Prostate Cancer: Blood, Urine and Tissue-based markers

Blood Based Biomarkers

PSA

• See Prostate Cancer: Diagnosis and Evaluation

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[1]
• 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[2][3]
• Circulates in bound/complexed (70-80%) and unbound/free (20-30%) forms
  • Bound/complexed 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)
         • Generally limited to transition zone tissue
      3. proPSA
         • Uncleaved free PSA molecule with a leader sequence (see above)
         • Evaluated as a marker with conflicting results
         • Exists in 3 forms: -2proPSA, -4proPSA, -7proPSA
      4. Normally, these isoforms exist in equal concentrations
         • BPSA is highly expressed in BPH
         • proPSA is highly expressed in prostate cancer

Landmark Studies

• 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 NEJM)
    • 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
      • Did not directly compare PSA between benign vs. malignant cases
      • Half-life: 12.6h+2.2 days; undetectable in most RP patients POD14; detectable PSA at 3 weeks = residual disease
    • 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 NEJM)
    • 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
      • Diagnosis of prostate cancer in all men (healthy and underwent biopsy):
        • PSA < 4.0: 11%
        • PSA 4-9.9: 24%
        • PSA ≥10: 65%
          • This demonstrates that:
            1. There is no "normal" value of PSA i.e. prostate cancer can be found with "low" (<4.0 ng/dL) PSA
            2. PSA is a continuum of risk i.e. the higher the value, the higher the risk
      • Multivariable analysis: PSA most predictive of cancer, followed by DRE > TRUS > age
    • Catalona, William J., et al. "Measurement of prostate-specific antigen in serum as a screening test for prostate cancer." New England Journal of Medicine 324.17 (1991): 1156-1161.
    • Study establishes role of PSA in screening (not role of prostate cancer screening)
• 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 JU)
    • 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

Prostate Health Index (PHI)

• Incorporates (3):
  1. fPSA
  2. tPSA
  3. -2proPSA
• Improved specificity compared to total PSA[4]
• 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
• Incorporates 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)

• See Prostate Cancer: Diagnosis and Evaluation Chapter Notes

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

1. What are the components of the Prostate Health Index? 4K score?
2. What does the PCA3 ratio represent?
3. DNA hypo vs hypermethylation, which is associated with silencing vs. activation?

Answers

1. What are the components of the Prostate Health Index? 4K score?
   • PHI: fPSA, tPSA, and -2proPSA
   • 4K: fPSA, tPSA, intact PSA, and hK2
2. What does the PCA3 ratio represent?
   • Urine PCA3 mRNA to urine PSA mRNA
3. DNA hypo vs hypermethylation, which is associated with silencing vs. activation?
   • Hypo: activation
   • Hyper: silencing

References

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