BIOMARKERS FOR DIAGNOSIS AND PROGNOSIS OF PROSTATE CANCER WITH RECENT UPDATE OF GRADING SYSYTEM

2. PROSTATE CARCINOMA
BIOMARKERS AND RECENT
GRADINGS
Dr. SUDIP ROY
JR, IPGME&R, KOLKATA

3. Overview:-
• Introduction
• Discovery
• PSA and related factors
• Newer biomarkers for diagnosis and prognosis
• New grading system
• Take home message

4. Introduction:-
• Prostate cancer is the 2nd most common
diagnosed malignancy (Incidence)
• 5th leading cause of cancer-related death
• Most prevalent cancer of men
• Newer biomarkers needed for early detection
and therapeutic targets for needed

5. Prostate-specific antigen (PSA)
• PSA is present in small quantities in the serum of
men with healthy prostates
• PSA is the most used test to detect carcinoma.
• limited specificity and an elevated rate of over
diagnosis

6. PSA elevated in
prostate cancer,
prostatitis,
irritation,
benign prostatic hyperplasia (BPH),
recent ejaculation and
Digital rectal examination(DRE)

7. PSA in Serum
Disruption of this epithelium due to disease
diffusion of the antigen
into blood
causes of elevated blood levels of PSA
o Obesity reduce serum PSA levels

8. Screening
• annual screening in men of age 50 and older
• Normally less than 4 ng/mL
• PSA levels between 4 and 10 ng/mL are suspicious
for malignancy
• >10ng/mL considered as high risk for malignancy

9. Risk categorization
D'Amico Criteria
• Low-risk: PSA < 10, Gleason score ≤ 6, AND clinical
stage ≤ T2a
• Intermediate-risk: PSA 10-20, Gleason score 7, OR
clinical stage T2b/c
• High-risk: PSA > 20, Gleason score ≥ 8, OR clinical
stage ≥ T3

10. Histology
• PSA is produced in the epithelial cells of the
prostate
• can be demonstrated in biopsy samples by using
IHC
• PSA remains present in malignant prostate cells
• Prostate cancer cells generally have variable or
weak staining for PSA due to the disruption of their
normal functioning

11. • individual prostate cancer cells produce less PSA
than healthy cells.
• increased number of cells
causes raised serum levels
• identify metastasis.
• some high-grade prostate
cancers may be entirely negative for PSA
Normal prostate tissue with PSA IHC

12. PSA kinetics:-
• PSA velocity
–rate of increase of PSA per year (ng/ml/year)
–valuable in prostate cancer prognosis.
–increased by more than 2.0 ng per milliliter
during the year -higher risk of metastasis
• PSA doubling time (PSA DT)
–identify life threatening disease before start of
treatment (n=>10 yrs)

13. Free PSA
• not protein bound - 'free PSA'.
• In prostate cancer the ratio of free (unbound) PSA
to total PSA is decreased.
• The risk of cancer increases if the free to total ratio
is less than 25%
• Help to eliminate unnecessary biopsies when PSA
levels between 4 and 10 ng/mL.

14. Inactive PSA
• Proteolytically active PSA has been shown to have
an anti-angiogenic effect
• inactive subforms are associated with prostate
cancer
• identified by MAb 5-D3-D11
• inactive proenzyme forms of PSA is another
potential indicator of disease.

15. PSA Density (PSA-D)
• PSA density =
total PSA (ng/ml)
prostate volume (ml)
• In benign diseases less than 0.06 ng/ml2
• Increased value associated with the risk prostatic
carcinoma
• Omitting prostate biopsy with Gleason Score ≥7
tumors with PSA-density
–≤0.07 ng/ml2 may miss 6.9%
–0.10 ng/ml2 may miss1.3%

16. • PSA-density
–Information about biopsy decisions,
–spare some men from unnecessary prostate
biopsy and
–diagnosis of low-grade prostate cancer.
BUT
• its use for biopsy decisions is conflicting
• not commonly recommended in guidelines
*Nordström, T., Akre, O., Aly, M., Grönberg, H., & Eklund, M. (2017, December 19).
Prostate-specific antigen (PSA) density in the diagnostic algorithm of prostate cancer.
https://www.nature.com/articles/s41391-017-0024-7

