Emerging Clinical Applications Of Selected Biomarkers In .
Clinical, Cosmetic and Investigational DermatologyDovepressopen access to scientific and medical researchReviewOpen Access Full Text ArticleEmerging clinical applications of selectedbiomarkers in melanomaThis article was published in the following Dove Press journal:Clinical, Cosmetic and Investigational Dermatology30 January 2015Number of times this article has been viewedMichael T Tetzlaff 1Carlos A Torres-Cabala 1,2PenvadeePattanaprichakul 1,3Ronald P Rapini 2Victor G Prieto 1,2Jonathan L Curry 1,2Department of Pathology, Sectionof Dermatopathology, 2Departmentof Dermatology, The University ofTexas MD Anderson Cancer Center,Houston, TX, USA; 3Department ofDermatology, Faculty of Medicine,Siriraj Hospital, Mahidol University,Bangkok, Thailand1Correspondence: Jonathan L CurryDepartment of Pathology, Unit 85,The University of Texas MD AndersonCancer Center, 1515 Holcombe Blvd,Houston, TX 77030, USATel 1 713 792 2956Fax 1 713 745 8228Email jlcurry@mdanderson.orgAbstract: Melanoma is a lethal skin disease with a mostly predictable clinical course accordingto a known constellation of clinical and pathologic features. The distinction of melanoma frombenign melanocytic nevus is typically unequivocol; however, there is a subset of tumors knownfor its diagnostic challenges, development of late metastases, and difficulties in treatment. Severalmelanocytic tissue biomarkers are available that can facilitate the histopathologic interpretationof melanoma as well as provide insight into the biologic potential and mutational status of thisdisease. This review describes the clinical application of some of these established and emerging tissue biomarkers available to assess melanocytic differentiation, vascular invasion, mitoticcapacity, and mutation status. The selected tissue biomarkers in this review include MiTF, Sox10,D2-40, PHH3, H3KT (anti-H3K79me3T80ph), anti-BRAFV600E, and anti-BAP-1.Keywords: immunohistochemistry, melanocytic differentiation, histone marks, BRAFV600EIntroductionMelanoma is a deadly skin disease known for its aggressive clinical behavior andhigh propensity for lethal metastasis.1,2 High throughput genomic analysis with nextgeneration sequencing technology has allowed us to interrogate whole exomes andgenomes in melanoma, which have provided insight to the key oncogenic pathwaysinvolved in melanomagenesis3,4 RAS/mitogen-activated protein kinase (MAPK)pathway and PI3 kinase/AKT pathways continue to emerge as key driver mutations inmelanomas.5,6 The most frequent oncogenic driver mutation identified in melanoma isin BRAF, which exists in approximately 50%–60% of patients with this malignancy.7,8Molecular techniques available for evaluation of BRAFV600E mutations in melanomaare further reviewed in detail elsewhere.9,10Regulation of melanogenesis in melanocytes involves a series of complex biochemical steps, with conversion of L-tyrosine to L-3,4 dihydroxyphenylalanine (L-DOPA)by tyrosinase to final melanin product.11 In fact, it is now clear that L-tyrosine andL-DOPA can act as positive regulators of melanogenesis and melanocyte function.12Tyrosinase is central to melanogenesis, and transcriptional regulation of tyrosinaseinvolves several MiTF-binding sites, further reviewed by Slominski et al.11 Melanogenicactivity is dependent on binding of MiTF to melanogenesis-related proteins; thus, thedevelopment of melanocytes in the epidermis and in the follicle is conserved as wellas melanin production in melanocytes and melanoma cells.12,13Corticotropin releasing hormone and proopiomelanocortin are known to beproduced in the skin in response to environmental stress.14 Melanocytes and melanoma cells predominantly express the α isoform of corticotropin releasing hormone35submit your manuscript www.dovepress.comClinical, Cosmetic and Investigational Dermatology 2015:8 35–46Dovepress 2015 Tetzlaff et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0)License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any furtherpermission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information onhow to request permission may be found at: doi.org/10.2147/CCID.S49578
