Prostate carcinoma- tumour markers

PROSTATE TUMOR MARKERS
Dept of Urology
Govt Royapettah Hospital and Kilpauk Medical College
Chennai
1

Moderators:
Professors:
• Prof. Dr. G. Sivasankar, M.S., M.Ch.,
• Prof. Dr. A. Senthilvel, M.S., M.Ch.,
Asst Professors:
• Dr. J. Sivabalan, M.S., M.Ch.,
• Dr. R. Bhargavi, M.S., M.Ch.,
• Dr. S. Raju, M.S., M.Ch.,
• Dr. K. Muthurathinam, M.S., M.Ch.,
• Dr. D. Tamilselvan, M.S., M.Ch.,
• Dr. K. Senthilkumar, M.S., M.Ch.
Dept of Urology, GRH and KMC, Chennai. 2

Biomarker
• A biological molecule found in blood, other body
fluids, or tissues that is a sign of a normal or
abnormal process, or of a condition or disease
• Tumour markers: biomarkers that can be found in
body when cancer is present
• Require special assay that is beyond routine clinical,
radiographic or pathologic examination
3
Dept of Urology, GRH and KMC, Chennai.

Biomarker Development
Early detection research network (EDRN) is a
National Cancer Institute (NCI) funded program
set forth with the objective to
- identify,
- develop, and
- validate promising biomarkers and
technologies for the early detection of cancer
4

The structure of the network consists of five scientific components.
1. biomarker development laboratories (BDLs)
- responsible for discovery of novel biomarkers and technologies
2. The biomarker reference laboratories (BRLs)
- facilitate the development & validation of assays that measure new biomarkers
3. clinical epidemiological validation centers (CEVCs)
- These are large clinical practices with infrastructure to procure ample clinical
samples and to lead clinical trials. They are responsible for procuring samples for the BDL.
4. data management and coordinating center (DMCC).
- provide all the logistical and statistical support for all phases of development
5. informatics center - responsible for developing software for information management.
5

Phases of biomarker development
Similar to the phases of drug development,biomarker developmenthas been broken
down into a number of sequential phases
- Phase 1 consistsof biomarker discovery in BDLs
- Phase 2 has the objective of measuring the sensitivityand specificity of the new
biomarker in its ability to differentiatecase statusfrom control. Done in BRLs
- Phase 3 investigators examine the ability of new biomarkers to detect preclinical
disease and evaluate how much lead time is provided by the biomarker relative to
clinical presentation
6

- Phase 4 indications for applying a new biomarker are clearly defined
- Phase 5 cancer control studies
The ultimate objective for new biomarkers is to reduce the burden of cancer.
Successful outcome in phase 5 requires reduction in cancer mortality that can be
attributed to the biomarker use without prohibitive costs or overdiagnosis.
This lofty goal has been achieved by few biomarkers.
7

FDA approval is considered to be the final end
point in the development process
Currently FDA-approved biomarkers include
1. PSA,
2. percent-free PSA,
3. PHI, and
4. PCA3
8

Prostate cancer biomarkers
1. Blood-Based Biomarkers
- Prostate-Specific Antigen (PSA or hK3)
- Free Prostate-Specific Antigen
- Free Prostate-Specific Antigen Isoforms -proPSA, BPSA, and
intact fPSA
- Prostate-Specific Membrane Antigen
- Human Kallikrein 2
- Circulating Tumor Cells and Circulating Tumor DNA
9

2. Urine-Based Biomarkers
- PCA3
- Gene Fusions in particular TMPRSS2:ERG
- HOXC6 and DLX1 mRNA levels
- Annexin A3
- miRNA
3. Tissue-Based Biomarkers
- α-Methylacyl Coenzyme A Racemase
- Epigenetic Modifications including changes in DNA
methylation and histone acetylation status
- Genomic Expression Profiles
- Inherited Genetic Markers
10

