The diagnosis and clinical management of patients with diffuse gliomas (DGs) have evolved rapidly over the past decade with the emergence of molecular biomarkers that are used to classify, stratify risk, and predict treatment response for optimal clinical care.
To develop evidence-based recommendations for informing molecular biomarker testing for pediatric and adult patients with DGs and provide guidance for appropriate laboratory test and biomarker selection for optimal diagnosis, risk stratification, and prediction.
The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. A systematic review of literature was conducted to address the overarching question, “What ancillary tests are needed to classify DGs and sufficiently inform the clinical management of patients?” Recommendations were derived from quality of evidence, open comment feedback, and expert panel consensus.
Thirteen recommendations and 3 good practice statements were established to guide pathologists and treating physicians on the most appropriate methods and molecular biomarkers to include in laboratory testing to inform clinical management of patients with DGs.
Evidence-based incorporation of laboratory results from molecular biomarker testing into integrated diagnoses of DGs provides reproducible and clinically meaningful information for patient management.
Diffuse gliomas (DGs) are primary central nervous system (CNS) neoplasms characterized by the widespread infiltration of individual tumor cells displaying cytologic and histologic features of glial differentiation.1 These tumors affect patients of all ages and arise throughout the neuro-axis but are most common in older adults and occur most frequently in the cerebral hemispheres. Clinical course and treatment vary based on tumor type and grade. Since the early 1900s, and until the 2016 revised 4th edition of the World Health Organization (WHO) Classification of Tumours of the Central Nervous System,1 DGs were classified based upon the morphologic features of neoplastic cells, with molecular testing playing an ancillary role.
During the past decade, numerous investigations have uncovered molecular genetic alterations that can be used to reliably and reproducibly classify DGs into clinically meaningful subsets, leading the 2016 WHO revised 4th edition update to incorporate diagnostic entities based on the integration of morphologic features with molecular biomarkers.1–6 More recent advances in our understanding of the pathogenesis and clinical behavior of specific DG subtypes has led to the inclusion of additional molecular biomarkers into clinical practice and the 2021 WHO 5th edition relies even more on molecular test results for diagnosis and grading.7–11 DNA methylome profiling continues to identify numerous tumor types with specific methylation patterns that have characteristic genetic alterations and clinical behavior.12 The increasing complexity and rapid pace of change in diagnostic criteria, relevant molecular biomarkers, laboratory testing platforms, and clinical practice warrant the development of evidence-based recommendations on biomarker testing for DGs.
DESIGN
This evidence-based guideline was developed following the standards endorsed by the National Academy of Medicine.13 A detailed description of the methods and the systematic review (including the quality assessment and complete analysis of the evidence) used to create this guideline can be found in the supplemental digital content (SDC) at https://meridian.allenpress.com/aplm in the May 2022 table of contents.
PANEL COMPOSITION
The College of American Pathologists (CAP) in collaboration with the American Association of Neuropathologists, Association for Molecular Pathology, and Society for Neuro-Oncology convened a multidisciplinary expert panel consisting of 13 practicing pathologists from a variety of specialties, institution types, and other professional groups, 2 oncologists who were representatives of the American Society of Clinical Oncology, 1 patient advocate, and a research methodologist consultant to develop this guideline. An advisory panel assisted the expert panel at specific key stages in the development of the guideline. All panel members, except for the methodologist consultant, volunteered their time and were not compensated for their involvement. Detailed information about the panel composition can be found in the SDC.
CONFLICT OF INTEREST POLICY
The collaborators agreed upon a conflict of interest policy (effective June 2017), and members of the expert panel disclosed all financial interests from 3 years before appointment through the development of the guideline. Individuals were instructed to disclose any relationship that could be interpreted as constituting an actual, potential, or apparent conflict. Complete disclosures of the expert panel members are listed in the Appendix. Disclosures of interest judged by the oversight group to be manageable conflicts are as follows: HC – Consultancies with F. Hoffman-La Roche, Ltd (Basel, Switzerland), Genentech USA, Inc. (South San Francisco, California), Upsher-Smith Laboratories, LLC (Grove, Minnesota), Novocure (St. Helier, Jersey), Insys Therapeutics (Phoenix, Arizona), Mateon Therapeutics (formerly OxiGENE) (Agoura Hills, California), CytRx Corp. (Los Angeles, California), Omniox Inc. (San Carlos, California), AbbVie Inc. (North Chicago, Illinois); EH – Consultancies with E.R. Squibb&Sons, LLC (New Brunswick, New Jersey), Honorarium from Arbor Pharmaceuticals, LLC (Atlanta, Georgia); MvdB – Consultancies with Bristol Myers Squibb (New York, New York), Celldex Therapeutics, Inc. (Hampton, New Jersey), Vaximm AG (Basel, Switzerland), Carthera, Nerviano, Agios, Honorarium from Merk Sharp & Dohme Corp (Kenilworth, New Jersey), Research Grants from AbbVie Inc (North Chicago, Illinois).
