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PROSTATE CANCER: PSA & OTHER MARKERS

Blood based biomarkers (PSA, PSA-derivatives, PHI, 4Kscore, PSMA)

PSA

PSA Basics
- Discovered in 1979
- Member of kallikrein gene family
- An enzyme (serine protease) which functions to liquify semen
- Molecular weight: 33-kD
  - Begins as 17-amino acid chain preproPSA --> cleavage results in inactive proPSA --> cleavage by hK2 results in active form PSA
- Primarily produced by prostatic luminal epithelial cells
  - Has been detected in other body fluids and tissues: in females, PSA is found in female ejaculate at concentrations roughly equal to that found in male semen; other fluids with high concentrations of PSA include breast milk and amniotic fluid.
  - Ectopic expression of PSA occurs in normal breast tissue, and tumours of the adrenals, kidneys, colon, ovaries, liver, and parotid§
- Expression is strongly influenced by androgens; becomes detectable at puberty with increasing levels of luteinizing hormone and testosterone
  - Clinical implication: in hypogonadal men with low testosterone levels, serum PSA level may be low because of decreased expression and may not reflect the presence of prostate disease such as cancer
- Half-life: 2-3 days§§
- Circulates in bound/complexed (70-80%) and unbound/free (20-30%) forms
  - Bound/complesed PSA
    - Proteins that bind PSA in blood (3):
      1. α1-antichymotripson (ACT)
        
        Binds most PSA in an irreversible fashion
        
        Detectable form of bound PSA
      2. α2-macroglobulin (A2M)
        
        Binds 5-10% of PSA
        
        PSA-A2M complex undetectable by most current assays
      3. α1-protease inhibitor (API)
        
        Binds 1-2% of PSA
        
        Detectable form of bound PSA (to a lesser extent than PSA bound to ACT)
  - Free PSA
    - Rendered inactive within the prostatic epithelial cell before release into the serum
    - Isoforms (3):
      1. Intact PSA
      2. BPH-associated PSA (BPSA)
      3. proPSA
      - Normally, these isoforms exist in equal concentrations
        
        BPSA is highly expressed in BPH
        
        proPSA is highly expressed in prostate cancer
PSA in Prostate Cancer Detection
- After PSA was discovered in 1979, small studies in the early 80's evaluated PSA as a marker, but it had not yet become widely accepted as a prostate cancer marker
- Stamey et al. (1987)
  - Population: Cohort study of 699 men aged 21-76 selected from an executive health-screening program
    - Excluded if abnormal DRE (including BPE), history of prostate disease, LUTS, or abnormal U/A
  - Results
    - PSA positively correlated with age, cancer stage, prostate volume, cancer volume
  - Stamey, Thomas A., et al."Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate." New England Journal of Medicine 317.15 (1987): 909-916.
  - Study establishes PSA as a marker for disease progression and response to treatment (not role in screening)
PSA in Prostate Cancer Screening
- Catalona et al. (1991)
  - Population:
    - Cohort study of 1953 men aged ≥50
      - 1653 healthy men, 300 men undergoing biopsies for symptoms or abnormal findings on rectal exam
        
        In healthy men, biopsy was recommended if they had two serum PSA ≥ 4.0 ng/dL and abnormal finding on DRE or TRUS
  - Results

Value of DRE in Prostate Cancer Screening

Prior to the introduction of the PSA test, prostate cancers were diagnosed more often in advanced stages (symptoms, abnormal DRE, etc.)
With the adoption PSA as a marker for prostate cancer, it has been questioned whether the DRE is still necessary i.e. will DRE detect cancers that are missed by PSA? If not, consideration should be given to omitting DRE due to likely patient preference for a blood test
Catalona et al. (1994)
- Population:
- Prospective study of 6630 volunteers aged ≥50 recruited from community via advertisement who underwent PSA test and DRE
  - If PSA >4.0 ng/dL OR abnormal DRE, underwent biopsy
- Results:
  - 18% (n=1167) underwent biopsy, of which 23% (n=264) were found to have cancer

	DRE+ (% (n))	DRE- (%(n))
PSA+	37 (98)	45 (118)	82 (216)
PSA-	18 (48)	0 (0)	18 (48)
	55 (146)	45 (118)	100 (264)

Detection rates are higher with PSA testing alone vs. DRE alone, and highest with the tests together

Catalona, William J., et al."Comparison of digital rectal examination and serum prostate specific antigen in the early detection of prostate cancer: results of a multicenter clinical trial of 6,630 men." The Journal of urology 151.5 (1994): 1283-1290.
Study establishes the role of PSA AND DRE in prostate cancer screening

