Disclosure Information. Lecture Outline. Lecture Outline. Introduction. Molecular Pathology of Cutaneous Melanoma. Nothing to disclose

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Molecular Pathology of Cutaneous Melanoma Disclosure Information Nothing to disclose Jonathan L. Curry, MD Assistant Professor of Pathology and Dermatology University of Texas-MD Anderson Cancer Center Houston, Texas Lecture Outline Melanoma diagnosis Mutations in melanoma Melanoma targeted therapy Lecture Outline Melanoma diagnosis Mutations in melanoma Melanoma targeted therapy Introduction Melanoma is the most deadly skin cancer Lifetime risk of developing melanoma 1:50 American Cancer Society projection for 2013 will be 76,690 new diagnosis and 9,480 deaths Photo provided by Dr. Kim 1

Distinction Among Melanocytic Nevi and Melanoma Morphologic features Immunohistochemical characterization HMB45 Mart-1/Ki-67 Molecular features Comparative genomic hybridization (CGH) Fluorescence in situ hybridization (FISH) Morphologic Features of Melanocytic Nevi vs. Melanoma Architecture asymmetric Border poor circumscription Cytology severe atypia Epidermal melanocytes confluent proliferation prominent Pagetoid extension single cell pattern Dermal melanocytes absence of maturation pattern presence of host response Increase mitosis Invasive Melanoma Distinction Among Melanocytic Nevi and Melanoma Melanocytic nevi Melanoma display between collagen push through dermis Immunohistochemical Features of Melanocytic Nevi vs. Melanoma HMB45 Glycoprotein 100 in stage I and II melanosomes Marker of melanocytic differentiation Mart-1/Ki-67 Mart-1 detects melanocytes Ki-67 labels proliferating cells HMB45 and Mart-1 Melanocytic nevus HMB-45 Mart-1 2

Immunohistochemistry: HMB-45 Immunohistochemistry: Mart-1/Ki-67 Diffuse Patchy Nevus Melanoma Immunohistochemistry: Mart-1/Ki-67 Comparative Genomic Hybridization (CGH) in Melanocytic Nevi vs. Melanoma Analyzed 186 melanocytic tumors by CGH Low in nevi High in melanoma Rudolph P et al. Am J of Path. 1995. 147 (6):1615-1625. 96% of melanomas carry some chromosomal copy aberration: Gains or losses in discrete fragments of chromosomes Bastian BC et al. American Journal of Pathology. 2003. 163 (5):1765-1770. Comparative Genomic Hybridization (CGH) Limitations of CGH as a Diagnostic Test Melanoma cell Normal cell Expensive, labor intensive and technical expertise. Requires large amount of pure tumor cell population. Melanoma DNA PBL NORMAL DNA Melanoma cell Normal cell Ratio (Mel/Norm) Region of loss Region of gain Identify regions of chromosomal gains/amplifications versus losses/deletions Dermatologic Therapy. 2003. 19 (1): 40-49. Region of loss NORMAL DNA NORMAL DNA NORMAL DNA Melanoma DNA NORMAL DNA NORMAL DNA Region of gain PBL NORMAL DNA 3

Fluorescence in situ hybridization (FISH) Allows evaluation of copy number at particular locus to distinguish nevi from melanoma Four FISH probe set commercially available: Melanocytic nevi FISH Melanoma Cen 6 6p25 6q23 RREB1 (6p25) CEN6 MYB (6q23) CCND1 (11q13) Probe Set Cut-Off RREB1 > 2 >16% RREB1 > CEN6 >53% MYB < CEN6 >42% CCND1 >2 >9% B 11q13 2 2 2 2 FISH Overall sensitivity approximately 80% in unambiguous melanocytic lesions 20% of melanomas may have negative FISH results FISH for ambiguous melanocytic lesions Sensitivity as low as 50-40% Awareness of polyploidy in Spitz tumors Gain in entire chromosome (6p25, 11q13, and 6q23) Misinterpreted as positive for melanoma Second Generation FISH Four FISH probe set commercially available: RREB1 (6p25) CDKN2A (9p21) MYC (8q24) CCND1 (11q13) Probe Set Cut-Off RREB1>2 >16% >29%* CDKN2A >10% >29%* MYC >2 >10% >29%* CCND1 >2 >19% >29%* Probe Set Sensitivity Specificity First Generation 75% 96% Second Generation 94%* 98%* Diagnosis of Melanoma Involves integration of Morphology Immunohistochemistry CGH or FISH Aware of polyploidy in Spitz nevi Reduce false positive rate of FISH Exclude tetraploid cells as abnormal cells (abnormal/total) Lecture Outline Melanoma diagnosis Mutations in melanoma Melanoma targeted therapy 4

