Mismatch Repair Deficiency Is an Uncommon Mechanism of Alkylator Resistance in Pediatric Malignant Gliomas: A Report From the Children's Oncology Group

Abstract

Background

Alkylating agents are commonly used in the treatment of childhood malignant gliomas. Overexpression of O⁶-methylguanine-DNA methyltransferase (MGMT)constitutes an important mechanism for resistance to such agents, and MGMT status has been associated with outcome in several recent trials. Deficiency in mismatch repair (MMR)function has been implicated in preclinical studies as an additional potential mechanism of resistance to methylating agents, such as temozolomide, independent of tumor MGMT status. However, the frequency of this abnormality as a clinical resistance mechanism in childhood malignant gliomas has not been well characterized.

Methods

To address this issue, we examined the frequency of microsatellite instability (MSI), a marker of defective MMR, in a series of 68 tumors, derived from newly diagnosed patients treated on the Children's Cancer Group 945 study, and the Children's Oncology Group ACNS0126 and 0423 studies. MSI was assessed using a panel of six microsatellite markers, including BAT-25, BAT-26, CAT-25, D2S123, D5S346, and D17S250. MGMT immunoreactivity was assessed in parallel to allow comparison of the relative incidence of MGMT overexpression and MSI.

Results

Only three tumors had high-level MSI involving three or more markers; the remainder had no MSI at any of the loci examined. These children did not have unusual features in terms of their outcome. In contrast to the infrequency of MSI, 25 tumors (37%)exhibited MGMT overexpression as assessed by immunohistochemistry. None of the tumors with MSI exhibited overexpression of MGMT.

Conclusion

MMR deficiency is an infrequent contributor to initial alkylator resistance in children with malignant gliomas. Pediatr Blood Cancer.

INTRODUCTION

High-grade gliomas are one of the most common malignant central nervous system tumors of childhood [1]. Although these tumors are morphologically similar to malignant gliomas that arise in adults, several studies have suggested that childhood lesions may differ biologically from their adult counterparts. In particular, childhood primary malignant gliomas rarely exhibit EGFR amplification or PTEN deletion [2–9], hallmarks of adult primary glioblastoma, and infrequently exhibit IDH1 mutations, which characterize adult glioblastomas that progress secondarily from lower grade lesions [10–14].

Despite these molecular differences, both adult and childhood malignant gliomas are generally treated with a combination of surgery, irradiation and alkylator-based chemotherapy, using agents such as temozolomide [15–17]. The Children's Cancer Group 943 study demonstrated a survival advantage for children treated with lomustine and vincristine in addition to irradiation compared to those treated with irradiation alone [15]. Recent studies in adults have also shown improved survival rates in patients treated with irradiation and temozolomide versus those treated with irradiation alone [16]. A comparable comparison has not been undertaken in the pediatric context, although a recently completed trial (ACNS0126) is comparing event-free survival of children treated with adjuvant temozolomide to those treated with lomustine and vincristine in the CCG-945 study. Unfortunately, in both studies a relatively small percentage of patients exhibited long-term survival, reflecting that many malignant gliomas have intrinsic or acquired resistance to these agents.

O⁶-Methylguanine-DNA methyltransferase (MGMT) constitutes a frequent mechanism for alkylator resistance by promoting the transfer of alkyl groups from the O⁶ position of guanine nucleotides in DNA to an alkyl group acceptor site within MGMT [18]. Studies in both children and adults have noted a significant survival advantage in alkylator-treated patients whose tumors had low MGMT levels, as defined by immunohistochemistry or gene inactivation by promoter methylation [19–26]. However, even in patients with favorable MGMT status, the likelihood of long-term survival is low, which suggests that other mechanisms of alkylator resistance are operational.

