Ⅰ Relation Between Promoter Methylation Of Mismatch Repair Gene HMLH1,hMSH2,hMSH3 And Transitional Cell Carcinoma Of The Bladder Ⅱ Clinical Study Of Retroperitoneal Laparoscopy For Treatment Of Adrenal Pheochromocytoma

Part One: Correlative studies of Promoter methylation and the expressions of mismatch repair gene hMLH1,hMSH2,hMSH3 in transitional cell carcinoma of the bladderObjective: To investigate the correlation between Promoter methylation and the expressions of mismatch repair gene hMLH1,hMSH2,hMSH3 in transitional cell carcinoma of the bladder(TCCB).Methods: In 13 cases of normal epithelial tissues of the bladder and 57 cases tissues of TCCB, the Promoter methylation status of hMLH1,hMSH2,hMSH3 was examined by means of methylation-specific PCR. Immunohistochemical staining was applied to detect the expression of hMLH1,hMSH2,hMSH3 genes. Results: Promoter methylation of hMLH1,hMSH2 and hMSH3 was not identified in the normal tissues. Promoter methylation of hMSH2 was not identified in TCCB tissues. Promoter methylation of hMLH1 and hMSH3 was present in 36.8%and 49.1% of TCCB tissues respectively. There was significant difference between the normal tissues and TCCB tissues respectively (P<0.01). But significant difference was not found in TCCB in regard to different histological grade and clinical stage (P>0.05). The positive protein expression rate of hMLH1,hMSH2 and hMSH3 was 100%,100%and 92.3% in the normal tissues respectively, while the positive expression rate was 59.6%,52.6% and 40.4% in TCCB respectively, which was significantly different between the normal tissues and TCCB tissues respectively (P<0.01). However there was no significant difference in TCCB at different clinical stage (P>0.05). The positive expression of hMSH2 and hMSH3 was negatively correlated to tumor grades. Promoter methylation of hMLH1 and hMSH3 genes inhibits the protein expressions of hMLH1 and hMSH3 in TCCB (P<0.01). Conclusions: The reduced protein expression of hMLH1 and hMSH3 was regulated by Promoter hypermethylation, except for the expression of hMSH2. The reduced expression of hMLH1,hMSH2 and hMSH3 might play an important role in carcinogenesis of TCCB. Part two: Effects of 5-Aza-CdR on growth,methylation and expression of mismatch repair gene hMLH1,hMSH2,hMSH3 in bladder carcinoma cell linesObjective: To study the effects of 5-Aza-CdR on growth,methylation and expression of mismatch repair gene hMLH1,hMSH2,hMSH3 in bladder carcinoma cell lines BIU-87 and T24.Methods: The bladder carcinoma cell lines BIU-87 and T24 were treated with 5-Aza-CdR. Cell morphology was observed under phase contrast microscope. The MTT assay was used to determine tumor cell growth. Apoptosis was assayed by flow cytometry and TUNEL (terminal deoxynucleotidyl transferase mediated nick end labeling). The genes expression level of mRNA before and after treated by 5-Aza-CdR was determined by reverse transcription-polymerase chain reaction (RT-PCR). The status of methylation of mismatch repair gene hMLH1,hMSH2,hMSH3 was analyzed by methylation-specific polymerase reaction (MSP).Results: 5-Aza-CdR inhibited the growth of bladder carcinoma cell lines BIU-87 and T24 in a time- and dose-dependent fashion. Apoptosis was observed after 5-aza-CdR treatment. The methylation ratio of the hMLH1 and hMSH3 promoter was significantly lower in 5-aza-CdR treated bladder cancer cells than in untreated bladder cancer cells (p<0.05). The expression level of hMLH1 and hMSH3 was higher than that before treatment (p<0.05). Conclusion: The hypermethylation of hMLH1 and hMSH3 promoter region is a powerful mechanism for the suppression of genes, the methylation of hMLH1 and hMSH3 promoter region can be removed and the express of genes can be reactivated by 5-aza-CdR. Paper two: Retroperitoneoscopic Adrenalectomy without Previous Control of Adrenal Vein is Feasible and Safe for PheochromocytomaObjectives: To evaluate the effectiveness and safety of retroperitoneal laparoscopic adrenalectomy for pheochromocytoma and report our experience in adrenalectomy without previous control of the adrenal vein.Methods: From January 2000 to December 2005, 56 patients underwent 58 retroperitoneal laparoscopic adrenalectomies for the treatment of pheochromocytoma. Adequate preoperative antihypertensive preparation was performed. Intraoperative hemodynamic changes were documented in detail. During surgery, the adrenal vein was identified and ligatured after dissection and mobilization of the adrenal gland. Results: One patient required conversion to open surgery. The mean operative time and estimated blood