Next-Generation Sequencing for Uveal Melanoma
The DecisionDx-UMSeq test is a 7-gene panel that uses next-generation sequencing (NGS) to identify somatic mutations relevant to UM. The test includes hotspot mutations in the genes GNAQ, GNA11, CYSLTR2, PLCB4, and SF3B1, mutations in exons 1-2 of EIF1AX, and all coding exons of the BAP1 gene.
This information, together with results from the DecisionDx®-UM and DecisionDx®-PRAME gene expression profile (GEP) tests, can help to build a comprehensive genomic profile of an individual UM tumor from a single biopsy. The genomic information can be used now to inform patient care and may become useful in the future as research and therapeutics evolve.
DecisionDx-UMSeq can be ordered for patients who are having DecisionDx-UM GEP testing performed. Castle Biosciences has optimized the sequencing so that it can be run using the same fine-needle aspiration biopsy (FNAB) tissue specimen submitted for GEP testing.
Mutations In 4 Genes Affect G-Protein-Coupled Receptor Signaling
In UM, mutations in GNAQ, GNA11, CYSLTR2, or PLCB4 result in constitutive activation of G-protein-coupled receptor signaling pathways, such as MAPK, PI3K, PKC, Hippo, etc. (Van Raamsdonk, 2009; 2011; Johansson, 2016; Moore, 2016).
While some of these pathways may be targeted with inhibitory therapies, these mutations are not currently markers for drug response. These mutations are also not known to be prognostic.
The majority of UM tumors will have a mutation in GNAQ or GNA11, while CYSLTR2 and PLCB4 mutations are less common. These mutations are usually mutually exclusive. The presence of one of these mutations may provide some confidence that a melanocytic tumor was sampled. However, other eye lesions can have these mutations, and some UM tumors will not have a mutation in any of these 4 genes. These mutations should not be used exclusively to rule-in or rule-out a UM diagnosis in the absence of other clinical/pathological features.
The GNAQ gene encodes for the alpha subunit of the heterotrimeric G protein complex that couples to seven transmembrane domain receptors (G-protein coupled receptors, GPCRs) to affect intracellular signaling. The alpha subunit has intrinsic hydrolase activity that regulates the availability of active GTP for GPCRs, influencing downstream signaling pathways, including MAPK, PKC, PI3K and Hippo. Mutations in GNAQ primarily affect two amino acids: Q209 (exon 5) or R183 (exon 4) (Van Raamsdonk, 2009). Both amino acid changes curtail the subunit’s GTPase activity and inhibit the conversion of GTP to inactive GDP. This results in the constitutive activation of the pathways downstream of the GPCR. Drugs targeting these various pathways are being explored as therapeutics for uveal melanoma (Shoushtari & Carvajal, 2014). Mutations in GNAQ occur in approximately 40-45% of uveal melanomas (Van Raamsdonk, 2009; Van Raamsdonk, 2010; Onken, 2008; Decatur, 2016) and are also found in blue nevi, cutaneous melanomas, mucosal melanomas, and other lesions. GNAQ is highly homologous to GNA11, and mutations in these genes are usually mutually exclusive with each other, as well as with CYSLTR2 and PLCB4 (Moore, 2016; Johansson, 2016). Approximately 85-90% of uveal melanomas will have a mutation in either GNAQ or GNA11 (Van Raamsdonk, 2010). GNAQ mutations are not associated with prognosis in uveal melanoma.
The GNA11 gene encodes for the alpha subunit of the heterotrimeric G protein complex that couples to seven transmembrane domain receptors (G-protein coupled receptors, GPCRs) to affect intracellular signaling. The alpha subunit has intrinsic hydrolase activity that regulates the availability of active GTP for GPCRs, influencing downstream signaling pathways, including MAPK, PKC, PI3K and Hippo. Mutations in GNA11 primarily affect two amino acids: Q209 (exon 5) or R183 (exon 4) (Van Raamsdonk, 2010). Both amino acid changes curtail the subunit’s GTPase activity and inhibit the conversion of GTP to inactive GDP. This results in the constitutive activation of the pathways downstream of the GPCR. Drugs targeting these pathways are being explored as therapeutics for uveal melanoma (Shoushtari & Carvajal, 2014). Mutations in GNA11 occur in approximately 40%-45% of uveal melanomas (Van Raamsdonk, 2010; Decatur, 2016), and are also found in other lesions, including blue nevi, cutaneous melanomas, and mucosal melanomas. GNA11 is highly homologous to GNAQ, and mutations in these genes are usually mutually exclusive with each other, as well as with CYSLTR2 and PLCB4 (Moore, 2016; Johansson, 2016). Approximately 85-90% of uveal melanomas will have a mutation in either GNAQ or GNA11 (Van Raamsdonk, 2010). GNA11 mutations are not associated with prognosis in uveal melanoma.
