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melanoma

3.1

Introduction

A significant number of patients who are potentially rendered disease-free by surgery remain at high-risk ffom recurrent melanoma. These include patients with deep primary melanomas (> 4mm in thickness) and those with lymph node metastasis who, following resection o f macroscopic disease, may harbour micrometastatic deposits o f melanoma in other sites. These groups o f patients have 5-year survival rates o f 47% and 10-46% (dependent on the number of nodes involved and other prognostic factors) respectively and may therefore potentially benefit from adjuvant treatment (Koh 1991).

To date the only adjuvant treatment licensed for use in melanoma patients is interferon following the publication of the ECOG 1684 trial (Kirkwood 1996). This trial demonstrated that patients with stage III melanoma, when treated with high-dose interferon, had an increase in relapse-free survival from 1.0 to 1.7 years and overall survival from 2.8 to 3.8 years at 7 years o f follow-up. However, the survival benefit shown by the high-dose interferon regimen used in this trial have not been demonstrated by the preliminary results of the subsequent ECOG 1690 trial or by the lower dose strategies used in other studies (Cascinelli 1995), (Creagen et al. 1995) (Grob et al. 1998, Pehamberger et al. 1998, Kirkwood 1999). In addition, interferon is expensive and its use is associated with a significant incidence and degree of toxicity. Therefore, its use as adjuvant therapy in melanoma is now controversial.

The mechanisms by which interferon exerts its anti-tumour effect have not been fully established. Administration of interferon to tumours has been shown to lead to down-regulation o f c-myc oncogene expression and up-regulation o f p21AVAF gene expression, together leading to Gl/S phase delay in the cell cycle and a reduction in tumour proliferation (Dron et al. 1986, Subramaniam et al. 1998). P21AVAF1

expression was shown to increase following the administration o f interferon a to Daudi human Burkitt Imphoma cells in-vitro. A large reduction in c-myc expression proceeded the increase in p21AVAF 1 expression. This was accompanied by their growth arrest in G1 phase resulting in terminal differentiation. Cell death by apoptosis followed, accompanied by the induction of the apoptotic ICE-family protease CPP32. However, this was not observed when the cells were exposed to interferon gamma to which they are resistant (Subramaniam et al. 1998). C-myc down-regulation and p21AVAFl up-regulation occur by independent mechanisms after exposure of cells to interferon, but both are thought to be required for interferon-induced GO/Gl arrest to occur (Resnitzky et al. 1992).

Further evidence of the role of c-myc down-regulation has been provided by experiments conducted with Balb/c 3T3 fibroblasts transfected with activated c-myc. The untransfected cells were sensitive to interferon a and p and the cell cycle was blocked in G1 phase following exposure o f these cells to either agent. However in the c-myc transfected line, a 3-10 fold increase in c-myc mRNA levels was noted and the cells were resistant to the actions o f the two agents. The transfected cells passed freely from G1 into S phase even in the presence o f interferon a or p (Einat et al. 1988). A similar effect was noted in Ml myeloblastic cells transfected with an SV-40 promoted c-myc plasmid (Resnitzky et al. 1991). Over-expression o f the c- myc gene, and the subsequent failure to down-regulate its expression after cell exposure to interferon, is therefore associated with interferon resistance. This has been demonstrated in experiments conducted with variant hairy cell leukaemia cell lines in-vitro (Lehn et al. 1986).

Down-regulation o f c-myc is thought to occur via a reduction in the half-life o f its mRNA, which was observed in Daudi cells treated with interferon. No effect on the c-myc transcription rate in isolated nuclei of the Daudi cells following exposure to interferon has been observed (Dani et al. 1985). Activation of the 2 ’,5’- oligoadenylate synthetase/RNAase L enzyme has been observed in the DLD-1 Clone A human colonic carcinona and Daudi cell lines, associated with a reduction in c- myc expression, following their exposure to interferon-p (Knight et al. 1985, Chattel]ee et al. 1992). Activation of this enzyme by interferon may therefore be the mechanism by which c-myc mRNA is degraded and the reduction in activity o f the c-myc gene produced by interferon occurs.

Interferon is also known to increase surface expression of HLA molecules either directly, or via down-regulation of c-myc oncogene expression, therefore leading to increase cell killing by cytotoxic T-cells. Stimulation of HLA class 1 mRNA transcription has been observed in Daudi lymphoma cells following their exposure to interferon a and p (Dron, 1986 ) (Dani, 1985 ). Reduced tumour c-myc oncogene expression is known to be associated with increased HLA expression on the surface of tumour cells (Grover et al. 1996), increasing their antigenicity.

It can therefore be postulated that c-myc down-regulation plays a key role in the anti-tumour effect of interferon, leading to both inhibition o f the cell cycle in the G1 phase, and up-regulation of HLA class 1 molecules on the cell surface. Cell death follows either by apoptosis or by killing ffom cytotoxic T cells. A postulated mechanism of action for interferon is shown in figure 3.1.

For interferon to be clinically useful as an adjuvant agent it must be effective against all melanoma tumours, including those with the worst prognosis as these are the group o f patients most likely to experience disease recurrence. However this group of patients has been shown to have tumours which express the highest levels o f the c- myc oncogene by a number of studies (Grover et al. 1997, Ghana et al. 1998, Ross et al. 1998). As one of the mechanisms that interferon uses to block tumour growth is to down-regulate c-myc expression, the anti-tumour effect of interferon may be least effective in the patients who have the tumours of worst prognosis. However, this is the group o f patients who need to obtain maximum benefit ffom interferon. This may potentially be a reason why interferon is of doubtful value as an adjuvant agent in melanoma.

The relationship between melanoma c-myc expression and tumour interferon sensitivity has therefore been investigated in this chapter using a series o f uveal melanomas.

Figure 3.1: Postulated Mechanism of Action of Interferon