Duplication and Polysomy: Novel Alternative Pathways for BRAF Activation in Primary Melanoma
Shivani R Kandukuri, Michael J McFall, Julie Wu, Jean Lopategui, Elizabeth Spiteri. Cedars-Sinai Medical Center, Los Angeles, CA
Background: Identification of the BRAF V600E activating mutation on chromosome 7q in a subset of melanomas (MM) has allowed for the application of targeted therapy. We utilized polymerase chain reaction (PCR) and fluorescent in-situ hybridization (FISH) to identify alternative mechanisms of BRAF activation with potential therapeutic implications.
Design: A search of our database identified 19 cases of primary MM for which hematoxylin and eosin slides were reviewed. Twenty cases of nevi served as controls. Two 10 µm and 4 µm unstained slides for PCR and FISH were analyzed per case. PCR analysis was performed with the FDA-approved Roche Cobas 4800 BRAF V600 mutation assay. For FISH analysis, 40 cells per case were interpreted for signals (gold-BRAF and green-centromere). A ratio of gold to green signals was calculated per 40 cells and interpreted as normal (2:2), amplification (≥2:1), and polysomy (≥3 green and gold signals with a ratio of less than 2:1 in ≥40% of the cells). Tandem duplication (TD) was defined as a ratio of 3:2 in ≥10% of the cells. Five cases of MM and 3 nevi were excluded due to inadequate specimens for a total of 15 MM and 17 nevi. Statistical analysis was performed with Fisher's exact test.
Results: V600E mutation was shown in 8/15 (53%) MM and in 14/17 (82%) nevi (p=0.128). FISH analysis showed polysomy of BRAF in 4/15 (27%) MM and none in nevi (p=0.038). TD was detected in 5/15 (33%) MM and none in nevi (p=0.015). Of the 7 cases negative for BRAF mutation, polysomy was observed in 1 (14%) case and TD in 2 (28%) cases. Neither MM nor nevi showed amplification. There were only 3 cases of MM with V600E and polysomy, one of which also showed TD. One case showed mutation and TD.
Conclusions: Polysomy and TD are present in 27% and 33% of MM, respectively. Concomitant with their notable absence in nevi, polysomy and TD may represent novel alternative pathways of BRAF activation in MM. The expected frequency of mutation in MM and nevi by PCR was confirmed. Neither MM nor nevi display amplification. TD, polysomy, and V600E are present in a single case, suggesting that these mechanisms may not always be mutually exclusive. As the sample size of MM cases is small, a significant p value was not reached for V600E mutation and polysomy. Additional research is needed to elucidate the biologic/therapeutic significance of these findings.
Tuesday, March 5, 2013 9:30 AM
Poster Session III # 40, Tuesday Morning