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| ORIGINAL ARTICLE |
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| Year : 2015 | Volume
: 4
| Issue : 2 | Page : 103-111 |
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Role of immunohistochemistry in diagnosis of brain tumors: A single institutional experience
Venugopal Madabhushi1, Renuka Inuganti Venkata1, Sailabala Garikaparthi1, Satya Varaprasad Kakarala2, Seshadri Sekhar Duttaluru2
1 Department of Pathology, Guntur Medical College, Guntur, Andhra Pradesh, India
2 Department of Neurosurgery, Government General Hospital, Guntur, Andhra Pradesh, India
| Date of Web Publication | 12-Jun-2015 |
Correspondence Address:
Venugopal Madabhushi
Department of Pathology, Guntur Medical College, Guntur, Andhra Pradesh
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/2277-8632.154262
Background: Central nervous system tumors which constitute 1-2% of all tumors pose diagnostic challenges because tumors of varying histogenesis show divergent differentiation and overlap in morphological features. In problematic cases, immunohistochemistry is done in addition to the routine histopathologic examination to overcome the diagnostic difficulties, since an accurate histologic diagnosis helps in predicting the clinical outcome of various brain tumors.
Materials and Methods: This study was carried out on a prospective basis in our institution from January 2009 to March 2012. During this period, a total of 246 neurosurgical specimens were received among which 118 brain tumors were diagnosed based on examination of Hematoxylin and Eosin stained sections of formalin-fixed and paraffin-embedded biopsy specimens. Immunohistochemical markers were applied in selective cases for an accurate diagnosis.
Results: In adults, astrocytomas occurred most frequently in the study, followed by meningiomas, nerve sheath tumors, metastatic deposits, glioblastomas, and gliosarcomas. Primitive neurectodermal tumors occurred frequently in children. Other rare tumors included lymphomas and mesenchymal tumors. Age and sex incidence and anatomic distribution of various tumors were studied. Grading of the tumors was done as per the revised World Health Organization criteria. The results of immunohistochemical study in selective cases were analyzed.
Conclusion: This study highlights the utility of immunohistochemistry as an adjunct in the histologic diagnosis of brain tumors in difficult cases. Keywords: Brain tumors, histopathology, immunohistochemistry
How to cite this article:
Madabhushi V, Venkata RI, Garikaparthi S, Kakarala SV, Duttaluru SS. Role of immunohistochemistry in diagnosis of brain tumors: A single institutional experience. J NTR Univ Health Sci 2015;4:103-11 |
How to cite this URL:
Madabhushi V, Venkata RI, Garikaparthi S, Kakarala SV, Duttaluru SS. Role of immunohistochemistry in diagnosis of brain tumors: A single institutional experience. J NTR Univ Health Sci [serial online] 2015 [cited 2023 Mar 28];4:103-11. Available from: https://www.jdrntruhs.org/text.asp?2015/4/2/103/154262 |
| Introduction | |  |
Brain tumors are a heterogeneous group as they differ in histogenesis and show a spectrum of morphological features. Though clinical data, imaging techniques, and peroperative findings offer some valuable clues to the diagnostic possibilities, histopathologic examination is the sine qua non of diagnosis of brain tumors. Nevertheless, histologic diagnosis of a brain tumor is not always straightforward and the pathologist faces diagnostic dilemmas not only because of overlap in morphological features among different categories of tumors but also due to divergent differentiation within the same tumor. Moreover, non-neoplastic lesions can mimic tumors. Hence, application of immunohistochemical markers has become imperative for an exact diagnosis and subtyping.
This study was taken up in our institution to assess the efficacy and utility of immunohistochemistry (IHC) as a rational supplementary technique in the diagnosis of brain tumors.
| Materials and Methods | | |
The present study was carried out in our department from January 2009 to March 2012. Neurosurgical specimens were received from the Department of Neurosurgery of our institution. Pertinent clinical data including details of imaging investigations and peroperative findings were obtained in all the cases.
