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Diagnostic algorithm for pathological evaluation of gliomas in a resource-constrained setting

1 Department of Pathology, ICMR-National Institute of Pathology, New Delhi, India
2 Department of Neurosurgery, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India

Date of Submission16-Jan-2021
Date of Acceptance19-Feb-2021
Date of Web Publication10-Jun-2022

Correspondence Address:
Fouzia Siraj,
Department of Pathology, ICMR-National Institute of Pathology, New Delhi - 110 029
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.jcrt_102_21

 > Abstract 

Introduction: Gliomas are the most common primary intracranial tumors. The current World Health Organization (WHO) classification of central nervous system tumors recommends integrated histo-molecular diagnosis of gliomas. However, molecular testing is not available in even most of the advanced centers of our country, and histopathology aided with immunohistochemistry (IHC) is still widely used for diagnosis. Immunohistochemical markers such as iso-citrate dehydrogenase1 (IDH1) and Alpha Thalassemia/Mental Retardation Syndrome X-linked (ATRX) can be reliably used for the correct diagnosis, prognosis, and treatment of gliomas.
Aim: We aimed to develop a diagnostic algorithm by integrating morphology, IDH1, and ATRX status of gliomas seen in our institute for 1 year.
Settings and Design: Analytical cross-sectional study.
Materials and Methods: This study included 60 histopathologically confirmed cases of astrocytic (n = 51) and oligodendroglial tumors (n = 9). Clinical, radiological, and histopathological features were noted and tumor grades assigned according to the WHO recommendations. IDH1 and ATRX mutation status was evaluated using IHC. The tumors were divided into three molecular groups on the basis of their IDH1 and ATRX mutation status: (1) Group 1: IDH1 negative and ATRX positive, (2) Group 2: IDH1 positive and ATRX positive, (3) Group 3: IDH1 positive and ATRX negative.
Results: The mean age of presentation was 45.0 ± 15.8 years with a male-to-female ratio of 2:1. Seizures, headache, and hemiparesis were the most common modes of presentation. The tumor subtypes studied were glioblastoma (n = 32), anaplastic astrocytoma (n = 7), diffuse astrocytoma (n = 6), oligodendroglioma (n = 6), pilocytic astrocytoma (n = 6), and anaplastic oligodendroglioma (n = 3). IDH1 mutation was present in 26 cases including anaplastic astrocytoma (n = 7), diffuse astrocytoma (n = 6), oligodendroglioma (n = 5), secondary glioblastoma (n = 5), and anaplastic oligodendroglioma (n = 3). ATRX mutation, i. e., loss of ATRX was observed in 17 cases including diffuse astrocytoma (n = 5), anaplastic astocytoma (n = 5), anaplastic oligodendroglioma (n = 3), oligodendroglioma (n = 3), and secondary glioblastoma (n = 1). All six cases of pilocytic astrocytoma were negative for IDH1 and ATRX mutation. There were 34 patients in Group 1 (IDH1– and ATRX +), nine cases in Group 2 (IDH1 + and ATRX +), and 17 patients in Group 3 (IDH1 + and ATRX-).
Conclusion: Diagnosis of gliomas should be based on a detailed clinicoradiological and histopathological assessment, followed by genotypic characterization. Evaluation for IDH1and ATRX status has both diagnostic and prognostic value as it helps in differentiating gliomas from reactive gliosis, primary glioblastoma from secondary glioblastoma, and pilocytic astrocytoma (WHO grade I) from diffuse astrocytoma (WHO grade II). Tumors with IDH1 mutations have a better outcome than those with wild-type IDH. IHC can serve as a useful surrogate to conventional molecular tests in resource-constrained settings. By devising an algorithm based on morphological and IHC features, we were able to stratify gliomas into three prognostic subgroups.

Keywords: Alpha Thalassemia/Mental Retardation Syndrome X-Linked, diffuse astrocytoma, glioblastoma, glioma, iso-citrate dehydrogenase1, iso-citrate dehydrogenase1 R132H, immunohistochemistry, oligodendroglioma, secondary glioblastoma

How to cite this URL:
Jain S, Gupta P, Shankar K B, Singh R, Siraj F. Diagnostic algorithm for pathological evaluation of gliomas in a resource-constrained setting. J Can Res Ther [Epub ahead of print] [cited 2022 Aug 19]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=347198

