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Address for correspondence: Aleodor A. Andea, MD, MBA, University of Michigan Department of Pathology, NCRC Bldg. 35, 2800 Plymouth Road, Ann Arbor, MI 48109, USA.
The diagnostic work-up of melanocytic tumours has undergone significant changes in the last years following the exponential growth of molecular assays. For the practising pathologist it is often difficult to sort through the multitude of different tests that are currently available for clinical use. The molecular tests used in melanocytic pathology can be broadly divided into four categories: (1) tests that predict response to systemic therapy in melanoma; (2) tests that predict prognosis in melanoma; (3) tests useful in determining the type or class of melanocytic tumour; and (4) tests useful in the differential diagnosis of naevus versus melanoma (primarily used as an aid in the diagnosis of histologically ambiguous melanocytic lesions). This review will present an updated synopsis of major molecular ancillary tests used in clinical practice.
The last years have witnessed an explosion in the field of molecular pathology. The number and type of tests available for various tumours, including melanocytic lesions has increased exponentially. For the surgical pathologist or dermatopathologist, keeping up with the rapid pace of development in this field may seem a daunting task. These challenges notwithstanding, it is often required presently to use molecular tools in the diagnosis of melanocytic tumours as certain molecular findings have significant implications for diagnosis, prognosis and/or treatment. The goal of this manuscript is to distil the significant advances in molecular testing for melanocytic tumours used in clinical practice and present them in a format that is relevant for the practising surgical pathologist.
Genetic evolution of melanocytic tumours
Significant advances have been made in the last decade in our understanding of the genetic foundation of melanocytic neoplasms. Current models of melanoma oncogenesis posit that tumour progression to melanoma occurs through multistep accumulation of genetic alterations involving multiple cellular pathways.
This process often follows discrete steps and progression can be permanently or temporarily halted at different stages leading to melanocytic tumours with variable biological potential from benign to malignant. It is now recognised that most benign melanocytic naevi are initiated by a single genetic alteration occurring in a precursor cell that activates the mitogen-activated protein kinase (MAPK) pathway. This single initiating event results in limited clonal expansion and is not sufficient for malignant transformation as uncontrolled growth is prevented by cellular safeguards.
In the last years it has been uncovered that some melanocytic tumours, formerly classified as naevi or borderline, harbour one or few additional oncogenic events, besides the MAPK pathway alteration seen in naevi, resulting in a morphologically distinct clonal expansion of the cells bearing the additional alteration(s). These lesions are now grouped under the umbrella term of melanocytoma in the revised World Health Organization classification, to acknowledge that at the molecular level they occupy an intermediate position in the progression sequence from naevus to melanoma.
Melanocytomas are often combined lesions, with a naevus carrying the initial alteration and a phenotypically distinct melanocytic population demonstrating the additional genomic event. Most melanocytomas have an indolent biological behaviour. They may disseminate to the local lymph node basin; however, distant spread is exceptionally rare. Due to their genetic profile, melanocytomas are expected to have a higher risk of transformation to melanoma compared to naevi; however, the absolute risk of transformation is not yet known.
Melanomas, in contrast, are characterised by multiple alterations affecting several biological pathways. Melanomas can arise within a naevus or an intermediate melanocytic tumour via additional stepwise genetic and epigenetic alterations, but most occur de novo without an associated precursor lesion. The alterations leading to melanoma usually involve corruption of pathways that prevent uncontrolled replication such as inactivation of CDKN2A, PTEN, TP53 or NF1 genes, TERT promoter (TERT-p) mutations and activation of additional oncogenes such as RAC1, ERBB2, MAP2K1, EGFR or MET among others.
Tests identifying some of these alterations, such as TERT-p mutations or homozygous deletion of CDKN2A, can be used to differentiate naevus from melanoma.
The spectrum of genetic alterations encountered in melanoma varies widely depending mainly on the anatomical site, age, or the presence of a specific benign precursor lesion. This variation is likely the result of interplay between the mutagenic processes, the state of the cell of origin and the microenvironment. The current classification of melanoma recognises eight pathways of cutaneous and mucosal melanoma development, some with associated benign and intermediate (melanocytoma) precursors (Table 1).
The 2018 World Health Organization classification of cutaneous, mucosal, and uveal melanoma: detailed analysis of 9 distinct subtypes defined by their evolutionary pathway.
Pathway 1 includes low cumulative sun damage (CSD) melanoma or superficial spreading melanoma. The benign precursor is represented by the conventional naevus carrying a BRAF or less common an NRAS gene mutation.
The melanocytomas associated with this pathway (and their defining secondary alterations) include deep penetrating naevus (DPN) (CTNNB1 or APC gene mutation), pigmented epithelioid melanocytomas (inactivation of PRKAR1A gene) and BAP1-inactivated melanocytic tumours (BAP1 gene inactivation).
Loss of expression of protein kinase a regulatory subunit 1alpha in pigmented epithelioid melanocytoma but not in melanoma or other melanocytic lesions.
