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 » Introduction
 »  Role of Immunohi...
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NI FEATURE: THE QUEST - COMMENTARY
Year : 2016  |  Volume : 64  |  Issue : 3  |  Page : 502-512

Role of immunohistochemistry in the diagnosis of central nervous system tumors


Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Date of Web Publication3-May-2016

Correspondence Address:
Dr. Sushila Jaiswal
Department of Pathology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Rae Bareli Road, Lucknow - 226 014, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0028-3886.181547

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 » Abstract 

Although the conventional hematoxylin and eosin (H and E) staining is vital for the histological diagnosis of lesions, the role of immunohistochemistry (IHC) is undeniable in surgical pathology. Morphology, which looks more or less similar on H and E staining, can be further differentiated by merely doing IHC. This, in turn, not only helps in rendering a definitive diagnosis but also helps in the selection of appropriate therapy for the individual patient. Thus, IHC has become an integral part of the armamentarium of neuropathology.


Keywords: Central nervous system tumor; immunohistochemistry; pathology


How to cite this article:
Jaiswal S. Role of immunohistochemistry in the diagnosis of central nervous system tumors. Neurol India 2016;64:502-12

How to cite this URL:
Jaiswal S. Role of immunohistochemistry in the diagnosis of central nervous system tumors. Neurol India [serial online] 2016 [cited 2020 Jan 29];64:502-12. Available from: http://www.neurologyindia.com/text.asp?2016/64/3/502/181547



 » Introduction Top


Immunohistochemistry (IHC) was originally described by Coons et al., in 1941. They developed an immunofluorescence technique to detect corresponding antigens in frozen tissue sections.[1] However, this method became popular in the 1990s in surgical pathology.[2] It is basically an amalgamation of immunology and histology and is based on the principle of localizing specific antigens in tissues or cells based on antigen-antibody recognition. It seeks to exploit the specificity provided by binding of an antibody with the corresponding antigen at the light microscopic level.

In IHC, one should have knowledge not only of the ability of a specific tissue to express an antigen but also of the exact localization of the antigen within the cell. Hence, different antibodies are used to distinguish the antigenic differences between the cells. The most commonly applied methods in IHC are the avidin-biotin method and the peroxidase anti-peroxidase immune complex method. The aim of the current article is to present an overview of different antibodies that are used routinely in IHC for the diagnosis of central nervous system (CNS) tumors.


 » Role of Immunohistochemistry in Neuropathology Top


In the last 50 years, IHC has tremendously revolutionized the field of diagnostic pathology. For example, apart from its cost effectiveness, it can also be applied to the routinely processed paraffin-embedded tissues even if the latter has been stored for long periods of time. Often, pathologists encounter difficulties in the interpretation of the tiny biopsies particularly with prominent artifacts. In these cases especially, IHC studies often come to the rescue by highlighting the cell type. For example, a diagnosis of carcinoma can be rendered on a severely crushed needle biopsy with the help of IHC study of cytokeratin (CK) whereas, in the past, pathologists would have considered it “biopsy inadequate for diagnosis.” The diagnosis and further classification of lymphomas, even on a small needle biopsy, has become much easier these days with the availability of IHC study of a broad spectrum of leukocyte antibodies reactive in paraffin sections. It would not be inappropriate to state that, if used wisely by a pathologist, IHC can, to some extent, compensate for his/her lack of sufficient experience and skills in morphological interpretation. For example, a biopsy which might otherwise have been interpreted as “suspicious of malignancy” can be given a definitive diagnosis if proper IHC studies can unequivocally demonstrate the neoplastic cells.

Recently, our knowledge regarding the genetics of central nervous system (CNS) tumors has expanded; hence, newer antibodies or molecular markers, which can be used in IHC, are continuously being developed. Some of these tumor markers have diagnostic importance while others are useful for prognostication of patient survival and therapeutic response. Some of these markers are so helpful that they are going to be considered as an integral part of WHO classification of CNS tumors. These antibodies or molecular markers, in addition, help to clarify the nature of cellular maturation, tissue differentiation, and tumor progression.

For the purpose of understanding, the IHC markers for CNS tumors can be broadly divided into three groups- (1) IHC markers used for diagnostic purpose, (2) IHC markers used for prognostic purpose, and (3) other IHC markers [Table 1].
Table 1: Immunohistochemistry markers for central nervous system tumors

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[Table 1] shows a large number of IHC markers described in the literature for various tumors. Many IHC markers which are less important have not been mentioned in this table. It is not possible to discuss each and every marker in this article; hence, only those IHC markers which are commonly used in routine practice are briefly described below.

