Update on Spitzoid and Blue nevus-like melanocytic lesions Emphasis on molecular studies informing diagnosis, prognosis and therapy Michael T. Tetzlaff MD, PhD Associate Professor Department of Pathology, Section of Dermatopathology Department of Translational and Molecular Pathology The University of Texas MD Anderson Cancer Center Executive Officer Translational Research Program The Alliance for Clinical Trials
Update on Spitz nevus and Spitzoid Melanoma Spitz nevus family of lesions Spitz nevus Atypical Spitz Tumor (AST) Spitzoid Melanoma????
Spitz nevus, Spitzoid Melanoma and Atypical Spitz Tumors Pink papule on the cheek of a 9 year old boy Spitz Nevus
Spitz Nevus
Spitz Nevus
Spitz nevus, Spitzoid Melanoma and Atypical Spitz Tumors Changing pink-red papule on the thigh of a 49 year old man
Mart-1/Ki-67
Invasive melanoma with Spitz features BAP1
Spitz nevus, Spitzoid Melanoma and Atypical Spitz Tumors Spitzoid lesions Spitz nevus Size <10 mm Symmetric Well-circumscribed Epidermal hyperplasia Kamino bodies common Rarely ulcerated Discontinuous intraepidermal proliferation Minimal pagetoid spread confined to center Well spaced cells in dermis Mitotic figures only superficial Monotonous atypia with maturation Spitzoid melanoma Size >10 mm Asymmetric Poorly circumscribed Minimal epidermal changes Kamino bodies uncommon Ulceration not uncommon Continuous intraepidermal proliferation Prominent pagetoid spread including at the edges Dense, sheet-like, expansile Mitotic figures common throughout Prominent cytologic atypia without maturation
Reproducible criteria for the distinction among Atypical Spitz Tumors have been historically difficult to establish
Predicting lymph node metastases and disease progression among Atypical Spitz Tumors has been historically difficult 57 patients alive without disease Median follow-up: 28.6 and 43.8 months
A molecular classification scheme for Spitzoid neoplasia? 11p amplified HRAS mutant Desmoplastic No metastases to date Spitzoid Lesion Molecular-genetic alteration? Predictable clinical course? Reproducible histopathologic criteria? TERT promoter mutant BAP1 deficient BRAFV600E Spitzoid lesions defined by FISH Homozygous deletion of 9p21 Relative loss of 6q23 Translocation associated ROS1 ALK NTRK1 NTRK3 MET BRAF RET
Identification of 11p amplified Spitz nevi Performed Comparative Genomic Hybridization (CGH) on 17 Spitz nevi 13 cases with no chromosomal copy number alterations 3 cases showed isolated gains of the entire short arm of chromosome 11 (11p) 1 case with gains at 7q21 No evidence of recurrence or metastasis in any of the lesions
Morphology of 11p amplified/hras mutant Spitz nevi FISH for 11p on 102 Spitz nevi: Identified 12 Spitz nevi (11.8%) with >3x gains of 11p HRAS mutations in 8/12 (67%) of 11p Spitz nevi HRAS mutations in 1/21 (5%) of Spitz nevi without 11p
11p amplified/hras mutant Spitz nevi HRAS sequencing on 170 Spitz tumors: 24/170 Spitzoid lesions (14%) with HRAS mutation HRAS mutated Spitzoid lesions histopathologically similar to 11p Spitz HRAS mutated Spitzoid lesions did not develop recurrence or metastasis (median follow-up: 10.5 y)
11p/HRAS correlates with benign clinical course HRAS sequencing on 170 Spitz tumors: 24/170 Spitzoid lesions (14%) with HRAS mutation HRAS mutated Spitzoid lesions histopathologically similar to 11p Spitz HRAS mutated Spitzoid lesions did not develop recurrence or metastasis (median follow-up: 10.5 y)
11p amplified Spitz nevi
BAP1 deficient BRAFV600E Spitz tumors Offspring with numerous papular melanocytic neoplasms with Spitzoid morphology Lesions analyzed by acgh Identified frequent deletions of 3p Sequencing revealed mutations in BAP1.
