Biomarker Testing For Gliomas (Details & References)

IDH MUTATIONAL TESTING BY IHC

IDH1 and IDH2 mutations result in a substitution for a key arginine at codons R132 and R172, respectively.¹ The most frequent IDH1 mutation is R132H, which accounts for 89% to 93% of all IDH1 and IDH2 mutations.^2,3,4,5,6 Immunohistochemistry (IHC) with highly sensitive and specific monoclonal antibody IDH1-R132H is a cost-effective and reliable first-line test for IDH1 mutation in supratentorial DGs.^{7, 8, 9,10,11,12} IDH1 R132H mutations are followed in frequency by R132C, R132S, R132G, and R132L.

IDH2 mutations represent approximately 3% of all IDH mutations and are more frequent in oligodendrogliomas than IDH-mutant astrocytomas. R172K is the most frequent, followed by R172M and R172W. Testing for these non–IDH1-R132H mutations is accomplished by DNA sequence analysis and is necessary when IHC for IDH1-R132H is negative in appropriate settings, with specific exceptions. Recent studies of primary infratentorial IDH-mutant astrocytomas have shown that they have a different spectrum of IDH mutations, with about 80% harboring non–IDH-R132H mutations, ¹³ suggesting that IDH sequencing may be required more frequently in this clinical setting.

Patients older than 55 years only rarely develop de novo glioblastomas (GBMs) that are IDH-mutant (4%–7%) and since the IDH1-R132H antibody recognizes the large majority of IDH mutations, the WHO has recommended that testing for non–IDH1- R132H mutations by sequence analysis may not be necessary in patients older than 55 years whose tumors are negative for IDH1-R132H by IHC.^{14, 15, 4 16, 17,18}

However, non-R132H mutations are sufficiently common in DGs of all grades in patients less than age 55, such that testing for these mutations is indicated when IHC does not detect the R132H mutation. For histologic grade 2 to 3 DGs that are IDH-WT, testing should be performed for whole chromosome 7 gain/whole chromosome 10 losses, EGFR amplification, and TERT promoter mutation to establish the molecular diagnosis of IDH-WT GBM, grade 4.

ATRX /TP53 STATUS (TESTING) BY IHC

Loss of nuclear ATRX protein expression in tumor cells of an IDH mutant DG as determined by IHC serves as a relatively (albeit not completely) sensitive and specific surrogate marker for astrocytic lineage. Because strong and diffuse nuclear p53 immunoreactivity is also commonly encountered in IDH-mutant astrocytomas, the ATRX immunostain is often run simultaneously with the IDH1-R132H and p53 stains. Loss of nuclear ATRX expression by IHC is strongly predictive of a 1p/19q noncodeleted status. Among IDH-mutant DGs, TP53 mutation and p53 overexpression are associated with astrocytic lineage and test results for these biomarkers are used to support the diagnosis of IDH-mutant astrocytomas, especially in the setting of ATRX loss or mutation.^{22, 23, 24,25,26,27}

H3K27M/H3K27Me3 ALTERATION TESTING BY IHC

H3 K27M testing must be performed in DGs that involve the midline in the appropriate clinical and pathologic setting. H3 K27M testing can be performed using anti-H3 K27M IHC, which displays strong nuclear staining with relatively high sensitivity for the mutation, ranging from approximately 71% to 100% when compared with sequencing methods. IHC can be particularly useful in small biopsy samples, when tissue is limited. As most of these tumors occur in the pediatric population, this recommendation for additional testing is essential for children and young adults. For older adults there is increasing evidence that the midline location of a DG is also tightly linked with H3 K27M mutation, warranting testing of all patients with midline gliomas to guide appropriate care.²⁸ Regardless of histologic features, the presence of an H3 K27M mutation in a diffusely infiltrative glioma involving the midline most often predicts clinically aggressive behavior and poor prognosis, leading to its designation of WHO grade 4.^29,30.

