The melanoma proteome

Malignant melanoma is the leading cause of skin-related death in Caucasians, but rare in populations with darker pigmented skin color. The incidence has increased dramatically during the last decades, with an almost four-fold increase in the Nordic countries in the time period 1964-2003. The increased incidence reflects in part different UV exposure behavior in the population, but hereditary risk factors including skin type are also important. Short intermittent exposure with sunburns appears to be the main risk factor. Other important risk factors include the number of melanocytic nevi and the number of dysplastic melanocytic nevi.

Primary cutaneous malignant melanoma is thought to develop in a multi-step process. Precursor lesions, such as dysplastic melanocytic nevi develop into a melanoma in situ stage and further into invasive melanoma and eventually metastatic melanoma. Invasive malignant melanoma is traditionally divided into four principal histopathological subgroups based on the microscopical appearance: superficial spreading melanoma (SSM), nodular malignant melanoma (NMM), lentigo maligna melanoma (LMM) and acral lentiginous melanoma (ALM).

Here, we explore the melanoma proteome using TCGA transcriptomics data and antibody-based protein data. 208 genes are suggested as prognostic based on transcriptomics data from 102 patients; 163 genes are associated with unfavorable prognosis and 45 genes are associated with favorable prognosis.

TCGA data analysis

In this metadata study, we used data from TCGA where transcriptomics data was available from 102 patients with skin cutaneous melanoma in total. The total dataset included 42 females and 60 males. Most of the patients (73 patients) were still alive at the time of data collection. The stage distribution was stage i) 2 patients, stage i/ii NOS) 1 patient, stage ii) 65 patients, stage iii) 27 patients, stage iv) 32 patients, and 4 patients with missing stage information.

Unfavorable prognostic genes in melanoma

For unfavorable genes, higher relative expression levels at diagnosis give significantly lower overall survival for the patients. There are 163 genes associated with an unfavorable prognosis in melanoma. In Table 1, the top 20 most significant genes related to an unfavorable prognosis are listed.

MCM6 is a gene associated with an unfavorable prognosis in melanoma. The best separation is achieved by an expression cutoff at 16.5 fpkm which divides the patients into two groups with 0% 3-year survival for patients with high expression versus 62% for patients with low expression, p-value: 1.09e-5. Immunohistochemical staining using an antibody targeting MCM6 (HPA004818) shows a differential expression pattern in melanoma samples.

p<0.001
MCM6 - survival analysis
MCM6 - high expression
MCM6 - low expression

TIMELESS is another gene associated with an unfavorable prognosis in melanoma. The best separation is achieved by an expression cutoff at 10.0 fpkm which divides the patients into two groups with 0% 3-year survival for patients with high expression versus 48% for patients with low expression, p-value: 1.06e-5. Immunohistochemical staining using an antibody targeting TIMELESS (HPA060655) shows a differential expression patterns in melanoma samples.

p<0.001
TIMELESS - survival analysis
TIMELESS - high expression
TIMELESS - low expression

Table 1. The 20 genes with highest significance associated with an unfavorable prognosis in melanoma.

