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Molecular Diagnostics for Melanocytic Neoplasms:

Moving towards a Revolution in the Management of Melanocytic Neoplasms

Pedr am Gerami MDAssociate Professor of Dermatology, Pathology and

Pediatrics at Northwestern UniversityCo-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

Melanoma Nevi

ClonalChromosomal Aberrations

95%Only 20% of Spitz Nevi

Common Gains

6p, 7q, 17q, 20q, 4q,8q, 1q, 11q

Isolated Gain in 11p

Common Deletions

9p, 10, 21q

Original ParadigmFISH

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Melanoma

Four P robe FISH A ssay:6p25

8q2411q13

9p21Sensitivity: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)Multi-center retrospective case controlled s tudy

2)Inclusion criteria:

Diagnosis of AST

5 years follow up no adverse event or tumor spread

beyond a sentinel lymph node or

less than 5 years of follow up if there was evidence of tumor spread beyond the sentinel lymph node

3)FISH evaluation with probes targeting 6p25, 6q23, C ep 6, 11q13, 9p21, and 8q24was performed on all

cases blinded to the c linical outcome

Data Collection and Analysis

The following data points were collected for each case:

1)Age2)Sex

3)Anatomic site

4)Breslow depth5)Mitotic count

6)Clark Level

7)Ulceration status8)Presence or Absence of kamino bodies

9)Expansile nodular growth

10)Epidermal Consumption11)Epithelioid versus spindle morphology

12)Complete clinical follow up including sentinel node

status 13)FISH data

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Clinical

Stage

N

75

Average

Age

(Yrs)

Sex

Ratio (M :F:NA)

Average

Breslow

(mm)

Ulceration

Status (Y: N)

Average

M itotic

Rate (/mm2 )

Average

Clark

Level

% Patients with

Positive

FISH

Average

FU Time

(months)

1 64 20.9 28M : 31F: 5NA 2.3 7Y: 57N 1.7 3.9 23.4 97.8

1a6

1b13

1x45

2 8 8.4 3M : 4 F: 1NA 3.2 4Y: 4N 6.1 4.2 100 22.9

4 3 36.3 2M : 1F 6.6 1Y: 2N 2.7 3.7 100 64

*NA- not available

Summary of clinical, histological, and molecular data by clinical stagePatient outcome

FISH result Good (1a,1b,1x) Bad (2,4)Fisher’s

exact test

6pPositive 8 5

0.02Negative 56 6

6qPositive 8 1

1.00Negative 56 10

6p Cep 6Positive 2 1

0.38Negative 62 10

11qPositive 8 5

0.02Negative 56 6

9pPositive 3 9

<0.0001Negative 61 2

8qPositive 1 1

0.27Negative 63 10

FISH

outcome

Positive 15 11<0.0001

Negative 49 0

FISH old

probe set

Positive 15 60.06

Negative 49 5

FISH new

probe set

Positive 9 11<0.0001

Negative 55 0

Cytogenetic

risk

Low risk 55 0

<0.0001Intermediate 6 2

High risk 3 9

Frequency of FISH results for good (1a,1b,1x) vs. bad (2,4) outcome Spitzoid patients

Variable Estimate Odds Ratio 95% CI for OR p-value

Mitotic 0.3334 1.40 1.04 – 1.87 0.03

F9P (+ vs. -) 2.0596 61.51 8.41 – 449.9 <0.0001

Footnote: backward elimination method was used to derive the final multivariable

models

Multivariate Analysis Comparing Variables for Group 1

versus Group 2 through 4

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ReeRecurring Themes Among ASTs With

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 deletionand likely to occur with a more protracted course compared to conventional melanomas

Chimeric proteins resulting from translocations involving receptor tyrosine kinases

Mutually exclusive fusion proteins identified in 72/140 (51%) Spitzoid neoplasms studied

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ALK positive Spitz Nevus

33 34

35

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NTRK positive Spitz tumor

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Figure 4

Figure 4. This boxplot demonstrates the range of lesion sizes by fusion group with the black bar representing the median and the overly ing boxes representing the 25th-75th

percentiles. As can be seen in the figure, the 25th-75th percentile ALK-fused cases do not

overlap with any of the other subgroups, indicating that the majority of ALK-translocations were s ignificantly larger than non-ALK-fusions.

