Diagnosis of melanocytic proliferations remains one of

Update on Fluorescence In Situ Hybridization in Melanoma State of the Art Pedram Gerami, MD; Artur Zembowicz, MD, PhD N Context. Recent advances in understanding the molecular basis of melanoma have resulted in development of fluorescence in situ hybridization (FISH) protocols designed to detect genetic abnormalities discriminating melanoma from nevi. The most extensively studied is a 4- probe multicolor FISH probe panel targeting chromosomes 6 and 11. Validation studies showed promising sensitivity and specificity for distinguishing benign nevi and malignant melanoma by FISH. Recent studies show that a melanoma FISH assay has great potential for becoming an important diagnostic adjunct in classification of melanocytic lesions and in diagnosis of melanoma. Objective. To present a comprehensive review of the science and practical aspects of FISH in melanoma for pathologists considering the use of melanoma FISH in their practice. Data Sources. Review of the literature and personal experience of the authors. Conclusions. Judicious use of a 4-probe multicolor melanoma FISH procedure can enhance accuracy for diagnosis of melanoma and improve classification of melanocytic proliferations. (Arch Pathol Lab Med. 2011;135:830 837) Diagnosis of melanocytic proliferations remains one of the most challenging areas in pathology. An unacceptably high number of lesions cannot be precisely and reproducibly classified as either entirely benign or malignant by routine histologic and immunohistochemical techniques. Recent advances in the understanding of molecular pathogenesis of melanocytic proliferations have revealed many genetic differences between benign nevi and melanoma, which could be exploited as targets for developing molecular diagnostic tests. It appears that most benign nevi are driven by point mutations in selected oncogenes but only exceptionally rarely do they show gross chromosomal abnormalities. 1 In contrast, tumor progression from a nevus to melanoma is associated with chromosomal instability (CI) resulting in gains, amplifications, and/or losses of specific chromosomal material, which can be detected by genetic techniques. 2,3 Because of its relatively low cost, the possibility of performing the test on archival material, and its increasing availability in pathology laboratories, fluorescence in situ Accepted for publication March 3, 2011. From the Department of Dermatology, Northwestern University and the Feinberg School of Medicine, Chicago, Illinois (Dr Gerami); and the Department of Pathology, Lahey Clinic, Burlington, Massachusetts, and www.dermatopathologyconsultations.com, Boston, Massachusetts (Dr Zembowicz). Dr Zembowicz serves as a clinical consultant to NeoGenomics Laboratories (Irvine, California). In this capacity he received honoraria for second-opinion consultations on cases submitted for melanoma FISH. The authors have no other relevant financial interest in the products or companies described in this article. Reprints: Artur Zembowicz, MD, PhD, Department of Pathology, www.dermatopathologyconsultations.com, 6th Floor, 133 Brookline Ave, Boston, MA (e-mail: dr.z@dermatopathologyconsultations.com). hybridization (FISH) has emerged as a preferred molecular technique to interrogate chromosomal abnormalities. Fluorescence in situ hybridization has proven its utility as a diagnostic adjunct in a number of lymphoid and solid tumors and has been recently developed and validated for diagnosis of melanoma. 3,4 The goal of this article is to describe the development of the first 4-probe melanoma FISH test currently commercialized by Abbott Molecular (Abbott Park, Illinois) and to assess its current place and future prospects in evaluation of melanocytic tumors. BACKGROUND Chromosomal instability has long been recognized as a hallmark of cancer. 4 There are a number of distinct mechanisms leading to CI, which include mutations or alterations in genes encoding centromeric proteins and regulators of the cell cycle and mitosis, as well as telomeric loss with fusion of chromatids and subsequent chromosome fragmentation during mitosis. 5 9 Aberrations in chromosomal copy numbers are among the most common of the myriad number and types of chromosomal aberrations that may occur as a result of CI. 2 There are several reasons why CI may contribute to the progression of cancer and result in clones of cells with copy number aberrations. Chromosomal instability results in genetic diversity and facilitates evolutionary selection. 2,10 Selection pressures result in growth advantage of tumor clones, which have copy number increases in oncogenes and copy number losses in tumor suppressor genes. Hence, cancers are frequently characterized by clones of cells with extra copies of chromosomal loci carrying oncogenes and with loss of tumor suppressor genes. 2 Conversely, the frequency of chromosomal copy number changes is significantly 830 Arch Pathol Lab Med Vol 135, July 2011 FISH in Melanoma Gerami & Zembowicz

