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Oncogenic GNAQ mutations are not correlated with disease-free survival in uveal melanoma

Background: Recently, oncogenic G protein alpha subunit q (GNAQ) mutations have been described in about 50% of uveal melanomas and in the blue nevi of the skin. Methods: GNAQ exon 5 was amplified from 75 ciliary body and choroidal melanoma DNAs and sequenced directly. GNAQ mutation status was correlated with disease-free survival (DFS), as well as other clinical and histopathological factors, and with chromosomal variations detected by FISH and CGH. Results: Of the 75 tumour DNA samples analysed, 40 (53.3%) harboured oncogenic mutations in GNAQ codon 209. Univariate and multivariate analysis showed that GNAQ mutation status was not significantly correlated with DFS. Conclusion: The GNAQ mutation status is not suitable to predict DFS. However, the high frequency of GNAQ mutations may render it a promising target for therapeutic intervention.

Cutaneous melanocytic nevi and melanomas show frequent oncogenic mutations in BRAF and NRAS, and consequent constitutive activation of the MAP-kinase pathway (Davies et al, 2002;Pollock et al, 2003). In contrast, melanocytic tumours such as uveal melanomas rarely show BRAF and NRAS mutations (Kilic et al, 2004;Saldanha et al, 2004;Janssen et al, 2008;Maat et al, 2008). Recently, somatic mutations in the heterotrimeric G protein alpha subunit q (GNAQ) have been found to be associated with dominant dark skin in a large-scale mutagenesis screen of inbred C3HeB/FeJ mice (Van Raamsdonk et al, 2004). In humans, GNAQ was found to be frequently mutated in the blue nevi of the skin (83%) and uveal melanoma (46%) (Van Raamsdonk et al, 2009). The mutations occur exclusively in codon 209 in the ras-like domain and result in constitutive activation, turning GNAQ into a dominant acting oncogene, activating the MAP-kinase pathway. We asked whether oncogenic GNAQ mutations in uveal melanoma are associated with patient survival.

Patients and tumour material
Ciliary body or choroidal melanomas were collected from patients who underwent enucleation of the tumour-containing eye at the Erasmus MC Rotterdam or Rotterdam Eye Hospital (Rotterdam, The Netherlands). Informed consent was obtained before enucleation and the study was performed according to the tenets of the Declaration of Helsinki. Fresh tumour material was obtained within 1 h after enucleation and processed for FISH and cytogenetic analysis as described (Kilic et al, 2005). A fraction of each tumour was snap-frozen and stored in liquid nitrogen. The remainder of the eye was embedded in paraffin and sections were stained with haematoxylin and eosin (H&E) for evaluation. All tumours were confirmed histopathologically as uveal melanomas (on the basis of H&E morphology, mitotic activity, and results of HMB45 and S100 staining). Seventy-five patients were selected from our extensive database with information on follow-up and clinical, cytogenetic and histopathological parameters.

FISH and CGH analysis
Dual-colour FISH on uncultured tumour material using centromeric and locus-specific cosmid, P1 or YAC probes for chromosome 1, 3, 6 and 8 was performed as described previously (Kilic et al, 2005). CGH analysis was performed on DNA from formalinfixed paraffin-embedded tumour material as described previously (Kilic et al, 2005).

Statistical analysis
The primary end point for disease-free survival (DFS) was the time to development of metastatic disease; death due to other causes was treated as censored. Statistical analyses were performed with SPSS software, release 16.0 (Munich, Germany). Actuarial probabilities of DFS (with an event defined as development of metastatic disease or death by disease) were estimated according to the Kaplan -Meier method. To examine the possibility that other clinical, histopathological or chromosomal variations affected the prognosis, we performed Cox proportional hazard analysis for each confounding variable. An effect was considered significant if the P-value was p0.05.

Tumour pathology and clinical outcome
A total of 75 uveal melanomas were included in the study. The median age of the patients at the time of enucleation was 62 years (range 21 -86). In all, 39 patients were female and 36 male. Based on their cell type 14 tumours were classified as epithelioid, 28 as mixed, and 33 as spindle cell type. The mean tumour diameter and thickness were 13.4 mm (range 7.0 -19.0) and 8.4 mm (range 1.5 -20.0), respectively. At the end of the follow-up (mean 56.1 and range 6.4 -136.4 months), 28 patients died of melanoma-related disease, 1 patient was diagnosed with metastases, 9 patients died due to other causes and 37 patients were still alive without metastases.

Molecular genetic analysis
All uveal melanomas were analysed for the oncogenic GNAQ mutation and chromosomal changes of chromosomes 3 and 8. Of the 75 tumour DNA samples analysed, 40 (53.3%) harboured oncogenic mutations in GNAQ codon 209. In detail, 29 cases showed a heterozygous Q209P mutation, 1 case a homozygous Q209P mutation, 9 cases a heterozygous Q209L and 1 case a Q209R mutation. The copy number of chromosome 3 was obtained in 74 uveal melanomas, and of chromosomal region 8q in 68 uveal melanomas.

Statistical analysis
Univariate analysis was performed for all parameters, showing a lower DFS for patients with loss of chromosome 3 and gain of chromosome 8q. Univariate analysis of GNAQ mutated cases compared with wild-type tumours did not show a significantly decreased DFS (P ¼ 0.273) (Figure 1). To examine the possibility that GNAQ mutations may affect the prognosis of patients with loss of one copy of chromosome 3, we calculated Kaplan -Meier survival curves of GNAQ status stratified for chromosome 3 status and performed log rank tests (P ¼ 0.559) (Figure 2). Disease-free survival was not modified by the presence of GNAQ mutations. In tumours with two copies of chromosome 3 the patients with a GNAQ mutation seemed to have a better prognosis, although it was not significant (P ¼ 0.097). Correlations between the clinical, chromosomal parameters and GNAQ mutation were calculated using Mann -Whitney and Fisher's exact tests (Table 1). We did not observe any significant correlations. When analysed in a multivariate model with GNAQ as a confounding variable, we obtained a hazard ratio of 1.07 with a P-value of 0.854 (data not shown).

DISCUSSION
In a cohort of 75 uveal melanoma patients, we could show that DFS was not significantly correlated with GNAQ mutation status. However, analysis stratified for loss of chromosome 3 as well as multivariate analysis was clearly limited by the small sample size, and hence the results should be interpreted with caution. The mutation frequency of GNAQ codon 209 (53%) was in the same range as that in a recent report by Onken et al (2008) (49%) and by Van Raamsdonk et al (2004) (46%), confirming the importance of oncogenic GNAQ mutations in uveal melanoma. However, GNAQ mutations have been shown to have similar frequencies at all clinical stages of uveal melanoma progression, and to be independent of chromosomal aberrations, hinting at GNAQ being an early or initiating oncogenic event (Onken et al, 2008). This is consistent with the assumption that frequent oncogenic mutations of BRAF and NRAS in cutaneous melanoma as well as in benign melanocytic nevi (Davies et al, 2002;Pollock et al, 2003), which also activate the MAP-kinase pathway, are early events and are not associated with clinical outcome (Shinozaki et al, 2004;Akslen et al, 2005;Edlundh-Rose et al, 2006).
In conclusion, we could show that oncogenic GNAQ mutations are not suitable to predict the clinical outcome in uveal melanoma. However, the high frequency of GNAQ mutations may render it a promising target for therapeutic intervention.