In this regard, defects in mismatch repair (MMR) can contribute to resistance to methylating agents, such as temozolomide. Under normal conditions, O⁶-methylated guanine nucleotides incorrectly pair with thymine during DNA replication, which activates the MMR system, leading to futile cycles of repair that culminate in apoptosis. Mutations of the genes involved in this process, such as hMLH1, hMSH2, hMSH3, hMSH6, hPMS1, and hPMS2, lead to deficient MMR [27,28], which allows the introduction of length abnormalities into tandemly repeated nucleotide sequences throughout the genome, so-called microsatellite instability (MSI). This phenomenon is commonly observed in hereditary non-polyposis colorectal cancer and Turcot's syndrome, and has also been noted in various sporadic tumors [29–32], including adult malignant gliomas, although the frequency of this finding has varied widely [28,33–39]. The reported incidence of MSI has also varied widely in pediatric malignant gliomas [5,27,28,40–42], but has to date not been examined in a large cohort of centrally reviewed tumors. We therefore examined MSI in a group of 68 childhood malignant gliomas, using a reference panel of six markers, including BAT-25, BAT-26, CAT-25, D2S123, D5S346, and D17S250. MSI was found to be uncommon in this cohort, much less frequent than the incidence of MGMT overexpression, suggesting that MMR deficiency is an infrequent mechanism of initial resistance to alkylator therapy.

METHODS

Tumor Samples

Tumor samples were obtained from children enrolled on the Children's Cancer Group (CCG)-945 study and the Children's Oncology Group (COG) ACNS0126 and 0423 studies, all of which involved post-surgical administration of alkylator-based chemotherapy and irradiation. Each study required institutional IRB approval for protocol enrollment. Children enrolled on CCG-945 were treated with lomustine, vincristine, and prednisone or the “eight-drugs-in-1-day” regimen [43]; those on ACNS0126 received temozolomide daily with radiation and on a 5-day per 28-day cycle post-irradiation; and those on ACNS0423 received daily temozolomide during irradiation followed by post-irradiation temozolomide and lomustine. Eligibility for all three studies required an institutional histopathologic diagnosis of high-grade glioma (i.e., glioblastoma (GBM), anaplastic astrocytoma (AA), or other eligible malignant glioma, such as gliosarcoma), and each incorporated central pathology review to refine classification.

Tissue accrual for the current study was coordinated by the Pediatric Branch of the Cooperative Human Tissue Network (CHTN). Specimens were de-identified by the CHTN to mask clinical and outcome data from investigators.

MSI Assay

Formalin-fixed paraffin-embedded (FFPE) tissue specimens were used for all analyses. Slides were reviewed by a neuropathologist (RLH) to confirm that tumor tissue of sufficient quantity was available for the planned studies. Tumor targets were manually microdissected from 4-μm unstained histologic sections under the guidance of a hematoxylin and eosin-stained slide using an Olympus SZ61 stereo microscope (Olympus, Hamburg, Germany). DNA was isolated from each target with the DNeasy Blood and Tissue kit on the automated QIAcube (Qiagen, Valencia, CA) instrument according to the manufacturer's instructions. The quantity of isolated DNA was assessed using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE). Detection of microsatellite instability was performed using a National Cancer Institute-recommended panel of microsatellite markers (BAT25, BAT26, D2S123, D5S346, D17S250) and an additional mononucleotide marker, CAT25, which was recently reported to be a sensitive and specific indicator for MSI detection [44] (Supplemental Table I). PCR amplification was performed with primers labeled either with TET, NED or HEX fluorophores at the 5′ end and carried out on a 9700 GeneAmp thermocycler (Applied Biosystems, Foster City, CA) using standard PCR profiles (denaturation at 94°C for 10 min followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 1 min). Post-PCR amplification products were detected using capillary gel electrophoresis on an ABI 3730 platform (Applied Biosystems). The relative fluorescence values (peak heights) and profiles of microsatellite peaks were analyzed using GeneScan 3.7 software (Applied Biosystems). Based on established criteria, if 0 out of 6 (0%) markers showed instability, the tumor was classified as microsatellite stable (MSS); if 1 out of 6 markers showed instability, the tumor was classified as having low-level microsatellite instability (MSI-L); and if at least 2 out of 6 (≥30%) markers showed instability, the tumor was classified as having high-level microsatellite instability (MSI-H) [45].