Gene	Description	Predicted location	mRNA (cancer)	p-value
SKP2	S-phase kinase associated protein 2	Intracellular	4.3	3.72e-7
PAPSS2	3'-phosphoadenosine 5'-phosphosulfate synthase 2	Intracellular	4.2	1.33e-6
TICRR	TOPBP1 interacting checkpoint and replication regulator	Intracellular	1.2	2.87e-6
TIMELESS	timeless circadian regulator	Intracellular	8.7	1.06e-5
MCM6	minichromosome maintenance complex component 6	Intracellular	14.2	1.09e-5
NUDT15	nudix hydrolase 15	Intracellular	11.0	1.25e-5
MED20	mediator complex subunit 20	Intracellular	6.9	1.48e-5
CKS1B	CDC28 protein kinase regulatory subunit 1B	Intracellular	9.8	3.12e-5
CCNB2	cyclin B2	Intracellular	8.8	3.21e-5
ZNF544	zinc finger protein 544	Intracellular	2.9	3.65e-5
KCTD14	potassium channel tetramerization domain containing 14	Intracellular	1.3	3.96e-5
MCM3	minichromosome maintenance complex component 3	Intracellular	50.2	4.40e-5
MAT2A	methionine adenosyltransferase 2A	Intracellular	33.1	5.94e-5
TDG	thymine DNA glycosylase	Intracellular	3.0	6.90e-5
CDC5L	cell division cycle 5 like	Intracellular	9.9	7.02e-5
PUS7	pseudouridine synthase 7	Intracellular	6.5	7.58e-5
ZNF551	zinc finger protein 551	Intracellular	1.2	7.68e-5
STK38	serine/threonine kinase 38	Intracellular	13.4	9.44e-5
SFSWAP	splicing factor SWAP	Intracellular	5.6	1.10e-4
MARK3	microtubule affinity regulating kinase 3	Membrane, Intracellular	6.9	1.14e-4
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Favorable prognostic genes in melanoma

For favorable genes, higher relative expression levels at diagnosis give significantly higher overall survival for the patients. There are 45 genes associated with a favorable prognosis in melanoma. In Table 2, the top 20 most significant genes related to a favorable prognosis are listed.

WIPI1 is a gene associated with a favorable prognosis in melanoma. The best separation is achieved by an expression cutoff at 14.0 fpkm which divides the patients into two groups with 52% 3-year survival for patients with high expression versus 0% for patients with low expression, p-value: 3.11e-4. Immunohistochemical staining using an antibody targeting WIPI1 (HPA007493) shows a differential expression pattern in melanoma samples.

p<0.001
WIPI1 - survival analysis
WIPI1 - high expression
WIPI1 - low expression

Table 2. The 20 genes with highest significance associated with a favorable prognosis in melanoma.

Gene	Description	Predicted location	mRNA (cancer)	p-value
SIRPG	signal regulatory protein gamma	Membrane	1.3	7.36e-7
TRBV28	T cell receptor beta variable 28	Intracellular	4.4	1.35e-5
HLA-G	major histocompatibility complex, class I, G	Secreted, Membrane	10.7	3.91e-5
CD2	CD2 molecule	Membrane, Intracellular	6.4	3.97e-5
OTUD7B	OTU deubiquitinase 7B	Intracellular	8.6	5.09e-5
LCK	LCK proto-oncogene, Src family tyrosine kinase	Intracellular	2.2	5.65e-5
GNLY	granulysin	Secreted, Intracellular	2.1	6.60e-5
NFKB2	nuclear factor kappa B subunit 2	Intracellular	12.9	8.45e-5
DLGAP4	DLG associated protein 4	Intracellular	19.1	9.88e-5
CD3E	CD3 epsilon subunit of T-cell receptor complex	Membrane	5.7	1.18e-4
KIF3C	kinesin family member 3C	Intracellular	9.5	1.22e-4
SLAMF6	SLAM family member 6	Membrane	1.2	1.25e-4
NR1D1	nuclear receptor subfamily 1 group D member 1	Intracellular	11.2	1.36e-4
TRBV20-1	T cell receptor beta variable 20-1	Intracellular	1.8	1.37e-4
PLA2G2A	phospholipase A2 group IIA	Secreted, Intracellular	3.6	1.49e-4
SPN	sialophorin	Membrane	1.1	1.88e-4
SDC3	syndecan 3	Membrane	102.2	2.36e-4
CCL5	C-C motif chemokine ligand 5	Secreted, Membrane	32.0	2.42e-4
CD7	CD7 molecule	Membrane, Intracellular	3.2	2.69e-4
TRBC2	T cell receptor beta constant 2	Membrane	10.7	2.81e-4
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The melanoma transcriptome

The transcriptome analysis shows that 68% (n=13794) of all human genes (n=20162) are expressed in melanoma. All genes were classified according to the melanoma-specific expression into one of five different categories, based on the ratio between mRNA levels in melanoma compared to the mRNA levels in the other 16 analyzed cancer tissues.