less in benign tumors and, when present, these changes typically involve very limited and/or specific chromosomal loci. This dichotomy in chromosomal copy number aberrations between benign and malignant tumors was efficiently exploited in 1992 with the introduction of a novel technique known as comparative genomic hybridization (CGH). 11 This technique allowed for evaluation of the entire genome of a tumor of interest for copy number aberrations in 1 experiment. In the traditional method, tumor DNA and reference DNA are labeled with different fluorochromes and are then simultaneously hybridized to immobilized and denatured chromosome metaphase spreads. 11,12 The fluorescence ratio along the axis of each chromosome can then be measured, indicating the presence of DNA gains or losses from the tumor sample at each chromosomal locus. The development of an array platform for CGH has since greatly increased the magnitude of resolution, allowing for significantly more precise definition of the area of chromosomal loss or gain. Array platforms may consist of DNA derived from bacterial artificial clones, complementary DNAs, selected polymerase chain reaction products, and oligonucleotides. 11,12 CHROMOSOMAL INSTABILITY IN MELANOCYTIC PROLIFERATIONS In 2003, Bastian and colleagues 1 published their findings regarding the evaluation of 132 melanomas and 54 benign nevi with CGH. This study showed that 96% of the melanomas had some type of chromosomal copy number aberration, while chromosomal copy number aberrations were only rarely seen in nevi. Frequent aberrations among the melanomas included gains in 6p, 1q, 7p, 7q, 8q, 17q, 11q, and 20q, and common losses included 9p, 9q, 10q, 10p, and 6q. Among the 54 nevi evaluated, only 7 lesions (13%), all Spitz nevi, had the same isolated gain in the entire short arm of chromosome 11. 1 Conversely, this particular aberration was not found in any of the 132 melanomas. Subsequent studies showed that the large bulky Spitz nevi, with sclerotic changes at the base, were morphologically the most likely type of Spitz nevus to carry this isolated gain in 11p. Also, these same tumors frequently had activating mutations in HRAS, which is located on 11p. 13,14 In subsequent studies, CGH demonstrated a distinct difference in the pattern of chromosomal aberrations seen in nodular proliferations within congenital nevi compared to melanoma arising in congenital nevi. In 6 of 10 proliferative nodules evaluated, whole chromosomal losses of either chromosome 9, 10, or 7 were noted. 15 In contrast, the melanomas showed mostly partial chromosomal gains and losses, typical of the findings seen in previous studies of melanoma with CGH. This finding showed not only that CGH may have utility as a diagnostic tool showing differences between these different types of melanocytic proliferations but also that the basic mechanism for genomic instability in these different tumor types may be different. Melanomas that have gains and losses in chromosomal fragments have most likely reached a crisis stage, with binding of chromatids and uneven breakage during mitosis. Conversely, changes in whole-chromosome copy number may result from the defects of the mitotic chromosome segregation apparatus. These findings laid the foundation for the development of CGH as a diagnostic tool. Comparative genomic hybridization is now used internationally in some selected centers as a diagnostic tool for histologically ambiguous melanocytic tumors. However, there are many drawbacks, mainly, the arduous and time-consuming nature of the procedure. Tumor cells must be microdissected from the specimen, which typically requires a paraffin block approximately 20 mm thick. For large bulky tumors, this can be done with a conventional dissecting microscope. However, as a relatively pure tumor population is required for the procedure, if the tumor is smaller or if a select group of cells is desired for evaluation, laser-capture microdissection may be necessary. The DNA then needs to be isolated and purified from the tumor sample, labeled with a fluorochrome, and hybridized. Additionally, the copy number change must be represented by at least 30% to 50% of the collected cells for it to be evident in the analysis. 16 Hence, CGH may yield a false-negative result in melanoma arising with a nevus in which the nevus component predominates. Conversely, if an aberration is identified, then the findings are typically very specific, since the aberration must be highly representative of the examined cell population to be identified in the analysis. Additional benefits obviously include the ability to scan for all possible copy number aberrations in a single experiment. PRINCIPLES OF FLUORESCENCE IN SITU HYBRIDIZATION Fluorescence in situ hybridization (FISH) is another method that can be used to look at chromosomal copy number aberrations. 