Disclosure Declaration Clinical Utility of Molecular Testing in Malignant Melanoma

Transcription

1 Disclosure Declaration Clinical Utility of Molecular Testing in Malignant Melanoma No Conflict of Interest Allison Cushman-Vokoun, M.D., Ph.D Associate Professor, Department of Pathology and Microbiology University of Nebraska Medical Center CLINICAL UTILITY OF MOLECULAR TESTING IN MALIGNANT MELANOMA Objectives Determine which genes are most likely to be mutated in various melanoma subtypes and benign nevi. Appreciate which gene mutations can currently direct targeted therapy in advanced melanoma. Understand how different mutations in the same gene may determine different therapy. Recognize the benefits and limitations of molecular pathology techniques in the determination of genetic mutations in solid tumors, including melanoma. 1

2 Outline BRAF NRAS KIT Genes on the Nebraska Medicine Melanoma Mutation Panel GNAQ GNA11 HRAS FISH Testing Solid Tumor Testing Caveats CDKN2A = pink4a (p16) and ARF From Miller, A. et al. (2006) NEJM 355:51 Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society. BRAF Mutations BRAF is a serine-threonine kinase Signal mediator in RAS-RAF-MAPK pathway BRAF p.v600e found in % of melanomas Missense mutation valine substituted by a glutamic acid Kinase Domain Constitutive activation and MEK phosphorylation Mimics phosphorylation site because of negative charge 2

3 EGFR EGFR 9/14/2016 EGF EGF NRAS GTP BRAF MEK MAPK Proliferation Cell Survival Nucleus BRAF and Cancer 7 % human cancers p.v600e accounts for >80 % of BRAF mutations in various cancers Melanoma Colorectal carcinoma Papillary Thyroid Carcinoma NSCLC Hairy Cell Leukemia Various CNS neoplasms (gliomas/astrocytomas) BRAF Mutations in Melanoma Activating BRAF mutations can occur in premalignant nevi More common in intermittently sun damaged skin (not chronic) 3

4 BRAF p.v600e and Vemurafenib Specific p.v600e BRAF inhibitor 80 % of BRAF-mutated tumors had a partial or complete response (26/32) WT BRAF (n=5) = no response From Flaherty, K. et al. (2010) NEJM 363:9 Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society. Other BRAF Mutations (Exon 15) Codon 600 p.val600lys (p.v600k) ~ 8% p.val600arg (p.v600r) ~ 1% Others Codon 601 (p.k601e) <1% Codons 594, 595, 597 <1% 4

5 EGFR EGFR 9/14/2016 From Long, G.V. et al. (2014) NEJM 371:1877 Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society. FDA-approved therapies Cobimetinib (MEKi) and vemurafenib (BRAFi) in combination for BRAF p.v600e and p.v600k Trametinib (MEKi) and Dabrafenib (BRAFi) in combination for BRAF p.v600e and p.v600k EGF EGF BRAF X MEK MAPK X Proliferation Cell Survival NRAS GTP Nucleus 5

6 FDA-approved Assays Both real-time PCR-based Limited Mutation detection BioMérieux THxID -BRAF V600E and V600K cobas 4800 BRAF V600 Mutation Test V600E may detect others Nebraska Medicine Assays Pyrosequencing covers codons 600 and 601 variants Next Generation Sequencing Melanoma Panel - Covers most of BRAF Exons 15 and 11 p.v600k p.g469e 6

7 NRAS Small GTPase, similar to KRAS 15-20% sporadic melanomas have activating NRAS mutations Possibly more often in chronically exposed sun damage-related lesions In congenital nevi also Mutually exclusive of BRAF mutations 60 year old Skin Left lower leg Superficial Spreading KIT Alterations Found in 20-30% of: Mucosal Melanomas Acral Melanomas Chronic sun damage Conjunctival Melanomas Mutations (10-20%), increased copy number, amplification Exon 11 (Juxtamembrane domain) Imatinib therapy useful for some mutations Durable response rate of 16% p.l576p or p.k642e Higher Mutant to wildtype ratios responded better Beadling, C. et al. (2008) Clin Can Res 14:6821 Caraval, R. et al. (2011) JAMA 305:2327 7

8 60 year old, ano-rectal melanoma, initially BRAF negative by single gene assay Uveal Melanomas 83% had somatic mutations in GNAQ or GNA11 alpha subunit for G-coupled protein receptors Q209 mutations of GNA11 7% blue nevi 32% primary uveal melanoma 57% metastases Q209 mutations of GNAQ 55% Blue nevi 45% uveal melanoma 22% metastases From Raamsdonk, C. et al. (2010) NEJM 363: Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society. 62 year old with liver mass and history of melanoma 8

9 HRAS and Spitz Nevus Useful in excluding a melanoma in the differential of a Spitz nevus/stump Mainly codon 61 mutations in HRAS See Dr. Kozel s discussion Van Engen-van Grusven, AC. et al. (2010) Am J Surg Pathol Oct;34(10): Melanoma Mutation Panel BRAF NRAS KIT GNAQ GNA11 HRAS Genes on the Nebraska Medicine Melanoma Mutation Panel FISH Analysis Neoplasm Chromosomal Gains Melanoma (NCSD) 6p, 7, 8q, 17q, 20q 9p, 10, 21q Chromosomal Losses Melanoma (CSD) 6p, 11q13, 17q, 20q 6q, 8p, 9p, 13, 21q Acral Melanoma Mucosal Melanoma 6p, 7, 8q, 17q, 20q Amplification (5q13, 5p15, 11q13, 12q14) 1q, 6p, 7, 8q, 11q13, 17q, 20q Amplification (1q31, 4q12, 12q14) 6q, 9p, 10, 11q, 21q 3q, 4q, 6q, 8p, 9p, 10, 11p, 21q Uveal Melanoma 6p, 8q 1p, 3, 6q, 16q Spitz Nevi 11p (~20%) Common Acquired, Dysplastic & Blue nevi None None Courtesy of Dr. Julia Bridge 9