Pathway 2 (high CSD melanoma or lentigo maligna melanoma), pathway 3 (desmoplastic melanoma), pathway 5 (acral melanoma) and pathway 6 (mucosal melanoma) are not associated with benign naevi (with some rare exceptions for acral melanoma) and the intermediate stage is represented by melanoma in situ. Pathway 4 includes Spitz melanoma with Spitz naevus as the benign lesion and atypical Spitz tumour (Spitz melanocytoma) as the intermediate precursor. HRAS mutations and rearrangements involving several tyrosine and serine-threonine kinase genes are associated with Spitz naevi.
Pathway 7 corresponds to melanoma arising from congenital naevus with NRAS gene mutations as initiating driver alterations and proliferative nodules as intermediate melanocytoma lesions (characterised by copy number alterations of whole chromosomes).
Finally, pathway 8 refers to melanoma arising in blue naevus with the atypical cellular blue naevus as an intermediate melanocytoma. The benign blue naevi are characterised by activating mutations in GNAQ and GNA11 and rarely in the CYSLTR1 and PLB4 genes.
Melanomas associated with blue nevi or mimicking cellular blue nevi: clinical, pathologic, and molecular study of 11 cases displaying a high frequency of gna11 mutations, bap1 expression loss, and a predilection for the scalp.
These remarkable advances in our understanding of the molecular underpinnings of melanocytic tumours outlined in the previous section have led to development of a broad spectrum of molecular tests aimed to characterise them. Evaluation of melanocytic lesions at molecular level is currently not just an academic endeavour but has entered clinical practice as specific molecular findings have implications for diagnosis, prognosis, and treatment. These tests can be broadly divided into four categories: (1) tests that predict response to systemic therapy in melanoma; (2) tests that predict prognosis in melanoma; (3) tests useful in determining the type or class of melanocytic tumour; and (4) tests useful in the differential diagnosis of naevus versus melanoma (primarily used as an aid in the diagnosis of histologically ambiguous melanocytic tumours).
Tests used to predict response to systemic therapy
The molecular tests used to predict response to systemic therapy are usually reserved for high-stage melanoma where such therapies are indicated. Presently, there are two types of systemic therapy in use for melanoma: targeted therapy agents that block the MAPK pathway using BRAF and MEC inhibitors, and immune checkpoint inhibitor therapy that modulates the immune response against melanoma cells.
The effectiveness of targeted therapy depends on the presence of a mutation in the BRAF gene. In fact targeted therapy may be detrimental in BRAF wild cases in which the use of BRAF-inhibitors can cause paradoxical activation of MAPK pathway.
For this reason, BRAF gene status should be evaluated for any melanoma in which BRAF-inhibitor therapy is considered. Currently next generation sequencing methods are replacing classical Sanger assays for BRAF sequencing; however, an immunohistochemical stain that detects the mutated BRAF V600E protein is available which is particularly important for practising surgical pathologists. The stain is useful in evaluating tumours that are small or are admixed with non-melanocytic cells for which sequencing by conventional methods could be difficult. BRAF immunohistochemistry shows good correlation with sequencing data.
To date, there is no successful specific treatment targeted against NRAS-mutated melanomas; however, trials with MEK combined with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are underway.
Less common, KIT gene mutations can be found in a minority of melanomas, more frequent in mucosal and acral melanoma, and these patients may benefit from KIT-inhibitor targeted therapy.
Response to immune modulators is more difficult to predict; however, it is important as these agents have significant side effects. Response to immune modulator therapy seems to correlate with the amount of foreign antigens that a tumour is presenting to the immune system which in turn correlates with the tumour mutation burden (TMB). Tests that evaluate TMB are currently in use with high TMB correlating with a better response to immunotherapy.
Recent predictive models using transcriptome (gene expression) data, such as the immuno-predictive score (IMPRES), are being investigated as potentially useful prognosticators of response to immune checkpoint inhibition therapy.
These assays aim to predict the risk of spread to the regional lymph node basin and/or the overall disease specific survival for melanoma. The goal is to better stratify the patients into risk groups and potentially select a low-risk group that does not need sentinel lymph node (SLN) biopsy. The tests employ gene expression profile (GEP) of selected sets of genes relevant for melanoma biology and various proprietary algorithms to generate a risk estimate for SLN positivity and/or disease progression.
Expert consensus on the use of prognostic gene expression profiling tests for the management of cutaneous melanoma: consensus from the skin cancer prevention working group.
One of these assays is a 31-GEP (DecisionDx-Melanoma; Castle Biosciences, USA). This is a commercially available assay that stratifies low-risk (class 1) and high-risk (class 2) cutaneous melanoma.
Recent studies evaluating the assay concluded that it predicts with reasonable accuracy the risk of regional and distant metastasis in univariate analysis; however, only one study reported a multivariate analysis accounting for some of the known clinicopathological variables associated with melanoma specific survival.
