2/6/2018. Original Paradigm. Clonal Chromosomal A berrations. Only 20% of Spitz Nevi 95% 6p, 7q, 17q, 20q, 4q,8q, 1q, 11q. Isolated Gain in 11p

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1 Molecular Diagnostics for Melanocytic Neoplasms: Moving towards a Revolution in the Management of Melanocytic Neoplasms Pedr am Gerami MD Associate Professor of Dermatology, Pathology and Pediatrics at Northwestern University Co-dir ector of Melanoma Program, Northwestern Skin Cancer Institute Disclosures: I have been a consultant to Abbott Molecular, Neogenomics, Castle Biosciences, Myriad Genomics, and Derm Tech Int. Summary 95% of melanomas have clonal chromosomal aberrations including gains on 7q, 8q, 6p, 1q, 20q, 17 and 3 as well as losses on 9p,10, 6q and 8p Conv ersely, nevi inf requently have chromosomal aberrations: 20% of spitz nevi have isolated gain in 11p Original Paradigm FISH Clonal Chromosomal A berrations Common Gains Common Deletions Melanoma 95% 6p, 7q, 17q, 20q, 4q,8q, 1q, 11q 9p, 10, 21q Nevi Only 20% of Spitz Nevi Isolated Gain in 11p 1

2 Melanoma Four P robe FISH A ssay: 6 p25 8 q q13 9 p21 Sensitivity:86% Specificity:95% Multi-center collaborative study on Spitz tumors involving Northwestern Univ, UCSF, Univ of Michigan, MD Anderson, Memorial Sloan Kettering and Sydney Melanoma Unit Study Design 1 )M ulti-center retrospective case c ontrolled s tudy 2 )I nclusion criteria: Diagnosis of AST 5 years f ollow up no adverse event or tumor spread beyond a s entinel lymph node or les s than 5 years of follow up if there was evidence of tumor s pread beyond the sentinel lymph node 3 )FISH evaluation with probes targeting 6 p25, 6 q23, C ep 6, 11q13, 9 p21, and 8 q24 was performed on all c ases blinded to the c linical outcome Data Collection and Analysis The following data points were collected for each case: 1)Age 2)Sex 3)Anatomic site 4)Breslow depth 5)Mitotic count 6)Clark Level 7)Ulceration status 8)Presence or Absence of kamino bodies 9)Expansile nodular growth 10)Epidermal Consumption 11)Epithelioid versus spindle morphology 12)Complete clinical follow up including sentinel node status 13)FISH data 2

3 Summary of clinical, histological, and molecular data by clinical stage Clinical N Average Sex Average Ulceration Average Average % Patients with Average Stage Age Ratio (M :F:NA) Breslow Status (Y: N) M itotic Clark Positive FU Time (months) (Yrs) (mm) Rate (/mm 2 ) Level FISH Frequency of FISH results for good (1a,1b,1x) vs. bad (2,4) outcome Spitzoid patients Patient outcome Fisher s FISH result Good (1a,1b,1x) Bad (2,4) exact test Positive 8 5 6p 0.02 Negative 56 6 Positive 8 1 6q 1.00 Negative p Cep 6 Positive 2 1 Negative M : 31F: 5NA 2.3 7Y: 57N a 13 1b 11q 9p Positive 8 5 Negative 56 6 Positive 3 9 Negative < x M : 4 F: 1NA 3.2 4Y: 4N q Positive 1 1 Negative FISH outcome Positive Negative 49 0 < M : 1F 6.6 1Y: 2N *NA- not available FISH old probe set Positive 15 6 Negative FISH new probe set Positive 9 11 Negative 55 0 < Cytogenetic risk Low risk 55 0 Intermediate 6 2 High risk 3 9 < Multivariate Analysis Comparing Variables for Group 1 versus Group 2 through 4 Variable Estimate Odds Ratio 95% CI for OR p-value Mitotic F9P (+ vs. -) < Footnote: backward elimination method was used to derive the final multivariable models 3

