Practical approach to the use of immunohistochemical markers used in neuropathology

Hannes Vogel, M.D. Director of Neuropathology Stanford University

Educational goals
Understand the role of IHC in neuropathology in:
Making the correct diagnosis Providing clinically useful information

Whats new in neuropathology IHC

Tumors
Neuroepithelial tumors
Astrocytomas Oligodendrogliomas and mixed gliomas Ependymomas Choroid plexus tumors Glioneuronal tumors Neuroblastic tumors Pineal parenchymal tumors Embryonal tumors

More tumors
Meningiomas Peripheral nerve tumors Lymphomas Germ cell tumors Sellar tumors Metastatic tumors

The basics
Glial differentiation
GFAP (glial fibrillary acidic protein)
Astrocytes Gliofibrillary oligodendrocytes Ependymocytes

S-100
Gliomas Chordomas Melanocytic tumors Schwannomas

The basics
Neuronal differentiation
Neurofilament Cytoplasmic and cell processes Synaptophysin Cytoplasmic and cell surface Neu-N Nuclear

The basics
EMA
Meningiomas Ependymomas

CD 20, CD3, CD138, etc.

Lymphomas, plasmacytomas Microglia and macrophages; CD68 or CD163

CD31 and CD34

SFT (solitary fibrous tumor) Hemangiopericytoma

Cytokeratins CK 20, CK7, TTF-1, etc.

Metastatic carcinomas

The basics
OCT4, CD30, alpha fetoprotein, beta HCG
Germ cell tumors

Inhibin-A
Capillary hemangioblastoma

MIB-1
Proliferative index

SV40
PML (progressive multifocal leukoencephalopathy)

Herpes Toxoplasmosis

The basics
GFAP S-100 Neurofilament Synaptophysin EMA CD 20, CD3, CD138, CD68 or CD163, etc. CD31 and CD34 Cytokeratins CK 20, CK7, TTF-1, etc. OCT4, CD30, alpha fetoprotein, beta HCG Inhibin-A MIB-1 SV40 Herpes Toxoplasmosis

Astrocytomas and glioblastoma

GFAP of limited usefulness (Vogel, unpublished) Highlights perivascular processes in ependymoma ?Exclude other diagnoses i.e. metastasis ?Coarse glial processes in neoplastic astrocytes vs. delicate in reactive Gliosarcomas are GFAP negative in sarcomatous regions

Gliosarcoma

Gliosarcoma GFAP

Gliosarcoma - reticulin

Differential dx: CNS clear cell tumors

Oligodendroglioma Neurocytoma Clear cell ependymoma Clear cell meningioma DNT (dysembryoplastic neuroepithelial tumor) Metastasis i.e. clear cell RCC, neuroendocrine tumors