BLOOD BASED BIOMARKERS
11

PSA (Prostate Specific Antigen)
• First demonstrated in 1979 in sera of Pca patients.
• Glycoprotein
• Act as serine protease
• Belongs to kallikerin family
• Also known as gamma semenogelin, hKL3
• Secreted by epithelial cells of prostatic acini and prostatic
duct
• Molecular weight is 34kda
• Contains 7 % carbohydrate
• Liquefies semen
• Gene located on long arm of chromosome-19 (locus q13.2-
q13.4)
12

• Contains 237 AA
• Synthesized as 261 AA preproPSA
↓ [ by cleaving 17 AA chain - dissociation]
proPSA [244AA ]
↓[ by cleaving 7 AA chain] (hk2 & hk4 are removed)
PSA [ 237 AA ] 13

• Concentration in semen is million fold higher
than serum
• In semen 0.5 -5.0 mg/ml
• Serum 1 – 4 ng/ml
• Serum exists in two forms
1.Complexed to proteases [c PSA]
2.Free PSA [f PSA]
• c PSA is cleared by LIVER, Half life is 2 -3 days
• f PSA half life is 2-3 hrs, excreted by KIDNEY.
14

MOLECULAR FORMS OF PSA
15

FREE PSA
• About 5 – 40 % 0f total PSA
• All the epitopes are free
• Immunoreactive
• Exist as 3 isoform: proPSA, BPSA, iPSA
• %fPSA is measured by dividing fPSA by total PSA
• fPSA is directly proportional to age and prostate
volume and inversely related to total PSA
16

FREE PSA ISOFORMS
17

Complexed PSA
• About 60- 95 % of total PSA
• Bound to protease inhibitors
• Inactive
• Immuno reactive
• Measured by BAYER immuno assay.
• c PSA volume based parameters offered equal to
superior results.
• Useful in cancer screening and staging.
18

PSA BINDING PROTEASES
19

MOLECULAR DERIVATIVES OF PSA
20

FACTORS ALTERING PSA
1.ANDROGENS
- PSA becomes detectableat puberty with increasinglevels of luteinizinghormone and
testosterone.
- In hypogonadal men serum PSA level may be low because of decreased expression
(may not reflect the presence of prostate cancer)
2. Obese
-men have slightly lower PSA levels than nonobese men (hemodilution)
3. Statin - use may reduce PSA levels
21

Elevated serum PSA levels
- Due to disruption of cellular architecture within the prostate gland
- loss of the barrier afforded by the basal layer and basement membranes is a
likely site for the egress of PSA into the circulation
Causes - BPH,
Urinary retention
prostatitis,
prostate cancer
prostate manipulation(prostate massage, prostate biopsy - takes 4 or more
weeks before returning to baseline
ejaculation – no effect < 30-40yrs, > 50yrs- rare increase-returns to normal with
in 24 hrs
22

EFFECTS OF PROSTATIC MANIPULATION
1.Digital Rectal Examinations
elevates PSA
but elevations fall within measurement
error ( < 0.26ng/ml)
2. Biopsy
elevates PSA
takes 4 weeks to return to baseline
23

OTHER FACTORS ALTERING PSA
3.Prostatic diseases
a. Inflammations
b.BPH
C.cancer
4.prostate disrupting treatment (decrease)
5 alpha reductase inhibitors
TURP
Catheterisation
Haemodialysis (free PSA)
Cystoscopy
24

Role of 5ARI such as finasteride and dutasteride
on PSA
- lower PSA levels by roughly 50% after 12 months of treatment
- doubling rule - PSA levels are multiplied by a factor of 2 in men
undergoing 5ARI therapy to guide decisions regarding prostate
cancer risk.
- the percentage of fPSA is not altered
25

PSA LEVELS
• PSA levels vary with
a.Age
b.Race (African-Americans)
c.Prostate volume
• Rate of change of PSA
In normal subjects- 0.04 ng/ml/yr
In BPH - 0.07 – 0.27 ng/ml/yr
In Ca. variable
• PSA increases by 4 % /ml of prostate volume /yr
26

PSA IN CANCER PROSTATE
• Prostate cancer cells does not produce more PSA in
fact produce less PSA
• Elevated levels in cancer is due to leak as a result of
architectural disruption.
• In cancer, basal cell and basement membrane barrier
become leaky.
• It is mainly complexed PSA – due to differential
expression of PSA isoform by PZ compared with TZ.
27

