2.2 URINARY MARKERS FOR DIAGNOSIS OF BLADDER CARCINOMA UNDER INVESTIGATION
3.2.2 SURVIVIN
TRAK in detection of bladder cancer by Abdel El- Gawad and co-workers 19, BTA TRAK showed highest sensitivity of 100% in bothbladder carcinoma group, schistosomal carcinoma subgroup and non-schistosomal carcinoma subgroup. The overall sensitivities of the other markers were, 54.3%, 91.3% and 80.4% for urine cytology, NMP22 and Telomerase respectively. Urine cytology showed highest specificity of 100% followed by Telomerase, BTA TRAK and NMP22 with specificity of 95%, 92.5% and 87.5% respectively. The use of combination of markers with cytology showed highest sensitivity and specificity. The markers showed highest sensitivity in bladder carcinoma group compared to benign or control group.
in review of urinary markers by Konety and co-workers 51, The mean sensitivity, and specificity of 68%, 66% and 74%, 74% were reported for BTA STAT and BTA TRAK respectively. BTA tests were more sensitive than cytology for low grade tumours but had equivalent sensitivity for high grade tumours. The overall sensitivities and specificities of BTA STAT and BTA TRAK were 58- 82.8%, 66-72% and 68-72%, 48-75% respectively 9. The sensitivity and specificity of urine cytology in this review were 20-53% and 83-99% respectively. False positive results occurred in the presence of haematuria from other GU diseases and BCG treatment within 2 years of testing, which decreased specificity by 25%. In a review of biomarkers for detection and surveillance of bladder carcinoma by Lorne et al 52, the highest sensitivity of 51-100% was by BTA TRAK followed by BTA STAT with sensitivity of 52.5-78%. The specificity of BTA TRAK was 73-92.5%, which was slightly lower than 78-100% of urine cytology. Both tests are approved by FDA only in combination with cystoscopy for the monitoring of bladder carcinoma.
Survivin is a member of the inhibitor of apoptosis proteins (IAP) family of molecules, a group of proteins involved in inhibition of cysteine proteases with aspartic acid specificity (Caspases) 3, 7 and 9 53. Survivin is 142-amino acid (16.5 kDa) protein produced by gene mapped to a chromosomal region 17q25, which encodes for a 1.9-kb transcript that contains 4 exons.
Alternative splicing of the human survivin gene can give rise to five different mRNA isoforms, wild-type survivin, survivin-2B, survivin-DEx3, survivin-3B and survivin-2a 54. Survivin is a multifunctional protein that has been demonstrated to inhibit apoptosis, regulate cell division and promote angiogenesis. Survivin inhibits apoptosis in response to a wide variety of apoptotic stimuli but the exact mechanism and precise effect of individual splice variants on apoptotic cascade have not been established. Recent evidence suggests that Survivin may inhibit apoptosis by binding to and inactivating second mitochondria-derived activator of caspases (SMAC), a mitochondrial protein that potentiates apoptosis by complexing with specific IAP, thus preventing their interaction with caspases 55. Some investigators have suggested that the primary function of Survivin is in controlling cell division rather than apoptosis inhibition. In several tumour models, survivin inhibition has been shown to produce defects in chromosome segregation, cytokinesis and ultimately cell division, without measurable impact on apoptosis 56. Significant positive correlation between Survivin levels and micro vessel density was discovered in various solid tumour models and its inhibition has led to measurable suppression of angiogenesis 57. It is normally expressed during embryonic and fetal development but undetectable in the adult 9. It controls mitotic progression and induces genes associated with tumour invasiveness.
Survivin has unique role in apoptosis, control of cell division and modulation of angiogenesis.
The selective expression of Survivin in malignant versus normal tissues has been explored as an
excellent therapeutic target in bladder carcinoma. Inhibition of Survivin expression and/or function by dominant negative mutants, antisense complementary DNA, antisense oligodeoxynucleotides (AS-ODN) or small interfering RNA (siRNA) constructs have been shown to inhibit tumour cell proliferation, and markedly induce spontaneous and radiotherapy or chemotherapy induced apoptosis 17.
Survivin messenger ribonucleic acid (mRNA) is over expressed in human cancers and can be detected in urine using a bio-dot immunoassay incorporating a rabbit polyclonal antisurvivin antibody, imunohistochemistry or RT-PCR.Urinary levels of Survivin gene activation, both at the protein and the mRNA level are associated with bladder carcinoma presence, higher grade, advanced pathologic stage and response to chemotherapy 12. Various applications of Survivin in diagnosis of bladder cancer include the following;
i In bladder carcinoma detection and surveillance
Selective expression of Survivin by malignant epithelium, and its detection in urine at mRNA or protein level makes it an attractive marker for detection of bladder cancer 17. In the first study on Survivin in the diagnosis of bladder carcinoma by Smith and co-workers 58 using RT-PCR, the sensitivity of Survivin protein and mRNA was 100%. Bladder carcinoma was detected in all the 47 patients with histological diagnosis of the tumour but in only 3 of 35 patients with negative histology. None of 16 healthy volunteers or 29 patients with other genitourinary cancers had detectable Survivin mRNA or protein, while 3 of the 30 patients with benign urinary tract disease, were positive for urinary Survivin. Moreover, patients with low-grade bladder carcinoma had significantly lower urinary Survivin levels than patients with carcinoma in situ 58. In a study by Horstmann and co-workers 59, the sensitivity and specificity of Survivin for
high-grade urothelial cell carcinoma were 83% and 88 % respectively. In the same study the sensitivity of Survivin for low grade was 35%.
