augmentation or inhibition in the future.

Circulating miRNA profiling in prostate cancer patients has been carried out by
various investigators, but studies thus far have involved small numbers of patients,
and a variety of methodologies, and have yielded heterogeneous results. However, the
potential to detect circulating miRNAs in serum and potentially, in urine, clearly
exists.

Furthermore, despite the variability of results, there are a number of miRNA targets
that have both a demonstrable functional role in prostate cancer pathogenesis and
have demonstrated over- or under-expression in serum samples in at least one study.
The investigation of these miRNAs, either singularly or as part of a panel, in larger,
prospective patient cohorts will help to define their potential role as diagnostic and
prognostic biomarkers in the future.

REFERENCES

Catto JW, Alcaraz A, Bjartell AS, De Vere White R, Evans CP, Fussel S, Hamdy FC,
Kallioniemi O, Mengual L, Schlomm T, Visakorpi T: MicroRNA in prostate, bladder,
and kidney cancer: a systematic review. Eur Urol. 2011, 59 (5): 671-681.
10.1016/j.eururo.2011.01.044.

Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, Guo J, Zhang Y, Chen J, Guo X, Li Q, Li

X, Wang W, Zhang Y, Wang J, Jiang X, Xiang Y, Xu C, Zheng P, Zhang J, Li R,
Zhang H, Shang X, Gong T, Ning G, Wang J, Zen K, Zhang J, Zhang CY:
Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis
of cancer and other diseases. Cell Res. 2008, 18 (10): 997-1006. 10.1038/cr.2008.282.

Heinlein CA, Chang C. 2004. Androgen receptor in prostate cancer. Endocr Rev 25:
276308.CA HeinleinC. Chang2004Androgen receptor in prostate cancer.Endocr
Rev25276308

Hessels D, Verhaegh GW, Schalken JA, Witjes JA. 2004. Applicability of biomarkers
in the early diagnosis of prostate cancer. Expert Rev Mol Diagn 4: 513526.D.
HesselsGW VerhaeghJA SchalkenJA Witjes2004Applicability of biomarkers in the
early diagnosis of prostate cancer.Expert Rev Mol Diagn4513526

Kumar-Sinha C, Rhodes DR, Yu J, Chinnaiyan AM. 2003. Prostate cancer

biomarkers: a current perspective. Expert Rev Mol Diagn 3: 459470.C. Kumar-
SinhaDR RhodesJ. YuAM Chinnaiyan. Prostate cancer biomarkers: a current
perspective.Expert Rev Mol Diagn3459470

Lin JF, Xu J, Tian HY, Gao X, Chen QX, et al. 2007. Identification of candidate
prostate cancer biomarkers in prostate needle biopsy specimens using proteomic
analysis. Int J Cancer 121: 25962605.JF LinJ. XuHY TianX. GaoQX
Chen2007Identification of candidate prostate cancer biomarkers in prostate needle
biopsy specimens using proteomic analysis.Int J Cancer12125962605

Lukk M, Kapushesky M, Nikkila J, Parkinson H, Goncalves A, et al. 2010. A global

map of human gene expression. Nat Biotechnol 28: 322324.M. LukkM.
KapusheskyJ. NikkilaH. ParkinsonA. Goncalves2010A global map of human gene
expression.Nat Biotechnol28322324

Meehan KL, Holland JW, Dawkins HJ. 2002.Proteomic analysis of normal and
malignant prostate tissue to identify novel proteins lost in cancer. Prostate 50: 54
63.KL MeehanJW HollandHJ Dawkins2002Proteomic analysis of normal and
malignant prostate tissue to identify novel proteins lost in cancer.Prostate505463

Mumenthaler SM, Yu H, Tze S, Cederbaum SD, Pegg AE, et al. 2008. Expression of
arginase II in prostate cancer. Int J Oncol 32: 357365.SM MumenthalerH. YuS.
TzeSD CederbaumAE Pegg2008Expression of arginase II in prostate cancer.Int J
Oncol32357365

Pritchard CC, Cheng HH, Tewari M: MicroRNA profiling: approaches and

considerations. Nat Rev Genet. 2012, 13 (5): 358-369. 10.1038/nrg3198.