Interim analysis of survival in a prospective, multi-center registry cohort of cutaneous melanoma tested with a prognostic 31-gene expression profile test.
In an attempt to improve performance, a combined prediction model for SLN positivity was developed that incorporates clinicopathological data with GEP. Results showed that for T1/T2 tumours, SLN biopsy reduction rate of 40% could be achieved.
A GEP panel developed in Europe (MelaGenix; Neracare, Germany) combines a gene expression risk score from an 11-gene panel with SLN status and was shown to improve prediction of relapse-free survival in patient with known SLN status.
These tests have the potential to improve management of patients with melanoma; however, to date, it is not clear if they add anything beyond conventional staging parameters.
Tests useful in determining the type or class of melanocytic tumour
Currently, the gold standard for diagnosing melanocytic lesions is still histological examination. However, there are certain types of melanocytic tumours that demonstrate overlapping histological features and in which a definitive classification can occasionally be challenging. In recent years, it has become evident that various classes of melanocytic lesions are characterised by specific molecular events which are now part of a two-dimensional classification (Table 1). These specific alterations can be used to assist in classification of melanocytic lesions when the histological features are not diagnostic. Most of these alterations represent initiating driver events that lead to the formation of a naevus or secondary events that lead to a melanocytoma. Because melanomas evolving from these naevi or melanocytomas carry the same abnormalities, their presence is often not useful in predicting malignant potential.
For the surgical pathologist it is important to recognise that some of these genomic events can be assessed using immunohistochemical stains including BRAFV600E, NRASQ61R, β-catenin, BAP1, PRKAR1, ALK, and pan-TRK (Table 2). Examples of lesions where these tests could be contributory include distinguishing deep penetrating naevi (DPN) from cellular blue naevi (CBN) and Spitz naevi, differentiating BAP1-inactivated melanocytic tumours (BIMT) from Spitz tumours, or diagnosing pigmented epithelioid melanocytoma (PEM).
Table 2Specific IHC stains helpful in determining the class of melanocytic tumour
IHC stain
Corresponding genetic alteration
Type of melanocytic tumour
Scenarios where may help in diagnosis
BRAFV600E
BRAFV600E activating point mutation
Acquired naevi Conventional naevi Small congenital naevi Melanoma BIMT DPN
SSM with spitzoid morphology (+) vs low-risk Spitz lesion (–)
NRASQ61R
NRASQ61R activating point mutation
Large congenital naevi Some conventional naevi Conventional melanoma
SSM with spitzoid morphology (+) vs low-risk Spitz lesion (–)
DPNs are now regarded as melanocytomas, being characterised by activation of the MAPK pathway via mutations in BRAF, MAP2K1 or HRAS and a second activating mutation in CTNNB1 gene encoding for β-catenin or in APC gene.
The presence of nuclear and/or cytoplasmic staining for β-catenin as opposed to membranous, is highly suggestive for CTNNB1 mutations and supports a diagnosis of DPN versus other entities in the differential such as CBN of Spitz lesions (Fig. 1A–E).
Fig. 1Examples of immunohistochemical stains (IHC) useful in classifying melanocytic lesions. (A–E) Atypical deep penetrating naevus (DPN). (A) Combined melanocytic proliferation involving deep dermis. (B) A conventional intradermal naevus is present in the upper dermis. (C) Epithelioid and spindle cells with abundant pale cytoplasm, nuclear atypia and numerous melanophages representing the atypical DPN component. (D) BRAFV600E IHC showing diffuse staining of both components. (E) β-Catenin IHC demonstrating abnormal cytoplasmic and nuclear staining in the DPN (lower right side) suggesting the presence of an activating mutation in CTNNB1 gene characteristic for this type of melanocytoma. Normal membranous staining in the conventional naevus (upper left side). (F–H) Atypical Spitz tumour with ALK gene rearrangement. (F) Polypoid dermal melanocytic proliferation with an expansile border and compact growth with no maturation. (G) Epithelioid and spindle fusiform cells with spitzoid morphology arranged in tightly packet fascicles. (H) ALK IHC showing diffuse positivity suggesting the presence of a rearrangement involving ALK gene. (I–L) BAP1-inactivated melanocytic tumour. (I) Large, predominantly intradermal tumour with biphenotypic morphology. (J) A conventional naevus component is noted in the superficial portion. (K) Epithelioid cells with large nuclei, prominent nucleoli and abundant eosinophilic cytoplasm with distinct cell membrane. (L) BAP1 IHC showing loss of nuclear staining in the epithelioid component (lower right side) and retained labelling in the conventional naevus (upper left side).
BIMT is another example of a melanocytoma in the differential diagnosis with Spitz lesions and demonstrates genomic alterations that include an activating mutation in MAPK pathway, usually a BRAF mutation combined with inactivation of BAP1 gene.
