Journal Information
Vol. 108. Issue 1.
Pages 17-30 (January - February 2017)
Share
Share
Download PDF
More article options
Visits
8961
Vol. 108. Issue 1.
Pages 17-30 (January - February 2017)
Review
Full text access
Use of New Techniques in Addition to IHC Applied to the Diagnosis of Melanocytic Lesions, With Emphasis on CGH, FISH, and Mass Spectrometry
Empleo de nuevas técnicas complementarias a la IHQ para diagnóstico de lesiones melanocíticas, con énfasis en HGC, FISH y espectrometría de masas
Visits
8961
P. Nagarajan, M.T. Tetzlaff, J.L. Curry, V.G. Prieto
Corresponding author
Vprieto@mdanderson.org

Corresponding author.
Department of Pathology, Unit 85, University of Texas MD Anderson Cancer Center, Houston, TX 77030, United States
This item has received
Article information
Abstract
Full Text
Bibliography
Download PDF
Statistics
Figures (9)
Show moreShow less
Abstract

Melanoma remains one of the most aggressive forms of cutaneous malignancies. While its diagnosis based on histologic parameters is usually straight forward in most cases, distinguishing a melanoma from a melanocytic nevus can be challenging in some instances, especially when there are overlapping clinical and histopathologic features. Occasionally, melanomas can histologically mimic other tumors and even demonstration of melanocytic origin can be challenging. Thus, several ancillary tests may be employed to arrive at the correct diagnosis. The objective of this review is to summarize these tests, including the well-established and commonly used ones such as immunohistochemistry, with specific emphasis on emerging techniques such as comparative genomic hybridization, fluorescence in situ hybridization and imaging mass spectrometry.

Keywords:
Melanoma
Diagnosis
Immunohistochemistry
Comparative genomic hybridization
Fluorescence in situ hybridization
Mass spectrometry
Resumen

Los melanomas continúan siendo unas de las neoplasias cutáneas más agresivas. Si bien su diagnóstico por medio de parámetros histológicos suele ser sencillo en la mayoría de los casos, la distinción entre un melanoma y un nevo melanocítico puede suponer un reto en ocasiones, sobre todo cuando las características histopatológicas y clínicas se solapan. A veces los melanomas pueden imitar histológicamente a otros tumores, e incluso demostrar el origen melanocítico resulta complicado. Por tanto, se deben realizar varias pruebas complementarias para alcanzar el diagnóstico correcto. El objetivo de la presente revisión es resumir dichas pruebas, entre las que se incluyen algunas de uso habitual como la inmunohistoquímica, enfatizando de manera específica en las técnicas emergentes como la hibridación genómica comparativa, la hibridación in situ con fluorescencia y la espectrometría de masas.

Palabras clave:
Melanoma
Diagnóstico
Inmunohistoquímica
Hibridación genómica comparativa
Hibridación in situ con fluorescencia
Espectometría de masas
Full Text
Introduction

Accurate diagnosis of melanoma is the most important prerequisite for optimal clinical management. Examination of hematoxylin and eosin (H&E) stained sections using conventional microscopy remains the gold standard for diagnosis of melanocytic lesions and as such most of the criteria used in the American Joint Committee on Cancer (AJCC)/College of American Pathologists (CAP) recommendations for reporting specimens with melanoma are based on routine examination of histologic slides.1 However, when lesions exhibit architectural and cytologic features that overlap both melanocytic nevi and melanomas, histologic examination alone may be insufficient for diagnosis. Being one of the ultimate histologic mimickers, melanoma may present in a range of histologic variants with spindle cell, desmoplastic, clear cell, balloon cell, signet ring cell, small cell, plasmacytoid and rhabdoid phenotypes.2–4 Rarely, even establishing melanocytic lineage of a tumor may become a challenging task, especially when the tumor cells are poorly differentiated and resemble multiple other tumors such as carcinoma, lymphoma, neuroendocrine carcinoma, angiosarcoma, fibrohistiocytic, and small round blue cell tumors.5 The objective of this article is to review the various ancillary tests that are available to pathologists that assist in the diagnosis of melanocytic lesions as well as their possible application to determine prognosis and therapy.

Immunohistochemistry

Immunohistochemistry (IHC) is arguably the most commonly employed ancillary test in pathology. This staining technique is used to detect the presence or absence of an antigen usually a protein.6,7 Introduced in the early 1940s, the current IHC protocols utilize specific antibodies tagged with a visible label or chromogenic agent against the target molecule.8 The presence of the antigen is revealed by serial amplification of the initial signal, thus enabling visualization of the protein and its distribution within the various cellular and extracellular components of the tissue as well as within the subcellular compartments.

Diaminobenzidine (DAB) is the most commonly used chromogen, which produces a brown precipitate. However, in heavily pigmented lesions, distinguishing the immunoreaction from melanin pigment can be challenging; therefore, other chromogens may be used including fast red or 3-amino-9-ethylcarbazole (AEC) or Texas red. An alternate approach would be to use a counterstain other than hematoxylin such as azure B or Giemsa to convert the brown pigment of melanin to green.9 Such method allows distinction between the brown DAB and the now green-colored melanin. Bleaching of melanin using potassium permanganate solution may affect the antigenicity of some epitopes and thus, affect immunodetection of certain markers.9 However, bleaching using hydrogen peroxide may be a better alternative.10 But we recommend avoiding such techniques.11

Though many of the common melanocyte specific markers are useful in the diagnosis of melanocytic lesions and primary melanomas,11,12 their expression may be lost focally or diffusely as the disease progresses, particularly in metastatic lesions. In addition, there might be acquired variable and aberrant expression of non-melanocytic markers such as cytokeratins, and neuroendocrine markers.13–15 This altered immunohistochemical profile may be accompanied by unusual tissue morphology as well. Therefore, one must exercise caution when examining a poorly differentiated neoplasm in a patient with prior history of melanoma. The following section will summarize commonly used diagnostic and prognostic markers of melanoma, not including the less commonly utilized markers such as PNL2, KBA.62, SM5-1, MC1R, NKI/C3, CSPG4 and CD146.5,16,17

Markers of melanocytic differentiationS100B

Since its expression in melanoma cell lines was documented 35 years ago, S100B, also referred to as just ‘S100’, is the most analyzed marker of melanocytic differentiation.18–21 It is a member of the S100 family of calcium binding heteromeric proteins that is expressed in a variety of tissues including melanocytes.22 In addition to being expressed intracellularly, and thus, being detectable by IHC, S100 is also secreted in the serum.23 The sensitivity of S100 in melanomas is very high in formalin-fixed tissues and thus, more than 90% of all primary melanomas, including desmoplastic variants, express S100 protein.24,25 However, its expression may be lost in metastases as well as in some spindle cell/desmoplastic melanomas.26,27 Also, since the specificity of S100 is not high, it is advisable to use it in conjunction with other melanocytic markers. Regarding tissue processing, S100 protein is soluble in organic solvents such as acetone and alcohol. Therefore, frozen tissue sections and cytology preparations utilizing alcohol based fixatives are likely to yield false negative results and are not recommended for S100 IHC.

HMB45

Human Melanoma Black-45 (HMB-45) is a mouse monoclonal antibody that recognizes gp100 or Pmel17, a premelanosome protein,28,29 which makes HMB45 very specific marker for melanocytic proliferations. Expression of HMB45 is directly proportional to melanin synthesizing capacity of the cells; therefore, melanocytes of blue nevi and some Spitz nevi exhibit uniform staining throughout the lesion,30 whereas the maturing dermal melanocytes of banal compound and intradermal nevi progressively lose their HMB45 expression.12,31 Most of the primary epithelioid melanomas, on the other hand are typically characterized by patchy expression of HMB45, while a majority of the desmoplastic melanomas tend to be negative for HMB45.32,33 As such, diffuse expression through the lesion or limited to the intra- and periepithelial melanocytes is usually associated with a diagnosis of nevus while patchy expression is more likely to be seen in melanomas.

MART1

Melanoma antigen recognized by T cells-1 (MART1/Melan-A) was identified as one of proteins recognized by tumor-infiltrating lymphocytes.34 MART1 interacts with gp100 and plays an important role in trafficking of gp100 to premelanosomes.35 It is one of the most widely used markers of melanocytic differentiation, recognized by 2 clones of antibodies: M2-7C10 and A103.36,37 MART1 is expressed by almost all benign melanocytic lesions as well as by the majority of epithelioid melanomas.38,39 However, desmoplastic melanomas are predominantly negative for MART1.38 One of the drawbacks consistently seen with MART1 is the over-estimation of junctional melanocytes in chronically sun-damaged skin.40–42 This is due to localization of MART1 to melanosomes and the plasma membrane, in addition to the Golgi apparatus and other organelles and thus, to the dendritic processes of the melanocytes.43 Therefore, extreme caution must be employed while interpreting intraepidermal pigmented lesions studied solely with anti-MART1.42

Tyrosinase

Tyrosinase is a copper-containing metalloenzyme that catalyzes three reactions in the melanin synthetic pathway, including hydroxylation of tyrosine.44Tyrosinase gene encodes for peptides recognized by cytolytic T-cells in melanoma patients.45 Tyrosinase is expressed in almost all epithelioid melanocytic lesions in a finely granular pattern within the cytoplasm. Though most melanocytic nevi express tyrosinase, the intensity of staining may decrease gradually as the melanocytes descend into the deep dermis.46 It is expressed in most epithelioid melanomas, where the pattern can be patchy, but the labeling is frequently weak, focal or absent in melanomas with spindled melanocytes and in metastatic melanomas.47,48 In general, tyrosinase is comparable to HMB45 in terms of expression and usefulness in the diagnosis of melanocytic lesions.12

MITF

Microphthalmia-associated transcription factor (MITF) is a dimeric transcription factor that plays a seminal role in melanocyte development, proliferation, function and survival.49–51 Though expressed in several other cells such as macrophages, fibroblasts, smooth muscle cells, Schwann cells and mast cells, the nuclear localization of this protein offers a major advantage in the detection of melanocytes52. This is particularly useful when the lesion is densely pigmented. Melanocytic nevi, epithelioid melanomas as well as metastatic melanomas with epithelioid morphology express MITF, while spindled and desmoplastic melanomas are typically negative (Fig. 1A).53–55 One of principal uses of MITF is the accurate enumeration of melanocytes in intraepidermal melanocytic proliferations to distinguish between pigmented actinic keratosis from lentigo maligna (Fig. 1B).56,57

Figure 1.

