Development of a Non-invasive Genomic-based Assay to Detect MelanomaDevelopment of a Non-invasive Genomic-based Assay to Detect MelanomaWilliam Wachsman1,2, Tara Palmer3, Lory Walls1,2, Sherman Chang3

1Research Service, VA San Diego Healthcare System, San Diego, CA; 2Division of Hematology-Oncology and Moores Cancer Center, UC San Diego;3DermTech International, San Diego, CA, USA

Materials, Methods and Clinical ProtocolAssay methods• EGIR specimens collected at 9 sites in U.S.• Shipped and stored at -800 C• MELT (Ambion, Inc.) used to extract RNA

- Only used tape demarcated over pigmented lesion- RNA product pooled from 4 tapes- Yield and quality assayed

• RNA (300 - 500 pg) amplified using WT-Ovation Pico kit (NuGen,Inc.)• GeneChip assay using U133 plus 2.0 microarray (Affymetrix, Inc.)

- Data QC using Simpleaffy (Bioconductor)- GCRMA used to normalize data

Class prediction algorithm• Microarray data divided into training and test sets• t-test with multi-testing correction to identify differentially expressed genesbetween melanoma and nevi• Training set: TreeNet® (Salford Systems) used for class selection• Test set: TreeNet used for class prediction

Clinical Protocol• Inclusion criteria

- Subjects 18 or older- Pigmented lesion, suspicious for melanoma that requires biopsy- Lesion size: 4 mm or greater- If 2 lesions, must have > 4 mm separation

• Exclusion criteria- Lesion that is ulcerated, bleeding or weeping- Use of topical moisturizer or sunscreen on sites within 24 h- Allergy to tape or latex

• Procedures- Tape stripping of lesion(s) and uninvolved, control skin- Demarcate lesion edge on tape- Biopsy lesion as per standard of care- Primary and central dermatopathology review

Specimens• Training set: 37 melanomas and 37 nevi

- Superficial spreading melanoma (33):in situ (14) and invasive (19)- Lentigo maligna (3) & lentigo maligna melanoma (1)- Nevi (37): Clark (29) and lentiginous (8)

• Test set: 39 melanomas and 89 atypical nevi- Superficial spreading melanoma (32): in situ (11) and invasive (21)- Lentigo maligna (2) & lentigo maligna melanoma (4)- Nodular melanoma (1)- Nevi (89): Blue nevus (1), Clark (67), congenital (12) and lentiginous (9)

Conclusions• EGIR-harvested stratum corneum RNA can be used for the detection of both in situ and invasive melanoma.• A 19-gene classifier, developed by microarray analyses of EGIR specimens, discriminates melanoma frombenign, pigmented nevi.• Testing of this classifier, shows it to be 100% sensitive and 88% specific for detection of in situ andinvasive superficial spreading melanoma and lentigo maligna – with a negative predictive value of 100% anda positive predictive value of 78%.• Most of the genes in the classifier are known to have a function in melanocyte development, melanoma, orcancer biology – suggesting that there is a direct or indirect effect of the malignant melanocyte uponsurrounding keratinocytes.• Further analyses of specimens, deemed to be false positive for melanoma by the 19-gene classifier, isneeded to determine whether it is more sensitive than standard histopathology.• Preliminary results indicate discovery data, obtained through microarray analyses of EGIR specimens,translates well onto a quantitative RT-PCR platform that can be used in the clinical setting.

ACKNOWLEDEGMENTSWe would like to acknowledge the following clinical sites for the melanoma study:• Dr. Tissa Hata, Department of Dermatology, UCSD, La Jolla, CA• Dr. Robert Scheinberg, Dermatologist Medical Group of North County, Oceanside, CA• Dr. Ken Gross, Skin Surgery Medical Group, San Diego, CA• Dr. Serena Mraz, Solano Dermatology Associates, Vallejo, CA• Dr. Harold Rabinovitz, Skin and Cancer Associates, Plantation, FL• Dr. Shondra Smith, Dermatology and Advanced Aesthetics, Lake Charles, LA• Dr. Howard Sofen, Dermatology Research Associates, Los Angeles, CA• Dr. James Zalla, Dermatology Associates, Florence, KY

DermTech International • Alejandra Ramos• Cheryl Peters• Kamaryn Peters• George Schwartz

Salford Systems• Dan Steinberg• Mikhail Golovnya

Translation of the Assay from Microarray to qRT-PCR Platform

Goals• To transition the 19-gene classifier from the microarray discovery platform to aquantitative RT-PCR platform that will improve clinical utilityMethods• Specimens: pre-amplified with WT-Ovation FFPE kit (NuGen, Inc)• qRT-PCR assay for expression of the 19-gene classifier in triplicate• Data normalized with a reference control (TRIM65)Results• Preliminary results (10 melanomas & 10 nevi) indicate that qRT-PCR recapitulatesdata obtained using the GeneChip microarray

