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Abstract
We evaluated the diagnostic efficacy of urinary CD44 and cytokeratin 20 (CK20) mRNA in comparison with voided urine cytology in the detection of bladder cancer patients. A total of 136 Egyptian patients provided a single voided urine sample for cytology, CD44 and CK20 mRNA before cystoscopy. Of the 136 cases, 111 were histologically diagnosed as bladder cancer; 55 with transitional cell carcinoma (TCC), 52 with squamous cell carcinoma (SCC) and 4 adenocarcinoma, whereas the remaining 25 had benign urological disorders. A group of 20 healthy volunteers were also included in this study. Voided urine was centrifuged and the urine sediment was used for cytology, estimation of CD44 by ELISA technique and RNA extraction. CK20 mRNA was detected by RT-PCR and real-time RT-PC.
This study revealed a statistically significant difference of the median level of CD44 (X =19.86, P=0.001) between the three investigated groups (bladder carcinoma, benign urological disorders and healthy volunteers). The best cutoff value for CD44 was calculated by a ROC curve as 141 ng/mg protein. The positivity rates for CD44 of the control, benign and malignant groups were 0%, 20% and 63.1%, respectively (X2 =36.4, P=0.001). There was a significant difference in positivity rate of urinary CD44 with respect to bilharziasis and urine cytology (P=0.006 and 0.001, respectively).
The best cutoff value for relative urinary CK20 mRNA expression by real-time RT-PCR calculated by a ROC curve was 100; while the positivity rate for urinary CK20 mRNA by either methods (RT-PCR and real-time RT-PCR) of the control, benign and malignant groups was 0%, 4% and 82%, respectively (X2 =88.541, P= 0.001). The overall sensitivity and specificity were 52.25% and 88.9% for voided urine cytology, 63.1% and 88.9% for urinary CD44, and 82% and 98% for CK20 mRNA. Combined sensitivity of voided urine cytology with urinary CD44 and CK20 mRNA together was 95.5.
CD44 can be measured reliably by ELISA in urine samples. The data presented demonstrate that detection of elevated levels of CD44 by ELISA; and expression of CK20 mRNA by RT-PCR and real-time RT-PCR in exfoliated urothelial cells are accurate and applicable to identification of bladder cancer. Comparing the diagnostic efficiency of the studied techniques revealed that CK20 mRNA detected by either RT-PCR or real-time RT-PCR achieved highest sensitivity. Since RT-PCR had the same diagnostic efficiency as real-time RT-PCR, RT-PCR will preferred as a routine test in terms of cost.