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. Histology is the gold standard for diagnosis. Molecular testing is helpful when planning targeted therapy.

  • Key Points
  • Diagnosis
  • Screening
  • Monitoring
  • Background
  • Pediatrics
  • Lab Tests
  • References
  • Related Topics
  • Videos
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.



Targeted Therapy


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


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


  • 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


  • 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


  • 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


  • 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



  • 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


  • 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


Indications for Testing

  • Refer to Diagnosis section

Laboratory Testing

  • Refer to Diagnosis section


  • 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


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

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

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


Mutations outside of targeted exons will not be detected

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


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

Melanoma Antibody, HMB45 by Immunohistochemistry 2003935
Method: Immunohistochemistry

Ki-67 with Interpretation by Immunohistochemistry 2007182
Method: Immunohistochemistry

Melan A by Immunohistochemistry 2003996
Method: Immunohistochemistry

S-100 Protein by Immunohistochemistry 2004127
Method: Immunohistochemistry

Vimentin by Immunohistochemistry 2004181
Method: Immunohistochemistry

p21 (Waf1/Cip 1) by Immunohistochemistry 2004067
Method: Immunohistochemistry

MITF by Immunohistochemistry 2011998
Method: Immunohistochemistry

Lactate Dehydrogenase, Serum or Plasma 0020006
Method: Quantitative Enzymatic


Not used for diagnosis

S-100B Protein, Serum 2001766
Method: Quantitative Enzyme-Linked Immunosorbent Assay

PD-L1 28-8 pharmDx by Immunohistochemistry with Interpretation, nivolumab (OPDIVO) 2013684
Method: Immunohistochemistry


Bichakjian CK, Halpern AC, Johnson TM, Hood AF, Grichnik JM, Swetter SM, Tsao H, Barbosa VH, Chuang T, Duvic M, Ho VC, Sober AJ, Beutner KR, Bhushan R, Begolka WS, American Academy of Dermatology. Guidelines of care for the management of primary cutaneous melanoma. American Academy of Dermatology. J Am Acad Dermatol. 2011; 65(5): 1032-47. PubMed

Choosing Wisely. An initiative of the ABIM Foundation. [Accessed: Nov 2017]

Dummer R, Hauschild A, Lindenblatt N, Pentheroudakis G, Keilholz U, ESMO Guidelines Committee. Cutaneous melanoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2015; 26 Suppl 5: v126-32. PubMed

Garbe C, Peris K, Hauschild A, Saiag P, Middleton M, Bastholt L, Grob J, Malvehy J, Newton-Bishop J, Stratigos AJ, Pehamberger H, Eggermont AM, European Dermatology Forum (EDF), European Association of Dermato-Oncology (EADO), European Organisation for Research and Treatment of Cancer (EORTC). Diagnosis and treatment of melanoma. European consensus-based interdisciplinary guideline - Update 2016. Eur J Cancer. 2016; 63: 201-17. PubMed

Guideline summary: Referral and follow-up surveillance of cutaneous melanoma. Agency for Healthcare Research and Quality (AHRQ); 2013 Feb 01. [c. 2013 Feb NGC: 010266

Hampel H, Bennett RL, Buchanan A, Pearlman R, Wiesner GL, Guideline Development Group, American College of Medical Genetics and Genomics Professional Practice and Guidelines Committee and National Society of Genetic Counselors Practice Guidelines Committee. A practice guideline from the American College of Medical Genetics and Genomics and the National Society of Genetic Counselors: referral indications for cancer predisposition assessment. Genet Med. 2015; 17(1): 70-87. PubMed

NCCN Clinical Practice Guidelines in Oncology, Melanoma. National Comprehensive Cancer Network. Fort Washington, PA [Accessed: May 2017]

Protocol for the Examination of Specimens from Patients with Melanoma of the Skin. Based on AJCC/UICC TNM, 7th ed. Protocol web posting date: Jan 2016. College of American Pathologists (CAP). Northfield, IL [Revised: Jan 2016; Accessed: Dec 2016]

US Preventive Services Task Force, Bibbins-Domingo K, Grossman DC, Curry SJ, Davidson KW, Ebell M, Epling JW, Garcia FA, Gillman MW, Kemper AR, Krist AH, Kurth AE, Landefeld S, Mangione CM, Phillips WR, Phipps MG, Pignone MP, Siu AL. Screening for Skin Cancer: US Preventive Services Task Force Recommendation Statement. JAMA. 2016; 316(4): 429-35. PubMed

Wong SL, Balch CM, Hurley P, Agarwala SS, Akhurst TJ, Cochran A, Cormier JN, Gorman M, Kim TY, McMasters KM, Noyes D, Schuchter LM, Valsecchi ME, Weaver DL, Lyman GH, American Society of Clinical Oncology, Society of Surgical Oncology. Sentinel lymph node biopsy for melanoma: American Society of Clinical Oncology and Society of Surgical Oncology joint clinical practice guideline. J Clin Oncol. 2012; 30(23): 2912-8. PubMed

General References

Grossmann AH, Grossmann KF, Wallander ML. Molecular testing in malignant melanoma. Diagn Cytopathol. 2012; 40(6): 503-10. PubMed

Igbokwe A, Lopez-Terrada DH. Molecular testing of solid tumors. Arch Pathol Lab Med. 2011; 135(1): 67-82. PubMed