17 Short DNA fragments (FISH probes) are hybridized to a 5 mm thick, formalin-fixed, paraffin-embedded section of a tumor of interest. Overlapping wavelength spectrums of the currently available fluorochromes limit to a maximum of 4 the number of probes that can be concurrently hybridized in a single experiment. Hence, in contrast to CGH, a maximum of 4 chromosomal targets can be evaluated in a single FISH test. Nonbound, fluorescently labeled DNA is washed away. The section on a slide can then be examined under a fluorescence microscope. Fluorescence in situ hybridization offers the possibility of examining individual tumor cells in different areas of a lesion and of correlating the results with tumor morphology under conventional light microscopy. Each fluorescently labeled fragment of DNA that hybridizes to a tumor cell nucleus will appear as a distinct fluorescent dot. Each dot identifies a single copy of the chromosomal locus with a homologous DNA. In an idealized scenario, each diploid nucleus will bind 2 probes, resulting in 2 dots (Figure 1, A). If a DNA region of interest is gained, more than 2 dots will be observed. If a DNA region of interest is deleted, less than 2 (1 or 0) signals will be identified. In a real-life scenario, for many perfectly normal diploid cells, probes will be outside the plane of section or overlapping, resulting in less than 2 dots per nucleus. Imperfect hybridization can also result in false-positive or false-negative signals. The number of dots per nucleus with a specific fluorescent color can then be detected either manually or with software designed for automated analysis. Because of intrinsic variability of FISH signals, a sufficient number of cells must be examined in each experiment and strict quality control measures Arch Pathol Lab Med Vol 135, July 2011 FISH in Melanoma Gerami & Zembowicz 831

Figure 1. Abbott Molecular s 4-probe melanoma fluorescence in situ hybridization (FISH) test. A, The figure shows a composite of 2 images for 2 representative normal nuclei from a benign nevus. Each of the images shows pairs of red, aqua, yellow, and green dots. Each signal represents a fluorochrome-labeled probe bound to a copy of a complementary DNA of a particular gene (see B for details). The images are from Metafer Slide Scanning System (MetaSystems, Altlussheim, Germany). The numbers in the left upper corner indicate image numbers and the aqua, red, yellow, and green numbers at the bottom of each image indicate number of signals for each probe, calculated by the automatic algorithm. B, The assay uses 4 fluorescent dye tagged probes, which are hybridized to 5-mm thick, formalin-fixed, paraffin-embedded sections. Three probes bind to loci on chromosome 6, and 1 binds to a locus on chromosome 11. RREB1 (ras responsive element binding protein 1) probe (labeled with a red fluorochrome) corresponds to 6p25; CEN6 (centromere 6) probe (labeled with an aqua fluorochrome) corresponds to a centromeric region of chromosome 6; MYB (v-myb myeloblastosis viral oncogene homologue) probe (labeled with a yellow fluorochrome) corresponds to 6q23; and CCND1 (cyclin D1) probe (labeled with a green fluorochrome) corresponds to 11q13 (original magnification 3640 under oil immersion [A]). must be implemented. The collected data are presented as a percentage of nuclei containing more than 2 copies of a particular locus or as a percentage of cells showing an increase or loss of signals in comparison to a probe directed against a centromere region of a chromosome. The signal cutoffs that is, a percentage of cells that must fulfill a particular FISH criterion for a result to be considered abnormal have to be determined and validated empirically for each probe, with appropriate controls. Having rigorously interrogated cutoff values is critical to the robustness of a FISH assay. Increasing the cutoff values will increase the specificity of a test at the expense of the sensitivity and vice versa. Minor variations in cutoff values between laboratories are expected. Fluorescence in situ hybridization became the favorite method of evaluation of CI by pathologists, as it is applied to standard 5 mm thick, paraffin-embedded sections and allows for histopathologic correlation. A pathologist can select a specific area or areas of tumor to be examined by FISH. Also, as the background fluorescence of DAPI (49,6- diamidino-2-phenylindole) allows visualization of the tumor s silhouette, with some experience one can easily make correlations between the histopathologic appearance and the chromosomal aberrations. Hence, the