Východiská: Cieľom pilotnej štúdie bolo zistiť, či expresiu génov pre UP-II a EGFR RT-PCR a imunohistochemicky (IH) na vzorkách odobratých pred a po operácii možno využiť ako potenciálny nádorový marker na detekciu cirkulujúcich nádorových buniek (CNB) v periférnej krvi pacientov s TCC a výsledky porovnať so štandardným histopatologickým vyšetrením. Materiál a metódy: 43 pacientov (33 mužov a 10 žien) s TCC o vo veku 37–85 rokov, priemer 65 ± 12 rokov. Z nich 40 (93 %) malo TCC močového mechúra, 2 (4,6 %) TCC obličkovej panvičky a 1 (2,3 %) TCC močového mechúra, močovodu a obličkovej panvičky. V rámci štúdie bolo odobratých 10 ml periférnej krvi pacientov pred a do 1 hod po operácii, imunomagneticky separované CNB a určená expresia génov EGFR a UP-II pomocou RT-PCR. Následne bolo histopatologicky vyšetrené TCC tkanivo, určená endolymfatická, intravaskulárna a perineurálna invázia, expresia CK-7, CK-20, stromelysinu, Ki-67, p 53. IH bola vykonané aj na vzorkách separovanej krvi pred a po operácii na prítomnosť CNB farbením hematoxylínom-eozínom (HE) a podľa Papanicolaua (PAP), ako aj na prítomnosť expresie CK-7, CK-20. Výsledky: EGFR a UP-II boli exprimované u 24 z 35 (68,6 %) a u 19 z 35 (54,3 %) vzoriek TCC tkaniva. EGFR nebol exprimovaný v žiadnej vzorke krvi ani imuno-separovaných buniek. UP-II bol exprimovaný v 1 z 19 (5,3 %) vzoriek imuno-separovaných CNB získaných pred operáciou, ale v žiadnej vzorke imuno-separovaných CNB získaných po operácii (P < 0,9999). Expresia UP-II bola detegovaná v 2 z 32 (6,3 %) vzoriek krvi odobratých pred operáciou a v 3 z 32 (9,4 %) vzoriek krvi odobratých do jednej hodiny po operačnom zákroku (P < 0,9999). U 11 zo 43 (25,6 %) pacientov sa pri histopatologickom vyšetrení zistila invázia TCC, z toho: u jedného intravaskulárna, u 6 endolymfatická, u jedného intravaskulárna a endolymfatická a u 3 intravaskulárna, endolymfatická a perineurálna. Vyšetrenie separovanej krvi pred a po operácii farbením HE a PAP a na CK-7, CK-20 bolo takmer vo všetkých vzorkách negatívne. Pri IH tkaniva TCC na CK-7, CK-20, stromelysin bola pozitivita v 97,7 %, 74,4 % a 97,7 %. Cytologické vyšetrenie moču bolo pozitívne u 19 (50 %) z pacientov a korelovalo s vyšším grade G3 u 20 (46,5 %) pacientov. Expresia Ki-67 bola u pacientov s G3 (31,15 %) signifikantne vyššia ako u pacientov s G1 (7,53 %) (p < 0,01). Pri skúmaní TCC tkaniva sa nenašiel signifikantný vzťah medzi grade a expresiou p53 a stromelysinu. Záver: Testy neukázali zmeny expresie génov pre EGFR a UP-II v periférnej krvi a v imuno-separovaných bunkách pacientov pred a po operačnom zákroku. Výsledky v tomto súbore pacientov s TCC s prevažne nižším štádiom pTNM nepotvrdili výskyt CNB TCC v periférnej krvi.

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.

Urologická klinika Univerzita P J Šafárika LF a Univerzitná nemocnica L Pasteura Košice

Detection of circulating tumor cells from peripheral blood in patients with transitional cell carcinoma – pilot study. Comparison with the standard histopathological staging

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