Professor of Clinical Pathology and Head Clinical Pathology Department, Faculty of Medicine, Mansoura University

Transcription

1 By Professor of Clinical Pathology and Head Clinical Pathology Department, Faculty of Medicine, Mansoura University

2 The first documented case of prostate cancer was reported in 1817.

3 In 1935 prostatic acid phosphate (PAP) levels were identified in the ejaculate of men, thus linking this enzyme to the prostate. Subsequent studies showed high PAP concentration in primary and metastatic prostate cancer tissues and in human serum, making it the first candidate marker for the diagnosis of prostate cancer.

4 Reductions in serum PAP levels were found to occur in response to antiandrogen therapy, whereas increasing serum levels were associated with treatment failure and relapse.

5 Meticulous sample collection and preparation are required because both platelets and leukocytes are contaminating sources of acid phosphatases and because PAP activity is rapidly lost at room temperature. Development of RIA for PAP in 1975 provided some improvement in test sensitivity, but the sensitivity levels were still inadequate for a detection of early-stage disease.

6 Therefore, it was clear that a more sensitive and robust indicator of disease presence would be required to detect prostate cancer in its earlier stages, when cure is more likely.

7 PSA is a kallekrein like serine protease that was first described in It is secreted from prostate epithelial cells and encoded by an androgen-responsive responsive gene located on chromosome 19q the main function of PSA is to liquefy human serum through its proteolytic action.

8 The target of PSA's enzymatic action are the three major gel-forming proteins, fibronectin, seminogelin I and seminogelin II. Their PSA-induced proteolysis causes liquefaction and subsequent release of motile spermatozoa. It is also secreted in small quantities from a number of other normal male tissues and even some female tissues.

9 It was first detected in the serum of prostate cancer patients in 1980, and a normal PSA serum concentration limit of 4 ng/ml for men was subsequently established. A serum level above 4 ng/ml was taken as an indicator of the trigger for further clinical evaluation.

10 False positive are a significant problem for the PSA test, elevations can occur due to benign prostatic hypertrophy (BPH) and prostatitis thus leading to unnecessary biopsies and other interventions. Of greater concern, 20-30% of men with prostate cancer have serum PSA levels in the normal range, resulting in undiagnosed disease.

11 Despite the drawbacks and criticisms, PSA is currently the best clinical marker available for prostate cancer and the only one approved by the FDA for both post-treatment treatment monitoring and, when combined with DRE, evaluation of asymptomatic men.

12 A common classification of total PSA concentration assigns risk ranges from 0-40 ng/ml, ng/ml and >10 ng/ml that are used to counsel patients on the likelihood of PCa and need to perform a prostate biopsy:

13 Total PSA<4 ng/ml: in the early stages of PSA application, men with PSA values below 4 ng/ml were assumed to be at low risk for PCa.. More recent data show that about 22% of men with a tpsa in the range ng/ml harbors significant PCa,, the majority of which are pathologically organ-confined and, hence, curable. An increase of detection rate by about 30% was observed when the PSA cutoff was lowered from 4 ng/ml to 3 ng/ml. the majority of these cancers were clinically significant and suitable for curative treatment. Based on these and other studies, lowering the PSA cutoff to 2.5 ng/ml has been suggested.

14 Total PSA range ng/ml is commonly refereed to as the diagnostic grey zone, and does not provide significant information as to whether an increased PSA is due to malignant or benign conditions. A prostate biopsy would reveal no evidence of PCa in three out of four men. Total PSA>10 ng/ml: show 50% likelihood of harboring PCa,, so it is widely accepted to perform biopsies on these men. The likelihood of organ- confined cancer may be as low as 20%.

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16 The majority of tpsa in serum is found in stable complexes with anti- proteases, whereas 5% to 25% of PSA occurs in the free, uncomplexed form. For still unknown reasons, men without prostate cancer have a higher ratio of free to tpsa when compared to men with PCa.

17 By measuring both free and total PSA, % fpsa can improve PCa detection. % fpsa cutoffs between 15% to 30% could eliminate 20% to 65% of unnecessary biopsies, while maintaining high sensitivity of PCa detection (70% to 100%).

18 Approximately 20% of men with tpsa from ng/ml harbor significant PSa.. Evaluation of a cutoff for % fpsa of 27% in this group demonstrated that it detected 90% of cancers, while 20% of unnecessary biopsies could be avoided.

19 Active PSA forms stable 1:1 complexes with alpha-1-antichymotrypsin antichymotrypsin (PSA-ACT), ACT), protein C inhibitor (PSA-PCI), PCI), alpha-1-protease protease inhibitor (PSA-API) API) and alpha-2-macroglobulin (PSA- AMG). PSA-ACT ACT is the major PSA form in serum and is strongly influenced by PCa.. Reports indicate that cpsa measurements enhance diagnostic performance compared to tpsa, which could translate into reduction of unnecessary biopsies, cost savings and reduced morbidity associated with biopsy.