Yang CH, Yue J, Fan M, Pfeffer LM: IFN induces miR-21 through a signal transducer
and activator of transcription 3-dependent pathway as a suppressive negative
feedback on IFN-induced apoptosis. Cancer Res. 2010, 70 (20): 8108-8116.
10.1158/0008-5472.CAN-10-2579.
 ENGRAILED-2 (EN2) AS A PROSTATE CANCER DETECTION
BIOMARKER

1. INTRODUCTION
Homeobox protein engrailed-2 is a protein that in humans is encoded
by the EN2 gene . Homeobox-containing genes are thought to have a role in
controlling development. In Drosophila, the 'engrailed' (en) gene plays an
important role during development in segmentation, where it is required for
the formation of posterior compartments. Different mutations in the mouse
homologs, En1 and En2, produced different developmental defects that
frequently are lethal. The human engrailed homologs 1 and 2 encode
homeodomain-containing proteins and have been implicated in the control of
pattern formation during development of the central nervous system.
(Poole et al. ,1989) .

A method for diagnosing prostate cancer by detection of EN2 in urine

has developed. The results of a clinical trial of 288 men suggest that EN2
could be a marker for prostate cancer which might prove more reliable than
current methods that use prostate-specific antigen (PSA). If effective, a urine
test is considered easier and less embarrassing for the patient than blood tests
or rectal examinations and, therefore, less likely to discourage early diagnosis.
At the time of the report, it was not clear whether or not the EN2 test could
distinguish between aggressive tumours that would require intervention and
relatively benign ones that would not
(Morgan R.et.al ,2011).

2. CHARACTERISTICS
Approved Symbol : EN2
Approved Name : engrailed homeobox 2
 HGNC ID : HGNC:3343
Locus Type : gene with protein product
Chromosomal Location : 7q36.3
Gene Family : NKL subclass homeoboxes , pseudogenes
HCOP : Orthology Predictions for EN2
(HGNC,2012).

Cytogenetic band:
7q36.3 by Ensembl

EN2 Gene in genomic location: bands according to Ensembl, locations

according to GeneLoc

3. JOURNAL REVIEW
Title : ROLE OF ENGRAILED-2 (EN2) AS A PROSTATE CANCER
DETECTION BIOMARKER IN GENETICALLY HIGH RISK MEN

The aim of this study was to assess the potential of EN2 urine for early
diagnosis of Prostate Cancer in the IMPACT study population. Based on the
review journal it has been made that the EN2 pee biomarker can identify
individuals with clinically relevant prostate cancer. BRCA1 and BRCA2 male
mutation attackers are at high risk of developing prostate cancer significantly
and can benefit from screening. Urine samples from BRCA1 and BRCA2
mutation carriers were evaluated. Based on EN2 screening results found EN2
levels were significantly higher in those diagnosed with cancer compared with
those who did not have a cancer diagnosis, p 0.001. Subjects underwent
annual PSA screening with diagnostic biopsy triggered by PSA> 3.0 ng / ml.
The EN2 level of urine was measured by ELISA and had a sensitivity of
66.7% and a specificity of 89.3% for cancer detection. There was no
statistically significant difference in EN 2 levels by genetic status or Gleason
score. EN2 urine is useful as a non-invasive early biomarker for the detection
of prostate cancer in genetically high-risk individuals.