Most of the expert panel (14 of 17 members) were assessed as having no relevant conflicts of interest. The CAP provided funding for the administration of the project; no industry funds were used in the development of the guideline. All panel members volunteered their time and were not compensated for their involvement, except for the contracted methodologist. See the SDC for complete information about the conflict of interest policy.
GUIDELINE OBJECTIVES
The expert panel addressed the overarching question, “What ancillary tests are needed to classify DGs to sufficiently inform the clinical management of patients?” To answer this, several more pointed key questions (KQs) were developed as follows:
- KQ 1a:
What genetic and molecular alterations should be included for optimal classification of DGs?
- KQ1b:
What are the acceptable techniques/methods for molecular genetic testing of DGs? What are the expected turnaround times for individual assays?
- KQ1c:
What are the acceptable techniques/methods for assessing whole genome copy number alterations?
- KQ 2:
What are the core molecular tests/findings that provide sufficient classifying information in the setting of discrete clinicopathologic entities?
- KQ 3:
What are the acceptable techniques/methods/criteria for determining MGMT promoter methylation status?
OUTCOMES OF INTEREST
The panel established outcomes of interest for both clinically based and pathology-based studies/articles. The clinical outcomes of interest included survival rates (overall, 1-year and 3-year survival, progression free), recurrence rates, response to treatment, and accuracy of diagnosis. The pathologic outcomes of interest included sensitivity, specificity, positive predictive value, negative predictive value, concordance, turnaround time, reproducibility of the various tests, as well as mutation/alteration/deletion status (percent, presence, frequency, and association with other alterations) for the molecular targets of interest.
The target audience of this review are those within the neuro-oncology community who care for patients with DGs, including pathologists, neuroradiologists, neurosurgeons, radiation oncologists, neuro-oncologists, medical oncologists, and other members of the patient care team. Recommendations will also be highly relevant to brain tumor investigators, epidemiologists, and cancer registrars.
LITERATURE SEARCH AND COLLECTION
Literature search strategies were developed in collaboration with a medical librarian for the concepts of DGs, molecular markers or gene alterations, and laboratory test methods. In consultation with the expert panel, the search strategies were created using standardized database terms and keywords. Databases searched included Ovid MEDLINE and Embase.com. Additional searches for unindexed literature were conducted in ClinicalTrials.gov, Cochrane Library, Guidelines International Network, National Guideline Clearinghouse, Trip search engine, University of York Centre for Reviews and Dissemination-PROSPERO, and applicable US and international organizational Web sites. In addition, expert panel members were surveyed for any relevant unpublished data at the onset of the project. Initial database searches were completed on November 13, 2017, and refreshed on September 3, 2019, and September 23, 2020. All searches were limited to English language and publication dates of January 1, 2008, to the date of search. Case reports, commentaries, editorials, conference abstracts, and letters were excluded. The Cochrane search filter for humans was applied in Ovid MEDLINE and Embase.com.14 MEDLINE and conference abstract records were excluded in the Embase searches. A targeted search was performed in Ovid MEDLINE on July 24, 2020, to ensure that emerging evidence about bithalamic glioma, infantile-type hemisphere glioma, and diffuse pediatric-type high-grade glioma and relevant mutations or amplifications was included in anticipation of the WHO 2021 update.7 The search was limited to publication dates January 1, 2016 to July 24, 2020, English language, and human studies. Case reports, commentaries, editorials, and letters were excluded. The Ovid MEDLINE and Embase search strings and Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) diagrams outlining details of the systematic and targeted review are provided in the SDC as Supplemental Figures 1 through 3.