17. Post-treatment monitoring
• PSA levels are monitored periodically (every 6–36
months) after treatment of high-risk disease
• less frequently in patients with lower-risk disease
• After successful therapy PSA becomes undetectable
within a few weeks.
• A subsequent rise above 0.2 ng/mL - recurrence

18. • After successful radiation therapy little PSA may be
detected
• recurrent prostate cancer - "biochemical
recurrence"

19. OTHER MARKERS

20. Newer Promising Biomarkers
PCA 3 score
PSA glycoforms,
TMPRSS2:ERG fusion gene,
microRNAs,
circulating tumor cells and
androgen receptor variants.

21. aggressiveness determination by
 PHI and
 the four kallikrein panel
assessment of cancer prognosis
Prolaris®
Oncotype DX®
Prostate-Specific Membrane Antigen (PSMA)
select the treatment by
 androgen receptor splice variant-7 (AR-V7)

22. PCA3
• only expressed in human prostate tissue
• the gene is highly overexpressed in prostate cancer
• because of its restricted expression profile, the
PCA3 RNA is useful as a tumor marker
• PCA3 is also known to as DD3

23. Discovery:-
• PCA3 was discovered in 1999 by MJ Bussemakers et.
al.
• done by comparing the mRNA expression patterns
of normal v/s tumor tissue of the human prostate
• then cDNA library of mRNA extracted

24. • six overlapping genomic phages that cover
the DD3 gene were isolated and characterized
• then structure of the DD3 transcription unit
identified
Genetic structure of PCA3 gene

25. Genetic structure:-
• DD3/PCA3 gene present in ch 9q21–22
• three reading frames
• DD3 cDNA has the high density of stop codons
• lack of an extensive open reading frame.
• the DD3gene consists of four exons

26. • alternative polyadenylation occurs at three different
positions in exon 4 (4a, 4b, and 4c)
• exon 2 is only present in 5% of the cDNA clones
• often skipped by alternative splicing
Structure of the DD3 transcription unit

27. • the DD3 gene is about 25 kb
• the first exon is relatively large (appr. 20 kb);
• whereas exon 2,3 and 4 are small
• several small open reading frames scattered
throughout the DD3 gene
• located in exons 3 and 4a
• So, the protein product is likely to be very small.

28. Relation of PCA3 with Prostate cancer:-
• 10–100 fold overexpression in the tumor areas than
adjacent non-neoplastic prostate tissue
• Expression is observed in almost all stages of the
tumors
• Up-regulation of DD3 expression is an early event in
prostate cancer development

29. • PCA3 expression is found in well-differentiated,
moderately differentiated and poorly differentiated
tumors
• Trend toward more expression in the poorly
differentiated tumors

30. Use as biomarker
• Urine-based PCA3 diagnostic tests is noninvasive
• Compared to serum PSA, urinary PCA3 has
–lower sensitivity but
–higher specificity and
–better positive and negative predictive value.
• PCA3 is independent of prostate volume compared
with PSA

31. • Suspected by DRE and PSA, approximately 60% of
cases 1st biopsy is normal
• On repeat testing, 20-40% have an abnormal biopsy
result
• PCA3 is useful to avoid these repeated biopsy
• Also found in metastatic lesions

32. PCA3 and prognosis:-
• PCA3 score and grading correlated statically with
prognosis
• PCA3 score lower than 51 and Gs ≤ 6 shows
comparatively good prognosis
• PCA3 score higher than 51 and a Gs ≥ 7 – poor
prognosis

33. Commercial availability:-
• A commercial kit called the Progensa PCA3 test is
marketed by Gen-Probe
• the first portion of urine
after prostate message
• The PCA3 Test measures
both PCA3 and PSA mRNA in a urine sample
• The PSA mRNA measurement ensures that prostate
cells were collected in the sample

34. • Using RT-PCR , PCA3 and PSA mRNA are amplified
from the sample
• The result is reported as a ratio of PCA3 mRNA to
PSA mRNA
• samples having cancer -higher PCA3/PSA ratios
• PSA mRNA measurements below a certain
detection amount produce an inconclusive test

