ex-blue nevus wiesner et al. pathology2015 21/07/2017 3 what is new? "realm of «molecular...
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Update on the Clinico-Pathological and Molecular
Diagnosis of Melanocytic Lesions
Belfast pathologyArnaud de la Fouchardière MD, PhD
Lyon, France
Conflicts of interest
• None to declare
What is new? Today’s Menu
• Realm of «Molecular Pathology»
• New concepts
• New techniques applied to FFPE
• New entities
What is new?
• Realm of «Molecular Pathology»
What is new?
• Realm of «Molecular Pathology»
• New concepts
What is new?
• Realm of «Molecular Pathology»
• New conceptsStep by step histo-molecular progression from nevusto melanoma
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In many lesions there is a progressive gain of molecular anomalies linked with incremental microscopic atypia
The «gray zone» of diagnosis is visible
• Nevus Atypical nevus Melanoma
• Blue nevus Atypical blue nevus
• Spitz Nevus Atypical Spitz tumor Spitzoid Melanoma
• DPN Atypical DPN Plexiform Melanoma
Atypical MalignantBenign
Melanomaex-blue nevus
Wiesner et al. pathology 2015
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What is new?
• Realm of «Molecular Pathology»
• New conceptsStep by step histo-molecular progression from nevusto melanoma« Integrative analysis »
Melanoledge projectIntegrative analysis
Embryogenesis
Clinical features
Microscopy/morphology
Immunophenotype
Genomic profile
Mutation status
Clinical evolution
« Integrative analysis »From concepts to tools
• Melanocytic tumors represent a wide variety of lesions.• There are many ways of viewing differences.• The goal is to combine as many «viewpoints» as
possible in order to ultimately visualize melanocytic lesions in a more accurate manner.
« Integrative analysis »From concepts to tools
• Melanocytic tumors represent a wide variety of lesions.• There are many ways of viewing differences.• The goal is to combine as many «viewpoints» as
possible in order to ultimately visualize melanocytic lesions in a more accurate manner.
Clinical features
• Location• Age of appearance• Size• Type of sun exposure/Phototype• Recent modifications:
• Color• Shape• Volume/ulceration• Pain/itching
M 0
M0: Giant congenital nevus
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M15 Back (Dr de Carrère): BAP1-inactivated melanocytic tumor
F34 Abdomen (Dr Bellili): Superficial Spreading Melanoma
F6 arm (picture Dr Dadban): Spitzoid melanoma
Microscopy/morphology• Low magnification (Silhouette)
• High magnification (cytology)
Molecular Pathology of melanocytic tumors Mutations
• « Driver »• BRAF• NRAS• NF1• HRAS• CKIT• GNAQ• GNA11• …
• « Oncogenetic »• CDKN2A• CDK4• MiTF• BAP1• …
• « Passenger »• PTEN• hTERT• p53• …
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Bastian B The molecular pathology of melanoma. Annu Rev Pathol Mech 2014
Main drivers SSM
Melanoma arising fromblue neviNMLMM
ALM
BRAF V600E
GNAQ/11 exon 4 or 5
NRAS exon 2 or 3BRAF V600K
Kit exon 11, 13, 17 or 18 Molecular Tools: use is guided by
clinical morphological features
IHC
FISH
NGS
CNV
What is new?
• Realm of «Molecular Pathology»
• New concepts
• New techniques applied to FFPE
What is new?
• Realm of «Molecular Pathology»
• New concepts
• New techniques applied to FFPEAntibodies, Next Generation Sequencing, RNA-seq
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Immunohistochemistry
It’s all about chosing the adapted antibody(ies) for the selected task(s)
Antibodies• « Melanocytic » antibodies
• S100• MelanA• HMB45• PNL2• MiTF• SOX10• …
• « Anomaly-specific » antibodies• BRAF V600E• NRAS Q61R• ALK• ROS1• NTRK1• MET• P16• BAP1• PDL1• …
• Other antibodies• D2-40• CD68• …
Specificity vs Sensitivity
HMB45
Antibodies• « Melanocytic » antibodies
• S100• MelanA• HMB45• PNL2• MiTF• SOX10• …
• « Anomaly-specific » antibodies• BRAF V600E• NRAS Q61R• ALK• ROS1• NTRK1• MET• P16• BAP1• PDL1• …
• Other antibodies• D2-40• CD68• …
NTRK1
Antibodies• « Melanocytic » antibodies
• S100• MelanA• HMB45• PNL2• MiTF• SOX10• …
• « Anomaly-specific » antibodies• BRAF V600E• NRAS Q61R• ALK• ROS1• NTRK1• MET• P16• BAP1• PDL1• …
• Other antibodies (DD mainly)• D2-40• CD68• …
Why perform IHC?
