Biomarkers in Ocular Disclosure of Relevant Melanoma ... · seen in low stage tumors, T2a or lower). BIOMARKERS IN OCULAR MELANOMAPRESENTATION TITLE Predictive markers NRAS

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Biomarkers in Ocular Melanoma

Patricia Chévez‐Barrios, MDProfessor of Pathology and Laboratory Medicine and Ophthalmology,

Weill Cornell Medical CollegePathology and Genomic Medicine, Houston Methodist Hospital

USCAP-AAOOP Companion Meeting

Disclosure of Relevant Financial Relationships

USCAP requires that all planners (Education Committee) in a position to

influence or control the content of CME disclose any relevant financial

relationship WITH COMMERCIAL INTERESTS which they or their

spouse/partner have, or have had, within the past 12 months, which relates to

the content of this educational activity and creates a conflict of interest.

PRESENTATION TITLEBIOMARKERS IN OCULAR MELANOMA

Disclosure of Relevant Financial Relationships

I do not have any Relevant Financial Relationships to Disclose

BIOMARKERS IN OCULAR MELANOMA PRESENTATION TITLEBIOMARKERS IN OCULAR MELANOMA

Ocular MelanomaIntroduction


Ocular Melanoma

• Skin of eyelids

• Conjunctiva

• Uvea


Melanoma - Eyelids

• Rare eyelid tumor

• Basal cell, squamous cell and sebaceouscarcinomas more frequent

• Associated with preexisting nevus or de novo

• Nevus > 0.5cm, irregular pigmentation and margins

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Melanoma - EyelidsTypes

Superficial spreadingMost common Radial growth beyond invasive component

Lentigo maligna melanomaFace of elderly (pre-invasive form Lentigo Maligna)

Nodular


Skin Melanoma

• Accounts for 75% skin-related deaths worldwide.

• Diagnosis is challenging due to large diversity of morphological patterns.

• Highest source of litigation in surgical pathology.


Melanoma - Diagnosis

PRESENTATION TITLEBIOMARKERS IN OCULAR MELANOMABIOMARKERS IN OCULAR MELANOMA

Biomarkers

Patient

DIAGNOSIS

PROGNOSISPREDICTIVE

Identification of conditions

Predicts patient’s response to a

treatment

Estimates progression or

indolent behavior


Biomarkers

Patient

DIAGNOSIS

PROGNOSISPREDICTIVE

Identification of conditions


Conventional Prognostic Markers

• Breslow thickness

• Clark level

• Growth phase (radial vs vertical)

• Tumor infiltrating lymphocytes (TILs) (density, type)

• Ulceration, present or absent

• Mitoses per mm2

• Status of sentinel lymph node (positive vs neg.)

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Histological staging

Breslow A. Thickness, cross-sectional areas and depth of invasion in the prognosis of cutaneous melanoma. Ann Surg. 1970 Nov;172(5):902-8.McGovern VJ, Mihm MC Jr, Bailly C, Booth JC, Clark WH Jr, et al. The classification of malignant melanoma and its histologic reporting. Cancer. 1973 Dec;32(6):1446-57.Clark WH Jr, From L, Bernardino EA, Mihm MC. The histogenesis and biologic behavior of primary human malignant melanoma of the skin. Cancer Res. 1969 Mar;29(3):705-27.


Prognostic Features

Depth of invasion (Breslow)

Clark Level

(mm)

1

2

3

4

5

10 YEAR SURVAVAL RATE

93%

71%

59%

36%

0 >

1>

2>

3>

4>

5>

94%

82%

63%

51%

40%


TILs


Mitoses per mm2


Immunohistochemical MarkersIdentification Biomarkers

Challenges :

• Melanin pigment (DAB chromogen is brown)

• Tumor associated lymphocytes, stromal cells (may mark with proliferation markers)

Options:

• Bleach prior to immunestaining (may alter results)

• Use of different chromogen (red, blue)

• Double staining with melanocytic marker (cytoplasmic) + proliferation marker (nuclear)



• S100: sensitive melanocytic marker but not specific. Excellent for desmoplastic melanoma.