From journal data it can be discussed that EN2 represents potentially

attractive biomarkers for early detection of cancer. Urine-based test is stable
for 4 days at room temperature, suitable for collection of post samples. Unlike
some other urinary markers such as PCA3 and TMPRSS2-ERG, prior DRE is
not required to improve the ability of acceptability to individuals and reduce
costs. Current lateral current strips in commercial development will increase
the speed and ease of investigation of EN2 as a starting point of early cancer
detection markers. The IMPACT study is expected to complete the
recruitment of operators and controls BRCA1 and BRCA2. It is expected to
provide further information on the usefulness of PSA screening in this high
risk group and also provide a useful sample bank for EN2 investigations and
new detection or prognostic biomarkers for PCs. As more recruits reach the
point of 5 years when an optional prostate biopsy is offered regardless of the
PSA information that will further be available regarding the true cancer status
of these individuals and the effectiveness of EN2 as the initial biomarker for
PCs.
REFERENCES :
HUGO Gene Nomenclature Committee (HGNC),2012
,http://www.genenames.org/cgibin/gene_symbol_report?
hgnc_id=HGNC:3343 [online at 29/April/2017]

Morgan R, Boxall A, Bhatt A, Bailey M, Hindley R, Langley S, Whitaker HC,

Neal DE, Ismail M, Whitaker H, Annels N, Michael A, Pandha H (March
2011). "Engrailed-2 (EN2): a tumor specific urinary biomarker for the early
diagnosis of prostate cancer". Clin. Cancer Res. 17 (5): 10908.

Poole, S. J., Law, M. L., Kao, F.-T., Lau, Y.-F. Isolation and chromosomal
localization of the human En-2 gene. Genomics 4: 225-231, 1989.
 Journal :
Role of Engrailed-2 (EN2) as a prostate cancer detection biomarker in
genetically high risk men
Emma Killick, Richard Morgan, Francesca Launchbury, Elizabeth Bancroft,
Elizabeth Page, Elena Castro, Zsofia Kote-Jarai, Armen Aprikian, Ignacio
Blanco, Virginia Clowes, Susan Domchek, Fiona Douglas, Diana Eccles, D.
Gareth Evans, Marion Harris, Judy Kirk, Jimmy Lam, Geoffrey Lindeman,
Gillian Mitchell, Nicholas Pachter, Christina Selkirk, Kathy Tucker, Janaz
Zgajnar, Rosalind Eeles, Hardev Pandha
Sci Rep. 2013; 3: 2059. Published online 2013 Jun 24. doi:
10.1038/srep02059
PMCID: PMC3690389

Prostate Cancer gene 3 (PCA3).

PCA3 (also known as DD3 or DD3PCA3) is located on chromosome 9 and is

transcribed into a non-coding prostate-specific mRNA which is overexpressed in tumor cells,
from 60 to 100 times, when compared to the normal prostate tissue (Bussemakers et. Al,
1999).

The PCA3 test is based on the quantification of the PCA3 mRNA on urine sample
after digital-rectal examination (DRE), using the methodology of the transcription mediated
amplification (TMA). The obtained result is then normalized to the amount of PSA mRNA,
evaluated in the same urine sample, in order to calculate the PCA3 score (PCA3 mRNA/PSA
mRNA 1000). To date, many studies have been performed and most of them showed how
the PCA3 test represents a useful tool to predict PCa, but questions about the optimal cut off
and the ability of PCA3 to predict tumor aggressiveness still remain highly controversial.
Thus, the PCA3 score tells the expression of PCA3 corrected for the background of normal or
BPH epithelial cells present in the specimen (Day et. al, 2011).
 PCA3 testing is highly dependent on the cutoff Score used to determine a "positive"
or "negative" test, because sensitivity and specificity vary reciprocally with the Score. The
higher the cutoff, the greater the specificity and the lower the sensitivity; the lower the cutoff,
the greater the sensitivity and the lower the specificity. Thus, although the test is now
available commercially, physicians must be cautious in interpreting the lab report and should
know the performance characteristics of the assay, before decisions are based on a "positive"
or "negative" test result (Bostwick et. al, 1996).