loss was 50.4±19.8 minutes (rang 25 to 150) and 76.4±23.5 mL (rang 20 to 300), respectively. A systolic blood pressure greater than 200 mm Hg or less than 80 mm Hg was observed in 6 and 3 patients, respectively. Moreover, an upward fluctuation of systolic blood pressure (20 mm Hg or greater) was recorded during laparoscopic manipulation in 21 patients (37.5%), and in 8 of them, it was 50 mm Hg or greater. The mean diameter of the excised masses was 4.6±1.7 cm (range 1.5 to 10.0). The mean hospital stay was 5.2±1.3 days (range 3 to 9). No patients had a major complication and none died. During the follow-up period of 5 months to 3 years, 36 patients recovered normal blood pressure without antihypertensive drugs. No tumor recurrences developed. Conclusions: Retroperitoneal laparoscopic adrenalectomy without previous control of the adrenal vein is effective and safe for ablation of pheochromocytoma. For experienced surgeons, the tumors larger than 6 cm in diameter can also be removed using the retroperitoneal endoscopic approach.INTRODUCTIONSince the first report on laparoscopic adrenalectomy (LA) by Gagner et al., [1] LA has become the preferred surgical management of benign adrenal tumors for its numerous advantages.[2-4] Pheochromocytoma was initially considered a contraindication to LA because of the perioperative medical management necessary, unique biologic behavior, and more difficult mobilization of catecholamine-secreting tumors.[5] However, more and more series of LAs using the transperitoneal or retroperitoneal approach for the treatment of pheochromocytomas have been reported.[6-11] This is largely owing to the advancement of techniques and accumulations of experience with laparoscopic operations and anesthesia. In the past 5 years, we have performed retroperitoneal laparoscopic adrenalectomy (RLA) on 56 patients with pheochromocytoma. In our technique, the adrenal tumor is dissected before control of the adrenal vein, and only 37.5 % of patients experienced significant intraoperative hemodynamic alterations. The goal of this study was to evaluate the effectiveness and safety of RLA for pheochromocytoma and to report our experience.METERIAL AND METHODSFrom January 2000 and December 2005, RLA for 56 consecutive patients with adrenal pheochromocytomas was performed by a single surgeon. This series consisted of 32 males and 24 females, with the mean age of 36.2±14.3 years (range 15 to 69). The medical history was 23±10.8 months long (range 10 days to 20 years). The preoperative diagnosis of pheochromocytoma was confirmed systematically by the clinical presentation and Paper two: Retrospective Comparison of Retroperitoneoscopic versus Open Adrenalectomy for PheochromocytomaObjective: To compare the clinical outcomes of of retroperitoneoscopic and open adrenalectomy for pheochromocytoma.Methods: The clinical data of 56 patients who underwent retroperitoneal laparoscopic adrenalectomy were retrospectively compared with those of 50 patients who underwent open adrenalectomy for pheochromocytoma in Tongji Hospital between January 1998 to December 2005. The differences between the 2 groups were studied on the patient demographic data, perioperative indexes and clinical outcomes. Differences were considered statistically significant at p<0.05.Results: Patient demographic data were similar between the 2 groups. In the retroperitoneoscopic group operative time (52±22 vs 120±42 minutes), estimated blood loss(74±34 vs 187±64 ml), return of bowel function(1 vs 2 days), length of postoperative hospital stay (5.2±1.7 vs 8.3±1.8 days), intraoperative incidence of hypertension (9 of 53, 17.0% vs 18 of 50, 36.0%)and number of patients requiring blood transfusion(1 of 53, 1.8% vs 8 of 50, 16.0%) were better than in the open group (P<0.05 for all). The incidence of systemic inflammatory response syndrome (SIRS) (11 of 53, 20.8% vs 21 of 50, 42.0%) is less in the retroperineoscopic group(P<0.05 ). However, the duration of SIRS and postoperative complications were equivalent in the 2 groups (p=0.110). In retroperitoneoscopic group, 1 haematoma and 1 wound infection were found. In open group, the complications included pleura injury in 2 patients and wound infection in 4 patients. 3 months after the operation, 81% in the retroperitoneoscopic group and 84% in the open group had normal blood pressure. During the follow-up of 5～36 months, there were no tumor recurrences and/or metastasis.Conclusions: When compared with open surgery, retroperitoneal laparoscopic adrenalectomy for pheochromocytoma is a safe, minimally invasive and effective procedure.