Immunohistochemical markers for CNS tumor diagnosis

Immunohistochemical markers
for CNS tumor diagnosis
한림대 성심병원
권미정
IHC markers in CNS tumors
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Few useful marker
Few specific marker
Variable expression
Difficult to interpret
IHC markers useful in D/Dx of CNS tumors
Tumor
IHC
Astrocytoma
GFAP, S-100
Oligodendroglioma
S-100, GFAP
Ependyomoma
S-100, GFAP, EMA(dot-like), CD99
Medulloblastoma
Synaptophysin
Central neurocytoma
Synaptophysin, Neu-N
Ganglioglioma
GFAP, synaptophysin, CD34, NF
Meningioma
EMA, Vimentin
Choroid plexus tumors
CK, S-100, Vimentin, Transthyretin
Metastatic carcinoma
EMA, CK
ATRT
INI1-loss
Hemangiopericytoma
Solitary fibrous tumor
CD34, CD99, Bcl-2
Schwannoma
S-100
Neurofibroma
S-100, NF
Germinoma
PLAP, c-Kit, OCT4
How to interpret?
What’s the meaning?
 Glial marker : GFAP vs. S-100
 Neuronal marker: SYP vs. NFP vs. Neu-N
» Chromogranin A, CD56 ??
 Epithelial markers: CK vs. EMA
Glial marker: GFAP vs. S-100
GFAP
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Glial fibrillary acidic protein
One of the intermediate filaments
Cytoplasmic stain only
Sensitive glial cell marker
 Glia
• True glia : astrocyte, oligodendrocyte, and ependyma
• Microglia : hematopoietic lineage
GFAP
 Strong expression in astrocytic neoplasm
• Astrocytoma, Ependymoma, Pituicytoma
 Focal GFAP expression : focal glial differentiation
• choroid plexus tumor, medulloblastoma, CNS PNET,
ATRT, ganglioglioma
 Negative in oligodendroglioma
• Negative in classic type oligodendrocyte
• Gliofibrillary oligodendrocytes (+), mini-gemistocytes (+)
GFAP in oligodendroglioma
Classic type
gliofibrillary type
Entrapped astrocytes
• Gliofibrillary
oligodendrocytes (+)
• mini-gemistocytes (+)
• Fried egg appearance
• Artifact occurring during tissue processing
• This feature should not be overrated diagnostically.
Diffuse astrocytoma with low cellularity and
minimal atypia vs. Reactive gliosis
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Radiographic feature of diffuse glioma
Nuclear enlargement & hyperchromasia
Nuclear clustering
Clustering of GFAP-positive cells
P53 immunoreactivity (+)
Ki-67↑↑
Ki-67 (+) in cytologically abnormal nuclei
WT-1(+)
Evenly spaced astrocytes
with radially arranged process
Diffuse astrocytoma
Reactive gliosis
GFAP
 Strongly positive in reactive astrocytes
• Stain “star-like” process
• Regular, even spacing and radially arranged process in gliosis
Diffuse astrocytoma
Gemistocytes in diffuse astrocytoma
S-100 Protein
 Comon to neuroectodermal cells
• Melanocyte, glia, Schwann cell, chondrocyte,
sustentacular cells of paraganglioma
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Highly sensitive “all-around” glial marker
Less specific
Nuclear and cytoplasmic staining
Positive: oligodendrocyte and astrocyte
• Oligodendroglioma (+)
Oligodendroglioma : S100(+)
GFAP vs S-100
GFAP
S-100
Glial marker
Yes
Yes
Staining
Cytoplasmic
Nuclear & cytoplasmic
Astrocyte
Positive
Positive
Oligodendrocyte
Negative
Positive
Oligodendroglioma
Negative
Positive
예외)
Gliofibrillary subtype
Mini-gemistocyte
Neuronal marker: SYP vs. NFP vs. Neu-N
Synaptophysin (SYP)
 Membrane protein of presynaptic vesicle in brain
and endocrine cell
 Sensitive marker of neuronal differentiation
 Typically found in most primitive tumors
• Medulloblastoma, PNET
 Cytoplasmic staining
• Granular or Membranous
Synaptophysin (SYP)
 Negative: normal cerebral cortical neurons
 Positive
• Neuropil background of brain
• Normally, neuropil of gray matter, rich in synaptic contacts :
diffuse, finely granular positive
• Neuronal tumors
• central neurocytoma
• neurons in ganglioglioma
• Primitive tumors: medulloblastoma, CNS-PNET
Medulloblastoma
Neurofilament (NF)
 Intermediate filament
 Specific to neuronal & neuroendocrine cells
 Normally expressed in most axonal process
• Axons in gray and white matters
 Antibody
• Unphosphorylated : Normal neuronal cell body (+)
• Phosphorylated: Normal neuronal cell body (-)
• Cortical dysplasia
Neurofilament (NF)
 Variable staining in neuronal or primitive tumors
• Ganglioglioma (±) : Neuronal cell body (±)
• Medulloblastoma (±): negative ~ patchy cytoplasmic (+)
