10/2/17. MELTUMP, SAMPUS, AST.An Algorithmic Approach to Challenging (Often Borderline) Melanocytic Tumors. An Introduction to SNP Arrays

Transcription

1 MELTUMP, SAMPUS, AST.An Algorithmic Approach to Challenging (Often ) Melanocytic Tumors An Introduction to SNP Arrays Rajiv M. Patel, M.D. RCPA NZ ASM 2017 (11:45-12:30pm, Saturday, ) Why do we need more stuff? A small proportion have ambiguous histology Common nevus Melanoma Common nevus? MELTUMP Molecular studies Any other ways to distinguish nevi from melanoma? 1

2 Molecular studies Chromosome T T T T Genomic instability in melanoma Chromosome Mitosis T T T Chromosome Mitosis T T p53, P53, RB, p16 Checkpoint Cell cycle arrest (Senescence) Chromosome Chromosome Mitosis T T T T T T T Nevi MAPK -B-RAF -N-RAS -H-RAS -GNAQ Chromosome Chromosome Mitosis Melanoma T T T T T T T MAPK -B-RAF -N-RAS -H-RAS -GNAQ Chromosome Mitosis T T p53, RB, p16 Chromosome Mitosis T T p53, P53, RB, p16 p16 inactivation: ~50% of MM Cell cycle arrest ( Oncogene-Induced Senescence) Nevus Chromosome Mitosis Mitosis DNA breaks, fusions -Re-stabilize telomeres -growth advantage Cell Crisis Cell Death Melanoma Gross chromosomal abnormalities Molecular studies Genomic instability in melanoma Detection of numerical abnormalities in the tumor genome (CGH/SNP and FISH) Molecular studies Detection of numerical abnormalities in the tumor genome Mass spectrometry Gene expression profile Identification of mutations (TERT gene promoter) 2

3 Comparative Genomic Hybridization (CGH)/ Single Nucleotide Polymorphism (SNP) arrays SNP arrays Screens the entire genome for gains and losses in DNA material in one experiment Variants: Array based CGH Gains and Losses Array based SNP Gains, Losses and Loss of Heterozygosity SNP arrays SNP arrays Copy number changes Copy number changes 11p gain SNP arrays SNP arrays Copy number changes Allele peak Copy number changes Allele peak 3

4 SNP arrays Copy number changes Allele peak Mutation data BRAF NRAS PTEN TP53 Advantages of SNP arrays over conventional CGH Detect Copy Neutral LOH Advantages of SNP arrays over CGH Detect Copy Neutral LOH Challenges with FFPE Input DNA Agilent > 500ng Affymetrix CytoScan > 250ng Challenges with FFPE Input DNA Agilent > 500ng Affymetrix CytoScan > 250ng DNA Quality 150 2,000 bp Molecular Inversion Probes (MIP) SNP array OncoScan TM Low DNA input 80ng DNA (as low as 20ng) 4

5 Molecular Inversion Probes (MIP) SNP array OncoScan TM Low DNA input 80ng DNA (as low as 20ng) Works with fragmented samples 40bp fragments Data Gains Losses LOH Mutations BRAF: V600E/K, G469A/E NRAS: Q61L/R/K, G12D/VS/C PTEN: R130*/G/Q/fs, R159S, R233*, P248fs, P267fs TP53: 16 variants University of Michigan Cohort University of Michigan Cohort Melanoma Melanoma Nevus Nevus University of Michigan Cohort University of Michigan Cohort Melanoma Melanoma Nevus Nevus 5

6 Compound nevus Primary melanoma No gains or losses BRAF V600E 18 CNA 11 losses 6 gains 1 CN-LOH BRAF wild NRAS wild PTEN wild TP53 wild Metastatic melanoma 30 CNA 25 gains 2 losses 3 CN-LOH BRAF V600K No DNA copy number changes Test for DNA copy number changes Multiple DNA copy number changes 40-year old woman with a papule on the right shoulder Clinical R/O BCC 35 6

7 HMB-45 Ki-67 Melanocytic neoplasm with borderline features between atypical nevus and melanoma Suspicious for nevoid melanoma p16 Chr 22 CN-LOH Chr 9p21 homozygous loss (CDKN2A) 7

8 Chr 1p gain (NRAS) Chr 13q loss (BRCA2) NRAS Q61R Nevoid melanoma Chr 9p 21 homozygous loss Chr 22 CN-LOH Chr 1p gain (NRAS) Chr 13q loss (BRCA2) NRAS Q61R This seems easy enough No abnormalities GOOD Abnormalities BAD Not so simple! Not all abnormalities are bad Some can be used to classify nevi 48 8