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21 PSA is far from a perfect tumor markers: Other frequent disorders of the prostate e.g. BPH (values overlap with those of PCa) ) and prostatitis (values may tenfold) are associated with high PSA. Factors as physical activity, ejaculation, cystoscopy,, and prostate biopsy can temporarily serum PSA.

22 Five-alpha alpha-reductase inhibitors e.g. finasteride serum PSA concentration by 50% without affecting % fpsa. Free PSA is sensitive to sample handling, therefore, serum needs to be separated within a few hours after sampling, and if not analysed on the same day, it should be stored frozen. DRE, prostate biopsy, urethral instrumentation should be avoided hr. before analysis of fpsa and % fpsa to prevent false elevations in fpsa.. Additionally, % fpsa increases with prostatic volume in patients with PCa.

23 Potential prostate cancer markers Marker Chromosom e locus M r a Subcellular location Biochemical function Biological/cellul ar function A2M 12p Secreted Protease inhibitor Protein carrier Akt-1 14q Nucleus/cytoplasm Protein kinase Apoptotic inhibition AMACR 5p13.2-q11 42 Mitochondria/peroxiso me Racemase Stereoisomerizat ion Annexin 2 1q21 11 Plasma membrane Calcium and lipid binding Membrane trafficking Bax 19q Cytoplasm/membrane Bcl-2 binding Apoptosis Bcl-2 18q Mitochondrial membrane Membrane permeability Apoptosis Cadherin-1 16q Plasma membrane Catenin/integrin binding Cell adhesion Caspase 8 2q Cytoplasm Protease Apoptosis Catenin 5q Cytoskeleton Cadherin binding Cell adhesion

24 Potential prostate cancer markers Cav-1 7q Plasma membrane Scaffolding Endocytosis/signaling CD34 1q32 41 Plasma membrane Scaffolding Cell adhesion CD44 11p13 82 Plasma membrane Hyaluronate binding Cell adhesion Clar1 19q Nucleus SH3 binding Unknown Cox-2 1q Microsomal membrane Prostaglandin synthase Inflammatory response CTSB 8p Lysosome Protease Protein turnover Cyclin D1 11q13 34 Nucleus CDK b regulation Cell cycle DD3 9q Nucleus/cytoplasm Noncoding Unknown DRG-1 22q Cytoplasm GTP binding Cell growth/differentiation EGFR 7p Plasma membrane EGF binding Signaling EphA2 1p36 11 Plasma membrane Tyrosine kinase Signaling ERGL 15q Plasma membrane Lectin/mannose binding Unknown ETK/BMK Xp Cytoplasm Tyrosine kinase Signaling EZH2 7q Nucleus Transcription repressor Homeotic gene regulation

25 Potential prostate cancer markers Fas 11q Plasma membrane Caspase recruitment Apoptosis GDEP 4q c Unknown Unknown Unknown GRN-A 14q32 50 Secretory granules Statin Endocrine function GRP78 9q Endoplasmic reticulum Multimeric protein assembly Cell stress response GSTP1 11q13 23 Cytoplasm Glutathione reduction DNA protection Hepsin 19q Plasma membrane Serine protease Cell growth/morph ology Her-2/Neu 17q Plasma membrane Tyrosine kinase Signaling HSP27 7q Cytoplasm Chaperone Cell stress response

26 Potential prostate cancer markers HSP70 6p Cytoplasm Chaperone Cell stress response HSP90 11q13 63 Cytoplasm Chaperone Cell stress response Id-1 20q Nucleus Transcription factor Differentiation regulator IGF-1 12q Secreted IGFR ligand Signaling IGF-2 11p Secreted IGFR ligand Signaling IGFBP-2 2q Secreted IGF binding Signaling IGFBP-3 7p Secreted IGF binding Signaling/apoptosis IL-6 7p Secreted Cytokine B-cell differentiation IL-8 4q Secreted Cytokine Neutrophil activation KAI1 11p Plasma membrane CD4/CD8 binding Signaling Ki67 10q25-ter 358 Nucleus Nuclear matrix associated Cell proliferation KLF6 10p15 32 Nucleus Transcription factor B-cell development KLK2 19q Secreted Protease Met-Lys/Ser-Arg cleavage

