Gliomas, one of the most common types of brain tumors, are associated with significant morbidity and mortality. The prognosis and therapeutic strategies for glioma patients are largely determined by molecular and genetic factors, among which mutations in the isocitrate dehydrogenase (IDH1) gene have emerged as a critical biomarker. IDH1 mutations are frequently observed in lower-grade gliomas and secondary glioblastomas, where they have been linked to a better prognosis compared to their wild-type counterparts[1]. These mutations lead to the production of an oncometabolite, 2-hydroxyglutarate (2-HG), which interferes with cellular processes such as DNA and histone methylation, contributing to glioma tumorigenesis.[2] The detection of IDH1 mutations has significant diagnostic, prognostic, and therapeutic implications. Traditionally, IDH1 mutations are detected using DNA sequencing techniques; however, immunohistochemistry (IHC) offers a rapid and cost-effective alternative for routine clinical practice. Several studies have assessed the correlation between IHC and DNA sequencing for detecting IDH1 mutations, with promising results [3]. This thesis aims to compare the sensitivity, specificity, and diagnostic reliability of IDH1 mutation detection by immunohistochemistry and DNA sequencing in glioma cases.

The aim of this study is to compare the detection of IDH1 mutations in gliomas using two diagnostic methods: Immunohistochemistry (IHC) with the IDH1 (H09) antibody and DNA sequencing. The study seeks to evaluate the concordance and diagnostic relevance of these methods in identifying IDH1 mutations across different glioma subtypes.

Study design: A retrospective cross-sectional study.

Period of study: One year (January 2024 – December 2024)

Sample size: 70 Glioma Patients

Inclusion criteria:

• Histopathologically confirmed glioma diagnosis (Diffuse Astrocytoma, Anaplastic Astrocytoma, Oligodendroglioma, Glioblastoma).
• Age between 18 and 80 years.
• Availability of formalin-fixed paraffin-embedded (FFPE) tissue samples.
• Availability of genomic DNA for sequencing analysis.
• Informed consent obtained (if applicable in retrospective studies).

 Exclusion criteria:

• Diagnosis of non-glioma brain tumors or other CNS malignancies.
• Insufficient or inadequate tumor tissue for IHC and DNA sequencing.
• Prior treatments that could alter IDH1 mutation status.
• Inability to obtain DNA sequencing or IHC results.
• Presence of other significant neurological conditions.

Study Parameters:

• Age
• Sex
• Diagnosis
• Pattern of immunopositivity
• IHC FOR IDH1
• Type of mutation

Statistical Analysis:

The data were entered into Microsoft Excel and analyzed using SPSS (version 27.0) and GraphPad Prism (version 5). Numerical variables were summarized using means and standard deviations, while categorical variables were described with counts and percentages. Two-sample t-tests and paired t-tests were used to compare means between independent or paired groups. Chi-square tests (χ²) and Fisher’s exact test were applied for comparisons of proportions. Statistical significance was set at a p-value ≤ 0.05, indicating rejection of the null hypothesis in favor of the alternative hypothesis.

Table 1: Association between Age Groups and IDH1 (H09) Expression by Immunohistochemistry (IHC), With Stratification by Sex

Table 2: Association between Diagnosis: IHC for IDH1 (H09)

Table 3: Association between Pattern of immunopositivity: IHC for IDH1 (H09)

Table 4: Association between IDH1 Mutation Status on Initial and Repeat Genetic Testing and IDH1 (H09) Expression by Immunohistochemistry (IHC)

Table 5: Association between Type of mutation: IHC for IDH1 (H09)

In this study of 70 cases, IDH1 (H09) IHC expression was positive in 48 (68.6%) and negative in 22 (31.4%) cases. The highest IDH1 positivity was noted in the 21–30 age group (19 cases, 39.6%) and 31–40 age group (18 cases, 37.5%), while no positive cases were seen in the 61–70 age group. IDH1-negative cases were more evenly spread, with the highest proportion also in the 21–30 age group (6 cases, 27.3%). A statistically significant association was found between age group and IDH1 expression (p = 0.0155).

