Melanoma

Primary Author: Wallander, Michelle L., PhD.

Disseminated melanoma is generally unresponsive to traditional chemotherapy and/or radiotherapy. With the advent of targeted therapy, molecular testing in melanoma has gained importance.

Mutation^a	Biology	Targeted Therapy	Comments
BRAF gene BRAF Codon 600 Mutation Detection by Pyrosequencing 2002498 BRAF V600E Mutation Detection in Circulating Cell-Free DNA by Digital Droplet PCR 2013921	BRAF is a protein involved in the MAPK pathway, which includes BRAF/MEK/ERK signaling Activates pathway, mediates growth signaling, links with factor receptors (eg, tyrosine kinases), leading to cell growth and depression of immune regulation of cancer cells >80% mutations substitute glutamic acid for valine at codon 600, resulting in V600E mutation Other mutations – V600K, V600R, V600D	Combining BRAF with MEK inhibition may improve outcome BRAF V600E inhibitors Vemurafenib Dabrafenib MEK inhibitors Trametinib Selumetinib Cobimetinib Binimetinib	~50% of melanomas (Long, 2011) Molecular genetic testing required if therapy directed at BRAF V600E mutation is being considered Most common in cutaneous melanomas derived from intermittent, acute sun-damaged skin, younger age (<55 yrs), and poorer prognosis compared to BRAF and NRAS wild type melanomas Resistance develops in all patients Resistance to BRAF inhibitors develops downstream, not at the initial mutation site
RAS family of genes (NRAS, KRAS, HRAS) NRAS Mutation Detection by Pyrosequencing 2003123	NRAS is a protein involved in the RAS/RAF/MAPK pathway Most mutations are in NRAS gene Mutation leads to defective GTPase activity with uncontrolled cell proliferation Most common mutations are located in codons 12, 13, 61 Mutation is generally mutually exclusive of BRAF mutation	Phase I and II clinical trials combining BRAF inhibitors with MEK, ATK, and PI3K inhibitors	15-20% of melanomas (Solus, 2013) Associated with thicker tumors, higher mitotic rate, and worse prognosis than BRAF mutation or wild type BRAF/NRAS genes
KIT gene KIT Mutations, Melanoma 2002695	KIT gene encodes for type III transmembrane receptor tyrosine kinase, which is involved in regulation of MAPK and PI3K pathways Mutations function as oncogene Exons 9, 11, 13, 14, 17, 18 Most common mutations located at exon 11	Tyrosine kinase inhibitors (TKIs) in clinical trials so far have demonstrated only minimal activity	Up to 15% of melanomas (Jiang, 2008) Rate of occurrence dependent on type of melanoma Most common in acral lentiginous, sun-damaged skin or mucosal melanomas Predicted to respond favorably to the TKI imatinib Molecular genetic testing should be performed if TKIs are being considered KIT IHC staining does not predict mutation status or sensitivity to TKIs Exons 11, 13 most likely to have response to TKIs Resistance develops in most patients with a secondary mutation in KIT gene
PDGFRA gene KIT Mutations, Melanoma 2002695	PDGFRA gene provides instructions to create platelet-derived growth factor Rare in melanoma Most common mutations in exons 12, 14, 18 Exons 12, 14 have more favorable prognosis	TKIs Best response for V658A Better response for crenolanib compared to imatinib	Exons 12, 14, 18 in PDGFRA gene may be associated with TKI sensitivity
PTEN gene	Functions as lipid phosphatase regulating PI3K/AKT pathways Mutation causes high-level activation of pathway with unregulated cell growth and proliferation Mutation is generally mutually exclusive of NRAS gene mutations, but usually inclusive for BRAF gene mutations	Multiple classes of inhibitors in phase I and II trials – PI3K, AKT, mTORC1, dual PI3K/mTOR inhibitors
^aTests listed are available at ARUP Laboratories.

Indications for Testing

Atypical melanocytic lesion (mole) requires histologic evaluation

Histology

Gold standard for diagnosis
- Immunohistochemistry may be required to distinguish poorly differentiated lesions of carcinoma, sarcoma, or lymphoma (B cell or T cell) from melanoma
- Stains to consider include cytokeratin 8,18 low molecular weight (CAM 5.2); melanoma antibody, HMB45; Ki-67 (Mib-1); melan A; p21 (Waf1/Cip 1); S-100 protein; vimentin; and MITF
Architectural and cytologic features of biopsy are interpreted in the context of clinical information
- Architecture – large, asymmetrical, poorly circumscribed lesion; pagetoid spread of melanocytes; effaced rete ridges and underlying dermal architecture; perineural invasion
- Cytology – large nuclei with prominent nucleoli; atypical mitosis (especially in dermis); lack of melanocyte maturation in deep portion of lesion
Metaplastic differentiation (nonmelanocytic cells or tissues) may be identified that may not be melanoma
Molecular testing – refer to Key Points
- Testing for BRAF and KIT mutations recommended by National comprehensive Cancer Network (NCCN, 2017) to guide choice of therapy
- PD-1 is being investigated as predictive marker for response to anti-PD-1 therapy (recommended first line at some centers)