Genetics of Melanoma Recent insights in melanoma genetics Rapid, evolving molecular testing platforms from single gene analysis to whole exome sequencing Provided an exciting era of targeted therapy Sporadic (~90%) Germline and Somatic Melanoma Mutations Hereditary (~6-12%) Somatic Mutations Cutaneous Melanoma G-protein Cell Pathway KIT GNAQ/GNA11 RTK RAS RAF PI3K/AKT PTEN Inhibition of apoptosis, cell cycle progression, differentiation MEK MAPK/ERK Inhibition of apoptosis proliferation Proliferation, cell survival, differentiation Bastian et al. J Clin Oncol. 2006 CDKN2A p16/rb BAP1 Somatic: Sporadic melanomas KIT NRAS BRAF GNAQ/GNA11 Germline: Hereditary melanoma CDKN2A/p16 Somatic: Sporadic melanomas KIT NRAS GNAQ/GNA11 BRAF Germline: Hereditary melanoma CDKN2A/p16 5

KIT Mutation KIT is a tyrosine kinase growth factor receptor KIT mutation promotes ligand-independent KIT dimerization and constitutive activation of MAPK and PI3K-AKT pathway to promote proliferative and survival advantage for melanoma cells KIT mutations and or increased copy numbers are seen in acral lentiginous/mucosal melanomas melanomas in skin with chronic sun damage (CSD) Melanoma exon 11 substitutions exon 13 exon 17 Up to 30% with KIT copy number or amplification KIT Mutation GIST exon 11 deletion or insertions exon 9 KIT copy number or amplification rare 15% 68% <1% GIST Ala502 Tyr503 dup F548S Y568C G565R V559D 559 del 6 W557R K550I 550 del 551 del K642E Exon 1 9 11 13 17 KIT Mutations Melanoma Del 554-559 Del 566-572 Y553N W557R K558N V559A V559N N566D V569G L576P Ins 583 R634W K642E N655K D816H D816V Y823D KIT Receptor JD TK1 TK2 Approximate Frequency of Somatic Histologic Type BRAF KIT NRAS GNAQ/ GNA11 BAP1 Superficial spreading 50-60 1 20 <10 Nodular 50 20-30 <10 Lentigo maligna 5 2-17 (amp 6) Acral lentiginous 13-20 15-20 (amp 30) Melanoma with blue nevus like features Atypical Spitzoid neoplasms 25-75% (89) reported 10 <4 10-15 <10 50 <1 reported ~11-28 Somatic: Sporadic melanomas KIT NRAS BRAF GNAQ/GNA11 Germline: Hereditary melanoma CDKN2A/p16 RAS The RAS oncogene family includes NRAS, HRAS, and KRAS Neuroblastoma rat sarcoma (NRAS) chr. 1p13 Harvey rat sarcoma (HRAS) chr.11p15 Kirsten rat sarcoma (KRAS) chr. 12p12 Share 90% identity with HRAS and KRAS Activated RAS recruits RAF which activates MEK and ERK and promotes cell proliferation, differentiation and survival Mutated NRAS demonstrates impaired GTP hydrolysis and constitutively activated 6

