!"#$%&'"()(*+$(,$-#)&.

(-&/$
0--1.("23'(4"#-23'5+$$

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

It’s all about chosing the adapted anAbody(ies)

 Immunohistochemistry

It’s all about chosing the adapted anAbody(ies)

for the selected task(s)
 AnAbodies
• « MelanocyAc » anAbodies
– S100
– MelanA
– HMB45
–
–
PNL2
MiTF Specificity vs SensiAvity
– SOX10
– …

• « Anomaly-specific » anAbodies
– BRAF V600E
– NRAS Q61R
– ALK
– ROS1
– NTRK1
– MET
– P16
– BAP1
– PDL1
– …

• Other anAbodies
–
–
D2-40
CD68
HMB45
– …
 AnAbodies
• « MelanocyAc » anAbodies
– S100
– MelanA
– HMB45
– PNL2
– MiTF
– SOX10
– …

• « Anomaly-specific » anAbodies
– BRAF V600E
– NRAS Q61R
– ALK
– ROS1
– NTRK1
– MET
– P16
– BAP1
– PDL1
– …

• Other anAbodies
–
–
D2-40
CD68
NTRK1
– …
 AnAbodies
• « MelanocyAc » anAbodies
– S100
– MelanA
– HMB45
– PNL2
– MiTF
– SOX10
– …

• « Anomaly-specific » anAbodies
– BRAF V600E
– NRAS Q61R
– ALK
– ROS1
– NTRK1
– MET
– P16
– BAP1
– PDL1
– …

• Other anAbodies (DD mainly)

– D2-40
– CD68
– …
 Why perform IHC?
• Confirm melanocyAc lineage
• Visualize the melanocytes
• Benign vs Malignant
• Molecular characterizaAon
 A. Confirm melanocyAc lineage
• Unpigmented dermal or ulcerated tumor (No
recognizable juncAonal melanocytes)
• Unpigmented metastases
• DesmoplasAc melanoma
 A1. Confirm melanocyAc 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 melanocyAc 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 melanocyAc lineage
Unpigmented dermal or ulcerated tumor

Sox10: heterogeneous nuclear posiAvity

 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 melanocyAc lineage
Unpigmented dermal or ulcerated tumor
• PS100 + MelanA - HMB45 -
 A1. Confirm melanocyAc lineage
Unpigmented dermal or ulcerated tumor
• PS100 + MelanA - HMB45 -
• MiTF + Sox10+ allows the diagnosis of a
melanoma
 A1. Confirm melanocyAc lineage
Unpigmented dermal or ulcerated tumor
• What about epithelial ab?
 A1. Confirm melanocyAc lineage
Unpigmented dermal or ulcerated tumor
• What about epithelial ab?

KeraAn AE1AE3: focal posiAvity EMA: focal posiAvity

 A1. Confirm melanocyAc lineage
Unpigmented dermal or ulcerated tumor
• What about epithelial ab?

KeraAn AE1AE3: focal posiAvity EMA: focal posiAvity

A2. Confirm melanocyAc lineage
Unpigmented metastases
 A2. Confirm melanocyAc lineage
Unpigmented metastases
• History of melanoma
• Unknown primary
 A2. Confirm melanocyAc lineage
Unpigmented metastases
• History of melanoma
• Unknown primary
• Always perform a panel including S100 Protein
 A2. Confirm melanocyAc lineage
Unpigmented metastases

History of lymphoma
 A2. Confirm melanocyAc lineage
Unpigmented metastases

CD138
 A2. Confirm melanocyAc lineage
Unpigmented metastases

HMB45
 A2. Confirm melanocyAc lineage
Unpigmented metastases

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

• S100 Protein
A3. Confirm melanocyAc lineage
DesmoplasAc 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 melanocyAc distribuAon
• Margin assessment
• Intra-epidermal ascension of cells
• Lympho-vascular invasion
• SLN
B1: Asymetric melanocyAc distribuAon

MelanA
B1: Asymetric melanocyAc distribuAon

MelanA
B1: Asymetric melanocyAc distribuAon
Also similarly useful for
• Breslow assessment
• Density evaluaAon in a hyperpigmented lesion
• JuncAonal interrupAon related to regression?
B1: Asymetric melanocyAc distribuAon

MelanA
B1: Asymetric melanocyAc distribuAon

Breslow assessment

MelanA
Density evaluaAon in a
hyperpigmented lesion
 Density evaluaAon in a
hyperpigmented lesion

Low density, regularly distributed juncAonnal nests of melanocytes: benign lesion

JuncAonal interrupAon
related to regression?
JuncAonal interrupAon
related to regression?