The biopsy specimens were fixed in buffered formalin, routinely processed, and sections were obtained from paraffin blocks. Histologic examination of hematoxylin and eosin (H and E) stained sections and immunohistochemical analysis were performed to diagnose and classify various brain tumors according to the World Health Organization (WHO) classification of central nervous system (CNS) tumors. [1] Primary brain tumors were graded as per the established criteria. [2]
IHC was performed on problematic cases where differential diagnosis was given on H and E sections. Using 3-μm-thick sections on poly-l-lysine coated slides, antigen retrieval was done using microwave in citrate buffer at pH 6. Selected markers from a panel including glial fibrillary acidic protein (GFAP), epithelial membrane antigen (EMA), HMB-45, S-100, pancytokeratin (panCK), vimentin, synaptophysin, CD99, leukocyte common antigen (LCA), CD20, and CD3 (DAKO) were used for antigen detection by standard avidin biotin kit.
| Results | | |
In the present study, a total of 118 brain tumors were diagnosed. Astrocytomas formed the major group among all the tumors with a frequency of 37.29%, followed by nerve sheath tumors (13.56%), meningothelial tumors (13.56%), and metastatic deposits (12.71%) [Table 1]. Pituitary tumors and embryonal tumors occurred with a frequency of 6.78% and 5.08%, respectively. Ependymal and oligodendroglial neoplasms, mesenchymal tumors, and malignant lymphomas were observed rarely.
The differential diagnosis offered on H and E sections and the analysis of immunohistochemical markers leading to the final diagnosis in difficult cases are shown in [Table 2]. Age and sex incidence of the tumors are shown in [Table 3] and [Table 4] respectively.
| Discussion | | |
CNS tumors, which account for less than 2% of all malignancies, are associated with guarded prognosis because of their location. [3] In the present study, 118 cases of brain tumors were reported out of a total of 246 neurosurgical specimens received. Microscopic examination of H and E-stained sections of routinely processed tissue samples and use of special stains will suffice for a specific diagnosis in majority of cases. However, in cases where morphological variations cause diagnostic dilemmas, IHC was applied to distinguish between different categories of lesions. [4]
Gliomas
Gliomas, the most common of the primary brain tumors in adults, are heterogeneous. Grading of gliomas is done as per the revised WHO criteria. [3] Circumscribed lesions of low proliferative potential are graded as Grade I. Infiltrative tumors are graded as Grade II, whereas infiltrating tumors with increased cellularity and mitotic activity are designated as Grade III. Grade IV is assigned to histologically malignant, mitotically active, and necrosis-prone tumors. Fibrillary astroglial neoplasms were designated as diffuse astrocytoma (WHO Grade II), anaplastic astrocytoma (WHO Grade III), or glioblastoma (WHO Grade IV) according to the histologic features. [5],[6] Diffuse fibrillary astrocytomas were the most frequent in the study with 20 cases. Seven cases were diagnosed as anaplastic astrocytomas.
Glioblastoma (WHO Grade IV) was diagnosed in 17 cases in the study with a frequency of 14.41% of all brain tumors and 38.64% of astrocytic tumors [Table 1]. Other workers reported higher frequency for glioblastoma in separate studies. [7],[8] Age distribution ranged from third to seventh decades with maximum number of cases in the fourth decade. The diagnosis of glioblastoma was based on the tissue pattern characterized by highly anaplastic cells, increased mitotic activity, microvascular proliferation with multilayering of endothelium, and wide areas of necrosis. In our study, accumulation of tumor cells around neurons and blood vessels was observed. In three cases of glioblastoma, features of diffuse astrocytoma of Grade II were also seen focally.
Primary variant of glioblastoma occurs de novo, while the secondary variant arises within pre-existent, differentiated astrocytic neoplasms. [5] Cellular polymorphism in a heterogeneous glioblastoma can simulate metastatic carcinoma or melanoma and IHC is necessary for confirmation in such cases. [9],[10] In four cases of our study, epithelial elements in the form of adenoid structures and mucinous background were observed [Figure 1]a. The positive expression of GFAP by epithelial structures confirmed unequivocally the diagnosis of glioblastoma [Figure 1]b and c. In our study, foci of lipidized cells were seen in a case of glioblastoma. Rosenblum et al. reported that expression of cytokeratins and EMA obscured the glial lineage of lipidized epithelioid glioblastoma. [11]  | Figure 1: Glioblastoma. (a) Cellular pleomorphism with focal epithelial features (H and E, ×400); (b) GFAP expression in tumor cells (IHC stain, GFAP, ×400); (c) EMA expression in epithelial elements (IHC stain, EMA, ×400)