Immunohistochemistry for BRAFV600E and BAP1 can be used to document absence of BAP1 staining in the nuclei of lesional cells and expression of BRAF (Fig. 1I–L). In contrast, Spitz tumours are characterised by HRAS mutations or fusions of kinase genes. Immunohistochemical stains are available for several of these fusion products including ALK, and pan-TRK and can be used as a surrogate for the presence of a rearrangement (Fig. 1F–H).
PEMs are dermal proliferations characterised by epithelioid cells with prominently pigmented cytoplasm. PEMs are characterised by activating MAPK pathway alterations and a second alteration involving loss of function of PRKAR1A gene.
Loss of expression of protein kinase a regulatory subunit 1alpha in pigmented epithelioid melanocytoma but not in melanoma or other melanocytic lesions.
In a subset of cases, PRKAR1α protein shows loss of expression by immunohistochemistry and this stain be used to diagnose them.
BRAFV600E and NRASQ61R stains are helpful in differentiating atypical Spitz naevus versus a superficial spreading melanoma with spitzoid morphology (spitzoid melanoma). Expression of BRAFV600E or NRASQ61R in a melanocytic lesion with spitzoid morphology helps to assign it to Pathway 1 of the classification (Low-CSD/SSM) rather than Pathway 4 (Spitz tumour). Often, the degree of cytological and architectural atypia that would be acceptable for a low-risk Spitz lesion in Pathway 4 is worrisome and more consistent with melanoma in Pathway 1 (Fig. 2).
Fig. 2(A) Broad, compound melanocytic proliferation. (B) Melanocytes have a spitzoid morphology and are arranged in nests with vertical and horizontal arrangement as well as a few single cells with occasional pagetoid spread. A Kamino body is noted (black arrow). Differential diagnosis includes melanoma versus atypical Spitz naevus. (C) BRAFV600E IHC demonstrates diffuse positivity. The presence of BRAFV600E mutation is incompatible with a Spitz tumour and a diagnosis of superficial spreading melanoma with spitzoid morphology was rendered.
Tests useful in the differential diagnosis of naevus versus melanoma
Most melanocytic tumours can be reliably classified as naevus or melanoma by conventional microscopic examination. However, there is a small but significant subset of melanocytic neoplasms that elude a definitive diagnosis as benign or malignant using histopathological criteria and routine immunohistochemistry alone and are often designated with terms that imply uncertainty regarding their biological potential.
These cases are usually sent in consultation; however, there is poor reproducibility even among experts in diagnosing them which opens the possibility for mismanagement including either under- or over-treatment. The tests discussed in this section are usually employed in this clinical context to help refine the diagnosis and biological potential of histologically ambiguous melanocytic tumours. Among the tests discussed in this review they are probably the most useful for practising pathologists.
Molecular tests based on assessment of genomic copy number abnormalities
Early research has uncovered that melanomas are characterised by an unstable genome with numerous copy number abnormalities (CNAs) while naevi lack or have a limited number of CNAs.
This pattern of genomic abnormalities led to development of diagnostic strategies based on evaluation of CNAs. The tests used currently for this purpose are comparative genomic hybridisation (CGH)/single nucleotide polymorphism (SNP) array and fluorescence in situ hybridisation (FISH).
CGH is a performed by hybridising the tumour DNA on microarrays and evaluates the entire genome for CNAs. The arrays are composed of a variable number of spots (from few thousands to over 4 million) containing DNA from a specific genomic locus to be interrogated; the resolution of the array is proportional to the number of spots. SNP arrays interrogate genomic loci centred on a specific SNP and can detect specific alleles. In addition to copy number changes, SNP arrays allow for the detection of loss of heterozygosity (LOH) events. CGH/SNP array testing from small formalin-fixed, paraffin-embedded (FFPE) biopsies which often yield low amounts of degraded DNA, presents challenges for most platforms.
Novel protocols such as those based on molecular inversion probes (MIPs) have improved the ability to analyse degraded DNA. MIPs are engineered oligonucleotides with ends being complementary to regions flanking SNPs and a footprint of only 40 bp which allows evaluation of fragmented DNA from FFPE tissue.
An advantage of this technique is that all the unused MIPs and the target DNA are removed from the reaction and an engineered tag sequence on the MIP is hybridised to the array which greatly improves the signal to noise ratio over that of conventional CGH/SNP arrays.
Fig. 3C shows a typical SNP array output. The upper panel shows copy number changes for each SNP with copy number or tumour to normal log ratio on the vertical axis and chromosomes on the horizontal placed in ascending order from p-ter to q-ter. DNA gains or losses are reflected by deflections of the average line above or below normal diploid status. The lower panel indicates the allele peak or B-allele frequency status for each SNP. For normal diploid state this panel has three tracks, a middle line composed of all the heterozygous SNPs and two outer lines containing SNPs that are homozygous for either allele. LOH is reflected by a split in the middle heterozygous line. Usually, LOHs are accompanied by DNA losses or gains; however, LOH events without associated copy number abnormalities (copy-neutral LOH) can also be detected.
