Classification of Glioblastoma Based on Immunohistochemical Expression of IDH-1, p53, and ATRX: A Study from a Tertiary Care Center in South India

Abstract

Introduction

Glioblastoma (GBM) is the most common malignant brain tumor in adults characterized by an extremely aggressive clinical phenotype. Understanding the molecular markers involved in GBM is critical for development of more effective and targeted therapies. The present study attempts to study the prevalence of immunohistochemical expression of IDH-1, p53, and ATRX in GBMs in our population.

Materials and Methods

The present study is a descriptive cross-sectional study conducted in the department of pathology, on a total of 95 cases of GBM during the study period of 5 years. The immunohistochemical testing was done and evaluated.

Results

Out of 95 cases of GBM, 74 were IDH wild-type (IDH-1 − ) and 21 were IDH mutant (IDH-1 + ). In our study, 37.9% of cases showed p53 overexpression (p53 + ) and 46.3% of cases showed loss of ATRX expression (ATRX − ). A statistically significant association was found in the expression of p53 and ATRX among IDH mutant cases on combined three-protein analysis (p-value = 0.01). Majority of them were p53 negative (p53 − ) and showed ATRX loss. Among IDH wild-type cases, majority were negative for p53 with retained ATRX expression (ATRX + ), which was in concordance with other studies.

Conclusion

These findings suggest that the identification of combinations of these protein markers may be useful for the classification and thus it may help to provide a deeper understanding of the molecular heterogeneity and aggressiveness of GBM and may help in the development of targeted therapies.

Keywords

ATRX - glioblastoma - IDH-1 - immunohistochemistry - p53

Introduction

Glioblastoma (GBM) is recognized as the most aggressive type of diffuse astrocytic glioma, characterized histologically by features such as nuclear atypia, cellular pleomorphism, brisk mitotic activity, microvascular proliferation, and/or necrosis. Brain tumors affect approximately 17,000 individuals annually, with gliomas accounting for nearly 60% of these cases.[1] Among gliomas, GBM is the most frequently diagnosed, showing a higher incidence in males.[1] It is associated with a dismal prognosis, and most patients succumb within a year of diagnosis.[2]

Although GBMs often arise sporadically, familial forms have also been documented. These tumors predominantly develop in the supratentorial compartment—especially the frontal lobe—and their highly infiltrative nature makes them difficult to distinguish from adjacent normal brain parenchyma.[3] In recent years, diagnostic approaches have expanded beyond histology to include immunohistochemistry (IHC), molecular pathology, and biomarker profiling, all of which now play a pivotal role.[4]

According to existing data, around 60% of GBMs originate de novo, while the remaining 40% evolve from lower grade gliomas such as diffuse or anaplastic astrocytomas, often over a period of 4 to 5 years.[5] These tumors commonly show altered expression of key regulatory genes and proteins, including ATRX (alpha-thalassemia/mental retardation syndrome X-linked), p53, and IDH-1 (isocitrate dehydrogenase 1).[1]

The significance of integrating molecular data with histopathological findings was emphasized in the revised 2016 World Health Organization (WHO) classification of central nervous system (CNS) tumors. Accurate classification now requires both morphological assessment and molecular profiling, as tumors with similar histology may behave differently depending on their genetic makeup and response to treatment.[6]

Standard treatment involves surgical resection followed by radiotherapy and chemotherapy. Additional strategies, including integrin inhibitors and immunotherapy, are under investigation. However, prognosis continues to depend on multiple variables, such as anatomical site, histological grade, molecular characteristics, proliferative index, and patient age. Although there has been a surge in research investigating the biology and treatment of GBM, comprehensive clinical analysis remains limited.[3] [7] [8]

Despite aggressive multimodal therapy, recurrence is common due to the molecular heterogeneity of GBM. Therefore, profiling protein expression of key molecular markers such as IDH-1, p53, and ATRX via IHC is crucial for subclassification and understanding tumor biology.

This study aims to classify GBM cases using an IHC-based algorithm, in accordance with the 2016 WHO classification of CNS tumors, which was the standard during the study period (March 1, 2016–March 1, 2021).