Atypical nevusMelanoma

Analysis of False Positive Nevi Identified by the 19-Gene Classifier

• The 19-gene classifier identified 11 nevi as melanoma (false positive)• Each false positive nevus was serial sectioned and re-reviewed

• 10 of 11 remain false positive• Specimen A (denoted below):

• Clark nevus on initial pathological review• Identified as melanoma when analyzed by the 19-gene classifier• Confirmed by both primary and central pathologists

Atypical nevusMelanoma

A

Introduction Melanoma incidence is increasing in the US population faster than all other cancers. It is already the seventh mostcommon serious cancer in the United States with a lifetime risk of 1 in 41 in men and 1 in 61 in women. Melanomaaccounts for more than 70% of skin cancer deaths, but when detected early it is considered highly curable. In currentclinical practice, the detection of melanoma is based upon visual cues, including the “ABCDE” criteria for pigmentednevi and results of optical imaging techniques, such as dermoscopy and confocal microscopy. However, only 2 to 10%of lesions biopsied for suspicion of melanoma are positive for this disease upon histopathologic examination. Inaddition, differences in histopathologic review of biopsy specimens by experienced dermatopathologists can result indiscordant tissue diagnoses for 10-35% of melanoma cases. Thus, there is a need for a melanoma test that improvesthe accuracy, objectivity and ease of detection in clinical practice.

The patented Epidermal Genetic Information Retrieval (EGIR) technology (DermTech International, Inc.) uses acustom adhesive film to non-invasively obtain RNA from stratum corneum (i.e. the upperrmost layer of the skin).Developed by Morhenn and Rheins (J Am Acad Dermatol 4: 687 [1999]). The EGIR methodology uses four 20 mmtapes (pictured below) to noninvasively sample stratum corneum. RNA so harvested has been found to be stable forassay, when kept at ambient temperature for at least 72 hours. This makes it an ideal method for use in the clinicalsetting.

Previous work showed that EGIR, non-invasive tape stripping of stratum corneum, identified 284 genes thatdifferentiated melanoma from atypical nevi and normal skin (p<0.001, false discovery rate q<0.05) . (Wachsman et al. JInvest Derm 128: S213 [2008]) (see Figure below). Several of the genes were found by Ingenuity Pathways analysis toplay a role in melanocyte development and function, as well as, skin development, cellular proliferation, and cancer.These findings further demonstrated that the presence of melanoma, directly or indirectly, alters the gene expressionprofile of stratum corneum.

Study Objective The goal of this study is to develop a more objective means for melanoma detection by use of the EGIR technologythat non-invasively samples stratum corneum by means of tape stripping.W

TreeNet analysis for class selection and prediction-Training set: 37 melanomas and 37 nevi

• Background subtraction: 100• Remove gene ≤ 2x difference• t-test with multi-testing correction identified 7,199 differentiallyexpressed genes (p < 0.05; FDR < 0.05)• TreeNet analyses of differentially expressed genes identified a19-gene classifier (sensitivity 100%, specificity 95%)

-Test set: an independent set of sample data (39melanomas and 89 nevi)

• PPV: 39/50 = 78%, NPV: 78/78 = 100%• Sensitivity 100%, specificity 88%

-The 19-gene classifier also distinguishes melanomafrom other pigmented lesions and normal skin

• Basal cell carcinoma (18)• Solar lentigines (12)• Non-lesional normal skin (73)

- Biological functions of the 19-gene classifier• 9/19 melanoma/melanocyte associated• 7 of remaining 10 are cancer associated

Characterization of a 19-Gene Classifier that Identifies Melanoma in Pigmented Lesions

Observed Melanoma Nevi Observed Melanoma Nevi

Melanoma Melanoma 39 0

Nevi Nevi 11 78

Total Total 50 78

Diagnosis Diagnosis

Predicted Class Predicted Class

37 0

2 35

39 35

Training Set Test Set

• Lentigo maligna/lentigo maligna melanoma (LM/LMM) and solarlentigo are often difficult to distinguish on sun damaged skin• Objective: To develop a classifier to differentiate LM/LMM from solarlentigo by EGIR-based genomic assay• Methods

• EGIR tape-stripped specimens were expression profiled by GeneChip assay• Lentigo maligna (N=5), lentigo maligna melanoma (N=5) and solar lentigo (N=12)

• GCRMA normalization• Background subtraction: 100• t-test between lentigo maligna and solar lentigo (p< 0.01)• Multiple testing correction (False discovery rate (q< 0.05))• 32 differentially expressed genes identified

Conclusion:• A 32-gene classifier distinguishes solar lentigo from both lentigo maligna andlentigo maligna melanoma.

A 32-Gene Classifier Differentiates Solar Lentigo from Lentigo Maligna/Lentigo Maligna Melanoma

Lentigo maligna Solar lentigo