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• PSA produced from malignant cells appears to
more frequently escape proteolytic
processing, resulting in a greater fraction of
serum PSA complexed to ACT and a lower
percentage of fPSA
30

CLINICAL APPLICATIONS
-SCREENING
-DIAGNOSIS
-STAGING
-PROGNOSIS
-FOLLOW UP
31

PSA IN SCREENING
• Leads to increased cancer detection
• More of organ confined early cancers
• DRE alone detects 56 % cancers
• PSA alone detects 83 % cancers
• DRE + PSA can act complimentarily
• PSA increased LEAD TIME from 5 to 10 years
• 2.5 to 10 ng/ml considered as DIAGNOSTIC
GREY ZONE
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New PSA Markers
• tPSA = total PSA, the standard measure
• fPSA = free PSA (unattached)
• % fPSA = ratio of free to total PSA
• iPSA = intact PSA
• 4K panel = tPSA, fPSA, iPSA, hK2
• [-2] proPSA or p2PSA= a precursor
(proenzyme) of PSA (239 AA)
• %p2PSA = [-2] proPSA/fPSA × 100
• PHI = ([-2] proPSA × √tPSA / fPSA)
35

AGE ADJUSTED PSA THRESHOLDS
• Morgan et al
4.0 ng/ml – 50 to 70 years
2.5 ng/ml – 40 to 50 years
• Low level of PSA in high risk patient should
be handled carefully
36

37

VOLUME BASED PSA PARAMETERS
• PSA DENSITY .
• PSA of transition zone DENSITY [TZ PSAD ]
38

PSA DENSITY
• Benson et al introduced the concept of PSAD.
• Defined as the Total PSA dividedby Prostatic Volume as assessed by TRUS.
(HtxBtxWdx0.52)
• Cut off value of 0.15 taken as significant.
• An advantage of PSAD is that it has been directly associatedwith prostate
cancer aggressiveness and therefore is currently used to help assess
eligibility for activesurveillance among men with prostatecancer
• Limitations :
1.Lack of reproducibility in measuring
2.Volume detection by TRUS.
39

PSAD OF TRASITION ZONE
• Djavan et al 1999,
• TZ PSAD is calculated by dividing PSA value
by transition zone volume [by TRUS ]
• Cut off value- 0.35 ng/ml
40

PSA VELOCITY
• Carter et al in 1992
• Defined as “ rate of change of PSA per year “
• PSAV= ½ [psa2 –psa1/time 1 in yrs + psa3-psa2/time 2
in yrs]
• PSA V > 0.75 ng/ml/yr is more specific for cancer
• Sensitivity is 72 %
• Only 5 % false positive rates
• Disadvantages
-minimum 18-24 month follow up
-minimum 3 assays needed.
41

Prostate Health Index (PHI)
- Calculation of PHI ( [−2]proPSA× square root of total PSA ) / free
PSA
- Prerequisites- men >50 yrs &
- Total PSA 4 to 10 ng/mL and
- DRE not suspicious for cancer interpretation
perform biopsies when the phi score
indicated an intermediateor
high probability of prostatecancer (phi ≥36).
42

4k score
- Prerequisites - free PSA, hK2 ,intact PSA
,total PSA ( Free HIT )and clinical information
(Age, DRE, and whether there has been a prior
negative Biopsy result) Free HIT + BAD
- The 4Kscore provides a percent risk (ranging
from 1-95%) of a patient having aggressive
prostate cancer on biopsy I.e., Gleason score 7
or higher
- Based on 4k score US validation study score
7 . 5 % or higher - biopsy adviced
43

% FREE PSA
• Ratio of free PSA
/total PSA
• Higher in BPH
• Cut off arbitrarily 18%
Free/total
PSA
Probability of
cancer
<10% 56%
10-15 28
15-20 20
20-25 16
>25 8
44

