Mismatch Repair Deficiency in Gliomas: A Rare Insight into Microsatellite Instability and Its Diagnostic Implications

Abstract

Objective

Mismatch repair deficiency (MMRD), a hallmark of microsatellite instability (MSI), has been extensively studied in gastrointestinal and endometrial cancers but remains underexplored in gliomas. Deficiencies in mismatch repair (MMR) proteins, such as MLH1, MSH2, MSH6, and PMS2, may contribute to tumor progression, treatment resistance, and responsiveness to immune checkpoint inhibitors. This study aimed to evaluate the expression of MMR proteins in gliomas using immunohistochemistry (IHC) and analyze their association with patient age, histological subtype, and central nervous system (CNS) World Health Organization (WHO) (2021) tumor grade.

Materials and Methods

A total of 64 glioma cases were retrospectively analyzed, including a range of histologic subtypes and grades. IHC for MLH1, MSH2, MSH6, and PMS2 was performed to detect MMR protein expression. Cases showing MMR deficiency by IHC were further evaluated using next-generation sequencing (NGS) for MSI and frameshift mutations in MMR genes. Statistical analyses were conducted to assess associations with clinicopathological parameters.

Analysis

Quantitative variables were expressed as mean and standard deviation. Quantitative variables were expressed as percentage or proportion. Chi-square test and Fisher's exact test were done to associate MMR protein deficiency with age, histopathological type, and CNS WHO grade of glioma. p-Value of <0.05 was considered significant.

Results

MMR deficiency was observed in 3 of 64 cases (4.69%), all showing isolated loss of MSH6 expression. These included two IDH-mutant astrocytomas and one pilocytic astrocytoma. No significant associations were found between MMRD and age (p = 1.000), histological subtype (p = 0.448), or WHO grade (p = 0.448). NGS revealed one MSI-high and one MSI-low tumor, both harboring frameshift mutations in multiple MMR genes.

Conclusion

MMR deficiency is rare in gliomas, with isolated MSH6 loss being the most common finding. While not significantly associated with tumor grade or patient demographics, MMRD may have clinical relevance in specific subgroups. NGS findings highlight the potential utility of integrating molecular diagnostics for identifying MSI and guiding immunotherapy decisions.

Keywords

mismatch repair protein - microsatellite instability - glioma - immunohistochemistry - astrocytoma

Introduction

Central nervous system (CNS) malignancies are among the top 10 causes of cancer-related deaths globally, with an incidence of 8 to 10 cases per 100,000 people each year.[1] [2] Among these, gliomas are the most common primary malignant brain tumors in adults, accounting for over 80% of cases.[3] Glioblastoma, the most aggressive glioma subtype, is marked by rapid progression and a poor median survival of only 15 months.[4]

The mismatch repair (MMR) system is a vital DNA repair pathway that maintains genomic stability by correcting base–base mismatches and insertion–deletion loops arising during DNA replication.[5] It relies on key genes—MLH1, PMS2, MSH2, and MSH6—which form functional protein complexes. Specifically, MSH2 pairs with MSH6 (MutSα) to recognize mismatches, while MLH1 partners with PMS2 (MutLα) to carry out the repair process.[6] [7] [8] When these proteins are deficient or dysfunctional, replication errors accumulate, resulting in a hypermutated phenotype and significantly increased risk of tumor development.[5] Microsatellites are short, repetitive DNA sequences found throughout the genome, typically made up of 1 to 6 base pair motifs repeated 10 to 60 times.[7] When MMR genes are mutated, replication errors accumulate at these sites, leading to microsatellite instability (MSI). MSI is classified as MSI-high (MSI-H), MSI-low (MSI-L), or microsatellite stable based on the number of unstable loci detected.[9] MSI is a hallmark of MMR deficiency (MMRD), resulting from defective DNA repair that causes insertions or deletions in microsatellite regions.[5] This phenomenon was first discovered in hereditary nonpolyposis colorectal cancer, also known as Lynch syndrome.[10] Since then, MSI has been identified in several other cancers, including endometrial, gastric, pancreatic, brain, biliary tract, urinary tract, and ovarian tumors, making it a valuable molecular marker for various malignancies.[11]

MMRD can be identified using immunohistochemistry (IHC), which assesses the loss of nuclear expression of MMR proteins such as MLH1, PMS2, MSH2, and MSH6. IHC offers several advantages: it is faster, easier to perform, and allows for the direct identification of the affected MMR protein, facilitating targeted genetic testing.[12] However, to definitively identify the causative mutation, sequencing of the MMR genes is necessary.[13]

