Detection of Circulating Tumor Cells from Peripheral Blood in Patients with Transitional Cell Carcinoma – Pilot Study. Comparison with the Standard Histopathological Staging

Česká verzia

Authors: V. Nagy ¹; J. Rosocha ²; J. Židzik ³; P. Bohuš ⁴
Authors place of work: Urologická klinika, Univerzita P. J. Šafárika, Lekárska fakulta a Univerzitná nemocnica L. Pasteura, Košice, Slovenská republika ¹; Združená tkanivová banka, Univerzita P. J. Šafárika, Lekárska fakulta a Univerzitná nemocnica L. Pasteura, Košice, Slovenská republika ²; Ústav lekárskej biológie, Univerzita P. J. Šafárika, Lekárska fakulta, Košice, Slovenská republika ³; Ústav patológie, Univerzita P. J. Šafárika, Lekárska fakulta a Univerzitná nemocnica L. Pasteura, Košice, Slovenská republika ⁴
Published in the journal: Klin Onkol 2011; 24(4): 287-292
Category: Original Articles

Summary

Backgrounds:
The aim of this pilot study was to investigate whether UP-II and EGFR genes expression detection with RT-PCR and the use of immunohistochemistry methods on patient samples taken before and after surgery could be used as a cancer marker for detection of circulating tumor cells in peripheral blood of patients with TCC. Another goal of this study was to identify whether surgery can influence the amount of circulating tumor cells and to correlate the samples with standard histopathological staging.

Materials and Methods:
A total of 43 patients with histologically proven TTC was enrolled in the study. There were 33 men and 10 women in the sample, mean age was 65 ± 12 years (range 37–85 years). Forty (93.0%) patients had TCC of the urinary bladder, 2 (4.6%) had TCC of renal pelvis and 1 (2.3%) had TCC of urinary bladder, urethra, and renal pelvis. A sample of 10 ml of peripheral blood was collected from each patient before and within 1 hour after a surgery. Blood samples were used for immunomagnetic separation of circulating tumor cells and determination of UP-II and EGFR genes expression. Subsequently, cancer tissue was processed, endolymphatic, intravascular and peritoneal invasion determined and CK-7, CK-20, stromelysin, Ki-67 and p53 expression evaluated. Blood samples taken before and after the surgery were also subjected to immunohistochemical analysis using hematoxylin-eosin (HE) staining and staining by Papanicolaus (PAP). CK-7 and CK-20 expression was also evaluated.

Results:
EGFR and UP-II were expressed in 24 of the 35 (68.6%) and in 19 of the 35 (54.3%) cancer tissues samples, respectively. EGFR was expressed neither in blood samples nor in immuno-separated cell samples. UP-II was expressed in 1 of the 19 (5.3%) samples of immuno-separated cells acquired before the surgery and in no sample of immuno-separated cells obtained after the surgery (P < 0.9999). Moreover, UP-II was expressed in 2 of the 32 (6.3%) whole blood samples taken before the surgery and in 3 out of 32 (9.4%) whole blood samples taken within an hour after the surgery (P < 0.9999). Histopathological examination showed TCC invasion in 11 of the 43 patients: 1 patient with intravascular, 6 with endolymphatic, 1 with intravascular and endolymphatic and 3 with intravascular, endolymphatic and perineural invasion. Immunohistochemical examination of separated blood before and after the surgery by PAP and HE staining, CK-7 and CK-20 expression were negative in nearly all samples. Immunohistochemical examination of TCC tissue showed positive results in 97.7% for CK-7expression, 74.4% for CK-20 and 97.7% for stromelysin. Cytological examination of urine was positive in 19 (50%) patients and correlated well with higher grade G3 in 20 (46.5%) patients. Ki-67 expression was significantly higher in patients with G3 (31.15%) in comparison to patients with G1 (7.53%) (p < 0.01). There was no significant association between grade and expression of p53 and stromelysin in cancer tissue.

Conclusion:
Our preliminary tests did not show any significant change to EGFR and UP-II expression in peripheral blood and in immuno-separated cells before and after a surgery. The results for a group of patients with lower pTNM grade did not confirm the presence of malignant urothelial cells in peripheral blood.

Key words:
urothelial cancer - RT PCR – beta-Actin – uroplakin II – EGFR genes – cytokeratins – circulating neoplastic cells – minimal residual disease

Zdroje

1. Nezos A, Pissimisis N, Lembessis P et al. Detection of circulating tumor cells in bladder cancer patients. Cancer Treat Rev 2009; 35(3): 272–279.

2. Desgrandchamps F, Teren M, Dal Cortivo L et al. The effect of transurethral resection and cystoprostatectomy on dissemination of epithelial cells in the circulation of patients with bladder cancer. Br J Cancer 1999; 81(5): 832–834.

3. Ghossein RA, Bhattacharya S. Molecular detection and characterization of circulating tumor cells and micrometastases in prostatic, urothelial, and renal cell carcinomas. Semin Surg Oncol 2001; 20(4): 304–311.