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Two cases of glioblastoma in our study showed biphasic tissue pattern comprising neoplastic astrocytes admixed with pleomorphic spindle-shaped cells and increased mitotic activity [Figure 2]. IHC revealed GFAP positivity in the glial elements [Figure 3]a. The spindle-shaped cells were negative for GFAP but positive for vimentin leading to the diagnosis of gliosarcoma [Figure 3]b and c. Literature shows that sarcomatous change occurs in approximately 2% of glioblastomas. [12],[13]  | Figure 2: Gliosarcoma neoplastic astrocytes along with pleomorphic spindle-shaped cells (H and E, ×200)
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 | Figure 3: Gliosarcoma. (a) GFAP positivity in neoplastic astrocytes (IHC stain, GFAP, ×400); (b) spindle-shaped cells negative for GFAP (IHC stain, GFAP, ×400); (c) vimentin expression by neoplastic spindle-shaped cells (IHC stain, vimentin, ×400)
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One case of oligodendroglial tumor was reported in the study with a frequency of 0.85%. Histology of a recurrent hemispheric tumor from a 15-year-old girl showed a diffusely infiltrating cellular tumor with rounded hyperchromatic nucleus surrounded by a halo and few cellular processes. Prominent mitotic activity, microvascular proliferation, and focal necrosis were present. The presence of a branching capillary network along with microcalcifications was helpful in clinching the diagnosis of anaplastic oligodendroglioma of WHO Grade III. [14]
Two cases of ependymal tumors were reported in the present work. A ventricular mass in a 46-year-old woman was diagnosed as subependymoma (WHO Grade I), as the histology showed clusters of monomorphic tumor cells around microcystic spaces, rosette pattern, and dense fibrillary matrix. A frontoparietal cystic lesion with a mural nodule in a 4-year-old girl was reported as anaplastic ependymoma (WHO Grade III) because of monomorphic cells arranged in perivascular pseudorosettes, mitotic activity, microvascular proliferation, and necrosis.
In the present study, histology of a frontoparietal mass in a 50-year-old woman showed a moderately cellular tumor with an intimate mixture of oligodendroglial and astrocytic tumor cells. Histologic diagnosis was oligoastrocytoma of WHO Grade II.
In our study, a rare case of site-specific tumor, chordoid glioma of the third ventricle, was reported in a woman in the third decade. [15] The lesion presented as a solid circumscribed mass in the third ventricle with attendant hydrocephalus. Histology showed clusters and nests of tumor cells with eosinophilic cytoplasm and round to oval nuclei in a vacuolated mucinous stroma. The differential diagnosis included metastatic carcinoma, intraventricular chordoma, and chordoid meningioma. But there was no evidence of true glands or physaliferous cells or cellular whorls. IHC showed diffuse cytoplasmic staining for GFAP and vimentin, thus confirming the diagnosis of chordoid glioma. [16],[17]
Two uncommon cases of neuronal and glioneuronal tumors were reported in our study. Histology of an intraventricular mass from a 21-year-old woman showed monomorphic cells in nests and sheets with intervening fibrillar matrix [Figure 4]a. Vacuolated cytoplasm and round nuclei with speckled chromatin were seen. The differential diagnosis was oligodendroglioma and central neurocytoma. IHC showed positivity of fibrillary matrix for synaptophysin which is diagnostic of central neurocytoma [Figure 4]b. [18],[19] GFAP positivity was observed in trapped neurocytes. Central neurocytoma carries a good prognosis and complete excision is sufficient in most cases. [20],[21] Histology of a parasellar lesion from a 16-year-old woman showed clusters of enlarged and multinucleated neurons admixed with small lymphocytes. The diagnosis was that of ganglioglioma of WHO Grade I. [22],[23] Ganglion cell like astrocytes are differentiated from neoplastic neurons by positive expression for GFAP. Neoplastic neurons are distinguished from native neurons by architectural disarray, abnormal clustering, pleomorphism, multinucleation, and diffuse cytoplasmic positivity for chromogranin. [24]  | Figure 4: Central neurocytoma. (a) Monomorphic cells in nests and sheets (H and E, ×400); (b) synaptophysin expression by tumor cells (IHC stain, synaptophysin, ×400)
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Embryonal tumors
Seven cases of embryonal tumors were reported in the study with a frequency of 5.08%. Posterior fossa was the most common location for embryonal tumors, but intraventricular and cerebellopontine lesions were also seen. The presence of small undifferentiated cells packed in sheets with hyperchromatic, round, and molded nuclei and scanty stroma was diagnostic of classic medulloblastoma. Synaptophysin expression was observed in these tumors.