Jiang X, Zhou J, Yuen NK, Corless CL, Heinrich MC, Fletcher JA, Demetri GD, Widlund HR, Fisher DE, Hodi S. Imatinib targeting of KIT-mutant oncoprotein in melanoma. Clin Cancer Res. 2008; 14(23): 7726-32. PubMed

Kashani-Sabet M. Molecular markers in melanoma. Br J Dermatol. 2014; 170(1): 31-5. PubMed

Khattak M, Fisher R, Turajlic S, Larkin J. Targeted therapy and immunotherapy in advanced melanoma: an evolving paradigm. Ther Adv Med Oncol. 2013; 5(2): 105-18. PubMed

Linos K, Slominski A, Ross JS, Carlson A. Melanoma update: diagnostic and prognostic factors that can effectively shape and personalize management. Biomark Med. 2011; 5(3): 333-60. PubMed

Long GV, Menzies AM, Nagrial AM, Haydu LE, Hamilton AL, Mann GJ, Hughes M, Thompson JF, Scolyer RA, Kefford RF. Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma. J Clin Oncol. 2011; 29(10): 1239-46. PubMed

Mills O, Messina JL. Pediatric melanoma: a review. Cancer Control. 2009; 16(3): 225-33. PubMed

Nalejska E, Mączyńska E, Lewandowska MA. Prognostic and predictive biomarkers: tools in personalized oncology. Mol Diagn Ther. 2014; 18(3): 273-84. PubMed

Ohsie SJ, Sarantopoulos P, Cochran AJ, Binder SW. Immunohistochemical characteristics of melanoma. J Cutan Pathol. 2008; 35(5): 433-44. PubMed

Palmer SR, Erickson LA, Ichetovkin I, Knauer DJ, Markovic SN. Circulating serologic and molecular biomarkers in malignant melanoma. Mayo Clin Proc. 2011; 86(10): 981-90. PubMed

Paradela S, Fonseca E, Prieto VG. Melanoma in children. Arch Pathol Lab Med. 2011; 135(3): 307-16. PubMed

Postow MA, Carvajal RD. Therapeutic implications of KIT in melanoma. Cancer J. 2012; 18(2): 137-41. PubMed

Solus JF, Kraft S. Ras, Raf, and MAP kinase in melanoma. Adv Anat Pathol. 2013; 20(4): 217-26. PubMed

Strouse JJ, Fears TR, Tucker MA, Wayne AS. Pediatric melanoma: risk factor and survival analysis of the surveillance, epidemiology and end results database. J Clin Oncol. 2005; 23(21): 4735-41. PubMed

References from the ARUP Institute for Clinical and Experimental Pathology®

Chandler WM, Rowe LR, Florell SR, Jahromi MS, Schiffman JD, South ST. Differentiation of malignant melanoma from benign nevus using a novel genomic microarray with low specimen requirements. Arch Pathol Lab Med. 2012; 136(8): 947-55. PubMed

Grossmann AH, Grossmann KF, Wallander ML. Molecular testing in malignant melanoma. Diagn Cytopathol. 2012; 40(6): 503-10. PubMed

Grossmann AH, Yoo JH, Clancy J, Sorensen LK, Sedgwick A, Tong Z, Ostanin K, Rogers A, Grossmann KF, Tripp SR, Thomas KR, D'Souza-Schorey C, Odelberg SJ, Li DY. The small GTPase ARF6 stimulates β-catenin transcriptional activity during WNT5A-mediated melanoma invasion and metastasis. Sci Signal. 2013; 6(265): ra14. PubMed

Hall BJ, Schmidt RL, Sharma RR, Layfield LJ. Fine-needle aspiration cytology for the diagnosis of metastatic melanoma: systematic review and meta-analysis. Am J Clin Pathol. 2013; 140(5): 635-42. PubMed

Lewis TB, Robison JE, Bastien R, Milash B, Boucher K, Samlowski WE, Leachman SA, Noyes D, Wittwer CT, Perreard L, Bernard PS. Molecular classification of melanoma using real-time quantitative reverse transcriptase-polymerase chain reaction. Cancer. 2005; 104(8): 1678-86. PubMed

Oakley GM, Curtin K, Layfield L, Jarboe E, Buchmann LO, Hunt JP. Increased melanoma risk in individuals with papillary thyroid carcinoma. JAMA Otolaryngol Head Neck Surg. 2014; 140(5): 423-7. PubMed

Szankasi P, Reading S, Vaughn CP, Prchal JT, Bahler DW, Kelley TW. A quantitative allele-specific PCR test for the BRAF V600E mutation using a single heterozygous control plasmid for quantitation: a model for qPCR testing without standard curves. J Mol Diagn. 2013; 15(2): 248-54. PubMed

Willmore-Payne C, Holden JA, Hirschowitz S, Layfield LJ. BRAF and c-kit gene copy number in mutation-positive malignant melanoma. Hum Pathol. 2006; 37(5): 520-7. PubMed

Willmore-Payne C, Holden JA, Tripp S, Layfield LJ. Human malignant melanoma: detection of BRAF- and c-kit-activating mutations by high-resolution amplicon melting analysis. Hum Pathol. 2005; 36(5): 486-93. PubMed

Medical Reviewers

Content Reviewed: 
May 2017

Last Update: October 2017