483MO - RNA-based multiplex polymerase chain reaction and sequencing to detect fusion genes in melanoma

Background

BRAF and MEK inhibitors are used to treat unresectable melanoma with BRAF mutations. BRAF-wild melanoma is usually treated with immune checkpoint inhibitors but treatment options are limited, indicating the need for alternative molecular targets. Fusion genes have been detected in a minority of driver mutation-negative melanoma cases and are possible treatment targets.

Methods

To detect fusion genes in driver mutation-negative melanoma, we used an Archer® FUSIONPlex® custom panel, which can detect LCK, MAP3K3, MAP3K8, MERTK, TRIM11, RASGRF1, and RASGRF2 fusions in addition to 60 fusion genes detectable with the Archer® FUSIONPlex® Sarcoma v2 panel. The Ion GeneStudio® G5 sequencer was employed for the sequencing. Detected fusion genes were validated using reverse transcription polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH).

Results

Among 104 Japanese melanoma tissues, single nucleotide variants and short insertions/deletions were analyzed using an AmpliSeq® custom DNA sequencing panel for melanoma, resulting in the identification of 21 melanomas without driver mutations such as BRAF, RAS, NF1, and KIT. Among the driver mutation-negative melanomas, 16 were tested for RNA quality. Twelve tumors that passed the quality control were analyzed for fusion genes using the Archer® FUSIONPlex® custom panel, identifying fusion genes in two tumors (17%): MAD1L1::BRAF and CIC::MEGF8. RT-PCR confirmed the existence of both fusion genes, and FISH confirmed the split of the 5’ and 3’ chromosomal regions of BRAF in the tumor cells bearing MAD1L1::BRAF. FISH could not validate CIC::MEGF8 because CIC and MEGF8 are close to each other (approximately 30 kb). Clinically, BRAF fusions are potential targets of MEK inhibitors. CIC::MEGF8 is a novel fusion gene. CIC is a transcriptional repressor of ETV1/4/5 and acts as a tumor suppressor gene. Homozygous deletion of CIC has rarely been seen in melanoma. Currently, no drugs targeting CIC fusions are known.

Conclusions

The Archer® FUSIONPlex® custom panel detected fusion genes in 10% of driver gene-negative melanomas. Combining DNA- and RNA-based multiplex sequencing is useful to identify potentially targetable gene alterations.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

JSPS Kakenhi, Maruho Takagi Dermatology Foundation, Suhara Memorial Foundation.

Disclosure

All authors have declared no conflicts of interest.