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Diagnostic
neuropathology has benefited tremendously in recent past
immunohistochemistry based techniques. Newer reagents are continuously
being developed against specific epitomes associated with stages of cell
lineage, cell cycle, oncogene and suppressor gene product or cell
activation. Use of these antibodies will help us to clarify the nature of
cellular maturation, tissue differentiation, tumor progression and
metastasis. The continuing refinement and evolution of reagents and
application of newer techniques result in revision of histological
classification. However, the all encompassing review of
immunohistochemistry is not possible here. We will concentrate on the
markers which are already well in use.
Before progressing further, a brief note about the mechanism
of immunohistochemistry seems indicated. Immunohistochemistry is an
amalgamation of immunology and histology. In immunohistochemistry, one
would know not only about the ability of a particular tissue to express an
antigen but also the exact cellular localization of the antigen. This
method employs different antibodies to distinguish the antigenic
differences between the cells. These antigenic differences can identify
- specific
cellular lineage
- different
subpopulation within one cell lineage
- functional
differences between the cells and even
- identify
infections.
The vast progress in the field of immunohistochemistry along
with the knowledge of cell and molecular biology allows the exploration
of the molecular phenotypes of the developing CNS tumors. A detailed
discussion of the methods is beyond the scope of this article. The reader
can refer to any standard text book for the same.
The most common methods
applied for immunohistochemistry are 1) Avidin-biotin method, and 2)
Peroxidase – antiperoxidase method.
The main key to this
excellent diagnostic modality is requisite antigen specific to particular
antibody.
The most important groups of antibodies are
- Intermediate filaments
- Neuroendocrine and
photoreceptor related proteins
- Markers associated with
suppressor genes, oncogenes and related gene products
- Markers with predominant
expression in CNS tumor
- Markers of historical
significance and
- Markers to detect cell
proliferation and cell death in CNS tumors
Intermediate filaments:
The intermediate filament proteins are intercellular
filaments measuring about 10mm in diameter. They form an important part
of the cytoskeleton.
There are 6 classes of intermediate filament proteins.
I & II -Keratin - identifies epithelium
-Vimentin -
identifies mesenchymal cells
o
primitive neuroepithelial cells
o
astrocytes
o
developing neuron
III -Glial fibrillary acidic protein
(GFAP) identifies
o
astrocytes
o
ependymal cells
o
nonmyelinating schwann cells
-Peripherin
identifies - neurones of CNS and peripheral nevous system
IV -Neuro-filament protein
identifies neurons and adrenal medullary low, medium and high cells
-Alpha – internexin identifies neurons
V -Lamin -
identifies nuclear membrane
VI -Nestin - identifies
- primitive neuroepithelium
o
developing astrocytes
o
developing neurons
o
schwann cells
Of these intermediate filaments, the * marked ones are used
for routine diagnostic immunohistochemistry on paraffin embedded
sections. The intermediate filaments not associated with central nervous
system are not mentioned here.
Nestin identifies the most
primitive neuroepithelium but also identifies many other embryonic
tissues. So it is not specific for CNS. Nestin expression is seen in
almost all GBMs and many melanomas (both primary and metastatic) but not
in any metastatic carcinoma.
Vimentin is most widely expressed antigen
in a variety of embryonic and mature tissues. Most mature neurons do not
express vimentin with two exceptions - (a) the horizontal cells of retina
and (b) the sensory neurons of olfactory epithelium.
Neuron specific intermediate filament proteins are
(a) neurofilament proteins, and (b) Alpha-internexin and peripherin.
The expression of these proteins signals the commitment of
primitive neuroepithelial cells to neuronal lineage.
(a) Neurofilament proteins (NFPs) are low,
intermediate and high molecular weight proteins which are expressed
exclusively by central and peripheral nervous system neurons and adrenal
medullary cells. The higher molecular weight NFPs appear with more
developed forms of neurons.
The tumors always expressing NFPs are
-
Ganglioneuroblastoma
Ganglioneuroma
Neurocytoma
PNET
Pinealblastoma
Extracranial neuronal tumours
The astrocytic tumors, other gliomas, ependymomas,
haemangioblastomas, pineocytomas and pituitary tumors are generally
negative for NFP expression.
(b) Alpha-internexin and peripherin: are two newly developed
neuron specific intermediate filaments. They are seen in CNS and
developing PNS and their tumors.
Glial fibrillary acidic protein(GFAP) is
a useful marker for astroglial cells. It is frequently co expressed with
vimentin and neurofilament in development and neoplasia of CNS. Although
it is a useful marker, there are certain disadvantages. Firstly it is not
specific for astroglial cells; secondly there is considerable interlaboratory
variation. Third problem is that the neoplastic astroglial cell and an
entrapped reactive one cannot be differentiated. Fourth, there is no
reliable correlation between the degree of GFAP expression and the tumor
anaplasia.