INCLUSION AND EXCLUSION CRITERIA
Studies were selected for inclusion in the systematic review of evidence if they met the following criteria: the study included human patients; the study was a systematic review with or without meta-analysis, a randomized clinical trial, or a comparative or noncomparative observational study (prospective or retrospective design); the study included a minimum sample size of at least 30, except for studies evaluating B-Raf proto-oncogene (BRAF) alterations, fibroblast growth factor receptor (FGFR) alterations, and MYB proto-oncogene (MYB) and MYB proto-oncogene like 1 (MYBL1) alterations, for which the expert panel determined all study sizes should be included based on a lower frequency of these alterations that occur in pediatric brain tumors; the study was published in English; the study addressed 1 of the key questions; the study included measurable data such as accuracy, sensitivity, specificity, negative predictive value, positive predictive value, time to appropriate testing, repeat procedures, overall survival, recurrence rates, turnaround time, concordance; and the study addressed DGs and included at least one of the following genetic and molecular alterations:
Isocitrate dehydrogenase (NADP(+))1 (IDH1) and isocitrate dehydrogenase (NADP(+))2 (IDH2) mutations
Histone H3 gene mutations
BRAF alterations
ATRX chromatin remodeler (ATRX) alterations
Tumor protein p53 (TP53) alterations
1p/19q codeletion
Chromosome 7 gain
Chromosome 10 loss
MYB and MYBL1 alterations
Telomerase reverse transcriptase (TERT) promoter mutations
FGFR alterations
Epidermal growth factor (EGFR) alterations
Platelet-derived growth factor receptor alpha (PDGFRA) alterations
MET proto-oncogene, receptor tyrosine kinase (MET) alterations
Cyclin-dependent kinase inhibitor 2A (CDKN2A) alterations
O-6-methylguanine-DNA methlytransferase (MGMT) promoter alterations
Phosphatase and tensin homolog (PTEN) alterations
Neurofibromin 1 (NF1) alterations
Microsatellite instability status
MDM2 proto-oncogene (MDM2) alterations
Cyclin-dependent kinase 4 (CDK4) alteration
Articles were excluded from the systematic review if they were meeting abstracts; case reports, consensus documents, editorials, commentaries, or letters; cell line or animal model studies; full-text articles not available in English; or studies that did not address at least 1 of the key questions with outcomes of interest as agreed upon.
QUALITY ASSESSMENT
A risk of bias assessment was performed for all fully published studies meeting inclusion criteria by the research methodologist. The methodologist assessed key indicators based on study design and methodologic rigor and a rating for the quality of evidence was designated. See Supplemental Tables 1 through 4 for the quality assessment for included studies by study design. See Supplemental Table 5 for the Quality of Evidence definitions. Using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach,15 an overall GRADE rating was given for each recommendation by outcome (Supplemental Table 6). Refer to the SDC for further details.
ASSESSING THE STRENGTH OF RECOMMENDATIONS
Following the quality of evidence assessment, completion of the GRADE Evidence to Decision framework,16 and discussion of the definitions and implications of strength of recommendation (Table 1), the expert panel designated the recommendations as either strong or conditional. Refer to the SDC for further details.
GUIDELINE REVISION
This guideline will be reviewed every 4 years, or earlier in the event of publication of substantive and high-quality evidence that could potentially alter the original guideline recommendations. If necessary, the entire panel will reconvene to discuss potential changes. When appropriate, the panel will recommend revision of the guideline to the CAP and its collaborators for review and approval.
RESULTS
A total of 4821 studies met the search term requirements. Based on review of these abstracts, 703 articles met the inclusion criteria and continued to full-text review. A total of 188 articles were included for data extraction, and 86 articles informed the recommendations. Excluded articles were available as discussion or background references. Additional information about the systematic review is available in the SDC. Note that only the studies listed in the quality summary were used to inform the recommendations. These are studies from our systematic review of the literature. All other references mentioned in the write-up of each recommendation were brought in to provide context or further support. For most recommendations, the evidence base consisted of studies reporting on the molecular alteration frequency of the recommended gene in defined WHO DG subtypes. To highlight this evidence as it relates to establishing an accurate diagnosis while maintaining brevity for publication, each recommendation is accompanied by a table of representative studies. The representative studies chosen for inclusion in the tables are those that the expert panel believed most important in summarizing the evidence for each recommendation. Refer to the diffuse gliomas guideline Web page (www.cap.org) for the complete set of data tables.