Unlike the well-established data on MMRD in colorectal and endometrial cancers, its prevalence in gliomas is poorly understood, with reported rates varying from 0 to 44% in literatures. MMRD appears more frequently in pediatric high-grade gliomas (pHGGs) than in adults. However, its clinical significance in gliomas is still uncertain, with studies offering inconsistent findings on its prognostic and predictive value.[6]

Some evidence links MMRD in gliomas to resistance against alkylating agents like temozolomide (TMZ), suggesting that MMR status may influence treatment decisions. Despite both MMRD and immune checkpoint molecules (PD-1/PD-L1) being biomarkers for immunotherapy response, no consistent relationship between them has been observed in gliomas. Identifying MMRD is crucial for diagnosing constitutional MMRD (CMMRD) syndrome, and it may support precision oncology approaches. These include avoiding TMZ in CMMRD-related gliomas, considering PD-1 inhibitors in resistant cases, and initiating family genetic counseling and surveillance programs.[6]

In view of the above context, the present study was undertaken to evaluate MMRD in gliomas through IHC analysis of key MMR proteins. Additionally, the study aimed to investigate the association of MMRD with patient age, histopathological subtype, and central nervous system (CNS) World Health Organization (WHO) 2021 tumor grade. Next-generation sequencing (NGS) was performed on cases demonstrating MMRD by IHC for further confirmation.

Materials and Methods

This cross-sectional study was conducted at a tertiary care center following approval from the Institutional Ethics Committee. Written informed consent was obtained from all participants. A total of 64 consecutive cases of gliomas, diagnosed over a period of 18 months, were included. All histopathologically proven cases were evaluated for histological subtype and classified according to the CNS WHO 2021 grading system. IHC analysis for IDH1, ATRX, TP53, and Ki67 was performed to support and confirm the histopathological diagnosis.

IHC for MMR proteins—MLH1, MSH2, MSH6, and PMS2—was performed using the following primary antibodies: anti-MLH1 mouse monoclonal antibody (BioGenex), anti-MSH2 rabbit monoclonal antibody (BioGenex), anti-MSH6 rabbit monoclonal antibody (BioGenex), and anti-PMS2 rabbit monoclonal antibody (BioGenex). Lymphocytes, stromal cells, and blood vessels served as internal controls, while colonic epithelium was used as the external control. Tumors were considered MMR-proficient if there was nuclear positivity (even focal) in tumor cells for all four MMR proteins. MMRD was defined as complete loss of nuclear staining for at least one MMR protein in tumor cells, with intact staining in both internal and external control tissues.[6] We used immunoreactivity score for interpretation of MMR protein expression ([Table 1]).

Table 1 Immunoreactivity score for IHC interpretation of MMR proteins

Score	IHC expression of MLH1, MSH2, MSH6, and PMS2
−	Tumor cells without staining.
+	Tumor cells without nuclear staining but positivity in cytoplasm or with <5% tumor cells with nuclear staining.
++	Tumor cells with 5–50% nuclear staining.
+++	Tumor cells with >50% nuclear staining.[9]

Abbreviations: IHC, immunohistochemistry; MMR, mismatch repair.

The cases that showed MMRD were confirmed by NGS. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue and assessed for concentration and purity using a Qubit Fluorometer and NanoDrop spectrophotometer, respectively. Only samples with DNA concentrations between 10 and 200 ng and A260/280 ratios of 1.8 to 2.0 were selected for further processing. The DNA was enzymatically fragmented and used to prepare sequencing libraries with the SureSelect XT HS2 DNA Library Preparation Kit, which included steps such as end repair, A-tailing, and adaptor ligation, with optional molecular barcodes.

Indexed libraries were generated via polymerase chain reaction (PCR) amplification using dual indexing primers. These libraries were then evaluated for quality and size, normalized, and pooled. Target enrichment was achieved by hybridizing the pooled libraries with custom biotinylated capture probes (designed using Agilent SureDesign) and enriching the bound fragments using streptavidin-coated magnetic beads. Post-capture PCR was performed to produce the final enriched library pool. The dual-indexed, SureSelect-enriched libraries (with or without molecular barcodes) were validated for fragment size and concentration. Sequencing was subsequently performed on an Illumina platform according to standard operating protocols.

Statistical Analysis

Data were entered in Microsoft Excel and analyzed using licensed SPSS software 25.0. Quantitative variables were expressed as mean and standard deviation. Quantitative variables were expressed as percentage or proportion. Chi-square test and Fisher's exact test were done to associate MMR protein deficiency with age, histopathological type, and CNS WHO grade of glioma. p-Value of <0.05 was considered significant.