Cytokeratins (CK) are the
most complex intermediate filament is a marker of epithelium and its
neoplasms. In CNS, it differentiates poorly differentiated metastatic
carcinoma from primary high-grade tumour. It also is seen in – chordomas,
meningiomas, gliosarcomas, many astrocytomas, and oligodendrogliomas.
So, to differentiate between a primary and a secondary CNS
tumor, the antibody panel should have CK, NF as well as GFAP.
Neuroendocrine & photo-receptor related proteins:
Neuroendocrine cells share the features of neuron and
endocrine cells. These cells have features of neurons but their secretory
products are stored like an endocrine pattern rather than synaptic
pathways. These cells show argentophyllic and argyrophyllic properties,
dense-core neuro-secretory granules and APUD phenotype.
Two proteins are rather consistently expressed by the
neuroendocrine lineage along with neuron specific enolase. a)
Synaptophysin b) Chromogranin.
- Neuron
specific enolase
is one of the first markers for neuroendocrine system and neuron is
the Gamma-subunit of NSE. However the extensive cross reaction of
Gamma-subunit with the Beta-subunit of NSE and abundant expression
of Beta-subunit is many non-neuroendocrine cells limit the
advantages of this antibody.
- Synaptophysin is a major calcium – binding protein of
synaptic-vesicle membrane. It cross-reacts with other granule
associated proteins. It is demonstrated in Medulloblastomas,
neurocytomas, pineocytomas, ganglioglioms, ganglioneuromas and some
oligodendrogliomas. Many peripheral neuroendocrine tumours e.g.,
pheochromocytoma, carcinoid, small cell carcinoma of lung and GI
tract and pituitary adenomas also express this antigen.
- Chromogranin
A is expressed by
intravesicular matrix of dense-core vesicles of neuroendocrine
cells. Only ganglioglioma within CNS consistently expresses this
antigen. While most neuroendocrine tumors outside CNS express this
antigen.
- Proteins by photoreceptor cells: Retinal photoreceptors and
pinealocytes express I) Retinal S-antigen (arrestin) II)
Rod-opsin, and III)
Inter-photoreceptor retinoid – binding Protein.
These are expressed with variable intensity in
retinoblastomas and medulloblastoma.
Tumor suppressor genes, oncogenes & related gene
products:
(a) p-53 protein – is enclosed in tumour
suppressor gene located in chromosome 17p. It s thought to be one of the
earliest alteration in human astrocytoma progression. This mutated gene
can be immunohistochemistry detected within the nucleus of the cells.
Sometimes cytoplasmic positivity also is seen but always in association
with the nuclear positivity. The positivity is seen in astrocytomas,
mixed astrocytoma and oligodendroglioma and gliosarcoma. The intensity of
expression is proportional to the degree of malignancy. It is doubtful
whether the intensity of the p53 expression has any prognostic
significance.
(b) Ongodenes: C-myc and N-myc protein-MYC
Amplificate is rare in astrocytic tumors. But sometimes present in
medulloblastomas. However, accumulation of c-myc protein in the nuclei
appears a separate event and denotes disease progression in astrocytic
tumors. N-myc is seen in some medulloblastomas.
Nerve growth factor receptor:
Nerve growth factor is the first to be associated with
neuroectodermal tumours and one of the most extensively studied proteins.
It has low and high affinity embryonic and adult CNS, medulloblastomas,
other pediatric CNS tumour like neuroblastomas, ganglioneuroblastoma and
ganglioneuroma. While peripheral tumours do not show such definite positivity.
Most of the astrocytomas also are positive.
Platelet derived growth factor receptor:
It is the first cellular growth factor which corresponds to
a known viral oncogene. These are expressed by gliomas at a much higher
concentration than in normal brain. These could be made into the target
of immunotherapy.
Epidermal growth factor receptor:
This growth factor receptor is encoded on the EGFR
cellular oncogene on chromosome 7. It has been observed in various
gliomas and some tumors outside CNS. Most of the markers discussed above
are intracytoplasmic. Where as there are some cells surface markers
including various growth factors. The intracytoplasmic proteins are
useful for diagnostic purposes while the cell-surface proteins can have a
widespread therapeutic application in not so far future.
Germ cell tumor markers:
Germ cell tumors are not so rare in central nervous system.
The primordial germ cell disseminates most frequently in mediastinum and
diencephalopineal region. Thus, the germ cell tumor markers are used in
CNS tumors not so infrequently. The markers are
- Placental
alkaline phosphatase – PLAP
- Alpha
feto protein – AFP
- Beta
Human chorionic gonadotrophin – BHCG
- Lectin-Dolichos
Biflora
More over Cytokeratin, Epithelial membrane antigen (EMA) and
Vimentin are often needed.
- Placental alkaline
phosphatase – PLAP usually seen in all geminomas and in some
choriocarcinomas focally.