DGs that have H3 K27M mutations also show loss of H3 K27 trimethylation (H3 K27me3), which can be detected by loss of nuclear immunoreactivity for H3 K27me3 by IHC. A smaller subset of diffuse midline gliomas that show loss of H3 K27me3 do not have H3 K27M mutations, but rather have EGFR mutations or overexpression of EZHIP.^{31, 32,33}

H3 G34 TESTING BY IHC

 H3 G34 testing may be performed in pediatric and young adults with IDH-WT DGs. A hotspot mutation in the histone gene H3F3A found within a subset of pediatric and young adult high-grade gliomas confers either a G34R or G34V substitution in the gene product. G34R/V mutant DGs occur in a somewhat older age group in comparison with K27M cases and tend to involve the cerebral hemispheres rather than midline locations. Prognosis for G34R/V-mutant gliomas is poor but somewhat better than the K27M-mutant gliomas (median survival of 12 months for K27M and 24 months for G34R or G34V).

BRAF (V600E) MUTATION TESTING BY IHC

BRAF V600E mutation occurs in a higher proportion of generally circumscribed neuroepithelial tumors, PXA, GG, and pilocytic astrocytoma. BRAF testing is most relevant to IDH-WT and H3-WT DGs in which BRAF V600E mutations have been shown to be enriched, including epithelioid GBMs and cases overlapping with PXA or anaplastic PXA; pediatric DGs or tumors overlapping with GG or pilocytic astrocytoma; and histologic grade 2 and 3 DGs lacking a ‘‘GBM molecular signature’’.

MAIN REFERENCE:

Brat DJ, Aldape K, Bridge JA, Canoll P, Colman H, Hameed MR, Harris BT, Hattab EM, Huse JT, Jenkins RB, Lopez-Terrada DH, McDonald WC, Rodriguez FJ, Souter LH, Colasacco C, Thomas NE, Yount MH, van den Bent MJ, Perry A. Molecular Biomarker Testing for the Diagnosis of Diffuse Gliomas. Arch Pathol Lab Med. 2022 Feb 17. doi: 10.5858/arpa.2021-0295-CP. Epub ahead of print. PMID: 35175291.