4. Hirata H, Hisatomi H, Kawakita M et al. Genetic detection for hematogenous micrometastasis in patients with various types of malignant tumor using Uroplakin II derived primers in polymerase chain reaction. Oncol Rep 2003; 10(4): 963–966.

5. Kinjo M, Okegawa T, Horie S et al. Detection of circulating MUC7-positive cells by reverse transcription-polymerase chain reaction in bladder cancer patients. Int J Urol 2004; 11(1): 38–43.

6. Gazzaniga P, Gandini O, Giuliani L et al. Detection of epidermal growth factor receptor mRNA in peripheral blood: a new marker of circulating neoplastic cells in bladder cancer patients. Clin Cancer Res 2001; 7(3): 577–583.

7. Lu JJ, Kakehi Y, Takahashi T et al. Detection of circulating cancer cells by reverse transcription-polymerase chain reaction for uroplakin II in peripheral blood of patients with urothelial cancer. Clin Cancer Res 2000; 6(8): 3166–3171.

8. Yuasa T, Yoshiki T, Isono T et al. Expression of transitional cell-specific genes, uroplakin Ia and II, in bladder cancer: detection of circulating cancer cells in the peripheral blood of metastatic patients. Int J Urol 1999; 6(6): 286–292.

9. Yuasa T, Yoshiki T, Tanaka T et al. Expression of uroplakin Ib and uroplakin III genes in tissues and peripheral blood of patients with transitional cell carcinoma. Jpn J Cancer Res 1998; 89(9): 879–882.

10. Yuasa T, Yoshiki T, Isono T et al. Molecular cloning and expression of uroplakins in transitional cell carcinoma. Adv Exp Med Biol 2003; 539(Pt A): 33–46.

11. Okegawa T, Kinjo M, Nutahara K et al. Value of reverse transcription polymerase chain assay in peripheral blood of patients with urothelial cancer. J Urol 2004; 171(4): 1461–1466.

12. Buchumensky V, Klein A, Zemer R et al. Cytokeratin 20: a new marker for early detection of bladder cell carcinoma? J Urol 1998; 160(6 Pt 1): 1971–1974.

13. Klein A, Zemer R, Buchumensky V et al. Expression of cytokeratin 20 in urinary cytology of patients with bladder carcinoma. Cancer 1998; 82(2): 349–354.

14. Inoue T, Nakanishi H, Inada K et al. Real time reverse transcriptase polymerase chain reaction of urinary cytokeratin 20 detects transitional cell carcinoma cells. J Urol 2001; 166(6): 2134–2141.

15. Retz M, Lehmann J, Röder C et al. Cytokeratin-20 reverse-transcriptase polymerase chain reaction as a new tool for the detection of circulating tumor cells in peripheral blood and bone marrow of bladder cancer patients. Eur Urol 2001; 39(5): 507–515.

16. Christoph F, Müller M, Schostak M et al. Quantitative detection of cytokeratin 20 mRNA expression in bladder carcinoma by real-time reverse transcriptase-polymerase chain reaction. Urology 2004; 64(1): 157–161.

17. Fujii Y, Kageyama Y, Kawakami S et al. Detection of disseminated urothelial cancer cells in peripheral venous blood by a cytokeratin 20-specific nested reverse transcriptase-polymerase chain reaction. Jpn J Cancer Res 1999; 90(7): 753–757.

18. Güdemann CJ, Weitz J, Kienle P et al. Detection of hematogenous micrometastasis in patients with transitional cell carcinoma. J Urol 2000; 164(2): 532–536.

19. Ribal MJ, Mengual L, Marín M et al. Molecular staging of bladder cancer with RT-PCR assay for CK 20 in peripheral blood, bone marrow and lymph nodes: comparison with standard histological staging. Anticancer Res 2006; 26(1A): 411–419.

20. Osman I, Kang M, Lee A et al. Detection of circulating cancer cells expressing uroplakins and epidermal growth factor receptor in bladder cancer patients. Int J Cancer 2004; 111(6): 934–939.

21. Naoe M, Ogawa Y, Morita J et al. Detection of circulating urothelial cancer cells in the blood using the Cell--Search system. Cancer 2007; 109(7): 1439–1445.

22. Gallagher DJ, Milowsky MI, Ishill N et al. Detection of circulating tumor cells in patients with urothelial cancer. Ann Oncol 2009; 20(2): 305–308.

23. Li SM, Zhang ZT, Chan S et al. Detection of circulating uroplakin-positive cells in patients with transitional cell carcinoma of the bladder. J Urol 1999; 162(3 Pt 1): 931–935.

24. Guzzo TJ, McNeil BK, Bivalacqua TJ et al. The presence of circulating tumor cells does not predict extravesical disease in bladder cancer patients prior to radical cystectomy. Urol Oncol. Epub ahead of print.

25. Karl A, Tritschler S, Hofmann S et al. Perioperative search for circulating tumor cells in patients undergoing radical cystectomy for bladder cancer. Eur J Med Res 2009; 14(11): 487–490.