- Alpha feto protin AFP –
Germinomas are negative for AFP. Endodermal sinus tumour and
embryonal carcinoma show strong positivity. Even the CSF level of
this marker is high in these two tumours.
- Human Chorionic
Gonadotrophic – HCG: It is seen in choriocarcinoma. Even the CSF
level of HCG is high. Multinucleate syncytiotrophoblastic cells seen
in germinoma as well as embryonal carcinoma show HCG positivity.
- Dolichos Biflora – This
lectin is seen in embryonal carcinoma.
Angiotensin 1 converting enzyme can be seen in suprasellar
germinoma. Many CNS germicell tumours are cytokeratin, EMA and vimentin
positive in contrast to their gonadal counterparts.
Pituitary tumor markers:
Normally cells of pituitary gland secrete hormones, and,
thus can be identified by the specific markers.
Somatotroph cells secrete growth hormone (GH)
Mammosomatotroph cells secrete both GH and prolactin (PRL).
Lactotroph cells secrete PRL only.
Thyrotroph cells secrete TSH.
Corticotroph cells secrete ACTH along with B-endorphin
Melanocyte secreting hormone (MSH)
Gonadotrophs secrete FSH and LH
They secrete many other hormones and peptides. However,
these hormones e.g. GH, PRL, TSH, FSH, LH & ACTH are mainly used to
identify the type of cells of pituitary adenoma including the clinically
nonfunctioning adenomas. This forms the basis of diagnosis and therapy from
a clinical point of view. Other markers – like – development regulatory
protein Pit-1, Keratins, receptors like estrogen receptor and
transcription factor SFI are also being tried in different pituitary
adenomas and their exact role in the prognosis and management are being
tested.
Miscellaneous markers:
a) S-100 protein: This is first isolated from CNS in 1965.
It is localized in the cytoplasm and nucleus of astrocytes,
oligodendrocytes and schwann cells. Few neurons also have this protein.
Its use is rather limited by the vastness of its neural positivity. The
main use is in identifying MPNST from therapeutic application in out so
far future.
b) Leu T(HNK-1): Oligodendrocytes and schwann cells exhibit
cell membrane staining. But many tumors in CNS show variable positivity.
Many tumors outside CNS also shows positivity and thus restrict the usage
of this antibody.
Cell proliferation and cell death markers:
The growth of any malignant neoplasm depends on the balanc3e
between the cell proliferation and cell death.
The cell proliferation markers are –
Brd UL,
Ki67, PCNA, and
Anti DNA prolymerase alpha
Of these BrdUL, and Ki67 are more consistent and their
results can be correlated with each cycle. These two antibodies give a
fairly good idea about the cell cycle. These correlative well with tumor
grade and survival. Higher the value of these two antibodies, worse is
the prognosis (it is necessary to administer the BrdUL intravenously
before the operation to allow its incorporation into the DNA.
The cell death is assessed by the passive process of
necrosis and active processes of apoptosis. A good morphological staining
detects these two processed fairly well. However, immunohistochemical
staining with bc12 protein detects the well population, which is rather
immune to apoptosis. This Bc12 over expression is seen in low grade
gliomas but net in GBMs.
The diagnostic pitfalls:
The tumor markers are very important diagnostic tools, but
there may be many pitfalls which one should be aware of. The
interpretation of any immunohistochemistry results should always be done
in accordance with the morphology and proper clinical and radiological
correlation.
The current trend:
Iimmunohistochemistry is one of the most important tools of
diagnostic histopathology. But now more stress in on finding tumor
markers of prognostic significance. Survival in astrocytic gliomas is
closely related to WHO tumor grade. Within one tumor grade, especially in
grade II and III tumors, the clinical course is variable and can hardly
be predicted by histological criteria. Neovascularization is a
neuropathological hallmark in high grade gliomas and angiogenic factors
may play an important role in malignant tumor progression. Vascular
endothelial growth factor (VEGF) expression, which is considered to
represent the main angiogenic factor in astrocytic gliomas is being
investigated immunohistochemically. A strong correlation between VEGF
expression and survival has been reported. In a multifactorial analysis
VEGF expression was not found to be an independent prognostic factor in
astrocytic gliomas.
The future of immunohistochemistry is aimed at not only the
diagnosis and prognostication of the tumors but also being able to
comment upon the probable response to various chemotherapeutic agents.
Conclusion:
The advent of immunochemistry has added to the accuracy of
diagnostic neuropathology which has previously concentrated on tumor
morphology. Immune stains may not be used for the identification of tumor
cell differentiation, but also for the analysis of proliferative activity
and the expression of oncoproteins, growth factors and receptors which
may more accurately reflect malignant potential. The above gives an idea
of the main important tumor markers used for the primary CNS tumors. The
list also contains numerous lymphoma markers used for primary CNS
lymphomas. For metastatic diseases, the various markers are used to
identify the type and, if possible, the source of the metastatic disease.
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