Test Interpretation

Clinical sensitivity/specificity 77.5% and 57.1% respectively (relative to prostate biopsy

outcome), based on a PCA3 ratio cut off value of 25.

PCA3 ratio Indicator Explanation

0-17 Negative Associated with decreased likelihood of a positive
biopsy for prostate cancer.
 18-24 Negative Should be interpreted with caution due to normal test
variability, specimens with ratios near the cut off may
yield a different overall interpretation upon repeat
testing.
25-31 Positive Should be interpreted with caution due to normal test
variability, specimens with ratio near the cutoff may
yield a different overall interpretation upon repeat
testing.
>31 Positive Increased probability of a positive biopsy for prostate
cancer.

Limitations

Sufficient number of prostate cells must be present in urine for analysis

PCA3 testing should not be used for men with atypical small acinar proliferation (ASAP)
on their most recent biopsy (Arup, 2013)

Journal:

Prostate Cancer Specificity of PCA3 Gene Testing: Examples from Clinical Practice by
Leonard S Marks, MD* and David G Bostwick, MD.

A specific marker for early detection of prostate cancer is useful in evaluation of the prostate
cancer antigen 3 (PCA3) gene vis--vis serum prostate-specific antigen (PSA) levels. PCA3
specificity for cancer is nearly perfect because of the gross overexpression of a segment of
noncoding messenger ribonucleic acid (mRNA) from chromosome 9q2122 by cancer cells.
The specificity is also seen in urine containing prostate cells from men with the disease.
PCA3 determination can separate benign from malignant prostate cells with an accuracy
approaching 100%.

Case 1: Elevated PSA Level, Biopsy Demonstrates Benign Prostatic Hyperplasia.

Biopsy is not supported parameter for prostate cancer. Prostate biopsies are performed
annually. The great majority of these biopsies are stimulated by increased serum PSA levels.
Theres a research show that some of the men with negative biopsy results, perhaps 25%, will
be found to harbor cancer upon subsequent biopsy. It is reported that a patient underwent 6
biopsies over 12 years because of PSA abnormalities. Each biopsy demonstrated benign
prostatic hyperplasia (BPH), with no evidence of cancer. Late in this mans story, the PCA3
gene test became available. The value of the PCA3 test in men with elevated PSA levels and
prior negative biopsies has been detailed previously.

Case 2: Normal PSA Level, Biopsy Reveals Cancer.

Statistics show that 15% of men found to have prostate cancer, followed with serial PSA
determination over years stated that serum PSA is not greater than 4.0 ng/mL. This is indicate
that serum PSA levels are not perfect screening tests for cancer, exhibiting false-negative and
false-positive values. The results are misleading with any PSA-based testing. A patient with
PSA level rising gradually over the next several years found that urinary PCA3 score was
71.7 ng/mL, which confirmed suspicion of increased PSA velocity, leading to discovery of
adenocarcinoma with a Gleason score of 7 on biopsy in both lobes; margins and lymph nodes
were clear. PCA3 scores, as opposed to serum PSA levels, do not increase with increasing
prostate volume.

Case 3: Elevated PSA Level, Irritative Voiding Symptoms (Prostatitis).

Acute prostatitis is known to cause marked elevation in serum PSA levels. 55-year-old man
undergoes observation over years with mild voiding symptoms (lower urinary tract
symptoms) not requiring treatment shows that high in serum PSA level. The PCA3 score was
22.5, which is below the cutoff of 35 for optimal sensitivity and specificity. Along with direct
trauma (eg, biopsy, cystoscopy), prostatitis is the most common cause of sudden, marked
elevation in serum PSA levels due to inflammation may be sufficient to alter cellular integrity
and cause leakage of PSA into serum.

Case 4: Microfocal Disease, Active Surveillance.