Differential dx of a clear cell tumor

Oligodendroglioma
GFAP, S-100 positive 1p 19q co-deletion by FISH

Neurocytoma
Synaptophysin positive, most GFAP negative

Clear cell ependymoma

GFAP positive, focally EMA positive

Clear cell meningioma

GFAP negative, EMA positive

Oligodendroglioma 1p19q co-deleted

Oligodendroglioma 1p19q co-deleted - GFAP

Differential dx of a clear cell tumor

Oligodendroglioma
GFAP, S-100 positive 1p 19q co-deletion by FISH

Neurocytoma
Synaptophysin positive, most GFAP negative

Clear cell ependymoma

GFAP positive, focally EMA positive

Clear cell meningioma

GFAP negative, EMA positive

Central neurocytoma

Central neurocytoma - synaptophysin

Central neurocytoma

Central neurocytoma

Differential dx of a clear cell tumor

Oligodendroglioma
GFAP, S-100 positive 1p 19q co-deletion by FISH

Neurocytoma
Synaptophysin positive, most GFAP negative

Clear cell ependymoma

GFAP positive, focally EMA positive

Clear cell meningioma

GFAP negative, EMA positive

Differential dx of a clear cell tumor

Oligodendroglioma
GFAP, S-100 positive 1p 19q co-deletion by FISH

Neurocytoma
Synaptophysin positive, most GFAP negative

Clear cell ependymoma

GFAP positive, focally EMA positive

Clear cell meningioma

GFAP negative, EMA positive

clear cell meningioma h&e

Clear cell meningioma WHO Grade II

Clear cell meningioma - EMA

Differential dx of a clear cell tumor

DNT (dysembryoplastic neuroepithelial tumor)
Rely on histology

Choroid plexus carcinoma

May need EM

Metastasis i.e. neuroendocrine tumors, clear cell RCC

Cytokeratins Chromogranin, other neuroendocrine markers

Dysembryoplastic neuroepithelial tumor (DNET) WHO Grade I

Dysembryoplastic neuroepithelial tumor (DNET) WHO Grade I

Differential dx of a clear cell tumor

DNT (dysembryoplastic neuroepithelial tumor)
Rely on histology

Choroid plexus carcinoma

May need EM

Metastasis i.e. neuroendocrine tumors, clear cell RCC

Cytokeratins Chromogranin, other neuroendocrine markers

Differential dx of a clear cell tumor

Beware of the greatest imitator of a CNS clear cell tumor! Pilocytic astrocytoma

Pilocytic astrocytoma mistaken as oligodendroglioma

Tumors
Neuroepithelial tumors
Astrocytomas Oligodendrogliomas and mixed gliomas Ependymomas Choroid plexus tumors Glioneuronal tumors Neuroblastic tumors Pineal parenchymal tumors Embryonal tumors

Oligoastrocytoma
Dx rests largely upon nuclear morphology
Oligodendrocytes: round, minigemistocytes Astrocytes: angular, hyperchromatic

GFAP brings out the neoplastic astrocytic component

Mixed oligoastrocytoma WHO Grade II

Mixed oligoastrocytoma WHO Grade II

Mixed oligoastrocytoma WHO Grade II GFAP

Tumors
Neuroepithelial tumors
Astrocytomas Oligodendrogliomas and mixed gliomas Ependymomas Choroid plexus tumors Glioneuronal tumors Neuroblastic tumors Pineal parenchymal tumors Embryonal tumors

Ependymoma
Clear cell and tanycytic variants are not obvious ependymomas Some ependymomas are cortical GFAP positive EMA: dot-like cytoplasmic positivity

Ependymoma WHO Grade II

Ependymoma WHO Grade II

Ependymoma WHO Grade II GFAP

Ependymoma WHO Grade II EMA

Tumors
Neuroepithelial tumors
Astrocytomas Oligodendrogliomas and mixed gliomas Ependymomas Choroid plexus tumors Glioneuronal tumors Neuroblastic tumors Pineal parenchymal tumors Embryonal tumors

Choroid plexus tumors

Transthyretin and S-100 positive, but less frequent in choroid plexus carcinomas GFAP positive in 25-55% of papillomas, 20% of carcinomas Synaptophysin positive Beware of other papillary tumors
Meningioma Ependymoma Metastases

Choroid plexus carcinoma WHO Grade III

Transthyretin

Synaptophysin

Tumors
Neuroepithelial tumors
Astrocytomas Oligodendrogliomas and mixed gliomas Ependymomas Choroid plexus tumors Glioneuronal tumors Neuroblastic tumors Pineal parenchymal tumors Embryonal tumors

Glioneuronal tumors
Gangliogliomas
Histology: dysplastic ganglion cells, EGBs (eosinophilic granular bodies), lymphocytic cuffing

DIG (desmoplastic infantile ganglioglioma)

Need to confirm glial (GFAP) and neuronal differentiation (NF, synaptophysin)

DNT
Oligodendroglioma-like cells: neu-N positive

Papillary or rosetted glioneuronal tumors

Neuropil islands positive for neuronal markers

Synaptophysin

GFAP

Neuroblastic tumors
Esthesioneuroblastoma, neuroblastoma Exclude:
Lymphoma - CDs SNUC (sinonasal undifferentiated carcinoma) Metastatic small cell carcinoma - cytokeratins Pituitary adenoma anterior pituitary markers Ewings and rhabdomyosarcoma

esthesioneuroblastoma h&e

Esthesioneuroblastoma

Esthesioneuroblastoma Synaptophysin

Esthesioneuroblastoma S-100

Embryonal tumors
Medulloblastoma
IHC not diagnostic IHC approaching utility in prognosis

Supratentorial PNET
Synaptophysin and GFAP positive

AT/RT
INI immunonegative

Atypical teratoid rhabdoid tumor (ATRT) WHO Grade IV BAF47/SNF5 Mab

Large cell medulloblastoma WHO Grade IV

Large cell medulloblastoma WHO Grade IV BAF47/SNF5 Mab

Meningiomas
EMA generally positive, but may be weak in:
Fibroblastic meningiomas Atypical meningiomas Malignant meningiomas