Intermediate filament proteins

The cytoskeleton of a cell is composed of actin filaments, microtubules, and intermediate filaments. Intermediate filaments help in the structural integrity of the cell and provide mechanical support to the plasma membrane. They are divided into six classes, and their characteristics and distributions are listed in [Table 2].
Table 2: Characteristics and distributions of various major types of intermediate filaments

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All intermediate filament proteins (IFPs) described in the [Table 2] are not important. Only those which are commonly used in routine diagnostic IHC on paraffin-embedded sections are described below.

Glial fibrillary acidic protein

This antibody was first reported by Eng et al.,[3] and was later described as a useful marker for astrocytes by Kleihues et al.[4] It is one of the major cytoplasmic intermediate filaments and is the principal cytoskeletal constituent of astrocytes.[5],[6] Glial fibrillary acidic protein (GFAP) positivity is seen not only in normal, reactive, and neoplastic astrocytes but also in developing, reactive, and neoplastic ependymal cells as well as developing and neoplastic oligodendrocytes.[7] Glial neoplasms can be differentiated from non-glial neoplasms by the former having GFAP positivity. GFAP is the only marker that can distinguish astrocytic tumors from nonglial tumors.[8],[9],[10] Most of the astrocytic tumors show GFAP positivity except for protoplasmic astrocytoma WHO Grade II where GFAP immunoreactivity is either scant or absent. It is also notable that as the WHO grade of a glial tumor increases, expression of GFAP decreases because the tumor tends to become poorly differentiated. That is why GFAP positive cells are lesser in number and focal in anaplastic astrocytomas and glioblastomas. In gliosarcomas, the glial component is GFAP-rich and reticulin poor whereas the sarcomatous component is reticulin-rich and GFAP-negative [Figure 1]. Glial neoplasms lack collagen, reticulin, and fibronectin.[11],[12]
Figure 1: Photomicrograph (H and E, ×200) showing a gliosarcoma disposed in sheets with pleomorphic cells having anisomorphic nuclei, frequent mitosis, areas of spindle tumor cells (a). IHC, ×200 showing GFAP positive tumor cells in a glial component and interspersed GFAP negative tumor cells

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Oligodendrogliomas (ODG) show a variable GFAP positivity.[8] In fact, for both ODGs and ependymomas, there is no specific and sensitive IHC marker especially related to their anaplastic variants. 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 tumor cells. These tumors are also positive for S-100 and vimentin and negative for CK and chromogranin. Hence, they can be distinguished from their differentials, i.e., chordomas, chondrosarcomas, paragangliomas, and papillary adenocarcinomas by having GFAP positivity and CK and chromgranin negativity. GFAP positivity is more prominent in pseudorosettes and variable in the ependymal rosettes, ependymal canals, and papillae in ependymoma grade II. Other markers that can be positive in ependymoma are S-100, CD99, and occasionally focal CK along with dot-like positivity of epithelial membrane antigen (EMA). EMA positivity is not specific for an ependymoma as it can also be seen in glioblastoma multiforme (GBM). A grade III anaplastic ependymoma shows the same IHC panel, but reactivity for GFAP is reduced in anaplastic cells, while in a ODG, it is variable. It is worth noting that an 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 are central primitive neuroectodermal tumors (PNETs).[13],[14],[15] Ependymal differentiation in PNETs, not picked up by light microscopy, may be revealed by the GFAP study. In classical medulloblastomas, glial differentiation in geographical areas usually shows GFAP positivity. Sometimes desmoplastic variant of a medulloblastoma displays GFAP reactivity in a reticulated pattern in “pale islands.” GFAP is also positive in the glial component of a ganglioglioma. GFAP positivity represents reactive entrapped astrocytes within the tumor rather than glial differentiation.

To summarize, GFAP study is particularly helpful in the diagnosis of gliomas at unusual sites and gliomas with atypical histological findings (such as a chordoid glioma and a xanthoastrocytoma); identification of glial components and glial differentiation in embryonal, neuronal, and mixed glio-neuronal tumors; and, the differentiation of high-grade glial tumors with undifferentiated or squamoid appearance from metastatic carcinomas.

There are certain limitations of GFAP. For example, its expression is not totally specific for glial cells. It has also been noted to be positive in schwannomas, choroid plexus tumors, and mixed tumors of salivary and sweat glands. Sometimes, the distinction between GFAP-positive neoplastic astrocytes and GFAP-positive reactive astrocytes becomes difficult.