Results

Out of 64 glioma cases, 21 cases were of the pediatric age group (<18 years) and 43 were adults (≥18 years). Furthermore, 14 (21.9%) patients belonged to age group of 41 to 50 years, followed by 11 (17.2%) patients in the age group of <10 years and 10 (15.6%) were in the age group of 11 to 20 years and 51 to 60 years each. There was only one patient (1.6%) in the age group of ≥71 years. The mean age of study participants was 35 ± 20.2 years, and the range was 3 to 78. In total, 47 (73.4%) were males and 17 (26.6%) were females. Out of 64 glioma cases, 5 cases (7.8%) had a history of recurrence. In addition, 62 (96.9%) gliomas were of brain and 2 (3.1%) gliomas were of spinal region. The most common location of gliomas in our study was the frontal region (26.6%), followed by the parietal region (18.8%) and posterior fossa (17.2%). The most common diagnosis among study subjects was astrocytoma, IDH (isocitrate dehydrogenase) mutant (45.3%), followed by glioblastoma, IDH-wildtype (20.3%), pilocytic astrocytoma (12.5%), ependymoma (9.4%), and others. The most common CNS WHO grade among study subjects was grade 4 (42.2%), followed by grade 2 (23.4%), grade 3 (20.3%), and grade 1 (14.1%). Most of the cases had a Ki67 proliferation index less than 20% (62.5%), followed by 21 to 40% Ki67 proliferation index in 31.3% cases.

Among 64 cases included in our study, only 3 (4.69%) showed MMRD. These cases showed isolated loss of MSH6 protein. However, these three cases did not show loss in the expression of other MMR markers (MLH1, MSH2, and PMS2). The remaining 61 (95.31%) cases examined were MMR-proficient as they showed positive staining for all MMR markers (MLH1, MSH2, MSH6, and PMS2).

In the present study, out of three (4.69%) cases which showed loss of MSH6, two were astrocytoma, IDH mutant, and one was pilocytic astrocytoma ([Figs. 1] [2] [3] [4]). However, MMRD had no statistically significant association with histological type (p = 0.448), age of patients (p = 1.000), and CNS WHO grade of glioma (p = 0.448).

Fig. 1 Pilocytic astrocytoma. (A, B) H&E sections show a grade 1 glioma (pilocytic astrocytoma; 100, 400× magnification). (C–F) IHC shows nuclear staining for MLH1 (C), MSH2 (D), PMS2 (F), and loss of nuclear staining for MSH6 (E) [100 × ].

Fig. 2 Myxopapillary ependymoma. (A, B) H&E sections show a grade 2 glioma (myxopapillary ependymoma; 100, 400× magnification). (C–F) IHC shows nuclear staining for MLH1 (C), MSH2 (D), MSH6 (E), and PMS2 (F) [100 × ].

Fig. 3 Grade 3 astrocytoma. (A, B) H&E sections show a grade 3 glioma (astrocytoma; 100, 400× magnification). (C–F) IHC shows diffuse nuclear staining for MLH1 (C), PMS2 (F), and focal nuclear staining for MSH2 (D) and MSH6 (E) [100 × ].

Fig. 4 Grade 4 astrocytoma. (A, B) H&E sections show a grade 4 glioma (astrocytoma; 100, 400× magnification). (C–F) IHC shows nuclear staining for MLH1 (C), MSH2 (D), PMS2 (F), and loss of nuclear staining for MSH6 (E) [100 × ].

NGS was performed on the three cases, which showed MMRD (MSH6 loss). The pilocytic astrocytoma sample demonstrated gene coverage of less than 30%, rendering it unsuitable for further analysis. In contrast, the remaining two cases exhibited gene coverage exceeding 30%, allowing for successful downstream evaluation. One IDH mutant astrocytoma showed MSI-H with frameshift mutations involving MLH1, MSH2, and MSH6 at locations Exon 1, Exon 1, and Exon 5, respectively. The other IDH mutant astrocytomas showed MSI-L with frameshift mutations involving MLH1, MSH2, MSH6, and PMS2 at location Exon 18, Exon 3, Exon 4, and Exon 11, respectively.