BAP1 deficient BRAFV600E Spitz tumors 32 sporadic Atypical Spitz Tumors assessed by BAP1 IHC: BAP1 negative by IHC in 9 BAP1 mutation in 5 Characteristic features: Trunk Dermal Epithelioid cells Prominent TILS BRAF V600E
BAP1 deficient BRAFV600E Spitz tumors
BAP1 deficient BRAFV600E Spitz tumors
BRAF BAP1
Translocation resulting in activating kinase fusions are frequent among Spitzoid lesions Different fusion partner gene Spanned the spectrum of Spitzoid neoplasia Each fusion occurred in mutually exclusive fashion of others Constitutively active kinase activity IHC+ Kinase Do specific translocations correlate with a predictable morphology or clinical course?
ALK kinase fusions Spitzoid lesions have distinctive histopathologic features Author Busam et al Yeh et al Amin et al Totals Gender Men 9 11 12 32 Women 8 21 5 34 Median age (range) 16 y (2-35y) 12y (5m-64y) 13y (1-38y) Location Extremity 8 18 11 37 Trunk/buttock 4 8 2 14 Head/Neck 3 6 4 13 Diagnosis Spitz nevus 5 6 NS 11 Atypical Spitz tumor 12 22 NS 34 Spitzoid melanoma 0 4 0 4 No discrete ALK-fusion genotype correlates with a particular clinical phenotype.
ALK-rearranged Spitz nevus with atypical features Compound or intradermal Exophytic and/or wedge-shaped silhouette Plexiform growth pattern Fusiform amelanotic melanocytes arranged in intersecting fascicles among dermal collagen bundles Rare cases with metastases restricted to sentinel lymph nodes (SLNs) identified, but long-term follow-up has not demonstrated further aggressive behavior. ALK
Translocation resulting in activating kinase fusions are frequent among Spitzoid lesions Early events in Spitzoid neoplasia seen across the spectrum Important oncogenic drivers Not sufficient for malignant transformation No evidence yet that a discrete fusion (5 partner with 3 kinase) corresponds with a predictable clinical phenotype Constitutively active kinase activity has critical therapeutic implications Important unanswered question: Do specific kinase fusions correlate with a discrete histopathologic and/or clinical phenotype?
Comparative genomic hybridization (CGH) systematically interrogates the whole genome The relative hybridization of tumor derived probe compared to normal defines the relative composition of tumor DNA throughout the genome Gains show net green signal Losses show net red signal
CGH confirms widespread genomic instability in melanoma compared to nevi Applied CGH to 186 melanocytic tumors 132 Melanomas 54 Melanocytic nevi 96% of melanomas carry some We chromosomal can exploit this copy aberration: difference between melanoma and nevi in a diagnostic assay? Gains or losses in discrete fragments of chromosomes
Fluorescence in situ hybridization (FISH): as a surrogate of CGH Hybridize fluorescently labeled probes: 11q13 (CCND1) 6p25 (RREB1) 6q23 (MYB) Cen6 11q13 6p25 6q23 Cen 6 Assess tumor cell nuclei for abnormalities in the number of fluorescent signals. Gerami P et al. Am J Surg Pathology. 2009. 33(8):1146-1156.
FISH assay as a surrogate of the genomic instability captured by CGH Cen 6 6p25 6q23 11q13 Gerami P et al. Am J Surg Pathology. 2009. 33(8):1146-1156.