Vimentin

Vimentin is another cytoplasmic intermediate filament protein. It is notoriously nonspecific, and its positivity is seen not only in the cells of mesenchymal origin, for example, fibroblasts, endothelial cells, and vascular smooth muscle cells but also in tumors of epithelial or neural origin. It is expressed in developing neurons and is not expressed in the mature neurons except in the horizontal cells of retina and the sensory neurons of olfactory epithelium. Apart from this, vimentin is also used as a control to check the reliability of tissue for the IHC reaction. Vimentin and GFAP are seen in a similar distribution in astrocytomas but vimentin staining is less prominent than GFAP. It is usually positive in the perinuclear region of the astrocytes. During astrogenesis, it is expressed earlier than GFAP. Hence, vimentin positive cells may be GFAP negative. Its expression in astrocytoma indicates a lower degree of differentiation. Moreover, it is consistently seen in high-grade astrocytomas. In GBMs, it has an inverse relationship with GFAP immunoreactivity. Its positivity in a GBM does not indicate stromal metaplasia or gliosarcomatous transformation. All meningiomas show vimentin positivity [Figure 2].[5],[8],[16]
Figure 2: Photomicrograph (H and E, ×200) showing a meningioma disposed in whorls and sheets with tumor cells displaying indistinct cellular boundaries, round to oval nuclei and finely stippled chromatin (a). IHC showing tumor cells positive for EMA (b: ×400), S-100 (c: ×200), and vimentin (d: ×200)

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Neurofilament protein

Neurofilament proteins (NFP) are the intermediate filaments of the neurons and their processes. They have three isoforms including the neurofilament-low (NF-L), neurofilament-medium (NF-M), and neurofilament-high (NF-H) isoforms having a molecular weight of 62 kD, 102 kD, and 110 kD, respectively. Each of these isoforms may further be phosphorylated or nonphosphorylated.[7] Their immunoreactivity is seen in tumors with neuronal differentiation, for example (e.g.), medulloblastomas, neuroblastomas, gangliogliomas, pineocytomas, neurocytomas, and retinoblastomas. NF-L and NF-M are usually seen in immature cells with neuronal differentiation while NF-H is associated with mature neuronal elements.[16]

The state of phosphorylation of NFPs, which in turn depends on the type of fixation, also determines the level of maturation in which they will be expressed. For example, heavily phosphorylated NF-H isoforms are expressed in mature neuronal differentiated tumors while, on the other hand, low phosphorylated NF-H isoforms, if demonstrated, indicate an immature neuronal state of the tumor. Moreover, 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 while less phosphorylated NF-H is localized to the perikarya and the dendrites.[17]

Hence, one should go for assessment of more than one neuronal marker for appropriate interpretation of neuronal differentiation of CNS tumors.

Other neuronal markers are microtubule-associated protein-2, which play a role in microtubule assembly and stabilization. They are specific markers of early neuronal differentiation and are usually used in embryonal tumors showing a neuronal differentiation.

Class III beta-tubulin is another specific marker of neuronal differentiation outside and inside the CNS except in  Sertoli cells More Details of the testis.

NFP positivity has also been seen in other non-CNS tumors like Merkel cell carcinomas of the skin, endocrine tumors of the pancreas, carcinoid tumors, and parathyroid tumors.

Cytokeratin

CK is a water-insoluble intermediate filament present intracellularly in almost all epithelia.[18] CK can be divided into at least 20 subtypes depending on their molecular weight. Apart from their presence in normal and neoplastic epithelium, they can also be seen in non-epithelial normal and neoplastic tissue, e.g., synovial sarcoma, uterine smooth muscle tumors, small round cell tumors, malignant melanomas, plasmacytomas, and even occasional malignant lymphomas. The primary utility of CK study is in differentiation between metastatic carcinomas (which are CK-positive) [Figure 3] from the primary CNS tumors (which are CK-negative). Sometimes, primary CNS tumors also show CK positivity. In gliomas, CK positivity is 60–80% and is due to nonspecific reactivity.[19],[20],[21] Occasionally, adenoid (glandular) or squamous metaplasia (more commonly seen in a gliosarcoma than in a GBM) also show CK positivity. Hence, to distinguish a GBM or a gliosarcoma from a metastatic carcinoma (both may show CK positivity and focal, weak, or absent GFAP immunoreactivity), one can further extend the IHC panel depending on the clinical history and morphology.
Figure 3: Photomicrograph (H and E, ×10) showing the fibrocollagenous tissue infiltrated by metastatic tumor arranged in islands and nests along with tumor emboli (a). On IHC, the tumor cells are positive for cytokeratin (b: ×200), EMA (c: ×200), and negative for vimentin (d: ×200)