Figure 1. The distribution of all genes across the five categories based on transcript abundance in melanoma as well as in all other cancer tissues.

319 genes show some level of elevated expression in melanoma compared to other cancers (Figure 1). The elevated category is further subdivided into three categories as shown in Table 3.

Table 3. The number of genes in the subdivided categories of elevated expression in melanoma.

		Distribution in the 17 cancers
		Detected in single	Detected in some	Detected in many	Detected in all	Total
Specificity	Cancer enriched	20	31	29	13	93
	Group enriched	0	48	60	13	121
	Cancer enhanced	12	14	33	46	105
	Total	32	93	122	72	319

Additional information

The histopathological features of malignant melanomas vary widely. A typical feature, often facilitating the diagnosis of melanoma, is the occurrence of a pagetoid growth pattern, characterized by the growth of melanoma cells in the upper layers of the epidermis from below, as opposed to the localization of normal melanocytes in basal layers of the epidermis. Melanoma cells can be large and rich in cytoplasm, small or even spindly. In a subset of melanomas, there are areas with abundant melanin and in other melanomas, melanin pigment is scarce or absent. Tumor cell nuclei are enlarged, often with a prominent nucleolus and mitoses are present at a variable degree.

Patients that present with an advanced tumor stage at the time for diagnosis and patients with relapse of melanoma after surgical removal generally have a poor prognosis. In patients diagnosed with localized disease, the most important prognostic indicator is tumor thickness at the time of diagnosis. The tumor thickness is measured in mm, according to a system initially described by Breslow, and is the dominating parameter for determining the tumor stage (T-stage) of melanoma. Other prognostic factors include mitotic rate and ulceration, as well as clinical factors such as age, gender and the anatomic site of the primary tumor. In Sweden, the 5-year melanoma-specific survival rate is 98% for patients in stage IA (tumor thickness <1mm without ulceration, and 49% for patients in stage IVB (tumor thickness >4 mm with ulceration).

Immunohistochemistry is often used to distinguish malignant melanoma from other tumor types. Traditionally, antibodies towards different S-100 proteins have been used as markers of melanocytes, however, these antibodies also stain in e.g. Langerhans cells and nerve fibers. Other markers, such as Melan-A (MART-1) and tyrosinase (TYR), stain melanocytes more specifically and are useful to determine if a tumor is of melanocytic origin. Proliferation markers are also widely used in the differential diagnostics of melanocytic lesions with uncertain malignant potential. The most accepted marker for determining mitotic cells is Ki-67 (MKI67). The presence of Ki-67 positive melanocytic cells is often used in routine pathology to distinguish malignant melanoma from benign melanocytic tumors.

Relevant links and publications

Uhlen M et al., A pathology atlas of the human cancer transcriptome. Science. (2017)
PubMed: 28818916 DOI: 10.1126/science.aan2507

Cancer Genome Atlas Research Network et al., The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet. (2013)
PubMed: 24071849 DOI: 10.1038/ng.2764

Uhlén M et al., Tissue-based map of the human proteome. Science (2015)
PubMed: 25613900 DOI: 10.1126/science.1260419

Edqvist PH et al., Expression of human skin-specific genes defined by transcriptomics and antibody-based profiling. J Histochem Cytochem. (2015)
PubMed: 25411189 DOI: 10.1369/0022155414562646

Ossio R et al., Melanoma: a global perspective. Nat Rev Cancer. (2017)
PubMed: 28450704 DOI: 10.1038/nrc.2017.43

Dermnet - Skin Disease Atlas

Histology dictionary - Melanoma