Another cohort study of 36,000 men participating in a prostate cancer screening study found that:
- 14% of prostate cancers were detected by DRE alone
- 64% by PSA only
- 22% by abnormal PSA and DRE

PSA Derivatives
- Concept is to adjust for known factors that can influence PSA (age, volume, race (BMI can also influence PSA)) and improve specificity
  - Patients with prostate cancer may have a “normal” PSA (i.e. false-negative) and patients without prostate cancer may have "elevated" PSA (i.e. false-positive); PSA is neither sensitive nor specific, particularly between 4.0-10.0 ng/dL.
- Volume-adjusted PSA
  - Concept is to reduce confounding from BPH
  - Includes PSA divided by prostate volume (PSA density, PSAD), complexed PSAD (cPSA divided by prostate volume), and PSA transition zone density (PSA divided by transition zone volume, PSAT)
    - A PSAD of ≥0.15 has been proposed for recommending prostate biopsy in men with PSA levels between 4-10 ng/mL and normal DRE
    - PSAT has been developed to adjust for the transition zone volume, the major determinant of serum PSA in men without prostate cancer
  - Prostate volume typically determined by US
- Age-/race- adjusted PSA
  - Race: African-American men without prostate cancer have higher PSA values than Caucasian men
  - Age: PSA normally increases with age
    - The mature prostate is between 20-25 g and remains relatively constant until ≈age 50, when the gland enlarges in many men; the average prostate volume in a 60-70 year old is ≈48 g
- PSA velocity
  - Short-term fluctuations in PSA can occur between measurements in the presence or absence of prostate cancer, primarily as a result of physiologic variation. However, the rate of change in PSA (PSAV)—PSA corrected for the elapsed time between measurements is associated with the risk for prostate cancer
  - PSAV > 0.75 ng/mL/year has been shown to be a specific marker for the presence of prostate cancer in men with PSA levels between 4-10 ng/mL
  - There are conflicting studies on whether PSAV provides more information than total PSA in predicting disease aggressiveness
- Free-PSA (fPSA)
  - %fPSA varies directly with age and volume, and indirectly with total PSA. Does not vary by race.
  - Low %fPSA is associated with increased risk of prostate cancer
    - PSA produced by malignant cells escapes proteolytic processing more frequently, resulting in a greater fraction of serum PSA complexed to α1-antichymotrypson and a lower %fPSA compared to men without cancer
    - Catalona et al. (1998)
      - Population:
        
        Prospective cohort study of 773 men aged 50-75 enrolled primarily through screening centers with PSA 4-10 and palpably benign gland that underwent diagnostic biopsy
        
        49% had cancer, 51% had benign disease
      - Primary outcome:
        
        %fPSA that maintained 95% sensitivity for PC detection
      - Results:
        
        %fPSA was inversely associated with risk of cancer
        
        AUC 0.72 %fPSA vs. 0.53 total PSA
        
        %fPSA cut-off:
        
        ≤25: sensitivity: 95%, specificity 20%
        
        ≤22: sensitivity 90%, specificity 29%

Catalona, William J., et al. "Use of the percentage of free prostate-specific antigen to enhance differentiation of prostate cancer from benign prostatic disease: a prospective multicenter clinical trial." Jama 279.19 (1998): 1542-1547.

%fPSA as a test is FDA-approved in men with a total PSA 4-10ng/mL and negative DRE
- %fPSA is most useful in the setting of PSA levels < 10ng/ml because the PPV of tPSA > 10- 20ng/ml has been shown to be ≈80%; it’s utility in PSA <4.0ng/ml is unknown
- No %fPSA threshold has been established, proposed cut points generally range from 15-25%
- Debate remains surrounding the utility of %fPSA as a prognostic biomarker

Complexed-PSA (as opposed to the free-PSA)
- Overall, at a high sensitivity, cPSA provides higher specificity than tPSA and comparable specificity to %fPSA in prostate cancer detection. A potential advantage of cPSA is the requirement for one assay

5ARIs lower total PSA levels by ≈50% after 12 months of treatment
- With the use of 5ARIs, fPSA decreases in a similar fashion to tPSA, and the %fPSA is not altered significantly
- Finasteride 1 mg (Propecia) used for male pattern hair loss (androgenic alopecia) results in the same decline in serum PSA levels as the 5-mg dosage used for the treatment of BPH
- In the PCPT trial, PSA had statistically significantly better sensitivity and AUC for detecting prostate cancer in the finasteride arm§