The expert panel convened 17 times (15 times by teleconference and 2 in-person meetings) to develop the scope, draft recommendations, review and respond to solicited feedback, and assess the quality of evidence that supports the final recommendations. A nominal group technique was used for consensus decision making to encourage unique input with balanced participation among group members. An initial open comment period was posted on the CAP Web site (www.cap.org) from September 9 to September 30, 2019, during which the draft recommendations were posted for public feedback. To allow for more responses, the CAP reopened the comment period from October 11 to October 31, 2019. Refer to the SDC for further details, including a list of organizations encouraged to participate. The expert panel approved the final recommendations with a supermajority vote.
An independent review panel, masked to the expert panel and vetted through the conflict of interest process, recommended approval by the CAP Council on Scientific Affairs. The manuscript was also approved by American Association of Neuropathologists, Association for Molecular Pathology, and Society for Neuro-Oncology. The final recommendations are summarized in Table 2. In addition, a visual representation of the guideline recommendations is provided to help laboratories and clinicians understand the testing involved that ultimately drives WHO-defined categorization. (Figure 1 shows the testing algorithm where IDH1/2 mutations are present. Figure 2 shows the testing algorithm where IDH1/2 mutations are not present.)
Testing algorithm for diffuse gliomas with IDH1/2 mutations. A, refer to Figure 2. Blue indicates WHO-defined entities; green indicates recommended tests. *Some institutions/laboratories may prefer to perform 1p/19q codeletion as the initial step for IDH-mutant gliomas. See recommendations 2 through 4. **Additional molecular biomarker testing and DNA methylation profiling may be helpful in establishing a diagnosis for challenging cases. Abbreviations: ATRX, ATRX chromatin remodeler; CDKN2A, cyclin-dependent kinase inhibitor 2A; CDKN2B, cyclin-dependent kinase inhibitor 2B; codel, codeletion; IDH, isocitrate dehydrogenase; MVP, microvascular proliferation; Mut, mutation; TERTp, telomerase reverse transcriptase promoter; TP53, tumor protein p53; WHO, World Health Organization.
Testing algorithm for diffuse gliomas with IDH1/2 mutations. A, refer to Figure 2. Blue indicates WHO-defined entities; green indicates recommended tests. *Some institutions/laboratories may prefer to perform 1p/19q codeletion as the initial step for IDH-mutant gliomas. See recommendations 2 through 4. **Additional molecular biomarker testing and DNA methylation profiling may be helpful in establishing a diagnosis for challenging cases. Abbreviations: ATRX, ATRX chromatin remodeler; CDKN2A, cyclin-dependent kinase inhibitor 2A; CDKN2B, cyclin-dependent kinase inhibitor 2B; codel, codeletion; IDH, isocitrate dehydrogenase; MVP, microvascular proliferation; Mut, mutation; TERTp, telomerase reverse transcriptase promoter; TP53, tumor protein p53; WHO, World Health Organization.
Testing algorithm for diffuse gliomas without IDH1/2 mutations. A, refer to Figure 1. Blue indicates WHO-defined entities; green indicates recommended tests; italic indicates good practice statements. **Additional molecular biomarker testing and DNA methylation profiling may be helpful in establishing a diagnosis for challenging cases. Abbreviations: ALK, ALK receptor tyrosine kinase; BRAF, B-Raf proto-oncogene; CNA, copy number alteration; EGFR, epidermal growth factor receptor; FGFR1, fibroblast growth factor receptor; H3, histone 3 gene mutation; H3 K27me3, H3 K27M trimethylation; IDH, isocitrate dehydrogenase (NADP(+)); IHC, immunohistochemistry; MAPK, mitogen-activated protein kinase; MET, MET proto-oncogene, receptor tyrosine kinase; MGMTp, O-6-methylguanine-DNA methlytransferase; Mut, mutation; MYB, MYB proto-oncogene; MYBL1, MYB-like; MYCN, N-myc proto-oncogene protein; NTRK, neurotrophic tyrosine receptor kinase; PDGFRA, platelet-derived growth factor receptor alpha; ROS1, ROS proto-oncogene 1; TERTp, telomerase reverse transcriptase promoter; WHO, World Health Organization.