Fig. 3Example of a low-risk atypical Spitz tumour with SNP array testing. (A) Predominantly dermal melanocytic proliferation showing compact growth and lack of maturation. (B) Epithelioid and spindle cells with spitzoid morphology, compact growth pattern and cytological atypia. (C) SNP array results. The upper panel shows copy number status (log ratio on the vertical and chromosome locus on the horizontal). Gains and losses are reflected by deflections of the average yellow line above or below 0, respectively. Black arrows indicate one copy number losses of chromosome 1p, 5p and entire chromosome 9. The lower panel shows the B-allele frequency for each SNP on the array (B-allele frequency on the vertical and chromosome locus on the horizontal). In the normal state this track consists of three lines, a middle heterozygous line and two outer lines which are homozygous for the A and B alleles. Red arrows indicate LOH events on chromosomes 1p, 5p and 9 associated with the losses. In this case, the presence of three CNAs is below the cut-off of four and favours a low risk for adverse outcome.
In their study, the authors found a striking difference between the frequency of CNAs in melanomas versus naevi (96.2% vs 13%, respectively). Melanomas demonstrated multiple CNAs, involving segments of chromosomes while the few naevi with CNAs had only isolated abnormalities. Subsequent studies using SNP and CGH-arrays have confirmed these results with 89.0–94.7% of melanomas demonstrating CNAs while 94.7–100% of naevi had normal chromosomal profiles.
The performance of CGH/SNP array has also been evaluated for specific subtypes of melanocytic tumours that are notoriously difficult to classify as benign or malignant by conventional microscopy alone, including the Spitz and blue naevus-like tumours.
Atypical cellular blue nevi (cellular blue nevi with atypical features): lack of consensus for diagnosis and distinction from cellular blue nevi and malignant melanoma ("malignant blue nevus").
The Spitz tumour group is composed of Spitz naevi (benign), atypical Spitz tumours (borderline/melanocytoma) and spitzoid melanomas (malignant). It has been shown that Spitz naevi have either no abnormalities or isolated CNAs including gains of 11p or 7p while spitzoid melanomas demonstrate multiple CNAs involving partial segments of chromosomes.
The blue naevus group is composed of cellular blue naevus (CBN) (benign), atypical CBN (borderline/melanocytoma) and melanoma arising in or resembling CBN (malignant).
Atypical cellular blue nevi (cellular blue nevi with atypical features): lack of consensus for diagnosis and distinction from cellular blue nevi and malignant melanoma ("malignant blue nevus").
Melanomas associated with blue nevi or mimicking cellular blue nevi: clinical, pathologic, and molecular study of 11 cases displaying a high frequency of gna11 mutations, bap1 expression loss, and a predilection for the scalp.
Melanomas arising in blue naevi were found to exhibit multiple chromosomal abnormalities while CBN and atypical CBN have no or a limited number of CNAs (<3).
From the initial studies it seemed that a dichotomic approach could be used in diagnosing naevi versus melanoma. However, recent studies have documented a gradual increase in the number of CNAs paralleling the progression of melanocytic tumours from benign to borderline, to melanoma and to metastatic melanoma.
A recent study by Alomari et al. found that the average number of CNAs increases from 0 in naevi to 0.6 in atypical naevi to 2.8 in borderline lesions and to 18.1 in melanoma.
It is apparent that there are naevi or melanocytomas that carry one or few CNAs but demonstrate benign or indolent biological behaviour. These abnormalities are not the result of genomic instability, as is the case in melanoma, but reflect DNA rearrangements specific for certain types of melanocytic lesions. Isolated CNAs can occur in Spitz naevi characterised by driver alterations which result in genomic rearrangements. The subgroup of Spitz naevi with HRAS gene mutations demonstrate a gain of 11p (the locus for HRAS).
In such cases, which may show atypical histological features, presence of 11p gain in the absence of other abnormalities supports a diagnosis of naevus (Fig. 4A–C). Another group of Spitz tumours including Spitz naevi, but also atypical Spitz tumours and spitzoid melanomas harbour tyrosine and serine-threonine kinase fusions including ROS1, NTRK1, NTRK3, ALK, BRAF, MET and RET as initiating driver mutations.
These abnormalities can present on CGH/SNP array as isolated CNAs involving the gene locus. Isolated CNAs can also occur in low-grade melanocytomas as a manifestation of the additional generic alteration.
One example is the BAP1-inactivated melanocytic tumours. These are lesions that develop in a pre-existent naevus in which a clone acquires a second abnormality causing inactivation of BAP1 gene usually by mutation coupled with deletion of the contralateral allele which results in a characteristic epithelioid morphology.
On CGH/SNP array, these tumours are characterised by a loss on chromosome 3 encompassing the BAP1 gene locus (3p21) (Fig. 4D–F). Proliferative nodules arising in congenital naevi are another example of a melanocytoma and are often difficult to differentiate form melanoma.