 Useful in distinguishing Grade II-III atrocytoma
from Grade I
• Confirmation of diffuse tumor infiltration in IHC preparations
Neurofilament
 Confirmation of diffuse tumor infiltration in IHC preparatio
ns
Preexisting axons
Neu-N
 Nuclear staining
 Expressed in mature & terminal neuronal cells
• Normal cortical neurons (+), Neuropil (-), Purkinje cell (-)
 Negative in most neoplastic ganglion cells
• Ganglioglioma (-)
 At least focally positive in primitive neuronal tumor
• Medulloblastoma, CNS PNET
 Positive
• Central/extraventricular neurocytoma
• almost all cases
Neu-N
Cerebral cortex
Neuronal cell bodies (+)
Glial cells (-)
Cerebellar cortex
Granular layer (+)
Purkinje cells (-)
Ganglioglioma
Synaptophysin(+)
Neu-N(-)
Central neurocytoma
Synaptophysin(+)
Neu-N(+)
Neurofilament(-)
Neu-N
 Useful in distinguishing
• tumoral ganglion cells vs. entrapped cortical neurons
(NeuN-)
(NeuN+)
• Entrapped cortical neurons (NeuN+) within a diffuse
glioma -> tumor infiltration
Neu-N negative neurons
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Cajal-Retzius neurons in layer 1 of cerebral cortex
Purkinje cells
Inf. Olivary and dentate nuclear neurons
Retinal photoreceptor cells
Mitral cells of olfactory tracts
Ganglion cells of sympathetic chain
Other neural markers
 Chromogranin A
• Neoplastic ganglion cell (+) : Ganglioglioma
• Neuroendocrine tumor (+)
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Metastatic small cell carcinoma
Pituitary adenoma
Carcinoid
Paraganglioma
• More specific than synaptophysin, but low sensitivity
 CD56
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Neural cell adhesion molecule expressed on the surface of neurons and glia
Positive : Normal cells of cortex and cerebellum
Not recommended in CNS
 NSE (neuron specific enolase)
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The earliest marker
Unreliable marker of neuronal differentiaion
Sensitive
Primitive
SYP
Specific
Mature
NFP
Neu-N
Staining
Cytoplasmic ±
Memb.
Cytoplasmic
Nuclear
Positive
neuropil
background
Axons
Normal cortical
neurons
Neuronal tumors
Constant (+)
Variable
Mostly (-)
Central neurocytoma
+
-
+
Ganglioglioma
+
+
-
Primitive tumor
Strong(+)
(-) ~ patchy(+)
At least focal(+)
Normal
cortical
neurons
Neuronal cell
body
Neuropil,
Neoplastic
ganglionic cells
(Medulloblastoma, PNET)
Negative
Epithelial markers: CK vs. EMA
Cytokeratin
 intermediate filament
 Cytoplasmic staining
 Demonstrate epithelial differentiation
• Most commonly used in the diagnosis of metastatic
carcinoma
• Identify primary epithelial or epithelium-containing CNS
neoplasm
• Craniopharyngioma, chordoma, teratoma
• epithelial cyst, choroid plexus tumor
Cytokeratin
 Cytokeratin AE1/AE3
• Cross-reactivity to GFAP
• Reactive astrocytes and astrocytoma
 Other CK Ab
• confirming metastatic carcinoma in brain
• CAM5.2
• Distinguish metastatic (CAM 5.2+, GFAP-) from primary (CA
M 5.2-, GFAP+) CNS tumors
• False positive in smooth muscle, myofibroblast, reticulum
cells in lymph node: CAM5.2 < CK AE1/AE3
• Brain: CAM5.2 > CK AE1/AE3
Pancytokeratin including CK AE1/AE3
Cross reactivity to glial fibrillary
acidic protein
False positive in Reticulum cells
Gemistocytic astrocytoma
CAM5.2(-)
CK AE1/AE3(+)
GFAP(+)
EMA
 Epithelial membrane antigen
 Commonly normal and neoplasitc epithelial cells
 Less specific
 Positive
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Normal or reactive arachnoidal cells (Membranous)
Meningioma (cytoplasmic)
Ependymoma (dot-like or ring-like)
Astroblastoma (dot-like)
Angiocentric glioma (dot-like)
EMA
 Useful marker in meningioma
 Most meningiomas only show patchy weak
expression, compared to carcinoma
 For typical EMA expression in meningioma
• Higher Ab concentration
• Enhanced antigen retrieval
EMA(+) in meningioma
EMA(+) in ependymoma
MIB-1/Ki-67
 Nuclear staining
 All active phases of cell cycle
– Non-G0 phases (G1, S, G2, and M phases)
 Negative
• Normal brain
• Reactive gliosis (1-2 cells possible)
 Very useful for grading of glioma and
meningioma
Diffuse astrocytoma
Ki-67
MIB-1 is important in assessing proliferation,
particularly among astrocytomas with inconspicuous mitoses.
