N-acetyltransferase 2 in the recurrence and progression of patients with bladder transitional cell carcinoma

N-acetyltransferase 1 and 2 are metabolic enzymes involved in the detoxification or bioactivation of aromatic amines. The development of transitional cell carcinoma of the bladder is associated with exposure to chemical carcinogens, either from occupational exposure or through smoking, and many chemical carcinogens are aromatic amines. The genes that encode the N-acetyltransferase enzymes are polymorphic, and particular alleles are associated with an increased incidence of bladder cancer. Transitional cell carcinoma of the bladder is the fourth commonest cancer in the UK and USA. Repeated recurrences (in up to 80% of patients), place an individual at increased risk of developing muscle invasion. However it is not possible to predict, from clinical and pathological data, which index (primary) carcinomas will recur. Due to the increased risk of recurrence and progression of non-muscle-invasive transitional cell carcinoma, patient management involves prolonged clinical follow-up as at present there are no reliable markers to predict the biological behaviour of an index, non-invasive transitional cell carcinoma. Intensive scientific research over the past decade into genetic mechanisms involved in transitional cell carcinoma has established two distinct genetic pathways to progression, and recently markers of recurrence have also begun to emerge. The aim of this study was to use a DNA probe for the N-acetyltransferase 2 gene (NAT2) and to investigate whether genetic abnormalities at this locus contribute to recurrence or progression in an index, non-muscle-invasive transitional cell carcinoma, and whether patients who present with muscle-invasive transitional cell carcinoma (stage pT2 and above) have an abnormal genotype. NAT2 is on chromosome 8p, a region commonly aberrent in many carcinomas including bladder. In order to achieve this aim, it was necessary to obtain a cosmid containing the NAT2 sequence, label the cosmid to produce a probe for fluorescence in situ hybridisation, and then to apply the technique to formalin fixed paraffin processed sections of transitional cell carcinomas. This process proved technically challenging, but once the methodology was working consistently well on formalin fixed paraffin processed control material, sections of transitional cell carcinomas were assessed in a dual labelling technique with the NAT2 probe and an alpha centromeric probe for chromosome 8. Nineteen patients were assessed, with full clinical follow-up (median=55 months), in eight of whom carcinomas recurred. Fluorescence in situ hybridisation was also applied to the recurrences, and a total of 37 carcinomas were assessed. Of the 19 primary carcinomas assessed, five were muscle-invasive at presentation (stage pT2 or above), the remainder were stage pTa or pT1. All five muscle-invasive carcinomas had polysomy of chromosome 8, four of whom also had abnormal NAT2 copy number. Another six primary carcinomas (all stage pTa or pT1) had abnormal chromosome 8 and/or NAT2 copy number. Of the 18 recurrences, 9 were abnormal for chromosome 8 and/or NAT2 copy number, eight of which were in patients who progressed at recurrence to muscle invasion (pT2 or above). Overall, a high proportion of carcinomas 54% (20/37) when assessed by mean chromosome or gene copy number, were abnormal for either gene or chromosome copy number or both, and this was significantly associated with high grade, p=0.014 and stage pT2 or above, p=0.039. There were more abnormalities in patients with detrusor muscle-invasive disease. Few studies of abnormalities of gene and/or chromosome copy number in transitional cell carcinoma of the bladder achieve full clinical follow-up, and are unable to relate their findings to the clinical course of the disease. The abnormalities of NAT2 demonstrated in this study were more common in patients with muscle-invasion at some point in their disease history, linking abnormal function of N-acetyltransferase 2 with aggressive disease course. Polysomy 8 was also common, and abnormalities of chromosome 8 have been previously been associated with late stage progression in transitional cell carcinoma. In conclusion, by studying the gene and chromosome copy number of NAT2 and chromosome 8 in index transitional cell carcinomas and any subsequent recurrences further understanding of the role of metabolic enzymes in transitional cell carcinoma has been realised. For molecular biology to have any impact on patient management, studies such as this one, recapitulated with larger numbers, will begin to become a much needed additional tool in the accurate diagnosis and management of this complex cancer