Fig. 4Specific CGH/SNP-array abnormalities in benign naevi or indolent melanocytic tumours/melanocytomas. (A–C) Desmoplastic Spitz naevus. (A) Intradermal melanocytic proliferation with pronounced desmoplastic stromal reaction and infiltrative growth pattern. (B) Epithelioid cells with large nuclei, prominent nucleoli and abundant amphophilic cytoplasm. (C) SNP-array showing a gain of 11p (black arrow) with no additional abnormalities suggesting a desmoplastic Spitz naevus. (D–F) BAP1-inactivated melanocytic tumour. (D) Large, predominantly intradermal tumour with biphenotypic morphology. (E) Upper panel: nests of bland cells with pigmented cytoplasm consistent with a conventional naevus. Lower panel: epithelioid cells with large nuclei, prominent nucleoli and abundant eosinophilic cytoplasm with distinct cell membrane. (F) SNP-array showing a loss of 3p21 (red arrow) with no additional abnormalities suggesting a low-risk BAP1-inactivated melanocytic tumour. (G–I) Proliferative nodule arising in congenital naevus. (G) Hypercellular dermal nodule composed of densely packed melanocytes. (H) Tightly packed spindle melanocytes with pigmented cytoplasm. (I) SNP-array showing gains of whole chromosomes 1, 6, 8 15, 16, 18, 20 and 22 suggestive of a proliferative nodule (black arrows).
Considering that low-risk melanocytic lesions can harbour a limited number of CNAs, it is important to establish a cut-off for the number of CNAs beyond which a borderline melanocytic tumour is concerning for melanoma. In a study by Alomari et al. the authors proposed that a SNP-array test showing three or less CNAs should be interpreted as reassuring for a low-risk melanocytic lesion. There are some exceptions noted: abnormalities involving genes important in melanoma progression, such as homozygous deletion of CDKN2A gene, are concerning for melanoma even in isolation. Conversely, a CGH/SNP array with four or more CNAs is worrisome for a high-risk melanocytic lesion (Fig. 3, Fig. 5). Again, exceptions are noted as proliferative nodules may show multiple gains and/or losses of entire chromosomes. Using this cut-off, the authors reported a sensitivity and specificity in diagnosing melanoma of 82.5% and 100% respectively.
Fig. 5Melanoma arising in cellular blue naevus. (A) Dermal melanocytic proliferation protruding into the subcutis. A more cellular nodule is noted in the upper portion of the lesion. B. High magnification showing ovoid melanocytes with pale cytoplasm arranged in interconnecting fascicles and associated with melanophages consistent with a cellular blue naevus. (C) High magnification from the cellular nodule showing tightly packed cells with cytologic atypia. (D) FISH with 6p25 (RREB1) probe from the cellular nodule. Most nuclei show more than two probe signals (red) consistent with a positive result. (D) SNP-array plot of melanoma from the cellular nodule showing numerous gains and losses of segments of chromosomes (black arrows) consistent with a positive result. A diagnosis of melanoma arising in cellular blue naevus was rendered.
As this test is usually employed for borderline melanocytic lesions it is important to evaluate in this group of tumours if number and pattern of CNAs correlate with clinical outcome. These studies are more difficult to perform, mostly due to the limited number of cases with long-term follow-up and adverse events. Alomari et al. reported that the number of CNAs in borderline melanocytic tumours without adverse events was lower compared to those with adverse events (3.7 versus 8.5, respectively); however, the number of cases was relatively low. Another study on blue naevus tumours with partial follow-up data showed that all three cases with adverse events showed abnormalities (100% sensitivity) while three of six cases with no adverse events did not show any CNAs (50% specificity).
Further studies are required to better define the number and pattern of abnormalities that correlate with poor outcome in histologically ambiguous melanocytic tumours.
Fluorescence in situ hybridisation
CGH/SNP array testing presents certain limitations related to the amount and purity of the tumour cells. The technique requires 10 unstained slides and a tumour purity over 25% to produce reliable results. Melanocytic proliferations that are very small, superficial or associated with a heavy inflammatory infiltrate are at times unsuitable for CGH/SNP array analysis. In addition, the turn-around time (TAT) is often one to several weeks. FISH has emerged as an alternative with the advantage of requiring only few sections, allowing analysis of lesions with low tumour content, and having a short TAT (usually few days to a week).
The assay was originally developed with data from CGH studies by evaluating FISH probes that target genomic areas frequently affected in melanoma.
The minimum number of probes allowing for best sensitivity and specificity in distinguishing melanoma from naevus were selected. The optimal set included four probes targeting 6p25 (RREB1), 6q23 (MYB), 11q13 (CCND1) and Centromere 6 with a sensitivity and specificity for diagnosing melanoma of 86.7% and 95.4%, respectively.