27 Potential prostate cancer markers Maspin 18q Extracellular Protease inhibitor Cell invasion suppressor MSR1 8p22 50 Plasma membrane LDL receptor Endocytosis MXI1 10q Nucleus Transcription factor Myc suppression MYC 8q Nucleus Transcription factor Cell proliferation NF-kappaB 10q24 97 Nucleus Transcription factor Immune response NKX3.1 8p21 26 Nucleus Transcription factor Cell proliferation OPN 4q Secreted Integrin binding Cell-matrix interaction p16 9p21 17 Nucleus CDK inhibitor Cell cycle p21 6p Nucleus CDK inhibitor Cell cycle p27 12p Nucleus CDK inhibitor Cell cycle p53 17p Nucleus Transcription factor Growth arrest/apoptosis PAP 3q Secreted Tyrosine phosphatase Signaling PART-1 5q Nucleus/cytoplasm Unknown Unknown PATE 11q Plasma membrane Unknown Unknown

28 Potential prostate cancer markers PC-1 5q35 32 Nucleus RNA binding Ribosome transport PCGEM1 2q32 0 Nucleus/cytoplasm Noncoding Cell proliferation/sur vival PCTA-1 1q Cytoplasm Unknown Cell adhesion PDEF 6p Nucleus Transcription factor PSA promoter binding PI3K p85 5q Cytoplasm Lipid kinase Signaling PI3K p110 1p Cytoplasm Lipid kinase Signaling PIM-1 6p Cytoplasm Protein kinase Cell differentiation/s urvival PMEPA-1 20q Plasma membrane NEDD4 binding Growth regulation PRAC 17q Nucleus Choline/ethanolamine kinase Unknown Prostase 19q Secreted Serine protease ECM degradation Prostasin 16p Plasma membrane Serine protease Cell invasion suppressor

29 Potential prostate cancer markers Prostein 1q a Plasma membrane Unknown Unknown PSA 19q Secreted Protease Semen liquification PSCA 8q Plasma membrane Unknown Unknown PSDR1 14q a Nucleus/cytoplasm Dehydrogenase reductase Steroid metabolism PSGR 11p15 35 Plasma membrane Odorant receptor Unknown PSMA 11p Plasma membrane Folate hydrolase Cell stress response PSP94 10q Secreted FSH inhibitor Growth inhibition PTEN 10q Cytoplasm Protein/lipid phopatase Signaling RASSF1 3p Cytoplasm Ras binding Signaling RB1 13q Nucleus E2F-1 inactivation Cell cycle RNAseL 1q Cytoplasm/mitoch ondria RNAse Viral resistance

30 Potential prostate cancer markers RTVP-1 12q Plasma membrane Unknown Immune response/apoptosis ST7 7q /85 Plasma membrane Unknown Cell proliferation STEAP 7q Plasma membrane Unknown Unknown TERT 5p Nucleus Reverse transcriptase Telomere synthesis TIMP 1 Xp Secreted Protease inhibitor Cell adhesion TIMP 2 17q25 24 Secreted Protease inhibitor Cell adhesion TMPRSS2 21q Plasma membrane Serine protease Unknown TRPM2 8p Plasma membrane Calcium channel Ion flux Trp-p8 2q Plasma membrane Calcium channel Ion flux UROC28 6q Nucleus/cytoplasm Choline/ethanolami ne kinase Unknown VEGF 6p12 27 Secreted VEGFR binding Angiogenesis

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32 The markers displayed in table represent a wide array of biochemical and cellular functions. These functions include these of transcription factor, prostease, kinase,, phosphatase, proteases inhibitor, cyclin-dependent kinase inhibitor, cytokine, reverse transcriptase, racemase, reducatse, synthase, hydrolase, RNase,, molecular chaperone, nuclear matrix, membrane scaffolding, and an assortment of other binding and permeability control proteins. The question is, which of these 91 molecules, if any, are candidates for advancement "from the laboratory bench to the clinic".

33 The most important item regarding the selection of a candidate marker m is: The quality of scientific and clinical data supporting its potential utility.these include: The functional role to the biology of the disease. Clinical data linking the marker with disease presence, alternations in stage, response to therapy and/or over all survival.

34 The marker should be measurable by a robust, reproducible, widely available assay that provides useful information that is readily interpretable by the clinician.

35 The ideal candidate for an early detection or disease monitoring marker would be one that is: Prostate specific Detectable in an easily accessible biological fluid e.g. serum, urine, prostatic fluid. Able to distinguish between normal, BPH, prostatic intraepithelial neoplasia,, and cancerous prostate tissues. The marker should have sufficiently convincing clinical correlation data from several different labs before it is brought forward for large-scale evaluation.

36 Hk2 and PSA (hk3) share 30% identity in protein sequence and both are expressed under androgen control. By immune histochemical studies, PSA was shown to be more expressed in BPH than in PCa and the expression is higher in low-grade compared to high-grade grade tumors. By contrast, expression of hk2 shows intense staining in high-grade grade cancers compared to low grade cancers and still weaker staining in BPH.