Caviate: Usually negative or faintly positive in uveal melanoma

• HMB-45: Helps differentiating benign nevi (decreased positivity with lesion depth/maturation) vs melanoma (consistent positivity in deeper part), not definitive in nevoid variants of melanomas.

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• MITF-1 & SOX-10: nuclear stains, best for lentiginous proliferations and pagetoid spread-difficult in-situ melanocytic lesions. Stain intraepithelial dendritic cells .

• SOX-10: High sensitivity for melanocytic differentiation. Used in desmoplastic melanoma along with S100 ( recently reported in scar). Caviate: metastatic vs. breast, salivary gland carcinomas, neural creasttumors and clear cell sarcomas.

• Ki-67 and pHH3: Estimates proliferation rate (inflammatory cells). Co-staining with a melanocytic marker (HMB-45) may improve accuracy.


ImmunohistochemistryHMB45 (red)/ Ki67 (brown)


Conjunctival Melanocytic Lesions


Challenge – histopathology characteristic of Conjunctival melanocytic lesions• Recent growth of lesion in bulbar conjunctiva (15 year-old)


ImmunohistochemistryMelan A (red)/ Ki67 (brown)


Clinical – Primary Acquire Melanosis(PAM)

Fair skin,unilateral, flat conjunctival pigmentationFair skin,unilateral, flat conjunctival pigmentation

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Histologic diagnosis

BiopsyPAM without atypia PAM with atypia

Clinical DiagnosisPAM

Unilateral, flat pigmented conjunctival lesion


Primary Acquired Melanosis (PAM)

PAM without Atypia

PAM with Atypia


No progression to melanoma

PAM without Atypia

25% Mortality

25-75% progression to

MelanomaPAM with Atypia

Ophthalmology. 1989 Feb;96(2):147-66. Review. PubMed PMID: 2649838.

Am J Surg Pathol. 2007 Feb;31(2):185-92. PubMed PMID: 17255762.


PAM with atypia to Melanoma - Clinical

PAM with atypia by bx.Nodule developed – Melanoma by bx.


Primary acquired melanosis (PAM) with atypia and Melanoma

Clinical and Experimental Ophthalmology 2008; 36: 786–795doi: 10.1111/j.1442-9071.2008.01888.x PRESENTATION TITLEBIOMARKERS IN OCULAR MELANOMABIOMARKERS IN OCULAR MELANOMA

Melanoma

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Clinical Approach to Patient with Melanoma

BiopsyMelanoma

Early Stage Late Stage

Microscopic Staging

Clinical staging

SLNBx Imaging LDH levels

Molecular staging

After tx

Targeted therapy

AJCC – staging and mutations


Sentinel Lymph Node Bx (Skin & Conjunctiva)

H&E Melan A (immunohistochemistry)



BiopsyMelanoma


Microscopic Staging

Clinical staging

SLNBx

LDH levelsImaging

Stage I and II (-)(localized)Stage 1=85%OS at 10 years

Stage III (+)Up to 40% upstage = micromets20-70%OS at 10 years

Stage IV (metastases)10 %OS at 10 years



BiopsyMelanoma


Microscopic Staging

Molecular staging

Clinical staging


Molecular Classification

• Highly heterogeneous disease

• Frequent driver mutations identified

• Regulate: • signal transduction pathways• developmental and transcriptional pathways• cell cycle


Prognostic markers

• Multimarkers assays• Single marker assays does not suffice in yielding enough prognostic or

predictive information• Multimarkers give highly accurate information about prognosis or

predictive response to therapy.• Prognostic multimarker signatures

Epithelial to sarcomatoidmorphology

Inflammatory/immune pathways

AngiogenesisMetastasis

and invasion

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Predictive markers

• Aim to predict patient response to treatment or combination of treatments.

• Could be very helpful in deciding personalized successful treatment

• Very few tests are available clinically.


BRAF• 50% of skin melanomas show mutations in BRAF gene.

