Melanocytic Lesions: Use of Immunohistochemistry and Special Studies Napa Valley 2018

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1 Melanocytic Lesions: Use of Immunohistochemistry and Special Studies Napa Valley 2018 Victor G. Prieto, MD, PhD Professor Depts. of Pathology and Dermatology University of Texas - MD Anderson Cancer Center Houston TX, USA Consultant for Myriad (MyPath)

2 Orange Poppy. State flower

3 Index Introduction Immunohistochemistry Newer techniques Conclusion

4 Introduction Growing incidence of melanoma Growing number of biopsies Gray areas (black / white) Need for additional techniques

5 Immunoperoxidase in Dermatopathology Multiple applications Several antibodies

6 Nevus Melanoma HMB45

7 MART1

8 MART1/Ki67 Ki67

9 MITF

10 SOX10

11 P16. Usually preserved in Spitz nevi and lost in melanoma

12 BAP1 BRAF V600E

13 H3Kme27. Preserved in most melanocytic lesions

14 IPOX in Dermatopathology: Special Situations Diagnosis (melanocytic differentiation) Depth of invasion Distinction between benign/ malignant

15 Detection of Level of Invasion Questionable dermal invasion Desmoplastic melanoma

16

17 Keratin

18 Differentiation between benign and malignant lesions

19 Wedge shape Papular

20 Large, regular junctional nests Maturation

21 Kamino bodies

22 HMB45 Maturation

23 Double MART1/Ki67 Spitz nevus

24 Differential Dx: Spitzoid melanoma Older patients (but not an exact science!) Asymmetry Poorly circumscribed Irregular nests No maturation

25 Marked pleomorphism Confluent pattern Atypical mitotic figures

26 HMB45: top bottom

27 MIB1 (deep) Spitzoid melanoma

28 Blue Nevus Family vs. Melanoma Mitotic figures Necrosis Cytologic atypia Biphenotypic pattern (nevus + mel.) Atypical BN: (Scalp) Scattered mitotic figures OR Necrosis OR Cytologic atypia Tran et l. J Cutan Pathol 1998; 25: Patchy HMB45 Increased MIB1

29 Large lesion Uniform pigmentation

30 Elongation of rete ridges, small junctional component

31 Uniform cells, small nucleoli

32 Ki67: rare cells HMB45 diffuse Cellular blue nevus

33 Irregular pigmentation

34 Pleomorphism, irregular pigmentation

35 Nuclear pleomorphism

36 Ki67/MART1 Cellular blue nevus with atypical features HMB45

37 Key Points Blue Nevus-Type Lesions Cellularity Distribution of pigment No or very rare mitotic figures Diffuse expression of HMB45 antigen and MART1 Low proliferation rate

38 Lentigo Maligna vs. Sun- Damaged Skin Diffuse, confluent growth of atypical melanocytes in the epidermis HMB45 / pan mel cocktail MiTF1 (nuclear)

39 MART1 HMB45

40 Rare cells: Actinic keratosis MiTF1 Nuclear: Melanoma in situ

41 Miami from the plane

42 IHC: Prognosis Mitotic figures ( /mm2) in the dermal component of the primary lesion (AJCC) Ki67 (metastasis) Vascular invasion (IHC) Petersson et al D2-40

43 Sentinel Lymph Nodes AJCC / CAP: Identification of tumor cells by IPOX is sufficient for a Dx. of metastatic melanoma

44 Immunohistochemistry: focal + MART1 HMB45

45 MART1 Majority of melanocytes (except for desmoplastic / spindle cell melanoma) MART1 artefactually positive: Macrophages (then use HMB45 or anti-mitf1) Diffuse labeling of dendrites in sundamaged skin Trejo O, Reed JA, Prieto VG. J Cutan Pathol 2002; 29: El Shabrawi-Caelen L, Kerl H, Cerroni L. Am J Dermatopathol. 2004;26:364-6 Wiltz KL et al. J Cutan Pathol. 2007;34:601-5

46 MART1 HE CD68 Multinucleated macrophages

47 Distinction between melanoma cells and macrophages SLN for deeply invasive melanoma. LN with highest count

48 Macrophage, not melanoma Panmel HMB45 Destained slide / SOX10

49 IHC for treatment purposes Panmel braf V600E

50 IHC for treatment purposes Figure 1. Immunohistochemical labeling for Programmed Death receptor-1 Ligand (PDL-1) performed with an anti-pd-l1 (clone [EPR1161(2)]) & PD-1 (clone [NAT105]) from Abcam (Cambridge, MA) with HIER using citrate buffer & LSAB method (Dako, Carpinteria, CA). (a) Melanoma cells showing strong, uniform reaction in tumor cells. (b) PD-L1 negative in tumor cells, there is a positive reaction in immune cells in the tumor stroma and TILs. a b Chakravarti and Prieto, 2015 magnification x 200