A recent literature survey from the United Kingdom found that microfocal disease does not
always equate with insignificant disease, with some patients demonstrating adverse outcomes
despite low-volume lesions on initial biopsy. A specific cancer marker in these patients would
be of immense value. The potential value of PCA3 in identifying insignificant disease is
shown. Two patients with well-differentiated microfocal lesions help to exemplify the value
of a low PCA3 score in confirming the presence of indolent disease.

Conclusion: A marker specific for early prostate cancer would fill an important void. In initial
evaluations of the PCA3 gene vis--vis serum PSA levels, the gene offers great promise.
PCA3 gene testing holds valuable potential in PSA quandary situations: (1) men with
elevated PSA levels but no cancer on initial biopsy; (2) men found to have cancer despite
normal levels of PSA; (3) men with PSA elevations associated with varying degrees of
prostatitis; and (4) men undergoing active surveillance for presumed microfocal disease. A
much larger experience will be required to identify regulators of the PCA3 gene and to fully
define sensitivity and specificity of PCA3 gene testing.

References

Arup, 2013. PCA3 Prostate Cancer Biomarke . Salt Lake City: University of Utah

Bostwick DG, Pacelli A, Lopez-Beltran A. Molecular biology of prostatic

intraepithelial neoplasia. Prostate. 1996;29:117134

Bussemakers MJ, van Bokhoven A, Verhaegh GW, Smit FP, Karthaus HF, Schalken JA,
et al. DD3: a new prostate-specific gene, highly overexpressed in prostate
cancer. Cancer Res. 1999;59(23):59759.
Day JR, Jost M, Reynolds MA, Groskopf J, Rittenhouse H. PCA3: from basic molecular
science to the clinical lab. Cancer Lett. 2011;301(1):16.

Marks, L. S., & Bostwick, D. G. (2008). Prostate Cancer Specificity of PCA3 Gene Testing:
Examples from Clinical Practice. Reviews in Urology, 10(3), 175181.

Topic : Annexin II

Annexin II (Annexin A2) is a protein in humans that is encoded by the

ANXA2 gene.

Annexin 2 is involved in diverse cellular processes such as cell motility

(especially that of the epithelial cells), linkage of membrane-associated protein
complexes to the actin cytoskeleton, endocytosis, fibrinolysis, ion channel formation,
and cell matrix interactionsThis protein is a member of the annexin family. Members
of this calcium-dependent phospholipid-binding protein family play a role in the
regulation of cellular growth and in signal transduction pathways

Annexin II is a pleiotropic protein meaning that its function is dependent on

place and time in the bodyThis protein functions as an autocrine factor which
heightens osteoclast formation and bone resorption.Epigenetic regulation of Annexin
A2 has been identified as a key determinant of mesenchymal transformation in brain
tumors.Annexin A2 has been proposed to function inside the cell in sorting of
endosomes and outside the cell in anticoagulant reactions.

Since annexin II is reduced in prostate cancer cells, the molecule may

normally suppress prostate cancer development by restricting cell proliferation,
promoting programmed cell death (apoptosis), or by limiting cell migration and
invasion.

Review Research :

Annexin II expression is reduced or lost in prostate cancer cells and its

re-expression inhibits prostate cancer cell migration

Jun-Wei Liu1, Jian-Jun Shen1, Angela Tanzillo-Swarts1, Bobby Bhatia1, Carlos M

Maldonado1,Maria D Person2, Serrine S Lau2 and Dean G Tang

The studying of the role of Bcl-2family proteins in regulating prostate cancer

cell apoptosis leads to studying Bim, a BH3-only proapoptotic protein, and identified
an B36 kDa protein, which was abundantly expressed in all five strains of primary
normal human prostate (NHP) epithelial cells but significantly reduced or lost in
seven prostate cancer cell lines.
 Annexins are a family of Ca2+-dependent, phospholipid- binding proteins.
More than 20 different annexin isoforms have been identified. Annexin II, also
known as p36, lipocortin II, and calpactin I heavy chain, was initially identified as
one of the major phosphorylation targets of pp60v-src