Testing algorithm for diffuse gliomas without IDH1/2 mutations. A, refer to Figure 1. Blue indicates WHO-defined entities; green indicates recommended tests; italic indicates good practice statements. **Additional molecular biomarker testing and DNA methylation profiling may be helpful in establishing a diagnosis for challenging cases. Abbreviations: ALK, ALK receptor tyrosine kinase; BRAF, B-Raf proto-oncogene; CNA, copy number alteration; EGFR, epidermal growth factor receptor; FGFR1, fibroblast growth factor receptor; H3, histone 3 gene mutation; H3 K27me3, H3 K27M trimethylation; IDH, isocitrate dehydrogenase (NADP(+)); IHC, immunohistochemistry; MAPK, mitogen-activated protein kinase; MET, MET proto-oncogene, receptor tyrosine kinase; MGMTp, O-6-methylguanine-DNA methlytransferase; Mut, mutation; MYB, MYB proto-oncogene; MYBL1, MYB-like; MYCN, N-myc proto-oncogene protein; NTRK, neurotrophic tyrosine receptor kinase; PDGFRA, platelet-derived growth factor receptor alpha; ROS1, ROS proto-oncogene 1; TERTp, telomerase reverse transcriptase promoter; WHO, World Health Organization.
RECOMMENDATIONS
Strong Recommendation: 1
IDH mutational testing must be performed on all DGs.
The quality of evidence to support this recommendation was assessed as moderate.
Quality Summary
The evidence base informing this recommendation comprises 38 studies.3–6,17–50 Four studies were genome sequencing studies,5,6,21,22 while the remaining 34 studies were retrospective cohort studies.3,4,17–20,23–50 All genome sequencing studies5,6,21,22 obtained samples retrospectively and were assessed as intermediate-low quality based on risk of selection bias, while no other forms of bias were identified. The retrospective cohort studies all suffered from risk of selection bias and were assessed as low* and very low quality4,18,20,27,29,30,34 based on risk of selection bias in addition to risk in performance, detection, and reporting domains. The aggregate risk of bias across the entire evidence base was serious but quality of evidence was upgraded based on a strong association between IDH mutational testing and DG diagnostic classification. Refer to the SDC for the quality assessment of included genome sequencing studies, retrospective cohort studies, and the GRADE Quality of Evidence Assessment. See Table 3 for a summary of the mutational status of IDH across all DG subtypes.
Representative Studies Reporting on IDH Mutation Testing and Status Across Diffuse Glioma (DG) Subtypes

The identification of an IDH mutation within a DG is required for the diagnosis of specific neoplastic types recognized by the WHO, including astrocytoma, IDH-mutant and oligodendroglioma, IDH-mutant, 1p/19q codeleted.1,7 More than 70% of histologic grades 2 and 3 infiltrating astrocytomas in adults are IDH-mutated. By definition, all oligodendrogliomas IDH-mutant, 1p/19q codeleted harbor IDH mutations.4–6,34 IDH-mutant DGs are distinct diseases that have specific genetic profiles, clinical courses, and therapeutic options that differ from other forms of DG, such as IDH wild-type (WT) and histone H3-mutant DGs.† Therefore, testing for IDH mutations must be performed on DGs to diagnose these tumor types for proper clinical care. Testing may not be necessary if a definitive diagnosis of an IDH-mutant glioma or another diagnosis that is mutually exclusive with IDH-mutant glioma has been previously established. IDH-mutant gliomas are uncommon in pediatric patients and testing may not be necessary in younger children.51 Testing for IDH mutations also may not be necessary if genetic alterations that are mutually exclusive with IDH mutations have been identified in a DG, such as histone H3 mutations. It should be noted that EGFR amplifications, gain of chromosome 7/loss of chromosome 10, and TERT promoter mutations have been documented in IDH-mutant astrocytomas, albeit at much lower frequency than GBM, IDH-WT, and their identification in a DG should not automatically rule out IDH testing.3,26,39,47,52 Other clinical or pathologic settings may be encountered in which the diagnosis of an IDH-mutant DG can be formally excluded without testing for the mutation.