technique is simple, rapid (can be completed in 2 days), fits into existing pathology laboratory workflows, and is relatively inexpensive. However, it requires highly trained technical staff. DEVELOPMENT AND VALIDATION OF FLUORESCENCE IN SITU HYBRIDIZATION TEST FOR MELANOMA The process of formulating a FISH test for melanoma was initiated by examining CGH data for potential highyield targets. A combinatorial analysis of the CGH data set of Bastian and colleagues 18 yielded 13 regions on 8 different chromosomes, namely, chromosomes 1, 6, 7, 9, 10, 11, 17, and 20, which in combination, yielded the best discriminatory tool for distinguishing between melanomas and nevi. Additionally, a probe targeting KIT on chromosome 4 was also added to the group of 13 regions because of its potential as a therapeutic target. Fluorescence in situ hybridization probes targeting these 14 regions were assembled. If the chromosomal locus was commonly gained, then a well-described oncogene from the region was selected as the target of the FISH probe. Conversely, if a chromosomal region was commonly deleted, then a tumor suppressor gene was selected as the target of the FISH probe. The FISH probes were arranged in panels consisting of either 3 or 4 probes, with each probe within a panel labeled with a different spectrally distinct fluorophore. Each panel was then hybridized to overlapping subsets of a group of 97 melanomas and 95 nevi. From this analysis, a combination of 4 probes was identified as having the best combined discriminatory ability for distinguishing melanoma from benign nevi. This panel consisted of ras responsive element binding protein 1 (RREB1, 6p25), v-myb myeloblastosis viral oncogene homologue (MYB, 6q23), CEN6 (centromere 6), and cyclin D1 (CCND1, 11q13) probes 18 (Figure 1, B). The new 4-probe panel was subsequently applied to an additional cohort of 58 melanomas and 51 nevi at the University of California at San Francisco (UCSF) to determine the ideal parameters and signal cutoffs resulting in the best sensitivity and specificity when discriminating between melanoma and nevi. From this analysis, the following 4 FISH criteria were identified: (1) if more than 38% of enumerated cells contained more than 2 signals for CCND1 (11q13) or (2) if more than 55% of nuclei contained more signals for 6p25 than for centromere 6 or (3) if more than 40% of nuclei contained fewer signals for MYB (6q23) than for centromere 6 or (4) if more than 29% of cells had more than 2 RREB1 (6p25) signals, then a case would be considered as positive for melanoma 18 (Table). To validate the UCSF cutoff parameters, they were then applied to a third cohort of 83 melanomas and 86 nevi at Northwestern University (Chicago, Illinois). The parameters resulted in a sensitivity of 86.7% and a specificity of 95.4%. 19 The above pioneering proof-of-concept studies demonstrated that a 4-probe FISH test targeting 6p, centromere 832 Arch Pathol Lab Med Vol 135, July 2011 FISH in Melanoma Gerami & Zembowicz

Criterion Abbott Molecular s 4-Probe Melanoma Fluorescence In Situ Hybridization Test Signal 6, 6q, and 11q can discriminate between nevi andmelanoma. This test has been patented by and is commercialized worldwide by Abbott Molecular. Efforts to develop other 4-probe FISH tests are underway, and other tests possibly may be developed and used at different institutions. However, the focus of this review is Abbott Molecular s melanoma FISH procedure. Unless specifically indicated, the subsequent discussion of FISH is restricted to this particular test. The principle of the Abbott melanoma FISH test is illustrated in Figure 1, B. ABBOTT MELANOMA FISH TEST: CRITICAL REAPPRAISAL AND EARLY EXPERIENCE For pathologists and clinicians contemplating incorporating melanoma FISH in their practice, it is critical to understand nuances of the FISH technique and potential pitfalls in order to appropriately interpret the results. The initial estimates of the sensitivity and specificity (even though relatively high for a laboratory test) of the melanoma FISH test are too low to satisfy the expectations for accuracy for a pathologic diagnosis of melanoma. Thus, melanoma FISH must not be used as a stand-alone test and has to be considered as a diagnostic adjunct to routine pathologic examination of tissue and clinicopathologic correlation. For example, in the Northwestern study, 18 there were a total of 4 false positives among the 86 nevi, including 2 lesions labeled as Spitz nevi and 2 dysplastic nevi. On subsequent retrospective review, one of the Spitz nevi showed considerable cytologic atypia even though overall it was a well-circumscribed small lesion. The false positivity in this case was a result of tetraploidy (discussed later). On retrospective review of the histologic appearance, the other lesion labeled