While the precise function of annexin II is unclear, it has been shown to be

involved in Ca2+-dependent exocytosis, endocytosis, cellcell adhesion,
proliferation, cell surface fibrinolysis, and osteoclast formation and bone resorption

Five primary strains of normal human prostate (NHP) epithelial cells, NHP1
NHP5, and seven prostate cancer cell lines, PPC-1, MDA PCa 2b (MDA 2b), LNCaP,
C4-2, C5,PC3, and Du145, were cultured as described previously for the
performance of this experiment

To identify this unknown protein, we employed proteomic approach. Whole

cell lysates from NHP2(that expressed abundant 36 kDa protein) and LNCaP (which
did not express the 36 kDa protein) cells were separated by 2D gel electrophoresis.

After transferring the proteins to nitrocellulose membrane, the blot was

probed with the polyclonal anti-Bim antibody, which detected a specific B36 kDa
protein spot in NHP2) but not in LNCaP cells

The duplicate 2D gels run in parallel for each sample were stained with
SYPRO Ruby protein stain. The protein spot in NHP2 cells that matched the spot
Annexin II inhibits prostate cancer cell migration detected by the anti-Bim antibody
was manually excised.

We focused our subsequent studies on annexin II, because it was initially

identified by the polyclonal anti-Bim antibody. To understand how reduced annexin II
expression may contribute to prostate cancer development, we first studied its normal
distribution in NHP cells and then compared with its expressions in prostate cancer
cells.
 Several lines of evidence suggest that annexin II may negatively regulate
prostate cancer cell motility and migration. First, annexin II has been shown to be
associated with the cytoskeleton. Subsequent mechanistic studies focused on annexin
II suggest that it probably inhibits prostate cancer development by inhibiting cell
migration without affecting proliferation or apoptosis. Exactly how the re-expressed
annexin II inhibits the migratory ability of prostate cancer cells remains to be
determined. It is possible that these annexin II molecules may form complexes with
p11 underneath plasma membrane and bind to cytoskeletal molecules, and the II2-
p112 complexes.

DAFTAR PUSTAKA

Aarli A, Kristofferson EK, Jensen TS, Ulvestad E and Matre R. (1997). Am. J.
Reprod. Immunol., 38, 313319
Chiang Y, Rizzino A, Sibenaller ZA, Wold MS and Vishwanatha JK. (1999).
Mol. Cell. Biochem., 199, 139147.
Lee SW, Tomasetto C, Swisshelm C, Keyomarsi K and Sager R. (1992). Proc.
Natl. Acad. Sci. USA, 89, 25042508.
Liu J-W, Chandra C, Tang S-H, Chopra D and Tang DG. (2002). Cancer Res.,
62, 29762982.
Ma ASP, Bell DJ, Mittal AA and Harrison HH. (1994). J. Cell. Sci., 107,
19731984.
Solito E, de Coupade C, Canaider S, Goulding NJ and Perretti M. (2001). Br.
J. Pharmacol., 133, 217228.
Srivastava M, Bubendorf L, Srikantan V, Fossom L, Nolan L, Glasman M,
Leighton X, Fehrle W, Pittaluga S, Raffeld M, Koivisto P, Willi N, Gasser TC,
Kononen J, Sauter G, Kallioniemi OP, Srivastava S and Pollard HB. (2001).
Proc. Natl. Acad. Sci. USA, 98, 45754580.
 Takahashi S, Reddy SV, Chirgwin JM, Devlin R, Haipek C, Anderson J and
Roodman GD. (1994). J. Biol. Chem., 269, 2869628701.
Vishwanatha JK, Chiang Y, Kumber KD, Holingsworth MA and Pour PM.
(1993). Carcinogenesis, 14, 25752579

Review Article

Human Kallikrein-2, Prostate Specific Antigen and Free-Prostate Specific

Antigen in Combination to Discriminate Prostate Cancer from Benign Diseases
in Syrian Patients

