The Association of Alpha-thalassemia X-Linked Intellectual Disability Mutation with Histopathological Grading in Isocitrate- Dehydrogenase-mutant Glioma

Abstract

Gliomas are the most common malignancies of the central nervous system. Two molecular profiles involved in gliomagenesis are isocitrate dehydrogenase (IDH) and alpha-thalassemia X-linked intellectual disability (ATRX). Inactive mutations in the ATRX gene are associated with tumorigenesis via the alternative telomere lengthening pathway as well as with IDH and tumor protein p53 mutation. The present study aims to determine the relationship between ATRX mutation and histopathological grading in IDH-mutant gliomas. This is a cross-sectional study using formalin-fixed paraffin-embedded blocks to examine the data of patients with an IDH-mutant glioma admitted to the Sardjito General Hospital between January 2017 and March 2022. The IDH-mutant status and ATRX mutation were determined via a polymerase chain reaction-sequencing examination and immunohistochemistry analysis. The association between ATRX mutation and histopathological grading was tested using the chi-square test. A total of 39 glioma samples from patients with IDH-mutant status were included in this study. Of these, 26 were obtained from men (66.67%). The age range of all patients was 20–66 years. A total of 19 samples (48.72%) showed immunopositivity to ATRX. The bivariate analysis revealed that ATRX mutation was not associated with histopathological grading (P > 0.05). The ATRX mutations were not associated with IDH-mutant glioma grading in the present study.

Introduction

Glioma is a broad term describing neuroepithelial tumors originating from the glial or supporting cells of the central nervous system (CNS). Gliomas account for 24% of all primary CNS tumors and vary widely histologically, from benign astrocytoma tumors to extremely aggressive and fatal grade IV glioblastomas (1).

Molecular biology studies have provided new insights into diffuse glioma carcinogenesis and progression, prompting neurologists to rethink the biology of diffuse gliomas. Therefore, in 2016, the World Health Organization (WHO) updated the classification of diffuse gliomas, using a new classification that integrates traditional histological and novel molecular biological features to provide a consistent prognosis.

Mutations in the isocitrate dehydrogenase (IDH) gene are estimated to occur in 70%–90% of low-grade diffuse gliomas, specifically in astrocytomas and oligodendrogliomas, and affect the epigenetic regulation of the genome. They are also strongly associated with tumorigenesis and prognosis (2). Inactivating mutations of the alpha-thalassemia X-linked intellectual disability (ATRX) gene corresponds with the alternative telomere lengthening (ALT) phenotype and are strongly associated with IDH and tumor protein p53 (TP53) mutation; however, they are commonly exclusive with 1p19q-codeletion (3).

The ATRX protein is an essential component of the chromatin remodeling complex that functions at the telomere level. The ATRX gene encodes the protein at Xq13 (4). The loss of ATRX causes telomere instability and elongation, resulting in genetic instability (3). Mutations in the gene abolish nuclear protein expression in tumor cells but retain expression in non-tumor cells (endothelial and pre-existing glial cells), thus serving as a positive internal control. In glioblastoma, the expression of ATRX is different. Low ATRX expression was observed more frequently in primary glioblastoma and anaplastic glioma than in grade II glioma, suggesting it is a malignancy marker (2).

A study was conducted on the association of ATRX mutation with the histopathological classification of glioma (4). However, most reports are correlative rather than mechanical (4,5,6). The present study aimed to evaluate the association of ATRX mutation with the histopathologic grading of patients with IDH-mutant glioma.

Material and Methods

The present study assumes a cross-sectional approach to assessing the association of variable-dependent ATRX mutation and other variables, including age, sex, and lobe involvement, with histopathological grading in patients with IDH-mutant glioma. Ethical eligibility approval was obtained from the Faculty of Medicine Ethics Committee, Gadjah Mada University (number KE/FK/0540/EC 2022).

The study sample comprised patients diagnosed with glioma (astrocytomas and oligodendrogliomas) between January 2017 and March 2022 at Sardjito General Hospital, Yogyakarta, Indonesia. All patients signed an informed consent form before participating in the study. Two independent pathologists reviewed and reclassified histopathology samples and clinical data based on the 2021 WHO classification of CNS tumors. Clinical data on age, sex, and tumor location were collected from the patient’s medical records.

The IDH mutation status was determined using a polymerase chain reaction-sequencing method, allowing the detection of IDH1 or IDH2 mutations, as described previously (7). A total of 39 patients with IDH-mutant glioma were included in the study.

The ATRX mutations were evaluated using an immunohistochemical examination. Tissue sections with a thickness of 3 µm were obtained from formalin-fixed paraffinembedded (FFPE) blocks from all samples and stained with anti-ATRX polyclonal antibodies (Sigma Aldrich HPA001906). Immunoreactive cells were visualized using diaminobenzidine chromogen. The immunohistochemistry staining results were assessed by two observers using an Olympus CX23 microscope. The positive controls for ATRX were normal brain cells and endothelial cells. The area of necrosis and many inflammatory cells were not assessed. The immunohistochemistry results were evaluated using an eyeballing technique with a cut-point value of 10%. Gliomas with an ATRX gene mutation were defined when immunohistochemistry examination showed a negative nuclear expression of <10 % in tumor cells.