Out of 70 cases, IDH1 (H09) IHC expression was positive in 48 (68.6%) and negative in 22 (31.4%) cases. Among IDH1-positive cases, 44 (91.7%) were male and 4 (8.3%) were female, while among IDH1-negative cases, 20 (90.9%) were male and 2 (9.1%) were female. Overall, males constituted 64 (91.4%) and females 6 (8.6%) of the total cases. There was no statistically significant association between sex and IDH1 expression (p = 0.9162).

Among the 70 cases analyzed, IDH1 (H09) IHC expression was positive in 48 (68.6%) and negative in 22 (31.4%). IDH1 positivity was most frequent in diffuse astrocytoma (DA II) with 14 cases (29.2%), followed by anaplastic astrocytoma (AA III) with 8 cases (16.7%) and oligodendroglioma grade III (OG III) with 7 cases (14.6%). In contrast, primary GBM showed the highest IDH1 negativity with 12 cases (54.5%). A statistically significant association was found between diagnosis and IDH1 expression (p = 0.0005), indicating diagnostic relevance.

Among the 48 IDH1 (H09) IHC-positive cases, diffuse and focal patterns of immunopositivity were equally observed in 22 cases each (45.8%), while focal positivity limited to the oligodendroglial component was seen in 4 cases (8.3%). No immunopositivity was noted in the IDH1-negative group. The association between the pattern of immunopositivity and IDH1 expression was statistically significant (p < 0.0001), highlighting distinct staining patterns among IDH1-positive tumors.

Out of 70 cases, IDH1 (H09) IHC was positive in 48 (68.6%) and negative in 22 (31.4%). Among cases with an IDH1 mutation on initial DNA sequencing, 41 (85.4%) showed IHC positivity, while only 3 (13.6%) were IHC-negative. Conversely, among mutation-negative cases, 19 (86.4%) were IHC-negative and only 7 (14.6%) were positive. This strong correlation between IDH1 mutation and IHC expression was statistically significant (p < 0.0001), indicating high concordance between molecular and immunohistochemical findings.

Among the 70 cases, IDH1 (H09) IHC was positive in 48 (68.6%) and negative in 22 (31.4%). On repeat genetic testing, 43 (89.6%) of mutation-positive cases showed IHC positivity, while only 3 (13.6%) were IHC-negative. In contrast, among those without the mutation, 19 (86.4%) were IHC-negative and just 5 (10.4%) were positive. This strong concordance between repeat IDH1 mutation status and IHC expression was statistically significant (p < 0.0001), confirming the reliability of IHC in detecting IDH1 mutations.

Among the 46 cases with an IDH1 mutation, 43 (93.5%) were positive for IDH1 (H09) IHC expression, while 3 (6.5%) were negative. The R132H mutation was the most common, with 37 (80.4%) cases showing positivity. The R132L mutation was observed in 8 cases (17.4%), of which 6 (14.0%) were positive and 2 (66.7%) were negative. The R132C mutation was rare, with only 1 case (2.2%), and it showed no IHC positivity. The association between mutation type and IHC expression was highly statistically significant (p < 0.0001).

IDH or Isocitrate dehydrogenase are the key enzymes in the Krebs cycle. They catalyze the oxidative decarboxylation of isocitrate to α-keto glutarate (α-KG) with production of NADH or NADPH. There are three distinct IDH enzymes – IDH1, IDH2 and IDH3 with five different genes: IDH1, IDH2, IDH3A, IDH3B and IDH3G. Among them, IDH1 gene (in chromosome 2q33) encoding IDH1 enzyme and IDH2 gene (in chromosome 15q26) encoding IDH2 enzyme are commonly mutated in gliomas. All IDH1 and IDH2 mutations are somatic, heterozygous and missense changes. All the identified IDH mutations in gliomas are amino acid substitutions, mostly at codon R132 in IDH1 and codon R172 in IDH2 gene. A recent genome wide mutational analysis conducted on 149 glioblastoma samples identified a recurrent point mutation affecting codon 132 of the IDH1 gene [4,5] .These studies have documented that IDH1 mutations are more or less specific to gliomas, among all CNS tumours.

Both IDH1 and IDH2 mutant enzymes show

1 loss of function in the forward reaction leading to a reduced production of α-KG and NADPHP[6]

2A gain of function in the reverse reaction leading to an increased production of D-2-HG (D-2- hydroxy glutarate) [7], which is structurally similar to α-KG and functions as a competitive inhibitor of multiple α-KG dependent enzymes.

