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

1. specific cellular lineage
2. different subpopulation within one cell lineage
3. functional differences between the cells and even
4. 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

1. Intermediate filaments
2. Neuroendocrine and photoreceptor related proteins
3. Markers associated with suppressor genes, oncogenes and related gene products
4. Markers with predominant expression in CNS tumor
5. Markers of historical significance and
6. 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. 

1. 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.
2. 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.
3. 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.
4. 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

1. Placental alkaline phosphatase – PLAP
2. Alpha feto protein – AFP
3. Beta Human chorionic gonadotrophin – BHCG
4. Lectin-Dolichos Biflora

More over Cytokeratin, Epithelial membrane antigen (EMA) and Vimentin are often needed.

1. Placental alkaline phosphatase – PLAP usually seen in all geminomas and in some choriocarcinomas focally.
2. 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.
3. 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.
4. 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.