35. • PCA3 levels were significantly higher among men
who were subsequently diagnosed
• the PCA3 score was significantly higher with
Gleason ≥7 tumors , clinical stage T2 disease

36. PSA glycoforms
• Based on different glycosylation patterns between
PCA patients and healthy subjects
• Significant increase of α2,3-sialic acid in malignancy
than BPH
• Identifies aggressive PCA and correlates with
Gleason score

37. • The cutoff value is 30%
• Helps in differentiates between high-risk PCa and
the groups of BPH, low- and intermediate-risk PCa
• PSA fucosylation also increases
• Decrease in core fucosylation of PSA in carcinoma

38. TMPRSS2:ERG fusion gene
• Transmembrane Protease; Serine 2
• Common in hematologic malignancies
• Can be detected in urine samples obtained after a
prostate massage
• RT-qPCR
• TMPRSS2:ERG score =
TMPRSS2:ERG mRNA
PSA mRNA

39. • TMPRSS2:ERG score and PCA3 score combined
significantly increased the predictive value of
diagnosis
• TMPRSS2:ERG score associated with –
the biopsy Gleason score and
tumor clinical stage.
• ExoDx Prostate IntelliScore measures
 PCA3, SPDEF, and ERG

40. MicroRNAs
• miRNAs are involved in all steps of carcinoma
development,
cell proliferation,
differentiation, and
progression.
• cancer stem cells proliferation and differentiation
miR-34 family and
let-7 family upregulated
• apoptosis
miR-21 is upregulated to reduce apoptosis

41. Circulating tumor cells
• Tumor cells can acquire the ability to pass vessel
walls and enter into the bloodstream.
• Facilitate the establishment of metastatic focus
• High proportions are commonly found in advanced
metastatic stages,
• Very rare in early stages

42. • CTCs present a heterogeneous phenotype, due to-
intratumor heterogeneity and
differences between primary tumor and
metastases
• Heterogeneity linked with therapy resistance
• Help to make therapeutic decisions
• No sufficient prognostic value in localized ca.

43. • CellSearch® platform has approved for monitoring
of metastatic breast, colon, and Prostate cancer
• Based on immunomagnetic capture followed by a
combination of positive and negative
immunological selections.
• In advanced stages, Androgen Deprivation Therapy
(ADT) help to slow carcinoma progression by CTC
identification

44. Androgen receptor(AR)
• SPOP or FOXA1 mutations
• Transcription factors are the main driver of CRPC
development
• AR gene amplification and overexpression,
• AR mutations, and
• expression of constitutively active AR variants
(AR-Vs).
• OncotypeDX AR-V7 Nucleus Detect

45. Prostate Health Index (PHI)
• PHI combines total PSA, fPSA, and p2PSA
•
• Useful in men >50 years, PSA between 4 and 10
μg/L, and a nonsuspicious digital rectal examination
(DRE).
• PHI demonstrated a higher accuracy than total PSA
and %fPSA
𝐏𝐇𝐈 =
𝐩𝟐𝐏𝐒𝐀
𝐟𝐏𝐒𝐀
𝐱 𝐭𝐨𝐭𝐚𝐥𝐏𝐒𝐀

46. • Significantly higher in patients with a Gleason score
≥7 than score 6.
• Values for PHI high in patients with clinical stage
T2–T3 than stage T1c.
• PHI density has a higher discriminative ability to
detect clinically significant PCa than PHI

47. Four-kallikrein panel
• Comprises
• total PSA,
• fPSA,
• iPSA, and
• human kallikrein 2
• Excellent diagnostic tool for high-grade carcinoma

48. • To reduce the over detection
• 30% of biopsies could be avoided
• able to predict the long term development of
distant metastasis

49. Prognostic factors
• Gleason score - most valuable and conventional
predictor
• Gleason score ≥7 require a radical treatment
• Gleason score 6 could probably have carcinoma
with low risk of progression
• Active surveillance is the best option for them.