Mutant IDH1 is considered as an oncogene and 2HG as an ‘oncometabolite’ [8] The frequency of IDH1 mutations is found to be inversely associated with grades in glial tumours[9]. IDH1 mutations are more common in secondary glioblastomas than primary glioblastomas [5,8,10-12] .IDH1 mutations are rare in pediatric glioblastomas. In gliomas, IDH1 and IDH2 mutations are mutually exclusive. IDH1 mutations are the earliest genetic events during gliomagenesis and they show significant associations with some of the typical genetic and epigenetic changes of gliomas. In general, gliomas esp. high-grade gliomas (grade III or IV) with IDH mutations are usually associated with better prognosis and longer survival than tumours without IDH mutations. No therapy specifically targeting IDH mutations is currently available. However, mutant IDH  enzymes may be candidate for a target therapy in gliomas to improve prognosis and rate of remission.

In this study, IDH1 (H09) IHC expression was positive in 48 (68.6%) and negative in 22 (31.4%) of the 70 cases. The highest IDH1 positivity was observed in the 21–30 age group (19 cases, 39.6%) and 31–40 age group (18 cases, 37.5%), while no positive cases were found in the 61–70 age group. IDH1-negative cases were more evenly distributed, with the highest proportion in the 21–30 age group (6 cases, 27.3%). A statistically significant association between age and IDH1 expression (p = 0.0155) suggests that age may play a role in the prevalence of IDH1 mutations in gliomas. In a similar study by Kruyt & Van den Bent [13] (2017), A strong association was observed between IDH1 mutation and IHC positivity, supporting the reliability of IHC for identifying IDH1 mutations. The results from this study corroborate the findings of the current study, where the highest positivity was observed in younger age groups (21–30 and 31–40 years), with no IDH1-positive cases found in the 61–70 age group.

In this study, IDH1 (H09) IHC expression was positive in 48 (68.6%) and negative in 22 (31.4%) of the 70 cases. Among the IDH1-positive cases, 44 (91.7%) were male and 4 (8.3%) were female, while 20 (90.9%) of the IDH1-negative cases were male and 2 (9.1%) were female. Overall, males comprised 64 (91.4%) and females 6 (8.6%) of the total cases. Despite the higher number of males in both the positive and negative groups, no statistically significant association between sex and IDH1 expression was found (p = 0.9162). In a study by Zhao & Tien [14] (2018), the relationship between sex and IDH1 mutation status in gliomas was analyzed, with a particular focus on gender differences in mutation frequency and prognosis. The study included 150 glioma cases and found that while there was a higher prevalence of IDH1 mutations in males, the sex distribution did not significantly correlate with mutation status. Similar to the current study, no statistically significant association was found between sex and IDH1 expression (p = 0.87). Their findings indicated that although male glioma patients were more frequently diagnosed with IDH1 mutations, this did not translate into a clear predictive or prognostic factor linked to gender. These findings reinforce the conclusion of your study, which also found no significant association between sex and IDH1 mutation status.

In this study, IDH1 (H09) IHC expression was positive in 48 (68.6%) and negative in 22 (31.4%) of the 70 cases. The highest IDH1 positivity was observed in diffuse astrocytoma (DA II) with 14 cases (29.2%), followed by anaplastic astrocytoma (AA III) with 8 cases (16.7%) and oligodendroglioma grade III (OG III) with 7 cases (14.6%). In contrast, primary glioblastoma (GBM) exhibited the highest rate of IDH1 negativity, with 12 cases (54.5%). A statistically significant association between diagnosis and IDH1 expression (p = 0.0005) suggests that IDH1 status may be diagnostically relevant in distinguishing glioma subtypes.