Melan A
 B2: Margin assessment

XP, F35, 5th resecAon of ALM, 4th finger

 B2: Margin assessment

XP, F35, 5th resecAon of ALM, 4th finger

 B2: Margin assessment

XP, F35, 5th resecAon of ALM, 4th finger

 B2: Margin assessment

MelanA
XP, F35, 5th resecAon of ALM, 4th finger
B3: Intra-epidermal ascent of cells
• HMB45 or melanA (A103) can target
melanosomes which are normaly transfered
to keraAnocytes
= risk of false posiAvity
• Prefer nuclear located anAbodies
B3: Intra-epidermal ascent of cells
• HMB45 or melanA (A103) can target
melanosomes which are normaly transfered
to keraAnocytes
= risk of false posiAvity
• Prefer nuclear located anAbodies
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: SenAnel Lymph Node evaluaAon
 C: Malignant vs Benign sekng
Lesions B>1mm

• 4 anAbody PANEL combinaAon analysis

– HMB45
– Melan-A (A103 clone)
– p16
– ProliferaAon index (ki-67/MIB1)
MelanA

HMB45

p16
p16
 Deviant IHC panel
HMB45 Melan A

p16 Ki67
 C: Malignant vs Benign sekng

• 4 anAbody PANEL combinaAon analysis

– HMB45: expression profiles (sAmulated melanocytes)
– Melan-A
– P16
– ProliferaAon 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 sekng

• 4 anAbody PANEL combinaAon analysis

– HMB45
– Melan-A: compared to HMB45 (all melanocytes)
– P16: clonal loss of expression
– ProliferaAon index (ki-67/MIB1)
 Melan A heterogeneous profile
« worrysome »

Nevoid melanoma
 C: Malignant vs Benign sekng

• 4 anAbody PANEL combinaAon analysis

– HMB45
– Melan-A
– P16: clonal loss of expression
– ProliferaAon index (ki-67/MIB1)
=HW$&.A@(7+$
 p16 staining paoerns
(useful even in thin lesions)
• Diffuse posiAvity
All melanocytes are stained
 p16 staining paoerns
(useful even in thin lesions)
• Diffuse posiAvity
• Checkerboard stain
Adjacent melanocytes are either totally stained or
not stained at all
%HW$&@$
["#4D#5@(&57$
=HW$3'&2.2.*$
 p16 staining paoerns
(useful even in thin lesions)
• Diffuse posiAvity
• Checkerboard stain
• Complete loss
 p16 staining paoerns
(useful even in thin lesions)
• Diffuse posiAvity
• Checkerboard stain
• Complete loss: check internal controls
p16 Complete loss
Eccrine sweat glands and pars recta
display focal p16 posiAvity
DysplasAc or regeneraAng epidermis
p16 posiAvity (inconstant)
p16 complete loss
Thin lesions
p16 complete loss
p16 complete loss
 p16 staining paoerns
(useful even in thin lesions)
• Diffuse posiAvity
• 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 paoerns
(useful even in thin lesions)
• Diffuse posiAvity
• Checkerboard stain
• Absence of staining
• Clonal loss
• Melanoma ex-nevus staining paoern
Different paoern in melanoma and nevus
%HW$-#)&.(-&$#fS.#]13$3'&2.2.*$%&o#5.$
$
 p16 staining paoerns
(useful even in thin lesions)
• Diffuse posiAvity
• Checkerboard stain
• Absence of staining
• Clonal loss
• Melanoma ex-nevus staining paoern
• Inverted gradient
Booom heavy staining
 Suspect p16 staining paoerns
(useful even in thin lesions)
• Diffuse posiAvity
• Checkerboard stain
• Absence of staining (check internal controls)
• Clonal loss
• Melanoma ex-nevus staining paoern
• Inverted gradient
 p16 staining paoerns
unknown benign/malignant significaAon
• Diffuse posiAvity
• Checkerboard stain
• Absence of staining
• Clonal loss
• Melanoma ex-nevus staining paoern
• Inverted gradient
 Malignant vs Benign sekng

