The adenomatous polyposis coli gene (APC) is located at human chromosome 5q21. Germline mutations in the APC gene characterise the Mendelian dominant inherited disorder, familial adenomatous polyposis coli (FAP). FAP patients develop numerous adenomas within the large intestine, some of which ultimately progress to carcinoma. A more general role for APC mutations in neoplasia is suggested by the fact that FAP patients are at risk of developing tumours of the brain, thyroid, bone and focal proliferative lesions ( "desmoid tumours ") of the connective tissue. Furthermore, mutations in the APC gene and / or loss of heterozygosity (LOH) at 5q21 have been reported in a range of sporadic tumours including tumours of the lung.
This thesis firstly investigated the occurrence of LOH at chromosome 5q21 in subtypes of human non -small cell lung cancer (NSCLC). Allele loss was compared in adenocarcinoma (ADC) and squamous cell carcinoma (SCC), the most common forms of NSCLC. Further sub -classification of the ADC subgroup was carried out to compare ADC arising in the bronchus versus the parenchyma, as determined by histology. Four genetic polymorphisms in the APC and MCC (mutated in colorectal cancer) genes at chromosome 5q21 were analysed for LOH in 92 consecutive primary SCC and ADC of the lung. Loss of heterozygosity was identified in at least two polymorphic loci in 40% (27/68) of informative cases. There was no significant difference in the frequency of LOH between SCC and ADC cases (Chi- squared test, df=1, p= 0.446) or within the ADC subgroups; bronchial and parenchymal origin (Fisher's exact test; two -tailed test p= 0.237). Results revealed that the frequency of LOH within the SCC group and the ADC remained similar at each tumour stage, Fisher's exact test (two -tailed test; p >0.2 in all cases). This result suggests that loss of 5q21 does not promote metastatic spread in these histological groups. Following the subdivision of ADC into site of origin, results revealed that LOH was not associated with increasing tumour stage in the parenchymal or bronchial subgroups, as determined by Fisher's exact test (two -tailed test; p >0.2 in all cases), or that the frequency of LOH differed between subgroups at each tumour stage,
Fisher's exact test, two tailed test; p >0.2 in all cases.
Thirty tumours showing LOH at one or more polymorphic sites were examined for mutations in the mutation cluster region (MCR) of APC by single- strand conformational polymorphism (SSCP) analysis. Mutations were not detected in any of these cases. These results suggest that it is likely that a tumour suppressor gene on 5q other than APC is involved in the pathogenesis of lung cancer.
Secondly, this thesis examined ADC of parenchymal or bronchial origin by the independent criterion of K -RAS mutation, a known feature in pulmonary ADC. Sixty -five surgically resected primary pulmonary adenocarcinomas were screened for mutations at codon 12 of the K -RAS gene by a PCR based method. Mutations in position 1 or position 2 of codon 12 were detected in 16 tumours (25 %). When analysed by site of origin, mutations were seen in 9/26 (35 %) parenchymal and in 0/12 bronchial ADC. This difference is significantly different, as determined by Fisher's exact test (two -tailed test; p= 0.0355). No association was noted between the presence ofK -RAS mutation at tumour stage, indicating that K -RAS mutations are not associated with metastatic spread. K -RAS mutations were also detected in 5 out of 32 foci of alveolar atypical hyperplasia (AAH), a lesion considered on histological grounds to be the precursor to parenchymal ADC. DNA sequencing showed that the great majority of mutations in both ADC and AAH were G -T transversions. These findings provide support for the classification of pulmonary ADC into bronchial and parenchymal subtypes and also provide molecular evidence to support the importance of AAH in the development of parenchymal cancers.
Finally, this thesis described the generation and initial characterisation of a murine APC transgenic founder line designed as a model to investigate the effects of aberrant expression of APC. Several Apc mutant murine models of FAP already exist. These all carry a heterozygous mutation in the Apc gene. The Min mouse (multiple intestinal neoplasia) is an example and carries a mutation at codon 850. All current models however are of limited use as mice homozygous for Apc mutations die at approximately
5 6.6 days post coitum, limiting analysis to Apc heterozygotes. Homozygous loss of Apc therefore depends upon additional somatic events that are not under direct experimental control and this may be associated with additional, undisclosed genetic events. Here a transgenic approach was taken to generate animals where the expression of the APC transgene is conditionally inactivated using the Cre -loxP recombination system of the bacteriophage P 1. APC cDNA was flanked by loxP sequences and the promoter sequence of the ubiquitously expressed murine phosphoglycerate kinase (Pgk) gene cloned upstream of the APC cDNA to drive the expression of the transgene. Pro -nuclear injection was used to deliver the APC transgene into oocytes of wildtype mice (F1 (C57BL /6 x CBA)). A transgene positive founder line was established and transcription of the APC cDNA confirmed in a wide variety of tissues using reverse transcription polymerase chain reaction (PCR). To date, transgene positive mice on an Apc wildtype background show no gross phenotype. Embryonic fibroblasts were derived and Cre- recombinase delivered by infection with a replication deficient adenovirus. APC cDNA excision was confirmed by PCR. The founder line was crossed with the C57BL /6 Apc +/min line. Transgene positive Apc +Irvin mice were interbred and offspring screened to identify whether the APC transgene can rescue the Apc M II lethal phenotype. To date embryonic rescue has not been identified.