DovepressTetzlaff et alreceptor-1 (CRHR-1). Upon CRHR-1α activation, cyclicadenosine monophosphate is produced, and throughrelease of proopiomelanocortin peptides (ACTH, α-MSH,β-endorphin), stimulates melanin production in melanocytes.15Activation of CRHR-1 inhibits melanocytes and melanomacell proliferation, making selective antagonists a rationaltarget for future melanoma targeted therapy.15The treatment for localized primary melanoma is surgicalexcision with adequate margins, and for a subset of patientswith high-risk tumors, lymphatic mapping and sentinel lymphnode biopsy is recommended. Adjuvant interferon alfa therapyand chemotherapy have been the standard treatment for patientswith advanced stage disease. However, more recently, targetedtherapy with small molecule inhibitors (eg, vemurafenib) has revolutionized melanoma treatment. In a Phase III trial (BRIM-3),patients who harbor BRAFV600E mutant melanomas whowere treated with vemurafenib had better clinical responseand overall survival rates than did patients treated with chemotherapy.16 Selective targeted therapy against other intracellularmolecules (eg, NRAS, MEK, KIT) are under clinical trials andhold promise for future melanoma therapy. Several publicationsreview current melanoma-targeted therapy.17–19Clinical response to vemurafenib therapy may be dramatic, with complete shrinkage of tumor burden in patients;however, the duration of response has been limited and eventual disease progression frequently occurs within months oftherapy.16 Resistant mechanisms have curtailed long-termtherapeutic benefit from vermurafenib therapy; thus, targetingmultiple pathways or combined therapy with immune checkpoint blockade (eg, anti-CTLA4 and anti-PD-L1) are underclinical investigation.20–23 Further review of resistant mechanisms via protective effects of insulin on melanoma cells orby activation of the PI3K/AKT pathway can be examinedin a study by Chi et al.24 Future application of nanotechno logy in melanoma to improve therapeutic efficacy is furtherreviewed by Chen et al.25Accurate diagnosis of melanoma remains critical tofurther clinical management. Melanoma can demonstrate awide range of morphologic features and may be misinterpreted as other human malignancies (eg, sarcomas, squamouscell carcinomas, Paget’s disease, and lymphomas). Thus,melanoma is known as the “great mimicker.”26 Diagnosisof melanoma can be further complicated since a subset ofambiguous melanocytic lesions may demonstrate featuresoverlapping with melanoma and benign nevi (in particular,Spitz nevi).27 These characteristics make the histologic diagnosis of melanoma challenging for even the most experienceddermatopathologists.36submit your manuscript www.dovepress.comDovepressTo help distinguish melanoma from its imitators,a variety of tissue biomarkers and ancillary techniques(eg, immunohistochemical [IHC] analysis or fluorescence insitu hybridization) are currently available. In fact, hundredsof tissue biomarkers are available in clinical laboratories fordiagnosing melanoma and determining the prognosis andmutation status of this devastating skin disease. This reviewprovides an update on the clinical applications of some ofthe established and emerging melanoma tissue biomarkers used at The University of Texas MD Anderson CancerCenter. Specifically, we will review the following melanomatissue biomarkers (Table 1) :1) melanocytic differentiation[MiTF and Sox10]; 2) vascular invasion [D2-40 and dualIHC marker with MiTF/D2-40 and Sox10/D2-40]; 3) mitoticfigures [PHH3, dual IHC marker with Mart-1/PHH3 andH3KT (anti-H3K79me 3T80ph)]; and 4) mutation status[anti-BRAFV600E, anti-BAP-1].Markers of melanocyticdifferentiationMiTFMiTF (microphthalmia-associated transcription factor) functions