Expression of MGMT

To compare the frequency of MMR deficiency with MGMT overexpression, MGMT status was assessed by immunohistochemistry in the samples subjected to MSI analysis, using techniques previously reported in studies from our group that examined the association between MGMT expression status and outcome in the CCG-945 and ACNS0126 cohorts [26,46]. Tumor-containing sections were baked at 60°C for 30 min, deparaffinized in xylene, and rehydrated in graded concentrations of ethanol. Endogenous peroxidase activity was quenched by incubation in 0.3% hydrogen peroxide solution. Antigen retrieval [47] was performed by heating the slides in 10 mmol citrate buffer (pH 6.0) for 20 min. Nonspecific antibody binding was blocked by incubation in Protein Blocking Reagent (Thermo Corp, Pittsburgh, PA) for 20 min. Sections were then incubated with mouse anti-MGMT antibody (mT23.2, Zymed Laboratories, San Francisco, CA, 1:100) [48,49] in Common Antibody Diluent (BioGenex, San Ramon, CA) at room temperature for 2 hr. Negative control sections were treated with diluent and mouse IgG (5 μg/ml, Dako Corporation, Carpinteria, CA) alone. Slides were then rinsed twice with PBS and antibody binding was localized using a Universal Labeled Streptavidin-Biotin 2 System (LSAB 2—HRP, Dako). Slides were incubated for 30 min in Biotinylated Link reagent at room temperature, followed by a 10 min PBS wash. Slides were then incubated in Streptavidin-HRP solution for 30 min at room temperature. Antibody binding was visualized using 3,3′-diaminobenzidine [50]. The slides were counterstained with Mayer's hematoxylin, dehydrated through graded concentrations of ethanol, cleared in xylene, mounted and examined using a light microscope. Positive controls (tonsil, ovary and a glioblastoma with known MGMT overexpression) and negative controls (normal brain and tonsil treated with diluent without primary antibody) were included with each batch of sections to confirm the consistency of the analysis. Specimens had all been examined independently for immunoreactivity using an antibody against a histologically verifiable internal positive control antigen (i.e., MIB1 staining of the KI67 antigen in tumor mitotic figures), to eliminate cases in which lack of immunoreactivity for MGMT might indicate problems in tissue preservation rather than lack of protein expression.

MGMT labeling was assessed semi-quantitatively, by examining stained and unstained cells in 5–10 high-power fields that incorporated the most anaplastic regions of the specimen. Only cells with dense nuclear staining were graded positive. Tumors were categorized as exhibiting little or no expression [0/1] or scattered positive cells [2], comparable to normal brain, versus overexpression, in which staining was observed in most or nearly all cells [3/4].

Central Pathology Review

Although eligibility for the CCG-945 clinical study was based on the institutional histopathologic diagnosis, it was subsequently recognized that a number of low-grade glioma variants may have been mistakenly classified as high-grade tumors prior to the adoption of contemporary classification criteria [43,51]. All cases from CCG-945 in the current study were independently reviewed in a masked fashion by a panel of five senior neuropathologists, and a consensus diagnosis was established if at least three of the five reviewers independently reached an identical histological diagnosis [51]. Only patients with consensus diagnoses of high-grade glioma were included in the current analyses. Both ACNS0126 and ACNS0423 used a prospective central review panel to restrict the eligible cohort to those cases with high-grade gliomas. The MSI and MGMT analyses were performed independently of this review, so the review diagnosis of each specimen was not known during the assessment.

RESULTS

Frequency of MSI

Sixty-eight patients had specimens that were available for both MSI and MGMT analysis in the current study—34 had tumors classified as GBM/grade IV, 30 as anaplastic astrocytoma/grade III, and 4 as other eligible malignant gliomas. Only three of these tumors (4.4%) had evidence of MSI. None of these patients had prior or subsequent colonic malignancies to suggest the existence of Turcot's syndrome. Figure 1 shows an illustrative example of a tumor with MSI in D5S346, D17S250, BAT25, and BAT26 loci as contrasted against another case lacking MSI.

Fig. 1.

An illustrative example of a tumor lacking MSI (A) and one with MSI in D5S346, D17S250, BAT25, and BAT26 loci (B). The arrows in (B) indicate aberrant microsatellite repeat elements, distinct in molecular weight and thus mobility on capillary electrophoretograms from the normal microsatellites, which are illustrated in (A). The length abnormalities of these nucleotide repeat sequences is indicative of microsatellite instability (MSI).

Two of the three tumors with MSI were noted to be glioblastoma on histological review, and one was an anaplastic astrocytoma. In all three cases, MSI was classified as high-level (MSI-H). One tumor had MSI involving BAT-25, BAT-26, D5S346, and D17S250, and two had MSI involving D2S123, D5S346, and D17S25, in one instance also involving CAT-25. No instances of low-level MSI were detected in this cohort.

