Role of IHC in CNS Tumours
- IHC was originally described by Coons et al., in 1941.
- It’s basically amalgamation of immunology and histology and is based on the principle of localizing specific antigens in the tissues or cells based on antigen-antibody recognition.
- For the purpose of understanding, the IHC markers for the CNS tumours can be broadly divided into three groups –
- IHC markers used for diagnostic purpose,
- IHC markers used for prognostic purpose and
- Other IHC markers
- Some of these IHC markers are so helpful that they are considered as an integral part of WHO Classification of CNS Tumours.
Table: IHC markers for CNS tumours
|IHC markers used for diagnostic purpose||IHC markers used for prognostic purpose||Other IHC markers|
|Markers for glial tumours
|Cell Cycle/proliferation markers
|– IDH-1 & IDH-2
|Markers for neuronal tumours
– GFAP +/-
|Tumour suppressor gene/oncogene protein
– p53 tumour suppressor gene
– Retinoblastoma tumour suppressor gene (Rb)
– c-myc oncogene
|Markers for meningeal tumours
|Markers for choroid plexus tumours
|Markers for lymphoma
– T-cell and B-cell markers
|Markers for Schwann cell tumours
|Markers for germ cell tumours
|Markers for melanocytic tumours
– MART-1 (Melan-A)
– Microphthalmia transcription factor
|Markers for vascular origin
– Factor VIII
– Ulex Europeans
|Markers for pituitary tumours
|Markers for neuroendocrine tumours
|Markers for ATRT
Glial fibrillary acidic protein (GFAP)
- This antibody was 1st reported by Eng et al., and later described as a useful marker for astrocytes by Kleihues et al.
- It’s one of the major cytoplasmic intermediate filaments.
- It’s principal cytoskeletal constituent of astrocytes
- GFAP positivity is seen in –
- Astrocytes – normal, reactive and neoplastic
- Ependymal cells – developing, reactive and neoplastic
- Oligodendrocytes – developing and neoplastic
- Glial neoplasms can be differentiated from non-glial neoplasms by the former having GFAP positivity.
- GFAP is the only marker that can distinguish astrocytic tumours from non-glial tumours.
- Most of the astrocytic tumours show GFAP positivity except for protoplasmic astrocytoma WHO grade II (scant or absent GFAP immunoreactivity).
- As the WHO grade of a glial tumour increases, expression of GFAP decreases because the tumour tends to become poorly differentiated.
- In gliosarcomas, the glial component is GFAP-rich and reticulin poor, whereas the sarcomatous component is reticulin-rich and GFAP-negative.
- Glial neoplasms lack collagen, reticulin and fibronectin.
- Oligodendroglioma (ODG) show a variable positivity.
- GFAP reactivity is seen in well-differentiated type of ODGs such as minigemistocytes, gliofibrillary oligodendrocytes and intermixed reactive astrocytes in ODGs, WHO grade II.
- In myxopapillary ependymomas, GFAP positivity is consistently seen in perivascular tumour cells.
- These tumours are also positive for S-100 and vimentin and negative for CK and chromogranin.
- Therefore, they can be distinguished from their D/D, i.e., chordomas, chondrosarcomas, paragangliomas, and papillary adenocarcinomas.
- GFAP positivity is more prominent in pseudorosettes and variable in the ependymal rosettes, ependymal canals, and papillae in ependymoma grade II.
- Other markers which can be positive in ependymoma are S-100, CD99 and occasionally focal CK along with dot-like positivity for EMA.
- Extensive GFAP reactivity in ODG should prompt the search for an alternative diagnosis.
- Non-glial neoplasms in the CNS that show a focal GFAP positivity
- Central primitive neuroectodermal tumours (PNETs) – ependymal differentiation
- Classical medulloblastomas – glial differentiation in geographical areas
- Sometimes desmoplastic variant of medulloblastoma
- Glial component of ganglioglioma
- GFAP is particularly helpful in –
- Diagnosis of gliomas at unusual sites and gliomas with atypical histological findings (such as chordoid glioma and xanthoastrocytoma)
- Identification of glial components and glial differentiation in embryonal, neuronal and mixed glioneuronal tumours
- Differentiation of high-grade glial tumours with undifferentiated or squamoid appearance from metastatic carcinomas.