Imaging Studies

Routine cross-sectional imaging (CT, PET, MRI) is not recommended in patients with localized melanoma due to low yield

Genetic Testing

Should be offered to individuals with (ACMG, 2015)
- ≥3 diagnoses of melanoma and/or pancreatic cancer in close relatives
- ≥3 primary melanomas in the same person
- Melanoma and pancreatic cancer in the same person
- Melanoma and astrocytoma in the same person or in 2 first-degree relatives

Prognosis

Depth of tumor invasion – used to determine prognosis
- Breslow thickness – most important factor contributing to T status in tumor, nodes, and metastasis staging
  - Measured from top of epidermal granular cell layer to deepest malignant melanocyte
  - 5-year survival rate – 95.3% with Breslow thickness ≤1.0 mm (corresponds to T1 lesion), 45.1% with Breslow thickness >4.0 mm
- Clark level – important in T1 lesions (<1 mm thick) (corresponds to 95% or better survival)
  - Clark I – melanoma in situ; does not cross basement membrane
  - Clark II – invasion of papillary dermis
  - Clark III – expansion of papillary dermis
  - Clark IV – invasion of reticular dermis
  - Clark V – invasion of subcutaneous fat
Additional histologic features used to determine prognosis
- Involvement of regional lymph nodes (regional nodal dissection or sentinel node testing)
  - Sentinel lymph node biopsy or other diagnostic tests for the evaluation of early, thin melanoma are not recommended as routine testing – these tests do not improve survival (Choosing Wisely, American Society for Clinical Pathology, 2016)
- Metastasis
- Growth phase
- Mitotic count
- Regression
- Ulceration
Additional serum testing
- Lactate dehydrogenase – prognostic factor in late-stage melanoma
  - Elevated levels likely correlate with tumor burden (NCCN, 2017)
  - Independent predictor of poor outcome
- S-100B, serum – elevated level associated with poor prognosis

Differential Diagnosis

Clinical – melanocytic nevi
Histologic
- Well differentiated – melanocytic nevi
- Poorly differentiated – carcinoma, sarcoma, lymphoma (T cell, B cell)

The American Academy of Dermatology (AAD) and American Cancer Society (ACS) recommend regular skin exams in all patients
The U.S. Preventive Services Task Force finds insufficient evidence to recommend regular skin exams

The National Comprehensive Cancer Network (NCCN) recommends skin examination at least once per year for life for melanoma patients (including those with stage 0, in situ melanoma)
- Patients with stage IA-IIA melanoma, no evidence of disease
  - Comprehensive exam every 3-12 months for five years and annually thereafter as clinically indicated
- Patients with stage IIB-IV melanoma, no evidence of disease
  - Comprehensive exam every 3-6 months for two years, then every 3-12 months for three years, and annually thereafter as clinically indicated
- Although not recommended at baseline, x-ray, CT/MRI, and/or PET/CT every 6-12 months can be considered to screen for recurrent or metastatic disease at discretion of physician
  - Most recurrences manifest within first five years – routine imaging not recommended beyond this period
S-100B, serum
- Rising concentrations after treatment indicate disease recurrence

Melanoma is a malignancy of the melanocytes. Because of the rising incidence worldwide, it now ranks second to adult leukemia in loss of years of potential life per death.

Epidemiology

Incidence – 22.3/100,000 (SEER, 2017)
- ~87,110 new melanomas in 2017 (estimated) (SEER, 2017)
- Tripled over the last 20 years
- Highest incidence worldwide is in Australia
Age – median is 64 years; rare in children (SEER, 2017)
Sex – M>F, 1.5:1
Ethnic – 10-fold increased incidence in Caucasians
- Acral lentiginous melanoma has equal distribution across all ethnic groups

Inheritance

Gene mutations associated with hereditary melanoma

Gene Symbol^a	Germline Mutations Increase Risk for the Following Cancer Types	Percentage of Hereditary Melanoma^b Attributed to Gene
BAP1	Mesothelioma, melanoma of the eye	Rare
CDK4	Melanoma	2%
CDKN2A	Melanoma, pancreatic	20-40%
PTEN	Thyroid, breast, renal, colorectal, endometrial, melanoma	Rare
RB1	Retinoblastoma, pinealoblastoma, sarcomas, melanoma	Rare
TP53	Li-Fraumeni syndrome, sarcoma, leukemia, breast, brain, adrenocortical, hepatocellular, melanoma	Rare
^aAll genes have autosomal dominant inheritance ^b~5-10% of melanomas are hereditary