9 32 y/o F, Face Desmoplastic Spitz nevus HRAS mutation 11p gain BRAF wild NRAS wild PTEN wild TP53 wild 28 y/o F, Lt Temple 9

10 BAP-1 negative nevus (BAP-oma) 10-day-old AA newborn with giant congenital nevus with several nodules 3p loss (BAP-1 locus) BRAF V600E Diagnosis: proliferative nodule Copy number Result: Losses of whole chromosomes 3, 4, 5, 10, 11, 13, 14, 16, 17, 18, 21 1 year old More complicating factors What to do with only a few abnormalities? 10

11 46 y/o M, Right chest 11p gain Chr 14 CN-LOH UM Cohort Melanoma Desmoplastic Spitz nevus with atypia Not enough for melanoma 11p gain Chr 14 CN-LOH Nevus 11

12 More problems How many abnormalities do we require for a melanoma diagnosis? Histological classification # of cases with at least one significant copy number variation Average # CNV Nevi 0/6 (0%) 0 Atypical nevi 3/15 (20%) 1.6 (1-2) Ambiguous 15/25 (60%) 6.3 (1-25) Melanoma 35/39 (90%) 21.7 (1-69) Sensitivity: 90% Specificity: 87% Alomari et al. Platform at USCAP meeting, Seattle WA, 2016 How many abnormalities do we require for a melanoma diagnosis? How many abnormalities do we require for a melanoma diagnosis? >=3 abnormalities significant (but with exceptions) Whole chromosomal abnormalities in proliferative nodules Isolated homozygous deletion of 9p21 favors melanoma Others to come. >=3 abnormalities significant (but with exceptions) Whole chromosomal abnormalities in proliferative nodules Isolated homozygous deletion of 9p21 favors melanoma Others to come. Molecular pathologist job: Provide a comprehensive interpretation Your job: Understand the report and communicate with your molecular pathologist Ultimate question Practical algorithm for use of molecular studies Can CNV number and/or pattern predict adverse outcome in borderline lesions? Unfortunately few studies 12

13 Melanocytic lesion Definitive diagnosis Melanocytic lesion Definitive diagnosis No further testing No further testing Molecular testing Mol - Mol - Mol + Mol - Mol + Mol + Mol - Mol - Mol + Mol - Mol + Mol + Mol - Mol - Mol + Mol - Mol + Mol + Nevus Melanoma favor nevus favor melanoma 13

14 Melanocytic lesion Definitive diagnosis No further testing Mol - Mol - Mol + Mol - Mol + Mol + Mol - Mol - Mol + Mol - Mol + Mol + Nevus favor nevus favor melanoma Melanoma Risk assessment Melanocytic lesion Definitive diagnosis Other findings on SNP arrays No further testing Detection of unbalanced rearrangements Mol - Mol - Mol + Mol - Mol + Mol + Nevus favor nevus favor melanoma Melanoma Excision with limited margins Excision with margins appropriate for depth +/- SLN Excision with margins appropriate for depth SLN 82 8 y/o girl, Right ear lobe 14

15 8 y/o girl, Right ear lobe Atypical Spitz nevus/tumor with LMNA- NTRK1 fusion LMNA NTRK1 Summary CGH/ SNP arrays remain valuable diagnostic ancillary tools in selected cases Information beyond CNV can be obtained Copy neutral LOH Mutation data Translocations Challenges Technical 30-50% pure tumor cells Tissue requirements Does not allow histologic correlation Biological More outcome studies are needed to define the CNV patterns associated with adverse outcome 89 15

16 Cases QUESTIONS? Case 1 28-year-old man 2-year history of lesion on the abdomen 1.2 cm pigmented papule with a surrounding rim of light brown pigment Symmetric Well circumscribed Hyperplastic epidermis Spindle melanocytes arranged in nests and fascicles Atypical junctional nevus at the periphery 16

17 Maturation Epithelioid and spindle melanocytes arranged in nests and fascicles Rare mitoses P16 Ki67/Mart1 HMB45 99 Favors nevus - Symmetry, well circumscribed - No significant atypia - Maturation - Stratification with HMB45 - Expression of p16 Favors melanoma - Slight increase in ki-67 - Mitotic activity Atypical Spitz tumor, favor atypical Spitz nevus 17

18 No abnormalities Mol - Nevus Atypical Spitz nevus Case 2 A 14-year old girl presents with a left foot cyst. FISH testing for melanoma is reported as borderline positive Gene expression profile is indeterminate Symmetric Well circumscribed Predominantly dermal Compact nests Epithelioid morphology No maturation 18