- Cytoplasmic intermediate filament protein.
- Positivity seen in cells of mesenchymal origin (fibroblasts, endothelial cells, vascular smooth muscle cells), tumours of epithelial or neural origin.
- It’s expressed in developing neurons and is not expressed in mature neurons except in the horizontal cells of retina and sensory neurons of olfactory epithelium.
- Vimentin is also used as a control to check the reliability of tissue for the IHC reaction.
- Vimentin & GFAP are seen in similar distribution in astrocytomas but vimentin staining is less prominent than GFAP.
- It’s usually positive in perineural region of astrocytes.
- During astrogenesis, it’s earlier than GFAP.
- Hence, vimentin positive cells may be GFAP negative.
- Its expression in astrocytoma indicates a lower degree of differentiation.
- It’s consistently seen in high-grade astrocytoma.
- All meningiomas show vimentin positivity.
Neurofilament protein (NFP)
- Intermediate filaments of the neurons and their processes.
- Three isoforms – neurofilament-low (NF-L), neurofilament-medium (NF-M) and neurofilament-high (NF-H)
- Their immunoreactivity is seen in tumours with neuronal differentiation, e.g. –
- Neurocytomas and
- NF-L and NF-M are usually seen in immature cells with neuronal differentiation while NF-H is associated with mature neuronal elements.
- State of phosphorylation of NFP determines the level of maturation.
- Heavily phosphorylated NF-H isoforms are expressed in mature neuronal differentiated tumours
- Low phosphorylated NF-H indicate an immature neuronal state of tumour
- Expression or distribution of this marker within the cells is also determined by its level of phosphorylation.
- Heavily phosphorylated NF-H is seen in axons
- Less phosphorylated NF-H is localized to perikaryal & dendrites.
- Other markers for neuronal differentiation –
- Microtubule-associated protein-2 – Role in microtubule assembly and stabilization, usually used in embryonal tumours showing a neuronal differentiation.
- Class III beta-tubulin
- Non-CNS tumours showing NFP positivity –
- Merkel cell carcinomas of the skin
- Endocrine tumours of the pancreas
- Carcinoid tumours
- Parathyroid tumours
- Water-soluble intermediate filament present intracellularly in almost all epithelia.
- CK can be divided into at least 20 subtypes depending on their molecular weight.
- In CNS, the primary utility of CK study is in differentiation between metastatic carcinomas (which are CK-positive) from the primary CNS tumours (which are CK-negative).
- Sometimes, primary CNS tumours also show CK positivity.
- Gliomas – CK positivity 60-80% (due to non-specific reactivity)
- Adenoid (glandular) or squamous metaplasia (more commonly seen in gliosarcoma than GBM) also show CK positivity.
- Choroid plexus tumours – CK positive.
- Difficult to distinguish them from metastatic papillary tumours.
- HEA125 & BerEp4 (both positive in metastatic papillary carcinoma) help in distinguishing the two.
- Synaptophysin – positive in choroid plexus papillomas and carcinomas, negative in metastatic papillary carcinomas.
- Meningiomas, especially meningotheliomatous, psammomatous and secretory subtypes may also show occasional CK positivity.
- Intermediate filament
- Has the largest molecular weight
- Term “Nestin” is derived from the location where it’s present, i.e., neural stem cells protein.
- In the developing nervous system, it’s expressed in the primitive neuroepithelial cells.
- As the fetal development proceeds, nestin is replaced by –
- Other NFPs in those cells which are committed to differentiate into neuronal lineage, and by
- GFAP in those cells which are committed to differentiate into astrocytes.