Immunohistochemical study for microphthalmia-associated transcription factor (MITF). (A) Invasive melanoma diffusely positive nuclear labeling in melanocytic lesions with epithelioid melanocytes (magnification 200×). (B) Melanoma in situ, with contiguous proliferation and suprabasal spread of atypical melanocytes (magnification 200×).

(0.32MB).
SOX10

Sex determinant region Y (SRY) related HMG-box gene 10 (SOX10), is a transcription factor that binds to and transactivates MITF gene promoter and regulates the expression of tyrosinase-related protein-1 among others and plays a critical role in melanocyte growth and function.58,59 SOX10 is a nuclear marker that can be expressed in Schwann cells, eccrine epithelium, myoepithelial cells and glial cells.60,61 Similar to MITF, SOX10 is a useful marker for the distinction of pigmented actinic keratosis from melanoma in situ (Fig. 2A).57. It is also more sensitive than MITF in that almost all spindle cell melanomas and most desmoplastic melanomas express SOX10 (Fig. 2B)60,62,63.

Figure 2.

Immunohistochemical study for SOX10. (A) Melanoma in situ, with contiguous proliferation and suprabasal spread of atypical melanocytes (magnification 200×). (B) Desmoplastic melanoma with diffusely positive nuclear expression (magnification × 100).

(0.25MB).
p75NGFR

Nerve growth factor receptor (molecular weight: 75kDa), also known as neurotropin receptor and CD271 belongs to the tumor necrosis factor receptor superfamily and is widely expressed in the skin. Transit amplifying keratinocytes of basal layer, follicular outer root sheaths, myoepithelial cells, fibroblasts and nerve fibers express NGFR.64,65 It is not expressed in epithelioid type-A melanocytes; but is induced during neurotization and as such is expressed in melanocytes with spindled morphology (type-C). NGFR has a higher sensitivity for detection of spindle cell and desmoplastic melanomas and has been touted to be a better marker than S100 for spindle cell melanomas.27 In addition, other spindle cells tumors of sun-damaged skin are frequently negative for NGFR.66,67 However, caution must be exercised while using NGFR as a sole marker of desmoplastic or spindle cell melanoma, since it can be expressed in dermatofibrosarcoma protuberans and scars.68,69

Melanocytic marker cocktails

Sometimes, a mixture of 2 or more monoclonal antibodies against melanocytic markers is used in immunohistochemistry.70,71 Though many of these cocktails are generated and validated in individual laboratories, some are also commercially available.16 The main advantage of this is the increased sensitivity for detection of melanocytes. For instance, combination of HMB45 and anti-tyrosinase can be used to determine the number and growth pattern of intraepidermal melanocytes and for detecting the presence of a dermal component as well (Fig. 3). The main application of such cocktails is in establishing a melanocytic origin in a given neoplasm and in the detection of microscopic metastases in sentinel lymph nodes.

Figure 3.

Immunohistochemical study using melanocytic cocktail (HMB45 and anti-tyrosinase) highlights intraepidermal and dermal components of this superficial spreading melanoma (magnification × 100).

(0.32MB).
Prognostic markers of melanomaKi67

Marker of proliferation Ki67 is a nuclear non-histone protein that is expressed in actively proliferating cells (not in G0 phase).72 The proliferative activity, as indicated by the percentage of lesional cells expressing Ki67 could be extrapolated to determine the biologic behavior of the melanocytic lesion. In general, fewer than 5% (usually 1%) of melanocytes comprising most benign lesions expressed Ki67,12,73,74 with a decreasing gradient toward the deeper aspect, reflecting maturation of melanocytes with progressive dermal descent.75 In melanomas, the Ki67 proliferation index is higher, ranging from 5% to 50%, with mitotic figurees distributed throughout the lesion.75 In some cases, foci of increased proliferation may be seen. Increased Ki67 proliferation rate has been correlated with recurrence and metastasis, particular in thick melanomas.76–78 To help distinguish between proliferating melanocytes and other cells at our institution, the growth fraction is determined by adding a cytoplasmic melanocytic marker such as MART1/Melan-A/tyrosinase to anti-Ki67 (Fig. 4A). This combination is particularly useful in lesions with a brisk inflammatory infiltrate. In addition, Ki67 positivity of intraepidermal melanocytes is seen more commonly in in situ melanomas, compared to nevi.79

Figure 4.

Immunohistochemical studies for prognostic factors. (A) Mart-1/Ki67 cocktail highlights the melanoma cells (red, cytoplasmic) with increased nuclear positivity for Ki67 (brown, nuclear) (magnification 200×). (B) Mart-1/PHH3 cocktail highlights a mitotic figure (brown) in a melanoma (red, cytoplasmic) (magnification 200×). (C) MITF/D2-40 reveals the presence of lymphovascular (red) space invasion by melanoma cells (brown, nuclear) (magnification × 100).

(0.75MB).
pHH3

Identification of mitotic figures is an important staging parameter in thin melanomas (Breslow thickness1.0mm).80 Phosphorylation of histone H3 begins in the late G2 phase of cell cycle. Phosphohistone H3 (pHH3) can be detected throughout the entire M-phase and thus, is a marker of cells undergoing mitosis.81–83 pHH3 expression in dermal melanocytes has been correlated with outcomes in melanoma.84–87 While manually identifiable mitotic figures do not include cells in prophase, pHH3 detects these cells as well. The AJCC-TNM (2009) staging system is based on mitotic figures detected on routine, H&E-stained slides, and thus immunedetection of pHH3 should not be used in routine examination of primary melanomas. Anti-pHH3 antibody may also be combined with a cytoplasmic melanocytic marker such as MART1 to enhance detection of actively dividing melanocytes (Fig. 4B).84,88 At our institution we use such a combination of anti-MART1 and anti-pHH3 in cases where we identify cells undergoing mitosis but we are not certain if they correspond to a melanocyte or to other dermal cells (e.g., endothelial cells, lymphocytes, macrophages).

D2-40

D2-40 is a monoclonal antibody that recognizes podoplanin, a transmembrane mucoprotein that is selectively expressed in lymphatic endothelium.89,90 Presence of lymphovascular space invasion (LVI) by melanoma has been correlated with metastases to sentinel and regional lymph nodes, and decreased disease-free and overall survival.91–93 Detection of LVI by examination of H&E stained sections alone may be difficult since filling up of small vessels with tumor cells may lead to flattening of endothelium, while retraction artifacts and dyshesive properties of the tumor cells may mimic a vascular space.94 Therefore, the use of lymphatic and vascular endothelial markers has become a common practice, particularly in thick melanomas. Dual immunostaining using S100/D2-40 cocktail did not correlate with sentinel lymph node status.95 However, a recent study from our group revealed that detection rates of LVI were increased by using MITF/D2-40 cocktail with positive correlation with sentinel lymph node metastasis (Fig. 4C).96

BRAFV600E

Somatic missense mutations in BRAF, a cytoplasmic serine/threonine kinase are common in cutaneous melanomas (50–60%).97 A single nucleotide mutation 1799 T>A constitutes more than 90% of these mutations, resulting in substitution of glutamic acid for valine (BRAF V600E). With the advent of BRAF inhibitors, detection of this mutation has acquired a paramount importance in management of patients with metastatic melanoma.98,99 Though the gold standard is molecular testing, immunohistochemical studies using anti-BRAFV600E antibody (clone VE1) have demonstrated high concordance with the results of molecular analysis.100–103 The staining is usually diffuse and cytoplasmic (Fig. 5); but, focal staining is also observed in several cases,83 which could be attributed in part to technical issues.104 Nevertheless, other studies have demonstrated that the heterogeneous BRAF V600E staining could indeed be a reflection of the polyclonal nature of melanoma.105 Caution must be exercised when analyzing the slides, especially in densely pigmented lesions, where the cytoplasmic melanin pigment may mimic positive staining. In such cases, counterstaining with Giemsa is warranted in order to avoid a false positive reading.106 Recent reports have demonstrated that the immunostain might be too sensitive in some cases,107 so more studies may be needed to determine the clinical application of immunohistochemical detection of BRAF V600E.