 > Introduction Top

Gliomas are the most common primary central nervous system (CNS) tumors.[1] Recent (2016) World Health Organization (WHO) classification of CNS tumors[2] incorporates gliomas into the following categories: Astrocytic tumors, oligodendroglial tumors, oligoastrocytic tumors, ependymal tumors, and neuronal and mixed neuronal-glial tumors. The current classification is based on integrated diagnosis comprising phenotypic-genotypic characteristics.[2] The prognoses of glial tumors depend on age at diagnosis, clinical status, histological type, tumor grade, genetic profile, and extent of tumor resection. Hence, a tumor should be primarily evaluated for its morphological features and grade followed by its genetic profile. The latter is especially important to understand the tumor pathophysiology and its management. According to WHO 2016 classification,[2] Isocitrate Dehydrogenase 1 (IDH1 R132H) and Alpha Thalassemia/Mental Retardation Syndrome X-linked (ATRX) are two key molecular markers which should be evaluated in glial tumors. Tumors with IDH1 mutations have a better outcome than those with wild-type IDH.[3],[4] Apart from this, IDH1 helps in differentiating true neoplasia from reactive gliosis.[5] Mutation/loss of ATRX acts as an accessory marker for tumor prognosis.[6],[7] Studies have shown that tumors with IDH1 and ATRX mutation have a significantly better prognosis than nonmutated tumors.[8] Nowadays, IDH1 testing is mandatory while reporting glial tumors and can be performed using DNA sequencing and/or immunohistochemistry (IHC). IHC is a cost-effective substitute to expensive molecular tests.

 > Materials and Methods Top

An analytical cross-sectional study was conducted at ICMR-National Institute of Pathology, New Delhi, India after obtaining the necessary ethical approval. The study group comprised 60 histopathologically confirmed cases of astrocytic and oligodendoglial tumors from patients who were operated in the Department of Neurosurgery, Safdarjung Hospital, New Delhi, India. The clinicopathological features of these tumors were studied. Ependymal and nonglial brain tumors were excluded from the index study because their IDH1 mutation status does not contribute significantly to tumorigenesis.[1] The surgical specimens were fixed in neutral buffered formalin and embedded in paraffin. Four to six micron thick sections were cut and stained with hematoxylin and eosin (H and E). Tumor characteristics were studied and graded according to WHO guidelines (Grade I to Grade IV). IHC was carried out using the standard protocol. Representative formalin-fixed paraffin-embedded sections of tumor and the adjacent normal brain parenchyma (internal control) were processed for IHC to assess their IDH1 and ATRX status. Antigen retrieval was done by TRIS EDTA buffer (PH = 9) and the slides stained with antibodies against IDH1 and ATRX receptors by LSAB (labeled streptavidin-biotin) system (IDH1 R132H clone H09, dilution 1:20; Dianova, Hamburg, Germany and ATRX clone Polyclonal IgG, dilution 1:500; Sigma-Aldrich, USA). All the immunostained slides were evaluated as follows: Tumor was reported IDH1 positive when tumor cells showed strong cytoplasmic positivity indicating the presence of IDH1 mutation. (Positive control; normal brain). For ATRX, nuclear staining in >10% of tumor nuclei were considered positive and loss of expression was reported when <10% nuclei were stained or no staining was observed. Loss of ATRX expression was labeled as ATRX mutation. (Positive control; endothelial cells of blood vessels). Based on the molecular signature, tumors were divided into three molecular groups: Group I: IDH1 negative and ATRX positive, Group II: IDH1 positive and ATRX positive, Group III: IDH1 positive and ATRX negative. In our study, there was no patient with a tumor negative for both IDH and ATRX.

Statistical analysis

Data are presented as number (%) and mean (±standard deviation), as appropriate. Association between IDH1 mutation and ATRX mutation was analyzed using Pearson's Chi-square test. A P < 0.05 was considered statistically significant.

 > Results Top

A total of 60 cases were studied. The mean age was 45 (±15.8) years, with a male-to-female ratio of 2:1. The most common site was frontal lobe (n = 29, 48.3%) followed by temporo-parietal lobe (n = 21, 35%), corpus callosum (n = 5, 8.3%) cerebellum (n = 3, 5%), thalamus (n = 1, 1.7%), and optic nerve region (n = 1, 1.7%). The most frequent mode of presentation was headache (n = 34, 56.7%) followed by episodes of seizures (n = 18) and vomiting (n = 6).

