Volume 52, Issue 5, Pages 1388-1397 (November 2007) Noninvasive Diagnosis of Bladder Cancer by Detection of Matrix Metalloproteinases (MMP-2 and MMP-9) and Their Inhibitor (TIMP-2) in Urine  Sanaa Eissa, Randa Ali-Labib, Menha Swellam, Manal Bassiony, Fathy Tash, Tarek Mostafa El-Zayat  European Urology  Volume 52, Issue 5, Pages 1388-1397 (November 2007) DOI: 10.1016/j.eururo.2007.04.006 Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 1 ROC curve analysis for MMP-2, MMP-9, and TIMP-2 to calculate the best cut-off point to discriminate between malignant and nonmalignant groups. Open circles denote best cut-off points of MMP-2 (solid line) as 1.9ng/mg protein (sensitivity=78% and specificity=75%; AUC [SE]=0.83 [0.04], 95% confidence limits range=0.724–0.875, p<0.01), and MMP-9 as 8.7ng/mg protein (sensitivity=77% and specificity=100%; AUC [SE]=0.95 [0.02], 95% confidence limits range, 0.91–0.99, p<0.01) and TIMP-2 as 4.49ng/mg protein (sensitivity=92% and specificity=68.3%; AUC [SE]=0.83[0.039], 95% confidence limits range, 0.75–0.90, p<0.01). ROC=receiver operating characteristic; MMP=matrix metalloproteinase; TIMP=tissue inhibitor of metalloproteinase; AUC=area under the curve. European Urology 2007 52, 1388-1397DOI: (10.1016/j.eururo.2007.04.006) Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 2 ROC curve analysis for MMP-2/TIMP-2 and MMP-9/TIMP-2 to calculate the best cut-off point to discriminate between malignant and nonmalignant groups. Open circles denote best cut-off points of MMP-2/TIMP-2 as 0.93ng/mg protein (sensitivity=99% and specificity=81.7%; AUC [SE]=0.93 [0.026], 95% confidence limits range, 0.87–0.98, p<0.01) and MMP-9/TIMP-2 as 3.81ng/mg protein (sensitivity=97% and specificity=88%; AUC [SE]=0.94 [0.04], 95% confidence limits range, 0.86–1.02, p<0.01). ROC=receiver operating characteristic; MMP=matrix metalloproteinase; TIMP=tissue inhibitor of metalloproteinase; AUC=area under the curve. European Urology 2007 52, 1388-1397DOI: (10.1016/j.eururo.2007.04.006) Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 3 Gelatin zymography for detection of the enzymatic activity of MMP-2 and MMP-9. Lane 1: Molecular weight standards (29–205kDa). Lanes 2–6: Urine supernatant of bladder carcinoma (lane 2: +ve bilharziasis, TCC, grade 2, stage 4; lane 3: +ve bilharziasis, TCC, grade 1, stage 1; lane 4: +ve bilharziasis, TCC, grade 2, stage 1; lane 5: +ve bilharziasis, SCC, grade 2, stage 4; lane 6: +ve bilharziasis, SCC, grade 2, stage 3). Lanes 7, 8: Urine supernatant of benign bladder lesions. Lanes 9, 10: Urine supernatant of normal healthy contols. Clear zones indicate gelatinolytic activity with molecular weights 92kDa corresponding to pro–MMP-9, 72kDa corresponding to pro–MMP-2, and 62kDa corresponding to active isoforms of MMP-2. MMP=matrix metalloproteinase; +ve=positive; TCC=transitional cell carcinoma. European Urology 2007 52, 1388-1397DOI: (10.1016/j.eururo.2007.04.006) Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 4 Gelatin zymography for detection of the enzymatic activity of MMP-2 and MMP-9. Lane 1: Molecular weight standards (29–205kDa). Lanes 2–10: Urine supernatant of bladder carcinoma (lanes 1, 2: +ve bilharziasis, TCC, grade 2, stage 1; lanes 3, 4: +ve bilharziasis, SCC, grade 1, stage 2; lanes 5, 6: +ve bilharziasis, SCC, grade 2, stage 2; lanes 7, 10: +ve bilharziasis, SCC, grade 2, stage 3; lanes 8,9: +ve bilharziasis, SCC, grade 2, stage 4). Clear zones indicate gelatinolytic activity with molecular weights 92kDa corresponding to pro–MMP-9, 72kDa corresponding to pro–MMP-2, and 62kDa corresponding to active isoforms of MMP-2. MMP=matrix metalloproteinase; +ve=positive; TCC=transitional cell carcinoma. European Urology 2007 52, 1388-1397DOI: (10.1016/j.eururo.2007.04.006) Copyright © 2007 European Association of Urology Terms and Conditions