NRAS in Melanoma: ESMO Biomarker Factsheet

Definition of NRAS

NRAS (neuroblastoma ras viral oncogene homolog) is a member of the Ras superfamily of low-molecular-weight plasma-membrane associated GTP-binding proteins. Ras proteins primarily regulate cell growth, differentiation and survival. They act as molecular switches that relay signals from activated receptor tyrosine kinases (RTK) at the cell surface to transcription factors and cell cycle proteins in the nucleus. Ligand binding to RTK signals the recruitment of proteins that catalyse the exchange of GDP to GTP on Ras, thereby activating it. Once activated, Ras recruits and stimulates a number of different intracellular signalling pathways that include the RAF-MEK-ERK mitogen activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K)/AKT pathway, as well as others.

The NRAS gene, together with two of its family members, HRAS (v-Ha-ras Harvey rat sarcoma viral oncogene homolog) and KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), are frequently mutated in human cancers. KRAS is most commonly mutated (>20% of all human cancers), followed by NRAS (8%) and HRAS (3.3%) [1]. Mutations in these RAS oncogenes can mediate cellular transformation through the constitutive activation of signal-transduction pathways independent of upstream RTK activation that leads to uncontrolled cell growth, motility and survival signalling. Single base substitutions in the RAS genes lead to the stabilisation of GTP binding, maintaining RAS in its active state and resulting in unopposed downstream signalling.

NRAS Mutations in Melanoma

NRAS was the first oncogene identified in melanoma [2] and mutations in NRAS, KRAS and HRAS are currently known to be present in 20%, 2% and 1% of all melanomas, respectively [3].

The majority of NRAS mutations (>80%) involve a point mutation leading to the substitution of glutamine to leucine at position 61. Such mutations result in impaired GTPase activity and the locking of NRAS into its activated (GTP-bound) state, independent of upstream RTK activation.

Activating mutations of NRAS in melanoma are generally mutually exclusive of BRAF mutations with NRAS mutation driving a large proportion of cutaneous melanomas unaccounted for by BRAFV600 mutation. 

The presence of frequent mutations in NRAS in melanoma suggests that its protein product might be an attractive target for melanoma therapy.

NRAS as a Prognostic Biomarker in Melanoma

NRAS-mutated melanoma is distinct from BRAF-mutated melanoma in clinical presentation and prognostic features. Patients who present with NRAS-mutated melanomas tend to be older (>55 years of age) with a more chronic pattern of ultraviolet exposure than patients with BRAF-mutated melanoma [4]. These patients tend to have thicker tumours at presentation, typically located at the extremities and have greater rates of mitosis [4,5].

NRAS mutation status is an independent prognostic factor in metastatic melanoma. Similar to BRAF mutations, NRAS mutations in metastatic melanoma have been associated with aggressive disease features and with shorter survival from the diagnosis of stage IV disease compared with tumours harbouring both wild-type NRAS and BRAF [5,6].  

NRAS as a Predictive Biomarker in Melanoma

Whilst NRAS is an attractive target for melanoma therapy, it is a challenging one. Little progress has been made in the development of direct inhibitors of Ras proteins, presumably due to the high affinity of RAS for GTP, limiting interference with its binding site [1]. 

An alternative approach has been to focus on targeting downstream signaling pathways that the NRAS protein activates such as the MAPK pathway and PI3K pathway. A further challenge however, is to understand which of the numerous pathways activated by NRAS (in addition to MAPK and PI3K) are important in melanoma. It may be necessary to identify molecular subgroups within NRAS-mutated melanomas that can predict response to different pathway-specific treatment strategies.

Despite their mutual stimulation of the MAPK pathway, NRAS signals through activation of CRAF as opposed to BRAF, and evidence suggests that BRAFV600 inhibition will have no effect in patients with melanomas driven by NRAS mutations [7,8].

NRAS targeted therapies are still in the clinical trial stage but so far inhibitors of activated MEK in the MAPK pathway have proved most successful as targeted therapy for NRAS-mutated melanoma. In a phase II study of MEK inhibitor, MEK-162, 6/28 (20%) patients with NRAS mutated advanced melanoma achieved an initial partial response to treatment with 13/28 (43%) showed stable disease [9]. A phase III trial is currently ongoing comparing MEK162 with dacarbazine specifically in NRAS-mutant metastatic melanoma patients (NCT01763164).