MIB-1/Ki-67 in meningioma
 Mean MIB-1 LI
• Benign: 1.5%
• Atypical: 8.1%
• Anaplastic: 19.5%
 Atypical meningioma criteria
• Increased mitotic activity: ≥4/10 HPFs
• ≥3 findings
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Increased cellularity
Small cells with high N/C ratio
Prominent nucleoli
Sheet-like, patternless growth
Spontaneous geographic necrosis
Atypical meningioma
Prominent nucleoli
Necrosis
Mitosis
MIB-1/Ki67
Counting
 Counting in highest MIB-1 labeling area (HL
method) vs. Randomly selected fields (RS
method)
 Focal accumulation of MIB-1(+) cells in
meningioma
• not likely to correlate with biologic aggressiveness
 RS method: a better predictor of recurrence and
tumor growth than HL method
Amatya Hum Pathol 2001;32:970
Nakasu Am J Surg Pathol 2001;25:472
PHH3
 Phospho-Histone H3
 Mitosis specific antibody
• only mitotic figure
 Useful to search mitosis
• in a clean background
• in glioma and meningioma
Atypical meningioma
Courtesy by Prof. Se Hoon Kim
P53 protein
 Any nuclear positivity is abnormal!
• 60% of all-grade astrocytomas
showing >5-10% p53 positivity
– A positive one: favoring Astrocytoma
 Useful
• Detection of Infiltrating astrocytoma nuclei
• at the edge of a diffuse astrocytoma
• D/Dx
• Reactive gliosis (p53-) vs. low-grade astrocytoma, gemistocytic astrocytoma
• Radiation effect (p53-) vs. recurrent astrocytoma
Diffuse astrocytoma vs. Gliosis
P53(+)
P53 protein
 Oligodendroglioma:
• Rarely / never staining with p53
• Strong & diffuse positive tumor
– unlikely to be oligodendroglioma
P53(-)
IDH1
 Isocitrate dehydrogenase (IDH) as an enzyme
 IDH1 mutations
• frequent (70%-80%) in grade II-III astrocytomas, oligo
dendrogliomas, oligoastrocytomas, secondary GBMs
 Over 90% of IDH1 mutations in diffuse gliomas occur at
a specific site
 R132H
 Base: Guanine -> Adenine within codon 132
 Amino acid: Arginine -> Histidine
IDH1
 mIDH1R132H
 Because of the consistent protein alteration, a monoclonal antibo
dy has been developed to the mutant protein
 장점: more sensitive than sequencing for identifying R13
2H mutant gliomas
 단점: Not detectable IDH2 and other IDH1 mutation
 D/Dx
• Gliosis (negative) vs. Diffuse glioma (positive)
Anaplastic oligodendroglioma
Courtesy by Prof. Se Hoon Kim
At tumor edge
IDH1(-)
No tumor involvement
IDH1(+)
Tumor involvement
At tumor edge
IDH1(+)
Tumor infiltrates the pia
Courtesy by Prof. Se Hoon Kim
IDH1
 Grade I and non-infiltrative glioma
• tend to not show IDH mutations: IDH1(-)
• not overexpress P53 gene product: p53(-)
• not overexpress EGFR : EGFR(-)
 In contrast, high-grade and infiltrative glioma ten
d to overexpress at least one of these.
INI1 (BAF47)
 Nuclear staining
 INI1 gene product
• Normally present in all cells
• Absent in malignant rhabdoid tumors
– due to gene deletion
 ATRT (atypical teratoid rhabdoid tumors)
• “negative” staining in tumor cells
• Retained positivity in vascular endothelium
ATRT
INI-1(-)
Internal control
Courtesy by Prof. Se Hoon Kim
Claudin-1
 Integral structural protein of tight junctions
 Marker of perineurial cells
 Positive
• Perineurium of normal nerves
 Negative
• Schwann cells, fat, and smooth muscle
 More reliable marker than EMA
• to distinguish perineurioma from mimics
Claudin-1
 Fibroblastic meningioma vs. Schwannoma
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EMA
Cerebellopontine angle
intradural, extramedullary regions of spinal canal
EMA: Faint and/or focal immunoreactivity
Overlap in S100 and EMA reactivity
S-100
Claudin-1 in meningioma
Granular staining in cytoplasm
감사합니다! ^^
“하루에 1시간씩 1년간 투자하면,
무엇이든 잘 할 수 있다”
“모차르트도 1만시간의 연습을 통
해 이루어졌다”
- 김난도 ‘아프니까 청춘이다’ 중에서

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