1. Introduction

Prostate cancer is the most prevalent cancer and the second leading cause of
cancer mortality among men in the western. In Middle East countries it may be less
common, but is still one of the most common cancers in men with elevated incidence.
The prostate cancer had few specific symptoms in its early stage, for that
reason, detecting cancer holds several difficult. The standard practice of screening for
prostate cancer includes PSA, with or without digital rectal exam test (DRE), starting
at the age of 50 years in all males or earlier in men with certain risk factors. However,
only according to the prostate biopsy results we can confirm the prostate cancer
diagnosis.
PSA levels influenced by many factors like age, race, the Metabolic Syndrome
or even Body Mass Index (BMI) and alcohol consumption. But the most important
disadvantage of the PSA test is its low specificity. This lack of specificity warrants
the use of new biomarkers to diagnose prostate cancer more accurately.
hK2, one of members of kallikrein, has a high level of significance in men with
prostate cancer. hK2 is a 28 kDa serine protease produced by prostatic epithelial cells.
Structurally, hK2 and PSA (hK3) share the highest sequence homology (in KLKS
family). The serum level of hK2 increases in many conditions including leaking from
the disrupted prostatic cells and over expression in cancerous prostate tissue which
increases with increasing grade.
Therefore, many studies indicate that measurement of serum hK2 with PSA can
improve diagnosis of prostate cancer, importantly, the ratio of hK2/fPSA ratio alone
or combined with fPSA/PSA ratio enhances the discrimination between prostate
cancer and BPH and may also be useful in grading the cancer.
Therefore, a study is conducted to investigate the role of hK2 and its
combinations with other markers to discriminate prostate cancer from benign diseases
in Syrian patients.

2. Materials and Methods

1. Patient selection
Included in this prospective oriented cross-sectional cohort study, were 70
patients who were referred to either the Urology department in Al-Bairouni university
Hospital or Al-Mouwasat university Hospital or Ibn Al-Nafees Hospital in Damascus,
Syria aged between 45 - 83 years old. These patients underwent prostatic TRUS
guided needle-core prostatic biopsies because of either PSA values of 4.0 ng/mL or
abnormal digital rectal examination (DRE), later they were diagnosed with either PCa
(35 patients) or BPH (35 patients) according to the histological tests results and Grade
was evaluated by the Gleason scoring system according to it patients classified into
three groups: low-grade group (GS=2-3-4), intermediate-grade group (GS=5-6-7) and
high-grade group (GS=8-9-10). Patients present with previous cancer, previous
prostate biopsy or metastatic diseases were excluded.
Blood samples were collected before DRE and biopsy procedure and any
treatment. Plasma was separated from blood samples and was stored at -70C. PSA,
fPSA and hK2 were analyzed at a later time in the frozen serum samples. Both fPSA
and PSA levels were analyzed with commercially ELISA kits (R&D Systems) and
hK2 levels were analyzed using a for-research only ELISA kit (Uscn Life Science
Inc.).

2. Statistical Analysis
Statistical analyses were done with commercially available software using
SPSS program version 19 and Microsoft Excel 2007. The analyses of differences
between these variables in the three groups were performed with the T-student test for
normally distributed parameters. Relationships between different variables were
assessed by the Spearman correlation coefficient. For all analyses, P< 0.05 was
considered statistically significant. ROC curves were constructed for PSA, fPSA, the
fPSA/PSA ratio, hK2 and hK2/fPSA ratio, plotting sensitivity vs. (1-specificity), and
the areas under the ROC curves (AUCs) were calculated.

3. Results

Table 1. Mean Values and Ranges of the Variables

 The results, as shown in table 1, indicate that the levels of PSA, fPSA/PSA ratio
and hK2/fPSA ratio were higher in cancer patients. However, the differences in
fPSA/PSA ratios values did not reach. It is also shown in tale 1 that hK2/fPSA ratio is
the highest among the other marker and the value is significantly higher in Pca than
BPH (P=0,01).