- By the end of gestation, nestin gets almost completely eliminated and is expressed only in endothelial cells of the mature human CNS & Schwann cells of the peripheral nervous system.
- Hence, nestin is regarded as a marker of embryonic CNS neuroectodermal cells and immature CNS precursor cells not yet committed to a neuronal or astrocytic lineage.
- Limitation – It’s not specific and is positive in many other tumours such as gliomas, medulloblastomas and meningiomas.
- Nestin positivity is seen not only in tumour cells but also in endothelial cells of gliomas.
- It is not expressed in metastatic carcinomas.
- Transmembrane glycoprotein
- It’s expressed in the normal, reactive and neoplastic cells of the neuroectodermal and neuroendocrine types.
- Most commonly used IHC marker for neuronal differentiated tumour.
- In the early stages of neurogenesis, synaptophysin is negative, but other markers such as Class III beta-tubulin isotype are expressed.
- In the white matter of the normal brain, it doesn’t stain the neuropil, while in grey matter, it stains neuropil.
- Synaptophysin is positive in CNS tumours with neuronal differentiation such as –
- Pleomorphic xanthoastrocytoma,
- Subependymal giant cell astrocytoma and is also positive in neuroendocrine tumours.
- It’s positive in almost all types of neuroendocrine tumours such as a paraganglioma.
- It’s named “S-100” because of its solubility in 100% saturated ammonium sulfate at a neutral pH.
- It’s an acidic, dimeric calcium-binding protein.
- In the normal brain, its immunoreactivity is seen in glial cells, i.e., astrocytes, oligodendrocytes, ependymal cells, Schwann cells and melanocytes.
- It’s also demonstrated in non-glial cells such as chondrocytes, myoepithelial cells, adipocytes and in the tumours derived from them.
- CNS tumours showing S-100 positivity are –
- Melanocytic tumours
- Peripheral nerve sheath tumours
- It’s expressed in both nuclei and cytoplasm, but nuclear staining is more specific.
- S-100 along with EMA is helpful in differentiating schwannoma from fibrous meningioma.
- Schwannoma – strong, diffuse positivity to S-100, EMA negative
- Meningioma – 20% positivity for S-100, EMA positive
Epithelial Membrane Antigen (EMA)
- Glycoprotein isolated from human milk fat globule.
- Marker of normal and neoplastic epithelium and perineural cells.
- It’s also expressed in variety of mesenchymal neoplasms, mesotheliomas & lymphomas.
- In CNS, it’s characteristically seen in –
- Metastatic carcinomas
- EMA helps in differentiating between meningiomas (EMA positive) and hemangiopericytomas (EMA negative)
- Meningioma vs metastatic carcinoma – Both EMA positive
- Extend the IHC panel for metastatic carcinoma depending upon the morphology as well as clinical profile, e.g., staining for CK 5/6, thyroid transcription factor-1, p63 etc.
- Metastatic RCC in the brain (EMA positive) vs. hemangioblastoma (in the Von Hippel-Lindau syndrome, EMA negative)
- Schwannoma (EMA negative) vs. meningiomas (EMA positive)
- Dot like positivity of EMA staining is seen in cases of low-grade ependymomas.
- Chordoma (EMA positive) vs. Chondrosarcoma (EMA negative)
- Differential diagnosis of round cell tumors
- Lymphoma – LCA/CD45RB positive
- All normal leukocytes except plasma cells (which are negative or variably positive) are also LCA positive; therefore, LCA positivity cannot differentiate normal lymphocytes from neoplastic lymphocytes.
- For the presence of a T cell lymphoma or a B-cell lymphoma, an IHC panel is performed.
- Most of the primary CNS lymphomas are of B-cell type.
- The recommended first-line antibodies for B-cell lineage are CD20 (or CD79a, PAX5), and for T-lineage are CD3 (or CD2).
- Some lymphomas – LCA negative, especially lymphoblastic lymphomas and anaplastic large cell lymphomas.