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Other CK-positive brain tumors are choroid plexus tumors, but it is difficult to distinguish them from metastatic papillary tumors. In this situation, HEA125 and BerEp4 help in distinguishing the two. These two markers are consistently positive in metastatic papillary carcinomas and rare in choroid plexus tumors. Moreover, synaptophysin is positive in choroid plexus papillomas and carcinomas and negative in metastatic papillary carcinomas.

Meningiomas, especially meningotheliomatous, psammomatous, and secretory subtypes may also show occasional CK positivity.

Nestin

Nestin is a recently identified member of intermediate filaments and has the largest molecular weight. The term nestin is derived from the location where it is present, i.e., neural stem cell protein.[22] It is widely expressed by various types of cells during embryogenesis. In the developing nervous system, it is 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 the neuronal lineage, and by GFAP in those cells which are committed to differentiate into astrocytes. Hence, there is a transitional phase in which both immature and mature cell markers are expressed.[23] By the end of the gestation, nestin gets almost completely eliminated and is expressed only in endothelial cells of the mature human CNS and 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. As nestin is expressed in many embryonic cells, it should not be considered as an unequivocal phenotypic marker of embryonic CNS neuroepithelial cells. This is its limitation, i.e., it is not specific and is positive in many tumors such as gliomas, medulloblastomas, and meningiomas.

Nestin expression has been seen in medulloblastomas with neuronal differentiation (nestin coexpression with NFP), medulloblastomas with glial differentiation (nestin coexpression with GFAP), astrocytic tumors of all grades (nestin coexpression with GFAP), gangliogliomas, and ependymomas.

Nestin positivity is seen not only in tumor cells but also in endothelial cells of gliomas. It is not expressed in metastatic carcinomas.

Synaptophysin

Synaptophysin is a major transmembrane glycoprotein, first isolated from bovine neuronal presynaptic vesicles.[24] It is expressed in the normal, reactive, and neoplastic cells of the neuroectodermal and neuroendocrine types. Synaptophysin is reliable, and hence, is the most commonly used IHC marker for neuronal differentiated tumor. However, its negativity at the same time does not rule out neuronal or neuroblastic differentiation, because in the early stages of neurogenesis, markers other than synptophysin such as Class III beta-tubulin isotype are expressed.[25],[26] In the white matter of the normal brain, it does not stain the neuropil while in gray matter, it stains it.

Synaptophysin is positive in CNS tumors with neuronal differentiation such as a ganglioglioma, pleomorphic xanthoastrocytoma, neurocytoma [Figure 4], medulloblastoma, pineocytoma, subependymal giant cell astrocytoma, and is also positive in neuroendocrine tumors [Figure 5].
Figure 4: Photomicrograph (H and E, ×200) showing the biphasic pattern of a liponeurocytoma comprising of small round isomorphic neuronal cells and focal lipomatous differentiation (a). IHC showing tumor cell positivity for synptophysin (b: ×200)

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Figure 5: Photomicrograph (H and E, ×200) showing a paraganglioma disposed in lobules or nests separated by fibrovascular connective tissue (a). Individual tumor cells show round to ovoid nuclei, finely granular chromatin and granular amphophilic cytoplasm (b). Tumor cells are positive for synptophysin (c: ×200) and chromogranin (d: ×200) on IHC

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Chromogranin

Chromogranin is positive in almost all types of neuroendocrine tumors such as a paraganglioma [Figure 5].

S-100

This protein was first isolated from the CNS.[27] It is named “S-100” because of its solubility in 100% saturated ammonium sulfate at a neutral pH.[28] It is an acidic, dimeric calcium-binding protein, the exact function of which is unknown. It exists in different combinations of alpha and beta subunits with a predominance of beta subunit in the CNS. In the normal brain, its immunoreactivity is seen in glial cells, i.e., astrocytes, oligodendrocytes, ependymal cells, Schwann cells, and melanocytes. It is also demonstrated in nonglial cells such as chondrocytes, myoepithelial cells, adipocytes, and other cells, and in the tumors derived from them.[29],[30] CNS tumors showing S-100 positivity are astrocytomas, ODGs, ependymomas, melanocytic tumors, histiocytosis, and peripheral nerve sheath tumors [Figure 6].[7],[31] It is expressed in both nuclei and cytoplasm but nuclear staining is more specific and cytoplasmic staining alone is questionable.
Figure 6: Photomicrograph (H and E, ×10) showing the normochromic spindle-shaped tumor cells of a schwannoma displaying round to elongated nuclei with tapered ends (a). On IHC, tumor cells are positive for S-100 (b: ×200)