Classification

Superficial spreading melanoma (~70%)
Nodular melanoma (10-15%)
Lentigo maligna melanoma (10%)
Acral lentiginous melanoma (~5%)
Mucosal lentiginous melanoma (3%)

Risk Factors

Sunlight – sunburns in childhood, intermittent UV exposure associated with higher risk
Blue or green eyes
Red or blond hair
Melanocytic nevus, dysplastic nevus
Family history – 2-fold risk if first-degree relative had melanoma
- Clustering of melanoma in familial retinoblastoma and Li-Fraumeni syndrome
- Increased risk of familial melanoma in the presence of family history of pancreatic cancer or astrocytoma
Immunosuppression
Prior melanoma

Clinical Presentation

82-85% of melanoma patients present with localized disease; 10-30% present with regional disease; 2-5% with distant metastatic disease
Nevus with Asymmetry, Border irregularity, Color variation, Diameter >6 mm, and Evolving changes (ABCDE criteria)
Head and neck most common sites
Ulceration, pigment loss
Rare sites – eye (iris, ciliochoroidal), mucosa, unknown primary

Clinical Background

Epidemiology

Incidence – 300-425 cases annually in the U.S. (SEER, 2017)
Age – 15-19 years most common
Sex – F>M (minimal)

Risk Factors

Family history – 2-fold risk if first-degree relative had melanoma
- Clustering of melanoma in familial retinoblastoma and Li-Fraumeni syndrome
- Increased risk of familial melanoma in the presence of family history of pancreatic cancer or astrocytoma
History of severe sunburns (>3 before 20 years)
Xeroderma pigmentosa
Immunosuppression
Dysplastic nevi (3- to 6-fold increased risk)

Clinical Presentation

Lesions frequently amelanotic or nodular
ABCDE criteria (Asymmetry, Border irregularity, Color variation, Diameter >6 mm, and Evolving changes) less reliable than in adults

Diagnosis

Indications for Testing

Refer to Diagnosis section

Laboratory Testing

Refer to Diagnosis section

Histology

Congenital nevus has slightly different histologic appearance
Refer to Histology in Diagnosis section

Imaging Studies

Refer to Diagnosis section

Differential Diagnosis

Spitz nevus
Blue nevus
Congenital nevus

Tests generally appear in the order most useful for common clinical situations. Click on number for test-specific information in the ARUP Laboratory Test Directory.

BRAF Codon 600 Mutation Detection by Pyrosequencing 2002498
Method: Polymerase Chain Reaction/Pyrosequencing

Recommended Use

Detect activating BRAF mutations at codon 600, which can indicate responsiveness to BRAF inhibitors in melanomas

Single gene assay

Limitations

Oncogenic mutations outside of codon 600 will not be detected

BRAF V600E Mutation Detection in Circulating Cell-Free DNA by Digital Droplet PCR 2013921
Method: Polymerase Chain Reaction

Recommended Use

Determine BRAF V600E mutation status in patients with solid tumors to select candidates for targeted therapy with kinase (BRAF and/or MEK) inhibitors

Monitor response to therapy and disease progression in patients carrying BRAF V600E mutation

NRAS Mutation Detection by Pyrosequencing 2003123
Method: Polymerase Chain Reaction/Pyrosequencing

Recommended Use

Predicts response to anti-EGFR and MAPK pathway therapies in melanoma

Single gene assay

Detects activating NRAS mutations (codons 12, 13, 61) associated with relative resistance to anti-EGFR therapy

Clinical sensitivity – oncogenic NRAS mutation found in a small percentage of melanomas

KIT Mutations, Melanoma 2002695
Method: Polymerase Chain Reaction/Sequencing

Recommended Use

Detect activating mutations in KIT and PDGFRA

Predict response to tyrosine kinase inhibitor (TKI) therapy

Refer to Additional Technical Information document for further content

Limitations

Mutations outside of targeted exons will not be detected

Solid Tumor Mutation Panel by Next Generation Sequencing 2007991
Method: Massively Parallel Sequencing

Recommended Use

Predicts prognosis and therapeutic response in patients with solid tumor cancers

Genes included – ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL

For a full list of the targeted regions of the above genes, click here

Refer to Additional Technical Information document for further content

Limitations

Does not detect translocations

Not intended to detect minimal residual disease

Cytokeratin 8,18 Low Molecular Weight (CAM 5.2) by Immunohistochemistry 2003493
Method: Immunohistochemistry

Recommended Use