- Plasma cell and plasmablastic neoplasms – negative or variable positivity for LCA.
- Reed–Sternberg cells seen in the classic Hodgkin’s lymphoma are typically CD45RB/LCA negative.
- Langerhans’s histiocytosis (LCH) –
- CD1a (most specific) – membrane bound antigen linked with macroglobulin.
- S-100 – Positive
- Primary melanotic neoplasms of the CNS are thought to arise from leptomeningeal melanocytes, and include –
- Diffuse meningeal melanocytosis,
- Rare meningeal melanocytoma, and
- Primary malignant melanoma
- The most common malignant melanocytic tumor of the CNS is metastatic melanoma.
- Most melanocytic neoplasms show a diffuse immunoreactivity with anti-melanosomal antibodies such as human melanoma black-45 (HMB-45) or MART-1 (Melan-A) and microphthalmia transcription factor.
- They also show S-100 positivity.
- Sometimes, primary brain tumors such as schwannomas, astrocytomas, ependymomas, medulloblastomas, and paragangliomas also demonstrate melanin and hence HMB-45 positivity.
- However, all these tumors do not show diffuse HMB-45 positivity like the melanocytic neoplasms of CNS.
Markers for pituitary tumor
- The cells of pituitary gland secrete various hormones such as prolactin, growth hormone, and adrenocorticotropic hormone.
- IHC marker studies of these hormones are mainly used to identify the characteristic type of cells of the pituitary adenoma.
- This forms the basis for the diagnosis and therapy of a pituitary tumor.
Germ cell markers (oncofetal markers)
- These markers are helpful in the diagnosis of germ cell tumors that may be either primary or metastatic to the CNS.
Marker for atypical teratoid/rhabdoid tumor
- INI-1/SMARCB-1 protein (coded on chromosome 22q) – seen in the nuclei of all the normal cells.
- Mutation of the INI-1/SMARCB-1 gene leads to loss of its expression in atypical teratoid/rhabdoid tumor (ATRT) cells.
- This is considered a specific and sensitive marker for ATRT.
- Other markers that are consistently seen in rhabdoid cells are EMA and vimentin.
- Less frequently – SMA. GFAP, NFP, keratin, and synaptophysin positive.
Cell proliferation markers
- Various antibodies used in IHC are available that evaluate the ongoing proliferation in the tumors.
- These antibodies correlate well with the tumor grade and survival.
- Higher the value of these antibodies, worse is the prognosis.
- An ideal proliferating marker is the one which can detect all the active parts of the proliferative cell cycle, i.e. G1, S, G2, and mitosis.
- Important cell proliferating markers are Mitotic figure, Molecular immunology borstel-1, Ki-67, Phosphohistone-3.
- On H&E staining, proliferative activity is determined by counting the number of mitoses seen
- Denote only the M phase of the cell cycle under light microscope.
Molecular immunology borstel-1 and Ki-67
- Molecular immunology borstel-1 (MIB-1) antibody is an improved version of Ki-67, which correlates best with the actual cellular proliferation.
- MIB-1 – Recognizes an antigen expressed in all the active phases of cell cycle, i.e. G1, S, G2, and M in paraffin-embedded tissue.
- It helps in measuring the growth rate of the tumors and hence indicates how aggressive the tumor is.
- It is used as a prognostic marker, and correlates with the prognosis, including time to recurrence and survival and also correlates well with the histological grade of the tumor.
- Phosphohistone-3 antibodies specifically target the phosphorylated version of core histone protein H3.
- Phosphorylation occurs almost exclusively during mitosis but is not exhibited during apoptosis.
- Tumor suppresser gene.
- It is also called as tumor protein 53 or TP53.
- It is coded by a tumor suppressor gene (p53 or TP53 gene) on chromosome 17p13.1.
- It is often considered the guardian of the cell because it is mainly responsible for the genomic stability of the cell.
- Marker of astrocytic tumor.