CEA positive in secretory meningiomas Beware of mimics:

SFT/hemangiopericytoma: CD31 and CD34 Schwannoma: nuclear S-100 positivity

Vascular tumors
Differential dx
Metastatic clear cell renal cell ca (CRCC): 70-90% are EMA and low-molecular-weight cytokeratin (CAM 5.2) positive Paraganglioma: chromogranin positive Angiomatous meningioma: EMA and vimentin positive SFT/hemangiopericytoma: CD31/34 positive Capillary hemangioma

Capillary hemangioblastoma
Inhibin A positive

Capillary hemangioblastoma WHO Grade I

Capillary hemangioblastoma WHO Grade I Inhibin

More tumors
Meningiomas Peripheral nerve tumors Lymphomas
B cell: CD20+ T-cell: CD3+; may appear reactive! EBV+ in immunocompromised MIB-1: prognostic?

Germ cell tumors Sellar tumors Metastatic tumors

CD20

CD3

Ki-67

CD20

CD3

cd3

Cd20

More tumors
Germ cell tumors
Germinoma: 100% OCT4+ vs. 92% PLAP+ Embryonal ca: CD30, PLAP, OCT4 Choriocarcinoma: HCG, +/- PLAP, cytokeratins Yolk sac tumor: AFP, +/- PLAP, cytokeratins Teratoma: AFP, cytokeratins

get pics from eyas

PLAP

OCT4

More tumors
Meningiomas Peripheral nerve tumors Lymphomas Germ cell tumors Sellar tumors
Adenomas cytokeratin and synaptophysin + Routine panel: prolactin, ACTH, GH, LH, FSH Beware of metastatic mimics of adenomas!

Metastatic tumors

More tumors
Meningiomas Peripheral nerve tumors Lymphomas Germ cell tumors Sellar tumors Metastatic tumors

Educational goals
Understand the role of IHC in neuropathology to:
Make the right diagnosis Provide clinically useful information

Whats new in neuropathology IHC

Survival by grade

Front Biosci. 2000 5:213-231

Grading gliomas
WHO Grade I a unique category
Pilocytic astrocytoma, ganglioglioma, DNT, choroid plexus papilloma, subependymoma, etc.

WHO Grade II nuclear atypia WHO Grade III add significant mitotic activity WHO Grade IV add vascular proliferation and/or necrosis

WHO and astrocytoma grading

The presence of a single mitosis in a resection specimen does not necessarily connote a worse behavior than that of grade II astrocytoma (might not be true for needle biopsies) High proliferation indices indicate more anaplastic, higher grade tumors, but both interobserver and interinstitutional variability precluded the MIB-1 index as a sole criterion used to distinguish grade II from grade III astrocytomas.

Proliferative index in gliomas

Grade 2: < 3mitoses or < 9% MIB-1+ cells/10 HPFs Grade 3: > 3mitoses or > 9% MIB-1+ cells/10 HPFs MIB-1 brings out infiltrating neoplastic astrocytes (Vogel, unpublished) Many MIB-1+ cells in pilocytic astrocytomas are endothelial Reactive gliosis generally less proliferative p53 not generally helpful in distinguishing reactive gliosis from neoplasia

Ki67

Ki67

Ki67

Ki67

Ki67

Grading meningiomas
Most are Grade 1 Atypical (Grade 2) Clear cell, chordoid 4-20 mitoses/10 hpf Other combined criteria (Perry et al, Cancer, 1999) Malignant (Grade 3) Rhabdoid, papillary >20 mitoses/10 hpf Carcinomatous or sarcomatous differentiation

Proliferation in meningiomas
% of MIB-1 positive cells has been reported to correlate well with histologic grade and recurrence Mean MIB-1 LI of benign, atypical, and anaplastic meningiomas was 1.5%, 8.1%, and 19.5%, respectively (Amatya, 2001) MIB-1 LI of 4.2% or more was strongly associated with decreased recurrence-free survival rate in gross, totally resected meningiomas (Perry, 1998) Use random fields, not highest staining areas

POP QUIZ!
Selected case(s) testing the use of a practical approach in the choice of immunohistochemical markers used in neuropathology.

Dx: Clear cell ependymoma

GFAP

GFAP

EMA

EMA

Vimentin

S-100

Dx: Myxoid (metaplastic) meningioma