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Molecular pathology of melanocytic tumors

Seminars in Diagnostic Pathology, 4, 30, pages 362 - 374

Abstract

Genetic and genomic analyses of melanocytic tumors have yielded new opportunities for improvements in diagnostic accuracy for the distinction of nevus from melanoma and better selection of patients affected by melanoma for targeted treatment. Since chromosomal copy number changes are commonly found in malignant melanoma, but rare in melanocytic nevi, cytogenetic assays have emerged as a promising ancillary study for the workup of melanocytic tumors with ambiguous light microscopic features. Comparative genomic hybridization (CGH) permits assessment of the full set of chromosomes, but requires a significant amount of lesional tissue, and may fail to detect aberrations in a minor subpopulation of tumor cells. Fluorescence in situ hybridization (FISH) is the cytogenetic assay of choice for limited amounts of tissue. FISH targets only specific chromosomes, with inherent limitations in test sensitivity and specificity. FISH analysis is also heavily dependent on individual experience. Molecular studies have identified distinct sets of mutations in melanoma and/or nevi. These mutations have become clinically relevant for targeted therapy of patients with advanced disease, especially for the treatment of patients with metastatic melanoma carrying the BRAFV600 or KIT mutations. However, mutation analysis can on occasion also be used for diagnostic purposes.

Keywords: Melanoma, Melanocytic nevus, Cytogenetics, Molecular dermatopathology, FISH.

Introduction

Histopathologic analysis of hematoxylin and eosin-stained tissue sections and correlation with the clinical context remains the gold standard for the diagnosis of melanoma. Experienced dermatopathologists can make a definitive, reliable, and reproducible distinction between a melanocytic nevus and malignant melanoma in most cases. However, there is a subset of lesions with unusual and/or conflicting histopathologic findings. Some parameters of a lesion (e.g., symmetric silhouette) may suggest a possible nevus, while others (e.g., cytologic atypia and mitoses) make one worry about a possible melanoma. The difficulty in classifying lesions with ambiguous features is reflected in terms such as “minimal deviation melanoma,” 1 “borderline melanocytic tumor,” 2 “prognostically indeterminate melanocytic tumor,” 3 “atypical Spitz tumor,”4 and 5 “atypical spitzoid melanocytic tumor,”6 and 7 “atypical Spitz tumor of uncertain malignant potential,”8, 9, and 10 and “atypical blue melanocytic neoplasms.”11 and 12

When pathologists disagree with each other or acknowledge the inability to render a definitive diagnosis based on routine sections, it poses a dilemma for clinicians and patients regarding prognosis and further management. 13 Therefore, pathologists have explored the use of ancillary techniques to improve diagnostic accuracy for problematic melanocytic tumors. Cytogenetic methods have recently gained popularity for the distinction of nevi from melanoma, including fluorescence in situ hybridization (FISH) analysis as well as comparative genomic hybridization (CGH).14, 15, 16, and 17

Even when the diagnosis of a melanoma has been established, it has become apparent that not all melanomas are the same. Different mutational subtypes have been identified. 18 This subclassification has become relevant for the optimal treatment selection for patients.

Cytogenetic studies

Comparative genomic hybridization

Basics

Comparative genomic hybridization (CGH) is a method to detect copy number changes throughout the genome.14, 18, 19, and 20 Total genomic DNA isolated from tumor, and normal tissue is labeled with different fluorochromes. The mixture is hybridized onto normal metaphase spreads from a healthy donor (classic CGH) or a microarray of mapped clones or genomic DNA (array CGH). Copy number gains or losses are identified based on differences in fluorescence intensities. Tumor cell heterogeneity can be a problem for the sensitivity of the test. If copy number changes are present only in a minor subpopulation of the total tumor from which DNA is extracted for CGH, they may not be detected. 21 In general, approximately one-third of the tumor should harbor copy number changes for them to be recognizable by CGH.

Clinical applications

Analysis of melanocytic tumors by CGH has revealed that nevi and melanoma differ cytogenetically.14, 15, 22, and 23 The vast majority of melanomas shows recurrent patterns of chromosomal aberrations, such as losses of chromosomes 6q, 8p, 9p, and 10q along with copy number gains of 1q, 6p, 7, 8q, 17q, and 20q.15, 16, and 18 In contrast, most melanocytic nevi lack copy number changes detectable by CGH ( Fig. 1 ). Exceptions include subtypes of Spitz nevi/tumors characterized by gains of 11p or loss of chromosome 3.14, 22, and 24 The lack of significant overlap in the patterns of chromosomal aberrations in melanoma and nevi provides the rationale for using CGH (or FISH; see below) as an ancillary diagnostic tool for diagnostically problematic melanocytic tumors. 17

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Fig. 1 Analysis of melanocytic nevus and malignant melanoma by array comparative genomic hybridization (CGH), there are no aberrations seen in a melanocytic nevus. Several copy number gains and losses are found in the melanoma.