Objectives of the Study

To subclassify GBM cases based on immunohistochemical expression patterns of IDH-1, p53, and ATRX.

Materials and Methods

This was a descriptive cross-sectional study conducted on histologically confirmed GBM cases received in the Department of Pathology over a 5-year period.

Inclusion Criteria

All newly diagnosed GBM cases (WHO Grade IV), classified as per the 2016 WHO classification of CNS tumors.

Exclusion Criteria

Recurrent GBMs were excluded, as comprehensive data on their primary tumor characteristics were often unavailable.

Tissue Processing and Immunohistochemistry

Representative tissue sections from tumor specimens were selected, processed, and stained with hematoxylin and eosin (H&E). Cases diagnosed as GBM on histopathology were included for further immunohistochemical analysis using antibodies against IDH-1, p53, and ATRX. IDH-1 showed cytoplasmic staining. It was assessed and categorized as either positive or negative. Antibody used was IDH-1 R132H (Clone QMOO2), a mouse monoclonal from Quartett. ATRX which showed nuclear staining was evaluated and classified as retained (positive in >10% of cells) or lost . Antibody used was Anti-ATRX, a rabbit polyclonal IgG (HPA001906) from Sigma Life Science-Prestige.

p53 showed nuclear staining which was semi-quantitatively scored based on the proportion of positive nuclei, using a four-tiered scale ([Table 1]). Antibody used was Anti-p53, a rabbit monoclonal (Clone QR025) from Quartett.

Table 1 Scoring of p53

Description	Score	Result
<10% of nuclei stained	0	Negative
10–30% of nuclei stained	1+	Positive
30.1–50% of nuclei stained	2+	Positive
>50% of nuclei stained	3+	Positive

Statistical Analysis

Data entry was performed using Microsoft Excel, and statistical analyses were conducted using SPSS software (version 23). A p-value less than 0.05 was considered statistically significant.

Results

This study aimed to classify GBM cases based on immunohistochemical markers as per the 2016 WHO classification of CNS tumors.

A total of 95 patients with histologically confirmed GBM were included. The mean age of the study population was 50.2 years, ranging from 10 to 70 years, with 53 patients above 50 years. The cohort comprised 56 males and 39 females.

Immunohistochemical Expression of IDH-1, p53, and ATRX

IDH-1 positivity was observed in 21 patients (22.1%). p53 overexpression was noted in 36 patients (37.9%). ATRX loss of expression was seen in 44 patients (46.3%; [Table 2]).

Table 2 Immunoexpression of IDH-1, p53, and ATRX in enrolled GBMs

IHC marker	Positive	Negative
IDH-1	21 (22.1%)	74 (77.9%)
P53	36 (37.9%)	59 (62.1%)
ATRX	51 (53.7%)	44 (46.3%)

Abbreviation: GBM, glioblastoma.

Analysis of Two-Marker Combinations

[Table 3] summarizes the results of dual-marker IHC. The most frequent IDH-1/ATRX combination was IDH-1–/ATRX+ (44.2%). For IDH-1/p53, IDH-1–/p53–(50.5%) was most common. Least prevalent patterns included IDH-1 +/ATRX+ (9.5%) and IDH-1 +/p53+ (10.5%). Statistical analysis showed no significant association between expression patterns in the two-marker combinations.

Table 3 Immunohistochemical results of various combinations of two protein pairs

IHC	Two-protein pairs	No. of cases (%)	p-Value
IDH-1/ATRX	IDH-1 +/ATRX−	12 (12.6%)	0.509
	IDH-1 +/ATRX+	9 (9.5%)
	IDH-1 −/ATRX−	32 (33.7%)
	IDH-1 −/ATRX+	42 (44.2%)
IDH-1/p53	IDH-1 +/p53+	10 (10.5%)	0.298
	IDH-1 +/p53−	11 (11.6%)
	IDH-1 −/p53+	26 (27.4%)
	IDH-1 −/p53−	48 (50.5%)
p53/ATRX	p53 +/ATRX−	13 (13.6%)	0.11
	p53 +/ATRX+	23 (24.2%)
	p53 −/ATRX−	31 (32%)
	p53 −/ATRX+	28 (29.4%)