Figure 5.

Immunohistochemical study for BRAF V600E. (A) Diffuse cytoplasmic positivity, indicating the presence of BRAF V600E mutation in the majority of cells (magnification 200×). (B) Negative stain, indicating wild type BRAF status (magnification × 200).

(0.34MB).
BAP1

Breast cancer type 1 susceptibility protein (BRCA1) associated protein-1 (BAP1), is a ubiquitin carboxy-terminal hydrolase that functions as a tumor suppressor.108,109 Individuals with heterozygous germline BAP1 mutations are at high-risk for developing a variety of tumors, including asbestos-associated mesothelioma, lung adenocarcinoma, meningioma, and renal cell carcinoma.110–112 These patients may also develop a gamut of melanocytic proliferations ranging from epithelioid nevi to spitzoid melanocytic proliferations (the so-called ‘BAPomas’) to melanomas (cutaneous and uveal).113–115 These tumors are characterized by loss of nuclear BAP1 staining, usually accompanied by of BRAF V600E expression (Fig. 6).116–119 Though BAP1 has traditionally been considered a tumor suppressor, recent studies have reported that BAP1 might play a role in survival of tumor cells.120

Figure 6.

Immunohistochemical studies for BAP1 and BRAF in a ‘BAPoma’. (A) Hematoxylin and eosin stained section of a dome-shaped BAPoma with dense proliferation of epithelioid melanocytes within the dermis (magnification 40×). (B) Typical nevus cells are also present at the peripheral of epithelioid proliferation (right side, magnification 100×). (C) Diffuse loss of nuclear BAP1 in most of the melanocytes; arrowhead indicates single positive cell. Of note, the background cytoplasmic reaction should not be interpreted as positive (magnification 200×). (D) These cells are diffusely positive for BRAF V600E protein (magnification × 200).

(1.06MB).
Additional techniques

While most melanocytic lesions can be characterized by microscopic examination of H&E-stained sections and immunostains, some melanocytic proliferations presenting with ambiguous features can be difficult to diagnose accurately. In such cases, molecular studies may provide additional diagnostic information. Though cutaneous melanomas represent a divergent group of diseases, they usually have recurrent, non-random chromosomal abnormalities,121,122 and detection of these aberrations may aid in the diagnostic process.123 The following section will summarize the basic principles and practical application of the various molecular tests for the diagnosis of melanocytic lesions.

Comparative genomic hybridization

Comparative genomic hybridization (CGH) is a cytogenetic assay that can detect losses and gains of genomic material and map them to specific chromosomes.124 CGH utilizes differential labeling of genomic DNA extracted from fresh or formalin-fixed tumor and normal tissue (usually peripheral blood leukocytes) with fluorochromes, followed by co-hybridization of equal amounts of each fraction on to metaphase spreads of chromosomes derived from a peripheral blood lymphocytes of a healthy donor (traditional CGH). DNA copy number changes can be determined by comparison of relative signal intensities of the different fluorochromes (Fig. 7). The main advantage of CGH is the ability to analyze the entire genome simultaneously and objectively, without specific probes. However, the utility of traditional CGH is restricted due to limited resolution of 3–10Mb.125 This can be overcome by utilizing an array of DNA targets covering the entire genome for hybridization (array CGH).126,127

Figure 7.

Schematic representing major steps in CGH. Left side demonstrates critical steps of a traditional CGH, while right side includes the commonly used array CGH.

(0.26MB).

The potential of CGH in the classification of melanocytic lesions began with the work of Bastian et al., who analyzed 32 primary melanomas.128 They identified loss of chromosome 9, particularly involving the short arm in majority of the melanomas. In addition, losses of parts of chromosomes 9 and 10 occurred in early lesions, whereas gains of chromosome 7 were late events in melanoma progression. A subsequent study from the same group revealed that chromosomal alterations could be identified in both melanomas and melanocytic nevi, albeit in a lower frequency and particularly in Spitz nevi. While cytogenetic alterations seen in melanomas involved portions of chromosomes, those in nevi typically involved the whole chromosomes or entire arms of the chromosomes.129,130

Metastatic melanomas have been shown to have a distinct set of chromosomal abnormalities, compared to primary melanomas.131 Some studies have revealed that loci of tumor suppressors are frequently deleted while those containing proto-oncogenes may be amplified.132–134 Among the amplified genes are BRAF and NRAS,134 somatic mutations of which have been implicated in early melanomagenesis. Melanomas of various histologic subtypes may be grouped together based on their cytogenetic abnormalities.135 For example, melanomas that arise in sun-exposed regions may exhibit NRAS and KIT mutations while most superficial spreading melanomas show BRAF mutations.

In addition to aiding in the diagnosis, CGH can also predict the biologic behavior of melanoma to some extent.123 Gains of 6p were seen exclusively in thicker melanomas, which along with gains at 1q were associated with a poor outcome.136 The total number of genomic aberrations in a tumor has been correlated with outcome: the higher the number of chromosomal alterations, the worse the prognosis. Some studies have demonstrated that the frequency of homozygous deletions might be a better predictor of metastasis to sentinel lymph nodes and distant sites.137

Thus, CGH may be a useful tool that can aid in distinguishing benign melanocytic proliferations from melanomas and, to some extent in predicting the outcome. However, the utility of CGH is limited by requiring a relatively high and “pure” number of tumor cells in the sample. Lesions with admixed tumor infiltrating lymphocytes, other inflammatory cells, and prominent intervening stroma are less frequently successfully studied by CGH. Also, when only a fraction of the cells (<30%) contain the cytogenetic abnormalities, they may evade detection by CGH, thus yielding a false negative result.130 In addition, the presence of balanced translocations cannot be detected by CGH.

Fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is a cytogenetic assay used to determine the copy number of specific genomic regions.138 Its application in the diagnosis of melanocytic lesions was realized only recently, after high-throughput use of CGH proved to be challenging for routine clinical use. In this assay, paraffin sections representing the tissue of interest are hybridized with a mixture of differentially labeled fluorescent probes targeting specific cytogenetic loci. The corresponding signals are enumerated per cell as the percentage of lesional cells containing altered number of signals. The advantages of FISH over CGH include the ability to examine the tumor cells specifically for the number of signals and thus, the feasibility for application in lesions with smaller tumor volume. However, since examination of immunofluorescence slides provides less defined morphologic detail than routine sections, dense lymphohistiocytic infiltrate and nevoid morphology of the tumor cells may result in difficult quantification of the FISH signals.

Genomic loci tested by FISH represent regions with the most frequent chromosomal aberrations, and 13 such hotspots were initially identified by analysis of the CGH assays.139 Of these, combination of four different probes produced the most distinct separation of melanomas from other melanocytic lesions. The initial 4-probe FISH assay utilized probes that target the following loci on chromosomes 6 and 11: 6p25 (RREB1, red), 6q23 (MYB, gold), 11q13 (CCND1, green), and centromere 6 (Cep6, aqua) (Fig. 8). A lesion was considered to be melanoma if at least one of the following chromosomal abnormalities was present: (i) 3 or more green dots per nucleus (11q13, CCND1) in at least 39% of tumor cells, (ii) 3 or more red dots (6p25, RREB1) in at least 30% of tumor nuclei, (iii) number of RREB1 signals (red dots) greater than the number of centromere 6 signals (aqua dots) in more than 55% of tumor nuclei and (iv) at least 41% of tumor cells with fewer number of MYB signals (gold dots), compared to centromere 6 signals (aqua dots). Using these criteria, nevi could be distinguished from melanomas with high sensitivity (90%) and specificity (95%). Of these, gain of 6p25 was found to have the highest sensitivity for this distinction.140,141

Figure 8.

Four-probe FISH assay. (A) Normal cell with two signals each of 6p25 (RREB1, red), 6q23 (MYB, gold), 11q13 (CCND1, green), and centromere 6 (Cep6, aqua). (B) Melanoma cell with an abnormal FISH result with multiple RREB1 (6 red dots) and CCND1 signals (7 green dots), indicating gains at 6p25 and 11q13, respectively.

(0.14MB).

The applicability of FISH with the 4-probe method has been examined in various settings including pigmented melanocytic lesions with blue nevus like morphology142,143; lesions with desmoplastic response144; lesions with nevoid melanocytes145,146; conjunctival melanocytic lesions147 and even intranodal melanocytic deposits148, as well as in distinguishing melanoma cells from associated nevocytes146. In addition, it is unknown how useful FISH is in ambiguous melanocytic lesions since most studies were done in histologically obvious nevi or melanoma and the presence of cytogenetic alterations did not always correlate with the clinical outcome.139,149–152 Furthermore, the presence of tetraploidy and polyploidy (common in Spitz and other melanocytic nevi) may yield false positive results.151,153–155

Additional genomic loci initially identified by CGH were examined to address the lack of sensitivity of conventional 4-probe FISH assay with respect ambiguous melanocytic lesions and in identifying polyploidy.156 Based on this, the current FISH panel includes probes against 6p25 (RREB1, red), 9p21 (CDKN2A, gold), 11q13 (CCND1, green), 8q24 (MYC, aqua) and centromere 9 (Cep9, green). The presence of at least 30% of tumor cells with gains of RREB1 (red), CCND1 (green) or MYC (aqua) with or without homozygous deletion of CDKN2A (gold) is considered to be a positive result. Use of this probe set has increased the sensitivity from 75% to 94% for the diagnosis of melanoma, in addition to eliminating most false positive results due to tetraploidy.157 Though this is a significant improvement from the initial panel, FISH assay should be used only to supplement the findings of a thorough histopathologic examination and the results from a FISH study should not influence the management of the patient.152

Imaging mass spectrometry

Mass spectrometry (MS) is a technique that utilizes molecule specific mass to charge (m/z) ratios to identify and quantify substances from various sources.141 MS involves generation of gas phase ions from the substance of interest, and then subject them to progressive fragmentation. The ions are separated based on their mass and charge and their amplitude of detection as ‘peaks’ would be directly proportional to their relative concentration in the parent substance. Each molecule has a specific m/z ratio, which will allow identification of the molecular composition of the parent substance.

