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High throughput mass spectrometry-based mutation profiling of primary uveal melanoma
Anthony B Daniels1, Joo-Eun Lee, Laura E MacConaill
1Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114-3096, USA.
Purpose:
We assessed for mutations in a large number of oncogenes and tumor suppressor genes in primary uveal melanomas using a high-throughput profiling system.
Methods:
DNA was extracted and purified from 134 tissue samples from fresh-frozen tissues (n = 87) or formalin-fixed, paraffin-embedded tissues (n = 47) from 124 large uveal melanomas that underwent primary treatment by enucleation. DNA was subjected to whole genome amplification and MALDI-TOF mass spectrometry-based mutation profiling (>1000 mutations tested across 120 oncogenes and tumor suppressor genes) using the OncoMap3 platform. All candidate mutations, as well as commonly occurring mutations in GNAQ and GNA11, were validated using homogeneous mass extension (hME) technology.
Results:
Of 123 samples, 97 (79%, representing 89 unique tumors) were amplified successfully, passed all quality control steps, and were assayed with the OncoMap platform. A total of 58 mutation calls was made for 49 different mutations across 26 different genes in 34/98 (35%) samples. Of 91 tumors that underwent hME validation, 83 (91%) harbored mutations in the GNAQ (47%) or GNA11 (44%) genes, while hME validation revealed two tumors with mutations in EGFR. These additional mutations occurred in tumors that also had mutations in GNAQ or GNA11.
Conclusions:
The vast majority of primary large uveal melanomas harbor mutually-exclusive mutations in GNAQ or GNA11, but very rarely have the oncogenic mutations that are reported commonly in other cancers. When present, these other mutations were found in conjunction with GNAQ/GNA11 mutations, suggesting that these other mutations likely are not the primary drivers of oncogenesis in uveal melanoma.
Insights
Uveal melanomas, a rare eye cancer, frequently exhibit mutations in GNAQ or GNA11 genes. Other common cancer mutations are rare and unlikely drivers in this cancer.
Area of Science:
- Oncology
- Genetics
- Ophthalmology
Background:
- Uveal melanoma is the most common primary intraocular malignancy in adults.
- Genetic alterations are key drivers in uveal melanoma pathogenesis.
Purpose of the Study:
- To comprehensively profile mutations in oncogenes and tumor suppressor genes in primary uveal melanomas.
- To identify frequently mutated genes and pathways in uveal melanoma using high-throughput screening.
Main Methods:
- DNA extraction from 134 primary uveal melanoma tissue samples.
- Whole genome amplification and MALDI-TOF mass spectrometry-based mutation profiling (>1000 mutations across 120 genes) using the OncoMap3 platform.
- Validation of candidate mutations and GNAQ/GNA11 mutations using homogeneous mass extension (hME) technology.
Main Results:
- Successfully profiled 97 out of 123 samples (79%).
- Identified 49 unique mutations across 26 genes in 35% of samples.
- Found GNAQ (47%) or GNA11 (44%) mutations in 91% of validated tumors; two tumors had EGFR mutations alongside GNAQ/GNA11 mutations.
Conclusions:
- The majority of primary uveal melanomas harbor mutually-exclusive GNAQ or GNA11 mutations.
- Oncogenic mutations common in other cancers are rare in uveal melanoma.
- Co-occurring mutations with GNAQ/GNA11 are unlikely primary drivers of uveal melanoma oncogenesis.