• as an independent prognostic marker has shown conflicting data ( found in benign nevi)

• vertical growth phase-melanomas = progression rather than genesis

• increased tumor thickness and ulceration

• Worse survival (5.7 months) for BRAF-mutant melanomas compared with BRAF-wild type cases (8.5 months)


BRAF• 95% of the mutations found are at aa 600 leading to a

constitutive MPAK/ERK pathway activation.

most commonly V600E (valine – glutamate).

sometimes V600K (valine – lysine)RAS

BRAF600

MEK

ERK

activates

Cell proliferation Evasion of senescence and apoptosis


Predictive markers• BRAF status to predict response to vemurafenib

• Vemurafenib improves PFS and OS in untreated melanomas carrying BRAF V600E mutation.

• Induced complete or partial tumor regression in 81% of BRAF +mutation patients.

RAS

BRAF600

MEK

ERK

VEM

X


BRAF

• Resistance due to upregulation of bypass pathways mediated by cancer Osaka thyroid kinase and the development of de novo NRAS or MEK mutations.


Predictive markersCombined treatment response

• BRAF mutation can be either favorable (with cisplatin-vinblastine-temozolomide when associated with low O-6-methylguanine-DNA methyltransferase expression)

OR

• BRAF mutation can be detrimental (melphalan & actinomycin-D).

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Predictive markersBRAF - Testing• BRAF mutations (V600E or V600K) can be detected from DNA

samples from FFPE biopsy tissue through PCR testing.

• IHC for BRAF (V600E) shows excellent correlation with molecular-based analysis.

• Other point mutations of BRAF (V600K, V600Q, and V600R) melanomas do not stain positively with BRAF VE1 antibody.


BRAF VE1 antibody

Homogeneous staining correlated well with BRAF mutation status

Heterogenous staining was found more often in metastatic advance tumors


Predictive markersNRAS• 20% of melanomas show mutation of NRAS gene.• NRAS as a prognostic indicator have shown mixed results

• no difference in melanoma-specific survival compared with wild type tumors

• NRAS mutations frequently found in nevi and early melanomas

• Tumors with NRAS mutations are thicker (75% of NRAS mutated tumor were >1mm thick and had more than 1 mitosis/mm2 )

• NRAS mutation is associated with poorer survival (this was not seen in low stage tumors, T2a or lower).


Predictive markersNRAS• NRAS & BRAF V600 mutations occur in an exclusive fashion.

• Direct inhibitors have not demonstrated significant clinical activity.

• IHC for NRAS (Q16R) shows excellent correlation with molecular-based analysis.

• Other point mutations of NRAS (Q61K, Q61L, Q61H) melanomas do not stain positively with NRAS Q61R antibody.


KIT (c-kit)

• Receptor tyrosine kinase that triggers downstream of different pathways (MAPK, PI3K, JNK and JAK/STAT) leading to cell growth, proliferation, migration and differentiation of melanocytes.

• Oncogenic potential of KIT in other tumors (GISTs, small-cell lung carcinomas and acute myeloid leukemia)


KIT (c-kit)

• KIT might have a melanoma-suppressing role. • KIT is hypermethylated in the majority of melanomas, causing gene

silencing• No correlation of KIT activation with BRAF/NRAS pathways - KIT

effects may be signaled through a different pathway• KIT-inhibitors could aggravate progression of melanomas in early stage

when growth is inhibited by KIT expression.

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KIT (c-kit)

• KIT mutated in less than 5% of melanomas: • 30% of mucosal(conjunctival), 20% acral and 20% of melanomas

arising in sun-damaged skin

• KIT in these specific scenarios behaves as an oncogene providing a window to treat specific cases with KIT inhibitors


KIT (c-kit)

• Imatinib is a c-KIT inhibitor that is currently on phase II/III trials for patients with metastatic melanoma with c-KIT mutation.


Predictive markers• Predictors of immunotherapeutic agents.

• No current biomarkers to predict success of immunotherapeutic agents (CTLA-4 (Ipilimumab) or PD-1/PD-L1 inhibitors (Atezolizumab))16.


