Immunohistochemistry Immunohistochemistry

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65.&17$7#$)&$8(14"&57295#$:;<$=";$ >+(.<$85&.4#$ Immunohistochemistry Immunohistochemistry

It’s all about chosing the adapted anbody(ies) Immunohistochemistry

It’s all about chosing the adapted anbody(ies) for the selected task(s) Anbodies • « Melanocyc » anbodies – S100 – MelanA – HMB45 – PNL2 – MiTF Speciﬁcity vs Sensivity – SOX10 – … • « Anomaly-speciﬁc » anbodies – BRAF V600E – NRAS Q61R – ALK – ROS1 – NTRK1 – MET – P16 – BAP1 – PDL1 – … • Other anbodies – D2-40 – CD68 HMB45 – …

Anbodies • « Melanocyc » anbodies – S100 – MelanA – HMB45 – PNL2 – MiTF – SOX10 – … • « Anomaly-speciﬁc » anbodies – BRAF V600E – NRAS Q61R – ALK – ROS1 – NTRK1 – MET – P16 – BAP1 – PDL1 – … • Other anbodies – D2-40 – CD68 NTRK1 – …

Why perform IHC?

• Conﬁrm melanocyc lineage • Visualize the melanocytes • Benign vs Malignant • Molecular characterizaon A. Conﬁrm melanocyc lineage

• Unpigmented dermal or ulcerated tumor (No recognizable junconal melanocytes) • Unpigmented metastases • Desmoplasc melanoma A1. Conﬁrm melanocyc lineage Unpigmented dermal or ulcerated tumor

M, 65 Back 6Ha$[(.T5-$-#)&.(4+A4$)2.#&*#$ b.%2*-#.'#7$7#5-&)$(5$1)4#5&'#7$'1-(5$$

b.%2*-#.'#7$.#3'$(,$#%2'"#)2(27$4#))3$ A1. Conﬁrm melanocyc lineage Unpigmented dermal or ulcerated tumor

S100 Protein 6Ha$[(.T5-$-#)&.(4+A4$)2.#&*#$ b.%2*-#.'#7$7#5-&)$(5$1)4#5&'#7$'1-(5$$

GHII$=5('#2./$"#'#5(*#.#(13<$4+'(%)&3-24$&.7$.14)#&5$ 6Ha$[(.T5-$-#)&.(4+A4$)2.#&*#$ b.%2*-#.'#7$7#5-&)$(5$1)4#5&'#7$'1-(5$$

:#)&.6/$.#*&A]#$d2'"$%(32A]#$2.'#5.&)$4(.'5()3$ 6Ha$[(.T5-$-#)&.(4+A4$)2.#&*#$ b.%2*-#.'#7$7#5-&)$(5$1)4#5&'#7$'1-(5$$

J:KLM/$.#*&A]#$d2'"$%(32A]#$2.'#5.&)$4(.'5()3$ 6Ha$[(.T5-$-#)&.(4+A4$)2.#&*#$ b.%2*-#.'#7$7#5-&)$(5$1)4#5&'#7$'1-(5$$

:2!8/$,(4&)$.14)#&5$&.7$4+'(%)&3-24$%(32A]2'+$e$2.'#5.&)$4(.'5()3$ 6Ha$[(.T5-$-#)&.(4+A4$)2.#&*#$ b.%2*-#.'#7$7#5-&)$(5$1)4#5&'#7$'1-(5$$

:2!8/$,(4&)$.14)#&5$&.7$4+'(%)&3-24$%(32A]2'+$e$2.'#5.&)$4(.'5()3$ A1. Conﬁrm melanocyc lineage Unpigmented dermal or ulcerated tumor

Sox10: heterogeneous nuclear posivity 6Ha$[(.T5-$-#)&.(4+A4$)2.#&*#$ b.%2*-#.'#7$7#5-&)$(5$1)4#5&'#7$'1-(5$$

G(fHI/$"#'#5(*#.#(13$.14)#&5$%(32A]2'+$ 6Ha$[(.T5-$-#)&.(4+A4$)2.#&*#$ b.%2*-#.'#7$7#5-&)$(5$1)4#5&'#7$'1-(5$$

G(fHI/$"#'#5(*#.#(13$.14)#&5$%(32A]2'+$ A1. Confirm melanocyc lineage Unpigmented dermal or ulcerated tumor • PS100 + MelanA - HMB45 - A1. Confirm melanocyc lineage Unpigmented dermal or ulcerated tumor • PS100 + MelanA - HMB45 - • MiTF + Sox10+ allows the diagnosis of a melanoma A1. Confirm melanocyc lineage Unpigmented dermal or ulcerated tumor • What about epithelial ab? A1. Confirm melanocyc lineage Unpigmented dermal or ulcerated tumor • What about epithelial ab?

