Abstract
Greater understanding of the pathological mechanisms underlying recurrent driver mutations is revolutionising the clinical management of melanoma. However given the association between UV radiation and UV-associated DNA mutagenesis, there are emerging indications that NZ may have a distinctive pattern of recurrent mutations more representative of high UV exposure. In order to gain insight into the frequency of recurrent mutations in NZ and how it may impact the clinical management of melanoma, this project encompassed three aims.
The first aim was to determine a robust protocol for DNA extraction from FFPE melanoma tissue, and then optimise PCR amplification and sequencing of BRAF, NRAS and RAC genes, and the TERT promoter. The Qiagen FFPE extraction kit provided DNA of sufficient quality and quantity. Optimal PCR amplification required the combined use of shorter amplicon fragments, KAPA2G Robust polymerase enzyme, a touchdown PCR cycle and nested PCR. Lastly, the purification of sequenced DNA products required either the EDTA-ethanol wash or the unifilter plate-sephadex resin method.
The second aim evaluated the analytical performance of the novel Immunohistochemistry (IHC)-based VEI monoclonal antibody test to detect the BRAF V600E mutation status in comparison to two reference methods, the Cobas assay, which is the only adopted test in NZ for BRAF mutation testing, and conventional Sanger sequencing. IHC demonstrated the highest sensitivity for the detection of BRAF V600E and V600E2 mutations, which supports its use as a supplementary screening assay for determining the BRAF-mutant genotype.
The final aim was to determine the frequency of recurrent mutations in the BRAF, NRAS and RAC1 genes and the promoter of TERT in South Island melanoma patients using 95 melanoma tissues from 87 patients with Sanger sequencing. A distinctive pattern of recurrent mutations was observed, which may be representative of chronic UV exposure. This included a relatively high frequency of melanomas wild-type for BRAF and NRAS mutations and a higher frequency of primary melanomas with TERT promoter mutations compared to studies in USA or Europe. This would then suggest that therapeutic regimes that target the non-BRAF-NRAS mutant subgroup and those with TERT promoter mutations may become more relevant in NZ.
The results from the BRAF mutation study were compared with 2 external studies, a study using North Island melanoma samples (Wellington) and a study carried out on a national scale (Auckland, Tauranga and Christchurch). The frequency of the BRAF V600E mutation was found to be relatively low in NZ compared to USA or Europe. This is consistent with BRAF V600E mutations having an inverse association with chronic UV damage. Moreover, the low BRAF V600E mutation rate indicates the clinically approved targeted inhibitors for BRAF-mutant melanomas may be less relevant in the NZ population. The BRAF V600K mutation rate was found to be higher in the North Island compared to the South Island, which highlights a potential difference in mutation frequency between geographical locations within NZ.
Overall, this study provides new insights of regarding a distinctive pattern of recurrent mutations in the South Island of NZ and NZ, more representative of chronic UV exposure. Furthermore this indicates the clinical management of melanoma may also be slightly different in NZ.