Shariat and colleagues 60, using a bio-dot microfiltration detection system (Bio-Rad, Hercules, California) using ELISA analysed voided urine from 117 bladder carcinoma patients undergoing cystoscopy and 92 controls for levels of Survivin, NMP22 and cytology. The overall sensitivity, specificity, and positive and negative predictive values of Survivin for diagnosis of bladder cancer was superior to those of NMP22 and cytology. Survivin had the highest specificity and positive predictive value for the detection of bladder carcinoma across tumour stages and grades.
The sensitivity, specificity, PPV and NPV of Survivin were 64%, 93%, 92% and 67%
respectively.
Wang and colleagues 61 using RT-PCR found sensitivity, specificity, NPV and PPV of 80%, 100, 100%, 86% respectively in a study involving 30 bladder carcinoma patients, 10 prostate cancer and renal cancer patients, and 20 healthy volunteers. Using the same method by Moussa and colleagues 16, the sensitivity and specificity of Survivin in bladder carcinoma detection in 167 patients were 94% and 95% respectively. When Real-time PCR was used by Kenny and colleagues 62, Survivin had sensitivity, specificity, PPV and NPV of 79%, 93%, 73% and 95%
respectively in 118 patients. The Survivin sensitivity and specificity were 69% and 100%
respectively in 89 patients using the Real-time PCR in a study by Weikert and colleagues 63. Survivin mRNA was detected in urine of 24/35 of patients with TCC and it was not detected in any of the healthy 33 volunteers. The sensitivity and specificity of voided urine cytology in the same study were 31.4% and 97.1% respectively 63. Use of molecular beacons (MBs) probes specific for Survivin mRNA was reported to be sensitive and specific and has potential use in early detection of bladder carcinoma and follow-up after surgery 64 .
ii In prognosis after treatment of Non-muscle invasive bladder carcinoma (NMIBC)
In a study by Swana et al 65, immunohistochemical staining for Survivin detected 28 out of 36 bladder tumours after transurethral resection. The rate of over expression of Survivin at 65%, 90% and 100% correlated with increasing grade of G1, G2 and G3 respectively. In a subgroup analysis of G1 tumours, Survivin over expression correlated with shorter mean time to recurrence of 12 months against 36 months for the patients without over expression. They proposed Survivin as a marker of high risk of recurrence but however their study was limited by small sample size and no control for other variables like intravesical therapy. Hausladen and collaborators 66 found urinary survivin protein levels measured prior to, during and after the completion of intravesical instillation of BCG or mitomycin C as a predictor of post-therapy recurrence in 25 patients. Presence of urinary survivin one month after the completion of treatment predicted probability of TCC recurrence at one year with 92% sensitivity and 78%
specificity. Survivin levels of <0.1 ng/ml in urine was detected in 12 out 14 patients who had negative cystoscopy/cytology immediately following BCG or mitomycin C and at 1-year after completion of the treatment. They concluded that urinary Survivin measurement may be a useful determinant of recurrence risk and can serve as a guide for post-treatment cystoscopic follow-up.
Survivin mRNA real- time PCR was found by Schult et al 67 to correlate with recurrence and progression in 32 patients with NMIBC. Using a normalised Survivin mRNA copy and a median of 0.13, the median time to recurrence of NMIBC above and below the median was 10 and 24 months respectively (p= 0.008). In a multivariate analysis by Ku et al 68, high expression of Survivin was associated with worse disease free survival compared to normal expression.There was trend toward reduced recurrence-free survival above and below normalised median Survivin mRNA of 27 and 48 months respectively. In subgroup analysis of 27 patients with pTa NMIBC,
the median time to first recurrence above and below the median was 29 and 39.3 months respectively.
iii In prognosis after treatment of muscle-invasive bladder carcinoma
Addition of Survivin to a model that included standard predictors significantly enhanced its predictive accuracy for disease recurrence and disease-specific survival 69. Use of global gene expression profiling by Als and colleagues 70, identified Survivin to correlated significantly and independently with progression free, disease-specific and overall survival following systemic chemotherapy for bladder carcinoma. The median survival times were 18.4 versus 9.8 months, and the 5-year overall survival rates were 27.8% and 5.1% for Survivin negative and Survivin positive tumours, respectively (p < 0.0001). In a study, presence of Survivin was associated with disease recurrence, disease specific mortality and overall mortality 71.
Srivastava et al 72 in a study of 191 patients, compared the performance of urinary survivin and cytology for detection of bladder cancer. Out 117 TCC patients urine cytology was positive in 78 patients (66.7%) and Survivin was positive in 96 patients (82.1%). The specificity of cytology and Survivin was 95.9% and 81.1% respectively. The urine survivin concentration correlated with stage and grade of the tumour. Urine survivin concentration was significantly higher in bladder cancer patients as compared to healthy volunteers and those with benign urologic conditions. The urine survivin concentration did not differ significantly between the healthy volunteers and those with benign urologic conditions. There is need for standardization in the methods of assay and cut off of Survivin for diagnosis of bladder tumour 17.
3.2.3 OTHER MARKERS FOR DIAGNOSIS OF BLADDER CARCINOMA