Next-Generation Sequencing for Cutaneous Melanoma

Castle Biosciences’ DecisionDx-CMSeq is a 3-gene test that uses next-generation sequencing (NGS) to identify somatic mutations relevant to cutaneous melanoma in melanoma tumor tissue. The test includes hotspot mutations in the genes BRAF, NRAS, and KIT.

Mutations in these genes are important to help inform therapy choices for melanoma patients in the adjuvant and metastatic settings, including clinical trial opportunities. There are FDA-approved therapies that target the BRAF mutation and downstream pathways in metastatic melanoma. The FDA has also approved adjuvant targeted combination therapy for resected Stage III melanoma based on recent evidence (Long 2017). Ongoing clinical trials evaluating new targeted therapies may also enroll patients with BRAF-, NRAS- or KIT-mutant melanoma.

The DecisionDx-CMSeq test can be ordered on formalin-fixed paraffin-embedded (FFPE) primary tumor tissue in conjunction with the DecisionDx-Melanoma gene expression profile (GEP) test to obtain both a prognostic and therapeutic profile of the tumor at the same time. The DecisionDx-CMSeq test can also be ordered separately on FFPE tissue from the primary tumor, lymph node, or metastasis.

BRAF, NRAS, KIT Mutations In Melanoma: Current Clinical Utility

In melanoma, tumor growth is predominantly driven by signaling in the mitogen-activated protein kinase (MAPK) pathway (Schadendorf 2015), which includes proteins encoded by the NRAS, BRAF, and KIT genes. In some melanoma tumors, mutations in members of the MAPK signaling pathway result in constitutively active signaling. Targeted therapies, such as BRAF and MEK inhibitors, are used to block an overactive MAPK pathway. They are often used in combination to target multiple components of the pathway in an effort to overcome resistance that can result with monotherapy.

The DecisionDx-CMSeq test evaluates mutations in exon 15 of BRAF, where the most common and clinically actionable mutations occur. BRAF is the most commonly mutated gene in melanoma with approximately 40-50% of cases harboring mutations (Hayward 2017; TCGA 2015). Mutations in BRAF are activating events and lead to stimulation of the MAPK pathway (Davies 2002; Holderfield 2012). The V600 codon is the most commonly mutated codon in the gene. Specifically, the V600E variant is present in ~90% of tumors with a BRAF mutation (Cheng 2017; Richtig 2017). There are FDA-approved BRAF and MEK inhibitors for patients with unresectable BRAF V600E and V600K-mutant Stage III or IV melanoma. Additionally, a recent clinical trial showed that combination targeted therapy is effective for resected Stage III patients, leading to the FDA approval of these therapies for resected Stage III patients with BRAF V600E/K-mutant melanoma. Less common mutations, such as V600R/D and nonV600, are also of potential significance as there are ongoing clinical trials enrolling patients with these mutations.

The DecisionDx-CMSeq test identifies mutations in codons Q61, G12, and G13 of NRAS, which occur in approximately 20% of cutaneous melanomas and also lead to MAPK pathway activation. Q61 mutations are the most common (~80-90%) (Posch 2016; Schubbert 2007; Fedorenko 2013). Secondary NRAS mutations have a role in the development of BRAF inhibitor resistance (Long 2014; Rizos 2014). While there are currently no FDA-approved targeted therapies for NRAS-mutant melanoma, a few clinical trials with MEK inhibitors have shown promise (Dummer 2017; Johnson 2015; Koelblinger 2017). There are some data to suggest that treatment with therapies that target NRAS may be an effective option. There are several ongoing clinical trials for the treatment of NRAS-mutant melanoma and these may be important to consider for patients whose tumors harbor these mutations.

The DecisionDx-CMSeq test identifies mutations across various positions in the KIT gene: exons 2, 9-11, 13-15 and 17 which include the most common mutations (L576P, V559A, W557R, V560D, K642E, D820Y, D816H, N822K). KIT mutations typically result in constitutive kinase activity, directly or indirectly, and subsequent downstream pathway activation (Reddy 2017). Mutations in KIT are less common (<10%) than BRAF and NRAS across melanoma broadly but are frequently found in acral, mucosal, and chronically sun-damaged melanoma (Dumaz 2015; Curtin 2006; Gong 2018). While there are currently no FDA-approved targeted therapies for KIT-mutant melanoma, clinical trials with tyrosine kinase inhibitors or MEK inhibitors have shown promise for some KIT-mutant melanoma (Hodi 2008; Lutzky 2008; Carvajal 2011; Guo 2011; Hodi 2013). There are several ongoing clinical trials for the treatment of KIT-mutant melanoma and these may be important to consider for patients whose tumors harbor these mutations.

Ordering DecisionDx-CMSeq

The DecisionDx-CMSeq test can be ordered for FFPE primary melanoma tumor tissue in conjunction with the DecisionDx-Melanoma GEP test. DecisionDx-CMSeq can also be ordered as a standalone test. If ordered together, all necessary slides will be requested at the same time. If ordered separately at different times, there will be an additional request of slides for sequencing. The DecisionDx-CMSeq test can also be ordered for FFPE metastatic tissue, including lymph node metastases.

To order the DecisionDx-CMSeq test, please complete the order form and check the “Additional Testing: DecisionDx-CMSeq Sequencing Test” option in the test menu.

Castle Biosciences, Inc. will bill your patient’s insurance for the DecisionDx-CMSeq test.

Understanding the DecisionDx-CMSeq Results

The DecisionDx-CMSeq report will tell you if clinically relevant mutations (variants) were found in the three genes targeted by the test. For each mutation found, the report will describe:

  • Genomic location of the mutation (where in the gene did it occur)
  • Type of mutation (missense, nonsense, etc.)
  • Functional change that occurs because of the mutation (i.e. 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 and relevant literature references

Tier and Level of Evidence, as defined by the College of American Pathologists (CAP), the American Society of Clinical Oncology (ASCO), and Association for Molecular Pathologists (AMP) guidelines (Li 2017), will also be reported for each mutation.

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