Discussion

The present study was designed to assess the MMR protein deficiency in gliomas by IHC with antibodies to MMR proteins and to study the association of its expression with the age, histological type, and CNS WHO (2021) grade of the gliomas. The age range of patients was 3 to 78 years, with a mean of 35 ± 20.2 years reflecting the known epidemiological variability of gliomas across age groups.[2] Most of the cases were adults in our study, 43 cases (67.2%), followed by pediatric, 21 cases (32.8%). Male predominance (M:F = 2.76:1) aligns with global epidemiological data, indicating higher glioma incidence in males across both low- and high-grade tumors.[14] [15] The frontal lobe was the most common tumor location, consistent with other studies that report a preference of gliomas for supratentorial sites, particularly in adults.[16] Interestingly, the posterior fossa was the third most common location in our study, which is in keeping with its frequent involvement in pediatric gliomas such as pilocytic astrocytomas and ependymomas.[17] Among histologic subtypes, astrocytoma, IDH mutant, was the most frequent diagnosis (45.3%), followed by glioblastoma, IDH-wild-type (20.3%), according to the revised WHO classification that emphasizes molecular profiling in diagnosis.[18] [19] The high prevalence of grade 4 tumors (42.2%) is also in keeping with the aggressive nature and clinical presentation of gliomas at later stages.

Among 64 cases included in our study, only 3 (4.69%) showed MMRD, as they showed loss of expression of MSH6. However, these three cases did not show loss in the expression of other MMR markers (MLH1, MSH2, and PMS2). The remaining 61 (95.31%) cases were MMR-proficient. The frequency of MMR protein deficiency in our study (4.69%) was lower than the frequency found in other studies. McCord et al studied 100 gliomas, out of which 8 cases (8%) showed loss of expression of at least one MMR marker.[20] Similarly, Tepeoglu et al evaluated 71 glioblastoma cases by IHC and found MMR protein loss in 9.9% cases.[12] Caccese et al observed MMRD in 12.1% of cases (43/355).[5]

Published research on MSI in high-grade glioma (HGG) has yielded contradictory findings. Several explanations have been provided for these variations, including the quantity of samples examined, the variability, and the precision of the techniques employed to detect MSI status. As per previous studies, a significant proportion of pHGGs were associated with cancer predisposition syndrome and showed MMR protein deficiency. Alphones et al studied nine pHGGs by IHC and found MMRD in 33% of cases.[6] Amayiri et al found MMRD in 39% of pHGGs.[21] Similarly, Carrato et al observed MMRD in 39% of pHGGs.[22] Almuhaisen et al observed MMRD in 6.25% of cases of HGGs in young adults.[23] In contrast to these studies, Vladimirova et al observed MSI in 3.2% pediatric malignant astrocytic tumors.[24] However, in the present study, we have included only 10 cases (15.62%) of pHGGs, but none of them showed MMRD. The rationale behind this might be small sample size or lack of any cancer predisposition syndromes in the current study. Also, the association of MMRD with the age of patients (p = 1.000) and CNS WHO grade of glioma (p = 0.448) was statistically insignificant. The lack of statistically significant associations between MMRD and age, histological type, or WHO grade may be due to the limited number of MMRD cases.

In the current study, out of three (4.69%) cases which showed loss of MSH6, two were astrocytoma, IDH mutant, and one was pilocytic astrocytoma. However, MMRD was found to have a statistically insignificant association with histological type (p = 0.448). In contrast to this finding, Caccese et al found a statistically significant association between MMRD and IDH-mutant gliomas.[5] Suwala et al observed MMRD in 32 IDH mutant astrocytoma cases.[25] Previous literature observed MMRD in pHGGs. Nonetheless, the presence of MMRD in one pilocytic astrocytoma is notable, as such tumors are typically considered low-grade and rarely exhibit this feature. A few recent studies, however, have documented similar findings, suggesting that MMRD can occasionally occur in otherwise indolent glioma subtypes, possibly due to underlying genetic predispositions.[26] Among the three cases showing MSH6 loss in our study, only one had a history of recurrence. In context to this finding, Cahill et al detected MSH6 mutations in 3 of 14 recurrent tumors but the mutations were not identified in 40 glioblastomas before treatment, suggesting that the recurrence may be due to loss of MSH6 following TMZ treatment.[27] This hypothesis is directly supported by the Hunter et al findings, in that MSH6 mutations were present in two recurrent cases but not in the pretreatment glioblastoma cases.[28]

