Molecular Markers in Diagnosis and Prognostication of Bladder Cancer Farhat Abbas M.D. Associate Professor Department of Surgery (Urology) The Aga Khan University, Karachi, PAKISTAN Bladder cancer ! ! ! ! ! 2nd most common GU tumor 15 per 100,000 persons USA: 53,200 new cases; 12,200 deaths / year 90% TCC 70% Superficial, 30% muscle-invasive Superficial bladder cancer ! ! ! ! ! 70% will recur 10 -15% will progress Grade 1: < 10% risk of recurrence and progression Grade 3: 70 - 80% recurrence 33-45% progression Recurrence and progression may occur with > 5 year interval Therefore there is a strong need for long-term surveillance Situation in underdeveloped world ! ! Most common urological tumor 30 – 65% present with invasive disease: ! ! ! ! ! ! inadequate diagnostic facilities Lack of screening of high-risk individuals lack of community awareness High bladder cancer-related morbidity & mortality Critical goal: to enhance early detection A case-control study of urinary molecular markers Objectives ! To compare the telomerase activity with other molecular markers in voided urine samples and urine cytology for the detection of bladder cancer ! To compare the telomerase activity in bladder tumor versus normal urothelium and correlate it to tumor grade Collaborators Farhat Abbas: Urology (P.I.) Jamsheer Talati: Urology Amanullah Memon: Urology Anwar Siddiqui: Biochemistry Naila Kiyani: Pathology Saeed Akhtar: Community Health Sciences Raziuddin Biyabani: Urology Hammad Ather: Urology Aga Khan University, Karachi Telomeres & Telomerase W hat are Telomeres ? Telomeres (TTAG G G) act as protective caps at end ofthe chromoso mes "To protect from exonuclease digestion "To prevent aberrant recombination "To prevent apoptosis • Each time a cell divides itloses 50100 base pairs atthe end ofits telomers • Once a certain amount oftelomeres are lost,the cellstops dividing and goes into apoptosis – senescence process Cell have evolved different mechanisms to prevent this progressive telomere loss "Co mplex recombination "Retrotransposition schemes "Telo merase: a specialized reverse transcriptase that adds back telo mere sequences that are lost during replication Telomerase ! a specialized reverse transcriptase that adds back telomere sequences that are lost during replication. ! Germ cells and cancer cells produce telomerase whereas normal somatic cells lack its activity 3’ 5’ G T T AG GG T T AG G G T T AG GG C CCAA T C CC Telomerase contains: "R NA molecule "Reverse transcriptase "Telomerase binds to 3’ end of DNA and extends them by copying R NA template in multiples of hexamer repeat sequences " Telomerase activityis turned offin cells from most tissues as they differentiate. " Itis reactivated in most human cancers. Type of cancer Type of sample/ Cancer No. of telomerase positive sa mples Head & Neck Oral rinse/ Squamous Cancer 14/44 Lung Bronchiallavage/ lung Cancer 29/37 Lung Pleuralfluid/lung Cancer 64/70 Pancrease Pancreaticjuice/ ductal Cancer 9/12 Colon Colonic wash/ colon Cancer 9/15 Thyroid FNA/ Papillary Cancer 9/14 Breast FNA/ Invasive Cancer 52/71 Prostate Prostate biopsy/ Prostate Cancer 17/19 Bladder Urine/ Bladder Cancer CIS Cystitis Normal 88/104 5/5 16/47 0/35 