Introduction 5 12 2 6 15 10 In this descriptive study the influence of different treatment modalities on bladder tumours (thermo-chemotherapy, chemotherapy and thermotherapy) was investigated by several immunohistochemical stainings. Methods Subjects 3 Group 3 patients were treated 2 days before cystectomy with two times 30 min intravesical MMC (20 mg in 50 ml sterile water) combined with local microwave induced hyperthermia delivered by the SB-TS 101 system. Group 4 patients were treated with a cycle of six thermo-chemotherapy treatments during the last 3 months. Finally, group 5 patients served as control group. These patients did not receive any intravesical instillations within 3 months prior to cystectomy. Immunohistochemistry All tumour tissue samples were fixed in a 10% buffered formaldehyde solution. The specimens were embedded in paraffin blocks and sections of 4 μm were cut. All specimens were deparaffinized and stained with Haematoxylin and Eosin. A microscopic examination of the samples was performed and the extend of inflammation (1+ to 3+) haemorrhage (1+ to 3+) were semi quantitatively scored. 11 2 2 2 2 7 2 2 Immunoreactivity scoring The screening of tumour tissue samples was performed by two independent investigators (CH and AH). The sections were screened for positive cells, defined as cells with nuclear staining. The amount of Ki67 or p53 staining is scored in percentages. The threshold for p53 “positivity” is ≥20% positive staining. The areas with maximal immunohistochemical staining were used for scoring. In total, 300–500 tumour cells were scored. In the visual estimation only definitely brown nuclei were recorded as positive. The results were expressed as percentage of immunoreactive tumour cell nuclei. Results 1 Table 1 Patient details containing number of previous occurrences, interval period between initial diagnosis and cystectomy in months, pathology data of biopsy and cystectomy (grading according to WHO 2002) Groups Patient no/age/sex N Interval biopsy–cystectomy (in months) Histopathology initial biopsy Histopathology cystectomy 1 (MMC) 1/75/M 0 2,0 ≥pT2aGIII pT3aGIIIN2 2/46/F 0 4,4 pT2GII pT3bGIIIN1 3/67/F 0 1,2 ≥pT2GIII ≥pT2GIII  2 (HT) 4/64/F 0 1,9 pT1GIII pT2aGIII 5/71/M 3 38,7 pT1GIII + CIS pT1GIII + CIS 6/42/M 10 121,9 pTaGII pTaGII 3 (MMC + HT) 7/55/M 0 2,6 pT1GIII ≥pT2GIII 8/67/M 0 1,9 pT2GIII pT3aGIIIN1 9/48/F 1 3,3 pT1GII pT2bGIIIN1 4 (History of MMC + HT) 10/54/M 18 107,5 pTaGII pTaGII 11/73/M 2 22,3 CIS pT4aGIIIN2 12/75/M 10 57,7 pTaGII pTaGII 5 (Control) 13/71/M 9 115,5 CIS pT2GIII 14/71/M 0 3,1 pT2GIII pT2GIII 15/71/M 0 3,3 pT2GIII pT2GIIIN1 HT 2 Table 2 The p53 and Ki67 immunoreactivity scorings in percentages of all patients divided in five different treatment groups Groups Material Inflammation Proliferation (%) P53 (%) Haemorrhage 1 (MMC)  a Biopsy +++ 30 20 + Cystectomy +++ 75 20 ++  b Biopsy ++ 20 75 − Cystectomy +++ 15 75 +  c Biopsy +++ 40 75 +++ Cystectomy NA NA NA NA 2 (HT)  a Biopsy + 60 80 − Cystectomy +++ 20 40 ++  b Biopsy + 30 90 − Cystectomy ++ 20 40 −  c Biopsy + 10 - − Cystectomy ++ 10 - + 3 (MMC + HT)  a Biopsy +++ 40 >75 − Cystectomy +++ 25 75 −  b Biopsy +++ >75 >75 + Cystectomy +++ 60 15 −  c Biopsy +++ 30 >75 + Cystectomy +++ 15 75 ++ 4 (History of MMC + HT)  a Biopsy + 10 75 − Cystectomy + 20 − −   b Biopsy +++ 30 25 − Cystectomy +++ 30 25 −  c Biopsy ++ 50 20 − Cystectomy ++ 20 10 + 5 (Control)  a Biopsy + 15 >75 − Cystectomy +++ 40 >75 −  b Biopsy ++ 40 20 − Cystectomy ++ 40 20 −  c Biopsy ++ 35 75 + Cystectomy +++ 35 75 − The extend of inflammation and haemorrhage is semi quantitatively scored (1+ to 3+) NA The intensity of inflammation increased in three out of nine patients treated with hyperthermia. In three out of five patients from the MMC group and control group an increase was seen. The intensity of haemorrhage increased in four out of nine patients treated with hyperthermia, one patient showed a decrease and four patients did not show any difference. In the MMC group and in the control group two patients showed an increase, one patient a decrease and two patients did not show any difference. 1 Fig. 1 a1 a2 b1 b2  1 Discussion 15 4 13 1 The sample size of the group studied is small due to the fact that recruitment of patients was difficult. Patients had to agree on an extra treatment session only two days before cystectomy, which did not give them any benefit at all. N N N At least one patient per group (except the control group) showed an increase in the degree of haemorrhage. Patients from group 1 (solely MMC) showed the highest degree of haemorrhage. 13 14 16 Furthermore, the group treated with solely MMC did not show a significant decrease in proliferation activity. This is probably due to the limited penetration properties of this intravesical used drug, especially after one single treatment. p53 is known to be responsible for repair or apoptosis in response to DNA damage. The p53 activity, in other words the expression of mutant p53, decreased exclusively in patients treated with hyperthermia with or without MMC. 8 9 8 Conclusion The degree of inflammation and haemorrhage in bladder tumours did not increase in patients treated with hyperthermia. This, in combination with a decrease in proliferation activity and a decrease in p53 activity, implies that thermo-chemotherapy is a safe and promising treatment.