- Low-grade gliomas carrying this positivity are associated with a shorter survival and a shorter time interval to progress to high-grade gliomas.
- Secondary GBMs commonly show p53 mutations while primary GBMs rarely show p53 mutations.
- p53 mutation is not seen in an ODG, ependymoma, medulloblastoma, and pineocytoma/pineoblastoma.
Epidermal growth factor receptor
- The epidermal growth factor receptor (EGFR) gene at 7p12, amplified and overexpressed gene in GBM
- The prognostic value of EGFR amplifications and mutations, especially the EGFRvIII mutation, is controversial.
- EGFR immunopositivity can be variable, and there might be discrepancies between EGFR amplification as determined by fluorescent in situ hybridization and IHC.
Isocitrate dehydrogenase-1 and -2
- Isocitrate dehydrogenases (IDHs) are the enzymes that are involved in tricarboxylic acid cycle.
- Decarboxylate isocitrate to α-ketoglutarate with the production of NADH and/or NADPH.
- When IDH gene is mutated, there is a reduction of α-ketoglutarate to 2-hydroxyglutarate, which is increased to about 10–100-fold in mutant gliomas.
- IDH-1 mutation is present in
- WHO Grades II & III diffuse gliomas,
- Anaplastic ODGs and
- Mixed oligoastrocytomas
- Well secondary GBM
- IDH-1 mutation is rare in primary GBM, pilocytic astrocytomas.
- IDH-1 is absent in ependymomas
- IDH-2 mutation is seen in a smaller proportion of gliomas, and that too mainly in oligodendroglial tumors.
- IHC using IDH-1 R132H mutation-specific antibody detects IDH-1 mutation.
- However, this method can miss about 10% of gliomas carrying an IDH-1 mutation and all gliomas with an IDH-2 mutation.
- Subsequent genetic analysis is recommended in the cases associated with a negative or inconclusive IDH immunostaining results.
- This antibody also helps in differentiating gliomas from reactive gliosis where it is immune-negative.
Alpha-thalassemia/mental retardation syndrome X-linked
- Similar to INI-1/SMARCB-1 protein in ATRT, α-thalassemia/mental retardation syndrome X-linked (ATRX) protein is also seen in the nuclei of all normal cells.
- Mutation of ATRX gene leads to loss of its expression in tumor cells.
- ATRX mutation is considered as a specific marker for astrocytic lineage including oligoastrocytomas and is thought to be mutually exclusive for the 1p19q co-deletion seen in an ODG.
- Gliomas have been divided into three prognostic groups based on the mutation in the ATRX gene, IDH-1 gene, and 1p/19q co-deletion.
- Tumors with IDH mutation and 1p/19q co-deletion but no mutation of the ATRX gene are classified as ODGs and oligoastrocytomas and have the best prognosis.
- The second group involving mutation of both the IDH and ATRX genes but no co-deletion of 1p/19q, are usually astrocytomas or oligoastrocytomas having prognosis intermediate between the two.
- The third group of tumors shows no IDH mutation. This group has a poor prognosis and behaves like a GBM.
- BRAF V600E mutation may be seen by IHC studies in pleomorphic xanthoastrocytoma or some cases of ganglioglioma.
- But, it cannot distinguish between pilocytic astrocytomas and low-grade gliomas.
- This mutation may be unfavourable.
Limitations of IHC Markers
- Most antigens are not restricted to one type of tumor.
- The amount of antigen present in the tumor is variable.
- The antigenic phenotype of tumor cells, as delineated by IHC and immunoreactivity of antibodies, are nonspecific.
- Moreover, often multiple IHC markers are used for the diagnosis of one tumor, which increases the cost.
- Hence, the interpretation of any IHC result should always be done in accordance with the morphology of the tumor and following proper clinical and radiological correlation.
Summarized IHC of common CNS tumors –
Simplified algorithm for classification of the diffuse gliomas based on histological and genetic features (WHO 2016)–