Imaging mass spectrometry (IMS) is a process where the MS analyses are performed directly on tissue sections. Therefore, the analysis can be restricted to specific regions of the sections, and thus, target only cells of interest with minimal contamination from the adjacent tissues.103,158 In contrast to routine immunohistochemical studies and other ancillary tests used in pathology, using IMS, the distribution and abundance of all proteins and peptides can be analyzed simultaneously. In situ imaging of proteins provides an unbiased view of molecular composition of tissues and has been used mostly in the research setting so far.159

In an attempt to facilitate accurate diagnosis of spitzoid lesions, Lazova and colleagues analyzed 114 cases,160 with 51 as the training set, and a validation set of 63 cases. Two serial sections of formalin fixed paraffin tissues were cut on to a glass side for H&E staining and on to a conductive glass slide for IMS. After marking areas of interest on the H&E slide, including dense areas of tumor devoid of other cells such as stroma or lymphocytes, the corresponding areas on the conductive slide were analyzed by IMS. Of the multiple models studied by the researchers, 5 peaks corresponding to tumor and 12 peaks corresponding to tumor microenvironment were found to provide the most discrimination between Spitz nevi (SN) and spitzoid melanoma (SMM). Tumor-associated peptides could distinguish the spitzoid lesion with a sensitivity of 97% and specificity of 90%, whereas the sensitivity and specificities of tumor microenvironment-associated peptides were 64% and 90%, respectively. Of the five differentially expressed tumor-associated peptides, the authors were able to identify actin and vimentin. Though IMS is at an early stage, it has shown promise in distinguishing between melanoma and congenital nevi.161

Conclusion

Melanoma continues to be a deadly disease and accurate diagnosis is critical for appropriate treatment of the patient. The various ancillary tests mentioned in this review serve as supplements to thorough histologic examination. In addition, they can also provide valid prognostic information and identify patients that may benefit from targeted therapy.

Conflict of interest

The authors declare no conflict of interest.