as a Spitz nevus was reclassified as atypical Spitz tumor. The reasons for false-positive results in dysplastic nevi were not determined. False positives have been reported by other groups as well. 20 Most false positives can be avoided by recognizing technical problems, such as poor hybridization, as well as familiarity with tetraploidy. Technical insufficiencies should be detected and, if possible, corrected by a technical staff during the quality control process. However, as many as 3% to 5% of samples may be inadequate for evaluation. Tetraploid cells are frequently found in Spitz nevi. An example of a spitzoid melanoma with tetraploid cells is illustrated in Figure 2, A through C. In 5% to 10% of lesions they are present frequently enough to cause a falsepositive result with the current FISH criteria if they are not identified as tetraploid cells by the interpreter of the assay. UCSF/Northwestern University Cutoff, % NeoGenomics Cutoff, % 6p25 gain.2 RREB1 (red).29.16 6p25 gain RREB1 (red). CEN6 (aqua).55.53 6p23 loss MYB (yellow), CEN6 (aqua).40.42 11q13 gain.2 CCND1 (green).38.19 Criteria and signal cutoffs were established by University of California in San Francisco UCSF/Northwestern University (Chicago, Illinois) studies and NeoGenomics Laboratories (Irvine, California). By NeoGenomics criteria, the fluorescence in situ hybridization test is considered abnormal if (1) 16% of cells show more than 2 red signals (RREB1 probe), indicating gain of 6p25 locus; (2) 53% of cells show more red (RREB1 probe) than aqua (centromere 6 probe) signals, indicating gain of 6p25 locus (RREB1); (3) 42% of cells show fewer yellow (MYB probe) than aqua (centromere 6) signals, indicating loss of 6q23 locus; and (4) more than 19% of cells show more than 2 green signals (CCND1 probe), indicating gain of 11p13 locus. For each of these 4 criteria, UCSF/Northwestern cutoffs were 29%, 55%, 40%, and 38%, respectively. CCND1, cyclin D1; CEN6, centromere 6; MYB, v-myb myeloblastosis virus oncogene homologue; RREB1, ras responsive element binding protein 1. Tetraploid cells can be readily identified by the pattern of increased copy number changes affecting all 4 probes (Figure 2, D). However, the presence of tetraploid cells is not diagnostic of Spitz nevus, as they can also be found in melanomas, including spitzoid melanoma, and other atypical nevi. Signal cutoffs for FISH are empirically derived values and may vary between different laboratories owing to different hybridization conditions, quantification methods, experience of staff and, importantly, desired sensitivity/specificity profile of the test. The number of falsepositive results can be decreased by increasing the signal cutoffs. However, this will result in an unavoidable decrease in the sensitivity of FISH. The signal cutoff will also depend on the scoring technique. The signal cutoffs recommended by NeoGenomics Laboratories (Irvine, California), which uses an automated method of scoring nuclei (company materials reviewed by the authors), are lower than those established in the initial studies. In their validation studies using a cohort of 157 nevi and 167 melanomas, signal cutoffs of 16% (RREB1, 6p25), 53% (RREB1:CEN6, 6p25:cen6), 42% (MYB:CEN6, 6q23:cen6), and 19% (CCND1, 11q13) were selected, resulting in 95% specificity and 84% sensitivity (Table). When Gerami s signal cutoffs were applied, the specificity was 98% and the sensitivity was reduced to 64%. It is understood that the signal cutoffs will have to be constantly validated in prospective studies and that the specificity and sensitivity of the assay may change over time. The most current NeoGenomics data indicate that, with their criteria, up to 25% of unequivocal melanomas are expected to show a normal FISH signal pattern. It is also likely that the sensitivity or specificity of the melanoma FISH test will depend on the histologic/molecular type of melanoma. For instance, with the current criteria, melanoma FISH results are negative in 50% of desmoplastic melanomas. 21 Validation studies in different tumor cohorts remain to be performed; as with nodular melanomas, the sensitivity may be as high as 90%. 22 It is currently unclear to what extent combining melanoma FISH with CGH can improve molecular diagnosis of melanoma. Fluorescence in situ hybridization is the only technique that can be applied to superficial melanocytic tumors or melanocytic tumors in which the malignant component may consist of a relatively small proportion of the overall cell population, as well as large bulky tumors. 23 It is important to remember that discrepancies between FISH and CGH results are expected. With FISH one can identify even small clones of chromosomally aberrant cells within a Arch Pathol Lab Med Vol 135, July 2011 FISH in Melanoma Gerami & Zembowicz 833