Predictive markersPD-L1 (programmed death receptor ligand 1)

• Negatively regulates T-cell function by binding to the PD-1 receptor on T cells = tumor evasion and proliferation

• Melanomas with aberrant expression of PD-L1 have poor prognosis

• Pembrolizumab and Nivolumab: human anti-PD-1 immune-checkpoint inhibitor antibody that blocks the interaction of PD-1 with PDL1 or another ligand PD-L2.

©2016 AstraZeneca. All rights reserved. 3265707 Last Updated 7/16


Furue M, Kadono T, Melanoma therapy: Check the checkpoints. J Dermatol. 2016 Feb;43(2):121-4.


UVEAL MELANOMA

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Clinical Approach to Patient with UvealMelanoma

Biopsy

Benign - Nevus

Melanoma

Clinical DX

Radiation plaque Enucleation

Molecular Prognostic testingConfirmation Dx

TX

Metastatic screening (liver)


Most Common Malignant Intraocular Tumors

Children

Retinoblastoma

Adult

Metastases

UvealMelanoma


Choroidal Melanoma• Predisposing factors

• melanosis oculi and Nevus of Ota• BAP1 associated tumor predisposition syndrome

• 6 cases per million in USA (60-70 years of age)

• 50% uveal melanoma patients will develop metastasis


Melanoma of the Uvea

• Location = symptoms specific to the location will develop and facilitate early or late diagnosis

• Ciliary body melanoma presents later because of location (hiding behind the iris/lens)

• Most frequent in whites and lightly pigmented individuals


Melanoma• Iris = nonaggressive

• 3.3 – 16.6% are from iris (~10 years earlier dx than choroidal)


Clinical Diagnosis

• Standard of care

• Patients treated based on clinical/imaging features

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Uveal Melanoma• Metastasis 80% to liver (90% in liver prior to death)

• 1/3 of metastatic MM solely to the liver

• No difference in survival: local treatment with radiation plaque vs. enucleation (COMS prospective trial)


High Risk Prognostic Factors


Malignant Melanoma – Size, location


Callender Classification

Spindle A Spindle B Epithelioid

Spindle Melanoma Epithelioid Melanoma

Mixed Melanoma


Vasculogenic Mimicry


Uveal MelanomaFive main genes are implicated in development

and progression in UM:

• BAP1, EIF1AX, GNA11, GNAQ, and SF3B1

Genetic features associated with metastasis include:

• monosomy 3 and gain of chromosome 8q

BAP1, SF3B1, and EIF1AX mutations occur during UM tumor progression

• Mutually exclusive manner = different levels of metastatic risk.

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Molecular Heterogeneity

FISH analysis on paraffin sections showed that heterogeneity of monosomy of chromosome 3 is a frequent phenomenon in uveal melanoma.

Matt W, et al. Arch Pathol Lab Med 2007;91:31-36


Molecular genetics in Uveal melanoma• GNAQ and GNA11 occur early in tumor formation and are not

associated with prognosis• Somatic mutations in the heterotrimeric G protein alpha-subunit,

GNAQ• Blue nevi (83%) and ocular melanoma of the uvea (46%). • The mutations occur exclusively in codon 209 in the Ras-like

domain and result in constitutive activation, turning GNAQ into a dominant acting oncogene.

• An alternative route to MAP kinase activation in melanocytic neoplasia

Nature. 2009 Jan 29; 457(7229): 599–602.


Molecular genetics in Uveal Melanoma

• BAP1, SF3B1, and EIF1AX occur later in tumor progression

• Prognostically significant


Molecular genetics in Uveal melanoma

• BAP1 (BRCA1-associated protein 1) is a tumor suppressor gene .

• BAP1 gene locus is on chromosome 3 (3p21.1).

• Deletions of the 3p21 region were commonly seen in small cell and nonsmall cell lung cancer cell lines, and breast cancers


BAP1 – Epithelioid phenotype


BAP1 • Inactivating mutations in BAP1 are seen in 47% (27/57) uveal

melanomas.

• Metastasis developed in the vast majority (26/27) of these tumors, many of which also showed monosomy of chromosome 3.

• These findings support the thesis that BAP1 is a classical tumor suppressor gene (Knudson’s two-hit model)

• One allele of BAP1 being lost via monosomy of chromosome 3 and the second allele being lost by inactivating BAP1 mutation(s).

