IDH-mutant astrocytoma with EGFR amplification—Genomic profiling in four cases and review of literature


 EGFR amplification is associated with aggressive glioma behavior and regarded as a molecular feature of glioblastoma. Although rare, IDH-mutant astrocytomas with EGFR amplification exist but remain poorly understood. We report the clinical and molecular profile of four grade 4 IDH-mutant astrocytomas with EGFR amplification, evaluated using histology, DNA sequencing, cytogenetics, and DNA methylation profiling. Other alterations included ATRX, TP53, and PIK3CA mutations; CDKN2A/B loss; PDGFRA+KIT amplifications; and MGMT methylation in two cases. None disclosed microsatellite instability. DNA methylation confidently classified all tumors as “IDH-mutant High-Grade Astrocytoma” (0.99 0.997, 0.98, and 0.99 scores), despite their EGFR-amplified status. Literature review indicated EGFR amplification occurs within 8-19% of IDH-mutant gliomas, with significantly lower overall survival only in the co-presence of CDKN2A/B loss and MET amplification. Our report suggests IDH-mutant astrocytomas with EGFR amplification are under-recognized and that EGFR amplification status alone does not carry diagnostic or prognostic significance in these tumors.

IDH-mutant astrocytomas carry significantly better prognosis compared to their IDH-wildtype grade 4 (glioblastoma) counterpart. Several molecular diagnostic markers have emerged in the 2021 CNS WHO classification, with powerful prognostic implications to consider when classifying diffuse IDH-mutant astrocytomas. 1-3 EGFR amplification status, associated with aggressive glioma behavior 4 and now regarded as a molecular feature of glioblastoma, is not currently a diagnostic consideration in IDH-mutant astrocytomas. 1 Although uncommon, IDH-mutant astrocytomas with EGFR amplification exist in large published datasets but their relevance has been underemphasized and remains poorly understood. [5][6][7][8] In an effort to better understand the biology of "IDH-mutant astrocytoma with EGFR amplification, " we present the clinical and molecular profiles in four such rare cases encountered at two institutions; evaluate them based on cytogenetics, DNA sequencing, and DNA methylation profiling; and scrutinize published datasets for this specific entity to gain further insight into its diagnostic and prognostic implications.

Clinical Presentation
We report four cases encountered at two institutions between 2015 and 2020, diagnosed as "Glioblastoma (Astrocytoma), IDH-mutant, WHO grade 4, " found to carry EGFR amplification, an alteration diagnostic of IDH-wildtype Glioblastoma. 1 Three patients were adults and one was pediatric. All presented with large and infiltrative, heterogeneously enhancing MRI lesions ( Figure 1A and B), and underwent gross total tumor resections followed by chemoradiation therapy. Case 1, a 59-year-old man with a large cystic right frontal mass, underwent resection and was discharged from the hospital on postoperative day 5. He passed away 16 days after his initial surgery. Case 2, a 15-yearold boy with a left frontal tumor, underwent sub-total resection and received standard therapy of Temozolomide and proton beam therapy along with Optune device. He had recurrent disease and progression through therapy 16 months after surgery, which was initially treated with Bevacizumab 17 months after surgery. He continued to progress and died of his disease 20 months after his initial resection. Case 3, a 28-year-old man with Charcot Marie Tooth syndrome and left frontoparietal tumor, was lost to follow-up a year after initial resection and Stupp-protocol treatment. Case 4, a 37-year-old man with a left parietal tumor, recurred a year after treatment with Procarbazine with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and adjuvant radiation, and is in stable condition at his most recent follow-up, 4 years after initial presentation.