Ovaries Peritoneal fluid/ Ovarian Cancer 37/42 Cervix Pap smear/ Cervical Cancer 15/17 Normal cell Telomere shortening Telomerase Telomere maintenance p53 Rb Immortalization Ras Oncogenic transformation Tumor cell Apoptosis (Senescence) Markers Diagnostic Telo merase N M P22 FDP BTA(stat) He moglobin dipstick Urine Cytology Prognostic Telo merase Ki 67 P53, MD M, p16 Selection criteria for the patients Exclusion Inclusion # Patients with known bladder tum or # Urinary instrumentation within 2 preceding weeks # Patients with suspected bladder tum or # Frank urinary tract infection # Patients on surveillance with prior resection of bladder tum or # Patients not consenting for additional tests to be performed on urine/ tissue sam ples Selection criteria for controls Inclusion # Rando mly selected patients undergoing cystoscopy for non m alignant cause Exclusion # # # # Current or prior history of urinary tract cancer Urinary instrumentation within 2 preceding weeks Frank urinary tract infection Patients not consenting for additional tests to be performed on urine/ tissue sam ples Urine sa mple collection and handling Voided urine/ Bladder washings He moglobin dipstick 2-3mls at roo m temp within3 hrs.. BTA STAT 5 drops at roo m temp within3 hrs.. N M P22 Telo merase FDP Cytology 20-30 mls in urine stabilizers at roo m temp. within 3 hrs. 50-100 mls im m ediately transferred on ice 1 ml within2 hrs. at roo m temp. 50 - 100 mls within 2 hrs at roo m temp. Tissue Sample Handling Bladder tum or patient Grossly norm al Bladder mucosa Control Patient Bladder tumor 10 % Formalin For Histopathology & Im munostaining Grossly norm al Bladder mucosa Im mediate storage in liquid nitrogen & transport to lab for storage at –80°° C Tissue Sa mple Collection Patient Control Bladder tumor Nor mal Bladder M ucosa Histopathology p53, MD M, p16 Ki-67 Telo merase activity Extraction from cells (voided Urine and bladder washings) Add PBS Cells were pelleted at 3000xg and PBS was added Add Lysis reagent W ashed 3X in PBS Resuspended in Lysis reagent and incubated on ice for 30 min. Centrifuged at 16000xg Supernatant was collected and stored At –70ºC Keep on ice Extraction from Tissues cryostat sections were prepared and ho mogenized Resuspended in lysis reagent & incubated on ice for 30 minutes Centrifuged at 16000xg Supernatant collected and stored at –70ºC Amplification To the reaction mixture PCR Program Step1: 10 min, 25ºC Te mplate was added nuclease free water Step2: 5 min, 94 º C 30s, 94 º C Add sa mple, negative control & Positive control 30s, 50 º C 30s, 72 º C Start PCR progra m m e Denaturation Add denaturation and incubate Step3: 10min, 72 º C Step4: hold, 4 º C Hybridization/ELISA Add hybridization buffer T Transfer to MTP wells and incubate Wash & add anti DIG-HRP and incubate Wash and add TMB substrate and incubate Add stop reagent and read Abs. at 450nm. 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F RESULTS Voided Urine Samples Urine Telomerase NMP22 BTA FDP Urine N=32 N=32 N=33 N=33 Dipstic Cytology k N=33 N=33 Positive 26 22 17 14 24 16 Sensitivity 81% 69% 52% 42% 73% 48% TRAP ASSAY ! 