Figure 2. Spitzoid melanoma with tetraploidy from the files of one of the authors (A.Z.). A, A superficial shave biopsy specimen of a rapidly growing pigmented lesion from a left ankle lesion in a 17-year-old adolescent girl. B and C, The tumor showed pagetoid spread, consumption of epidermis, and severe cytologic epithelioid cell atypia, allowing diagnosis of malignant melanoma. D, Composite fluorescence in situ hybridization (FISH) images of 4 tetraploid nuclei from this case, each showing 3 to 4 signals for CCND1 (cyclin D1, 11q13, green), RREB1 (ras responsive element binding protein 1, 6p25, red), MYB (v-myb myeloblastosis viral oncogene homologue, yellow, 6q23) and centromere 6 (aqua). The images are from Metafer Slide Scanning System (MetaSystems, Altlussheim, Germany). The numbers in the left upper corner indicate image numbers and the aqua, red, yellow, and green numbers at the bottom of each image indicate number of signals for each probe, calculated by the automatic algorithm (hematoxylin-eosin, original magnifications 320 [A], 3100 [B], and 3400 [C]; FISH, original magnification 3640 under oil immersion [D]). larger tumor, while CGH can detect chromosomal abnormalities only if they involve a high percentage of cells. For example, a melanoma with a predominant copy number aberration involving 7q, but only a small focus of abnormality in 6p, if studied by FISH and CGH may have different results. The CGH study may only identify the aberration in 7q because this abnormality is represented by greater than 30% of the tumor cells, while the aberration in 6p is sufficiently present to be detected by FISH but not by CGH. This explains a highly interesting finding: by targeting just 4 probes, the sensitivity for detecting melanoma can be as high as 85%.22 The authors cannot overemphasize the fact that evaluation of routine sections to select the most appropriate area of the tumor, and subsequent correlation between FISH results and available clinicopathologic information, are an integral part of the melanoma FISH test. 834 Arch Pathol Lab Med Vol 135, July 2011 FLUORESCENCE IN SITU HYBRIDIZATION IN HISTOLOGICALLY AMBIGUOUS MELANOCYTIC PROLIFERATIONS Validation studies established sensitivity and specificity of melanoma FISH test in distinguishing between undisputed benign nevi and conventional melanomas. Obviously, FISH is not needed to confirm the diagnosis in histologically unambiguous cases. In routine practice, diagnostic utility of FISH will eventually depend on whether it can help to classify histologically ambiguous/ borderline lesions and/or provide additional prognostic or management information. Several proof-of-principle studies23 26 have shown potential applications of FISH for solving a variety of diagnostic dilemmas in the evaluation of melanocytic tumors, including differentiating blue nevus like metastasis from blue nevus, mitotically active nevus from nevoid FISH in Melanoma Gerami & Zembowicz

melanoma, and dysplastic nevi from superficial spreading melanoma. Fluorescence in situ hybridization abnormalities characteristic of melanoma were identified in lentiginous melanoma, supporting this entity as a variant of melanoma. 27 These studies compared unequivocal benign and malignant counterparts and showed that the signal cutoffs established in validation studies can be applied to different types of melanocytic lesions. Very few studies have examined application of FISH in diagnosis of histologically ambiguous/borderline lesions. In the last phase of the UCSF/Northwestern validation studies, 27 melanocytic tumors, deemed histologically ambiguous by expert dermatopathologists, were evaluated by FISH. The differential diagnosis in most cases consisted of atypical Spitz tumor versus melanoma. All cases had 5 years of clinical follow-up, and presence or absence of bulky or distant metastasis was the primary end point. All 6 metastatic lesions (100%) had a positive FISH result, while only 6 of 21 remaining nonmetastatic lesions (29%) had FISH-positive results. The correlation between FISH and metastatic behavior was statistically highly significant. 