CGH has been and is currently the most widely used tool clinically for the workup of diagnostically ambiguous/problematic spitzoid melanocytic proliferations and is also applicable to non-spitzoid lesions (e.g., atypical blue nevus-like, sclerosing, or deep penetrating nevus-like melanocytic proliferations). It can be applied to archival material (formalin-fixed and paraffin-embedded tissue). If a spitzoid lesion harbors a copy number gain of 11p, for example, the diagnosis of Spitz nevus can be made with reasonable confidence.17 and 22 On the other hand, the detection of multiple copy number changes would represent strong evidence in support of a diagnosis of malignant melanoma.

Pitfalls
CGH-negative melanomas

While the vast majority of melanomas show chromosomal aberrations detectable by CGH, not all of them do. A rare melanoma may be CGH negative. This may be related to test sensitivity or to intrinsic biologic reasons. Test sensitivity is an issue when, for example, the quality or amount of extracted DNA is insufficient or melanocytes with chromosomal aberrations represent only a minor component of the entire tissue used for DNA extraction. Admixed precursor nevomelanocytes, stromal, or inflammatory cells may dilute the cytogenetically aberrant melanoma cells thereby leading to a false-negative CGH result. However, on rare occasion, even a uniform malignant tumor cell population may be CGH negative due to the lack of chromosomal copy number changes. I have seen a rare case of metastasizing and lethal melanoma with a negative CGH test result (no detectable copy number gains or losses).

Benign lesions with cytogenetic abnormalities

Although the presence of multiple gains and/or losses of different chromosomes argues against a diagnosis of a benign melanocytic nevus, the detection of a copy number change in a single chromosome should not be regarded as a definite proof for malignancy. A Spitz nevus or tumor, for example, may harbor a gain in 11p 22 or loss of chromosome 3.24 and 25 Other isolated copy number changes have also been found in spitzoid melanocytic proliferations lacking overt malignant features and behaving clinically in an indolent fashion. Thus, caution is necessary when one is confronted with an isolated aberration. Cytogenetic findings need to be evaluated on a case-by-case basis, taking into account the specific alteration present, the clinical setting, light microscopic findings, and at times also immunohistochemical and clinical findings (e.g., proliferative nodules in a congenital nevus).

A further limitation of CGH is differentiating heterozygous deletions from homozygous deletions. For example, a loss of a single copy of 9p21 can be seen not only in melanoma but occasionally may also be seen in melanocytic nevi or borderline tumors with indolent behavior (K.J. Busam, personal observations). Alternatively, homozygous deletion of 9p21 tends to be a much more specific finding for melanoma 26 ( Fig. 2 ). This distinction is not always readily apparent by CGH. Hence, detection of a single copy number aberration including loss of 9p21 should be interpreted with caution, and it should be investigated further by fluorescence in situ hybridization analysis. 26

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Fig. 2 Spitzoid melanoma: (A) silhouette of a spitzoid melanocytic proliferation; (B) the tumor cells are atypical and lack maturation; (C) CGH reveals loss on chromosome 9; and (D) FISH reveals homozygous loss of 9p21 (only the centromeric probe in aqua color is seen; there should be two additional red signals per nuclei in normal cells).

Fluorescence in situ hybridization (FISH) analysis

Basics

Fluorescence in situ hybridization (FISH) targets individual chromosomes or specific regions within a chromosome. Fluorescence-labeled oligonucleotide probes bind to their complementary DNA sequence and label that region, which can then be visualized under a fluorescence microscope ( Fig. 3 ). Two types of probes are currently relevant for the workup of melanocytic tumors. Centromeric probes that identify the centromeric region of a specific chromosome and thus help in enumerating the number of copies of that chromosome, and allele-specific probes that adhere to a specific target sequence such as cyclin D1 on chromosome 11q or CDKN2A (also known as p16 by its protein name) on 9p21. 23

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Fig. 3 Analysis of melanocytic nevus and malignant melanoma by fluorescence in situ hybridization (FISH). The probes include the following: (1) RREB1 (ras-responsive element-binding protein 1) on 6p25 (Vysis LSI RREB1-SpectrumRed), (2) MYB (V-myb myeloblastosis viral oncogene homolog) on 6q23 (Vysis LSI MYB-SpectrumGold), (3) CCND1 (cyclin D1) on 11q13 (Vysis LSI CCND1-SpectrumGreen), and (4) the centromeric enumeration probe control for chromosome 6 (Vysis LSI CEP6-SpectrumAqua). (A) There are two signals per probe in the cell nucleus of a nevomelanocyte, indicating a normal diploid set of chromosomes. (B) Multiple copy number changes are seen in the majority of melanoma cells.