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Recently a number of familial melanoma syndromes involving BAP1 have been described:

1)Wiesneret al described 2 families with uvealmelanoma, cutaneous melanoma, nevi with atypical

epithelioid cell component and grey zone lesions with atypical epithelioid cell component

Family1: c.1305 del6Family2: c.2057-2A>G

2)Testa et al described 2 families with uvealmelanoma and mesothelioma

Family 1: p.GIn684XFamily 2: p.IIe72fsX7

3)Abdel-Rahmandescribed 1 family with uvealmelanoma, lung adenocarcinoma and meningiomas

Family 1: c. 799 C->T

Associated Malignancies

Patients with germline mutations in BAP1 were found to be at increased risk for:

1. Uveal melanoma2. Mesothelioma (no asbestos exposure)

3. Cutaneous Melanoma4. Renal Cell Carcinoma

5. Atypical Spitz Tumors/BAPomas6. 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.

BAP1 = BRCA1-associated protein-1

Image: Carbone M, Yang H, Pass HI, Krausz T, Testa JR,

GaudinoG. BAP1 and cancer.

Nat Rev Cancer. 2013 Mar;13(3):153-9. doi:

10.1038/nrc3459. Review.

PubMed PMID: 23550303; PubMed Central PMCID:

PMC3792854.

• 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

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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 5Median Age of Onset and Prevalence of Characteristic Tumors in BAP1 Patients

Tumor/Malignancy

Number of Cases

EstimatedPenetrance

Median Age Literature

Median Age Our Study

Median Age General

Population

UvealMelanoma 61/215 cases 28% 53 59 55

Mesothelioma 48/215 cases 22% 56 46 69

Cutaneous Melanoma 38/215 cases 18% 41 43 53

BAP1 Deficient Tumors 33/215 cases 17% 32 31 23.7

Renal Cell Carcinoma 20/215 cases 9% 47 51 64

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.

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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

Conventional Melanoma

Most Aggressive

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

Spitzoid Melanoma with Homozygous

9p21 Deletion

Intermediate

Hierarchy of Risk for Distant Metastasis in Melanocytic Neoplasms with Spitzoid Morphology

Melanoma Spitz tumors

Chromosomal Aberrations

95%Chimeric Fusion Proteins: Ros, AlK, NTRK1, BRAF, RET

Common Gains6p, 7q, 17q, 20q, 4q,8q, 1q, 11q

Isolated Gain in 11p, can have gains in 7q

Common Deletions

9p, 10, 21q

3p21, 6q23, heterozygous loss of 9p21

New Paradigm Cellular functions represented in GEP signature

54 g enes initially assessed and then narrowed to 28 with 3 additional controls

Gerami, Clin Cancer Res 2013

Migration/chemotaxis/metastasis

CXCL14SPP1CLCA2S100A9S100A8

Differentiation/proliferation

CRABP2SPRRIBBTG1

Chemokine/secreted molecules

CCL14MGPSPP1

Cell surface receptors

TACSTD2CLCA2ROBO1

Gap junction/cellular adhesion

GJA1DSC1PPL

Structural proteins MGPSPP1CST6

Lymphocytic invasion

LTA4H Angiogenesis regulator

CXCL14

Transcription factor TRIM29 Extracellular functions

KRT6BKRT14

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1st intended use: Identify the node negative patients

who have aggressive disease

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Patients with Stage I

and II

melanoma

Class 1 test result:

Low risk of metastasis within 5

years

Class 2 test result:

High risk of metastasis within 5

years

Quantifies expression of 31 genes from primary tumorApplies a validation algorithmClassifies patients as low vs high risk with strong accuracy

Identification of high risk patients allows them the opportunity to access further evaluation, treatment, and monitoring with the goal of improving long-term survival

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Validation Study #1: Background demographics

Gerami, Clin Cancer Res 2015

Censor date: May 2013

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 (0.0-13.7) 5.7 (0.0-11.9)

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 (0.15-16.0) 1.4 (0.1-14.0)

≤ 1mm 46 (28%) 45 (43%)

> 1mm 101 (62%) 58 (56%)

Mitotic Index

≤ 1/mm2 43 (26%) 29 (28%)