in the development and differentiation of a variety ofcell types, including melanocytes.28 There are ten isoformsof MiTF, with the M isoform specifically expressed in melanocytes.29 MiTF regulates the transcription of genes(eg, tyrosinase, tyrosinase-related protein 1 and 2) involved inmelanin synthesis and survival of postnatal melanocytes.28,30,31Thus, MiTF is critical for pigment synthesis and melanocytedifferentiation. MiTF protein functions in the nucleus ofmelanocytes and can be recognized with antibodies directedagainst it. The D5 antibody recognizes human MiTF.32The sensitivity of MiTF in melanocytic lesions exceeds80% and is similar to that of HMB45.33 In desmoplasticmelanomas, however, the sensitivity of MiTF dramaticallydecreases to less than 55% according to some studies.32,34The low sensitivity of MiTF in desmoplastic melanomais comparable to that of HMB45, a marker of premelanosomal glycoprotein 100. Therefore, MiTF appears to exhibitsensitivity comparable to that of HMB45 in melanocyticneoplasms. MiTF and HMB45 differ, however, in theirspecificity. HMB45 is a highly specific marker with greaterthan 97% specificity for melanocytic differentiation.35,36In contrast, the specificity of MiTF in melanocytic lesionsis less, and the widespread use of this biomarker alone inevaluating melanocytic lesions is an important pitfall. MiTFhas been shown to highlight cells other than melanocytes andnonmelanocytic neoplasms. In particular, MiTF reactivityClinical, Cosmetic and Investigational Dermatology 2015:8
DovepressMelanoma biomarkers, clinical applicationTable 1 List of selected tissue biomarkers in melanomaMarkerPropertyClone (vender)ConcentrationIHCpatternInternal controlClinical applicationMiTFNuclear transcriptionfactorD5(Thermo uclear transcriptionfactorPolyclonal(Cell Marque)1:50NEpidermalmelanocytes,adnexae, nerveD2-40O-linkedsialoglycoproteinon lymphatic endotheliumRegulation of xaeDistinction frommelanocytic hyperplasiaand LM confirmationof melanocyticdifferentiationDistinction frommelanocytic hyperplasiaand LM confirmationof melanocyticdifferentiationEvaluate for LVIHistone H3(Ser10) (Millipore)1:400NH3KTRegulation of rotein kinaseUbiquitin hydrolaseand enhance BRCA-1tumor suppressionHistone H3(H3K79me3T80ph)(Millipore)VE1(Spring Bioscience)C-4(Santa Cruz)1:50CMitotic figures inepidermis, adnexa,inflammatory cellsMitotic figures inepidermis, adnexa,inflammatory cellsNone available1:150NKeratinocytesPHH3BAP-1Evaluate or confirmmitotic figureEvaluate or confirmmitotic figureDetect the presence ofBRAFV600E mutationDetect the presence ofBAP-1 mutationAbbreviations: C, cytoplasmic; IHC, immunohistochemistry; LM, lentigo maligna; LVI, lymphovascular invasion; N, nuclear.can be seen in macrophages, fibroblasts, and mast cells andin a variety of spindle cell tumors in the differential diagnosis of melanoma (eg, dermal scar, dermatofibrosarcoma,leiomyosarcoma, neurofibroma, malignant peripheral nervesheath tumor).37 In our experience, MiTF has clinical utility if incorporated with a panel of biomarkers in certainclinical scenarios:38 first, to enumerate melanocytes in theepidermis on chronic sun-damaged skin; second, to detectmelanocytic differentiation in tumors negative for more conventional melanocytic markers (HMB45, anti-tyrosinase, and anti-Mart1); and third, to confirm melanocytic differentiationin rare isolated cells in vascular channels and the presenceof lymphovascular invasion (LVI).Since MiTF is expressed in the nucleus of melanocytes, itfacilitates and accurately highlights the number of melanocytesseen in the epidermis (Figure 1A and B) and lesions of lentigomaligna (Figure 1C).39 This is particularly useful in evaluating melanocytes on atrophic sun-damaged skin that typicallyharbors increased numbers of melanocytes in the epidermis,where perceived melanocytes numbers may be overestimatedand their presence may be misinterpreted as lentigo malignawith Melan A since this marker labels the