Sandra Medic

Date of Award

1-1-2006

Document Type

Thesis - ECU Access Only

Publisher

Edith Cowan University

Degree Name

Master of Science

School

School of Exercise, Biomedical and Health Sciences

Faculty

Faculty of Computing, Health and Science

First Supervisor

Dr Melanie Ziman

Abstract

Cutaneous Malignant Melanoma (CMM) is the most aggressive form of skin cancer, with high mortality rate in patients with metastatic spread. The focus of recent research has been to find molecular markers of melanoma that can be used to detect metastatic cells in the blood of patients with CMM and to assist in diagnosis and staging as well as to predict the outcome of the disease. PAX3, a transcription factor encoding gene that regulates melanocyte migration, proliferation and differentiation during development is found to be highly expressed in melanomas and melanoma cells, particularly its alternate transcript PAX3d. Therefore, the aim of this study was to establish an assay that can be used to detect occult melanoma cells in peripheral blood of patients with CMM and to assess PAX3d as a melanoma cell marker.

We used staged formalin-fixed paraffin-embedded tissue and metastatic lymph node tissue to assess the expression of PAX3 transcripts in primary and metastatic melanoma tissue as well as in naevi and normal skin. Although problematic we were able to establish a technique for isolation of total RNA from archival formalin-fixed tissue slides. Furthermore, we perfected blood collection procedures and methods of total RNA isolation from peripheral blood. We also demonstrated that real time qRT-PCR provides a significantly higher marker detection rate relative to conventional gel-based RT-PCR and therefore suggest its use as a more sensitive and accurate detection method.

Analysis of PAX3 expression in peripheral blood of patients diagnosed with CMM showed significantly higher marker expression frequency in patient blood samples in contrast to no expression in blood from healthy volunteers. Results also showed that the alternate transcript PAX3d was detected at significantly higher frequencies in peripheral blood of melanoma patients than the PAX3c alternate transcript. Moreover PAX3d was more frequently expressed in patients diagnosed with metastatic disease compared to those diagnosed with in-situ and invasive melanoma.

Frequent expression of PAX3d in patients with in-situ melanoma even after more than one year since diagnosis was interesting, and suggests the presence of circulating melanoma cells even at this early stage of the disease. Since recent studies have found that melanoma cells with high metastatic potential have a different gene expression signature compared to highly proliferative cells with low metastatic potential, it is still to be clarified whether PAX3d is a marker of metastatic cells with low or high metastatic potential. Nevertheless, results presented here show that PAX3d is a sensitive and useful marker of melanoma cells in peripheral blood of melanoma patients.

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