4-Thiothymidine closely resembles thymidine but has distinct photochemical properties. The molecule absorbs light in the UVA range and readily undergoes photochemical reactions. Thiothymidine can be incorporated into the DNA of replicating cells and subsequent exposure to UVA light results in the formation of lethal DNA adducts. This project aims to investigate whether the combination of thiothymidine and UVA may offer a selective treatment for bladder cancer. The rhymidylate synthetase inhibitor, raltitrexed, is hypothesised to increase thiothymidine incorporation into cellular DNA thus potentiating the efficacy of this treatment.

Certain membrane bound mucins such as MUC1, MUC4, and MUC12 were expressed in normal bladder urothelium.  MUC1 expression was the highest in normal bladder urothelium and was present in almost all samples. In superficial bladder cancer, MUC1 demonstrated a pattern of expression that increased in proportion to the increase in tumour grade from Grade 1 to Grade 3 and carcinoma <i>in situ</i>.  Utilising TMAs, membrane-bound mucins such as MUC1, <i>MUC3, </i>MUC4, MUC12 and MUC 16 were shown to have increased incidence of expression with tumour stage progression  to muscle-invasive bladder cancer (stage T2 and above).  Secretory mucins showed a more varied pattern of expression.  <i>MUC2</i> mRNA was up-regulated in progressive muscle-invasive bladder cancer.   MUC5AC and MUC5B displayed a low incidence of expression in superficial tumours and muscle invasive bladder cancer. <i>MUC7</i> mRNA expression, however, was limited to only muscle-invasive disease similar to the membrane-bound mucins described above. In a small proportion of patients in the recurrent progressive TMA, expression of MUC4, MUC5AC and <i>MUC</i>5B in penultimate superficial tumours prior to tumour progression allowed prediction of impending disease progression to muscle invasive disease.  MUC5B, MUC12 and MUC13 were expressed at higher incidence in the single non-recurrent TMA, compared with the incidence of expression in the recurrent non-progressive TMA. The expression of these mucins in newly diagnosed superficial bladder tumours may thus favour better prognosis, with a subsequent reduced risk of tumour recurrence.

Hypoxia has been evaluated in a series of human bladder carcinomas using pimonidazole. The relationships of hypoxia with vascularity and with proliferation have been explored. Two intrinsic markers of hypoxia have been validated against pimonidazole and then used to assess the impact of hypoxia on a cohort of bladder tumours treated with a hypoxia modifying treatment stratagem. Thirty-one patients with confirmed transitional cell carcinoma of the bladder received pimonidazole before transurethral resection of the tumour. Sections from 26 tumours were stained for pimonidazole. The median hypoxic fraction was 9% (range 0 to 38%). Twenty-one tumours were double stained for pimonidazole with Ki67 (proliferation), and pimonidazole with CD31/34 (vascularity). Most staining was distant from vessels (median 100um). Some hypoxia was close to vessels (< 40um), suggesting a perfusion limiting mechanism in these areas. Dual staining for pimonidazole and proliferation showed a predominantly inverse relationship between these factors. However, some hypoxic regions of less than 40um from a vessel had a higher proliferative index than those further away from vessels Having confirmed GLUT1 and CA9 as appropriate intrinsic markers for hypoxia, archived samples of bladder carcinoma from 64 patients treated with ARCON were obtained. Sections were stained for GLUT1, CA9, Ki67 and CD31/34 and analysed as above. The median follow up time was 24 months The median stained fraction for GLUT1 was 6.5% (range 0 to 62%) and for CA9 was 3.5% (range 0 to 67%). Those patients above the median (more hypoxic tumours) for each marker had a statistically significantly poorer cause specific survival (GLUT1 p < 0.005, CA9 p < 0.003) but not local recurrence or metastasis free survival, analysed with a log rank test. A multivariate analysis, accounting for stage, age, grade, Ki67 index and vascularity confirmed hypoxic fraction as an independent factor in cause specific survival (GLUT1 p < 0.02, CA9 p < 0.03). In conclusion, hypoxia is a significant factor in bladder cancer, and is quantifiable with both extrinsic and intrinsic markers. Staining patterns are consistent with diffusion limited and perfusion limited mechanisms of hypoxia. Proliferation is reduced in hypoxic areas but less so in those that are acutely hypoxic. In a cohort of patients with bladder cancer treated with ARCON, patients with more hypoxic tumours, assessed with intrinsic markers, tend to a poorer cause specific survival.

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