50. Prolaris test
Oncotype DX test
• Recommends for very-low and low risk PCa in
patients
• Choice between active surveillance and definitive
therapy.
• Prolaris evaluate of 31 cell cycle progression genes
related to cancer proliferation
• Oncotype DX test evaluate 5 housekeeping genes
related to metastasis.

51. Prostate-Specific Membrane Antigen
• Transmembrane protein
• Expressed in all types of prostatic tissue
• Acts as a glutamate-preferring carboxypeptidase
• Upregulated in androgen deprivation
• Independent indicator of poor prognosis
• Possible target for chemotherapy

52. Recent Biomarkers
• Prostate Stem Cell Antigen - difficult
• AMACR (Alpha Methyl Acyl Coenzyme A Racemase)
• TFF3
–high expression in prostate cancers but
statistically insignificant predictors by using urine
samples
• GOLPH2 (Golgi phosphoprotein 2) – in study

53. Limitations
• Performance has not been established in patient
had biopsy less than three months ago
• Effect of medications unknown
• Other diagnostic procedures as radiation may affect

55. PROGNOSTIC SCORRINGS

56. Gleason grading system
• Original scheme established in the 1960–70s
• Incorporated into the WHO classification of prostate
cancer
• New “grade group” system proposed by the 2014
ISUP consensus
• Adopted by the who classification of tumours of the
prostate in 2016

57. The classical Gleason system
• Five histological growth patterns (grades) based on
architectural patterns
• Gleason 1 represents the best differentiated
most favorable prognosis,
• Gleason 5 the least differentiated
poor prognosis.

58. • Gleason pattern 1 - well-circumscribed, nodular
lesion- VERY RARE
• Gleason pattern 2 –
–variations in sizes of the neoplastic glands,
–slightly increased stroma between the glands,
and
–slight irregularity at the periphery of the nodule
–rare

59. • Gleason pattern 3 –
–most common pattern,
–distinct neoplastic glands,
–typically small, but often of variable sizes and
–infiltrating into the stroma in between the benign
glands.
• Gleason pattern 4 –
• fused glands, no individual or distinct gland,
• broad, irregular cribriform patterns

60. • Gleason pattern 5 –
–Comedo type necrosis
• Gleason score = the sum of the primary and
secondary patterns (grades)
• With just one pattern, the primary and secondary
patterns are considered the same

61. • The major changes in the 2016 WHO blue book are
–Cribriform glands should be assigned Gleason
pattern 4.
–Glomeruloid glands should be assigned Gleason
pattern 4.
–Mucinous carcinoma should be assigned Gleason
pattern 4.
• According to the 2004 WHO blue book, rare
cribriform glands diagnosed as pattern 3

62. Grade groups:
• By 2014 ISUP consensus conference on Gleason
grading of prostatic carcinoma
• These grade groups are as follows:
–Grade group 1: Gleason score ≤6
–Grade group 2: Gleason score 3 + 4 = 7
–Grade group 3: Gleason score 4 + 3 = 7
–Grade group 4: Gleason score 4 + 4 = 8, 3 + 5 = 8,
5 + 3 = 8
–Grade group 5: Gleason scores 9–10

64. Take home message
• Prostate cancer is the 2nd most common diagnosed
malignancy (Incidence); Most prevalent cancer of
men
• PSA remains the most used marker to detect
carcinoma
• Most important limitation is over diagnosis
• D'Amico Criteria Risk categorization is done by
combination of PSA, Gleason score, clinical stage

65. • PSA velocity >2.0 ng /ml -higher risk of metastasis
• The risk of having cancer increases if the free to
total PSA ratio is less than 25%
• Post-treatment PSA becomes undetectable within a
few weeks
• PCA3 is highly overexpressed in prostate cancer
(10–100-fold)
• Urine-based PCA3 estimation by RT-qPCR

66. • TMPRSS2:ERG fusion gene estimated from urine by
RT-qPCR
• Therapy resistance can determined by CTC
• Prostate-Specific Membrane Antigen is a marker of
prognosis
• Gleason grading system based on architectural
patterns
• WHO Grading 2016 based on modified Gleason
grading, the grade groups

67. REFERENCES