FOR DETAILED REFERENCES REFER TO RGCIRC WEBSITE –DEPTT OF LAB SERVICES

• Parsons DW, Jin G, et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med. 2009;360(8):765–773. doi:10.1056/NEJMoa0808710
• Ceccarelli M, Barthel FP, Malta TM, et al. Molecular profiling reveals biologically discrete subsets and pathways of progression in diffuse glioma. Cell. 2016;164(3):550–563. doi:10.1016/j.cell.2015.12.028
• Brat DJ, Verhaak RGW, Aldape KA, et al; Cancer Genome Atlas Research Network. Comprehensive, integrative genomic analysis of diffuse lower-grade gliomas. N Engl J Med. 2015;372(26):2481–2498. doi:10.1056/NEJMoa1402121
• Felsberg J, Wolter M, Seul H, et al. Rapid and sensitive assessment of the IDH1 and IDH2 mutation status in cerebral gliomas based on DNA pyrosequencing. Acta Neuropathol. 2010;119(4):501–507. doi:10.1007/ s00401-010-0647-4 28. Fontana L, Tab
• Labussiere M, Idbaih A, Wang XW, et al. All the 1p19q codeleted gliomas are mutated on IDH1 or IDH2. Neurology. 2010;74(23):1886–1890. doi:10. 1212/WNL.0b013e3181e1cf3a 37. Lewandowska MA, Furtak J, Szylberg T, et al. An analysis of the prognostic value of IDH1 (isocitrate dehydrogenase 1) mutation in Polish glioma patients. Mol Diagn Ther. 2014;18(1):45–53. doi:10.1007/s40291-013-0050-7
• Sanson M, Marie Y, Paris S, et al. Isocitrate dehydrogenase 1 codon 132 mutation is an important prognostic biomarker in gliomas. J Clin Oncol. 2009; 27(25):4150–4154. doi:10.1200/JCO.2009.21.98
• Mellai M, Piazzi A, Caldera V, et al. IDH1 and IDH2 mutations, immunohistochemistry and associations in a series of br
• Rajeswarie RT, Rao S, Nandeesh BN, Yasha TC, Santosh V. A simple algorithmic approach using histology and immunohistochemistry for the current classification of adult diffuse glioma in a resource-limited set-up. J Clin Pathol. 2018;71(4):323–329. doi:10.1136/jclinpath-2017-204638 51. Pollack IF, Hamilton RL, Sobol RW, et al. IDH1 mutations are common in malignant gliomas arising in adolescents: a report from the Children’s Oncology Group. Childs Nerv Syst. 2011;27(1):87–94. doi:10.1007/s00381-010-1264-
• Capper D, Reuss D, Schittenhelm J, et al. Mutation-specific IDH1 antibody differentiates oligodendrogliomas and oligoastrocytomas from other brain tumors with oligodendroglioma-like morphology. Acta Neuropathol. 2011;121(2):241– 252. doi:10.1007/s00401-010-0770-2
• DeWitt JC, Jordan JT, Frosch MP, et al. Cost-effectiveness of IDH testing in diffuse gliomas according to the 2016 WHO classification of tumors of the central nervous system recommendations. Neuro Oncology. 2017;219(12):1640– 1650. doi:10.1093/neuonc/nox120
• Preusser M, Wohrer A, Stary S, Hoftberger R, Streubel B, Hainfellner JA. Value and limitations of immunohistochemistry and gene sequencing for detection of the IDH1-R132H mutation in diffuse glioma biopsy specimens. J Neuropathol Exp Neurol. 2011;70(8):715–723. doi:10.1097/NEN. 0b013e31822713f0
• Pyo JS, Kim NY, Kim RH, Kang G. Concordance analysis and diagnostic test accuracy review of IDH1 immunohistochemistry in glioblastoma. Brain Tumor Pathol. 2016;33(4):248–254. doi:10.1007/s10014-016-0272-
• Yang RR, Shi ZF, Zhang ZY, et al. IDH mutant lower grade (WHO grades II/III) astrocytomas can be stratified for risk by CDKN2A, CDK4 and PDGFRA copy number alterations. Brain Pathol. 2020;30(3):541-553. doi:10.1111/bpa.12801
• Louis DN, Oghaki H, Wiestler OD, Caenee WK, eds. Who Classification of Tumours of the Central Nervous System. 4th ed. World Health Organization Press; 2016. World Health Organization Classification of Tumours, vol 1.
• Ebrahimi A, Skardelly M, Bonzheim I, et al. ATRX immunostaining predicts IDH and H3F3A status in gliomas. Acta Neuropathol Commun. 2016; 4(1):60. doi:10.1186/s40478-016-0331-6
• Jha P, Suri V, Sharma V, et al. IDH1 mutations in gliomas: first series from a tertiary care centre in India with comprehensive review of literature. Exp Mol Pathol. 2011;91(1):385–393. doi:10.1016/j.yexmp.2011.04.017
• Nobusawa S, Watanabe T, Kleihues P, Ohgaki H. IDH1 mutations as molecular signature and predictive factor of secondary glioblastomas. Clin Cancer Res. 2009;15(19):6002–6007. doi:10.1158/1078-0432.CCR-09-0715