The CYSLTR2 gene encodes for a G-protein coupled receptor in the rhodopsin-like family called cysteinyl leukotriene receptor 2. The receptor is stimulated by leukotrienes to mediate downstream signaling and has been shown to activate the Gaq subunit. A hotspot mutation in CYSLTR2 has been identified in uveal melanoma that results in an amino acid change at a L129 (Moore, 2016). This mutation results in constitutive activation of the receptor through the Gaq subunit, and may activate downstream pathways similar to those activated by GNAQ and GNA11 mutations in uveal melanoma, including the MAPK, PKC, PI3K, and Hippo pathways. CYSLTR2 mutations have been shown to occur in approximately 3% of uveal melanomas and are mutually exclusive with GNAQ, GNA11, and PLCB4 mutations (Moore, 2016). The CYSLTR2 mutation is not associated with prognosis in uveal melanoma.
The PLCB4 gene encodes for phospholipase C, beta 4, which is a downstream target of the Gaq and Ga11 subunits of G-protein-coupled receptors. PLCB4 catalyzes the formation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAC) from phosphatidylinositol 4,5-bisphosphate (PIP2) and thus plays a role in calcium-mediated intracellular signaling. A hotspot mutation resulting in an amino acid change at D630 in PLCB4 is thought to disrupt the catalytic core of PLCB4 and result in aberrant signaling downstream of the Ga subunits (Johansson, 2016). PLCB4 mutations occur in 4-7% of uveal melanomas and are mutually exclusive with mutations in GNAQ, GNA11, and CYSLTR2 (Moore, 2016; Johansson, 2016) PLCB4 mutations also occur in cutaneous melanoma but at different genetic locations than in uveal melanoma. The PLCB4 mutation is not associated with prognosis in uveal melanoma.
Mutations in 3 Genes Are Associated With Differential Clinical Outcomes
Mutations occurring in EIF1AX, SF3B1, and BAP1 are usually mutually exclusive:
- The EIF1AX protein is involved in translation (from mRNA to protein) initiation
- SF3B1 is a component of the spliceosome, which regulates transcript usage
- BAP1 is a tumor suppressor gene on chromosome 3 that encodes for a deubiquitinating enzyme that forms protein complexes with BRCA1, BARD1, and ASXL1
It is well known that mutations in BAP1 in UM are associated with a higher risk of metastasis and there is considerable overlap with a Class 2 GEP (Harbour, 2010). Some BAP1 mutations are germline and can be inherited (Abdel-Rahman, 2011; Gupta, 2015), but the majority are somatic and thus confined to the tumor.
In retrospective studies, having an EIF1AX or SF3B1 mutation has been shown to be associated with a better prognosis than having a BAP1 mutation (Martin, 2013; Ewens, 2014; Decatur, 2016; Furney, 2013; Harbour, 2013; Yavuzyigitoglu, 2016). Tumors with EIF1AX mutations may have the lowest risk of metastasis compared to those with SF3B1 mutations, which may be prone to late metastasis.
Importantly, in a recent retrospective study comparing the prognostic value of these 3 mutations when using the DecisionDx-UM GEP test, a Class 2 result was the only significant, independent predictor of metastasis and UM-related mortality in multivariate analysis (Decatur, 2016). Therefore, the GEP test currently provides the best prognostic information about the tumor.
The EIF1AX gene encodes for the eukaryotic translation initiation factor 1A, x-linked protein. This protein plays a role in the initiation of translation of mRNA to protein. In uveal melanoma, mutations in EIF1AX have been reported in the first two exons of the gene, which encode for the unstructured N-terminal tail of the EIF1AX protein (Martin, 2013). EIF1AX mutations occur in approximately 17-24% of uveal melanomas and are usually not found in conjunction with SF3B1 or BAP1 mutations (Decatur, 2016). In small, retrospective, single center studies, mutations in EIF1AX have been associated with a good prognosis in uveal melanoma (Ewens, 2014, Yavuzyigitoglu, 2016).