IDH1 and IDH2 mutations result in a substitution for a key arginine at codons R132 and R172, respectively.53 The most frequent IDH1 mutation is R132H, which accounts for 89% to 93% of all IDH1 and IDH2 mutations.5,6,27,35,47,53 A highly sensitive and specific monoclonal antibody that recognizes the IDH1-R132H mutant protein is widely used in diagnostic practice. Immunohistochemistry (IHC) for IDH1-R132H is a cost-effective and reliable first-line test for IDH1 mutation in supratentorial DGs.39,50,54–57 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 (see above). 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,58 suggesting that IDH sequencing may be required more frequently in this clinical setting.
Sequence analysis for IDH mutations can be performed by pyrosequencing, Sanger sequencing, polymerase chain reaction (PCR), or next-generation sequencing (NGS).27,34,59–61 Because patients older than 55 years only rarely develop de novo glioblastomas (GBMs) that are IDH-mutant (4%–7%) and 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.1,26,27,32,43,55 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. As above, IDH testing may also be unnecessary if other mutually exclusive genetic alterations are identified.
The finding of an IDH mutation in the setting of glial proliferation strongly supports the diagnosis of a DG because this event does not occur in nonneoplastic diseases and is only rarely, if ever, found in CNS neoplasms other than DGs.27,46,62–64 IDH mutations are almost always stable and persist through the course of disease, which can be exploited to evaluate residual/progressive tumor33,65,66 ; however, loss of the IDH-mutated allele has been documented in some cases at the time of recurrence and may be associated with greater cell proliferation or high-grade behavior.67,68
Public Comment Response to Recommendation 1
There were 92 respondents, of whom 84 (91.31%) agreed or agreed with modifications, 6 (6.52%) disagreed, and 2 (2.17%) were neutral. There were 31 written comments, including many suggesting that the phrase “appropriate clinical and pathologic setting” needed to be further explained. There were also suggestions that the statement should recommend that IDH testing “should” be performed rather than “must” be performed. Others indicated that the methods of testing (eg, IHC versus sequencing) should be described. These comments were taken into consideration. While the recommendation remained the same, the comments were addressed in the text above.
Strong Recommendation: 2
ATRX status should be assessed in all IDH-mutant DGs unless they show 1p/19q codeletion.
Conditional Recommendation: 3
TP53 status should be assessed in all IDH-mutant DGs unless they show 1p/19q codeletion.
Strong Recommendation: 4
1p/19q codeletion must be assessed in IDH-mutant DGs unless they show ATRX loss or TP53 mutation.
Recommendations 2 through 4 are tightly linked to one another and should be considered together. Once the initial testing of a DG has revealed that the tumor is IDH-mutant, further testing for ATRX, TP53, and 1p/19q support the diagnosis of IDH-mutant astrocytoma or oligodendroglioma, IDH-mutant, and 1p/19q codeleted. Testing algorithms and workflows differ across practices, institutions, and countries. Some perform IHC for ATRX and p53 first, with follow-up studies for 1p/19q only if these tests are negative. Others perform 1p/19q testing as the initial step, followed by ATRX and/or p53 testing if the test is negative. Multigene panels represent another approach that provides test results simultaneously; proponents assert that it provides information on other biomarkers relevant to classification and grade.69 Because positive test results from ATRX/p53 and 1p/19q testing are nearly mutually exclusive, it is not necessary to perform additional testing if the initial test results are sufficient to establish a diagnosis. Recommendations 2 through 4 are written in a manner to reflect these considerations.
The quality of evidence to support Recommendations 2 and 4 is moderate, the quality of evidence to support Recommendation 3 was assessed as low.