An advantage of FISH is that it permits detection of abnormal subpopulations within a heterogeneous tissue mixture. 21 Hence, identification of much smaller populations of emerging clones of chromosomally aberrant cells is possible compared to CGH. Visual correlation between a cell nucleus with an abnormal number of chromosomes and cytologic features is possible, which permits the verification of the identity of an affected cell. Only small amounts of tissue are necessary for FISH. For skin biopsies, for example, one or, depending on the number of probes being tested, two to three unstained 5-μm thin sections of formalin-fixed and paraffin-embedded material is usually sufficient.

There are two major drawbacks of the FISH test. First, it only tests for aberrations in the targeted areas, which is usually limited to four chromosomal loci, i.e., it does not analyze the entire set of chromosomes as CGH does. Second, depending on the probe set utilized, the enumeration requires some level of expertise. 21 For example, with the current commercially available melanoma probe set that targets three loci on chromosome 6 and one on chromosome 11, tetraploidy can often result in the false impression of copy number gains. Newer probe sets testing a broader number of chromosomal loci may help diminish this problem.

There are a number of FISH assays currently available for us in dermatopathology. The so-called “melanoma FISH test” uses four probes targeting ras-responsive element-binding protein 1 (Vysis®LSI® RREB1-SpectrumRed), myeloblastosis (Vysis®LSI® MYB-S Gold), cyclin D1 or chromosome 11q (Vysis®LSI® CCND1-SpectrumGreen™), and centromeric enumeration probe control for chromosome 6 (Vysis®LSI® CEP6-SpectrumAqua) from Abbot Molecular, Inc. 23 The enumeration protocol requires that 30 lesional melanocytes should be analyzed per case. A lesion is considered as having a positive FISH result if any of the following criteria are met: (1) gain in 6p25 (RREB1) relative to CEP6 greater than 55%, (2) gain in 6p25 (RREB1) greater than 29%, (3) loss in 6q23 (MYB) relative to CEP6 greater than 42%, or (4) gain in 11q13 (CCND1) greater than 38%. 23 Variations in the cutoff values have been suggested (Kutzner, 2012). Additional probes are currently being incorporated into clinical utility, such as probes targeting 9p21, which are useful for the diagnosis of both conventional and spitzoid melanomas, 26 and probes targeting 8q24, 27 which are useful for nodular amelanotic and nevoid melanomas.

Other FISH probes that are of interest for the diagnosis of melanoma include probes targeting chromosome 3 (centromeric probe and bcl2) for uveal melanoma 28 and 22q21 (EWSR1) for the diagnosis of cutaneous clear cell sarcoma 29 as well as miscellaneous other probes for the distinction of melanocytic tumors from soft tissue tumors.

Clinical applications
Distinction of nevus from melanoma

Because conventional nevi tend to lack chromosomal aberrations, but the majority of melanomas harbor copy number changes, a FISH test, using probes for chromosomes commonly altered in melanoma, should have potential value as an adjunct test for the distinction of nevi from melanomas. Gerami et al. documented that such a test could be established. Using probes targeting loci on 6p, 6q, 6cent, and 11q, they determined cutoff values (see above), which permitted a test sensitivity of 85% and specificity of 95% for histopathologically obvious/non-controversial benign and malignant lesions. 16 Other investigators have reported similar results FISH analysis by Vergier et al. 30 of 43 non-equivocal melanomas and nevi revealed a sensitivity of 85% and specificity of 90%. In a review of 500 cases from a commercial laboratory, 83.8% of melanomas were FISH positive. 31 Another commercial laboratory analyzed 32 nevi and 31 melanomas by FISH and reported an overall sensitivity of 94% and specificity of 94%. 32 In a recent study from Memorial Sloan-Kettering Cancer Center, test specificity was 98% and sensitivity was 82%. 33

A number of studies confirmed that the FISH test correlates well with consensus diagnoses separating conjunctival nevus from conjunctival melanoma, 34 sclerosing nevus from desmoplastic melanoma, 35 and blue nevus from blue nevus-like melanoma. 36 The test has also been used for the diagnosis of Spitz nevi.37 and 38 While Spitz nevi usually show negative result, a significant subset of Spitz nevi is tetraploid, 38 which may lead to a false-positive enumeration (see below). For the distinction of Spitz nevus from spitzoid melanoma, the addition of a probe for 9p21 (CDKN2A) has been shown to increase sensitivity and specificity. 26 In fact, a more recently validated probe set targeting 6p25, 11q13, 8q24, and 9p21 has been shown to have an overall improved sensitivity and specificity in distinguishing melanoma from benign nevi, with the probe targeting 9p21 being particularly helpful in spitzoid neoplasms. 26