• Confirm melanocytic lineage• Visualize the melanocytes• Benign vs Malignant• Molecular characterization
IHC to confirm melanocytic lineage
• Unpigmented dermal or ulcerated tumor (No recognizable junctional melanocytes)
• Unpigmented metastases• Desmoplastic melanoma
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IHC to confirm melanocytic lineage
• Unpigmented dermal or ulcerated tumor (No recognizable junctional melanocytes)
• Unpigmented metastases• Desmoplastic melanoma• If usual melanocytic markers are negative• Combination of SOX10 and MiTF nuclear positivity
favors melanocytic origin
M69 Torso. No MM history
M69 Torso. No MM history S100Protein
S100Protein MelanA/Mart1/A103
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MelanA/Mart1/A103 HMB45
HMB45 SOX10
SOX10 MiTF
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MiTF IHC for Molecular characterization
• Point mutations• Gene fusions• Loss of function (tumor suppressor genes)« Theragnostic » tools
Point mutations
• BRAF V600E• NRAS Q61R
BRAF VE1
BRAF VE1 NRAS Q61R
Melanoma arising from a large congenital nevus
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Kinase Fusions IHCScreening tool
• ALK• ROS1• NTRK1• Pan-NTRK• MET• Currently no reliable BRAF or NTRK3 fusion Ab
Spitzoid tumors with kinase fusions
Wiesner et al. pathology 2015
IHC Gene FusionsNTRK1
ROS1
MET
ALK
Spitzoid tumors with ALK fusions
Yeh et al Am J Surg Pathol 2015
Spitzoid tumors with kinase fusions
• Lesional spectrum of low grade tumors• Kinase fusions allow a subclassification of tumors
with (but not limited to) Spitzoid morphology• Genotype/morphotype link? Work in progress• Potential therapeutic targets• Potential prognostic factors (BRAF fusion worse?)
Drivers are mutually exclusiveNevoid Vs Spitzoid?
BRAF V600E mutation vs Kinase fusions
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« Raise your hand » vote
BRAF mutation Fusion
Raise
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BRAF VE1 AbIHC to explore loss of gene function
(tumor suppressor genes)
• BAP1• p53• …
Usefulness of molecular pathology in cutaneous melanocytic tumors
• Level 1
100%
Morphological analysis with knowledge of essential clinical information: age of patient, size, localization and evolution of the tumor (recent modification for instance)
• Level 2
10%IHC screening if «benign vs malignant» doubt : 4 Ab panel (anti-MelanA, HMB45, anti-p16 et Ki67) with a red chromogene for thick lesions (>1 mm).
Usefulness of molecular pathology in cutaneous melanocytic tumors
• Level 3
5%
Complementary IHC study searching for a specific anomaly guided by morphology and 4 Ab panel (anti-BAP1, anti-ALK, etc.).
Usefulness of molecular pathology in cutaneous melanocytic tumors
• Level 4
Extensive molecular analysis in the event of a wide surgery (digital amputation , etc.), in pediatric variants of melanoma or rare melanocytic tumors (unclassified)Case specific combination of techniques :• FISH : 4 color, CDKN2A (P16), fusions specific probes (ALK, ROS1, etc.) ;• Gene sequencing accessing somatic mutations (BRAF, NRAS, GNAQ, GNA11, HRAS, etc.) ;• CGH-array; RNA-seq analysis.1%
Usefulness of molecular pathology in cutaneous melanocytic tumors
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Hyperbole of uncertainty/technique
Technique
Uncertainty
Clinical evolution
• Local relapse• Nodal extension• Metastatic dissemination
• Site• Biological comparison to primary tumor
Melanoledge projectConvergence of viewpoints
Embryogenesis
Clinical features
Microscopy/morphology
Immunophenotype
Genomic profile
Mutation status
Clinical evolution
What is new?
• Realm of «Molecular Pathology»
• New concepts
• New techniques applied to FFPE
• New entities
What is new?