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5 Recurring Themes A mong A STs With Ree Aggressive Clinical Course : 1) 9 of 11 cases with disease progression beyond the sentinel had homozygous 9p21 deletions 2) 4 of 9 patients with ASTs with homozygous 9p21 deletion developed recurrent in transit metastasis in the skin in addition to sentinel and non-sentinel lymph node involvement all of whom are still alive. One patient with up to 8 years of follow up. 3) Distant metastasis and death from disease from ASTs uncommon but when it does happen most cases likely to have homozygous 9p21 deletion and likely to occur with a more protracted course compared to conventional melanomas Chimeric proteins resulting f rom translocations involving receptor tyrosine kinases Mutually exclusive fusion proteins identified in 72/140 (51%) Spitzoid neoplasms studied 5

6 ALK positive Spitz Nevus

7 NTRK positive Spitz tumor 7

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9 Figure 4 Figure 4. This boxplot demonstrates the range of lesion sizes by fusion group with the black bar representing the median and the overlying boxes representing the 25 th -75 th percentiles. As can be seen in the figure, the 25 th -75 th percentile ALK-fused cases do not overlap with any of the other subgroups, indicating that the majority of ALK-translocations were significantly larger than non-alk-fusions. 9

10 Recently a number of familial melanoma syndromes involving BAP1 have been described: 1)Wiesneret al described 2 families with uveal melanoma, cutaneous melanoma, nevi with atypical epithelioid c ell component and grey zone lesions with atypical epithelioid cell c omponent Family1: c.1305 del6 Family2: c a>g 2)Testa et al described 2 families with uveal melanoma and mesothelioma Family 1 : p.gin684x Family 2 : p.iie72fsx7 3 )Abdel-Rahman described 1 family with uveal melanoma, lung adenocarcinoma and meningiomas Family 1 : c. 799 C->T Associated Malignancies BAP1 = BRCA1-associated protein-1 Patients with germline mutations in BAP1 were found to be at increased risk for: 1. Uveal melanoma 2. Mesothelioma (no asbestos exposure) 3. Cutaneous Melanoma 4. Renal Cell Carcinoma 5. Atypical Spitz Tumors/BAPomas 6. Basal Cell Carcinoma *Other tumors such as meningioma, cholangiocaricinoma, breast and lung carcinoma have been seen in multiple carriers and may be associated with the syndrome. Image: Carbone M, Yang H, Pass HI, Krausz T, Testa JR, Gaudino G. BAP1 and cancer. Nat Rev Cancer Mar;13(3): d oi: /nrc3459. Review. PubMed PMID: ; PubMed Central PMCID: PMC Nuclear deubiquitinating protein that interacts with several other proteins, including BRCA1 Structural architecture in interaction with other proteins not completely understood but has been shown to be involved with: DNA damage response Cell cycle regulation Cell growth Chromatin dynamics 10

11 8 New Families Identified 4 families identified after a dermatologist diagnosed a BAP-1 Deficient Tumor in a young patient (ages 10-32) and asked about family history 1 family identified after a patient in her 40 s was diagnosed with a BAP1 associated nevoid melanoma Family 4 Family 5 Median Age of Onset and Prevalence of Characteristic Tumors in BAP1 Patients Tumor/Malignancy Number of Cases Estimated Penetrance Median Age Literature Median Age Our Study Median Age General Population UvealMelanoma 61/215 cases 28% Mesothelioma 48/215 cases 22% Cutaneous Melanoma 38/215 cases 18% BAP1 Deficient Tumors 33/215 cases 17% Renal Cell Carcinoma 20/215 cases 9% Median Ages of Onset of Associated Tumors Prevalence in Patients with Skin Examinations Of the 53 patients with a documented TBSE by a dermatologist, 40 (75%) were found to have at least one BDT on clinical exam. The number of BDTs in BAP1 patients thought to increase as the patient ages. 11