10 Acral-lentiginous Melanoma Most common amplified region = 11q13 (CCND1) Amplification also found in isolated cells in histologically normal appearing skin adjacent to the melanomas permitting the detection of occult tumor cells CEP 11 CCND1 Courtesy of Dr. Julia Bridge 4-Color Melanoma FISH Probe Set Some melanocytic tumors are ambiguous; reproducible histologic dx of benign or malignant is difficult FISH exploit genetic differences between benign nevi and melanoma Low cost, perform on archival FFPE, a useful tool in reinforcing dx of melanoma (not standalone test) Courtesy of Dr. Julia Bridge Melanoma FISH Probe Set 6p25 RREB1 CEP q23 MYB 11 11q13 CCND1 RREB1 (6p25) Ras responsive elem binding protein 1 MYB (6q23) v-myb myeloblastosis viral oncogene CCND1 (11q13) cyclin D1 CEP (6p11.1-q11.1) Courtesy of Dr. Julia Bridge 10

11 Normal Abnormal CEP6 RREB1 (6p25) MYB (6q23) CCND1 (11q13) Courtesy of Dr. Julia Bridge Scoring Criteria RREB1 (6p25)>2 RREB1 (6p25)>CEP6 MYB (6q23)<CEP6 CCND1 (11q13)>2 FISH Utility in Melanoma 9p21, 6p25, 11q13, and 8q24 as a probe set improved discriminatory power in differentiating melanomas from nevi: sensitivity of 94% and specificity of 98% By incorporating 9p21 (CDKN2A) into the 4-probe FISH assay improved sensitivity for detecting spitzoid melanomas High incidence of 9p21 loss in melanoma-associated nevi in contrast to relative absence of chromosomal aberrations in common nevi; 9p21 loss regarded as cytogenetic marker of melanocytic nevi likely to progress Gerami, P. et al. (2012) Am J Surg Pathol Jun;36(6):808-17; Casorzo, L. et al. (2005) Melanoma Res 15: Courtesy of Dr. Julia Bridge Molecular MelanomaTesting-Caveats Frozen or Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Frozen Tissue can t estimate tumor FFPE DNA somewhat degraded No Decalcified tissue (DNA degraded by acid solution) EDTA may work but slow decalcification Melanin is an inhibitor of PCR, can be overcome by albumin addition Know you are testing the tumor Enrich for at least 20 % tumor Assays usually 1-5% sensitivity for mutated allele 11

12 References Miller, AJ., Mihm, MC. Jr New England Journal of Medicine 355 (1), Flaherty, KT., Puzanov, I., Kim, KB., Ribas, A., McArthur, GA., Sosman, JA., O'Dwyer, PJ., Lee, RJ., Grippo, JF., Nolop, K., Chapman, PB New England Journal of Medicine 363 (9), Long, GV., Stroyakovskiy, D., Gogas, H., Levchenko, E., de Braud, F., Larkin, J., Garbe, C., Jouary, T., Hauschild, A., Grob, JJ., Chiarion Sileni, V., Lebbe, C., Mandalà, M., Millward, M., Arance, A., Bondarenko, I., Haanen, JB., Hansson, J., Utikal, J., Ferraresi, V., Kovalenko, N., Mohr, P., Probachai, V., Schadendorf, D., Nathan, P., Robert, C., Ribas, A., DeMarini, DJ., Irani, JG., Casey, M., Ouellet, D., Martin, AM., Le, N., Patel, K., Flaherty, K New England Journal of Medicine 371 (20), Beading, C., Jacobson-Dunlop, E., Hodi, FS., Le, C., Warrick, A., Patterson, J., Town, A., Harlow, A., Cruz, F. 3 rd, Azar, S., Rubin, BP., Muller, S., West, R., Heinrich, MC., Corless, CL Clinical Cancer Research 14 (21), Carvajal, R, Antonescu, C, Wolchok, J, Chapman, P, Roman, R-A, Teitcher, J, Panageas, K, Busam K, Chmielowski, B, Jose Lutzky, J, Pavlick, A, Fusco, A, Cane, L, Takebe, N, Vemula, S, Bouvier, N, Bastian, B, Schwartz, G JAMA 305 (22): Van Raamsdonk, CD., Griewank, KG., Crosby, MB., Garrido, MC., Vemula, S., Wiesner, T., Obenauf, AC., Wackermagel, W., Green, G., Bouvier, N., Sozen, MM., Baimukanova, G., Roy, R., Heguy, A., Dolgalev, I., Khanin, R., Busam, K., Speicher, MR., O Brien, J., Bastian, BC New England Journal of Medicine 363 (23), van Engen-van Grunsven, AC., van Dijk, MC., Ruiter, DJ., Klaasen, A., Mooi, WJ., Blokx, WA American Journal of Surgical Pathology 34 (10), Gerami, P., Li, G., Pourvazdanparast, P., Blondin, B., Beilfuss, B., Slenk, C., Du, J., Guitart, J., Jewell, S., Pestova, K., American Journal of Surgical Pathology 36 (6), Casorzo, L., Luzzi, C., Nardacchione, A., Picciotto, F., Pisacane, A., Risio, M., Melanoma Research 15,