The NGS analysis conducted on the three glioma cases with immunohistochemically confirmed MMRD offers critical insights into the underlying molecular alterations and their potential clinical significance. Although limited in number, these cases highlight the relevance of integrating genomic profiling into glioma diagnostics, particularly in cases exhibiting MMR protein loss. NGS revealed that two of the three MMR-deficient tumors had sufficient gene coverage (>30%) for successful genomic analysis. These included two IDH-mutant astrocytomas, which demonstrated MSI—one being MSI-H and the other MSI-L. The MSI-H tumor harbored frameshift mutations in three key MMR genes: MLH1 (Exon 1), MSH2 (Exon 1), and MSH6 (Exon 5). These early exonic mutations likely result in nonfunctional truncated proteins, directly impairing the DNA MMR mechanism. Similarly, the MSI-L tumor exhibited frameshift mutations in MLH1 (Exon 18), MSH2 (Exon 3), MSH6 (Exon 4), and PMS2 (Exon 11), further supporting the notion of a defective MMR system. On IHC, loss of protein expression other than MSH6 could not be identified in these two cases. This may be attributed to heterogeneous or weak staining patterns.[29] In the present study, even focal nuclear positivity in tumor cells was interpreted as MMR proficiency, whereas some other studies have classified such cases as exhibiting partial MMR loss.

These findings are in line with previous studies that have shown that MSI-H status, while rare in gliomas (<2% overall), is enriched in subsets such as pHGGs, treatment-related gliomas, and those with underlying germline predisposition syndromes (e.g., Lynch syndrome or CMMRD).[30] [31] The presence of frameshift mutations in multiple MMR genes in both tumors strongly supports a bona fide MMR-deficient phenotype, which can have significant prognostic and therapeutic implications. Previous research has shown that IDH-mutant gliomas with additional hypermutator phenotypes due to MMR gene mutations can display more aggressive clinical behavior, higher recurrence rates, and resistance to TMZ.[32] Moreover, MMR-deficient gliomas, especially those that acquire these mutations post-therapy, often show clonal evolution and genomic instability, which complicates treatment and may lead to more aggressive, therapy-resistant recurrences.[33]

A particularly important clinical implication of identifying MSI-H status in gliomas is its relevance to immune checkpoint inhibitor (ICI) therapy. Tumors with dMMR/MSI-H are characterized by a high tumor mutation burden, which increases neoantigen presentation and enhances immunogenicity—rendering them susceptible to PD-1/PD-L1 blockade therapies.[34] In fact, the Food and Drug Administration has approved pembrolizumab for any solid tumor with MSI-H or dMMR status, regardless of tumor site, based on the success seen in colorectal and other carcinomas.[35]

Though the effectiveness of ICIs in gliomas remains under investigation, emerging reports have shown clinical benefit in MMR-deficient and hypermutated gliomas.[36] [37] For instance, a study by Bouffet et al demonstrated partial responses in pediatric hypermutated gliomas treated with nivolumab or pembrolizumab.[37] [38] Therefore, the identification of MSI-H status in one of our glioma cases, along with a clear MMR mutation pattern, underscores the potential eligibility of such patients for immunotherapy, which could offer a novel line of treatment where standard therapies fail. Although routine MSI testing is not standard for all gliomas, our findings support incorporating IHC-based MMR screening in selected glioma cases, especially those with unusual clinical or histological features, pediatric onset, or recurrence after therapy.[39]

The main limitations of our study were small sample size and a single center study. Pediatric gliomas were underrepresented in our cohort, despite existing literature suggesting higher MMRD prevalence in this subgroup. None of the pHGGs demonstrated MMRD in our study. IHC was used as the primary method to detect MMR protein loss, and not all cases underwent confirmatory molecular testing (NGS), which may have led to underestimation of MMRD. Furthermore, the retrospective design may have introduced selection bias, and clinical outcome data (e.g., treatment response, survival) were not systematically evaluated, limiting insights into the prognostic or predictive significance of MMR status.

Conclusion

The findings of the current study, coupled with low prevalence and discordant literature reports, suggest that MMRD is not a major driver in gliomagenesis. Given the potential responsiveness of MSI-H gliomas to immunotherapy, even rare identification of such cases may warrant genomic testing and consideration for targeted treatment.

IHC is a practical first-line tool for MMR screening, but confirmation by sequencing is recommended. Larger, multicenter studies are required to clarify MMRD's true prevalence and prognostic value in gliomas, and to establish standardized protocols for its detection and integration into clinical decision-making.

Conflict of Interest

None declared.

Ethical Approval

The study was performed after ethical approval from Institutional Ethics Committee (IEC/VMMC/SJH/Thesis/06/2022/CC-227).

Patients' Consent

Informed patient consent was taken.

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Publication History

Article published online:
09 September 2025