• DeWitt JC, Jordan JT, Frosch MP, et al. Cost-effectiveness of IDH testing in diffuse gliomas according to the 2016 WHO classification of tumors of the central nervous system recommendations. Neuro Oncology. 2017;219(12):1640–1650. doi:10.1093/neuonc/nox120
• Hewer E, Vajtai I, Dettmer MS, Berezowska S, Vassella E. Combined ATRX/IDH1 immunohistochemistry predicts genotype of oligoastrocytomas. Histopathology. 2016;68(2):272–278. doi:doi.org/10.1111/his.12743
• Wiestler B, Capper D, Holland-Letz T, et al. ATRX loss refines the classification of anaplastic gliomas and identifies a subgroup of IDH mutant astrocytic tumors with better prognosis. Acta Neuropathol. 2013;126(3):443– 451. doi:10.1007/s00401-013-1156-z
• Sahm F, Reuss D, Koelsche C, et al. Farewell to oligoastrocytoma: in situ molecular genetics favor classification as either oligodendroglioma or astrocytoma. Acta Neuropathol. 2014;128(4):551–559. doi:10.
• McLendon R, Friedman A, Bigner D, et al; Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008;455(7216):1061–1068. doi:10.1038/ nature07385
• Bienkowski M, Wohrer A, Moser P, et al. Molecular diagnostic testing of diffuse gliomas in the real-life setting: a practical approach. Clin Neuropathol.2018;37(4):166–177. doi:10.5414/NP301110
• Shao LW, Pan Y, Qi XL, et al. ATRX loss in adult supratentorial diffuse astrocytomas correlates with p53 over expression and IDH1 mutation and predicts better outcome in p53 accumulated patients. Histol Histopathol. 2016; 31(1):103–114. doi:10.14670/HH-11-664 1007/s00401-014-1326-
• Pessoa IA, Amorim CK, Ferreira WAS, et al. Detection and correlation of single and concomitant TP53, PTEN, and CDKN2A alterations in gliomas. Int J Mol Sci. 2019;20(11):2658. doi:10.3390/ijms20112658
• Yang RR, Shi ZF, Zhang ZY, et al. IDH mutant lower grade (WHO grades II/ III) astrocytomas can be stratified for risk by CDKN2A, CDK4 and PDGFRA copy number alterations. Brain Pathol. 2020;30(3):541–553. doi:10.1111/bpa.12801
• Reis GF, Pekmezci M, Hansen HM, et al. CDKN2A loss is associated with shortened overall survival in lower-grade (World Health Organization Grades II/III) astrocytomas. J Neuropathol Exp Neurol. 2015;74(5):442–452. doi:10.1097/NEN
• Solomon DA, Wood MD, Tihan T, et al. Diffuse midline gliomas with histone H3-k27m mutation: a series of 47 cases assessing the spectrum of morphologic variation and associated genetic alterations. Brain Pathol. 2016; 26(5):569–580. doi:10.1111/bpa.12336
• Khuong-Quang DA, Buczkowicz P, Rakopoulos P, et al. K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas. Acta Neuropathol. 2012;124(3):439–447. doi: 10.1007/s00401-012-0998-0
• Castel D, Philippe C, Calmon R, et al. Histone H3F3A and HIST1H3B  K27M mutations define two subgroups of diffuse intrinsic pontine gliomas with different prognosis and phenotypes. Acta Neuropathol. 2015;130(6):815–827. doi:10.1007/s00401-015-1478-0
• Venneti S, Santi M, Felicella MM, et al. A sensitive and specific histopathologic prognostic marker for H3F3A K27M mutant pediatric glioblastomas. Acta Neuropathol. 2014;128(5):743–753. doi:10.1007/s00401-014-1338-3
• Mondal G, Lee JC, Ravindranathan A, et al. Pediatric bithalamic gliomas have a distinct epigenetic signature and frequent EGFR exon 20 insertions resulting in potential sensitivity to targeted kinase inhibition. Acta Neuropathol. 2020;139(6):1071–1088. doi:10.1007/s00401-020-02155-5
• Castel D, Kergrohen T, Tauziede-Espariat A, et al. Histone H3 wild-type DIPG/DMG overexpressing EZHIP extend the spectrum diffuse midline gliomas with PRC2 inhibition beyond H3-K27M mutation.
• Brat DJ, Aldape K, Bridge JA, Canoll P, Colman H, Hameed MR, Harris BT, Hattab EM, Huse JT, Jenkins RB, Lopez-Terrada DH, McDonald WC, Rodriguez FJ, Souter LH, Colasacco C, Thomas NE, Yount MH, van den Bent MJ, Perry A. Molecular Biomarker Testing for the Diagnosis of Diffuse Gliomas. Arch Pathol Lab Med. 2022 Feb 17. doi: 10.5858/arpa.2021-0295-CP. Epub ahead of print. PMID: 35175291.