Methods: The present study was performed on DNA extracted from archival paired tumours of 9 male and female patients. It utilised (a) NSA and allelic imbalance (AI) analysis with a total of 7 microsatellite markers on 3p, 8p, 9p and 17p; (b) p53 mutation analysis by the direct sequencing of exons 4-10 of the gene, and (c) X-chromosome inactivation analysis in females. HPV16 analysis and p53 immunohistochemistry (IHC) were also employed.
Results: The presence of a shared NSA in the paired tumours of 3/6 females confirmed the value of these aberrations for identifying clonally related lesions. Another set of paired tumours was shown to arise independently based on discordant X-chromosome inactivation. Unexpectedly, no p53 mutations were detected in the 6 index tumours from females, although stabilised p53 was detected by IHC and 2 tumour pairs were HPV16-positive. Second lung tumours arising in 2 males with UAT primary lesions were shown to be metastases, based on concordant AI patterns, shared NSAs and concordant p53 mutations. In a further second lung tumour case, with concordant AI at 2 microsatellite markers, the paired tumours bore different p53 mutations, highlighting the difficulties in interpretation inherent in clonality analysis.
Conclusions: These findings provide substantial molecular evidence that a proportion of UAT second tumours are recurrences of an index lesion. In addition, the combination of AI at 2 or more loci at separate chromosomal arms and concordant p53 mutations can act as a surrogate marker of clonality when NSAs are not detected.