> 1/mm2 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/ acral lentiginous

25 (15%) 10 (10%)

n=104

5-yr DFSClass 1 = 91%Class 2 = 25%

ROC = 0.9052Accuracy = 83%Sensitivity = 85%

Tr aining Set Validation Set

5-yr DFSClass 1 = 97%Class 2 = 31%

ROC = 0.9089Accuracy = 86%Sensitivity = 89%

Gerami, Clin Cancer Res 2015

Censor Date: May, 2013

GEP Accuracy:

Disease-free survival prediction – all cases

n=104

p<0.0001

n=164

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Patients with SLN Procedure (n=217)

SLNB vs. GEP – 1st and 2nd Validation Studies with SLNB

procedure

SLN positive =

58

SLN negative =

159

Met = 37

Non = 21

Met = 70

Non = 89

PPV = 64% NPV = 56%

SLN Results

Class 2 =

141

Class 1 =

76

Met = 91

Non = 50

Met = 16

Non = 60

PPV = 65% NPV = 79%

GEP Results 100%

75%

50%

25%

0%

1086420

DMFS

n=217

p<0.0001

SLNB-

SLNB+

Time (years)

% f

ree

of

me

tas

tas

is

100%

75%

50%

25%

0%

1086420

SLNB

DecisionDx in SLNB- Patients

n=159

p<0.0001

Time (years)

% fre

e

of

meta

sta

sis

Class 1/ SLNB-

Class 2/ SLNB-

Class 1/SLNB-

(n=67)

Class 2/SLNB-

(n=92)

Events 10 43

5-yr DMFS 86% 49%SLNB- (n=159) SLNB+ (n=58)

Events 53 32

5-yr DMFS 64% 42%

DecisionDx-Melanoma Improves Prediction Over SLNB Negative Status for Distant Metastasis-Free Survival

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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

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Independent cohort of 93 previously unreported stage II

cases

166 previously unreported stage III patients

DMFS by SLN Status

DecisionDx-Melanoma Identifies ~70% of SLNB Negative

Patients who had Distant Metastasis

Zager et al. J Clin Oncol 2016;34 (suppl; abstr 9581); manuscript in preparation

SLNB-

SLNB+

n=368

p<0.0001% f

ree

of

me

tas

tas

is

Time (years)

SLN-5-yr

DMFSEvents

n=216 80% 42

5-yr

DMFSEvents

Class 1/SLN-

(n=106)90% 13

Class 2/SLN-

(n=110)71% 29

DecisionDx in SLNB- Patients

SLN+5-yr

DMFSEvents

n=152 53% 69

Identified ~70% (29/42) events

Adhesive Patch Testing

EGIR

Continue observation

Excis ional Biopsy

RNA Expression

Non-invasive biopsy (tape strip)

Highly accurate technologyObjective findings

Clinically Suspicious Melanocytic Lesion (mole)ABCDE criteria or Dermascope

Scientific Rationale

89

Results of Validation Study:

Total series sensitivity:91%Total series specificity:69%

Sensitivity in consecutive series: 79%Specificity in consecutive series: 80%

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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

Housekeeper Group

Algorithm

Score

Immune

Signaling Group

IRF1CCL5

CXCL9CXCL10

CD38LCP2

PTPRCSELL

S100A Group

S100A7S100A8S100A9S100A12

PI3

PRAMEPRAME (2 Amplicons)

Scale and Threshold Values

Training set of 464 and Validation set of 395

58 year old male, upper back

Case 1201

Pretest: “Dysplastic nevus with moderate to severe atypia”

+2.2 (Malignant) 3/3: Melanoma in situ

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97 98

99 100

Pretest: Desmoplastic melanoma

Case 1825212

71 year old male; face

Score: +3.8

71 year old male, face: Excision

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H&E

S100 Sox-10

Melan-A

Excision Specimen (Block A4) 74 year old female, cheek

Score: +3.1 = False positive

Pretest: “Inflamed nevus”

51 year old female, mid back

Score: - 5.1 =False negative

Immune: -2.4 S100A: -7.5 PRAME: +3.3Case 0720.

Pretest: “Melanoma” 34 year old femal, My histologic diagnosis: “Spindle Cell nevus of Reed”

Case 2077866Score: +6.5Immune: - 0.1 S100A: +3.4

PRAME: +2.3

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