Histology of two cases revealed nodular, pale islands of reduced cellularity [Figure 5]a. GFAP positivity was expressed in the pale central areas, whereas EMA was negative [Figure 5]b. These tumors were reported as desmoplastic/nodular variant of medulloblastoma. One of these cases presented as an intraventricular mass in a 12-year-old girl, while the other tumor presented as a cerebellopontine lesion in a 60-year-old man. Review of literature shows that desmoplastic/nodular medulloblastomas arise laterally in the cerebellar hemispheres and occur at all ages. [25],[26] Medulloblastomas with extensive nodularity may resemble desmoplastic/nodular variant, but exhibit a more prominent lobular microarchitecture and elongated reticulin-free zones in fibrillary matrix. [27] An extracerebellar mass in an 18-year-old adolescent was diagnosed as a primitive neurectodermal tumor (PNET) which on IHC showed positivity for CD99 and was negative for LCA, excluding a malignant lymphoma [Figure 6]a and b. | Figure 5: Desmoplastic/nodular variant of medulloblastoma. (a) Nodular tumor with pale islands of reduced cellularity (H and E, ×200); (b) GFAP expression in pale areas (IHC stain, GFAP, ×400)
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 | Figure 6: Primitive neurectodermal tumor. (a) Uniformly small hyperchromatic cells in sheets (H and E, ×200); (b) CD99 expression by tumor cells (IHC stain, CD99, ×200)
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A ventricular mass from a 9-year-old girl with a clinical impression of ependymoma and choroid plexus papilloma showed dense cellularity with rosettes and areas of hyalinization, leading to the differential diagnoses of anaplastic ependymoma, ependymoblastoma, and medulloblastoma. In contrast to multilayered true rosettes in ependymoblastoma, anaplastic ependymoma shows perivascular pseudorosettes with single circumferential layering of cells. The present case showed true rosettes with multilayering of cells, but there was no evidence of neuroepithelium [Figure 7]a. IHC demonstrated diffuse positivity for GFAP, whereas vimentin was negative confirming the diagnosis of ependymoblastoma [Figure 7]b.  | Figure 7: Ependymoblastoma. (a) Tumor cells disposed in multilayered true rosettes (H and E, ×200). (b) Diffuse positivity for GFAP expression (IHC stain, GFAP, ×200)
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Nerve sheath tumors
In the present study, 16 cases of schwannoma were reported with a frequency of 13.56% [Table 1]. Most of the cases presented as cerebellopontine angle lesions in adults [Table 3]. Other sites included brain stem, jugular bulb, acoustic nerve, and spinal cord. These findings are in accordance with literature. Schwannoma, the most common variant of nerve sheath tumors in the central neuraxis, occurs in adults in the cerebellopontine angle or lumbosacral spinal extramedullary space. [29] Schwannomas arising at spinal levels typically involve the posterior roots and assume a "dumb bell" configuration. In most of the cases, characteristic Antoni A and B areas, nuclear palisading, and vascular hyalinization were sufficient for a diagnosis of schwannoma. However, in some cases, IHC is required to differentiate between meningiomas and schwannoma. In contrast to meningothelial tumors, schwannomas show diffuse cytoplasmic S-100 protein expression, but they are negative for EMA. [29],[30]
In our study, a dumb bell-shaped spinal cord mass at C2-C3 level showed prominent dark pigment and psammomatous calcification on histology. IHC demonstrated positive expression for S-100 and HMB-45 and the tumor was diagnosed as melanotic schwannoma of psammomatous variety. Literature shows that melanotic variant of schwannoma exhibits a predilection for the spinal nerve roots. [31],[32]
Meningeal tumors
Meningiomas accounted for 16 cases in our study with a frequency of 13.56%. The age incidence varied from second decade to eighth decade with six cases occurring in the fourth decade. Female preponderance was seen with 9 out of 16 cases. These findings are in accordance with other studies. [33],[34],[35] In our study, meningiomas mostly presented within the cranial cavity and were dura-based. Uncommon sites included sphenoid ridge, cerebellopontine angle, and extracranial locations. Arrangement of tumor cells in concentric whorls and presence of clear nuclei with pseudoinclusions and psammoma bodies were the striking histologic features. IHC showed positive expression for EMA and vimentin, whereas GFAP was negative. All the 16 cases in the study were diagnosed as meningiomas of WHO Grade 1, based on these findings. Meningothelial and transitional meningiomas were the common variants followed by angiomatous and fibroblastic meningiomas. [36]