51 Miami from the plane

52 New Techniques CGH FISH Mass spectrometry

53 CGH 127 (96.2%) melanomas showed at least one abnormality 7 nevi also had abnormalities (Spitz nevi gains in 11p)

54 CGH in Melanocytic Lesions Requirements Relatively large amount of pure tumor cells (conflict with histologic analysis) Difficult if numerous admixed inflammatory or stromal cells The abnormality must be present in a sufficient number of tumor cells DNA must be suitable for subsequent enzymatic manipulations for fluorescent labeling reactions

55 FISH in Melanocytic Lesions Allows evaluation of copy number aberrations at particular loci (specific fluorescent probe) in relation to cellular morphology under light microscopy

56 FISH in Melanocytic Lesions Five probes: CEN9 (centromere 9) RREB1 (6p25) MYB (6q23) CCND1 (11q13) p16 (9p21) Gerami et al. Am J Surg Pathol 2013; 37:

57 FISH in Melanocytic Lesions FISH + should not modify treatment FISH - does not exclude melanoma (if there are histologic features of melanoma) Tetzlaff et al Am J Surg Pathol 2013;37: Homozygous deletions of 9p21 in spitzoid lesions is associated with malignant behavior Gerami et al Am J Surg Pathol 2013;37: Amelanotic melanomas with amplification of MYC 8q24 Pouryazdanparast et al Modern Pathology 2012;25,

58 Possible pitfall: Increase of RREB1 y CND1 signals with normal RREB1 to CEP9 ratio probably indicates polyploidy (benign spitzoid lesions) Patient with multiple Spitz nevi. Boone et al. Arch Dermatol 2011; 147: 227

59 Mass Spectrometry Use of mass spectrometry to determine the protein profile (MALDI: Matrixassisted laser desorption ionization) 114 cases of Spitz nevus and spitzoid melanoma Lazova R, et al. Am J Dermatopathol 2012; 34: 82-90

60 Mass Spectrometry 5 peptides were differentially expressed in the 2 groups, including actin and vimentin

61 Mass Spectrometry Separation between SN and SM with 97% sensitivity and 90% specificity Using only the information from the dermis: Correct classification of 28/31 melanomas

62 Gene Arrays (Prognosis) Recent application to histology and cytology specimens from pigmented lesions in the eye Decision Dx (class 1/2)

63 Decision Dx Analysis of 31 genes (gene expression profile): Class 1: low risk of metastasis at 5y Class 2: 69% recurrence/metastasis at 5y Similar to breast carcinoma More significant than SLN? More studies and follow-up

64 MyPath (Myriad) Expression of 14 genes: Inflammatory Melanocytic Prame Very useful in sun-damaged and in spitzoid lesions

65 Application of Techniques to a Demonstrative Case

66 22 yo man, leg lesion Shave biopsy

67 Compound melanocytic proliferation

68 Intermediate-powered view Large junctional nests

69 Irregular dermal nests of hyperchromatic spindle melanocytes

70 Junctional nests

71 Irregular dermal nests but with maturation (change in size with

72 Outside Diagnosis Invasive melanoma, Clark IV Re-excision and sentinel lymph node

73 S100 Sentinel Lymph Node

74 S100

75 Outside Diagnosis Metastatic melanoma to one of one lymph node Patient referred to UT-MDACC for evaluation of further therapy (completion lymphadenectomy)

76 Immunohistochemical analysis at MDACC HMB45 Maturation of the dermal component (decreased labeling with d Minimal pagetoid migration

77 HMB45 Maturation of the dermal component (decreased labeling with d

78 Double immunoreaction: MART1 (cytoplasmic, red) and Ki67 (nuclear, brown) Intraepidermal component with focal (central) pagetoid migratio Decrease in size of dermal nests

79 Intraepidermal component: scattered cells expressing both mark Only focal pagetoid cells

80 Very rare cells in dermis proliferating (expressing both markers)

81 S100 Back to the Sentinel Lymph Node

82 MART1. Same cells as shown in the S100 slide

83 HMB45: Negative

84 Double IPOX: MART1 and Ki67. No proliferation

85 FISH Analysis Report Final diagnosis: Spitz nevus with atypical features One SLN negative for melanoma (Intraparenchymal nodal nevus)

86 Spectrum of Melanocytic Lesions Nevus Favor Nevus??? Favor Mel Mel Other lesions?: Borderline melanoma? Histologic features Clinical features IHC Newer techniques FISH, CGH, etc.

87 Summary Importance of histologic examination: Breslow thickness Ulceration Mitotic counts (hot spot) Sentinel lymph node

88 Summary Immunohistochemistry: Patterns of HMB45 (maturation and diffuse vs. patchy) Low proliferation with Ki67 Preserved expression of MART1 CGH FISH: 9p21, MYC 8q24 Mass spectrometry (Adjunct to the diagnosis)

89 Silverado Grove Thank you

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