1 2 3 4 5 6 PM1 E PM3 G2,G4 G3 SI NRAS SII 6 exons spanning 12,438 bp Oncogenic mutations in NRAS are commonly activating mutations Occur in ~15-25% of cutaneous melanoma Mutations frequently involve exon 2 and 3 HV RAS KRAS and HRAS mutations are infrequent in cutaneous melanoma HRAS isoform is more commonly mutated in Spitz nevi nearly 30% increase copy number of chr. 11p in 20% Congenital melanocytic nevi demonstrate the most prevalent NRAS mutations approximately 65% of these mutations occur at codon 61 of exon 3 NRAS The most frequent mutations involve codon 12 exon 2 amino acid substitution of glycine (G) with glutamic acid (D) or serine (S) at position 12 G12D G12S codon 61 exon 3 amino acid substitution of glutamine (Q) with lysine (K) or arginine (R) at position 61 Q61K Q61R G12D G12S G12C NRAS Protein G13D G13R 1 2 3 4 5 6 PM1 E PM3 G2,G4 G3 SI Q61R Q61K Q61L Q61H Q61P Q61E SII HV Approximate Frequency of Somatic Histologic Type BRAF KIT NRAS GNAQ/ GNA11 BAP1 Superficial spreading 50-60 1 20 <10 Nodular 50 20-30 <10 Lentigo maligna 5 2-17 (amp 6) Acral lentiginous 13-20 15-20 (amp 30) Melanoma with blue nevus like features Atypical Spitzoid neoplasms 25-75% (89) reported 10 <4 10-15 <10 50 <1 reported ~11-28 Somatic: Sporadic melanomas KIT NRAS BRAF GNAQ/GNA11 Germline: Hereditary melanoma CDKN2A/p16 7

RAF Kinase Family RAF is a serine-threonine protein kinase in the MAPK pathway involved in cell proliferation, differentiation, and transformation. Three isoforms ARAF, chromosome Xp11 BRAF, chromosome 7q34 CRAF (RAF1), chromosome 3q34 Mutation in ARAF and CRAF genes are rare events in human cancer BRAF mutations are more frequent in cancer higher basal kinase activity preferred mutation target BRAF Gene 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Ras Binding Domain C-rich Catalytic loop Kinase Domain Activation segment 18 exons spanning 190,284 bp Oncogenic mutations in BRAF are commonly activating mutations Occur in ~50-60% of cutaneous melanoma Mutations frequently involve exon 15 BRAF Mutation BRAF V600E seen in over 90% of the cases single point mutation with a DNA base substitution from T (thymine) to A (adenine) or T to A valine to glutamic acid at the 600 position of the amino acid (BRAF V600E) BRAF Mutation BRAF V600K is second most frequent mutations, 5-10% two DNA base pair change from G (guanine) and T (thymine) to A (adenine) and A (adenine) or GT to AA valine to lysine at the 600 position of the amino acid (BRAF V600K) V600K more associated with chronic sun exposure than V600E Others combine for less than 1% BRAF V600R, K601N, L597R, L597Q, G596R, and D594N N BRAF Protein K439Q G466A G464E G466E G464V G466V V600E V600K V600D V600R L579Q K601E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Ras Binding Domain C-rich Catalytic loop Kinase Domain Activation segment C BRAF Mutant Clinical <55 years old Non-sun damage site Acquired nevi Nodal nevi Histologic Pagetoid: greater Size of intraepithelial nests: larger Pigment: heavy Circumscription: greater Solar elastosis: less Melanoma BRAF Wild Type Clinical Older patients Sun damage sites Congenital nevi May demonstrate NRAS and KIT mutations Histologic Pagetoid: less Size of intraepithelial nests: smaller Pigment: light Circumscription: less Solar elastosis: greater Broekaert et al. Pigment cell Melanoma Res. 2010 8