Harbour JW, Onken MD, Roberson ED, et al. Frequent mutation of BAP1 inmetastasizing uveal melanomas. Science 2010; 330: 1410–3.

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Molecular genetics in Uveal melanoma• BAP1 mutations recently reported to increase susceptibility for the

development of uveal melanoma, cutaneous atypical and epithelioid melanocytic lesions, clear cell renal cell carcinoma, mesotheliomas and other tumors.


BAP1 testing• Direct (Sanger) sequencing of the BAP1 gene using blood or salivary

DNA from the individual(s) of interest (suspect of BAP1 related predisposition syndrome).

• BAP1 testing in tumors by RT-PCR

• BAP1 IHC in paraffin embedded tumor tissue (BAP1 loss of staining by IHC)

• Equivocal IHC results may undergo subsequent confirmatory sequencing.


Uveal Melanoma

• Gene-expression signatures (15 genes) have also been identified that accurately distinguish tumors at low metastatic risk (class 1 signature) and high metastatic risk (class 2 signature)

Gene Symbol Gene Name Direction of Change in Class 2 Tumors

CDH1 E-cadherin Up

ECM1 Extracellular matrix protein 1

Up

E1F1B Eukaryotic translation initiation factor 1B

Down

FXR1 Fragile X mental retardation autosomal homolog 1

Down

HTR2B 5-hydroxytryptamine (serotonin) receptor 2B

Up

ID2 Inhibitor of DNA binding 2

Down

LMCD1 LIM and cysteine-rich domains 1

Down

LTA4H Leukotriene A4 hydrolase

Down

MTUS1 Microtubule-associated tumor suppressor 1

Down

RAB31 RAB31, member RAS oncogene family

Up

ROBO1 Roundabout, axon guidance receptor 1

Down

SATB1 SATB homeobox 1 Down


Gene expression profile analysis of primary uveal melanomas reveals two distinct tumor classes.

Michael D. Onken et al. Cancer Res 2004;64:7205-7209

©2004 by American Association for Cancer Research


GEP – Uveal Melanoma

• Based upon the clinical outcomes from the prospective, 5-year multi-center Collaborative Ocular Oncology Group (COOG) study, the test reports Class 1A, Class 1B and Class 2 phenotype:

• Class 1A: Very low risk, with a 2% chance of the eye cancer spreading over the next five years;

• Class 1B: Low risk, with a 21% chance of metastasis over five years;

• Class 2: High risk, with 72% odds of metastasis within five years.


Choroidal Melanoma

Future Oncol. 2008 October ; 4(5): 629–636.

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Genes and GEP groupJAMA Ophthalmol. 2016 Jul 1;134(7):728-33. doi: 10.1001/jamaophthalmol.2016.0903


Genes and GEP group• BAP1 mutations were associated with class 2 GEP (P < .001) and

older patient age (P = .007).

• EIF1AX mutations were associated with class 1 GEP and the absence of ciliary body involvement (P = .03 for both).

• SF3B1 mutations were associated with younger patient age (P = .006).

• GNAQ mutations were associated with the absence of ciliary body involvement (P = .008) and greater largest basal diameter (P = .04).

• GNA11 mutations were not associated with any of the analyzed features.

JAMA Ophthalmol. 2016 Jul 1;134(7):728-33. doi: 10.1001/jamaophthalmol.2016.0903


PRAME

• Small percentage of Class 1 tumors result in metastatic disease.

• Cancer-testis antigen PRAME (preferentially expressed antigen in melanoma) = biomarker for increased metastatic risk in Class 1 tumors.

• Increased PRAME mRNA expression in Class 1 UM associated with transcriptional up-regulation of key genes involved in chromosome maintenance and stability (1q and 6p)

Clinical cancer research : an official journal of the American Association for Cancer Research 22.5 (2016): 1234–1242.


Molecular Mechanisms in UM

Feed-forward mechanism = progressively increasing PRAMEexpression and specific chromosomal gains mutually reinforce one another to promote Class 1 tumor progression.