12 Histologic Presentation Mesothelioma Truncating Mutations Of the 48 patients diagnosed mesothelioma, 96% (n=46) carried truncating mutations in BAP1. The 2 mesothelioma patients without truncating mutations were diagnosed at age 71 and 72 (median age of diagnosis = 56) Of the 29 patients age >56 without a mesothelioma diagnosis, 66% had truncating mutations (n=19) Truncating mutation occurs before the nuclear localization sequence BAP1 protein accumulates in cell cytoplasm Abberrant BAP1 protein has been shown to form amyloid aggregates in cell cytoplasm Inflammation and cytotoxicity involved in mesothelioma pathogenesis? Reported Exonic Mutations in BAP1 Hierarchy of Risk f or Distant Metastasis in Melanocytic Neoplasms with Spitzoid Morphology Conventional Melanoma Most Aggressive Spitzoid Melanoma with Homozygous 9p21 Deletion Intermediate ASTs with no evidence of copy number aberrations Low Risk ASTs with 6q23 Deletion Low Risk ASTs with 3p21 Deletion/BAPomas Low Risk ASTs with 11p gains Low Risk Chromosomal Aberrations New Paradigm Melanoma 95% Spitz tumors Chimeric Fusion Proteins: Ros, AlK, NTRK1, BRAF, RET Cellular functions represented in GEP signature 54 g enes initially assessed and then narrowed to 28 with 3 additional controls Migration/chemotaxi s/metastasis CXCL14 SPP1 CLCA2 S100A9 S100A8 Differentiation/ proliferation CRABP2 SPRRIB BTG1 Common Gains 6p, 7q, 17q, 20q, 4q,8q, 1q, 11q Isolated Gain in 11p, can have gains in 7q Chemokine/secreted molecules Gap junction/cellular adhesion CCL14 MGP SPP1 GJA1 DSC1 PPL Cell surface receptors Structural proteins TACSTD2 CLCA2 ROBO1 MGP SPP1 CST6 Common Deletions 9p, 10, 21q 3p21, 6q23, heterozygous loss of 9p21 Lymphocytic invasion Gerami, Clin Cancer Res 2013 LTA4H Angiogenesis regulator Transcription factor TRIM29 Extracellular functions CXCL14 KRT6B KRT

13 % free of metastasis % free of metastasis 1 st intended use: Identify the node negative patients who have aggressive disease Validation Study #1: Background demographics Characteristics Training Set (n = 164) Validation Set (n = 104) Age, median yrs (range) 61 (23-89) 58 (18-94) Follow-up, median yrs (range) 4.9 ( ) 5.7 ( ) Patients with Stage I and II melanoma Quantifies expression of 31 genes from primary tumor Applies a validation algorithm Classifies patients as low vs high risk with strong accuracy Class 1 test result: Low risk of metastasis within 5 years Class 2 test result: High risk of metastasis within 5 years Identification of high risk patients allows them the opportunity to access further evaluation, treatment, and monitoring with the goal of improving long-term survival 73 AJCC Stage n (%) n (%) 0 15 (9%) 0 (0%) I 63 (38%) 56 (54%) II 67 (41%) 34 (33%) III 18 (11%) 12 (11%) IV 1 (1%) 2 (2%) Breslow Thickness Median mm (range) 1.86 ( ) 1.4 ( ) 1mm 46 (28%) 45 (43%) > 1mm 101 (62%) 58 (56%) Mitotic Index 1/mm 2 43 (26%) 29 (28%) > 1/mm 2 82 (50%) 53 (51%) Ulceration Absent 104 (63%) 65 (63%) Present 46 (28%) 28 (27%) Growth Pattern Superficial spreading 75 (46%) 56 (54%) Nodular 47 (29%) 25 (24%) Desmoplastic/lentigo maligna/ 25 (15%) 10 (10%) acral lentiginous Gerami, Clin Cancer Res 2015 Censor date: May GEP A ccuracy: Disease-free survival prediction all cases Tr aining Set n=104 p< Validation Set n=164 n=104 5-yr DFS Class 1 = 91% Class 2 = 25% ROC = Accuracy = 83% Sensitivity = 85% Gerami, Clin Cancer Res 2015 Censor Date: May, yr DFS Class 1 = 97% Class 2 = 31% ROC = Accuracy = 86% Sensitivity = 89% 75 SLNB v s. GEP 1 st and 2 nd Validation Studies with SLNB pr ocedure Patients with SLN Procedure (n=217) SLN positive = 58 Met = 37 Non = 21 SLN Results SLN negative = 159 Met = 70 Non = 89 PPV = 64% NPV = 56% Class 2 = 141 Met = 91 GEP Results 100% Non = 50 Class 1 = 76 Met = 16 Non = 60 PPV = 65% NPV = 79% DecisionDx-Melanoma Improves Prediction Over SLNB Negative Status for Distant Metastasis-Free Survival DMFS 100% 75% 50% 25% 0% 0 n=217 p< SLNB 4 6 Time (years) 8 SLNB- SLNB+ 10 SLNB- (n=159) SLNB+ (n=58) Events yr DMFS 64% 42% 75% 50% 25% 0% DecisionDx in SLNB- Patients 0 n=159 p< Time (years) Class 1/SLNB- (n=67) Class 1/ SLNB- Class 2/ SLNB Class 2/SLNB- (n=92) Events yr DMFS 86% 49% 13