Approximate Frequency of Somatic Histologic Type BRAF KIT NRAS GNAQ/ GNA11 BAP1 Superficial spreading 50-60 1 20 <10 Nodular 50 20-30 <10 Lentigo maligna 5 2-17 (amp 6) Acral lentiginous 13-20 15-20 (amp 30) Melanoma with blue nevus like features Atypical Spitzoid neoplasms 25-75% (89) reported 10 <4 10-15 <10 50 <1 reported ~11-28 Somatic: Sporadic melanomas KIT NRAS BRAF GNAQ/GNA11 Germline: Hereditary melanoma CDKN2A/p16 GNAQ/GNA11 GNAQ (guanine nucleotide binding protein (G protein), alpha, q polypeptide) on chr. 9q21 GNA11 (guanine nucleotide binding protein (G protein), alpha 11) on chr. 19p13 Encode for the α-subunit of G-protein-coupled receptor GNAQ and GNA11 share 90 % homology Mutations in GNAQ/GNA11 in melanocytic lesions subsequent amino acid changes of glutamine to leucine or proline (Q209L or Q209P) GTP hydrolysis is prevented in mutant forms of GNAQ, thus resulting in a constitutive activation GNAQ/GNA11 GNAQ mutations 83% of blue nevi 50% of melanoma with blue nevus features 46% of uveal melanoma 4% of melanomas on chronic sundamage skin GNA11 mutations 7% of blue nevi 32% of uveal melanomas Approximate Frequency of Somatic Histologic Type BRAF KIT NRAS GNAQ/ GNA11 BAP1 Superficial spreading 50-60 1 20 <10 Nodular 50 20-30 <10 Lentigo maligna 5 2-17 (amp 6) Acral lentiginous 13-20 15-20 (amp 30) Melanoma with blue nevus like features Atypical Spitzoid neoplasms 25-75% (89) reported 10 <4 10-15 <10 50 <1 reported ~11-28 Somatic: Sporadic melanomas KIT NRAS BRAF GNAQ/GNA11 Germline: Hereditary melanoma CDKN2A/p16 9

BAP1 BAP1 BAP1 (BRCA1 associated protein 1) on chr. 3p21 Tumor suppressor functions Ubiqutin properties Interactions with BRCA1 and BARD1 Two families with recurring deletions involving BAP1 Autosomal dominant inheritance Each family had one individual with uveal melanoma One family had three members with cutaneous melanoma Affected individuals with multiple (5-50) skin colored, red to brown dome shaped papules Beginning in the second decade Clinically, reminiscent of Spitz nevus differ molecular profile Histologically, dermal tumors Composed of epithelioid shaped melanocytes Large nuclei, prominent nucleoli Cytologically cells appear Spitzoid Absence of hyperplasia, Kamino bodies, clefting Atypical features included Increase cellularity Nuclear pleomorphism Chromosomal abnormalities seen in melanoma (loss of 1,3, 6, 9, and 22) BAP1 BAP1 Sporadic melanocytic lesions evaluated for BAP1 mutation: Common nevi Spitz nevi Atypical spitz tumors 2/18 (11%) Acral lentiginous 1/15 (7%) Mucosal CSD Non-CSD 2/15 Uveal 13/33 (40%) Atypical Spitz Tumors Examination of 32 sporadic atypical spitz tumors (AST) 28% AST with loss of BAP1 89% concomitant loss of BAP1 and with BRAF V600E mutation Three category of spitzoid tumors 1) HRAS mutant 2) BRAFV600E/BAP1 mutant 3) Unknown genetics Wiesner et. al. Am J Surg Pathol. 2012 Approximate Frequency of Somatic Histologic Type BRAF KIT NRAS GNAQ/ GNA11 BAP1 Superficial spreading 50-60 1 20 <10 Nodular 50 20-30 <10 Lentigo maligna 5 2-17 (amp 6) Acral lentiginous 13-20 15-20 (amp 30) Melanoma with blue nevus like features Atypical Spitzoid neoplasms 25-75% (89) reported 10 <4 10-15 <10 50 <1 reported ~11-28 10