cytoplasm ofFigure 1 MiTF accurately enumerates the melanocytes in the epidermis of chronic sun exposed skin and in lesions of lentigo maligna.Notes: (A) Chronic sun-damaged skin with atrophic epidermis and solar elastosis (*) with background increase in melanocytes along the dermal–epidermal junction (DEJ)(arrows) (H&E stain, 100); (B) MiTF labels nuclei of melanocytes and allows for better enumeration of melanocytes along the DEJ (IHC stain, 100); (C) Area of lentigomaligna with atypical melanocytes with back-to-back nuclei and pagetoid spread highlighted by MiTF (IHC stain, 100).Abbreviations: H&E, hematoxylin and eosin; IHC, immunohistochemistry.Clinical, Cosmetic and Investigational Dermatology 2015:8submit your manuscript www.dovepress.comDovepress37
DovepressTetzlaff et almelanocytes.40 One factor contributing to the overestimationof melanocytes with Melan A is the cytoplasmic labeling ofdendritic processes; also, Melan A has been shown to labeloccasional keratinocytes as “pseudomelanocytic nests.”41When we evaluate Melan A in this clinical setting, attentionto labeling of only the cell bodies helps prevent overestimation of the number of melanocytes. A study by Nybakkenet al39 demonstrated that the use of Melan A resulted in anestimated 23.3% higher melanocyte count in the epidermiscompared with use of hematoxylin and eosin (H&E)-stainedsections in atypical intraepithelial melanocytic proliferations(lesions of concern for diagnosis of melanoma in situ); use ofMiTF resulted in estimates of melanocytes similar to thoseobserved with H&E-stained sections. Thus, MiTF can be useful to distinguish pigmented actinic keratosis from melanomain situ.42 In fact, compared with Melan A and HMB45, MiTFallowed greater distinction of melanocytes from keratinocyteswith melanin pigment.43 The mean density of intraepithelialmelanocytes in a 1.0 mm segment of epidermis was reportedto be 27 positive MiTF cells in solar lentigines compared to111.9 in melanoma in situ.43 Morphometric analysis identifiedgreater than ten MiTF-positive nuclei per 200 µm segment ofH&E-stained, section-favored melanoma in situ over solarlentigo.44 Although quantitative assessment is valuable, we alsofind it helpful to compare the background melanocytes withthe central lesion in skin ellipse specimens. Typically, thereshould be crescendo-decrescendo density of melanocytes in theepidermis from the periphery–center of the lesion–peripheryin examined cross-sections (either by H&E or IHC) (Figure 2).Discrimination from background increase density of melanocytes in chronic sun damage skin from lentigo maligna maybecome more challenging in en face margin evaluation.PeripherySox10The Sox (Sry-related box) family of genes is involved in thedevelopment processes of testis, oligodendrocytes, the centralnervous system, and chodrocytes.45 Sox10 plays a pivotal rolein neural crest cell development, and mutations in Sox10 resultin Waardenburg–Shah syndrome. Afflicted individuals exhibitpigmentary alterations and nervous system defects.46 Sox10directly regulates expression of MiTF and other genes essentialin melanin synthesis.47,48 A study by Bondurand et al49 showedthat Sox10 in synergy with PAX3 bind directly to the MiTFpromoter; thus, regulating MiTF expression. Consequently,Sox10 has emerged as a useful tissue biomarker in melanocyticneoplasms.50,51 Similar to MiTF, Sox10 protein is expressedin the nucleus, and the utility of Sox10 in evaluation of melanocytic lesions parallels that of MiTF. In particular, likeMiTF, Sox10 facilitates the enumeration of melanocytes inthe epidermis on chronic sun-damaged skin, and minimizesthe overestimation of melanocytes. Sox10 also facilitates thedistinction of pigmented actinic keratosis and melanocytichyperplasia from lentigo maligna.42Sox10 is expressed in Schwann cells, myoepithelialcells, granular cells, adnexal structures, and salivaryglands. Since the expression of Sox10 has been limitedto tumors of melanocytic, Schwannian, or