18 A similar conclusion was reached by a European group led by Vergier et al. 28 The authors compared 113 ambiguous melanocytic tumors with and without evidence of recurrence at 5 years and found that FISH analysis improved the sensitivity and specificity of diagnosis as compared with expert opinion alone. Conversely, in evaluating 12 ambiguous melanocytic tumors using clinical outcome as a the final end-point measure, Gaiser et al 29 concluded that FISH assay did not reach a clinically useful sensitivity and specificity. It should be noted that Vergier and colleagues used the same criteria developed in the multi-institutional study from UCSF and Northwestern University for determining a positive or negative FISH result, while Gaiser et al used a different set of criteria developed earlier that were based on a preliminary analysis of a more limited number of cases that never reached a validation stage. Thus, it is not surprising that the study of Gaiser et al also reported a lower correlation between FISH results and histologic analysis of unambiguous nevi and melanomas than other validations studies. They also showed low correlation between FISH results and CGH results. However, as discussed earlier, this is not an unexpected finding and does not disqualify FISH results. The above studies demonstrated that, when performed as described in the original validation studies and using the same cutoffs, the Abbott Molecular s melanoma FISH test has potential to become a useful adjunct in evaluation of ambiguous melanocytic tumors. Yet, further studies and experience is needed to determine the best practices and parameters for its usage. It is unreasonable to expect that objective validation data, similar to those for unequivocal nevi and melanoma, can be obtained for histologically ambiguous lesions. As decades of inconclusive clinicopathologic and consensus studies have shown, research on controversial melanocytic lesions is very difficult. 30 33 These lesions are morphologically diverse and there are no established and validated case sets of histologically ambiguous lesions that could serve as a reference. Studies comparing sets of lesions with known long-term follow-up are important but also have their limitations. Recent studies have established that the group of histologically ambiguous lesions contains distinct entities that can metastasize to lymph nodes but have limited ability for distant spread. A prototype of such an entity is pigmented epithelioid melanocytoma. 34 36 Published evidence from a number of institutions from Europe, the United States, and Australia indicates that most atypical Spitz tumors display similarly limited malignant potential. 30,37 39 Zembowicz and Scolyer 40 recently went as far as to propose that a dichotomous classification mandating classification of melanocytic tumors as either benign (nevi) or malignant (melanoma) might be replaced with a classification scheme containing a third diagnostic category of melanocytoma, reserved for tumors with malignant potential restricted to lymph node metastases in most cases. Although highly desirable, even a long-term follow-up using tumor-related death as an end point has limited value, as a significant percentage of unambiguous melanomas are not lethal. Thus, unless coupled with new, currently nonexisting technologies capable of providing objective information about benign or malignant potential of a lesion, it is unlikely that clinicopathologic correlation studies will establish objective specificity and sensitivity of the melanoma FISH test in borderline melanocytic tumors in the near future. Thus, while the melanoma FISH assay is very likely to significantly advance our diagnostic armamentarium, melanoma FISH is unlikely to entirely solve the problem associated with controversial melanocytic proliferation and differences of opinion about a particular lesion. FLUORESCENCE IN SITU HYBRIDIZATION IN HISTOLOGICALLY AMBIGUOUS MELANOCYTIC LESIONS: PERSONAL APPROACH The authors routinely use Abbott Molecular s melanoma FISH test as a diagnostic adjunct in virtually all borderline cases with sufficient tissue. Examples of 2 unusual melanomas with positive FISH results are illustrated in Figure 3, A through F. The process starts with examination of routine sections, formulation of a preliminary diagnosis, and selection of an area, and in some cases, multiple areas, to be interrogated by FISH. After FISH is performed and the results are interpreted, we reexamine the case to reconcile the findings and to formulate the final interpretation. If the FISH results are abnormal and the histologic appearance is compatible with melanoma, a definitive diagnosis of melanoma is usually made. Determination of FISH positivity certainly helps in making a diagnosis for a suspected spitzoid melanoma. However, we usually do not automatically reclassify an atypical Spitz tumor as an outright melanoma but rather make a correlative diagnosis including both the histopathologic and molecular features. If the FISH result is normal and the initial impression was that of a severely atypical but benign lesion, the FISH findings are interpreted as reassuring, and in most cases a biopsy is reported as consistent with an atypical nevus. In the NeoGenomics study, up to 25% of unequivocal melanomas have yielded a FISH-negative result, which is least helpful in a context of a highly suspicious lesion. In such tumors, the initial diagnosis of a histologically ambiguous/borderline melanocytic tumor of uncertain malignant potential is not changed. Cases with an abnormal FISH result, but showing histologic features at the low end of the spectrum of atypia, need to be carefully evaluated. This is especially true in a clinical context with a low Arch Pathol Lab Med Vol 135, July 2011 FISH in Melanoma Gerami & Zembowicz 835