Sequencing Platforms in Mutation Analysis of Melanoma Early sequencing technology Sanger Sequencing Pyrosequencing Cobas 4800 BRAF BRAFV600E Mutation Detected Next generation sequencing technology Limitations of Molecular Test Amplification of DNA by PCR remains central in evaluating for genomic mutations in melanoma Melanin pigment is a known inhibitor of PCR reaction May yield no results due to nonamplifiable DNA Limitations of Molecular Test This may become an issue with melanomas that are heavily pigmented Repeat mutational analysis on tumor specimens from different blocks and/or different tissue sources from a patient if material is available Molecular Analysis of Primary vs. Metastatic Melanoma High consistency of mutation status in primary melanomas and metastasis Lymph node Visceral organs Consistency of mutation status appears to decrease when metastasis include Skin Brain Frequency of BRAF/NRAS Mutations in Paired Melanoma Samples 99 patients with paired primary and metastatic melanoma 84/99 (85%) had same BRAF or NRAS mutation pattern in paired primary and metastatic samples Brain and skin metastasis demonstrated lower mutation pattern of 80% and 75% Colombino J Clin Oncol 2012 11

Discrepant Mutation Patterns in Paired Tissue Samples 15/99 (15%) paired primary and metastatic lesions were discrepant in BRAF or NRAS mutation status 8 mutated primary and wild type metastatic lesions (7 BRAF and 1 NRAS) In different metastatic lesions, discrepant WT and BRAF/NRAS mutations observed (7 BRAF and 2 NRAS) Subclones of tumors generated in some patients Immunohistochemical Detection of BRAFV600E Mutation Anti-BRAF V600E (VE1) 37/38 known BRAFV600E mutant melanomas by genetic analysis positive with BRAFV600E immunostain All 11 BRAF mutant variants negative for BRAFV600E stain 47/48 BRAF wild type by genetic analysis were negative with BRAF antibody Sensitivity 97%, Specificity 98% Tumor Heterogeneity for Mutant BRAFV600E BRAFV600E in Acral Lentiginous Melanoma A B C North America Asia South America BRAFV600E Expression Pattern Mutant BRAFV600E in Acral Lentiginous Melanoma Lecture Outline North America U.S.A. Asia Melanoma diagnosis Mutations in melanoma Melanoma targeted therapy Thailand Peru Brazil Positive, Diffuse Positive, Focal Negative South America 12

Targeted Therapy Small molecule inhibitors target genomic abnormality of cancer cell and function Monoclonal antibodies (MABs) Target extracellular receptors Prevent growth, incite death Small molecule inhibitors (NIBs) Target cellular signaling pathways Prevent growth, tumor cell survival Melanoma Targeted Therapy KIT inhibtior Tyrosine kinase inhibitor (TKI) Sorafenib MEK inhibitor Selumetinib, Trametinib AKT inhibitor PI3K inhibitor RAF inhibitor Vemurafenib Imatinib, Phase II Study 42% patients had regression of tumor mass after initiation of therapy 6 month progression free survival of 36.6% V600-Selective BRAF Inhibitors: Vemurafenib Vemurafenib/PLX4032/RG7204 Day 15 Day 1 Guo et. al. J Clin Oncol. 2011 Flaherty NEJM, 2010 Chapman NEJM, 20111 48% with confirmed objective response Improved overall survival 84% vs. 65% Dabrafenib, Phase I Trial Toxicities of Cancer Therapy Inhibitor of BRAF Targets mutant V600E and non- V600E (e.g. V600K) Penetrates bloodbrain barrier Cutaneous toxicities with and KA Falchook et. al. Lancet. 2012 Dermatologic toxicities (DT) is ten most common adverse reaction to adjuvant therapy Examination of DT will become increasingly important component of our daily clinical practice Era of oncodermatology and oncodermatopathology 13