Keran AE1AE3: focal posivity EMA: focal posivity A1. Conﬁrm melanocyc lineage Unpigmented dermal or ulcerated tumor • What about epithelial ab?

Keran AE1AE3: focal posivity EMA: focal posivity A2. Confirm melanocyc lineage Unpigmented metastases A2. Confirm melanocyc lineage Unpigmented metastases • History of melanoma • Unknown primary A2. Confirm melanocyc lineage Unpigmented metastases • History of melanoma • Unknown primary • Always perform a panel including S100 Protein A2. Confirm melanocyc lineage Unpigmented metastases

History of lymphoma A2. Conﬁrm melanocyc lineage Unpigmented metastases

CD138 A2. Conﬁrm melanocyc lineage Unpigmented metastases

HMB45 A2. Conﬁrm melanocyc lineage Unpigmented metastases

Sox10 6ga$[(.T5-$-#)&.(4+A4$)2.#&*#$ ;#3-(%)&3A4$-#)&.(-&$ A3. Conﬁrm melanocyc lineage Desmoplasc melanoma A3. Conﬁrm melanocyc lineage Desmoplasc melanoma

• S100 Protein A3. Conﬁrm melanocyc lineage Desmoplasc melanoma

MelanA HMB45 6ga$[(.T5-$-#)&.(4+A4$)2.#&*#$ ;#3-(%)&3A4$-#)&.(-&$ •! 0J[$23$-&.7&'(5+$ •! =GHIIe$ •! J:KLMS$ :#)&.6S$ •! SMA •! G(fHI$She$ •! :2!8$She$ •! G:6$ehS$ •! [;W\$e$ CD68 (KP1) B.Visualize the melanocytes

• Asymetric melanocyc distribuon • Margin assessment • Intra-epidermal ascension of cells • Lympho-vascular invasion • SLN B1: Asymetric melanocyc distribuon

MelanA B1: Asymetric melanocyc distribuon

Also similarly useful for • Breslow assessment • Density evaluaon in a hyperpigmented lesion • Junconal interrupon related to regression? B1: Asymetric melanocyc distribuon

MelanA B1: Asymetric melanocyc distribuon

Breslow assessment

MelanA Density evaluaon in a hyperpigmented lesion Density evaluaon in a hyperpigmented lesion

Low density, regularly distributed junconnal nests of melanocytes: benign lesion Junconal interrupon related to regression? Junconal interrupon related to regression?

Melan A B2: Margin assessment

XP, F35, 5th resecon of ALM, 4th ﬁnger B2: Margin assessment

MelanA XP, F35, 5th resecon of ALM, 4th ﬁnger B3: Intra-epidermal ascent of cells

• HMB45 or melanA (A103) can target melanosomes which are normaly transfered to keranocytes = risk of false posivity • Prefer nuclear located anbodies B3: Intra-epidermal ascent of cells

• HMB45 or melanA (A103) can target melanosomes which are normaly transfered to keranocytes = risk of false posivity • Prefer nuclear located anbodies B3: Intra-epidermal ascent of cells B3: Intra-epidermal ascent of cells

HMB45 B3: Intra-epidermal ascent of cells

MelanA B3: Intra-epidermal ascent of cells

MiTF B4: Lympho-vascular invasion B4: Lympho-vascular invasion

PMID: 21881483

B4: Lympho-vascular invasion

D2-40 PMID: 21881483 B5: Sennel Lymph Node evaluaon C: Malignant vs Benign seng Lesions B>1mm

• 4 anbody PANEL combinaon analysis – HMB45 – Melan-A (A103 clone) – p16 – Proliferaon index (ki-67/MIB1)

MelanA

HMB45

p16 p16 Deviant IHC panel HMB45 Melan A

p16 Ki67 C: Malignant vs Benign seng

• 4 anbody PANEL combinaon analysis – HMB45: expression proﬁles (smulated melanocytes) – Melan-A – P16 – Proliferaon index (ki-67/MIB1) HMB45 top heavy Proﬁle « reassuring »

Congenital-like type nevus J:KLM$72n13#$%(32A]2'+$%5(T)#$$ E$5#&33152.*$F$

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6'+%24&)$;=N$ HMB45 heterogeneous proﬁle « worrysome »

Nevoid melanoma HMB45 heterogeneous proﬁle « worrysome »