19 Lymphocytic infiltrate Mitoses, some deep Spitzoid morphology Cytologic atypia HMB-45 p16 Ki-67 / Mart-1 19

20 Favors nevus - Symmetry - Well circumscribed - Expression of p16 Favors melanoma - Cytological atypia - Lack of maturation - Mitotic activity - Abnormal HMB45 pattern - Increased ki-67 - Positive FISH - Indeterminate Gene Expression Atypical Spitz tumor, favor Spitzoid melanoma No abnormalities Mol Final diagnosis: Atypical Spitz tumor of uncertain malignant potential Initial FISH Repeat FISH Initial FISH 6p % 8q % 11q13 21% Interpretation positive 6p % 6.7% 8q % 0% 11q13 21% 6.7% Tetraploidy 20% Interpretation positive Negative 119 Tetraploidy may increase false-positive FISH results

21 Case 3 Asymmetric Well-circumscribed 60-year old male Left lateral shoulder lesion Clinical impression: irritated benign nevus. Epithelioid cells Eosinophilic cytoplasm Distinct cytoplasmic borders Nuclear atypia No maturation Banal intradermal nevus

22 Banal intradermal nevus HMB45 p16 Ki67 / Mart-1 BAP-1 BAP-1 BRAF V600E Favors nevus - Well circumscribed - No significant atypia - No mitoses - Negative HMB45 - Expression of p16 Favors melanoma - Asymmetric - Focal cytological atypia - Focal increase ki-67 22

23 3p Deletion Atypical Spitz tumor, favor atypical BAP-1 negative nevus 134 BAP1 gene Mol - Nevus Final diagnosis: BAP1 negative nevus 135 BAP1 Nuclear ubiquitin carboxy-terminal hydrolase (UCH) Interactions with BRCA1, polycomb repressor complex, others Germline mutations associated with: Malignant mesothelioma Cutaneous and uveal melanoma Carcinomas (lung, breast, ovarian, renal) Atypical Spitz Tumors 23

24 BAP1 loss in melanocytic lesions Diagnostic/prognostic significance: likely indolent behavior Risk of germline mutation: Suggest genetic consult H&E H&E 33 AST 28% BAP1 25% BRAF+/BAP1- H&E BAP1 Wiesner et al. Am J Surg Pathol, August 2012 Case 4 Small 2 y/o boy Lesion on left thigh Asymmetric Lack of maturation 24

25 Lack of maturation Spitzoid cytology Atypia Mitoses Ki P16 Case 4 FISH: No abnormalities

26 Favor nevus - Small - Infant - Nega*ve FISH Favor melanoma - Asymmetry - No maturation - Cytological atypia - Mitotic activity - Increased ki-67 - Diffuse loss of p16 Atypical Spitz tumor CDKN2A 0.6 MB Homozygous deletion at 9p21 Losses of chormosome 9 and Heterozygous loss of chromosome 9 CDKN2A 9p21 FISH probe 9p21 CEN 9 FISH probe produces a signal: False positive result

27 Case 5 16 y/o male with lesion on the arm Mol + favor melanoma Final diagnosis: Atypical Spitz Tumor, favor spitzoid melanoma Compound melanocytic proliferation Bulges into the subcutis dumbbell profile Extends along neurovascular bundles Courtesy of Dr Guido Massi 160 Junctional component 27

28 Plexiform architecture Epithelioid to spindled Large ovoid nuclei with occasional nucleoli and nuclear pseudoinclusions Irregular nuclear border with hyperchromasia and nuclear pleomorphism Pale cytoplasm with occasional balloon cell changes HMB-45 Ki-67 / Mart-1 28

29 P16 BRAF V600E 170 Favor nevus - Symmetric - Well circumscribed - DPN morphology - Expression of p16 Favor melanoma - Cytological atypia - Mitotic activity - Focal increased ki-67 - BRAF V600E Favor atypical DPN. Mol + Chromosome Type or Size Relevant genes abnormality 7q Gain 42.7 BRAF (also with V600E mutation) MET possible fusion 9 (whole Loss CDKN2A chromosome) 14q32.12 Loss 0.25 GOLGA5 possible fusion 21 (whole Gain chromosome) Final diagnosis: DPN-like melanocytic tumor of uncertain malignant potential Possible associated translocation 29

30 Case 6 25 y/o man with nevus on the arm No gains or losses BRAF -wild 30

31 Dermatopathology Molecular Diagnostics and Research Laboratory Dx: Deep penetrating nevus Min Wang Dr. Doru Andea Acknowledgments U of M Dermatopathology Section Douglas Fullen Lori Lowe May Chan Alex Hristov Paul Harms U of M Melanoma Clinic Tim Johnson Chris Bichakjian Alison Durham Kelly Harms QUESTIONS? 31