Quality Summary
Recommendation 2 was informed by 12 retrospective cohort studies.24–26,49,50,70–76 Eight of these studies were assessed as low quality,24–26,49,50,70,74,75 and 4 were assessed as very low quality.71–73,76 All included studies were limited by a critical risk of selection bias, plus individual studies were further limited by risk of bias in performance,26,72,73 detection,24,26,71–76 and reporting50,71–74,76 domains. Although the aggregate risk of bias across the evidence base was very serious, the evidence was upgraded based on a strong association between ATRX assessment and DG WHO classification. Recommendation 3 focuses on the need for TP53 assessment and was informed by 2 genome sequencing studies,6,22 2 prospective cohort studies,77,78 and 15 retrospective cohort studies.‡ The retrospective cohort studies were assessed as low§ and very low quality29,71,82 based on risk of bias in selection,‖ performance,19,29,79 detection,¶ and reporting29,42,71,80,82–85 domains. The aggregate risk of bias for the evidence base was very serious and evidence was not further downgraded or upgraded based on any domain. The evidence base supporting Recommendation 4 comprises one genome sequencing study6 and 11 retrospective cohort studies.# All retrospective cohort studies carry a critical risk of selection bias, plus individual studies were further limited by risk of bias in performance,4 detection,4,20,42,63,74–76,86 and reporting** domains. Although the aggregate risk of bias across the evidence base was very serious, the evidence was upgraded based on a strong association between 1p/19q codeletion status and diffuse glioma WHO classification. Refer to the SDC for the quality assessment of included genome sequencing studies, retrospective cohort studies, and the GRADE Quality of Evidence Assessment. See Table 4 for a summary of the mutational status of ATRX, TP53, and 1p/19q across all DG subtypes.
Recommendation 2
As with all cancers, DGs must employ a mechanism to prevent telomeres from shortening to escape cellular senescence. In the case of IDH-mutant astrocytomas (WHO grades 2–4), this is primarily accomplished by alternative lengthening of telomeres via inactivation of the ATRX gene.87 Most oligodendrogliomas, IDH-mutant, 1p/19q-codeleted, and IDH-WT GBMs accomplish this via overexpression of telomerase resulting from activating TERT promoter mutations. Owing to these associations, 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 (Recommendation 1) and p53 stains (discussed in Recommendation 3). Less common astrocytoma subtypes, which are more common in children, may also show loss of ATRX expression, including diffuse midline gliomas with H3 K27M mutation (∼15% of cases), diffuse hemispheric gliomas with H3 G34 mutation (nearly all cases), and anaplastic astrocytoma with piloid features.26,88–93 Consistent with their associations with glial lineage, there is a strong inverse relationship among IDH-mutant DGs between ATRX loss and the presence of 1p/19q codeletion (ie, molecularly defined oligodendrogliomas).49,50,72,76,94 Only rare examples of “dual genotype” IDH-mutant DGs have been described in which ATRX loss, TP53 mutation, and 1p/19q codeletion were identified in the same neoplasm.95–97 For this reason, it has been recommended that 1p/19q testing need not be pursued in IDH-mutant gliomas with immunohistochemically identified ATRX loss or p53 overexpression.98 By the same token, ATRX testing is not considered necessary for IDH-mutant DGs with established codeletion of chromosome 1p/19q.
Beyond these basic testing recommendations, a few practical issues are worth highlighting. First, the ATRX immunostain can be technically challenging and appropriate positive and negative controls are therefore critical for accurate assessment. Internal positive controls, such as entrapped neurons, nonneoplastic glia, and endothelial cells, should show retained expression within the same regions where tumor nuclei are immunonegative. If all nuclei (including nonneoplastic cells) are immunonegative, this should be interpreted as a technical failure of the immunostain rather than true loss of expression in the tumor. A mosaic reactivity pattern, wherein some tumor nuclei appear positive and others negative, often represents a technical failure as well and as such, should not be interpreted as loss of expression within just a subset of tumor cells. Regardless, in the case of equivocal immunostaining or in diagnostic practices that do not initially screen for ATRX inactivation using IHC, ATRX gene alterations can be identified instead using other molecular techniques (eg, NGS). Second, because only 70% to 80% of IDH-mutant astrocytomas (WHO grades 2–4) show ATRX inactivation, retained ATRX immunoexpression does not exclude the possibility of this diagnosis. Infratentorial IDH-mutant astrocytomas show loss of ATRX in only approximately 50% of cases.58 For cases suspected to be IDH-mutant astrocytoma based on morphology, but showing retention of ATRX on IHC, evidence of astrocytic lineage should depend instead on other findings, such as p53 protein