Microstaging of primary melanoma

When a melanoma arises in association with a melanocytic nevus, precise assessment of tumor thickness can be problematic. If melanoma cells are distinctly different in appearance from adjacent nevus cells, measurement of Breslow thickness is straightforward. However, thickness may be wrongly measured, i.e., overestimated or underestimated, if the melanoma does not contrast well cytologically with the nevus cells and the pathologist is uncertain regarding how much of the intradermal melanocytic proliferation is nevus versus invasive melanoma. This does not only affect the patient's overall prognosis but may also impact immediate surgical management and staging, for example, eligibility for sentinel lymph node biopsy. Using the FISH assay may help in this regard by identifying intradermal melanoma cells with chromosomal gains. 39

Primary nevus versus nevus-like metastatic melanoma

Metastatic melanoma can on occasion simulate the appearance of a melanocytic nevus. An example is a blue nevus-like melanoma metastasis from primary cutaneous or ocular melanoma. Using the four-color FISH test, Gerami et al. documented that detection of chromosomal changes was found in 9 of 10 blue nevus-like melanoma metastases but not in a single case of conventional blue nevus. 36 Thus, a positive FISH test result in this setting can confirm malignancy. However, clinical and pathologic correlation is required for staging. The FISH test per se cannot distinguish primary from metastatic melanoma.

While the conventional melanoma FISH test is suitable for confirmation of metastatic cutaneous melanoma, a different probe set is needed for the detection of blue nevus-like uveal melanoma. Since monosomy 3 and amplifications of MYC are common findings in metastasizing uveal melanoma, FISH probes targeting chromosomes 3 and 8 may be useful in establishing a diagnosis of metastatic blue nevus-like uveal melanoma. Caution is necessary, however, since cutaneous melanoma as well as a subtype of Spitz nevi may be associated with loss of chromosome 3. Furthermore, cutaneous melanomas may also have 8q24 gains, particularly amelanotic nodular melanomas, in areas of non-chronically sun-damaged skin. Although the majority of metastatic melanomas will show copy number changes detectable by the FISH test, not all metastatic lesions do. 33

Nodal nevus versus metastatic melanoma

Melanoma may also be confused with a melanocytic nevus in the lymph node with serious implications for prognosis (stage) and further management. 40 Although conventional microscopic analysis and immunohistochemical findings usually suffice to distinguish a nodal nevus from metastatic melanoma, it has be shown that the FISH test can have value for this diagnostic problem. A major limitation of using the test is the fact that the distinction of nevus from melanoma is most problematic when only very few melanocytes are available for routine light microscopic review and/or immunohistochemical studies. Not uncommon is that fact that there is insufficient tissue remaining for analysis of the melanocytes by FISH.

Histopathologically ambiguous melanocytic tumors

When the FISH test was developed, the main purpose was to use it eventually as an ancillary tool for cases that were difficult to solve by morphologic analysis alone, i.e., cases with unusual or conflicting morphologic features, where some features (e.g., circumscribed wedge-shaped silhouette) favor a nevus, while others (e.g., mitotic figures, incomplete maturation, and cytologic atypia) suggest a possible melanoma. This is the group of melanocytic tumors generally referred to as “melanocytic tumor of uncertain malignant potential,” 41 “borderline tumors,” or referring to more specific findings “atypical Spitz tumors”9 and 42 or “atypical cellular blue tumors.” 11 Most of the ambiguous lesions for which the FISH test has been used fall into the category of “atypical Spitz tumors.” In selected cases, the use of the FISH test, if a clearly positive result is obtained, can settle diagnostic controversy and permit reclassification of an atypical Spitz tumor as “spitzoid melanoma” ( Fig. 4 ).

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Fig. 4 Spitzoid melanoma: (A) silhouette of a pigmented Spitz tumor; (B) junctional nest of pigmented fusiform melanocytes; (C) mitotic figures at the base of the lesion; (D) regional lymph node metastasis; and (E) positive FISH test.