4. Results

Other studies indicate that no single marker could be used to detect PCa, and
combination of markers improves specificity of detecting PCa.
Previous studies have indicated that hK2 has a valuable importance since its
levels are significantly higher in men with prostate cancer; based in on these findings
and considering many factors in the patients, the hK2 role and its combinations with
PSA and fPSA in the detection of PCa program applied in some Syrian hospitals,
which include PSA tests with or without DRE examination is investigated. The
results were compatible with the previous studies as hK2, fPSA, and PSA
combinations are valuable in predicting PCa in our population.
As shown in table 1, ratio hK2/fPSA is distinguish significantly between PCa
and BPH patients (P=0.01).
Another studies reported that that some kallikreins are elevated in many
malignancies, especially hK2 which increase in prostate cancer so it may be used as a
marker in this cancer. It is also confirms that ratio hK2/fPSA was a better diagnosis
marker than fPSA/PSA and has higher specifity, which proves that the results of this
study is compatible.
 An explanation of this effect may be found in the position of fPSA as
denominator and hK2 as numerator. Because the levels of fPSA are lower in men with
PCa and the levels of hK2 is higher in men with PCa, the ratio could result in a test
with more discriminating power.
Several limitations of our study deserve mentioning. The small number of
patients included in our study mainly caused by financial limitations was an obstacle
especially in having a more reliable statistical analysis data.

Also, the levels of fPSA may be influenced by freezing our samples at -80 oC;
although the freezing was as soon as possible after centrifuging and the analysis was
made immediately after thawing in order to avoid fPSA degradation in vitro.

5. Conclusion

Based of the study, we can conclude that the use of ratio of hK2/fPSA is better
than the use of fPSA/ PSA alone since hK2/fPSA offered the most statistical power in
detecting prostate cancer, with the result shown is higher than other marker, with PCa
mean value 126,6 (6,11-539,8) and BPH mean value 51,9 (1,39-42,7), with the value
of PCa higher than BPH and the significance higher than other marker (P=0,01).

Reference
Bachour, DM., Chahin, E., and Al-Fahoum, S. 2015. Human Kallikrein-2, Prostate
Specific Antigen and Free-Prostate Specific Antigen in Combination to
Discriminate Prostate Cancer from Benign Diseases in Syrian Patients. Asian
Pac J Cancer Prev. 16(16): 7085-88.
 TUGASAN BIOTEKNOLOGI FARMASI
PROSTATE CANCER BIOMARKERS
Kelompok 2
FARMASI KPBI 2014
DOSEN: IBU YUNI ELSA HADISAPUTRI

NAMA NPM BIOMARKER

BIBI YASIMAH BT MD 260110142022 PROSTATE SPECIFIC
YUSOF ANTIGEN (PSA)
FARKHANAH RAHMI 260110142023 PROSTATE SPECIFIC
BT GHOLIB SANTOSA ANTIGEN (PSA)
TAN MEI LEE 260110142014 MicroRNA
JACKIE KANG SING 260110142017 MicroRNA
LUNG
FATIN ZAFIRAH BT 260110142021 ENGRAILED-2 (EN2)
RUHAIYEM
 VITHYA LAKSHMI 260110142018 ENGRAILED-2 (EN2)
NUR FARAH 260110142020 PROSTATE CANCER
FARHANAH BT ISMAIL GENE 3(PCA3)
MAISARAH BT 260110142019 PROSTATE CANCER
GHAZALI GENE 3(PCA3)
EU JOHNNY 260110142016 HUMAN KALLIKREIN-
2(hK-2)
GUNTUR ASTAWA 260110130110 HUMAN KALLIKREIN-
2(hK-2)

FALKUTAS FARMASI
UNIVERSITAS PADJADJARAN JATINANGOR
2017