Statistical Analysis

The data obtained were evaluated using the Kappa test. Analysis of the association between the results of the ATRX immunohistochemistry examination and histopathological grade was performed using the chi-square test via a computerized statistical test because of the two category-scale variables. Existing data were presented in the form of text and tables. A P value of <0.05 was considered statistically significant.

Results

The present study involved patients with IDH-mutant glioma from Sardjito General Hospital, Yogyakarta. Clinically, the recruited patients experienced headaches (56%), seizures (21%), and other neurological deficits (32%). The radiologic results showed brain tumors located in the frontal lobe (46%), parietal lobe (38%), temporal lobe (28%), and brainstem (5%), with single lobe involvement in 59% of cases and multilobe involvement in 41% of cases. Histopathologically, most tumors were diagnosed as astrocytoma (74%), followed by oligodendroglioma (26%) Table 1. Most participants were men (66.67%) aged 20–66 years, with a mean age of 41.07. The complete characteristics of the participants are shown in Table 2.

The immunohistochemistry results in this study were assessed by two observers (pathologists); the coefficient value of Cohen’s Kappa for suitability between observers was 0.84. Representative images of the immunohistochemistry results are shown in Figure 1.

Figure 1. Results of ATRX immunohistochemistry examination. (A) IDH-mutant oligodendroglioma, WHO grade 2. (B) ATRX retained as >10% of tumor cell nuclei were positively stained (brown color). (C) Grade IV IDH-mutant astrocytoma. (D) ATRX loss due to cell nuclei not being positively stained (blue color; 400x magnification)
ATRX: Alpha-thalassemia X-linked intellectual disability, IDH: isocitrate dehydrogenase, WHO: World Health Organization

Bivariate analysis was conducted on independent variables of the ATRX mutation status assessed through immunohistochemistry examination and dependent variables, such as sex, age, and histopathological morphology. The chi-square test was performed to evaluate the association of each variable with the dependent variable histopathologic grading (Table 3).

The bivariate analysis showed no statistical association between patients’ sex, age, tumor location, ATRX mutation, and histopathological grading (P > 0.05).

Discussion

The present study used 39 FFPE specimens from patients with IDH-mutation glioma ranging from grade 2 to 4. IDH-mutant glioma samples were used because approximately 75%–90% of ATRX mutations were found in IDH-mutant gliomas (8).

In the present study, the bivariate analysis of the association of ATRX mutation with histopathological grading showed no significant association (P > 0.05). This finding is inconsistent with previous studies, which found that ATRX mutation was associated with glioma grading and was commonly found in lowgrade gliomas (4,5,6). This was because the mutation of ATRX and IDH occurred in the early stages of gliomagenesis (9). The incompatibility of the results with previous studies may be caused by the poor distribution of patients in the present study, which was dominated by patients with high-grade (grade 3 and 4) gliomas (n = 22).

Although ATRX mutations are more commonly found alongside IDH mutations occurring at the beginning of gliomagenesis, they are also found in patients with high-grade glioma, grade 3 covering astrocytoma, and secondary glioblastoma (grade 4 astrocytoma). Research has uncovered a significant correlation between canonical IDH mutations and ATRX mutation, allowing neuropathologists to discriminate between astrocytic and oligodendrocytic tumors (10).

In gliomas with IDH1/2 mutations, TP53 mutations are associated with ATRX changes. Most grade II/III gliomas with ATRX loss had IDH1/2 mutations; furthermore, TP53 mutation and p53 overexpression were significantly correlated with ATRX loss. According to a report revealing the relationship between ATRX, IDH1/2, and TP53 mutations (3), ATRX changes are likely required alongside TP53 mutations in IDH1/2-mutant astrocytomas (astrocytic lineage) to increase oncogenesis in adult gliomas.

This study is a single-institution study based in a referral hospital; thus, the sample has relatively low heterogeny. Further research involving patients without an IDH mutation status in various levels of health facilities is necessary.

Conclusion

There was no association between ATRX mutation, sex, age, involved lobes, or histopathological morphology with histopathological grading in patients with IDH-mutant glioma from the Sardjito General Hospital, Yogyakarta. ATRX mutations occur not only in low-grade gliomas but also in high-grade gliomas, which are secondary to low-grade gliomas. Although the mechanism of how ATRX mutations induce ALT is still unclear, it is believed that ATRX deficiency alone is insufficient for ALT induction. ATRX gene mutations are often found alongside IDH and TP53 mutations in gliomagenesis. ATRX, TP53, and IDH combined mutations stimulate neoplastic growth in astrocytomas.

Ethical eligibility approval was obtained from the Faculty of Medicine Ethics Committee, Gadjah Mada University (number KE/FK/0540/EC 2022).

Informed consent was taken from all participants.

Externally and internally peer-reviewed.

Surgical and Medical Practices: R.G.M., R.A.H., R.C., Concept: E.K.D., R.G.M., R.A.H., Design: E.K.D., R.G.M., Data Collection or Processing: E.K.D., R.C., A.S.A., Analysis or Interpretation: E.K.D., A.S.A., Literature Search: E.K.D., A.S.A., Writing: E.K.D., A.S.A., Y.P.K.

No conflict of interest was declared by the authors.

The authors declared that this study received no financial support.

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