The experience of various investigators in using the current commercially utilized melanoma FISH test for diagnostically ambiguous melanocytic neoplasm has been variable. Gerami et al. examined a set of 27 ambiguous melanocytic tumors. FISH test results correlated well with outcome: all six tumors, which metastasized, showed FISH positive results. 16 Groups led by Vergier 30 and Massi 7 found similar utility in the test. In contrast, Raskin et al. 42 and Gaiser et al. 43 had a less successful experience in their utilization of the FISH test. Gaiser reported a specificity of the test of only 50% for metastasis and a sensitivity of 60%. However, their FISH methodology differed significantly from that of Gerami et al. Furthermore, when evaluating the specificity of the FISH test, it is important to take into account what parameter the test result is compared to (histopathologic consensus diagnosis versus adverse event). For example, metastasis is not the only valid parameter of malignancy. A melanoma may be unequivocally malignant but can be cured by surgical removal. Thus, failure to metastasize during a given follow-up period does not imply a tumor is benign. Otherwise, histopathologic analysis would have low specificity, since the majority of melanomas judged clinically and by conventional light microscopic criteria as malignant do not metastasize.

A major limitation of the studies assessing the diagnostic value of the FISH assay is the limited number of documented patients with ambiguous melanocytic neoplasms resulting in distant metastasis or death of the patient. For years it has been recognized that isolated lymph node involvement without disease progression could be seen in Spitz tumors. More recently, Ludgate et al. 9 demonstrated in their experience of 67 cases of ambiguous Spitz tumors with follow-up that lymph node involvement was seen in 47% of their cases and that none of these cases developed distant metastasis or death. Hence, a paramount objective of any FISH assay for diagnosis of ambiguous melanocytic neoplasms is to be able to successfully identify and discriminate those cases that are capable of and most likely to result in distant metastasis or death and whether or not it can do this better than light microscopic analysis alone.

Further improvements in the efficiency and diagnostic utility of FISH may develop as tailored probe sets emerge. For example, it has been recently recognized that in cases with a concern for nodular amelanotic/nevoid melanoma in areas of non-chronically sun-damaged skin, a probe targeting 8q24 is highly useful, while in spitzoid neoplasms, 9p21 is highly pertinent.26 and 27

Prognosis of melanoma

Preliminary evidence has also been presented that the FISH status of a melanoma is prognostically relevant. North et al. 44 analyzed 144 primary melanomas with a minimal tumor thickness of 2 mm and compared the development of metastatic disease and melanoma-specific mortality as well as relapse-free and disease-specific survival between FISH-positive and FISH-negative cases. The risk of metastasis or melanoma-related death was higher for patients with FISH-positive primary tumors compared to FISH-negative cases. The FISH status remained prognostically significant even after adjustment for other known prognostic parameters. In another case–control study of 97 melanomas (55 metastasizing and 43 non-metastasizing tumors), Gerami et al. 27 documented that copy number gains in 11q13 and 8q24 were predictive of metastatic disease. Interestingly, independent studies have shown that 8q24 has prognostic value in uveal melanomas as well. Thus as in uveal melanomas, cytogenetic analysis of cutaneous melanomas may assist in a more accurate identification of tumors with metastatic potential. However, further studies are needed to determine whether any of such prognostic refinements have an impact on current clinical management. Moreover, limitations in the prognostic value of the FISH test are already apparent. Patients with a FISH-negative melanoma may die of metastatic disease, while others with FISH-positive melanoma may survive their melanoma for 5 years or longer. 33

Diagnosis and prognosis of uveal melanoma

Uveal melanoma (UM) is the most common extracutaneous melanoma. Nearly half of all patients with UM eventually die of metastatic disease. Non-random chromosomal alterations are present in the majority of cases.45 and 46 The most common characteristic and prognostically powerful aberration is monosomy 3, which is seen in approximately half of the cases of uveal melanoma.28 and 47 Copy number gains of 8q (MYC) are also not uncommon and appear prognostically relevant. 48 Accordingly, FISH analysis of uveal melanomas for monosomy 3 has become part of the workup of patients at tertiary care centers. Although the presence of monosomy 3 is typical of uveal melanoma, it is not specific. Correlation with clinical and other histopathologic findings is needed to distinguish uveal from cutaneous melanoma or nevi, occasional cutaneous melanomas or rare nevi may also show loss of chromosome 3. 25

Diagnosis of melanoma of soft parts (clear cell sarcoma)

Clear cell sarcoma (CCS), also known as melanoma of soft parts, is a unique malignant neoplasm initially described by Enzinger in 1965. 49 It is generally classified as “sarcoma” because of its typical presentation as a tumor nodule in association with tendons and aponeuroses of distal extremities. However, the tumor shows evidence of melanocytic differentiation by light and electron microscopy as well as immunohistochemically. 50 Cytogenetic studies have shown that most tumors harbor a characteristic reciprocal translocation, t(12;22)(q13;q12), which to date has not yet been found in cutaneous or mucosal melanomas. In most cases of CCS, the gene fusion product involves the EWS (22q12) and ATF1 (12q13) genes. However, another fusion product of EWS to CREB1 on 2q13 has recently been found in a subset of tumors. 51