Analysis of Three-Marker Combinations

[Table 4] outlines the results of combined expression for IDH-1, p53, and ATRX. The most common profile was IDH-1–/p53–/ATRX + , seen in 27.3% of cases ([Fig. 1]). The least common profile, IDH-1 +/p53 +/ATRX–, was found in only 3.1% of cases ([Fig. 2]). A statistically significant correlation was found between p53 and ATRX expression in IDH-1 mutant tumors (p = 0.01).

Table 4 Immunohistochemical results of various combinations of 3 proteins

IDH-1	p53	ATRX	Frequency	%	p-Value
IDH-1+	p53+	ATRX−	3	3.15	0.01
	p53+	ATRX+	7	7.4
	p53−	ATRX−	9	9.5
	p53−	ATRX+	2	2.1
IDH-1−	p53+	ATRX−	10	10.5	0.54
	p53+	ATRX+	16	16.8
	p53−	ATRX−	22	23.1
	p53−	ATRX+	26	27.3

Fig. 1 The figure shows representative pictures of one of the GBM cases with IDH-1-/p53-/ ATRX + . (A) H&E (20 × ), (B) IDH-1-, (C) p53-, (D) ATRX + , (B–D) 40 × . GBM, glioblastoma.

Fig. 2 The figure shows representative pictures of one of the GBM cases with IDH-1 +/ p53 +/ATRX-. (A) H&E (20 × ), (B) IDH-1 + , (C) p53 score 2, (D) ATRX-, (B–D) 40 × . GBM, glioblastoma.

Histological Subtypes of Glioblastoma

The distribution of histological subtypes is shown in [Table 5]. Classic GBM was the most common histological subtype ([Fig. 3]).

Table 5 Morphological subtypes of GBM cases

Morphological subtypes	Frequency	Percentage
Classical GBM	71	74.7
Giant cell GBM	18	18.9
Gemistocytic GBM	2	2.1
Oligodendroglial	1	1.05
Primitive neuronal	1	1.05

Abbreviation: GBM, glioblastoma.

Fig. 3 The figure shows (A) microvascular proliferation (B) pseudopalisading necrosis in classical GBM (A, B) 20 × . GBM, glioblastoma.

A total of 18 cases were giant cell GBM ([Fig. 4A]), 2 cases were gemistocytic GBM, 1 case was GBM with primitive neuronal component ([Fig. 4B]), and 1 case was GBM with oligodendroglial component.

Fig. 4 (A) Giant cell glioblastoma (20 × ) and (B) GBM with primitive neuronal component (40 × ). GBM, glioblastoma.

Among giant cell GBM cases, 72.2% were IDH-1 negative, 61.1% showed p53 positivity, and 55.5% had loss of ATRX expression.

Discussion

GBM is the most malignant and aggressive primary brain tumor, known for its resistance to standard treatment modalities. This resistance is largely attributed to the tumor's molecular heterogeneity, involving variations in key regulatory genes. Our study aimed to assess the immunohistochemical expression of IDH-1, p53, and ATRX in GBM cases, and to evaluate marker combinations that could effectively classify tumors based on their molecular profile as per the 2016 WHO classification of CNS tumors.

Out of 95 GBM cases analyzed, 56 (59%) were males and 39 (41%) were females, closely aligning with the findings of Chaurasia et al, who reported a similar male predominance (58.2%) in their cohort of 163 cases.[1] Lee et al observed an equal gender distribution (75 males and 75 females out of 150 cases).[2]

Regarding patient age, our cases ranged from 10 to 70 years with a mean of 50.2 years. This finding corresponds to studies by Chaurasia et al (mean: 49.4 years; range: 21–79)[1] and Lee et al (mean: 58.5 years; range: 19–85).[2]