References
[1]
D.P. Frishberg, C. Balch, B.L. Balzer, A.N. Crowson, M. Didolkar, J.M. McNiff, et al.
Protocol for the examination of specimens from patients with melanoma of the skin.
Arch Pathol Lab Med, 133 (2009), pp. 1560-1567
[2]
S.S. Banerjee, B. Eyden.
Divergent differentiation in malignant melanomas: a review.
Histopathology, 52 (2008), pp. 119-129
[3]
S.S. Banerjee, M. Harris.
Morphological and immunophenotypic variations in malignant melanoma.
Histopathology, 36 (2000), pp. 387-402
[4]
C.M. Magro, A.N. Crowson, M.C. Mihm.
Unusual variants of malignant melanoma.
Mod Pathol, 19 (2006), pp. S41-S70
[5]
S.J. Ohsie, G.P. Sarantopoulos, A.J. Cochran, S.W. Binder.
Immunohistochemical characteristics of melanoma.
J Cutan Pathol, 35 (2008), pp. 433-444
[6]
L.L. Matos, D.C. Trufelli, M.G. de Matos, M.A. da Silva Pinhal.
Immunohistochemistry as an important tool in biomarkers detection and clinical practice.
Biomark Insights, 5 (2010), pp. 9-20
[7]
S. Jaffer, I.J. Bleiweiss.
Beyond hematoxylin and eosin – the role of immunohistochemistry in surgical pathology.
Cancer Invest, 22 (2004), pp. 445-465
[8]
A.H. Coons, H.J. Creech, R.N. Jones.
Immunological properties of an antibody containing a fluorescent group.
Proc Soc Exp Biol Med, 47 (1941), pp. 200-202
[9]
C.J. Kligora, K.P. Fair, M.S. Clem, J.W. Patterson.
A comparison of melanin bleaching and azure blue counterstaining in the immunohistochemical diagnosis of malignant melanoma.
Mod Pathol, 12 (1999), pp. 1143-1147
[10]
M. Momose, H. Ota, M. Hayama.
Re-evaluation of melanin bleaching using warm diluted hydrogen peroxide for histopathological analysis.
Pathol Int, 61 (2011), pp. 345-350
[11]
V.G. Prieto, C.R. Shea.
Use of immunohistochemistry in melanocytic lesions.
J Cutan Pathol, 35 (2008), pp. 1-10
[12]
V.G. Prieto, C.R. Shea.
Immunohistochemistry of melanocytic proliferations.
Arch Pathol Lab Med, 135 (2011), pp. 853-859
[13]
E.M. Bekers, A.C. van Engen-van Grunsven, P.J. Groenen, H. Westdorp, R.H. Koornstra, J.J. Bonenkamp, et al.
Metastatic melanoma mimicking solitary fibrous tumor: report of two cases.
Virchows Arch, 464 (2014), pp. 247-251
[14]
H. Lee, F.X. Torres, S.A. McLean, R. Chen, M.W. Lee.
Immunophenotypic heterogeneity of primary sinonasal melanoma with aberrant expression of neuroendocrine markers and calponin.
Appl Immunohistochem Mol Morphol, 19 (2011), pp. 48-53
[15]
R.C. Romano, J.M. Carter, A.L. Folpe.
Aberrant intermediate filament and synaptophysin expression is a frequent event in malignant melanoma: an immunohistochemical study of 73 cases.
Mod Pathol, 28 (2015), pp. 1033-1042
[16]
N.G. Ordonez.
Value of melanocytic-associated immunohistochemical markers in the diagnosis of malignant melanoma: a review and update.
Hum Pathol, 45 (2014), pp. 191-205
[17]
D. Weinstein, J. Leininger, C. Hamby, B. Safai.
Diagnostic and prognostic biomarkers in melanoma.
J Clin Aesthet Dermatol, 7 (2014), pp. 13-24
[18]
R. Gaynor, R. Irie, D. Morton, H.R. Herschman.
S100 protein is present in cultured human malignant melanomas.
Nature, 286 (1980), pp. 400-401
[19]
T. Nakajima, S. Watanabe, Y. Sato, T. Kameya, Y. Shimosato, K. Ishihara.
Immunohistochemical demonstration of S100 protein in malignant melanoma and pigmented nevus, and its diagnostic application.
Cancer, 50 (1982), pp. 912-918
[20]
J. Palazzo, P.H. Duray.
Typical, dysplastic, congenital, and Spitz nevi: a comparative immunohistochemical study.
Hum Pathol, 20 (1989), pp. 341-346
[21]
R. Gaynor, H.R. Herschman, R. Irie, P. Jones, D. Morton, A. Cochran.
S100 protein: a marker for human malignant melanomas?.
Lancet, 1 (1981), pp. 869-871
[22]
A.R. Bresnick, D.J. Weber, D.B. Zimmer.
S100 proteins in cancer.
Nat Rev Cancer, 15 (2015), pp. 96-109
[23]
F. Egberts, W.N. Hitschler, M. Weichenthal, A. Hauschild.
Prospective monitoring of adjuvant treatment in high-risk melanoma patients: lactate dehydrogenase and protein S-100B as indicators of relapse.
Melanoma Res, 19 (2009), pp. 31-35
[24]
S.S. Fernando, S. Johnson, J. Bate.
Immunohistochemical analysis of cutaneous malignant melanoma: comparison of S-100 protein, HMB-45 monoclonal antibody and NKI/C3 monoclonal antibody.
Pathology, 26 (1994), pp. 16-19
[25]
D. Nonaka, L. Chiriboga, B.P. Rubin.
Differential expression of S100 protein subtypes in malignant melanoma, and benign and malignant peripheral nerve sheath tumors.
J Cutan Pathol, 35 (2008), pp. 1014-1019
[26]
D.L. Aisner, A. Maker, S.A. Rosenberg, D.M. Berman.
Loss of S100 antigenicity in metastatic melanoma.
Hum Pathol, 36 (2005), pp. 1016-1019
[27]
R. Lazova, I. Tantcheva-Poor, A.C. Sigal.
P75 nerve growth factor receptor staining is superior to S100 in identifying spindle cell and desmoplastic melanoma.
J Am Acad Dermatol, 63 (2010), pp. 852-858
[28]
G.J. Adema, A.J. de Boer, A.M. Vogel, W.A. Loenen, C.G. Figdor.
Molecular characterization of the melanocyte lineage-specific antigen gp100.
J Biol Chem, 269 (1994), pp. 20126-20133
[29]
A.M. Gown, A.M. Vogel, D. Hoak, F. Gough, M.A. McNutt.
Monoclonal antibodies specific for melanocytic tumors distinguish subpopulations of melanocytes.
Am J Pathol, 123 (1986), pp. 195-203
[30]
W.S. Wood, V.A. Tron.
Analysis of HMB-45 immunoreactivity in common and cellular blue nevi.
J Cutan Pathol, 18 (1991), pp. 261-263
[31]
H.G. Skelton 3rd, K.J. Smith, T.L. Barrett, G.P. Lupton, J.H. Graham.
HMB-45 staining in benign and malignant melanocytic lesions. A reflection of cellular activation.
Am J Dermatopathol, 13 (1991), pp. 543-550
[32]
S.E. Weissinger, J.K. Lennerz.
Comparison of MelanA/MART-1 and HMB45 labeling in desmoplastic melanoma.
Mod Pathol, 27 (2014), pp. 1421-1423
[33]
X. Jing, C.W. Michael, C.G. Theoharis.
The use of immunocytochemical study in the cytologic diagnosis of melanoma: evaluation of three antibodies.
Diagn Cytopathol, 41 (2013), pp. 126-130
[34]
Y. Kawakami, S. Eliyahu, C.H. Delgado, P.F. Robbins, L. Rivoltini, S.L. Topalian, et al.
Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.
Proc Natl Acad Sci U S A, 91 (1994), pp. 3515-3519
[35]
T. Hoashi, H. Watabe, J. Muller, Y. Yamaguchi, W.D. Vieira, V.J. Hearing.
MART-1 is required for the function of the melanosomal matrix protein PMEL17/GP100 and the maturation of melanosomes.
J Biol Chem, 280 (2005), pp. 14006-14016
[36]
K.J. Busam, Y.T. Chen, L.J. Old, E. Stockert, K. Iversen, K.A. Coplan, et al.
Expression of melan-A (MART1) in benign melanocytic nevi and primary cutaneous malignant melanoma.
Am J Surg Pathol, 22 (1998), pp. 976-982
[37]
K.J. Busam, A.A. Jungbluth.
Melan-A, a new melanocytic differentiation marker.
Adv Anat Pathol, 6 (1999), pp. 12-18
[38]
A.A. Jungbluth, K.J. Busam, W.L. Gerald, E. Stockert, K.A. Coplan, K. Iversen, et al.
A103: an anti-melan-a monoclonal antibody for the detection of malignant melanoma in paraffin-embedded tissues.
Am J Surg Pathol, 22 (1998), pp. 595-602
[39]
G.F. Hofbauer, J. Kamarashev, R. Geertsen, R. Boni, R. Dummer.
Melan A/MART-1 immunoreactivity in formalin-fixed paraffin-embedded primary and metastatic melanoma: frequency and distribution.
Melanoma Res, 8 (1998), pp. 337-343
[40]
L. El Shabrawi-Caelen, H. Kerl, L. Cerroni.
Melan-A: not a helpful marker in distinction between melanoma in situ on sun-damaged skin and pigmented actinic keratosis.
Am J Dermatopathol, 26 (2004), pp. 364-366
[41]
H. Beltraminelli, L.E. Shabrawi-Caelen, H. Kerl, L. Cerroni.
Melan-a-positive pseudomelanocytic nests: a pitfall in the histopathologic and immunohistochemical diagnosis of pigmented lesions on sun-damaged skin.
Am J Dermatopathol, 31 (2009), pp. 305-308
[42]
K.L. Wiltz, H. Qureshi, J.W. Patterson, D.C. Mayes, M.R. Wick.
Immunostaining for MART-1 in the interpretation of problematic intra-epidermal pigmented lesions.
J Cutan Pathol, 34 (2007), pp. 601-605
[43]
A.M. De Maziere, K. Muehlethaler, E. van Donselaar, S. Salvi, J. Davoust, J.C. Cerottini, et al.
The melanocytic protein Melan-A/MART-1 has a subcellular localization distinct from typical melanosomal proteins.
Traffic, 3 (2002), pp. 678-693
[44]
A. Korner, J. Pawelek.
Mammalian tyrosinase catalyzes three reactions in the biosynthesis of melanin.
Science, 217 (1982), pp. 1163-1165
[45]
V. Brichard, A. Van Pel, T. Wolfel, C. Wolfel, E. De Plaen, B. Lethe, et al.
The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas.
J Exp Med, 178 (1993), pp. 489-495
[46]
G.F. Hofbauer, J. Kamarashev, R. Geertsen, R. Boni, R. Dummer.
Tyrosinase immunoreactivity in formalin-fixed, paraffin-embedded primary and metastatic melanoma: frequency and distribution.