Although the deep location of the tumor, its predilection for adolescents and young adults, and some fairly characteristic histopathologic features (e.g., the presence of multinucleate wreath-like giant cells, cells with clear cytoplasm, and sclerotic stroma) usually suffice to distinguish it from primary nodular or metastatic melanoma, a rare tumor of CCS may involve or even be centered in the dermis thereby leading to possible diagnostic confusion (primary or metastatic melanoma or monophasic cellular blue nevus). In problematic cases, FISH analysis, for example, by using 22q12 (EWS) and 12q13 (ATF1) probes and documenting the presence of a translocation, can provide a decisive diagnostic result 29 ( Fig. 5 ).

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Fig. 5 Melanoma of soft parts (clear cell sarcoma). (A) Amelanotic spindle cell proliferation on the lip of a 13-year-old boy. (B) Fascicles of melanocytes with focal clear cell changes. (C) Positive labeling of the tumor cells for S100 protein: FISH confirms translocation of ATF1 (D) and EWSR1 (E).

Pitfalls and limitations of the FISH test
FISH-negative malignant melanomas

As documented in most studies using FISH as a diagnostic tool for melanocytic tumors, not all malignant melanomas are FISH-positive.33 and 52 An example of a FISH-negative melanoma is illustrated in Fig. 6 . A negative FISH test in spite of an unequivocally malignant tumor by morphology with documented adverse outcome may be due to the fact that the melanoma may have copy number changes of chromosomes other than those targeted by the assay. Furthermore, not all melanomas need to be associated with copy number changes. Thus, one needs to be cautious when cytogenetic results are used for the interpretation of difficult melanocytic lesion. Although the inclusion of additional probes, such as 9p21, increases the sensitivity of the test,26 and 53 a negative FISH result does not exclude the diagnosis of malignant melanoma.

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Fig. 6 (A) Lentigo maligna melanoma with a 2.2-mm thick invasive melanoma. (B) The invasive melanoma is epithelioid in appearance and involves a peripheral nerve trunk. (C) The FISH test result (performed for investigational purposes) was negative (normal).

FISH-positive melanocytic nevi

The less than perfect specificity of the FISH test implies that some nevi are reported as FISH-positive.16, 33, and 52 This is likely due to a combination of incorrect interpretations of the test as well as a true biologic phenomenon resulting in less than perfect diagnostic criteria. 52 One pitfall with regard to a false-positive FISH result is polyploidy. For example, it has been documented that a minority of Spitz nevi are tetraploid.37 and 38 Thus, correction for tetraploidy may be necessary for the interpretation of a FISH result. Tetraploidy is apparent when there are four copies per nucleus of any chromosome tested. The issue of whether to discount tetraploid nuclei, however, is not always straightforward, especially when confronted with a mixture of signals, including gains of three and four copies, and when outside material is sent with no specifications on how thick the sections are (truncated tetraploid nuclei may falsely appear to have only three copies of a chromosome). Although tetraploidy may be the most common reason for false-positive FISH results of melanocytic nevi, it does not explain all cases of melanocytic nevi with copy number changes meeting FISH criteria for melanoma. 33 A common interpretative error leading to a (false) positive FISH result of a nevus is related to “cherry-picking” of abnormal nuclei (large nuclei at different foci in a lesion are selectively counted, instead of counting all nuclei in a given area). However, it may happen that a positive FISH result is documented for a histopathologically and clinically clearly benign melanocytic without any methodological flaw in how the FISH test is performed and enumerated. 33 Such a case simply reflects the fact that some numerical chromosomal aberrations indeed occur in benign lesions, thereby setting inherent biological limits to the specificity of empirical threshold or cutoff values. The basic principle that a chromosomal copy number gain can be found in a benign melanocytic proliferation is already accepted: it is known that a subtype of Spitz nevus may have an isolated copy number increase of chromosome 11p where the HRAS gene is located and the majority of these cases will also have an activating mutation in HRAS.14 and 54 Another Spitz nevus/tumor composed of large epithelioid cell melanocytes has been found to be associated with loss of BAP1. 24 There are also reports of partial loss of chromosome 9p21 in melanocytic nevi.