Gains of 6p25 and 11q13 and losses of 6q23 exceeding the cut-off values are regarded as positive result and favour a diagnosis of melanoma or high-risk melanocytic lesion (Fig. 5D). Later studies evaluating this assay on different cohorts of naevi and melanomas found similar results with sensitivity and specificity ranging from 75–100% and 89–100%, respectively.
Similar to CGH/SNP arrays, it is important to evaluate the performance of this test in predicting clinical outcome for histologically ambiguous melanocytic tumours. In the original study by Gerami et al., a cohort of ambiguous melanocytic tumours with follow-up was analysed and the test performed with a sensitivity and specificity of 100% and 71%, respectively.
Similar results were obtained by Massi et al., while a study by Geiser et al. showed a lower sensitivity and specificity of only 60% and 33%, respectively.
Subsequent studies have evaluated the effect of including additional probes, as the original probe set demonstrated a lower sensitivity for spindle and spitzoid melanoma.
A large study using this expanded probe set was conducted on a series of borderline atypical Spitz tumours with follow-up data. The study found that the assay had a sensitivity and specificity of 100% and 76%, respectively, in diagnosing atypical Spitz tumours with adverse events.
Telomerase reverse transcriptase (TERT) encodes the catalytic subunit of telomerase, the enzyme preventing cellular senescence due to telomere attrition by adding nucleotide repeats to the ends of telomeres.
Horn et al. described for the first time a germline TERT promoter (TERT-p) mutation in a melanoma-prone family and ultraviolet-induced mutations in 74% of investigated melanoma cell lines and 33% of primary melanoma tumours.
In contrast, acral melanomas show a lower incidence of TERT-p mutations (4.2–19% of cases); however they demonstrate frequent TERT copy number gains and amplifications.
However, a study found that two of 14 naevi harboured TERT-p mutations (specificity of 85.7%) at a very low allelic frequency (0.2%), suggesting the existence of mutated subclones possibly representing early foci of transformation towards melanoma.
Another study found TERT-p mutations in 77% of melanocytic lesions classified as intermediate or melanoma in situ but in none of the benign lesions. This seems to indicate that TERT-p alterations occur early in the evolution of melanoma.
Several studies evaluated the feasibility of TERT-p mutational status as an ancillary diagnostic tool to separate naevi from melanoma. Thomas et al. in a study on 86 melanomas, 72 naevi, and 40 uncertain melanocytic proliferations reported TERT-p mutations in 77.9%, 1.4% and 5% of melanomas, naevi and uncertain melanocytic proliferations, respectively, for a sensitivity, specificity and overall accuracy of 77.9%, 98.6% and 87.3%, respectively, in diagnosing melanoma.
In contrast, in a Korean cohort of melanomas Roh et al. found TERT-p hotspot mutations in only 33.3% and 22.2% of CSD and low-CSD tumours, respectively. In the same study the frequency of TERT-p mutations in acral melanoma was 10.9%.
In a study evaluating the use of TERT-p mutations in differentiating recurrent naevi versus recurrent melanoma, the authors found a sensitivity of 44% and specificity of 100%, while in the control group of naevi and melanoma the sensitivity and specificity were 65% and 90.5%, respectively.
One study correlated TERT-p mutations with outcome in a series of 56 atypical Spitz tumours and spitzoid melanomas. The authors found TERT-p mutations in all four patients with fatal outcome but in none of the patients with a favourable clinical course, for a sensitivity and specificity of 100%.
GEP involves extracting RNA, reverse transcribing it into cDNA and performing real-time PCR. A set of genes differentially expressed in naevi versus melanoma is selected from large scale gene expression studies and validated on a cohort of naevi and melanoma. One of the panels that is currently commercially available is composed of 23 genes including one gene related to melanoma tumourigenesis (PRAME), eight genes involved in immune signalling (CCL5, CD38, CXCL10, CXCL9, IRF1, LCP2, PTPRC, and SLL), five genes with multifunctional roles (S100A9, S100A7, S100A8, S100A12 and PI3) and nine housekeeping genes.
Gene expression levels are converted into a score using a proprietary weighted algorithm that separates malignant cases (>0) from benign (<–2). Scores between –2 and 0 are considered as indeterminate. In the initial validation studies, the test showed a sensitivity and specificity of 90–93% and 91–96%, respectively, in diagnosing melanoma.
An independent study found a lower sensitivity of only 62% and specificity of 95% in a series of unequivocal naevi and melanomas and a correlation with FISH results of 80%.
Another commercially available GEP test is based on a 35 gene panel (32 discriminant genes and 3 control genes) and employs a proprietary neural network algorithm to classify cases.
The GEP assays will likely have a role as ancillary tests for difficult melanocytic tumours; however, more research correlating test results with outcome in ambiguous melanocytic lesions is needed.