Pathological Evaluation
Histologic examination in all cases revealed moderate-tohighly cellular, diffusely infiltrative glial neoplasms composed of pleomorphic glioma cells with astrocytic appearance, including angulated nuclei, fibrillary cytoplasm, and occasional IDH-mutant astrocytoma with EGFR amplification-Genomic profiling in four cases and review of literature gemistocytic morphology ( Figure 1C1 and D1). Nuclear atypia was consistently seen and mitotic figures were readily apparent with proliferation index ranging between 10% and 40%. Grade 4 histological features, including microvascular proliferation and palisading-type tumor necrosis, were present in all cases ( Figure 1C1 and D1). In immunohistochemical studies, cases showed diffuse positivity for GFAP, mutant IDH1R132H expression ( Figure  1C2 and D2), nuclear loss of ATRX ( Figure 1C3 and D3), and strong nuclear P53 ( Figure 1C4 and D4), suggestive of mutant IDH1R123H, ATRX, and TP53 status, respectively. None of the cases were positive for mutant BRAFV600E or H3K27M. As part of routine clinical workup, analysis for EGFR amplification was performed using multiplex dual color DNA probe chromogenic in situ hybridization (CISH) or fluorescent in situ hybridization (FISH). These assays identified focal and/or diffuse amplification of EGFR at chromosome 7 in all cases: case 1 with 10 average EGFR/ CEP7 CISH signal ratio ( Figure 1E); case 2 with 5 average EGFR/CEP7 CISH signal ratio ( Figure 1F); case 3 with 2.1 FISH ratio and 5 EGFR copies on average; and case 4 with 3.6 FISH ratio and 9.7 EGFR copies on average.
The presence of both EGFR amplification and IDH mutation in these high-grade astrocytoma tumors was unusual and raised questions within our clinical team in regards to the tumors' correct nomenclature, biological behavior, possible syndromic genomic instability, and, ultimately, patient prognosis.
We considered the possibility that excessive genomic instability may have led to EGFR amplification in our cases and therefore also tested the four cases on a classifier that includes primary mismatch repair-deficient IDH-mutant astrocytomas (PMMRDIA) as a separate category. PMMRDIA encompasses a small subset of IDH-mutant astrocytomas, which have hereditary mismatch repair (MMR) deficiency and worse clinical outcome. 10 Importantly, none of the four cases clustered with "PMMRDIA IDH-mutant astrocytomas" (data not shown). Furthermore, DNA methylation-based prediction of the MLH1 gene status, one of the most commonly mutated DNA MMR complex members, as well as immunohistochemical studies for loss of expression of MLH1, MSH2, MSH6, and PMS2 MMR proteins, did not reveal evidence for microsatellite instability (data not shown). Overall, despite the presence of EGFR amplification in all four cases, a diagnostic molecular marker in IDH-wildtype glioblastoma and a predictor of its aggressive behavior, IDH-mutant status was the determinate factor for an epigenetics-based classification of these high-grade astrocytomas.

Literature Review
Limited by long-term survival data in our prospective cases, we performed retrospective review of the literature to explore further the prognostic significance of EGFR amplification within IDH-mutant astrocytomas. No studies were found specifically focused on EGFR-amplified IDHmutant astrocytomas. Thus, we focused on large glioma dataset studies, searching for the specific co-occurrence of IDH mutation and EGFR amplification. [5][6][7][8] In the Li et al's study examining 57 IDH-mutant GBM, 7 (12.3%) had EGFR amplification. 7 Of these, only three cases (40%) had a glioma methylator (G-CIMP) high phenotype typically found in IDH-mutant grade 4 tumors with more favorable prognosis (Table 1). Other copy number alterations within this series included deletion of CDKN2A and amplification of CCND2, PDGFRA, MYC, CDK4, and MET (Table  1). All MET-amplified tumors belonged to the G-CIMP-low subgroup and exhibited CDKN2A alterations. Importantly, worse overall survival (OS) was associated within the group of tumors with G-CIMP-low, CDKN2A deletion, and MET amplification status (median OS of 252 days). In the Brennan et al's study profiling 332 GBM tumors, 5 out of 30 IDH-mutant GBM cases were identified to be EGFRamplified (16.7%), all of which were G-CIMP-high, with 2   (Table 1). Although EGFR status showed statistically significant lower survival within grade 2-3 tumors, MYC amplification, PTEN loss, and CDKN2A loss status were associated with tumor progression. 8 Generally, none of the studies found EGFR amplification to be an independent marker of worse OS within grade 4 IDH-mutant astrocytomas, unless it co-occurs with CDKN2A/B loss, MET amplification, and/or G-CIMP-low status.