On microscopy, histopathological features were studied and grades (I to IV) were assigned based on microvascular proliferation, necrosis, and mitosis, according to the WHO guidelines [Table 1]. The most common tumor was glioblastoma/glioblastoma multiforme (GBM) (n = 32, 53.3%) followed by anaplastic astocytoma (n = 7, 11.7%), diffuse astrocytoma (n = 6, 10%), oligodendroglioma (n = 6, 10%), pilocytic astocytoma (n = 6, 10%), and anapalstic oligodendroglioma (n = 3, 5%). The IDH1 and ATRX status were evaluated using IHC. IDH1 mutation was seen in 26 (43.3%) and ATRX mutation (ATRX loss) was seen in 17 (28.3%) cases [Table 1]. Tumors with IDH1 mutations were diffuse astrocytoma, anaplastic astrocytoma, anaplastic oligodendroglioma, oligodendroglioma, and secondary glioblastoma. These IDH1 positive GBM (secondary glioblastoma) cases developed from diffuse/anaplastic astocytoma. IDH1 mutation was absent in pilocytic astrocytoma and primary GBM. ATRX loss was seen in anaplastic oligodendroglioma, diffuse astrocytoma, and anaplastic astocytoma whereas ATRX was retained in primary GBM. A significant association (P ≤ 0.001) between IDH1 mutation and ATRX mutation was noted in various types of gliomas [Table 1].
Table 1: Expression and association between isocitrate dehydrogenase and alpha thalassemia/mental retardation syndrome X-linked in gliomas

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The molecular subtyping of the studied tumors is provided in [Table 2]. The molecular subtypes were IDH1 – and ATRX + (group 1, n=34 [56.7%]) [Figure 1], IDH1 + and ATRX + (group 2, n=9 [15.0%]) [Figure 2] and IDH1 + and ATRX – (group 3, n=17 [28.3%]) [Figure 3]. There was no patient with a tumor negative for both IDX and ATRX.
Table 2: Molecular subtyping of glial tumors in index study

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Figure 1: Microphotograph showing primary glioblastoma (Group 1) (a) Highly pleomorphic tumor cells with endothelial proliferation (long arrow) and increased mitosis (short arrow); (H and E, ×400) (b) ATRX positivity in nuclei of tumor cells (arrow), ×400 (c) IDH 1 negative tumor cells, ×400

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Figure 2: Microphotograph showing secondary glioblastoma (Group 2) (a) Highly pleomorphic tumor cells with palisading necrosis, H and E (400X) (b) Tumor cells showing nuclear positivity for ATRX (arrow), 200X (c) Tumor cells showing cytoplasmic positivity for IDH1 (arrow), 400X

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Figure 3: Microphotograph showing diffuse astrocytoma (Group 3) (a) Infiltrating tumor cells showing minimal nuclear atypia, H and E (400X), (b) ATRX negative tumor cells, 400X (c) Tumor cells showing cytoplasmic positivity for IDH1 (arrow), 200X

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Based on the results of our study, we proposed a diagnostic algorithm for pathological evaluation of gliomas in resource-constrained settings [Figure 4].
Figure 4: Diagnostic algorithm for pathological evaluation of gliomas. AA = Anaplastic astrocytoma, AO = Anaplastic oligodendroglioma, ATRX = Alpha Thalassemia/Mental Retardation Syndrome X-Linked, DA = Diffuse astrocytoma, GBM = Glioblastoma, H and E = Hematoxylin and eosin, IDH1 = Iso-citrate dehydrogenase1, OD = Oligodendroglioma, PA = Pilocytic astrocytoma; WHO = World Health Organization

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 > Discussion Top

The term glioma incorporates a group of heterogeneous tumors characterized by a common origin from progenitor glial cells. As per the 2016 WHO classification of CNS tumors,[2] gliomas can be further categorized into astrocytic tumors, oligodendroglial tumors, oligoastrocytic tumors, ependymal tumors, and neuronal and mixed neuronal-glial tumors. This classification is based on phenotypic and genotypic characteristics of the tumor. Phenotypic analysis is done by routine histopathology on H and E-stained sections. The histopathological analysis is essential for grading the tumor, and therefore, is of prime importance in the evaluation of such tumors. The grading of gliomas is based on cellularity, atypia, mitotic activity, microvascular proliferation, and necrosis.[2] Tumor grade not only predicts response to therapy but also the overall outcome. On the other hand, genotypic characteristics of the tumor can be determined through genetic analysis performed using Sanger sequencing, next-generation sequencing, and IHC.[9] Sanger sequencing has a low limit of detection and next-generation sequencing is very expensive. IHC is a fast, cost-effective, and reliable method to evaluate IDH1 R132H and ATRX gene mutation status.[10],[11]