In terms of IDH-1 expression, we found 22.1% positivity, which was higher than those of Chaurasia et al (10.4%)[1] and Lee et al (11.1%),[2] but comparable to that of Dahuja et al, who reported 31.1% positivity in a smaller cohort of Indian patients.[4]

p53 overexpression was identified in 37.9% of cases in our study, aligning closely with Dahuja et al (40%).[4] Higher rates were reported by Chaurasia et al (48.4%) and Lee et al (49.5%).[1] [2]

Loss of ATRX expression was seen in 44 cases (46.3%). This was higher than the 15.3% reported by Chaurasia et al,[1] 4.8% by Gülten et al,[9] and 24.4% by Dahuja et al.[4] Other studies such as Reuss et al (18%)[10] and Liu et al (26%)[11] have shown intermediate values. These findings reinforce the role of IHC in detecting ATRX mutations, as it correlates well with molecular alterations.[12] [13] [14]

In the analysis of dual-marker combinations, IDH-1–/p53–was the most prevalent pattern (50.5%), similar to Chaurasia et al (46%).[1] This may be due to limitations of IHC in detecting non-R132H mutations of IDH-1, which require gene sequencing for confirmation. Also, not all TP53 mutations lead to protein accumulation; nonsense mutations may yield negative IHC results.

The combination IDH-1–/ATRX+ was seen in 44.2% of our cases, whereas Chaurasia et al found this pattern in 78.5% of cases. For p53 and ATRX, our data showed that 29.4% had p53–/ATRX + , compared to 42.9% in Chaurasia et al's cohort.[1] These dual-marker combinations did not yield statistically significant associations in our study.

When evaluating all three markers (IDH-1, p53, ATRX) together, we identified eight molecular profiles. The most frequent was IDH-1–/p53–/ATRX + , seen in 27.3% of cases, mirroring Chaurasia et al's result, where 39% had the same profile. Among IDH-mutant cases, IDH-1 +/p53–/ATRX– was the most common combination in our series (9.5%), higher than the 2.5% reported by Chaurasia et al.[1] A statistically significant association between p53 and ATRX expression in IDH-1 mutant cases was observed (p = 0.01), in line with other reports.

Regarding histological subtypes, the classical GBM was most common, followed by giant cell GBM. In giant cell GBMs, most cases were IDH-1-negative, p53-positive, and showed ATRX loss. These findings are consistent with Cantero et al, who noted IDH-1 negativity in 97.2%, p53 expression in 89%, and ATRX loss in 24% of giant cell GBM or GBMs with a giant cell component.[15]

Conclusion

Our study revealed distinct immunohistochemical expression patterns among GBM subtypes. Among IDH-wild-type GBMs, the predominant phenotype included negative p53 expression with retained ATRX staining. In contrast, IDH-mutant tumors most frequently exhibited negative p53 expression along with ATRX loss.

These patterns suggest that analyzing the combined expression of IDH-1, p53, and ATRX proteins may provide meaningful subclassification of GBM cases. This approach can aid in diagnosis, influence treatment strategies, and may potentially offer prognostic value.

Our findings contribute to a deeper understanding of the molecular diversity and biological aggressiveness of GBM. By identifying molecular profiles through IHC, this study supports efforts to develop more targeted and individualized treatment options.

Limitations

A key limitation of this study is its relatively small sample size. Larger studies involving broader populations are essential to validate and generalize these findings. Additionally, while IHC serves as a practical tool in resource-limited settings, it cannot detect all forms of IDH and TP53 mutations—particularly non-R132H IDH-1 variants, IDH-2 mutations, and null mutations of p53. These require molecular confirmation via gene sequencing, which was not feasible in our setting.

Future research incorporating the 2021 WHO classification and molecular diagnostic techniques will be crucial for refining GBM subtyping, enhancing prognostication, and improving therapeutic outcomes.

Conflicts of Interest

None declared.

Ethical Approval Statement

Ethical approval was obtained from the Institutional Ethics Committee prior to the initiation of the study.

• References

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Publikationsverlauf

Artikel online veröffentlicht:
19. August 2025