Genetic alterations in RAS regulated pathway in Acral Lentiginous Melanoma

Abstract

Studies integrating clinicopathological and genetic features have revealed distinct patterns of genomic aberrations in Melanoma. Distributions of BRAF or NRAS mutations and gains of several oncogenes differ among melanoma subgroups while 9p21 deletions are found in all melanoma subtypes.

In the study, status of genes involved in cell cycle progression and apoptosis were evaluated in a panel of 17 frozen primary acral melanomas.

NRAS

mutations were found in 17% of the tumours. In contrast, BRAF mutations were not found. Gains of AURKA gene (20q13.3) were detected in 37.5% of samples, gains of CCND1 gene (11q13) or TERT gene (5p15.33) in 31.2% and gains of NRAS gene (1p13.2) in 25%. Alterations in 9p21 were identified in 69% of tumours. Gains of 11q13 and 20q13 were mutually exclusive; and 1p13.2 gain was associated with 5p15.33.

Our findings showed that alterations in RAS related pathways are present in 87.5% of acral lentiginous melanomas.

Introduction

Molecular studies have revealed the existence of different biological subsets of melanomas based on the patterns of alterations identified (1–3), some of which correlated with degree of chronic sun-induced damage and site of origin (3). Furthermore, such molecular differences could result in clinical and histopathological differences among lesions (4, 5).

Melanomas classified as acral lentiginous melanomas (ALM) develop on volar skin, usually unexposed to UV radiation and are characterized by the presence of an atypical lentiginous proliferation. ALM carry a high number of genomic alterations compared to other melanoma subtypes and most of them account for a smaller proportion of genome (1, 3). The molecular hallmarks of ALM are CCND1 amplifications (1, 6, 7) or somatic mutations in c-KIT (8).

Deletions in the 9p21 region where the CDKN2A gene is located are widely detected (9–11). However, other genes from this region could be implicated in melanoma since retention of the CDKN2A locus has been found in tumours with deletions at one or both sides of CDKN2A (10). Other reported aberrations include large amplifications of 12q (1, 3), 7q or 20q and gains localized at 5p15, 11q13, 11q14 (3) and 22q11-13 (1).

Questions addressed

To characterize acquired molecular genomic alterations in a set of ALM from Spanish patients. The study was focused on specific chromosomal regions where genes involved in signalling pathways, cell cycle progression and apoptosis are located.

Experimental design

Seventeen fresh-frozen histopathologically confirmed primary ALMs based on Clark’s classification were included. Sampling was guided by ex vivo dermoscopy (12) and documented by photography without altering the specimen, immediately fixed in formalin and embedded in paraffin (FFPE) for conventional histopathological diagnosis following the step sectioning protocol for melanoma. Clinical data is described in Table S1. Genomic characterization of the BRAF, NRAS, CDKN2A and MC1R genes was performed by PCR-direct sequencing. Deletions of the 9p21 region and gains of regions wherein oncogenes of interest are localized were carried out by the Multiplex Ligation-dependent Probe Amplification (MLPA) approach (Data S1). Significance of AURKA gain into protein was evaluated by inmunohistochemistry method (Data S1).

Unsupervised hierarchical clustering of amplified regions detected was carried out using Cluster 3.0 developed by Eisen Lab (University of California, Berkeley, California, USA) using Pearson’s correlations distance and average linkage clustering. The study was approved by the institutional review board of Hospital Clinic of Barcelona (Spain) and tumours were from the sample collection of Melanoma Unit at the Hospital Clínic of Barcelona.

Results

MC1R variants were found in 62.5% of patients (Table S1). NRAS missense mutations were detected in three out of 17 ALMs. In contrast, activating BRAF mutations were not present (Table S1).

9p21 alterations were detected in 69% of tumours, one sample failed to yield a result (Table S1). Whilst five tumours carried focal deletions of CDKN2A, CDKN2B and/or MTAP, 6 showed loss of the 9p21 region. No mutations were detected in CDKN2A.

Copy number gains were detected in 12 different loci (Table 1). All melanomas showed at least one altered locus; 43.7% of tumours presented two and 25% presented ≥ three loci. Four alterations were detected in at least 25% of samples: 1p13.2 (NRAS gene), 5p15.33 (TERT gene), 20q13.3 (AURKA gene) and 11q13 (CCND1 and contiguous genes).