The SF3B1 gene encodes for splicing factor B3 subunit 1. This protein is a component of the U2 small nuclear ribonucleoprotein complex and plays a role in splicing of pre-mRNA. In uveal melanoma, SF3B1 mutations usually result in an amino acid change at R625 (Harbour, 2013; Martin, 2013) that may result in differential splicing of protein coding genes (Furney, 2013). SF3B1 mutations occur in 18-24% of uveal melanomas (Harbour, 2013; Furney, 2013, Decatur, 2016), and can also occur in other malignancies, including breast cancer, pancreatic cancer, chronic lymphocytic leukemia, and myelodysplastic syndrome. Mutations in SF3B1 are usually not found in conjunction with EIF1AX or BAP1 mutations. In small, retrospective, single center studies, SF3B1 mutations have been associated with a favorable prognosis but may be linked to a risk for late metastasis (Yavuzyigitoglu, 2016; Harbour, 2013).
The BAP1 gene is a 17-exon gene located on chromosome 3 and encodes for BRCA1-associated protein 1, a ubiquitin carboxy-terminal hydrolase (deubiquitinating enzyme) that can interact with multiple factors, including BRCA1, BARD1, ASXL1, and HCFC1, and functions as a tumor suppressor. BAP1 mutations can occur anywhere along the gene and generally result in loss of expression and/or functional inactivation. BAP1 mutations occur in approximately 40-45% of uveal melanomas (Harbour, 2010; Decatur, 2016) and are not usually found in conjunction with SF3B1 or EIF1AX mutations. In uveal melanoma, retrospective studies have shown that BAP1 mutations are associated with an increased risk of metastasis (Decatur, 2016) and are strongly correlated with other unfavorable prognostic characteristics, such as a Class 2 gene expression profile and monosomy 3 (Harbour, 2010; Ewens, 2014).
BAP1 mutations can be somatic, meaning they arose spontaneously in the tumor, or germline, meaning they are present in every cell in the body and can be heritable. Most BAP1 mutations in uveal melanoma are somatic in origin, and only a very small percentage (2-5%) of uveal melanoma tumors will have a germline BAP1 mutation (Aoude 2013; Gupta 2015). Germline BAP1 mutations are associated with BAP1 tumor predisposition syndrome and patients with this syndrome may be at an elevated risk for uveal melanoma, cutaneous melanoma, mesothelioma, renal cell carcinoma, and other conditions (Abdel-Rahman, 2011; Njauw, 2012; Gupta, 2015; Rai, 2016).
Understanding the DecisionDx-UMSeq Results
The DecisionDx-UMSeq test results can be expected in 2-4 weeks after receipt of the sample in our laboratory.
The DecisionDx-UMSeq report will tell you if clinically relevant mutations (variants) were found in any of the 7 gene targets. For each mutation found, the report will describe:
- Genomic location of the mutation (where in the gene it occurred)
- Type of mutation (eg, missense, nonsense)
- Functional change that occurs because of the mutation (ie, an amino acid change in the protein)
- Frequency that the mutation was detected in the sample (variant allele frequency)
- Potential consequences of that mutation on gene function, as well as relevant literature references
The Tier and Level of Evidence will also be described for each mutation, as recommended by the College of American Pathologists (CAP), the American Society of Clinical Oncology (ASCO), and the Association for Molecular Pathologists (AMP) [Li, 2017].
DecisionDx-UMSeq is available for patients undergoing DecisionDx-UM GEP testing or those who have already received their GEP results. DecisionDx-UMSeq can be run using the same biopsy taken for the DecisionDx-UM GEP test.
DecisionDx-UMSeq can be ordered in conjunction with DecisionDx-UM by electing sequence testing in the test menu on the DecisionDx-UM order form
Castle Biosciences will submit the cost for DecisionDx-UMSeq to your patient’s insurance and will track the claim on behalf of your patient throughout the process. The company also sponsors an industry-leading Patient Assistance Program for both insured and uninsured patients with the belief that quality care should not depend on financial considerations. You can get more information about insurance coverage, claims processing, and financial assistance by calling 866-788-9007 and selecting option #3.
BAP1 Germline Testing Information
If your patient has a detected BAP1 mutation and is interested in germline testing, Castle Biosciences can provide information to assist with finding testing services and genetic counseling. Please call Castle Biosciences at 866-788-9007, option 1 for these resources.