IDH has two tumor-associated isoforms: IDH1 and IDH2. The IDH1 gene on chromosome 2q33.3 encodes for IDH1, located in the cytoplasm and the peroxisomes of a cell.[12] The IDH1 R132H accounts for 90% of all glioma-associated IDH mutations. Studies have shown that IDH1 mutation is an early step in gliomagenesis and has been reported in grades II and III astrocytomas, oligodendrogliomas, oligoastrocytomas, and secondary GBM.[13],[14],[15] Similar findings were noted in the index study. Genotypic assessment of gliomas by IDH1 not only helps in diagnosis, prognosis, and prediction of response to therapy but also in differentiating gliomas from nonneoplastic CNS lesions,[16] diffuse astrocytoma (grade II) from pilocytic astrocytoma (grade I), primary from secondary GBM, anaplastic astrocytomas (grade III) from primary GBM (grade 1V), astrocytomas from ependymomas and oligodendroglial tumors from neurocytomas and dysembryoplastic neuroepithelial tumor.[17],[18] Clinical and preclinical trials suggest that genetic and pharmacological inactivation of IDH1 results in the promotion of differentiated phenotype and increased apoptosis of tumor cells in response to targeted therapies.[19] In large studies by Hartmann et al. and Wick et al., it was found that patients with anaplastic astrocytoma, anaplastic oligodendrogliomas, and secondary GBM with IDH1 mutation had significantly longer survival and good prognosis, compared to those without.[20],[21]

The ATRX gene is located on chromosome Xq21.1, and encodes a protein that belongs to the H3.3-ATX-DAXX chromatin remodeling pathway. ATRX expression is lost in tumors with ATRX mutation. In gliomas, ATRX mutation has been associated with a better prognosis in grade II, III, and IV glial tumors.[22] ATRX mutation is seen in association with IDH1 mutation and indicates a better prognosis and survival. This is in accordance with our study which found a significant association between IDH1 and ATRX mutation (P < 0.001).[22],[23] Thus, we suggest that ATRX can be used in conjugation with IDH1as a potential prognostic biomarker.

In the index study, the most common tumor was glioblastoma, grade IV (n = 32). Out of 32 cases of GBM, 27 had a history of surgery for low-grade glioma and all these patients were relatively older (age >50 years). These were the clinical clues indicating secondary GBM, the diagnosis of which was later confirmed by positive IDH1 mutation status. Similarly, few cases of pilocytic astrocytoma and diffuse astrocytoma with overlapping morphological features on H and E stained sections were differentiated by their IDH1 status.

The findings of our study suggest that IHC may serve as a potential tool to assess the genetic profile of astrocytic and oligodendroglial tumors in a resource constraint neuropathology laboratory. The strengths of this study are: (a) the use of a uniform and standard protocol at each diagnostic step, (b) a detailed evaluation of clinicopathological correlation, and (c) proposal of an IHC-based diagnostic algorithm for evaluation of gliomas in resource-constrained settings. We acknowledge certain limitations. Wild-type IDH analysis, which is a known independent poor prognostic factor, was not performed in our study. Wild-type IDH promotes transcription and expression of podoplanin which is involved in the migration of glioma cells. Future studies should focus on the evaluation of both wild-type and mutant IDH for a better understanding of epigenetic regulation and tumorigenesis.[24]

 > Conclusion Top

IDH1 and ATRX mutation status assessment is helpful in the stratification of patients with glioma. IHC may serve as a reliable and cost-effective method to assess these mutations. Based on the findings of this study, the authors propose an IHC-based diagnostic algorithm for the evaluation of gliomas in a resource-constraint neuropathology laboratory.

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Conflicts of interest

There are no conflicts of interest.

 > References Top

Yan W, Zhang W, You G, Zhang J, Han L, Bao Z, et al. Molecular classification of gliomas based on whole genome gene expression: A systematic report of 225 samples from the Chinese Glioma Cooperative Group. Neuro Oncol 2012;14:1432-40.  Back to cited text no. 1
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Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009;360:765-73.  Back to cited text no. 3
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Gupta P, Siraj F, Malik A, Shankar KB. Clinical and histopathological profile of dysembryoplastic neuroepithelial tumor: An experience from a tertiary care center. J Can Res Ther 2020. DOI: 10.4103/jcrt.JCRT_632_19.  Back to cited text no. 17
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Wick W, Hartmann C, Engel C, Stoffels M, Felsberg J, Stockhammer F, et al. NOA-04 randomized phase III trial of sequential radiochemotherapy of anaplastic glioma with procarbazine, lomustine, and vincristine or temozolomide. J Clin Oncol 2009;27:5874-80.  Back to cited text no. 21
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Abedalthagafi M, Phillips JJ, Kim GE, Mueller S, Haas-Kogen DA, Marshall RE, et al. The alternative lengthening of telomere phenotype is significantly associated with loss of ATRX expression in high-grade pediatric and adult astrocytomas: A multi-institutional study of 214 astrocytomas. Mod Pathol 2013;26:1425-32.  Back to cited text no. 23
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2]


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