Table 1.

List of oncogenes analyzed by the MLPA approach

Chromosomic Region	Oncogenes	Frequency of tumours with amplifications
r01p13.2	NRAS	25%
r01p22.1	BCAR3	0
r01q32	MDM4	0
r03q27	BCL6	0
r05p15.33	TERT	31.2%
r05q13	BIRC1	12.5%
r06p21	CCND3	0
r07q21.3	CDK6	6.2%
r08q11	MOS	18.8%
r11q13	RELA, GSTP1, CCND1, EMS1, FGF3,	31.2%*
r11q22	BIRC3	6.2%
r12p13.32	CCND2	0
r12p13.2	BCLG	0
r12q14	CDK4	12.5%
r12q14.3	MDM2	12.5%
r13q12.3	CCNA1	0
r14q32.33	AKT1	0
r17q25	BIRC5	6.2%
r18q21.3	BCL2	0
r19q12	CCNE1	6.2%
r20q11.1	BCL2L1	0
r20q13.1	PTPN1	0
r20q13.3	AURKA	37.5%
r20q13.33	FLJ20517	0
Xq25	BIRC4	0

^*Frequency regarding gains of the CCND1 gene

Unsupervised hierarchical clustering was carried out to analyze the distribution of these recurrent alterations. Although the analysis was not statistically significant, it suggested the presence of different patterns (Fig. 1). Any sample with 20q13.3 gain (37.5%) presented 11q13 gain (31.25%). Furthermore, gains of 1p13.2 (25%) were more associated with 5p15.33 gains. Deletions in 9p21 were associated with any specific gained region.

Figure 1.

Hierarchical Clustering Subgroups of Acral lentiginous melanoma: Distribution of recurrent gains (11q13, 20q13.3, 5p15.33, 1p13.2). Amplification of a given region is indicated by grey box.

Aurora A protein was evaluated in the corresponding FFPE biopsies of 12 samples. Among samples harbouring AURKA gains (6 tumours) inmunohistochemistry failed in 2. Protein expression was detected in 3 out of 4 tumous in a range of 10% of cells up to 75% of cells (Figure S1). In contrast; Aurora A expression was not detected in samples without alteration.

Conclusion

Although the study showed a high proportion of patients harbouring germinal MC1R variants, the frequency of them did not differ from the frequency detected in the control population (data not shown). Activating NRAS mutations were detected in ALM as previously described (7, 13–15). BRAF mutations were not present in any sample which could be explained by the large proportion of samples carrying other deregulated genes located downstream of the MAPK pathway (CCND1 or CDK4) as described by Curtin et al. (3).

Focal amplifications have been described as molecular markers of ALM (1, 3). The most frequently gained loci were at 1p13.2, 5p15.33, 11q13 and 20q13.3. Our data suggests the existence of different ALM subgroups based on distribution of these alterations. Although a large set of tumours should be analyzed to obtain statistical power to detect such profiles, there is plausible biological evidence to support the existence of such patterns. A group of tumours carried 20q13.3 gains (AURKA gene) which has been reported previously in melanoma (16). AURKA overexpression which has been closely related to gene amplification or genetic instability (17, 18), could be implicated in promoting cancer cell survival activating Akt and stimulating the PI3K pathway (19). Interestingly, AURKA may converge upon oncogenic Ras signalling through the RALGEF pathway (20, 21). Another subgroup presented 11q13 gains including CCND1 which is a frequent initial molecular event in ALM (2, 6, 7, 22). Gains of AURKA and CCND1 were mutually exclusive, suggesting that both genes would lead to deregulation of cell proliferation in the same way. Revalidation of these results has been carried out by FISH methodology in a large set of ALM obtaining concordant findings (manuscript in preparation).

The third subgroup presented 1p13.2 gains (NRAS), frequently associated with 5p15.33 (TERT) alterations. Increased copy number of NRAS has been described previously (9, 23, 24).

In summary, based on these results, we hypothesize that alterations in cell progression genes (NRAS, AURKA or CCND1) could play similar roles as driver alterations in ALM. Further studies have to be performed on a large set of Acral lentiginous melanoma in order to elucidate the existence of these patterns and also to evaluate the cross-talk between downstream NRAS pathways and melanoma development.