Figure 3. Two malignant melanomas from the files of one of the authors (P.G.). A, Low-magnification view of a spitzoid melanoma from a 25-year-old woman. B, Higher-magnification view of this lesion showing lack of maturation and sheetlike growth of monotonous appearing melanocytes. C, Fluorescence in situ hybridization (FISH) image shows increased copies of RREB1 (ras responsive element binding protein 1, red) and decreased copies of MYB (v-myb myeloblastosis viral oncogene homologue, yellow), indicating a clonal chromosomal imbalance involving chromosome 6. D, Low-magnification view of a melanoma in a 2-year-old child. E, Higher-magnification view of this lesion shows ulceration of the dermis and sheetlike proliferation of atypical melanocytic cells with no maturation. F, A corresponding FISH image shows normal numbers of RREB1 signals (red) with most cells showing loss of MYB, characterized by only a single copy of the yellow signal in most cells (hematoxylineosin, original magnifications 320 [A], 3400 [B and E], and 340 [D]; FISH, original magnifications 3400 [C and F]). probability of melanoma, such as in young children. As explained earlier, such cases have to be carefully examined to rule out a false-positive result due to tetraploidy, which, in the authors experience, is by far the most common source of an abnormal FISH result in this scenario. From a theoretic standpoint, there is no question that malignant tumors have greater genomic instability than benign tumors and, hence, should have more measureable chromosomal copy number aberrations. Thus, when debating how much weight to put on a positive FISH result in a lesion initially thought to most likely represent an atypical nevus, FISH data are harder to ignore if a large percentage of cells show chromosomal abnormalities or if a tumor shows positivity by multiple FISH criteria. One also has to bear in mind that FISH can detect a focus of melanoma too small to produce a histologically diagnostic morphology. Thus, very cautious judgment has to be exercised when reporting FISHpositive ambiguous lesions in which routine histologic analysis does not allow for an outright diagnosis of melanoma. Yet, as shown by the validation studies, true false-positive FISH results can occur. Such lesions have to be reported as benign. IMPLEMENTATION OF MELANOMA FISH TEST IN PATHOLOGY LABORATORIES Outside of the United States, Abbott Molecular s melanoma FISH assay is available as a diagnostic kit, and the test can be performed in-house if technical expertise and equipment are available. In the United 836 Arch Pathol Lab Med Vol 135, July 2011 States, melanoma FISH is only performed at the academic centers involved in the development of the test (UCSF, Northwestern University, and Memorial Sloan-Kettering Cancer Center in New York, NY). The test is also available as a commercial service for the NeoGenomics Laboratories. NeoGenomics Laboratories offers it as a technical component only for pathologists wishing to interpret the results themselves and as a global interpretation where both the technical and professional components of the FISH test are performed by NeoGenomics consulting physicians. The authors believe that FISH is not a standalone test and must be interpreted in conjunction with evaluation of routine sections; thus, we only do FISH as part of comprehensive consultations sent to us. References 1. Bastian BC, Olshen AB, LeBoit PE, et al. Classifying melanocytic tumors based on DNA copy number changes. Am J Pathol. 2003;163(5):1765 1770. 2. Albertson DG, Collins C, McCormick F, et al. Chromosome aberrations in solid tumors [review]. Nat Genet. 2003;34(4):369 376. 3. Bastian BC. Understanding the progression of melanocytic neoplasia using genomic analysis: from fields to cancer [review]. Oncogene. 2003;22(20):3081 3086. 4. Sandberg AA, Chen Z. Cytogenetics and molecular genetics of human cancer. Am J Med Genet. 2002;115(3):111 112. 5. Boland CR, Komarova NL, Goel A. Chromosomal instability and cancer: not just one CINgle mechanism. Gut. 2009;58(2):163 164. 6. Cheung AL, Deng W. Telomere dysfunction, genome instability and cancer [review]. Front Biosci. 2008;13:2075 2090. 7. Ganem NJ, Godinho SA, Pellman D. A mechanism linking extra centrosomes to chromosomal instability. Nature. 2009;460(7252):278 282. 8. Murnane JP. Telomere loss as a mechanism for chromosome instability in human cancer [review]. Cancer Res. 2010;70(11):4255 4259. FISH in Melanoma Gerami & Zembowicz

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