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Immunohistochemistry Immunohistochemistry !"#$%&'"()(*+$(,$-#)&.(-&/$ 0--1.("23'(4"#-23'5+$$ 65.&17$7#$)&$8(14"&57295#$:;<$=";$ >+(.<$85&.4#$ Immunohistochemistry Immunohistochemistry It’s all about chosing the adapted anbody(ies) Immunohistochemistry It’s all about chosing the adapted anbody(ies) for the selected task(s) AnAbodies • « Melanocyc » anbodies – S100 – MelanA – HMB45 – PNL2 – MiTF Specificity vs Sensivity – SOX10 – … • « Anomaly-specific » anbodies – BRAF V600E – NRAS Q61R – ALK – ROS1 – NTRK1 – MET – P16 – BAP1 – PDL1 – … • Other anbodies – D2-40 – CD68 HMB45 – … AnAbodies • « Melanocyc » anbodies – S100 – MelanA – HMB45 – PNL2 – MiTF – SOX10 – … • « Anomaly-specific » anbodies – BRAF V600E – NRAS Q61R – ALK – ROS1 – NTRK1 – MET – P16 – BAP1 – PDL1 – … • Other anbodies – D2-40 – CD68 NTRK1 – … AnAbodies • « Melanocyc » anbodies – S100 – MelanA – HMB45 – PNL2 – MiTF – SOX10 – … • « Anomaly-specific » anbodies – BRAF V600E – NRAS Q61R – ALK – ROS1 – NTRK1 – MET – P16 – BAP1 – PDL1 – … • Other anbodies (DD mainly) – D2-40 – CD68 – … Why perform IHC? • Confirm melanocyc lineage • Visualize the melanocytes • Benign vs Malignant • Molecular characterizaon A. Confirm melanocyc lineage • Unpigmented dermal or ulcerated tumor (No recognizable junconal melanocytes) • Unpigmented metastases • Desmoplasc melanoma A1. Confirm melanocyc lineage Unpigmented dermal or ulcerated tumor M, 65 Back 6Ha$[(.T5-$-#)&.(4+A4$)2.#&*#$ b.%2*-#.'#7$7#5-&)$(5$1)4#5&'#7$'1-(5$$ 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 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 XP, F35, 5th resecon of ALM, 4th finger 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$ ;=N$/$;##%$%#.#'5&A.*$.#]13$ J:KLM$"#'#5(*#.#(13$%5(T)#$$ E$d(55+3(-#$F$ 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
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