J Cutan Pathol, 25 (1998), pp. 204-209
[47]
A.A. Jungbluth, K. Iversen, K. Coplan, D. Kolb, E. Stockert, Y.T. Chen, et al.
T311--an anti-tyrosinase monoclonal antibody for the detection of melanocytic lesions in paraffin embedded tissues.
Pathol Res Pract, 196 (2000), pp. 235-242
[48]
J.L. Boyle, H.M. Haupt, J.B. Stern, H.A. Multhaupt.
Tyrosinase expression in malignant melanoma, desmoplastic melanoma, and peripheral nerve tumors.
Arch Pathol Lab Med, 126 (2002), pp. 816-822
[49]
N. Fuse, K. Yasumoto, K. Takeda, S. Amae, M. Yoshizawa, T. Udono, et al.
Molecular cloning of cDNA encoding a novel microphthalmia-associated transcription factor isoform with a distinct amino-terminus.
J Biochem, 126 (1999), pp. 1043-1051
[50]
C. Levy, M. Khaled, D.E. Fisher.
MITF: master regulator of melanocyte development and melanoma oncogene.
Trends Mol Med, 12 (2006), pp. 406-414
[51]
G.G. McGill, M. Horstmann, H.R. Widlund, J. Du, G. Motyckova, E.K. Nishimura, et al.
Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability.
Cell, 109 (2002), pp. 707-718
[52]
K.J. Busam, K. Iversen, K.C. Coplan, A.A. Jungbluth.
Analysis of microphthalmia transcription factor expression in normal tissues and tumors, and comparison of its expression with S-100 protein, gp100, and tyrosinase in desmoplastic malignant melanoma.
Am J Surg Pathol, 25 (2001), pp. 197-204
[53]
M. Miettinen, M. Fernandez, K. Franssila, Z. Gatalica, J. Lasota, M. Sarlomo-Rikala.
Microphthalmia transcription factor in the immunohistochemical diagnosis of metastatic melanoma: comparison with four other melanoma markers.
Am J Surg Pathol, 25 (2001), pp. 205-211
[54]
S.R. Granter, K.N. Weilbaecher, C. Quigley, C.D. Fletcher, D.E. Fisher.
Microphthalmia transcription factor: not a sensitive or specific marker for the diagnosis of desmoplastic melanoma and spindle cell (non-desmoplastic) melanoma.
Am J Dermatopathol, 23 (2001), pp. 185-189
[55]
J. Clevenger, C. Joseph, M. Dawlett, M. Guo, Y. Gong.
Reliability of immunostaining using pan-melanoma cocktail, SOX10, and microphthalmia transcription factor in confirming a diagnosis of melanoma on fine-needle aspiration smears.
Cancer Cytopathol, 122 (2014), pp. 779-785
[56]
G.E. Nybakken, M. Sargen, R. Abraham, P.J. Zhang, M. Ming, X. Xu.
MITF accurately highlights epidermal melanocytes in atypical intraepidermal melanocytic proliferations.
Am J Dermatopathol, 35 (2013), pp. 25-29
[57]
J.N. Buonaccorsi, V.G. Prieto, C. Torres-Cabala, S. Suster, J.A. Plaza.
Diagnostic utility and comparative immunohistochemical analysis of MITF-1 and SOX10 to distinguish melanoma in situ and actinic keratosis: a clinicopathological and immunohistochemical study of 70 cases.
Am J Dermatopathol, 36 (2014), pp. 124-130
[58]
M. Lee, J. Goodall, C. Verastegui, R. Ballotti, C.R. Goding.
Direct regulation of the Microphthalmia promoter by Sox10 links Waardenburg-Shah syndrome (WS4)-associated hypopigmentation and deafness to WS2.
J Biol Chem, 275 (2000), pp. 37978-37983
[59]
R. Mollaaghababa, W.J. Pavan.
The importance of having your SOX on: role of SOX10 in the development of neural crest-derived melanocytes and glia.
Oncogene, 22 (2003), pp. 3024-3034
[60]
M. Miettinen, P.A. McCue, M. Sarlomo-Rikala, W. Biernat, P. Czapiewski, J. Kopczynski, et al.
Sox10-a marker for not only schwannian and melanocytic neoplasms but also myoepithelial cell tumors of soft tissue: a systematic analysis of 5134 tumors.
Am J Surg Pathol, 39 (2015), pp. 826-835
[61]
A. Mohamed, R.S. Gonzalez, D. Lawson, J. Wang, C. Cohen.
SOX10 expression in malignant melanoma, carcinoma, and normal tissues.
Appl Immunohistochem Mol Morphol, 21 (2013), pp. 506-510
[62]
D. Tacha, W. Qi, S. Ra, R. Bremer, C. Yu, J. Chu, et al.
A newly developed mouse monoclonal SOX10 antibody is a highly sensitive and specific marker for malignant melanoma, including spindle cell and desmoplastic melanomas.
Arch Pathol Lab Med, 139 (2015), pp. 530-536
[63]
B. Palla, A. Su, S. Binder, S. Dry.
SOX10 expression distinguishes desmoplastic melanoma from its histologic mimics.
Am J Dermatopathol, 35 (2013), pp. 576-581
[64]
F. Truzzi, A. Saltari, E. Palazzo, R. Lotti, T. Petrachi, K. Dallaglio, et al.
CD271 mediates stem cells to early progeny transition in human epidermis.
J Invest Dermatol, 135 (2015), pp. 786-795
[65]
P.G. Chesa, W.J. Rettig, T.M. Thomson, L.J. Old, M.R. Melamed.
Immunohistochemical analysis of nerve growth factor receptor expression in normal and malignant human tissues.
J Histochem Cytochem, 36 (1988), pp. 383-389
[66]
A.C. Sigal, M. Keenan, R. Lazova.
P75 nerve growth factor receptor as a useful marker to distinguish spindle cell melanoma from other spindle cell neoplasms of sun-damaged skin.
Am J Dermatopathol, 34 (2012), pp. 145-150
[67]
C. Bull, M. Mirzabeigi, W. Laskin, M. Dubina, T. Traczyc, J. Guitart, et al.
Diagnostic utility of low-affinity nerve growth factor receptor (P 75) immunostaining in atypical fibroxanthoma.
J Cutan Pathol, 38 (2011), pp. 631-635
[68]
S. Otaibi, D.M. Jukic, L. Drogowski, J. Bhawan, A. Radfar.
NGFR (p75) expression in cutaneous scars; further evidence for a potential pitfall in evaluation of reexcision scars of cutaneous neoplasms, in particular desmoplastic melanoma.
Am J Dermatopathol, 33 (2011), pp. 65-71
[69]
J.C. Fanburg-Smith, M. Miettinen.
Low-affinity nerve growth factor receptor (p75) in dermatofibrosarcoma protuberans and other nonneural tumors: a study of 1,150 tumors and fetal and adult normal tissues.
Hum Pathol, 32 (2001), pp. 976-983
[70]
Z. Krizan, J.L. Murray, E.M. Hersh, M.G. Rosenblum, H.J. Glenn, C.R. Gschwind, et al.
Increased labeling of human melanoma cells in vitro using combinations of monoclonal antibodies recognizing separate cell surface antigenic determinants.
Cancer Res, 45 (1985), pp. 4904-4909
[71]
G. Orchard.
Evaluation of melanocytic neoplasms: application of a pan-melanoma antibody cocktail.
Br J Biomed Sci, 59 (2002), pp. 196-202
[72]
C. Schluter, M. Duchrow, C. Wohlenberg, M.H. Becker, G. Key, H.D. Flad, et al.
The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins.
J Cell Biol, 123 (1993), pp. 513-522
[73]
P. Rudolph, C. Schubert, B. Schubert, R. Parwaresch.
Proliferation marker Ki-S5 as a diagnostic tool in melanocytic lesions.
J Am Acad Dermatol, 37 (1997), pp. 169-178
[74]
M.R. Nasr, O. El-Zammar.
Comparison of pHH3, Ki-67, and survivin immunoreactivity in benign and malignant melanocytic lesions.
Am J Dermatopathol, 30 (2008), pp. 117-122
[75]
L.X. Li, K.A. Crotty, S.W. McCarthy, A.A. Palmer, J.J. Kril.
A zonal comparison of MIB1-Ki67 immunoreactivity in benign and malignant melanocytic lesions.
Am J Dermatopathol, 22 (2000), pp. 489-495
[76]
J.A. Ramsay, L. From, N.A. Iscoe, H.J. Kahn.
MIB-1 proliferative activity is a significant prognostic factor in primary thick cutaneous melanomas.
J Invest Dermatol, 105 (1995), pp. 22-26
[77]
S. Moretti, A. Spallanzani, A. Chiarugi, M. Fabiani, C. Pinzi.
Correlation of Ki-67 expression in cutaneous primary melanoma with prognosis in a prospective study: different correlation according to thickness.
J Am Acad Dermatol, 44 (2001), pp. 188-192
[78]
R.G. Ladstein, I.M. Bachmann, O. Straume, L.A. Akslen.
Ki-67 expression is superior to mitotic count and novel proliferation markers PHH3, MCM4 and mitosin as a prognostic factor in thick cutaneous melanoma.
BMC Cancer, 10 (2010), pp. 140
[79]
B.J. Hall, P.E. LeBoit.
Suprabasal spread of melanocytes in dysplastic nevi and melanoma in situ: Ki-67-labeling rate of junctional melanocytes and suprabasal cells may be a helpful clue to the diagnosis.
Am J Surg Pathol, 38 (2014), pp. 1111-1117
[80]
C.M. Balch, A.C. Buzaid, S.J. Soong, M.B. Atkins, N. Cascinelli, D.G. Coit, et al.
Final version of the American Joint Committee on Cancer staging system for cutaneous melanoma.
J Clin Oncol, 19 (2001), pp. 3635-3648
[81]
M.J. Hendzel, Y. Wei, M.A. Mancini, A. Van Hooser, T. Ranalli, B.R. Brinkley, et al.
Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation.
Chromosoma, 106 (1997), pp. 348-360
[82]
B. Perez-Cadahia, B. Drobic, J.R. Davie.
H3 phosphorylation: dual role in mitosis and interphase.
Biochem Cell Biol, 87 (2009), pp. 695-709
[83]
M.T. Tetzlaff, C.A. Torres-Cabala, P. Pattanaprichakul, R.P. Rapini, V.G. Prieto, J.L. Curry.
Emerging clinical applications of selected biomarkers in melanoma.
Clin Cosmet Investig Dermatol, 8 (2015), pp. 35-46
[84]
M.T. Tetzlaff, J.L. Curry, D. Ivan, W.L. Wang, C.A. Torres-Cabala, R.L. Bassett, et al.