Cutaneous Toxicities Tyrosine Kinase Inhibitors Kerationcytic proliferations Sorafenib therapy Invasive ( with KA features) 64% of lesions AK 2% of lesions Dubauskas et. al. Clin Genitourin Cancer. 2009 MEK Inhibitor Selumetinib and Trametinib Suppurative Folliculitis Desar et. al. Acta Oncologica. 2010 Querfeld et. al. J Am Acad Dermatol. 2011 AKT and PI3K Inhibitors Skin Toxicities Perivascular lymphocytic infiltrate = dermal hypersensitivity reaction (DHR) Responsive to anti-histamines and/or topical steroids Bendell et. al. J Clin Oncol. 2011, Yap et. al J Clin Oncol. 2011 Vemurafenib Associated Toxicities Most common adverse events Arthralgia Fatigue Cutaneous events All cutaneous epithelial proliferations 54% 18% developed either a or KA or both 14

Dermatologic Toxicities with Vemurafenib Adverse event Vemurafenib (N=336) N Dacarbazine (N=282) N Rash 165 (49) 9 (3) Pruritus 74 (22) 4 (1) Xerosis 16 (54) 3 (1) Erythema 38 (11) 4 (1) HFSR 22 (7) 1 (<1) Photosensitivity 101 (30) 10 (4) Sunburn 31 (9) 0 Alopecia 117 (35) 6 (2) Seborrheic Keratosis 24 (7) 1 (3) Hyperkeratosis 6 (21) 0 Skin papilloma 62 (18) 0 Actinic keratosis 21 (6) 9 (3) Keratoacanthoma 27 (8) 0 Squamous carcinoma 40 (12) 1 (<1) Chapman et.al. Mabs Nibs Non-selective Sorafenib Sunitinib Vemurafenib Selective Dabrafenib Inhibition of apoptosis, cell cycle progression, differentiation Therapeutic Targets G-protein GNAQ/GNA11 KIT RTK RAS RAF MEK MAPK/ERK Imatinib Selumetinib Trametinib Proliferation, cell survival, differentiation CDKN2A p16/rb BAP1 PI3K/AKT BKM120 MK-2206 PTEN Inhibition of apoptosis proliferation Clinical and Histologic Features Clinical and Histologic Features Class Drug Clinical Histology KITRi Imatinib Maculopapular rash Perivascular dermatitis Multi-TKi Sorafenib Keratosis, warty papules, Multi-TKi Sorafenib HFSR Hyperkeratosis, MEKi Selumentinib, Papulopustular rash Suppurative folliculitis Trametinib PI3Ki BKM120 Maculopapular rash Perivascular dermatitis AKTi MK-2206 Maculopapular rash Perivascular dermatitis BRAFi Vemurafenib Keratosis, warty papules, BRAFi Vemurafenib HFSR Hyperkaratosis, Class Drug Clinical Histology KITRi Imatinib Maculopapular rash Perivascular dermatitis Multi-TKi Sorafenib Keratosis, warty papules, Multi-TKi Sorafenib HFSR Hyperkeratosis, MEKi Selumentinib, Papulopustular rash Suppurative folliculitis Trametinib PI3Ki BKM120 Maculopapular rash Perivascular dermatitis AKTi MK-2206 Maculopapular rash Perivascular dermatitis BRAFi Vemurafenib Keratosis, warty papules, BRAFi Vemurafenib HFSR Hyperkaratosis, Clinical and Histologic Features Clinical and Histologic Features Class Drug Clinical Histology KITRi Imatinib Maculopapular rash Perivascular dermatitis Multi-TKi Sorafenib Keratosis, warty papules, Multi-TKi Sorafenib HFSR Hyperkeratosis, MEKi Selumentinib, Papulopustular rash Suppurative folliculitis Trametinib PI3Ki BKM120 Maculopapular rash Perivascular dermatitis AKTi MK-2206 Maculopapular rash Perivascular dermatitis BRAFi Vemurafenib Keratosis, warty papules, BRAFi Vemurafenib HFSR Hyperkaratosis, Class Drug Clinical Histology KITRi Imatinib Maculopapular rash Perivascular dermatitis Multi-TKi Sorafenib Keratosis, warty papules, Multi-TKi Sorafenib HFSR Hyperkeratosis, MEKi Selumentinib, Papulopustular rash Suppurative folliculitis Trametinib PI3Ki BKM120 Maculopapular rash Perivascular dermatitis AKTi MK-2206 Maculopapular rash Perivascular dermatitis BRAFi Vemurafenib Keratosis, warty papules, BRAFi Vemurafenib HFSR Hyperkaratosis, 15