Mutational analysis of melanocytic nevi and melanoma

A number of mutations have been identified in melanoma.55, 56, 57, 58, and 59 Most of them are point mutations. Somatic and genomic aberrations have been found in genes such as BRAF, NRAS, KIT, GNAQ/GNA11, PTEN, and MAP2K1/2. Some of these mutations are not only restricted to malignant melanoma but can also be detected in melanocytic nevi ( Table ).

Table Mutations in melanocytic nevi and malignant melanoma.

Mutation Nevus type Melanoma type
BRAF Acquired nevi (ordinary and “atypical”/“dysplastic”) Usually superficial spreading melanoma a
NRAS Congenital nevi Usually nodular melanoma a
HRAS Spitz nevi  
GNAQ/GNA11 Blue nevi Blue nevus-like melanoma
BAP1 Epithelioid Spitz nevi Uveal melanoma
KIT   Subset of acral and mucosal melanomas and melanomas of sun-damaged skin

a Mutation is not restricted to one melanoma subtype.

Testing for genetic changes

Gene mutations are detected by the determination of the order of the nucleotides adenine, guanine, cytosine, and thymine. There are different analytical methods available for mutation analysis. A technical discussion of different methods goes beyond the scope of this review. Current commonly used methods for mutation analysis in solid tumors such as melanoma include direct sequencing (traditional chain-termination, also known as Sanger sequencing), pyrosequencing, or single nucleotide extension assays. 59 The advantage of direct sequencing is that it can identify all point mutations in a given stretch of DNA. However, it is less sensitive than other methods. For Sanger sequencing, for example, the mutant DNA needs to amount on average to at least 25% of the sample DNA, while in pyrosequencing, mutations can be detected even when the mutant DNA constitutes only 5% of the total DNA. Pyrosequencing uses an enzymatic assay to detect pyrophosphate after nucleotide incorporation into a growing DNA chain. It permits mutation analysis in a read length of 300–500 nucleotides. Single nucleotide extension assays are used for detecting a specific point mutation. Two commonly used techniques include iPlex (Sequenom, Inc., San Diego, CA) and SNaPshot (Applied Biopsystems, Inc, Foster City, CA). Roche has a real-time PCR-based assay for detecting BRAFV600E mutations known as the Cobas 4800, which is the only FDA-approved test, and it is the companion test to go along with their FDA-approved BRAF-inhibitor drug vemurafenib. 60

Common mutations in melanoma
BRAF

BRAF encodes a serine/threonine protein kinase downstream of the epidermal growth factor receptor (EGFR) and the RAS family of small G-proteins. It is mutated in approximately 8–10% of human tumors and 40–60% of melanomas.57 and 60 Many different BRAF mutations have been identified. Most melanomas have a V600 mutation. Among them, 70–85% are V600E mutations, in which a thymidine at nucleotide 1799 on exon 15 is replaced by adenine, resulting in the substitution of glutamic acid for valine at residue 600. This mutation leads to greater than 10-fold activity of the kinase domain and subsequent downstream activation of the MAP kinase pathway. Another not infrequent mutation is V600K. In this mutation, adenine is replaced by lysine. The incidence of the V600K mutation ranges from 5% to 30% of patients with BRAF mutations.61, 62, and 63 The highest rates have been reported in Australia. 61 BRAVFV600 mutations among primary tumors tend to be associated with melanomas occurring in areas of intermittently sun-damaged skin such as on the trunk or extremities. Pyrosequencing, nucleotide extension assays, as well as the Cobas 4800 real-time PCR assay are commonly used methods to determine BRAF mutation status. Recently, a monoclonal antibody (VE1) has become available, which recognizes mutant BRAFV600E ( Fig. 7 ) 64 . It has been found to be highly sensitive and specific for the detection of BRAFV600E in formalin-fixed and paraffin-embedded tissue of metastatic melanoma. 65 Current studies are underway to determine the potential clinical utility of this antibody. It will likely be helpful in establishing BRAF mutation status for small tumor cell volume in a mixed cell background (e.g., few isolated tumor cells in a lymph node).

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Fig. 7 Metastatic melanoma: (A) hematoxylin and eosin-stained sections shows extensive involvement of the lymph node and (B) the melanoma is strongly and diffusely positive for BRAFV600E (red chromogen, using the antibody VE1).

NRAS mutations

NRAS is an isoform of the RAS family of GTPase proteins involved in cell growth and differentiation. NRAS affects a number of different pathways, including MAP kinase. Activating mutations of NRAS have been identified in 15–20% of melanoma samples. 59 The most common mutations are located in exon 2 at codon 61. NRAS mutations tend to occur in nodular melanomas and melanomas of sun-damaged skin. 66 They are usually mutually exclusive of BRAF mutations. NRAS mutations are also the predominant mutation identified in large congenital nevi. 67 Pyrosequencing and nucleotide extension assays are commonly used to test for NRAS mutations.