Immunodetection of phosphohistone H3 as a surrogate of mitotic figure count and clinical outcome in cutaneous melanoma.
Mod Pathol, 26 (2013), pp. 1153-1160
[85]
P.S. Nielsen, R. Riber-Hansen, T.O. Jensen, H. Schmidt, T. Steiniche.
Proliferation indices of phosphohistone H3 and Ki67: strong prognostic markers in a consecutive cohort with stage I/II melanoma.
Mod Pathol, 26 (2013), pp. 404-413
[86]
D.J. Casper, K.I. Ross, J.L. Messina, V.K. Sondak, C.N. Bodden, T.W. McCardle, et al.
Useof anti-phosphohistone H3 immunohistochemistry to determine mitotic rate in thin melanoma.
Am J Dermatopathol, 32 (2010), pp. 650-654
[87]
T.T. Schimming, F. Grabellus, M. Roner, S. Pechlivanis, A. Sucker, N. Bielefeld, et al.
pHH3 immunostaining improves interobserver agreement of mitotic index in thin melanomas.
Am J Dermatopathol, 34 (2012), pp. 266-269
[88]
K. Ikenberg, M. Pfaltz, C. Rakozy, W. Kempf.
Immunohistochemical dual staining as an adjunct in assessment of mitotic activity in melanoma.
J Cutan Pathol, 39 (2012), pp. 324-330
[89]
H.J. Kahn, D. Bailey, A. Marks.
Monoclonal antibody D2-40, a new marker of lymphatic endothelium, reacts with Kaposi's sarcoma and a subset of angiosarcomas.
Mod Pathol, 15 (2002), pp. 434-440
[90]
M. Fukunaga.
Expression of D2-40 in lymphatic endothelium of normal tissues and in vascular tumours.
Histopathology, 46 (2005), pp. 396-402
[91]
F. Niakosari, H.J. Kahn, D. McCready, D. Ghazarian, L.E. Rotstein, A. Marks, et al.
Lymphatic invasion identified by monoclonal antibody D2-40, younger age, and ulceration: predictors of sentinel lymph node involvement in primary cutaneous melanoma.
Arch Dermatol, 144 (2008), pp. 462-467
[92]
F. Petersson, A.H. Diwan, D. Ivan, J.E. Gershenwald, M.M. Johnson, R. Harrell, et al.
Immunohistochemical detection of lymphovascular invasion with D2-40 in melanoma correlates with sentinel lymph node status, metastasis and survival.
J Cutan Pathol, 36 (2009), pp. 1157-1163
[93]
K. Doeden, Z. Ma, B. Narasimhan, S.M. Swetter, M. Detmar, S.S. Dadras.
Lymphatic invasion in cutaneous melanoma is associated with sentinel lymph node metastasis.
J Cutan Pathol, 36 (2009), pp. 772-780
[94]
N. Thompson, S. Storr, S. Zhang, S. Martin.
Lymphovascular invasion: assessment and prognostic impact in melanoma and breast cancer.
Histol Histopathol, 30 (2015), pp. 1001-1009
[95]
M. Petitt, A. Allison, T. Shimoni, T. Uchida, S. Raimer, B. Kelly.
Lymphatic invasion detected by D2-40/S-100 dual immunohistochemistry does not predict sentinel lymph node status in melanoma.
J Am Acad Dermatol, 61 (2009), pp. 819-828
[96]
L. Feldmeyer, M.T. Tetzlaff, P. Fox, P. Nagarajan, J.L. Curry, D. Ivan, et al.
Prognostic significance of lymphovascular invasion detected by double immunostaining for D2-40 and MITF1 in patients with cutaneous melanoma.
Am J Dermatopathol, (2015),
[Epub ahead of print]. doi:10.1097/DAD.0000000000000453
[97]
H. Davies, G.R. Bignell, C. Cox, P. Stephens, S. Edkins, S. Clegg, et al.
Mutations of the BRAF gene in human cancer.
Nature, 417 (2002), pp. 949-954
[98]
P.A. Ascierto, J.M. Kirkwood, J.J. Grob, E. Simeone, A.M. Grimaldi, M. Maio, et al.
The role of BRAF V600 mutation in melanoma.
J Transl Med, 10 (2012), pp. 85
[99]
J. Martin-Liberal, J. Larkin.
Vemurafenib for the treatment of BRAF mutant metastatic melanoma.
Future Oncol, 11 (2015), pp. 579-589
[100]
G.V. Long, J.S. Wilmott, D. Capper, M. Preusser, Y.E. Zhang, J.F. Thompson, et al.
Immunohistochemistry is highly sensitive and specific for the detection of V600E BRAF mutation in melanoma.
Am J Surg Pathol, 37 (2013), pp. 61-65
[101]
D. Capper, M. Preusser, A. Habel, F. Sahm, U. Ackermann, G. Schindler, et al.
Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody.
Acta Neuropathol, 122 (2011), pp. 11-19
[102]
C.A. Routhier, M.C. Mochel, K. Lynch, D. Dias-Santagata, D.N. Louis, M.P. Hoang.
Comparison of 2 monoclonal antibodies for immunohistochemical detection of BRAF V600E mutation in malignant melanoma, pulmonary carcinoma, gastrointestinal carcinoma, thyroid carcinoma, and gliomas.
Hum Pathol, 44 (2013), pp. 2563-2570
[103]
M.T. Tetzlaff, P. Pattanaprichakul, J. Wargo, P.S. Fox, K.P. Patel, J.S. Estrella, et al.
Utility of BRAF V600E immunohistochemical expression pattern as a surrogate of BRAF mutation status in 154 patients with advanced melanoma.
Hum Pathol, 46 (2015), pp. 1101-1110
[104]
E. Riveiro-Falkenbach, C.A. Villanueva, M.C. Garrido, Y. Ruano, R.M. Garcia-Martin, E. Godoy, et al.
Intra- and inter-tumoral homogeneity of BRAF(V600E) mutations in melanoma tumors.
J Invest Dermatol, 135 (2015), pp. 3078-3085
[105]
M. Yancovitz, A. Litterman, J. Yoon, E. Ng, R.L. Shapiro, R.S. Berman, et al.
Intra- and inter-tumor heterogeneity of BRAF(V600E)mutations in primary and metastatic melanoma.
[106]
S. Ravishankar, P. Nagarajan, J.L. Curry, M.T. Tetzlaff, D. Ivan, C.A. Torres-Cabala, et al.
Giemsa is the optimal counterstain for immunohistochemical detection of BRAF V600E mutation status in pigmented melanomas.
J Cutan Pathol, (2016),
[in press]. doi:10.1111/cup.12714
[107]
L. Ehsani, C. Cohen, K.E. Fisher, M.T. Siddiqui.
BRAF mutations in metastatic malignant melanoma: comparison of molecular analysis and immunohistochemical expression.
Appl Immunohistochem Mol Morphol, 22 (2014), pp. 648-651
[108]
D.E. Jensen, F.J. Rauscher 3rd.
BAP1, a candidate tumor suppressor protein that interacts with BRCA1.
Ann N Y Acad Sci, 886 (1999), pp. 191-194
[109]
F. Baumann, E. Flores, A. Napolitano, S. Kanodia, E. Taioli, H. Pass, et al.
Mesothelioma patients with germline BAP1 mutations have 7-fold improved long-term survival.
Carcinogenesis, 36 (2015), pp. 76-81
[110]
J.R. Testa, M. Cheung, J. Pei, J.E. Below, Y. Tan, E. Sementino, et al.
Germline BAP1 mutations predispose to malignant mesothelioma.
Nat Genet, 43 (2011), pp. 1022-1025
[111]
R. Murali, T. Wiesner, R.A. Scolyer.
Tumours associated with BAP1 mutations.
Pathology, 45 (2013), pp. 116-126
[112]
M.H. Abdel-Rahman, R. Pilarski, C.M. Cebulla, J.B. Massengill, B.N. Christopher, G. Boru, et al.
Germline BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers.
J Med Genet, 48 (2011), pp. 856-859
[113]
T. Wiesner, A.C. Obenauf, R. Murali, I. Fried, K.G. Griewank, P. Ulz, et al.
Germline mutations in BAP1 predispose to melanocytic tumors.
Nat Genet, 43 (2011), pp. 1018-1021
[114]
Z. Marusic, M. Buljan, K.J. Busam.
Histomorphologic spectrum of BAP1 negative melanocytic neoplasms in a family with BAP1-associated cancer susceptibility syndrome.
J Cutan Pathol, 42 (2015), pp. 406-412
[115]
T. Wiesner, R. Murali, I. Fried, L. Cerroni, K. Busam, H. Kutzner, et al.
A distinct subset of atypical Spitz tumors is characterized by BRAF mutation and loss of BAP1 expression.
Am J Surg Pathol, 36 (2012), pp. 818-830
[116]
A. Piris, M.C. Mihm Jr., M.P. Hoang.
BAP1 and BRAFV600E expression in benign and malignant melanocytic proliferations.
Hum Pathol, 46 (2015), pp. 239-245
[117]
K.J. Busam, H. Kutzner, L. Cerroni, T. Wiesner.
Clinical and pathologic findings of Spitz nevi and atypical Spitz tumors with ALK fusions.
Am J Surg Pathol, 38 (2014), pp. 925-933
[118]
K.J. Busam, J. Sung, T. Wiesner, A. von Deimling, A. Jungbluth.
Combined BRAF(V600E)-positive melanocytic lesions with large epithelioid cells lacking BAP1 expression and conventional nevomelanocytes.
Am J Surg Pathol, 37 (2013), pp. 193-199
[119]
M. Llamas-Velasco, Y.C. Perez-Gonzalez, L. Requena, H. Kutzner.
Histopathologic clues for the diagnosis of Wiesner nevus.
J Am Acad Dermatol, 70 (2014), pp. 549-554
[120]
R. Kumar, M. Taylor, B. Miao, Z. Ji, J.C. Njauw, G. Jonsson, et al.
BAP1 has a survival role in cutaneous melanoma.
J Invest Dermatol, 135 (2015), pp. 1089-1097
[121]
G. Balaban, M. Herlyn, R. Guerry Dt Bartolo, H. Koprowski, W.H. Clark, et al.
Cytogenetics of human malignant melanoma and premalignant lesions.
Cancer Genet Cytogenet, 11 (1984), pp. 429-439
[122]
J. Limon, P. Dal Cin, S.N. Sait, C. Karakousis, A.A. Sandberg.
Chromosome changes in metastatic human melanoma.
Cancer Genet Cytogenet, 30 (1988), pp. 201-211
[123]
J. March, M. Hand, A. Truong, D. Grossman.
Practical application of new technologies for melanoma diagnosis: Part II. Molecular approaches.
J Am Acad Dermatol, 72 (2015), pp. 943-958
[124]
A. Kallioniemi, O.P. Kallioniemi, D. Sudar, D. Rutovitz, J.W. Gray, F. Waldman, et al.
Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.
Science, 258 (1992), pp. 818-821
[125]
M. Kirchhoff, T. Gerdes, J. Maahr, H. Rose, M. Bentz, H. Dohner, et al.