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Morphologically, sometimes a fibrous meningioma mimics schwannoma. In that case, schwannomas usually shows a strong, diffuse positivity to S-100 while meningiomas show positivity in only 20% cases, and that too is focal and less intense,[32],[33],[34] EMA further helps in differentiating the two, as unlike in schwannomas, this antibody is positive in meningiomas.[35]

Epithelial membrane antigen

This is a glycoprotein isolated from human milk fat globule.[36] It is considered as a marker of normal and neoplastic epithelium and perineural cells.[37],[38] It is also expressed in a variety of mesenchymal neoplasms, mesotheliomas, and even in lymphomas.[39],[40] In CNS, it is characteristically seen in meningiomas, chordomas, metastatic carcinomas, and ependymomas.

In meningiomas, EMA membranous positivity is seen in 70–80% of cases of meningothelial and transitional variants and only focal or negative staining is seen in atypical and malignant variants [Figure 2].[32],[33],[34] Hemangiopericytomas are EMA-negative and this helps in differentiating them from meningiomas.

Another differential diagnosis of a dura-based meningioma is metastatic carcinoma. Both of these show EMA positivity. In such cases, further extending 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., helps in excluding meningiomas.

Metastatic renal cell carcinoma in the brain can be differentiated from a hemangioblastoma (in Von Hippel–Lindau syndrome) morphologically by doing an EMA staining. A hemangioblastoma is consistently EMA negative while a metastatic renal cell carcinoma is positive for EMA.[41],[42]

Apart from this, as mentioned earlier, EMA immunoreactivity also helps in differentiating meningiomas from schwannomas, where it is consistently negative in the later condition.

EMA staining shows a dot like positivity in cases of low-grade ependymomas. Similarly, a chordoma also shows EMA immunoreactivity. Hence, it is helpful in distinguishing a chordoma from a chondrosarcoma, as in the later condition, EMA is usually negative, except in a small percentage (6%) of cases.[43]

Lymphoid markers

Lymphoma is the common differential diagnosis of round cell tumors. Leukocyte common antigen (LCA/CD45RB) is positive in lymphoma. All normal leukocytes except plasma cells (which are negative or variably positive) are also LCA positive; hence, LCA positivity cannot differentiate normal lymphocytes from neoplastic lymphocytes. Hence, for further characterization of the types of T cell or B cell predominance to assess for the presence of a T cell lymphoma or a B-cell lymphoma, an IHC study 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).[44] One should also remember that there are some lymphomas which are LCA negative, especially lymphoblastic lymphomas and anaplastic large cell lymphomas. Plasma cell and plasmablastic neoplasms also show a negative or variable positivity for LCA. Reed–Sternberg cells seen in the classic Hodgkin's lymphoma are typically CD45RB/LCA negative. One of the histiocytic tumors that can affect the CNS is Langerhans's histiocytosis (LCH). The most specific IHC marker for Langerhans's cell is CD1a, which is a membrane bound antigen linked with macroglobulin. This marker is also seen in immature thymocytes and rarely in Rosai–Dorfman disease and acute T-cell lymphoblastic lymphoma. Another IHC marker that is characteristically positive in LCH is S-100.[45],[46]

Melanotic markers

Primary melanotic neoplasms of the CNS are thought to arise from leptomeningeal melanocytes, and include diffuse meningeal melanocytosis, the rare meningeal melanocytoma, and primary malignant melanoma. The most common malignant melanocytic tumor of the CNS is metastatic melanoma. Most melancytic neoplasms [Figure 7] 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.[19] Sometimes, primary brain tumors such as schwannomas, astrocytomas, ependymomas, medulloblastomas, and paragangliomas also demonstrate melanin and hence HMB-45 positivity. However, all of these tumors do not show diffuse HMB-45 positivity like the melanocytic neoplasms of CNS.
Figure 7: Photomicrograph (H and E, ×200) showing a melanoma disposed in sheets showing focal melanin pigment and round to ovoid pleomorphic tumor cells, numerous mitotic figures, and a large, nucleoli (a). On bleaching, melanin pigment got bleached (b). Tumor cells are positive for S-100 (c: ×200) and HMB-45 (d: ×200)