37 This suggests that hk2 measurements may provide information independent of PSA, however, it cannot currently replace tpsa or fpsa for the early diagnosis of cancer. Limitations of immunodetection include different types of immunoassays, no standardization and careful pre-analytical sample workup.

38 BPSA: associated with BPH and is degraded form of free PSA that contains peptide bond cleavages at Lys 145 and Lys 182. It is a form that reflects benign prostatic hyperplasia and may have the potential to monitor BPH under surgical or medical treatment.

39 Truncated pro PSA forms: these are variants of free PSA characterized by incomplete removal of the seven-amino aminoacid propeptide that is normally cleaved to convert pro PSA to its mature active form. Examples are a form with removal of only three amino0acids, termed (-4)pPSA, and a form with removal of five amino-acids acids (-2)pPSA,( both have been linked with PCa.

40 Intact PSA: this cancer-associated associated form of free PSA which lacks the internal cleavage site of multichain free PSA forms, such as BPSA, and also has undergone complete removal of the propsa,, which differentiate it from the truncated propsa isofoms.

41 GRA-A A is a member of the granin family of proteins and acts as a prohormone,, which, after proteolytic processing, results in the generation of multiple peptides with biological activity. It is stored in the dense core secretory granules of most endocrine and neuroendocrine differentiation. Serum levels do not accurately distinguish BPH from prostate cancer very well, but they correlate with tumor stage and grade.

42 In addition, it has the capability to detect neuroendocrine cells and thus has the potential to identify androgen-independent disease. Two significant weakness of GRA- A as a marker are: not all prostate tumors contain neuroendocine cells. GRA-A A is unable to detect very early stage disease.

43 GSTP-1 1 is a member of a large family of glutathione transferases that function to protect cells from oxidative stress, thus the biological rationale for selecting this marker is its role in preventing damage to cells by neutralizing free radicals. This marker is also unique in its capacity to provide a methylation based detection method since in cancer prostate, its reduced expression due to promotor hypermethylation represents the most common epigenetic alteration associated with the disease.

44 Strengths of GSTP-1 1 as a clinical marker are The ability to quantitate the methylation status of the GSTP-1 1 gene in biopsy prostatectomy tissues and in cells derived from serum, urine and seminal plasma. Its high prevalence in this disease. It can be used as a sensitive marker for prostate cancer in men with clinically localized disease. The possible availability in the near future of drugs that can reverse hypermethylation make this marker a good candidate for further evaluation as an early detection marker, as well as a marker for post treatment monitoring of disease.

45 PSCA is a glycosyl phosphatidyl inositol- anchored call surface antigen that is found predominantly in prostate and may play a role in stem cell functions such as proliferation or signal transduction.

46 PSCA is highly expressed in the majority of prostate cancers and this is correlated with Gleason score and more advanced tumor stage, and progression to androgen-independent prostate cancer. It is also highly expressed in metastatic disease. Three weaknesses of PSCA are: The limited number of published reports supporting its value as a clinical marker. The need for better quantitation methods. The need for further evaluation as a therapeutic target.

47 PSMA is a type II intergral membrane protein that displays multiple enzymatic activities. It has been detected in prostate tissues (IHC-Western analysis), in circulating prostate cancer cell (RT- PCR), and in serum (ELISA). A weakness of PSMA as a clinical marker for early diagnosis is that it is elevated in breast cancer patients. In addition, serum level PSMA have been shown to increase with increasing age, which could be a confounding factor in a disease that most often occurs late in life.

48 New technologies are being developed that allow quantitative high-throughput analysis of this marker in biological fluids to evaluate its use in prostate cancer detection or treatment monitoring.

49 The TERT gene encodes the reverse transcriptase component of telomerase that maintains the telomeric ends of chromosomes and has been associated with senescence and cancer. It is expressed in cells that exhibit telomerase activity and is undetectable in most benign tissues. The biological rationale for selecting TERT is its ability to confer cellular immortalization, a major step in the process of malignant transformation and consequently provide a very sensitive means for detecting infiltrating cancer cells in benign tissue.

50 Although TERT activity level appear to be independent of PSA, the "value added" of TERT for early detection, staging, on prognosis and the overall clinical utility of TERT remain to be fully uncovered.

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52 Conclusion The goal of this effort is not to dictate the optimal markers or the methodologies for their verification, but to provide a framework from which the general research community can work toward achieving the goal of bringing new prostate cancer marker forward for clinical use.

53 Recently, methodologies as divers as positional cloning, differential display, comprehensive DNA expression analysis and serum proteomic analysis have provided some preliminary yet exciting prostate cancer marker candidates.

54 Candidate markers identified by these new technologies will require confirmation and correlation with disease formation or progression or with patient survival or response to therapy before they can be considered for validation studies.

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