Discussion
The field of melanocytic pathology has experienced a substantial increase in the number and availability of molecular tests. The tests related to prognosis and treatment of melanoma are usually used by surgeons and oncologists; however, the assays employed for diagnosis and classification are helpful in the work-up of difficult melanocytic lesions and thus are relevant for surgical pathologists and dermatopathologists.
The most impactful tests in clinical practice are those helping differentiate melanoma from naevus in histologically ambiguous cases. Several tests are available for diagnostic purposes including CGH/SNP array, FISH, GEP, and TERT-p mutation analysis.
Choosing between the multitude of available tests can be a difficult task and it is important for the pathologist to understand the advantages and limitations of each assay. CGH/SNP arrays and FISH are the tests with the longest history of clinical use. CGH/SNP arrays and FISH testing are usually offered by large reference laboratories or academic institutions. If available, CGH/SNP array is usually the preferred test due to coverage of the entire genome which confers higher sensitivity compared to FISH which interrogates only few selected loci. CGH/SNP array may also be more specific due to the possible false positive FISH results in the setting of tetraploidy, encountered especially in Spitz naevi.
A recent study comparing FISH and CGH/SNP array testing found that for borderline melanocytic lesions FISH has a sensitivity of only 61% and specificity of 84% when compared to CGH/SNP array.
In addition, the accuracy of FISH results is also dependent on the experience of the person enumerating the signals, which is less of a problem for CGH/SNP array testing. Notwithstanding these limitations, there are instances where FISH is preferred. Cases in which only a limited amount of material is available or when tumours are infiltrated by other cell types may be unsuitable for CGH/SNP array but amenable for FISH. Also FISH has a lower TAT and is less expensive. The 23- and 35-GEP assays are available commercially (myPath Melanoma and DiffDx-Melanoma; Castle Biosciences). These tests are promising; however, more experience with them is needed before a clear recommendation can be made.
Appropriate use criteria for ancillary diagnostic testing in dermatopathology: new recommendations for 11 tests and 220 clinical scenarios from the American Society of Dermatopathology appropriate use criteria committee.
TERT-p mutation can be easily performed in most molecular laboratories and thus it is more widely available. TERT-p mutation status can differentiate between unequivocal naevi and melanomas; however, sensitivity for detecting melanoma is relatively low in some studies. Similar to GEP, TERT-p testing is relatively new, and more research is needed before clear recommendations can be made.
Appropriate use criteria for ancillary diagnostic testing in dermatopathology: new recommendations for 11 tests and 220 clinical scenarios from the American Society of Dermatopathology appropriate use criteria committee.
While the role of these tests in clinical practice is still being refined, a study by Emanuel et al. surveying a group of dermatopathologists revealed that a great majority (92%) use molecular studies for diagnostically challenging melanocytic lesions (54% reported routine use while 34% reported rare use).
In two recent studies evaluating appropriate use criteria for molecular tests in dermatopathology, a group 17 experts were asked to rate the appropriateness of these tests in various clinical scenarios. The results revealed that CGH/SNP arrays and FISH were considered appropriate to be used for melanocytic tumours in which the histology is not conclusive and, as expected, there was no indication for use when a definitive diagnosis could be rendered by histology. There was no consensus on the use of GEP and TERT-p mutation assays due to the lack of sufficient evidence.
Appropriate use criteria for ancillary diagnostic testing in dermatopathology: new recommendations for 11 tests and 220 clinical scenarios from the American Society of Dermatopathology appropriate use criteria committee.
Deciding when to employ the diagnostic molecular tests and how to use the results in clinical practice is not always straightforward. A recent study proposed an algorithm for the integration of molecular studies in the diagnosis of melanocytic tumours (Fig. 6).
As a general rule, molecular testing should only be employed in conjunction with histology and clinical presentation and the results should be used to support initial histological impression. If conventional microscopic examination and routine immunohistochemical stains allow for a definitive diagnosis, no additional molecular test is indicated. For borderline melanocytic lesions that elude a definitive diagnosis an effort should be made to classify them into three categories: (1) favour benign, (2) borderline, or (3) favour maligant. The classification should be based on all available data including histology, immunohistochemistry as well as demographic data and clinical presentation; however, it is inherently subjective. At this point the molecular test is performed. If the lesion is in category 1 or 2 and the test is negative, a diagnosis of naevus/low-risk tumour can be rendered. If the lesion is in category 2 or 3 with a positive test, a diagnosis of melanoma/high-risk tumour can be made. For cases with discrepant results in which a category 1 was favoured and test was positive or category 3 with a negative test, the lesion should be left as borderline and the test regarded as non-contributory.
Fig. 6Algorithm for integrating histology with molecular data in the diagnosis of melanocytic tumours.
The last decade has seen significant advances in molecular evaluation of melanocytic lesions resulting in improved diagnostic precision. However, there is a need for more outcome-based research to better define and improve the performance of these assays for borderline melanocytic tumours.
Conflicts of interest and sources of funding
The authors state that there are no conflicts of interest to disclose. No special funding was received by the authors of this review.
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