Deletions below 10 megabasepairs are detected in comparative genomic hybridization by standard reference intervals.
Genes Chromosomes Cancer, 25 (1999), pp. 410-413
[126]
H. Fiegler, P. Carr, E.J. Douglas, D.C. Burford, S. Hunt, C.E. Scott, et al.
DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones.
Genes Chromosomes Cancer, 36 (2003), pp. 361-374
[127]
A.S. Ishkanian, C.A. Malloff, S.K. Watson, R.J. DeLeeuw, B. Chi, B.P. Coe, et al.
A tiling resolution DNA microarray with complete coverage of the human genome.
Nat Genet, 36 (2004), pp. 299-303
[128]
B.C. Bastian, P.E. LeBoit, H. Hamm, E.B. Brocker, D. Pinkel.
Chromosomal gains and losses in primary cutaneous melanomas detected by comparative genomic hybridization.
Cancer Res, 58 (1998), pp. 2170-2175
[129]
B.C. Bastian, A.B. Olshen, P.E. LeBoit, D. Pinkel.
Classifying melanocytic tumors based on DNA copy number changes.
Am J Pathol, 163 (2003), pp. 1765-1770
[130]
J. Bauer, B.C. Bastian.
Distinguishing melanocytic nevi from melanoma by DNA copy number changes: comparative genomic hybridization as a research and diagnostic tool.
Dermatol Ther, 19 (2006), pp. 40-49
[131]
M. Balazs, Z. Adam, A. Treszl, A. Begany, J. Hunyadi, R. Adany.
Chromosomal imbalances in primary and metastatic melanomas revealed by comparative genomic hybridization.
Cytometry, 46 (2001), pp. 222-232
[132]
A. Gast, D. Scherer, B. Chen, S. Bloethner, S. Melchert, A. Sucker, et al.
Somatic alterations in the melanoma genome: a high-resolution array-based comparative genomic hybridization study.
Genes Chromosomes Cancer, 49 (2010), pp. 733-745
[133]
G. Jonsson, C. Dahl, J. Staaf, T. Sandberg, P.O. Bendahl, M. Ringner, et al.
Genomic profiling of malignant melanoma using tiling-resolution array CGH.
Oncogene, 26 (2007), pp. 4738-4748
[134]
M. Stark, N. Hayward.
Genome-wide loss of heterozygosity and copy number analysis in melanoma using high-density single-nucleotide polymorphism arrays.
Cancer Res, 67 (2007), pp. 2632-2642
[135]
J.A. Curtin, J. Fridlyand, T. Kageshita, H.N. Patel, K.J. Busam, H. Kutzner, et al.
Distinct sets of genetic alterations in melanoma.
N Engl J Med, 353 (2005), pp. 2135-2147
[136]
T. Namiki, S. Yanagawa, T. Izumo, M. Ishikawa, M. Tachibana, Y. Kawakami, et al.
Genomic alterations in primary cutaneous melanomas detected by metaphase comparative genomic hybridization with laser capture or manual microdissection: 6p gains may predict poor outcome.
Cancer Genet Cytogenet, 157 (2005), pp. 1-11
[137]
S. Boi, T. Tebaldi, A. Re, C. Cantaloni, V. Adami, M. Barbareschi, et al.
Increased frequency of minimal homozygous deletions is associated with poor prognosis in primary malignant melanoma patients.
Genes Chromosomes Cancer, 53 (2014), pp. 487-496
[138]
P. Gerami, A. Zembowicz.
Update on fluorescence in situ hybridization in melanoma: state of the art.
Arch Pathol Lab Med, 135 (2011), pp. 830-837
[139]
P. Gerami, S.S. Jewell, L.E. Morrison, B. Blondin, J. Schulz, T. Ruffalo, et al.
Fluorescence in situ hybridization (FISH) as an ancillary diagnostic tool in the diagnosis of melanoma.
Am J Surg Pathol, 33 (2009), pp. 1146-1156
[140]
P. Gerami, M. Mafee, T. Lurtsbarapa, J. Guitart, Z. Haghighat, M. Newman.
Sensitivity of fluorescence in situ hybridization for melanoma diagnosis using RREB1, MYB, Cep6, and 11q13 probes in melanoma subtypes.
Arch Dermatol, 146 (2010), pp. 273-278
[141]
R. Matthiesen.
Useful mass spectrometry programs freely available on the Internet.
Methods Mol Biol, 367 (2007), pp. 303-305
[142]
P. Pouryazdanparast, M. Newman, M. Mafee, Z. Haghighat, J. Guitart, P. Gerami.
Distinguishing epithelioid blue nevus from blue nevus-like cutaneous melanoma metastasis using fluorescence in situ hybridization.
Am J Surg Pathol, 33 (2009), pp. 1396-1400
[143]
B. Gammon, B. Beilfuss, J. Guitart, K.J. Busam, P. Gerami.
Fluorescence in situ hybridization for distinguishing cellular blue nevi from blue nevus-like melanoma.
J Cutan Pathol, 38 (2011), pp. 335-341
[144]
P. Gerami, B. Beilfuss, Z. Haghighat, Y. Fang, S. Jhanwar, K.J. Busam.
Fluorescence in situ hybridization as an ancillary method for the distinction of desmoplastic melanomas from sclerosing melanocytic nevi.
J Cutan Pathol, 38 (2011), pp. 329-334
[145]
P. Gerami, A. Wass, M. Mafee, Y. Fang, M.P. Pulitzer, K.J. Busam.
Fluorescence in situ hybridization for distinguishing nevoid melanomas from mitotically active nevi.
Am J Surg Pathol, 33 (2009), pp. 1783-1788
[146]
M.D. Newman, T. Lertsburapa, M. Mirzabeigi, M. Mafee, J. Guitart, P. Gerami.
Fluorescence in situ hybridization as a tool for microstaging in malignant melanoma.
Mod Pathol, 22 (2009), pp. 989-995
[147]
K.J. Busam, Y. Fang, S.C. Jhanwar, M.P. Pulitzer, B. Marr, D.H. Abramson.
Distinction of conjunctival melanocytic nevi from melanomas by fluorescence in situ hybridization.
J Cutan Pathol, 37 (2010), pp. 196-203
[148]
S.R. Dalton, P. Gerami, N.A. Kolaitis, S. Charzan, R. Werling, P.E. LeBoit, et al.
Use of fluorescence in situ hybridization (FISH) to distinguish intranodal nevus from metastatic melanoma.
Am J Surg Pathol, 34 (2010), pp. 231-237
[149]
B. Vergier, M. Prochazkova-Carlotti, A. de la Fouchardiere, L. Cerroni, D. Massi, V. De Giorgi, et al.
Fluorescence in situ hybridization, a diagnostic aid in ambiguous melanocytic tumors: European study of 113 cases.
Mod Pathol, 24 (2011), pp. 613-623
[150]
T. Gaiser, H. Kutzner, G. Palmedo, M.D. Siegelin, T. Wiesner, T. Bruckner, et al.
Classifying ambiguous melanocytic lesions with FISH and correlation with clinical long-term follow up.
Mod Pathol, 23 (2010), pp. 413-419
[151]
A. Zembowicz, S.E. Yang, A. Kafanas, S.R. Lyle.
Correlation between histologic assessment and fluorescence in situ hybridization using MelanoSITE in evaluation of histologically ambiguous melanocytic lesions.
Arch Pathol Lab Med, 136 (2012), pp. 1571-1579
[152]
M.T. Tetzlaff, W.L. Wang, T.L. Milless, J.L. Curry, C.A. Torres-Cabala, M.S. McLemore, et al.
Ambiguous melanocytic tumors in a tertiary referral center: the contribution of fluorescence in situ hybridization (FISH) to conventional histopathologic and immunophenotypic analyses.
Am J Surg Pathol, 37 (2013), pp. 1783-1796
[153]
Y. Fang, S. Dusza, S. Jhanwar, K.J. Busam.
Fluorescence in situ hybridization (FISH) analysis of melanocytic nevi and melanomas: sensitivity, specificity, and lack of association with sentinel node status.
Int J Surg Pathol, 20 (2012), pp. 434-440
[154]
V. Martin, S. Banfi, A. Bordoni, S. Leoni-Parvex, L. Mazzucchelli.
Presence of cytogenetic abnormalities in Spitz naevi: a diagnostic challenge for fluorescence in-situ hybridization analysis.
Histopathology, 60 (2012), pp. 336-346
[155]
A.K. Isaac, T. Lertsburapa, J. Pathria Mundi, M. Martini, J. Guitart, P. Gerami.
Polyploidy in spitz nevi: a not uncommon karyotypic abnormality identifiable by fluorescence in situ hybridization.
Am J Dermatopathol, 32 (2010), pp. 144-148
[156]
P. Gerami, G. Li, P. Pouryazdanparast, B. Blondin, B. Beilfuss, C. Slenk, et al.
A highly specific and discriminatory FISH assay for distinguishing between benign and malignant melanocytic neoplasms.
Am J Surg Pathol, 36 (2012), pp. 808-817
[157]
B. Gammon, B. Beilfuss, J. Guitart, P. Gerami.
Enhanced detection of spitzoid melanomas using fluorescence in situ hybridization with 9p21 as an adjunctive probe.
Am J Surg Pathol, 36 (2012), pp. 81-88
[158]
M.M. Gessel, J.L. Norris, R.M. Caprioli.
MALDI imaging mass spectrometry: spatial molecular analysis to enable a new age of discovery.
J Proteomics, 107 (2014), pp. 71-82
[159]
W.M. Hardesty, R.M. Caprioli.
In situ molecular imaging of proteins in tissues using mass spectrometry.
Anal Bioanal Chem, 391 (2008), pp. 899-903
[160]
R. Lazova, E.H. Seeley, M. Keenan, R. Gueorguieva, R.M. Caprioli.
Imaging mass spectrometry – a new and promising method to differentiate Spitz nevi from Spitzoid malignant melanomas.
Am J Dermatopathol, 34 (2012), pp. 82-90
[161]
A.K. Alomari, E.J. Glusac, J. Choi, P. Hui, E.H. Seeley, R.M. Caprioli, et al.
Congenital nevi versus metastatic melanoma in a newborn to a mother with malignant melanoma - diagnosis supported by sex chromosome analysis and Imaging Mass Spectrometry.
J Cutan Pathol, 42 (2015), pp. 757-764
Copyright © 2016. AEDV
Idiomas
Actas Dermo-Sifiliográficas
Article options
Tools
es en

¿Es usted profesional sanitario apto para prescribir o dispensar medicamentos?

Are you a health professional able to prescribe or dispense drugs?