GNAQ/GNA11 mutations

GNAQ and GNA11 encode for alpha subunits of G-protein-coupled receptors. Mutations result in constitutive activation. Downstream pathways include MAP kinase and PLC/PKC. Van Raamsdonk et al. found somatic mutations in GNAQ or GNA11 in 83% of uveal melanomas. 68 They are mutually exclusive. The most common mutation affects exon 5 (Q209). GNAQ and GNA11 mutations have also been identified in blue nevi and blue nevus-like melanomas.55 and 69

KIT mutations

KIT is a cell surface receptor tyrosine kinase that is involved in a number of intracellular signaling pathways, including MAP kinase and PI3K. Kit mutations tend to occur in approximately one-third of acral and mucosal melanoma, 70 but can also be found in a minor subset of melanomas arising on chronically sun-damaged skin. Mutations have been found in exons 9, 11, 13, and 17. There is no predominant mutation. Therefore, molecular testing for KIT mutations must evaluate multiple regions of the gene. Traditional Sanger sequencing is often used for this purpose.

Mutations in melanocytic nevi

Except for KIT mutations, all of the above-mentioned mutations can also be found in melanocytic nevi. 71 BRAFV600 mutations predominantly occur in acquired melanocytic nevi, but have also been reported in a subset of congenital, Spitz, and blue nevi. The recent availability of immunohistochemical reagents to confirm BRAFV600E mutation status suggests that BRAF mutant nevi are clonal, since they label uniformly with the antibody VE1. 72 NRAS mutations are predominantly found in congenital melanocytic nevi, especially medium and large congenital nevi.67 and 73GNAQ/GNA11 mutations have been found in blue nevi of various types (common, cellular, sclerosing, and plaque type) as well as nevus of Ota. 55 Other mutations that have been reported to occur in nevi include HRAS, another member of the RAS family. 54 The most common mutation involves Q61 of exon 3, with replacement of glutamine by lysine. HRAS mutations predominantly affect Spitz nevi. 14 However, less than 20% of Spitz nevi carry HRAS mutations, and this tends to be limited to large bulky Spitz nevi with sclerosis of the deep dermal component. 54 Additionally, on rare occasion, an HRAS mutation may be found in a melanoma.

Clinical utility of mutation analysis

The identification of distinct genetic changes has led to the development of targeted therapies. The main clinical role of mutation analysis is for selection of patients for treatment to which they are most likely to respond. Vemurafenib, for example, is a targeted therapy against BRAFV600 mutant melanomas. 60 It has been approved by the FDA for the treatment of patients with metastatic melanoma known to have the V600 mutation. 60 In limited studies, patients with KIT mutated melanomas have shown responses to treatment with imatinib 70 or other tyrosine kinase inhibitors. 74

However, mutation analysis is not only relevant for treatment selection. It may also be useful for diagnostic purposes. For example, the detection of a GNAQ/GNA11 mutation in a sclerosing melanocytic tumor with a differential diagnosis of amelanotic sclerosing blue nevus versus desmoplastic melanoma would support the diagnosis of a blue nevus. 69 Likewise, the detection of GNAQ/GNA11 mutation may be helpful for the distinction of a pauci-melanotic blue nevus from a pigmented dermatofibrosarcoma protuberans. Mutation analysis may also be helpful for staging purposes, for example, in a patient with a history of acral melanoma and new tumor with a differential diagnosis of epidermotropic metastasis versus second primary superficial spreading melanoma. If the primary acral melanoma was KIT positive and the new tumor KIT negative, but BRAF mutated, this would be a clinically valuable information.

Mutation analysis may also have prognostic value. NRAS mutation status has recently been suggested to predict shorter survival after a diagnosis of stage IV melanoma. 58

Conclusion

Cytogenetic and mutation analyses have led to improved understanding of the biology of melanocytic tumors and the development of new diagnostic and therapeutic opportunities. Some of the tests, such as the documentation of EWS rearrangement for CCS, have become diagnostic gold standard, or others, such as BRAF mutation analysis, have become routine in the workup of patients with advanced melanoma for treatment selection. CGH and FISH analysis are increasingly being used at specialized centers as ancillary tools for the workup of diagnostically problematic melanocytic tumors. The field is rapidly advancing. There are issues remaining regarding test sensitivity and specificity for diagnostically ambiguous tumors as more experience is gained with these novel techniques. An important issue is also a cost–benefit analysis, i.e., to what extent the added cost from using these tests can be justified in a health care system with limited resources and conflicting needs.

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Footnotes

Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, New York 10065