Exploration of a Genotype-Phenotype Correlation in a Panel of Metastatic Human Melanoma Cell Lines
Andrea Boni, Marc S Ernstoff, Mary C Schwab, Jan L Fisher, Laura J Tafe, Gregory J Tsongalis. Dartmouth Hitchcock Medical Center, Lebanon, NH
Background: Metastatic malignant melanoma remains an incurable disease, but recent advances in innovative therapeutic modalities such as molecular targeted therapy offer exciting new opportunities. These advances have created the clinical need to assess the genetic makeup of tumors. Although genetic testing is becoming widely available it would be an advantage to rely on more widespread technologies, like morphology and immunohistochemistry (IHC) to screen melanomas for common mutations. The focus of our study was to establish if any IHC staining pattern could be used to infer the genotype of melanoma cell lines.
Design: We tested 11 melanoma and 1 dermal fibroblast cell line that we had previously analyzed with a Qiagen qBiomarker™ Somatic Mutation PCR Array interrogating common point mutations in the AKT1, BRAF, cKIT, KRAS, HRAS, NRAS, MEK1, PIK3CA and PTEN genes (Qiagen). We than stained our cells for melanocytic markers (gp100, MELAN-A, tyrosinase), cancer testis antigens (NY-ESO-1, MAGE-A1), metalloproteinases-1 (MMP-1) and protease activated receptor -1(PAR1), using IHC methodology on agar embedded cell pellets.
Results: Three cell lines were wild type for all 85 mutations tested. Eight cell lines harbored various mutations, with all 8 including the common BRAF V600E. Most of the tested markers showed a highly heterogeneous expression with no correlation between IHC staining pattern and underlying genotype. Protease activated receptor -1(PAR1) was consistently strongly positive in all cell lines. Because many of the mutations present in our cell lines act primarily on the MAPK pathway we also analyzed the levels of pERK1/2, pMEK1/2 and pBRAF as a surrogate marker for the activation status of the pathway. Again no positive correlation could be made between the phosphorylation status of key molecules in the pathway and the IHC phenotype.
Conclusions: In light of these results, changes in expression levels of specific molecules upon inhibition at various points of the MAPK pathway might be more revealing of its importance in their regulation. Future studies will help us determine what phenotypic changes might be expected in patients that are being treated with new drugs targeting this pathway.
Wednesday, March 21, 2012 1:00 PM
Poster Session VI # 109, Wednesday Afternoon