Prostate Health Index (PHI)
- Incorporates (3):
  1. fPSA
  2. tPSA
  3. -2proPSA
- Improved specificity compared to total PSA§
- FDA approved for males age ≥ 50 with tPSA 4-10 ng/ml and negative DRE
4kscore
- Human kallikrein 2 (hK2) is a closely related serine protease in the PSA/kallikrein gene family
  - In benign epithelium, PSA is intensely expressed compared with hK2
  - In prostate cancer, hK2 is expressed more intensely
- Incororates 4 kallikrein forms:
  1. fPSA
  2. tPSA
  3. Intact PSA
  4. hK2
- Intended use for men with elevated PSA considering biopsy
Prostate-Specific Membrane Antigen (PSMA)

Urine-based biomarkers

Prostate Cancer Antigen (PCA) 3

Long non-coding RNA on chromosome 9q21-22 that is not expressed outside of the prostate
Function remains unknown
Increased in malignant tissue compared to benign tissue; independent of prostate size
Urine samples are collected after an “attentive” DRE involving 3 firm strokes on each lobe of the prostate towards the median sulcus. The first 20-30 mL of voided urine should be collected within 1 hour.
The PCA3 score is reported as the ratio of urine PCA3 mRNA to urine PSA mRNA x 1000, normalizing PCA3 expression to PSA expression.
- Represents a continuum of risk; different thresholds lead to difference in sensitivity and specificity. Multiple cutoffs have been proposed, most commonly 10, 25, and 35.
FDA approved for use after negative biopsy and in initial screening

Gene fusions

TMPRSS2:ERG gene fusion
- One of the earliest events that occurs in prostate carcinogenesis
- Close to 100% specific for prostate cancer, when present. However, only present in approximately 50% of PSA-screened prostate cancers, and therefore its sensitivity is substantially lower
- Most promising application currently appears to be as a urine biomarker as part of a multiplex assay with PCA3

Others: ExoDX Prostate, MiPS, SelectMDX

Tissue-based biomarkers

Epigenetic modifications

Epigenetic events affect gene expression without altering the actual sequence of DNA. Known mechanisms include hypomethylation, chromatin remodeling, and miRNA and lncRNA regulation.
Segments within the gene promoter that are composed of glycine-cytosine-rich regions are termed CpG islands. Alterations in the methylation status of these regions may affect gene expression and have been shown to play a role in carcinogenesis

DNA hypermethylation generally causes gene silencing

Key hypermethylated genes that may have a role in prostate cancer include GSTP1, APC, RARβ2, and RASSF1A

DNA hypomethylation causes activation of oncogenes and leads to genetic instability

ConfirmMDx uses a quantitative methylation assay and has been shown to have a negative predictive value of 0.90 and sensitivity of 0.68 for the presence of cancer in tissues from “negative”-biopsies

Gene expression profiles

Polaris assesses 31 cell cycle progression genes and demonstrated an association between this gene signature and risk of progression and death from prostate cancer
OncotypeDx uses gene sets in several biologic pathways and predicts the likelihood of aggressive pathologic features at the time of prostatectomy
Decipher test predicts development of metastatic disease in men with high-risk disease and men with biochemical recurrence

Others: Promark

Questions

What is the function of PSA? What is the half life?
How does PSA circulate in the blood?
When does serum PSA become detectable?
What are the components of the Prostate Health Index? 4K score?
What does the PCA3 ratio represent?
DNA hypo vs hypermethylation, which is associated with silencing vs. activation?

Answers

What is the function of PSA? What is the half life?
- Liquify semen
- 2-3 days
How does PSA circulate in the blood?
- Circulates free (20-30%) and bound (70-80%).
- Bound to 3 proteins: α1-antichymotripson, α2-macroglobulin, α1-protease inhibitor
When does serum PSA become detectable?
- Puberty
What are the components of the Prostate Health Index? 4K score?
- PHI: fPSA, tPSA, and -2proPSA
- 4K: fPSA, tPSA, intact PSA, and hK2
What does the PCA3 ratio represent?
- Urine PCA3 mRNA to urine PSA mRNA
DNA hypo vs hypermethylation, which is associated with silencing vs. activation?
- Hypo: activation
- Hyper: silencing

Next Chapter: Biopsy

References

Wein AJ, Kavoussi LR, Partin AW, Peters CA (eds): CAMPBELL-WALSH UROLOGY, ed 11. Philadelphia, Elsevier, 2015, chap 108