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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. [Table 3] summarizes the details of these markers.
Table 3: Immunohistochemical markers for germ cell tumors

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Marker for atypical teratoid/rhabdoid tumor

INI-1/SMARCB-1 protein (coded on chromosome 22q) is 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 and less frequently, smooth muscle actin. GFAP, NFP, keratin, and synptophysin are also shown to be positive.[19]

Cell proliferation markers

Various antibodies used in IHC are available that evaluate the ongoing proliferation in the tumors [Table 4]. 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.[47] Important cell proliferating markers are described below.
Table 4: Various methods for the detection of proliferation of cells in tissue sections

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Mitotic figure

On H and E staining, proliferative activity is determined by counting the number of mitoses seen, which 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. It recognizes an antigen expressed in all the active phases of cell cycle, i.e. G1, S, G2, and M in paraffin-embedded tissue.[48] 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

Phosphohistone-3 antibodies specifically target the phosphorylated version of core histone protein. The phosphorylation of histone protein H3 occurs almost exclusively during mitosis but is not exhibited during apoptosis. Hence, studies have shown that it is a very effective and a better mitotic marker than MIB-1 labeling index in differentiating Grade II from Grade III astrocytomas in males.

p53

p53 is also known as tumor suppresser gene. Its name is based on its chemical nature, i.e. phosphoprotein and its molecular mass, i.e., 53 kD. 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.

It is considered as a marker of astrocytic tumor with a frequency that is in the range of 58–83%. 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 has been described as the most frequently amplified and overexpressed gene in approximately 60% of GBMs and has been associated with shorter survival times.[49] The prognostic value of EGFR amplifications and mutations, especially the EGFRvIII mutation, is controversial because several studies have shown contradictory results. The EGFRvIII mutation might be helpful in the identification of a subgroup of tumors with more malignant behavior than suggested by their histopathology alone. 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. They decarboxylate isocitrate to α-ketoglutarate with the production of NADH and/or NADPH. In most of the IDH mutated genes, only one copy of IDH gene is mutated. 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. 2-hydroxyglutarate may be an onco-metabolite responsible for the malignant progression of the tumor.[50]

IDH-1 mutation is present in 70-80% of WHO Grades II and III diffuse gliomas, ODGs (80%), anaplastic ODGs (85%), and mixed oligoastrocytomas (71%), as well secondary GBMs (82%). IDH-1 mutation is rare in primary GBMs (5%), pilocytic astrocytomas (10%), and is absent in ependymomas. IDH-2 mutation is seen in a smaller proportion of gliomas, and that too mainly in oligodendroglial tumors.

Several studies have shown that IDH-1 mutation is associated with a longer survival. 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.[51] 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 immunonegative [Figure 8].
Figure 8: Photomicrograph (H and E, ×400) showing a low-grade glial tumor infiltrating the brain parenchyma. On IHC, IDH-1 stain highlights positive tumor cells amidst the normal glial cells (×40)

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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.[52]

BRAF

BRAF V600E mutation may be seen by IHC studies in pleomorphic xanthoastrocytoma (80%) or ganglioglioma (25%) but cannot distinguish between pilocytic astrocytomas and low-grade gliomas. This mutation may be unfavorable and may be a therapeutic target in the future. Anti-BRAF V600E clinical trials are ongoing.[53]

[Table 5] summarizes the immunoreactivity of some common CNS tumors. Based on these IHC studies, various common brain tumors can be differentiated from each other.
Table 5: Immunoreactivity of common central nervous system tumors

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 » Limitations of Immunohistochemistry Markers Top


Although IHC is very useful diagnostic tool, it has its own set of limitations which one should be aware of. For example, 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.


 » Conclusions Top


IHC has a definite and important role to play in the field of diagnostic neuropathology. It is not only used for diagnostic purposes for the identification of tumor cell differentiation but is also used for prognostic purposes in the form of the analysis of proliferative activity and the expression of oncoproteins and growth factor receptors, which may more accurately reflect the malignant potential of the tumor. Proper clinical, radiological, and morphological correlation is mandatory for accurate interpretation of any IHC study.

Acknowledgments

We would like to acknowledge Mr. Vishvakarma, IHC technician, Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, for his help in performing the IHC tests. The image collection by Dr. Krishna Pani and Dr. Azfar Neyaz, residents in the Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, is also appreciated.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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