Three cases of meningeal mesenchymal tumors were also reported in our study. The differential diagnosis of a vascular lesion in a 70-year-old man included angiomatous meningioma and meningeal hemangiopericytoma. Psammomatous calcification, tumor cells in whorls, and intranuclear pseudoinclusions are the clues to the possibility of meningiomas with pericytomatous growth pattern, which were absent in our case. In contrast to meningioma, hemangiopericytoma shows a network of reticulin around individual tumor cells. [37],[38] Immunohistochemical study demonstrated positivity for CD34 and vimentin in our case, clinching the diagnosis of meningeal hemangiopericytoma. An extremely rare diagnosis of giant intracranial osteochondroma was made in a frontal lobe tumor in a 24-year-old man [Figure 8]a and b. Literature shows that osteochondromas may bulge into the cranial cavity from its floor or from the dura. [39],[40] A cerebellar cystic lesion in a 40-year-old man in our study was reported as cavernous hemangioma.  | Figure 8: Giant intracranial osteochondroma. (a, b) Benign chondroid and osteoid elements (H and E, ×200). (Inset) Computerized tomography scan showing a frontal lobe mass with calcifications
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Malignant lymphomas
Malignant lymphoid neoplasms, restricted at presentation to the brain, spinal cord, or meninges, are termed as primary CNS lymphoma (PCNSL). Involvement of the CNS by node-based non-Hodgkin's lymphoma is usually limited to permeation of the leptomeninges. In our study, two cases of primary malignant lymphoma were diagnosed with a frequency of 1.69%. One case presented as a frontal mass in a 60-year-old woman, while the other case presented as a frontal extradural mass in a 30-year-old man. Histology of both the lesions revealed characteristic vasocentric growth pattern of tumor cells with infiltration of walls of blood vessels and Virchow-Robin spaces [Figure 9]a and b. Positive expression for CD45 and CD20 was demonstrated, whereas GFAP and CD3 were negative, thus confirming the diagnosis as primary malignant lymphoma of B cell origin. Majority of PCNSLs are situated in the supratentorium and exhibit a B-cell immunophenotype. [41],[42],[43] | Figure 9: Non-Hodgkin's lymphoma. (a) Vasocentric growth pattern of small blue round cells (H and E, ×400). (b) CD20 expression by tumor cells (IHC stain, CD20, ×400)
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Tumors of sella
In our study, two cases of craniopharyngioma and six cases of pituitary adenomas were reported with a frequency of 1.69% and 7.8%, respectively. Pituitary adenomas constitute 10-15% of all intracranial neoplasms and have a prevalence of 1 per 1000 individuals. [44] Five of six cases in the study occurred in fourth and fifth decades, with males outnumbering females in the ratio 4:2. This is in accordance with other studies. [45],[46]
Metastatic tumors
Secondary involvement of the CNS by direct extension or hematogenous metastasis is a common complication of systemic cancer. In the present study, 15 cases of metastatic tumors were reported with a frequency of 12.71%. Adenocarcinoma was the most common metastatic deposit in our study, found in eight cases. Brain metastases in adults usually derive from carcinomas of the lung and breast, followed by malignant melanomas, renal carcinomas, and colorectal adenocarcinomas. [47],[48] In our study, 12 out of 15 metastatic lesions were located in cerebral hemispheres, while corpus callosum, cerebellum, and dura accounted for the remaining lesions. These observations are in accordance with literature. [48]
Metastatic nodules are sharply circumscribed with pushing margins and usually reflect the histology of the donor lesions. In case of well-differentiated deposits, the histologic diagnosis was straightforward. Poorly differentiated carcinomas were distinguished from anaplastic gliomas by cohesive architecture, abrupt interface with adjacent neural tissue, and peritheliomatous pattern of tumor cell preservation about stromal blood vessels. Complex microvascular hyperplasia was absent in secondary deposits. In one of our cases, a mass lesion presented in corpus callosum in an 80-year-old woman. The differential diagnosis included malignant melanoma and pigmented ependymoma. Strong positivity was expressed for HMB-45, whereas GFAP was negative, thus clinching the diagnosis of a metastasis from malignant melanoma. In another case, differential diagnosis of a temporal lobe lesion included metastatic carcinoma and glioblastoma. Strong positivity for panCK was demonstrated, while GFAP was negative supporting the diagnosis of metastasis.
| Conclusion | | |
The present work shows that histopathologic examination is the mainstay in the diagnosis and grading of majority of brain tumors, but IHC plays a crucial supplementary role in resolving diagnostic dilemmas in the routine practice of neurosurgical pathology.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
[Table 1], [Table 2], [Table 3], [Table 4]
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