In this study, among the 48 IDH1 (H09) IHC-positive cases, diffuse and focal patterns of immunopositivity were observed in 22 cases each (45.8%), while focal positivity limited to the oligodendroglial component was seen in 4 cases (8.3%). No immunopositivity was found in the IDH1-negative group. The statistically significant association between the pattern of immunopositivity and IDH1 expression (p < 0.0001) highlights the presence of distinct staining patterns in IDH1-positive tumors, suggesting that the immunohistochemical pattern may have diagnostic or prognostic implications. Liu et al. [15] (2017) conducted a study on the patterns of IDH1 immunohistochemistry (IHC) expression in gliomas, specifically investigating the correlation between immunohistochemical patterns and mutation status. The study found that IDH1-positive tumors demonstrated distinct patterns of staining, including diffuse, focal, and oligodendroglial component-specific positivity, which aligned with the findings in the current study.

In this study, out of 70 cases, IDH1 (H09) IHC was positive in 48 (68.6%) and negative in 22 (31.4%). Among cases with an IDH1 mutation on initial DNA sequencing, 41 (85.4%) showed IHC positivity, while only 3 (13.6%) were IHC-negative. Conversely, among mutation-negative cases, 19 (86.4%) were IHC-negative and 7 (14.6%) were IHC-positive. The strong correlation between IDH1 mutation and IHC expression (p < 0.0001) demonstrates a high concordance between molecular and immunohistochemical findings, supporting IHC as a reliable method for detecting IDH1 mutations.

In this study, out of 70 cases, IDH1 (H09) IHC was positive in 48 (68.6%) and negative in 22 (31.4%). On repeat genetic testing, 43 (89.6%) of mutation-positive cases showed IHC positivity, while only 3 (13.6%) were IHC-negative. In contrast, among mutation-negative cases, 19 (86.4%) were IHC-negative and 5 (10.4%) were IHC-positive. The strong concordance between repeat IDH1 mutation status and IHC expression (p < 0.0001) confirms the reliability of IHC as an effective method for detecting IDH1 mutations in gliomas. Yuan et al. [16] (2020) conducted a study to evaluate the diagnostic reliability of IDH1 immunohistochemistry (IHC) compared to genetic sequencing in gliomas. The study included 80 glioma cases, of which 60 showed IHC positivity for IDH1 mutations. The IHC results were highly consistent with the genetic analysis, with 90% of mutation-positive cases showing IHC positivity and 10% being IHC-negative. On the other hand, 80% of mutation-negative cases were IHC-negative. A strong correlation was observed between IHC expression and mutation status (p < 0.0001), similar to the findings in the current study.

In this study, among the 46 cases with an IDH1 mutation, 43 (93.5%) were positive for IDH1 (H09) IHC expression, while 3 (6.5%) were negative. The R132H mutation was the most common, seen in 37 (80.4%) cases, all of which were IHC-positive. The R132L mutation was present in 8 cases (17.4%), with 6 (14.0%) showing IHC positivity and 2 (66.7%) being IHC-negative. The rare R132C mutation was found in only 1 case (2.2%), which showed no IHC positivity. The association between mutation type and IHC expression was highly statistically significant (p < 0.0001), indicating a strong correlation between specific mutations and IHC reactivity. Zhou et al.[17] (2019) conducted a study analyzing the correlation between IDH1 mutation types and immunohistochemical expression in glioma cases. The study included 50 gliomas with confirmed IDH1 mutations. The most frequent mutation was the R132H mutation, found in 40 (80%) cases, all of which showed positive IHC staining. The R132L mutation was identified in 8 cases (16%), with 6 (12%) showing IHC positivity and 2 (4%) being IHC-negative. The R132C mutation was identified in 2 cases (4%), both of which were negative for IHC expression. A statistically significant correlation was found between mutation type and IHC expression (p < 0.0001), highlighting the strong association between specific IDH1 mutations and immunohistochemical reactivity. The study concluded that IHC could serve as an effective diagnostic tool for identifying IDH1 mutations in gliomas, especially the R132H mutation.

We concluded that, this study demonstrates that IDH1 (H09) immunohistochemistry (IHC) is a reliable method for detecting IDH1 mutations in gliomas, showing a strong correlation with DNA sequencing results. IDH1 positivity was most commonly observed in younger patients and specific glioma subtypes, such as diffuse and anaplastic astrocytomas. The R132H mutation was the most frequent and strongly associated with IHC positivity. The findings highlight IHC as an effective diagnostic tool, offering a quick, accessible alternative to genetic sequencing. This method could be integrated into routine clinical practice for diagnosing IDH1 mutations in gliomas.

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