- FDA has approved fPSA use in men with a serum total PSA level
of 4 to 10 ng/mL and a negative DRE or elevated PSA with prior
negative biopsy
- The NCCN Prostate Cancer Early Detection Guidelines
incorporate %fPSA as an option to help with decision making
before biopsy and recommend a threshold of 10%
45

PSA doubling time (PSA-DT): which measures the
exponential increase in serum PSA over time
Prostate specific antigen velocity and PSA-DT may
have a prognostic role in treating PCa but have
limited diagnostic use
46

47

PSA IN SCREENING
• All four methods – age-adjusted PSA, free/total PSA
ratio, complexed PSA, and PSA/TZ PSAD – can be used to
improve the sensitivity (detect more cancers) and/or
specificity (avoid unnecessary biopsies) of PSA testing.
• An important recent development has been the
increasing availability and usage of predictive tools that
incorporate multiple clinical variables such as total PSA,
%fPSA, and DRE findings eg prostate cancer prevention
trial (PCPT) risk calculator
48

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THERE IS ROLE OF
OPPORTUNISTIC/INDIVIDUALIZED SCREENING
• Pt should have atleast10-15yrs of exp survival to benefit by
screening- don’t screen above 70yrs
• Do PSA before any medical/surgical intervention for prostate(
usually symptomatic)
• Early screening depending on racial/familial predisposition(
40-45yrs)
• Shared decision making: pts are encouraged to decide for
themselves whether the benefits of screening outweigh
harms
• Further evaluation by smart screening may reduce
overtreatment by identifying more of high risk pts
51

PSA IN STAGING
..,
52

PSA IN STAGING
• Serum PSA levels correlate with the risk of
extra-prostatic extension, seminal vesicle
invasion, and lymph node involvement.
• Patients with serum PSA levels of less than
10.0 ng/mL are most likely to respond to local
therapy.
53

PSA IN STAGING
• PSA less than 4 ng/ml → 80% organ confined disease.
• PSA 4- 10ng/ml → 60 % organ confined .
• PSA more than 10ng/ml → less than 50 % organ
confined .
• PSA <10 ng/ml → 5% pelvic lymph node involvement.
• PSA 10-20 ng/ml → 18 % pelvic lymph node
involvement.
• PSA more than 50 ng/ml → 75 % pelvic lymph node
involvement.
54

Prostate specific Membrane Antigen
(PSMA)
• Glycoprotein, 3 parts, located on short arm of chrom-11.
• Expressed in secretory acinar epithelium of prostate
• Elevated in CaP
• During cancer progression differentially expressed variants of PSMA
have been identified. Of the three alternatively spliced variants, one
known as PSM’ is differentially expressed in normal tissue, BPH &
prostate cancer.
• PSMA/PSM’ ratio is upregulated 3 to 6 fold in prostate ca compared
with BPH and it correlates with cancer progression.
(PSM is N-terminally truncated PSMA variant)
55

HUMAN KALLIKREIN
• hK2 shares 80% AA homology with PSA & exhibits similar
specificity for prostate tissues & are hormonally regulated by
androgens
• The conc of hK2 is <2% of that of tPSA
• Most exist in free form
• tPSA 4-10 ng/ml >> hK2/fPSA , to differentiate CaP from BPH.
• Also tells about prognosis
• hK2 expression varies independent of PSA expression .In
benign epithelium, PSA is intensely expressed than hK2 . In
contrast in cancerous tissue hK2 is expressed intensely.
56

ENDOGLIN
• CD 105
• Cell surface coreceptor for TGF beta 1& 3
• Key role in angiogenesis
• Assoc. with node positivity
• Also expressed in urine of CaP
57

Circulating Tumor Cells and Circulating Tumor DNA
- CTCs are defined as being CD45− and positive for an epithelial
marker such as epithelial cell adhesion molecule (EpCAM)
and/or cytokeratin
- Cd45 is expressed on all Hematopoietic cells except mature RBC
- EpCAM is expressed in normal epithelium and epithelial
derived cancers. However in normal epithelium it is expressed
in basolateral membrane
- Currently, there is only one FDA-approved methodology for
identifying CTCs: CellSearch
58