These tumors did not have unusual properties in terms of outcome. All three children progressed, 4, 11, and 25 months, respectively, after study enrollment. Two patients subsequently died (7 and 12 months from study enrollment). One patient was alive at last contact at 32 months.

Expression of MGMT and Relationship to MSI

Overexpression of MGMT proved to be substantially more common in this cohort than MSI. Of the 68 tumors examined, 25 (37%) showed overexpression of MGMT, with dense staining in more than 25% of cells, whereas 43 showed little or no MGMT immunoreactivity (Fig. 2). None of the tumors with MSI demonstrated MGMT overexpression.

Fig. 2.

An example of a pediatric malignant glioma showing absent MGMT expression (left) and overexpression (right). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

DISCUSSION

The treatment of malignant gliomas incorporates multimodality therapy with surgery, irradiation and chemotherapy, generally including an alkylating agent such as temozolomide. Unfortunately, the majority of patients succumb to disease progression, reflecting intrinsic or acquired resistance of the tumor to adjuvant therapy. The adverse impact of MGMT overexpression on response to temozolomide and nitrosoureas has been well described in both adult [19–25] and pediatric [26,46] malignant gliomas, although the high frequency of disease progression in tumors that do not overexpress MGMT highlights the potential contribution of other resistance mechanisms to treatment failure. Defects in MMR, either by mutation or epigenetic inactivation of key MMR proteins, has been implicated in the resistance of glioma cell lines to alkylator treatment, independent of MGMT status [52–54], although the contribution of such abnormalities to clinical tumor resistance is less well established.

A role for germline MMR defects has been noted in gliomas arising in the context of hereditary colon cancer [32,39,40], but such tumors make up a small subset of malignant gliomas, and may well arise by a different mechanism than sporadic tumors. Studies that have examined MSI in adult malignant gliomas have noted a wide range in the frequency of this finding at diagnosis [28,33–39], which may in part reflect differences in the composition of study populations. For example, Leung et al. [39] observed an 18% incidence of high-level MSI, although several of their patients met some of the criteria for Turcot's syndrome, whereas Maxwell et al. [38] found only a 3% incidence of MSI, and the majority of recent studies have found comparably low rates of this abnormality [28,35].

Similarly conflicting results have been obtained regarding the incidence of MSI in pediatric malignant gliomas. Alonso et al. [27] observed MSI in 12 of 45 cases (27%), although it is unclear if these all represented newly diagnosed tumors and whether any cases were associated with Turcot's syndrome. Kanamori et al. [40] noted MSI in 2 of 6 pediatric cases examined (33%), although at least one of the two patients met criteria for Turcot's syndrome. Likewise, Cheng et al. [5] found MSI was present at multiple loci in 2 of 24 cases and at isolated loci in an additional two cases (17%). In contrast, Amariglio et al. [42] found no MSI among seven pediatric high-grade gliomas; Blaeker et al. [41] found this in none of nine cases, and Eckert et al. [28] in none of 41 cases.

Given the wide discrepancy in the frequency of MSI, the current study sought to address this issue conclusively using a multi-locus panel of established MSI markers in a well characterized group of newly diagnosed patients. The results from the current study, in a large series of children with histologically reviewed high-grade gliomas, suggests that the frequency of MSI at diagnosis is quite low, in agreement with the findings of Eckert et al. [28] using a smaller panel of markers. In addition, this alteration is substantially less common than MGMT overexpression as a potential mechanism of resistance to alkylating agents, which fits with recent findings in adult malignant gliomas [38].

Although the current study indicates that MMR defects are uncommon in newly diagnosed pediatric malignant gliomas and are unlikely to contribute to intrinsic temozolomide resistance, this does not rule out the involvement of this mechanism in tumor progression. Recent studies have demonstrated that MMR gene abnormalities can be acquired in malignant gliomas with initially intact MMR as a somatic mutation following alkylator therapy [55,56].

In addition to MGMT overexpression and MMR, there are undoubtedly a host of molecular mechanisms that contribute to the resistance of pediatric glioma cells to adjuvant therapy. These include mutations of tumor suppressor genes, such as TP53 [3,8] and PTEN [6], that influence apoptotic signaling pathways, as well alterations in other molecular mediators of DNA damage repair, including base excision repair pathways [57,58]. Identifying strategies to circumvent or reverse this resistant phenotype remains an ongoing focus in the management of these tumors.