Clinical and Histologic Features Class Drug Clinical Histology KITRi Imatinib Maculopapular rash Perivascular dermatitis Multi-TKi Sorafenib Keratosis, warty papules, Multi-TKi Sorafenib HFSR Hyperkeratosis, MEKi Selumentinib, Papulopustular rash Suppurative folliculitis Trametinib PI3Ki BKM120 Maculopapular rash Perivascular dermatitis Dermatologic Toxicities with Selective RAF Inhibitor Vemurafenib MDACC Experience AKTi MK-2206 Maculopapular rash Perivascular dermatitis BRAFi Vemurafenib Keratosis, warty papules, BRAFi Vemurafenib HFSR Hyperkaratosis, Evaluation of 141 DTs During Vemurafenib Therapy Clinically Distinct Characteristics Sex Male Female Age (in years) at initiation of VT Median Range Tumor type treated Melanoma Papillary thyroid cancer Onset (in months) of first DT(s) Median Range No. of DT(s)/patient 1-5 6-10 >10 No. of Patients 13 20 29 (88) 4 (12) 26 (79) 2 (6) 5 (a) (15) 56 27-84 3.0 0.3-36 BRAFi Induced Cutaneous Epithelial Proliferations Clinical distinct lesions Papules/ with keratosis Size range: 0.3-1.0 cm Peripheral rim of erythema Diagnostic Category of Cutaneous Epithelial Proliferations Category of DTs No. of Lesions Diagnosis CEP 48 (a,c) (40) VV 29 (24), invasive 25 (a, b) (21) AK 6 (5) SCIS 4 (3) FAD 4 (3) KA 1 (<) KP 1 (<1) BLK 1 (<1) FC 1 (<1) IFK Total 120 16

* * A B C KA VV * D E F HAK VV * A B G H I 17

Summary of Histologic Features with BRAFi Papillomatosis Hyperkeratosis with parakeratosis Hypergranulosis, hemorrhage in stratum corneum Changes of verruca Associated with abnormal follicle/folliculcentric Endophytic/exophytic In situ component prominent Collaret of epidermis without significant atypia BRAFi Induced Cutaneous Epithelial Proliferations Histologically reproducible architectural appearance on scanning magnification Crateriform proliferation with histologic changes of verruca Associated with abnormal follicle Range of diagnosis Keratosis to Mechanisms of Vemurafenib Induced Squamous Cell Carcinoma Vemurafenib Associated Melanocytic Lesions Melanocytic lesions 4 (36) DN 3 (27) CMN 1 (9) AMP 1 (9) IDN 1 (9) Melanoma, metastatic 1 (9) MIS+nevus Total 11 Oberholzer et.al. J Clin Oncol. 2011 Morphologic Changes Melanoma with Associated Nevus Involution-regressed features Alteration in color and size-increase pigment and size Development of new nevi Development of melanoma 18

Conclusion Examined diagnosis of melanoma Incorporation of IHC and FISH Reviewed frequent mutations in cutaneous melanoma Described the range of cutaneous toxicities from melanoma targeted therapy Thank You Acknowledgements Section of Dermatopathology Dr. Prieto Dr. Torres-Cabala Dr. Tetzlaff Dr. Ivan Division of Dermatology Dr. Duvic Dr. Tsai Department of Melanoma Medical Oncology Dr. Kim 19