14 Previously unreported validation cohort of 523 patients Cox Regression Analysis of 523 Patients Comparison of GEP and SLN in 523 patients GEP plus SLN in combination Independent Validation of 357 Previously Unreported Stage I and II patients RFS in validation cohor t of 264 pr eviously unr eported stage I patients 14

15 % free of metastasis Independent cohort of 93 previously unreported stage II cases 166 previously unreported stage III patients DecisionDx-Melanoma Identifies ~70% of SLNB Negative Patients who had Distant Metastasis Adhesive Patch Testing DMFS by SLN Status DecisionDx in SLNB- Patients Clinically Suspicious Melanocytic Lesion (mole) ABCDE criteria or Dermascope 5-yr SLN- Events SLNB- DMFS n=216 80% 42 5-yr SLNB+ SLN+ Events DMFS Class 1/SLN- (n=106) Class 2/SLN- (n=110) 5-yr DMFS Events 90% 13 71% 29 Identified ~70% (29/42) events E G I R Non-invasive biopsy (tape strip) Highly accurate technology Objective findings Excisional Biopsy n=368 p< n=152 53% 69 Time (years) RNA Expression Continue observation Zager et al. J ClinOncol 2016;34 (suppl; abstr 9581); manuscript in preparation Scientific Rationale Results of Validation Study: Total series sensitivity:91% Total series specificity:69% Sensitivity in consecutive series: 79% Specificity in consecutive series: 80% 89 15

16 mrna Expression for Diagnosis of Melanocytic Neoplasms From Myriad Evaluated 859 total lesions by mrna expression profiling focusing on 23 genes Gene Signature 1) 13 immune related genes 2) 1 cell differentiation gene 3) 9 housekeeping genes A 23 gene expression signature for differentiating melanoma from nevi PRAME (2 Amplicons) S100A7 S100A8 S100A9 S100A12 PI3 S100A Group PRAME Housekeeper Group Algorithm Score Immune Signaling Group IRF1 CCL5 CXCL9 CXCL10 CD38 LCP2 PTPRC SELL Scale and Threshold Values Training set of 464 and Validation set of year old male, upper back Pretest: Dysplastic nevus with moderate to severe atypia +2.2 (Malignant) 3/3: Melanoma in situ Case

17 year old male; face Pretest: Desmoplastic melanoma 71 year old male, face: Excision Score: +3.8 Case

18 Excision Specimen (Block A4) H&E Melan- A 74 year old female, cheek Pretest: Inflamed nevus S100 Sox-10 Score: +3.1 = False positive 51 year old female, mid back Pretest: Melanoma 34 year old femal, My histologic diagnosis: Spindle Cell nevus of Reed Score: = False negative Immune: -2.4 S100A: -7.5 PRAME: +3.3 Case Score: +6.5 Immune: S100A: +3.4 PRAME: +2.3 Case Myriad Score 4.2 Conclusions from metaanalysis: most useful for predicting prognosis in stage III patients. Most studies show relatively low detection rate of patients with relapse in stage I or II patients Best results in stage III patients but more data needed and more standardization as far as markers assessed and timing of the assessment 18