Second generation FISH: New probes expand and improve the diagnostic utility of FISH Gerami P et al. Am. J. Surg Pathol. 2012. 36(6):808-817. cen9 cen9 9p21 9p21 6p25 6p25 8q24 8q24 11q13
FISH as a prognostic marker in Spitzoid lesions Assessed 75 Atypical Spitz Tumors by FISH (First and Second generation probes)
FISH as a prognostic marker in Spitzoid lesions Assessed 75 Atypical Spitz Tumors by FISH (First and Second generation probes) SLN- SLN+ SLNx SLN+/LN+ Death
FISH as a prognostic marker in Spitzoid lesions Assessed 75 Atypical Spitz Tumors by FISH (First and Second generation probes) SLN- SLN+ SLNx SLN+/LN+ Death
Homozygous deletion of 9p21 correlates with aggressive behavior in Spitzoid melanoma 9p21 9/11 patients with spread beyond sentinel node and or death, showed homozygous deletion of 9p21 compared to 3/64 in group 1.
Case example 38 year old man with an enlarging tan-brown lesion on the shoulder
HMB-45
Mart-1/Ki67
Case example 9p21 cen9 6p25 8q24 11q13 Results Low stringency cut off High stringency cut off Chrom 6: RREB1 (6p25) 6.0% >16% 29% >29% Chrom 8: cmyc (8q24) 4.3% >10% 29% >29% Chrom 9: CDKN2A (9p21)/CEN9 55.0% >10% 29% >29% Chrom 11: CCND1 (11q13) 11.1% >19% 29% >29% Patient with positive SLN (2/3). Currently free of disease (10 mos)
TERT promoter mutations occur in melanoma and associate with aggressive clinical course Oncology Reports. 2012. 28: 1945-1952.
Spitzoid lesions with TERT promoter mutations 56 patients with AST and follow-up 21/40 (50%) with at least one positive SLN 9 with extensive nodal metastases 4 with hematogenous metastases who died of disease Mean follow up for remaining 52 patients=32.5 months Clinical Features Age (years) (P =0.03)* <10 10 Gender (P =0.64) Female Male Location (P=0.27) Lower extremity Upper extremity Face Ear Scalp Trunk Race (P=0.70) White Black Other Not specified Lesional diameter (P=0.054) 5 mm 6-10 mm >10 mm Favorable (n=52) 31 (60%) 21 (40%) 30 (60%) 22 (40%) 25 9 6 5 2 5 30 (81%) 2 (5%) 5 (14%) 15 22 (43%) 23 (45%) 6 (12%) 1 Unfavorable (n=4) 0 (0%) 4 (100%) 3 (75%) 1 (25%) 1 0 1 0 1 1 4 (100%) 0 (0%) 0 (0%) 0 1 (33%) 0 (0%) 2 (67%) 1
Spitzoid lesions with TERT promoter mutations 4 patients who died of disease had TERT promoter mutations 0/52 patients alive had TERT promoter mutations 12/49 with favorable outcome had homozygous deletion of 9p21 2/4 patients who died of disease had homozygous deletion of 9p21 All 4 had loss of p16 protein by IHC
Spitzoid lesions with TERT promoter mutations TERT promoter mutations are not always associated with poor prognosis in atypical spitzoid tumors. Requena C, Heidenreich B, Kumar R, Nagore E. Pigment Cell Melanoma Res. 2016. Spitz/Reed Nevi (n=15) AST/SMM (n=9) htert promoter mutations 0/15 2/9 1 of 2 AST/SMMs with htert promoter mutations had positive SLN Both patients alive and without disease (60 and 66 months) htert promoter mutations as marker of bad prognosis could not be demonstrated in our series
A molecular classification scheme for Spitzoid neoplasia 11p amplified HRAS mutant Desmoplastic No metastases to date Spitzoid Lesion Molecular-genetic alteration? Predictable clinical course? Reproducible histopathologic? criteria TERT promoter mutant*** BAP1 deficient BRAFV600E Spitzoid lesions defined by FISH Homozygous deletion of 9p21 Relative loss of 6q23 Translocation associated ROS1 ALK NTRK1 NTRK3 MET BRAF RET
Spitzoid melanomas share molecular-genetic similarity to conventional melanoma Spitzoid melanomas share significant mutational signature with conventional melanoma and little overlap with Spitz nevi