• Realm of «Molecular Pathology»
• New concepts
• New techniques applied to FFPE
• New entitiesKinase fusions in spitzoid tumorsMelanocytic tumors with loss of BAP1 expression
BAP1-inactivated melanocytic tumors
(BAPimt)Arnaud de la Fouchardière, MD, PhD
Lyon, France
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M Carbone et al. 2013
BAP1: BRCA1- Associated Protein 1A gene with multiple faces
BAP1 IHCNormal staining
Compound nevus
Loss of nuclear BAP1 expression= loss of gene function
Loss of BAP1 expression in melanocytic lesions of the skin
Distinct scenarii
• Solitary BAPimt• BAPimt(s)/ melanoma(s) in the context of a BAP1
cancer predisposition syndrome (germline mutation)• Sporadic epidermal–linked melanomas (DM)• Melanomas arising from/mimicking cellular blue nevus
BAPimtsSomatic > germline mutation of BAP1
• Children or young adults• Female>Male• Sun-exposed areas• Modification of a nevus• Growing unpigmented nodule <1cm• Inflammatory features• Multiple lesions: germline
Eur J Dermatol. 2015 25:201-2.
F, 14 back
BAPimts
M,56 Arm
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BAPimts
• F 16, cheek M 10 ; shoulder
M 27 with uveal melanoma
F25 multiple lesions since age 11
BAPimts keep the morphologicfeatures of the nevi they arised from
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Congenital-like nevus with nodule
Lipidic inclusions Lipidic inclusions
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F 8: Ear; familial history of uveal melanoma
Lateralcombinedaspect
commun nevus
M, 15 Back
F, 19 Back
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Nests of nevoid cellsNests of nevoid cells
Large epithelioid and nevoidmelanocytes +/- lymphocytes
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Large epithelioid and nevoidmelanocytes +/- lymphocytes
Clonal large unpigmentedmelanocytes
Clonal large unpigmentedmelanocytes Multinucleations
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BAP1 IHC BAP1 IHC
BAP1 IHC
BAP1 IHC
BAP1 IHC BAP1 IHC
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BAP1 IHC BRAF V600E mutation association (> 80%)
GC AG A AA A
c.1799T>A
W
BRAF mutation IHC (VE1) BAPimtsNo formal guidelines
• Unknown prognosis• Complete surgical removal• Follow-up• Identify patients needing oncogenetic counseling
Tumors associated with a BAP1 germline mutation
• Atypical cutaneous melanocytic tumors (BAPimts )• Uveal melanoma• Cutaneous melanoma• Leptomeningeal melanoma• Mesothelioma (pleural and abdominal)• Clear cell renal cancer• Meningiomas• Multiple basal cell carcinomas• These cancers arise at a younger age• Maybe other cancers: lung, stomach, pancreas, CholangioK• Probably incomplete cancer spectrum : ongoing work
Frequency of germline mutations?
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Estimated a <16% frequency of germline mutation
• We recommend, following the diagnosis of aMelanocytic tumor with loss of BAP1 expression,performing a BAP1 immunohistochemistry in allother cutaneous melanocytic tumors removedpreviously or simultaneously and all skinmelanomas.
Malignant transformation of BAPimts?
• M43, history in childhood of BAPimt on the neck• 3cm buttocks mass that grew in a few months
Malignant transformation of BAPimts?
• M43, history in childhood of BAPimt on the neck• 3cm buttocks mass that grew in a few months
Malignant transformation of BAPimts?
• M43, history in childhood of BAPimt on the neck• 3cm buttocks mass that grew in a few months
Malignant transformation of BAPimts?
• M43, history in childhood of BAPimt on the neck• 3cm buttocks mass that grew in a few months
Malignant transformation of BAPimts?
BAP1
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Malignant transformation of BAPimts?
BAP1
Malignant transformation of BAPimts?
Ki67
Differential Diagnosis
• Spitz nevus• Nevoid melanoma• Dystrophic nevus• BAPimt-like melanocytic tumors
DD: Spitz nevus
DD: Spitz nevus DD:Spitz nevus
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M, 64 Neck BAP1
Nevoid melanoma ex-nevus Dystrophic nevus
« BAPimt-like» lesions
BAP1
F29, shoulder
BAP1 take home messages• BAPology is a fascinating new science• BAP1-inactivated melanocytic tumors can identify carriers
of a germline syndrome with cancer-predisposition. • Isolated cases with somatic mutations prevail.• Exophytic combined architecture• Large unpigmented epithelioid and nevoid dermal
melanocytes +/- lymphocytes.• IHC is an excellent technique to identify loss of function.
Global Take home messages
• Extremely active field for molecular pathology• Molecular techniques are strongly dependant on
high quality clinical and morphological analysis• Enhancing classifications improves patient care
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Definition of Primary Tumor (T) - AJCC 8th Edition
Gershenwald, Scolyer, et al. Melanoma. In Amin, M.B., Edge, S.B., Greene, F.L., et al. (Eds.) AJCC Cancer Staging Manual. 8th Ed. New York: Springer; 2017