29 / 32 +ve in TCC samples – 92% sensitivity ! Mean Telomerase activity: - Voided urine in TCC cases = 52.9 + 25.3 - Normal urothelium = 20.7 + 7.8 - Bladder tumor = 561 + 231.5 (p=0.001) Telomerase activity in urine samples in patients with bladder carcinoma References Sample Size (n) telomerase activity (sensitivity) Muller et al 1996 30 0% Kinoshita et al 1997 42 55% Yoshida et al 1997 26 62% Linn et al 1997 12 0% Dalbagni et al 1997 63 35% Kavaler et al 1998 104 85% Yokota et al 1998 29 86% Muller et al 1998 30 7% Ramakumar et al 1999 57 70% Kitsukawa et al 1999 26 85% Rahat et al 1999 21 81% Gelimi et al 2000 33 94% Arai et al 2000 19 21% Bailkowska – Hobrozanka et al 2000 35 49% Muller M, Oncogene, 2002 Telomerase activity in tissue samples in patients with bladder carcinoma References Sample Size (n) telomerase activity (sensitivity) Muller et al 1996 75 96% Lin et al 1996 40 97% Kamata et al 1996 13 100% Kinoshita et al 1997 42 98% Yoshida et al 1997 56 86% Linn et al 1997 12 92% Kyo et al 1997 13 100% Lee et al 1998 23 96% Yokota et al 1998 26 100% Ito et al 1998a 22 100% Mayfield et al 1998 52 81% Rahat et al 1999 29 90% Gelimi et al 2000 33 94% Lancelin et al 2000 14 78% Muller M, Oncogene, 2002 Commercial Assays (FDA Approved) Sensitivity Overall Grade Specificity Stage Cytology 26% G1 - 6% G2 - 36% G3 - 79% PTis - 67% PTa - 26% PT1-4 64% > 93% BTAstat 65-79% G1 - 57% G2 - 81% G3 - 80% PTis - 100% PTa - 53% PT1 - 70% PT2-4 88% 60 – 64 % BTAtrak 66% G1 - 48% G2 - 59% G3 - 88% PTis - 60% PTa - 51% PT1 - 88% PT2-4 88% 62 – 69% NMP22 73-100% G1 - 57 % G2 - 81 % G3 - 80% PTis – 66% PTa-1 71% PT2-4 92% 85 – 97 % AccuDx (FDPs) 68% G1 - 63% G2 - 88% G3 - 95% PTis - 100% PTa – 2 75-85% PT3 - 100% 96 % ImmunoCyt 86% G1 - 84% G2 - 84% G3 - 89% PTis - 100% PTa - 86% PT1 - 85% PT2 - 3 83% 79% Saad A, Eur Urol, 2001 Research – Based Assays Sensitivity Overall Specificity Grade Stage TRAP 62% G1 - 79% G2 - 84% G3 - 87% PTis - 100% PTa -1 75% PT2-3 84% 96% CD44 91% NA NA 83% HA 83% G1 - 80% G2 - 80% G3 - 85% PTis - 87% PTa - 76% PT1 - 87% PT2-3 92% 90% HAase 81% G1 - 22% G2 - 82% G3 - 81% PTis - 77% PTa - 42% PT1 - 87% PT2-3 86% 84% HA-HAase 92% G1 - 86 % G2 - 95 % G3 - 93% PTis – 93% PTa-1 87% PT1 - 93% PT2-3 92-100% 85% Cadherine-E 35% NA PTa – T1 - 53% PT2-3 – 25% NA Lewis X 85% NA PTis – 100% 85% Microsatellite assay 83% G1 - 50% G3 - 81% G3 - 81% G2 - 50% G2 - 50% PTa – T1 60% 72% PT2 -4 72% PT2-4 10% Saad A, Eur Urol, 2001 Cytokeratins Sensitivity Overall Specificity Grade Stage UBC-EISA Test 64% G1 - 50% G2 - 50% G3 - 68% PTis - 100% PTa - 62% PT1- 53% PT2 – 80% UBC-Rapid Test 66% G1 - 44% G2 - 78% G3 - 75% PTis - 50% PT1- 69% PTa - 60% PT2 - 80% 83% 83-96% G1 - 75% G2 - 90% G3 - 92% PTis - 100% PTa - 75% PT1 - 86% PT2 - 72% PT3-4 100% 90% CYFRA-21-1 96% TPS 64% NA NA 84% CK20 86% G1 - 71 % G2 - 80 % G3-4 100% NA 85% TPA 80% G1 - 75 % G2 - 87 % G3 - 80% Ptis - 66% PTa -75% PT1 - 84% PT2 - 54% NA 486 P 3/12 89% NA NA 85% AN43 & BB369 47% NA NA 10% DD23 85% NA NA Saad A, 2001 95% Conclusions ! Our interim results and the literature substantiates the potential usefulness of urinary telomerase in detection of bladder cancer. ! Further studies on the specificity of these markers, prognostic markers, and sub units of telomerase are being done.