- The anti-EpCAM ferrofluid captures the cells, and they are then
validated with cytokeratin-positive and CD45negative staining.
- With this system, a CTC count of 5 or more cells per 7.5 mL of
blood at any time during the course of the disease has been
associated with a poor prognosis in prostate, breast, and
colorectal cancers
CellSearch
59

CTCChip-
- One of the platforms for CTC detection
- Developed in Harvard Medical School
- a high level of sensitivity, able to detect a single EpCAM-
positive cell among 1 billion blood cell
Detection of the androgen receptor splice variant 7 (AR-V7) in CTCs
predicts resistance to abiraterone and enzalutamide in metastatic
CRPC & responds better to taxaes
[Going forward, the use of CTCs as a biomarker will likely revolve less around
simple enumeration and more around the specific molecular alterations that can
be identified—providinga real-time “liquid biopsy” in men with prostate cancer]
60

Stockholm3 blood test
Stockholm3 is a blood test that increases the detection of
aggressive prostate cancers [gleason 7 or more ] by 20% and, at the
same time, reduces the number of unnecessary biopsies by 50%.
Combining five protein markers(tPSA, fPSA, fhK2, PSP94, GDF-15),
over 100 genetic markers, clinical data and a proprietary algorithm,
Stockholm3 predicts the risk of aggressive prostate cancer.
61

URINE BASED BIOMARKERS
62

Prostate Cancer Antigen 3 (PCA3)/
Progensa test
• Long noncoding RNA that is not expressed outside of the prostate
prostate.its gene is located on chromosome 9q21-22
• Prostate cancer cells express 60 to 100 times more PCA3 RNA than
normal cells
• Unlike PSA, PCA3 levels are independent of prostate size
• PROGENSAPCA3 Assay is the first FDA-approved urine-based molecular
test that detects the over-expression of the PCA3 gene
• to decide whether a repeat biopsy is necessary in men with one or
more previous negative biopsies.
• but its clinical effectiveness for this purpose is uncertain.
63

Sample Preparation for PCA3
1. Conduct a DRE ,from the base to the apex,from the lateral to
the median line,exactly three strokes for each lobe
2. approximately 20 to 30 mL of the initial urine stream
at least 2.5 mL is required to run the PROGENSA PCA3 Assay
1. Unprocessed urine specimens, if not immediately processed,
must be maintained at 2°C to 8°C or kept on ice
64

Interpretation of PROGENSA PCA3 Assay Results
PCA3 Score is calculated as the ratio of PCA3 RNA copies to PSA
RNA copies, multiplied by 1000
Currently, the main indication for the Progensa test is to determine
whether repeat biopsy is needed after an initially negative biopsy
65

66

Gene Fusions-
- Gene fusion between transmembrane protease, serine 2 (TMPRSS2)and v-ets avian
erythroblastosisvirus E26 oncogene homolog (ERG) was reported as a recurring
event in PCa.
- mutated protein results in aberrant expression of ERG, which contributesto
oncogenesisby forcing progression through the cell cycle
- Highly-specific PCa biomarker that is presentin at least 50% of PCa cases
- Associatedwith more aggressive forms of PCa and poorer clinical prognosis (higher
PSA level, Gleason score and/or tumour stage)
- Detectablein urine as a potential non-invasive and convenientbiomarker for early
PCa-detection.
- Most importantly,TMPRSS2:ERGis a binary biomarker which is commonly associated
with premalignantPCa but not benign conditions,thus making it the most specific
PCa biomarker to date
- Also can be used as a tissue based marker
67

• Mi(chigan)ProstateScore [MiPS]: combining serum PSA, urine PCA3, urine
TMPRSS2-ERG – increases the likelihood of detecting prostae cancer
• SelectMDX test:
- is based on mRNA biomarker isolation from urine.
- presence of HOXC6 and DLX1 mRNA levels is assessed
- When additional risk factors (namely age, PSA, PSA density, family history
and rectal examination) are added to the calculation, the accuracy of
the test (negative predictive value) for excluding prostate cancer
rises to 98%,
- estimates risk of both presence of PCa on biopsy as well as presence of
high-risk cancer.
68