• 4 anAbody PANEL combinaAon analysis

– HMB45
– Melan-A
– P16: clonal loss of expression
– ProliferaAon index (ki-67/MIB1) : 20% threshold
MM high proliferaAon index
 Ki-67 can be low
in malignant melanoma
«Hotspot area» visualizaAon
 D Molecular characterizaAon
• Point mutaAons
• Gene fusions
• Loss of funcAon (tumor suppressor genes)
« TheragnosAc » tools
 D1 Point mutaAons
• 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 confirmaAon
 D3 Loss of funcAon
(tumor suppressor genes)
• BAP1
• p53
• …
 Loss of BAP1 expression
in melanocyAc lesions of the skin
DisAnct scenarii
• Solitary BAPoma
• BAPoma(s) / melanoma(s) in the context of a
BAP1 cancer syndrome (germline mutaAon)
• 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 funcAon
 BAP1 IHC False negaAvity
Always check internal controls

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

BAP1 IHC
26645730

Melanoma arising from a blue nevus

or mimicking a cellular blue nevus

PMID: 26645730
BAP1 IHC
 Take home messages
• IHC is a powerful tool that relies on the careful
choice of anAbodies adapted to a specific
situaAon
• To confirm melanocyAc lineage always perform a
panel of anAbodies (S100 Protein mandatory)
• MelanA (A103) is the most adapted anAbody to
vizualize the distribuAon of a melanocyAc lesion
• A 4 anAbody panel (MelanA, HMB45, p16, ki67) is
a good screening tool in the benign/malignant
diagnosAc sekng
• IHC is a potenAal molecular screening tool
 Take home messages
• IHC is a powerful tool that relies on the careful
choice of anAbodies adapted to a specific
situaAon
• To confirm melanocyAc lineage always perform a
panel of anAbodies (S100 Protein mandatory)
• MelanA (A103) is the most adapted anAbody to
vizualize the distribuAon of a melanocyAc lesion
• A 4 anAbody panel (MelanA, HMB45, p16, ki67) is
a good screening tool in the benign/malignant
diagnosAc sekng
• IHC is a potenAal molecular screening tool
 Take home messages
• IHC is a powerful tool that relies on the careful
choice of anAbodies adapted to a specific
situaAon
• To confirm melanocyAc lineage always perform a
panel of anAbodies (S100 Protein mandatory)
• MelanA (A103) is the most adapted anAbody to
vizualize the distribuAon of a melanocyAc lesion
• A 4 anAbody panel (MelanA, HMB45, p16, ki67) is
a good screening tool in the benign/malignant
diagnosAc sekng
• IHC is a potenAal molecular screening tool
 Take home messages
• IHC is a powerful tool that relies on the careful
choice of anAbodies adapted to a specific
situaAon
• To confirm melanocyAc lineage always perform a
panel of anAbodies (S100 Protein mandatory)
• MelanA (A103) is the most adapted anAbody to
vizualize the distribuAon of a melanocyAc lesion
• A 4 anAbody panel (MelanA, HMB45, p16, ki67) is
a good screening tool in the benign/malignant
diagnosAc sekng
• IHC is a potenAal molecular screening tool
 Take home messages
• IHC is a powerful tool that relies on the careful
choice of anAbodies adapted to a specific
situaAon
• To confirm melanocyAc lineage always perform a
panel of anAbodies (S100 Protein mandatory)
• MelanA (A103) is the most adapted anAbody to
vizualize the distribuAon of a melanocyAc lesion
• A 4 anAbody panel (MelanA, HMB45, p16, ki67) is
a good screening tool in the benign/malignant
diagnosAc sekng
• IHC is a potenAal molecular screening tool
Many thanks to
our IHC and
diagnosAc team