Update on blue nevi and blue nevus-like Melanoma Blue nevus family of lesions Common blue nevus Cellular blue nevus Melanoma, blue nevus type Atypical Cellular blue nevus
Common blue nevus Dark blue-black (4-10 mm) papule with ill-defined border Anywhere on the body (extremities most common) Variable density of spindled/dendritic melanocytes in the dermis Separated by dermal collagen bundles Abundant coarse intracytoplasmic melanin pigment Considerable range in cellularity and pigmentation
Cellular blue nevus Large tumor (>1 cm) raised papule with ill-defined border Lower back and buttocks in young adult Pandermal melanocytic proliferation with a characteristic dumb-bell configuration Plaque/nodule in the upper dermis connected to a nodule in the lower dermis Typically bi-phasic spindled/ pigmented melanocytes admixed with epithelioid melanocytes containing less pigment
Melanoma, blue nevus type
Distinguishing among blue nevus-like lesions Blue nevus Cellular Blue nevus Atypical Cellular Blue nevus Melanoma, Blue nevus type Location Mid- to upper dermis Pan-dermal Pan-dermal Pan-dermal and may involve subcutis Cellularity Dendritic melanocytes with coarse pigment Biphasic with dendritic and epithelioid melanocytes Biphasic with dendritic and epithelioid melanocytes May contain or resemble blue nevus/cellular blue nevus Size Small (<10 mm) Can be >10 mm Can be >10 mm >10 mm Symmetry Present Present Present/Absent Usually absent Cytologic atypia Absent Absent Increased Prominent Mitotic figures Usually absent Infrequent Increased High, atypical Necrosis Absent Absent Absent Present HMB-45 Strong, diffuse Strong, diffuse Variable Heterogeneous Ki-67 Low absent Low Slightly increased Elevated (>5-10%) BAP-1 Preserved Preserved Preserved Lost FISH Normal Normal Normal Abnormal (6p25)
Chromosomal instability is common in Blue nevus-like melanoma: CGH identifies numerous alterations Copy number changes Cellular Blue nevus Melanoma, Blue nevus type 3/17 (17%) 6/9 (67%) Blue nevi show few (isolated when present) copy number alterations Melanoma, blue nevus type show complex copy number alterations
Chromosomal instability is common in Blue nevus-like melanoma: FISH studies confirm 100% (12/12) Blue nevi FISH negative 100% (5/5) Melanoma, blue nevus type FISH positive Gains of 6p25 most common
GNAQ and GNA11 mutations in blue nevus-like melanomas
GNAQ and GNA11 mutations, loss of BAP1 and chromosomal instability in blue nevus-like melanomas Loss of nuclear BAP1 IHC GNAQ mutation (Exon 5) GNA11 mutation (Exon 5) Cellular Blue nevus Melanoma, Blue nevus type 0/24 (0%) 7/11 (64%) 16/18 (89%) 1/11 (9%) 1/18 (6%) 8/11 (73%) GNAQ/GNA11 mutations in benign and malignant blue nevus-like proliferations BAP1 loss typical in melanoma, blue nevus type Chromosomal abnormalities frequent in melanoma, blue nevus type
Targeted sequencing facilitates distinction among cellular blue nevi and blue nevus like melanomas Mutations in BAP1 (thus far) only in blue nevus-like melanomas GNAQ/GNA11 mutations across the spectrum of blue nevus-like lesions BAP1 and SF3B1 mutated in melanoma, blue nevus type Sequencing analyses likely to be informative in diagnosis of blue nevus like lesions.
Questions
Thank you Victor G. Prieto MD PhD Jonathan L. Curry MD Carlos A. Torres-Cabala MD PhD Priya Nagarajan MD PhD Phyu Aung MD PhD Doina Ivan MD Department of Pathology, Cleveland Clinic Steven D Billings MD