• association with SF3B1 mutations (mutually exclusive with EIF1AX mutations) →bifurcated pathway.

• This Class 1 metastatic pathway distinct from the bi-allelic loss of BAP1 and acquisition of the Class 2 gene expression profile.



PRAME




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Requirement of Tissue for Molecular Testing• Adequacy of sampling

• GEP does not discriminate between normal, benign or malignant melanocytic lesion

• Blood elements = Class 1• Some metastatic carcinomas = Class 2

• Confirmation of diagnosis

• FNABx before radioactive plaque

• Tissue retrieval at time of enucleation

• FFPE tissue acceptable


Cytology : Sampling prior to plaque • Diagnosis and molecular prognosis of uveal

melanoma

Adequacy check in OR Adequacy check in OR

RPMIRPMIMolecularMolecular

Cell blockCell block

Intraocular biopsyIntraocular biopsy


Cytology : Sampling prior to plaque • Diagnosis and molecular prognosis of uveal

melanoma

Adequacy check in OR Adequacy check in OR

RPMIRPMIMolecularMolecular

Cell blockCell block

Intraocular biopsyIntraocular biopsy

HMB45/Ki67


UVEAL MELANOMA BIOMARKERS• Uveal melanoma does not have

effective treatment for metastatic phase

• GEP or other prognostic molecular testing shows no detrimental psychological effect in most patients

• Early treatment of localized metastasis and possible enrollment in clinical trials Cook SA, Damato B, Marshall E, Salmon P (2009) Psychological aspects of

cytogenetic testing of uveal melanoma: preliminary findings and directions for future research. Eye (Lond) 23: 581-585.


Examples of Current TrialsMD Anderson Cancer CenterElizabeth Grimm, MD ; Sapna Patel, MD

• Not stratifying uveal melanomas based on any biomarkers

• Usually test for GNAQ and GNA11 and often do a post-hoc look at responses in these groups.

• To date, targeted therapy trials do not show a difference in the GNAQ/11 wild-type or mutated populations.

• Other melanomas, next generation panel (BRAF, NRAS, CKIT, GNAQ and GNA11) .

• Standard of care and clinical trial options based on mutation status.

• PD-L1 status on a metastatic specimen to drive treatment decisions regarding single-agent versus combination checkpoint blockade (selective cases)

• Trial using iNOS as a biomarker for entry onto an anti-inflammatory/NF-KB inhibitor study in combination with checkpoint blockade for melanoma (Elizabeth Grimm, MD).

• Engineered T cell trials based on HLA-A02:01 status, and MART-1 expression in the tumor tissues.

Massachusetts General Hospital Cancer CenterKeith T. Flaherty, M.D.

• Uveal - Developing clinical trial to combine intralesional oncolytic virus with PD-1/CTLA-4 therapy, given the relative lack of efficacy for immune checkpoint antibody therapy in this population to date.

• Cutaneous, mucosal, and acral melanomas large clinical trial portfolio aiming to

• (1) expand the benefits of immune checkpoint antibody therapy by targeting novel immune targets,

• (2) extending the benefits of BRAF inhibitor-based therapy by targeting additional vulnerabilities outside of the MAP kinase pathway with triplet drug regimens

• (3) developing molecular targeted therapy strategies pertinent to BRAF wild type melanomas that build on the modest efficacy of single agent MEK inhibition


Conclusions• Oncogenes in melanoma are relevant for prognosis and

therapeutic biomarkers• Immunohistochemistry as surrogate for mutations (BRAF,

BAP1, PRAME)• Noninvasive biomarkers needed (blood circulating tc)• Immunoprofiling is a valid form of biomarker• Need for predicting response of immuno-based therapies• Epigenetic alteration are an expanding group of potential

biomarkers• Special type melanomas have different tumorogenesis and

prognostic biomarkers.• Development of combination therapies appear to be important

in melanoma

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Biomarkers in Ocular Disclosure of Relevant Melanoma ... · seen in low stage tumors, T2a or lower). BIOMARKERS IN OCULAR MELANOMAPRESENTATION TITLE Predictive markers NRAS