Hepatocellular carcinoma (HCC) is one of the most common internal malignancies worldwide. There are a number of well-recognised risk factors for HCC including cirrhosis of any cause, male gender and specific liver disease such as hepatitis B infection. However, relatively little is known about the genetic alterations that occur during hepatocarcinogenesis. Recently a family of enzymes, the DNA mismatch repair (MMR) enzymes, has been implicated in a variety of human cancers. Inactivation of DNA MMR results in the accumulation of mutations during cell division.
Scattered throughout the genome, principally in non-coding regions are polymorphic markers called microsatellites. These are composed of variable number of repeat units, with up to 6 nucleotides per repeat. In cells with defective MMR, strand slippage during DNA synthesis results in the addition or loss of repeat units from microsatellites, resulting in microsatellite instability (MSI) in the cancer. These cancers develop similar changes in mononucleotide repeat sequences in coding regions of genes such as TGFBR2 and IGF2R. Defective DNA MMR is due to inactivation of one or more of the family of proteins normally involved in MMR.
A second major mechanism of malignant transformation involves the loss or duplication of large portions of chromosomal arms during cell division. Loss of chromosomal material containing tumour suppressor genes can offer growth advantages to affected daughter cells. Microsatellites can be used to recognise alterations in chromosome copy number, a change described as loss of heterozygosity (LOH). IGF2R has previously been identified as a major target for LOH in HCC.
The role of MSI in hepatocarcinogenesis has not previously been established. The aims of the studies in this thesis were to determine i) the prevalence of MSI in HCCs, ii) the relationship between MSI and the MMR genes, hMLH1 and hMSH2, iii) the relationship between defective MMR and alterations in IGF2R and TGFBR2.
HCC is relatively uncommon in Western countries. Initially DNA was extracted from microdissected formalin-fixed tissues obtained in the USA and studied with a panel of 16 microsatellites. MSI was present in 31 of 46 (67%) North American HCCs, although this was relatively low-level MSI. MSI was associated with LOH of the MMR genes hMLH1 and hMSH2 in 9 of the 46 liver tumours. In the HCCs with LOH there was generally a consistent pattern of LOH throughout the cancer (in 8 of 9) and this was associated with LOH in adjacent non-malignant liver tissue.
Subsequent experiments examined DNA from frozen liver tissue for LOH and MMR-specific mutations of IGF2R and TGFBR2, and the relationship of these to MSI. LOH at chromosome 6q25-27 in the vicinity of IGF2R was detected in 17 of 41 (41%) Australian and South African HCCs. IGF2R appeared to be the target of LOH at 6q25-27 and the prevalence of this was significantly higher than for LOH of TGFBR2, confirming the importance of LOH of IGF2R. However, MSI was detected in only 3 (7%) HCCs and no MMR-specific mutations in the coding regions repeats of IGF2R and TGFBR2 were detected. Loss of hMLH1 protein expression was detected in only 1 HCC.
The reason for the discrepancy in the prevalence of MSI in North American and Australian and South African HCCs was not clear. One possibility was that this was related to the tissue source of DNA (formalin-fixed versus frozen). Another possibility was that there were fundamental differences in the pathogenesis of HCC in the two groups. To address this experiments using DNA from microdissected formalin-fixed tissues were repeated with Australian HCCs. MSI was detected in 16 of 44 (37%) HCCs. This was similar to the prevalence detected in North American HCCs. One possible explanations for the higher prevalence of MSI in formalin-fixed tissues compared to frozen tissue include the presence of subclones with varying degrees of MSI within the HCC. Alternatively this MSI could be an artefact related to the use of formalin-fixed tissue as a source of DNA. Four HCCs were microdissected and PCRs repeated using the same DNA extraction to determine the reproducibility of results. This demonstrated a) that LOH detected in frozen liver tissue will generally also be detected in formalin-fixed tissue, and b) DNA quality may be associated with the detection of MSI and/or a chaotic pattern of LOH in microdissected tissues. The studies in this thesis have demonstrated that when MSI is detected in HCCs, this is low-level and not associated with MMR-specific mutations of TGFBR2 and IGF2R, or with loss of hMLH1 protein expression. This suggests MMR function is intact in these tissues. Further studies are necessary to determine the significance of MSI detected in HCC. This thesis has also confirmed the importance of LOH of IGF2R in hepatocarcinogenesis in HCCs from diverse ethnic and geographical backgrounds.