Atypical spitz nevus C: Malignant vs Benign seng

• 4 anbody PANEL combinaon analysis – HMB45 – Melan-A: compared to HMB45 (all melanocytes) – P16: clonal loss of expression – Proliferaon index (ki-67/MIB1) Melan A heterogeneous proﬁle « worrysome »

Nevoid melanoma C: Malignant vs Benign seng

• 4 anbody PANEL combinaon analysis – HMB45 – Melan-A – P16: clonal loss of expression – Proliferaon index (ki-67/MIB1)

=HW$&.A@(7+$ p16 staining paerns (useful even in thin lesions) • Diﬀuse posivity All melanocytes are stained

p16 staining paerns (useful even in thin lesions) • Diffuse posivity • Checkerboard stain Adjacent melanocytes are either totally stained or not stained at all %HW$&@$ ["#4D#5@(&57$ =HW$3'&2.2.*$ p16 staining paerns (useful even in thin lesions) • Diffuse posivity • Checkerboard stain • Complete loss p16 staining paerns (useful even in thin lesions) • Diffuse posivity • Checkerboard stain • Complete loss: check internal controls p16 Complete loss Eccrine sweat glands and pars recta display focal p16 posivity Dysplasc or regenerang epidermis p16 posivity (inconstant) p16 complete loss Thin lesions p16 complete loss p16 complete loss p16 staining paerns (useful even in thin lesions) • Diffuse posivity • Checkerboard stain • Complete loss • Clonal loss Only an area of the tumor has lost p16 staining p16 Clonal loss p16 Clonal loss p16 clonal loss p16 staining paerns (useful even in thin lesions) • Diffuse posivity • Checkerboard stain • Absence of staining • Clonal loss • Melanoma ex-nevus staining paern Different paern in melanoma and nevus %HW$-#)&.(-&$#fS.#]13$3'&2.2.*$%&o#5.$ $ p16 staining paerns (useful even in thin lesions) • Diffuse posivity • Checkerboard stain • Absence of staining • Clonal loss • Melanoma ex-nevus staining paern • Inverted gradient Boom heavy staining

Suspect p16 staining paerns (useful even in thin lesions) • Diffuse posivity • Checkerboard stain • Absence of staining (check internal controls) • Clonal loss • Melanoma ex-nevus staining paern • Inverted gradient p16 staining paerns unknown benign/malignant significaon • Diffuse posivity • Checkerboard stain • Absence of staining • Clonal loss • Melanoma ex-nevus staining paern • Inverted gradient Malignant vs Benign seng

• 4 anbody PANEL combinaon analysis – HMB45 – Melan-A – P16: clonal loss of expression – Proliferaon index (ki-67/MIB1) : 20% threshold MM high proliferaon index Ki-67 can be low in malignant melanoma «Hotspot area» visualizaon D Molecular characterizaon

• Point mutaons • Gene fusions • Loss of funcon (tumor suppressor genes) « Theragnosc » tools D1 Point mutaons

• BRAF V600E • NRAS Q61R BRAF V600E NRAS Q61R D2: Fusions

• Screening tool IHC Fusions

NTRK1 MET

ROS1 ALK These anomalies are mutually exclusive 6>Z$ N!UZH$

UPGH$ Weak stain in ROS1 FISH conﬁrmaon D3 Loss of funcon (tumor suppressor genes)

• BAP1 • p53 • … Loss of BAP1 expression in melanocyc lesions of the skin Disnct scenarii

• Solitary BAPoma • BAPoma(s) / melanoma(s) in the context of a BAP1 cancer syndrome (germline mutaon) • Sporadic epidermal–linked melanomas (DM) • Melanomas arising from/mimicking cellular blue nevus BAP1 IHC Normal staining

Compound nevus Loss of nuclear BAP1 expression = loss of gene funcon BAP1 IHC False negavity Always check internal controls

Dermal nevus Melanoma arising from a blue nevus or mimicking a cellular blue nevus

BAP1 IHC

PMID: 26645730

Melanoma arising from a blue nevus or mimicking a cellular blue nevus

BAP1 IHC PMID: 26645730 Take home messages

• IHC is a powerful tool that relies on the careful choice of anbodies adapted to a speciﬁc situaon • To conﬁrm melanocyc lineage always perform a panel of anbodies (S100 Protein mandatory) • MelanA (A103) is the most adapted anbody to vizualize the distribuon of a melanocyc lesion • A 4 anbody panel (MelanA, HMB45, p16, ki67) is a good screening tool in the benign/malignant diagnosc seng • IHC is a potenal molecular screening tool Take home messages