Discussion/Conclusion
Identifying prognostically relevant subgroups of astrocytic gliomas is extremely important for clinical trial inclusion and patient care. EGFR amplification has long been established as an independent and significant factor associated with shorter survival time and poorer prognosis in patients with glioblastoma, and therefore its co-occurrence in the setting of IDH-mutant astrocytomas warrants closer examination. 4 In the four cases presented, the co-existence of both alterations challenged the diagnostic and prognostic stratification of these tumors, eliciting uncertainty as to the tumors' clinical behavior. Reports of IDH-mutant astrocytomas with EGFR amplification are rare in the literature. 5,8,11 Indeed, at one of the authors' institutions, only two such cases were detected out of 56 IDH-mutant astrocytomas between 2019 and 2020. Consequently, the clinical relevance of EGFR amplification in IDH-mutant astrocytomas has not been established. In the most recent CNS WHO classification, mutant IDH is a diagnostic molecular marker of diffuse astrocytomas, whereas EGFR amplification is a diagnostic molecular marker of glioblastoma. [1][2][3] In cIMPACT-NOW update 5, many molecular alterations were considered to identify IDH-mutant astrocytomas with a clinical course corresponding to WHO grade 4, including CDKN2A/B homozygous deletion, CDK4 amplification, RB1 mutation, or homozygous deletion, PIK3CA or PIK3R1 mutations, PDGFRA amplification, MYCN amplification, global DNA methylation levels, chromosome 14 loss, and genomic instability. 3 However, EGFR amplification was not included in this evaluation.
Our literature review analysis establishes that IDHmutant astrocytomas with EGFR amplification are not as rare as previously considered. By mining several large glioma datasets for this specific subtype, we uncovered a relatively high occurrence of EGFR amplification/copy number gain in IDH-mutant astrocytomas, 8%-19% depending on the study. [5][6][7][8] The discrepancy between the rare clinical recognition of these tumors and their more frequent appearance in published datasets may be related to the lack of additional molecular testing in astrocytomas, once they are diagnosed as IDH-mutant using immunohistochemistry.
DNA methylation-based classification analysis in our case series showed consistent clustering of tumors based on IDH-mutant status rather than on EGFR amplification, indicating that these tumors should be considered as "IDHmutant astrocytomas" for diagnostic purposes, despite the presence of EGFR amplification. Furthermore, albeit limited to a single study, current literature suggests that EGFR amplification in grade 4 IDH-mutant astrocytomas is not associated with worse OS, unless CDKN2A/B loss is also detected. 7 This is consistent with current CNS WHO recommendations, which recently established homozygous loss of CDKN2A/B as an independent marker of poor prognosis   within IDH-mutant astrocytomas, sufficient to diagnose a tumor as CNS WHO grade 4 in the absence of microvascular proliferation and tumor necrosis. 1,12 It also correlates with the poor outcome observed in case 1 and case 2, both of which displayed concurrent CDKN2A/B loss. Although none of the cases in our series contained microsatellite instability, co-occurrence of IDH and EGFR alterations in an astrocytoma may indicate a hypermutated phenotype and/or hereditary MMR deficiency (PMMRDIA). Such tumors have more aggressive clinical behavior but may respond to specific immune checkpoint therapies. 10 Therefore, the presence of mutant IDH plus EGFR amplification in a diffuse glioma should alert clinicians to rule out MMR deficiency and/or microsatellite instability. Further investigation is warranted to explore the impact of EGFR amplification on the biology and clinical progression of IDH-mutant astrocytomas, including processes, such as tumor growth, invasion, recurrence, and on patients' response to specific therapeutic regimens. Despite significant preclinical evidence for the therapeutic potential of monoclonal antibodies and tyrosine kinase inhibitors (TKIs) that target EGFR, clinical trials thus far have failed to demonstrate significant survival benefits in patients with glioblastoma, regardless of EGFR amplification and mutation status. 13,14 Ongoing studies continue to explore EGFR amplification status as a potentially targetable marker and our report flags an important subgroup of IDH-mutant astrocytomas that may respond to such therapy if and when it becomes actionable.

Supplementary Material
Supplementary material is available at Neuro-Oncology Advances online.

Funding
This work was supported by the National Institutes of Health (R01NS106229 to N.M.T.).