69

ExoDx Prostate IntelliScore
● Simple, non-DRE, urine-based, liquid biopsy test indicated for men 50 years
of age and older with a PSA 2-10ng/mL, or PSA in the “grey zone”,
considering an initial biopsy.
● Returns a risk score that determines a patient’s risk of clinically significant
prostate cancer (Gleason Score ≥7) on prostate biopsy.
● A score above the validated cut-point of 15.6 is associated with an increased
likelihood of GS≥7 PCa on a biopsy
● Help reassure a patient to avoid a prostate biopsy or improve patient
compliance with physician recommendation.
70

Other Urine Biomarkers
• Metabolomics: sarcosine
- the most prominent metabolite is sarcosine, a metabolite of glycine
- distinguishes malignant from benign prostate tissue
- also associated with progression to metastasis.
- Prostarix assay (Metabolon) is a commercial metabolomics panel
derived from this research that uses post-DRE urine.
• Annexin A3:
-inversely related to the presence of prostate cancer
-protein is part of a family of calcium and phospholipid binding
proteins that have been shown to be altered in cancer
• MicroRNA: small noncoding, single-strand RNAs
miR-141 & miR-187
71

Tissue based biomarkers
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• Alpha Methylacyl Co A Racemase (AMACR) :
upregulated, located on chr 5, 97% sensitivity and
100% specificity.
• Epigenetic modifications: hypermethylation of
GSTP1, APC, RARbeta2, RASSF1A
• Genomic expression profiles: Prolaris test,
OncotypeDx, Decipher test(22 genes).
• Gene susceptibility loci: BRCA, HOXB13, MSH2
73

Epigenetic
Modifications
- Genetic changes - alterations in DNA
sequence
- Epigeneticchanges - changes in DNA
methylation and histone acetylation status
- Segments within the gene promoter
( where transcription of gene is
initiated)that are composed of GCrich
regions are termed CpG islands
- Epigenetic modifications:
hypermethylationof GSTP1, APC, RARbeta2,
RASSF1A
74

ConfirmMDx
● Individuals presenting with risk factors, including abnormal
DRE, elevated/rising prostate specific antigen (PSA),
elevated/high risk biomarkers such as SelectMDx, in
combination with one or multiple negative prostate biopsies
● By testing for the presence of hypermethylation of GSTP1,APC,
RASSF1A in previous prostatic biopsy tissue able to identify
likelihood of detecting cancer in subsequent biopsy and which
area(s) of the prostate may be “hot spots” for prostate cancer,
subsequently targeting these zone(s) of suspicion at the time of
repeat biopsy
75

Genomic Expression Profiles
-Here a large set of genes will be evaluated together as a single
biomarker assay
-TESTS
1. Prolaris (Myriad Genetics, Inc.),
2. Oncotype Dx Prostate (Genomic Health, Inc.), and
3. Decipher (GenomeDx Biosciences, Inc).
help with management decisions in patients with newly
diagnosed prostate cancer regarding active surveillance or
primary treatment
77

1.Prolaris (Myriad Genetics, Inc.),
assesses 31 cell cycle progression
(CCP) genes and 15 housekeeping
genes
Accurately Predicts Risk of Prostate
Cancer Progression , biochemical
recurrence and metastasis, prostate
cancer– specific mortality
78

2.Oncotype Dx®:
• is a RNA-based test based on twelve
carcinoma-associated genes and five reference
genes .
• determines the aggressiveness of the
carcinoma.
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3.Decipher assay
patients classified by Decipher as low-, intermediate-, and high-risk categories
shows a 10-year cumulativeincidence of metastases after radical prostatectomy
of 5.5%, 15.0%, and 26.7%
Uses-
- In post-prostatectomysetting, Decipher may be used to guide decision
making regarding adjuvant or salvage radiation therapy and retrospective
data that patients with higher Decipher scores (≥0.4) may benefit from
postoperative radiation
- Decipher is now also available for clinical use in patients with newly
diagnosed prostate cancer to potentiallyassist with initial management
decisions,
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THANK YOU…
83

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