Effort is also being focused on combination therapies for NRAS-mutated melanoma. In a phase Ib/II study, combination of MEK162 with the selective CDK4/6 inhibitor LEE011, achieved a 43% partial response rate in patients with NRAS-mutated melanoma [10].

Intriguingly, a retrospective study has recently suggested that patients with NRAS mutant advanced melanoma have an increased benefit from immune-based therapies compared with other genetic subtypes [11]. 

Whilst clinical trials are still ongoing, preliminary data suggest that molecularly targeted therapies will become available for this subgroup of melanoma patients.

NRAS Testing Recommendations in Melanoma

NRAS mutation status should be confirmed by a validated test carried out in an accredited (certified) institute that includes appropriate quality controls.

Several methods are available for detecting NRAS mutations including, mutation-specific real-time polymerase chain reaction (RT-PCR), Sanger sequencing, pyrosequencing, conformation analysis and high-resolution melting analysis. There are several manufacturers of targeted genetic tests that can detect NRAS mutations in melanoma tumour samples.

Which Technique and Which Algorithm Should be Used for the Analysis of the NRAS Status in Melanoma?

Parameters that should be considered when choosing a suitable companion diagnostic test include sensitivity, specificity, limit of analytical sensitivity and failure rates [12].

Methods to increase the sensitivity and specificity and to reduce the failure rates include:

• validating the chosen method thoroughly through comparison with 'gold-standard' methods,
• performing macrodissection of specimens to increase the sensitivity of the technique,
• choosing small amplicons for PCR amplification to reduce the failure rate due to DNA degradation,
• ongoing validation of the method through application of best practice and participation in external quality controls [12].

Patient Selection

As NRAS-targeted therapies are still in clinical trials, many clinicians only test for NRAS mutations if a patient is found to be BRAF wild-type. European guidelines for cutaneous melanoma recommend NRAS mutation testing (preferentially of metastatic lesions) as optional in cases of metastatic (stage IV) melanoma, to help direct patients to appropriate clinical trials and in the long term to validate the prognostic relevance of this biomarker. Mutation testing of primary tumours in patients without metastases is not recommended [13].

Key References

1. Baines AT, Xu D, Der CJ. Inhibition of Ras for cancer treatment: the search continues. Future Med Chem 2011;14:1787-808.
2. Albino AP, Le Strange R, Oliff AI, et al. Transforming ras genes from human melanoma: a manifestation of tumour heterogeneity? Nature 1984;308(5954):69-72.
3. Milagre C, Dhomen N, Geyer FC, et al. A mouse model of melanoma driven by oncogenic KRAS. Cancer Res 2010;70:5549-57.
4. Devitt B, Liu W, Salemi R, et al. Clinical outcome and pathological features associated with NRAS mutation in cutaneous melanoma. Pigment Cell Melanoma Res 2011;24:666-72.
5. Ellerhorst JA, Greene VR, Ekmekcioglu S, et al. Clinical correlates of NRAS and BRAF mutations in primary human melanoma. Clin Cancer Res 2011;17:229-35.
6. Jakob JA, Bassett RL Jr, Ng CS, et al. NRAS mutation status is an independent prognostic factor in metastatic melanoma. Cancer 2012;118:4014-23.
7. Nazarian R, Shi H, Wang Q, et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 2010;468:973-7.
8. Dumaz N, Hayward R, Martin J, et al. In melanoma, RAS mutations are accompanied by switching signaling from BRAF to CRAF and disrupted cyclic AMP signaling. Cancer Res 2006;66:9483-91.
9. Ascierto PA, Schadendorf  D, Berking C, et al. MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF mutations: a non-randomised, open-label phase 2 study. Lancet Oncol 2013;14:249-56.
10. Sosman J, Kittaneh M, Lolkema M, et al. A phase 1b/2 study of LEE011 in combination with binimetinib (MEK162) in patients with NRAS-mutant melanoma: early encouraging clinical activity. J Clin Oncol 2014;32(Suppl.):abstract 9009.
11. Gonzalez D, Fearfield L, Nathan P, et al. BRAF mutation testing algorithm for vemurafenib treatment in melanoma: recommendations from an expert panel. Br J Dermatol 2013;168:700-7.
12. Johnson DB, Lovly CM, Flavin M, et al. Impact of NRAS Mutations for Patients with Advanced Melanoma Treated with Immune Therapies. Cancer Immunol Res March 2015 3; 288.
13. Dummer R, Hauschild A, Guggenheim M, et al. ESMO Guidelines Working Group. Cutaneous melanoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2012;23(Suppl 7):vii86-91.