myoepithelialdifferentiation, Sox10 has become useful in differentialdiagnosis of melanoma. The sensitivity of Sox10 inmelanoma with various morphologies and patterns (eg,spindle, nodular, desmoplastic, metastatic) is comparableto that of S100 and is greater than 95%.50,52,53 Sox10 andMiTF differ, however, in specificity. Sox10 expression isabsent in fibroblasts and macrophages and thus can aid inevaluating excision specimens for residual tumor.54 Sox10CenterCrescendoPeripheryDecrescendoFigure 2 Evaluation of bread-loafed cross-sections of peripheral margins allows for comparison of the background increased density of melanocytes, reflective of chronicsun damage (at the periphery), with area of lentigo maligna (at the center).38submit your manuscript www.dovepress.comDovepressClinical, Cosmetic and Investigational Dermatology 2015:8
DovepressMelanoma biomarkers, clinical applicationFigure 3 Sox10 identifies rare tumor cells in heavily regressed areas of melanoma.Notes: (A) Melanoma nodule with extensive regression with melanophages (H&E stain, 40); (B) Sox10 labels nucleus (arrows) of scattered viable melanoma cells in theregressed nodule with an absence of labeling in pigmented macrophages (IHC stain, 400).Abbreviations: H&E, hematoxylin and eosin; IHC, immunohistochemistry.has also been reported to be a reliable marker in evaluatingsentinel lymph nodes for metastatic melanoma.55 In addition to these uses for Sox10, we have found this tissuebiomarker to be helpful in identifying rare tumor cellsin a heavily regressed area of metastatic melanoma withabundant melanophages (Figure 3A). The nuclear labeling of Sox10 (preferably with red chromogen to contrastmelanin pigment in the cytoplasm of the macrophages)confirms the presence of isolated melanoma cells (Figure3B) but not macrophages, and thus may have importantclinical implications in staging.Markers of vascular invasionD2-40The endothelial marker D2-40 (podoplanin) is a highlyspecific monoclonal antibody for lymphatic endothelium butdoes not react with blood vessel endothelium; thus, D2-40is a selective marker to highlight LVI by tumor cells.56 Useof D2-40 in cutaneous melanoma, compared with use of HEstain alone, increased the frequency of both intratumoraland peritumoral LVI detection.57,58 In fact, detection of LVIwith D2-40 in cutaneous melanoma correlated with positivesentinel lymph nodes and worse overall and disease-freesurvival (Figure 4A and B).58,59 The importance of LVIdetection with D2-40 is not limited to cutaneous melanomasbut offers prognostic value in other human cancers such asendometrial, breast, esophageal, colorectal, and Merkel cellcancers.60–63CD34 and CD31 are panvascular markers that label bothlymphatic and blood vessel endothelium. The sensitivity ofCD34 and CD31 in identifying lymphatic endothelium isClinical, Cosmetic and Investigational Dermatology 2015:8lower than that of D2-40.64,65 In addition, CD31 is reactivein macrophages. These qualities make CD34 and CD31suboptimal markers in the detection of LVI in cutaneousmelanoma.Dual IHC marker with MiTF/D2-40or Sox10/D2-40The use of double IHC stains to evaluate melanocytic lesionsis generally available in academic settings and some private laboratories.66 At our institution, to confirm histologicsuspicion of LVI in tumors that histologically harbor poorprognostic attribute(s) (eg, ulceration, mitotic figures, thicktumors) in patients with cutaneous melanomas, we routinelyevaluate for LVI with D2-40 in lesions with adequate dermalcomponent amenable to further IHC studies. Since a vasc
a variety of tissue biomarkers and ancillary techniques (eg, immunohistochemical [IHC] analysis or fluorescence in situ hybridization) are currently available. In fact, hundreds of tissue biomarkers are available in clinical laboratories for diagnosing melanoma and determining the prognosis and mutation status of this devastating skin disease.
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