overexpression, TP53 gene mutation, or a lack of 1p/19q codeletion. Last, ATRX loss may also be encountered in rare IDH-WT GBMs of older adults and in several pediatric/young adult IDH-WT astrocytoma subtypes, as already discussed. As such, ATRX loss is not entirely specific for IDH-mutant astrocytomas and this finding should not be interpreted in isolation, but rather in the context of other alterations, including IDH mutation. DNA methylation profiling has rapidly emerged as a platform that is capable of reproducibly identifying IDH-mutant astrocytomas, as well as IDH-mutant subtypes that are infratentorial, supratentorial low grade and supratentorial high grade. Although the technology is not widely available at this time, methylation profiling may be useful in diagnostically challenging cases and will likely emerge as an important diagnostic platform.12,58
Public Comment Response to Recommendation 2
There were 92 respondents, of whom 78 (84.8%) agreed or agreed with modification, 12 (13.1%) disagreed, and 2 (2.2%) were neutral. There were 37 written comments, including many requesting clarification of recommended testing methodologies and a specific algorithm outlining the recommended order of testing, including the relation of ATRX to 1p/19q, p53, histone H3, or TERT promoter analyses. One respondent commented that ATRX status is not needed if 1p/19q is done routinely, whereas another stated that 1p/19q results can be misleading, and as such, ATRX data provide useful quality assurance. Although some of these issues are beyond the scope of this paper, a few practical guidelines for testing were nonetheless discussed in the prior paragraph. A few respondents also commented that they did not believe ATRX testing was sufficiently cost effective or diagnostically informative to warrant its routine use, whereas a few felt that Recommendation 2 did not go far enough, because ATRX testing could be potentially useful for all gliomas, including nondiffuse and pediatric astrocytoma variants. These useful comments were taken into consideration and while the final recommendation remained the same, some of these specific concerns were addressed in the text above.
Recommendation 3
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.22,49,75,78,82,85,99 IDH-mutant gliomas that have whole-arm 1p/19q codeletion (ie, oligodendroglioma, IDH-mutant, 1p/19q codeleted) only rarely (2%–3%) exhibit TP53 mutation or strong p53 overexpression.19,42,71,83
In one large series, TP53 mutations were noted in 94% of IDH-mutant DGs that did not demonstrate 1p/19q codeletion.6 Thus, 1p/19q codeletion and TP53 mutations are nearly, but not entirely, mutually exclusive in IDH-mutant DGs. In IDH-mutant DGs with TP53 mutations, the 17p-arm typically exhibits copy neutral loss of heterozygosity (cnLOH); thus, there are generally 2 identical copies of the mutant TP53 gene.4–6,65,100 In a small percentage of IDH-mutant astrocytomas there are 2 distinct TP53 mutations; 1 involves each of the TP53 alleles on the two 17p arms.4–6,65,100 TP53 mutations that occur in IDH-mutant astrocytomas tend to activate the expression and/or enhance the stability of the p53 protein. Thus, p53 IHC is an excellent surrogate of TP53 mutation in IDH-mutant gliomas, especially if the expression of ATRX is not retained.101,102 Unlike IHC for ATRX, however, p53 expression is more of a continuum, and the interpretation may not be straightforward. For higher sensitivity and specificity, more than 10% of tumor cells must show strong nuclear positivity, although most TP53-mutant gliomas show even more widespread (>50%) p53 expression.102 Using this threshold, only a small subset of DGs with strong p53 immunoreactivity in more than 10% of nuclei were found to be TP53 WT by sequencing.102 If tumor DNA sequencing is performed, the combination of IDH, TP53 and ATRX mutations and the associated copy number patterns are distinctive for IDH-mutant astrocytomas.4–6,65,100 It should be noted that TP53 mutation is also encountered in other DGs, especially IDH-WT GBMs.24,29,41,42,86,103 In IDH-WT gliomas, one TP53 gene typically undergoes mutation, and the WT TP53 gene is lost through deletion. In IDH-WT gliomas, TP53 mutation less predictively generates p53 overexpression.75,83,104 Nevertheless, p53 overexpression is encountered in up to 30% of IDH-WT GBM, so this finding should not be used as support for IDH-mutant status in isolation.
Public Comment Response to Recommendation 3
There were 87 respondents, of whom 64 (73.6%) agreed or agreed with modifications, 16 (18.4%) disagreed, and 7 (8.0%) were neutral. There were 35 written comments received during the public comment period. These comments could be classified as follows: (1) the word “should” could be replaced with “must” or “may.” The “should” language of the recommendation was retained based on the strength of the evidence in the literature. (2) The method of TP53 mutation assessment should be addressed. As described in the text above, IHC can be used to assess the expression of p53 protein in the context of IDH-mutant gliomas based on its correlation with TP53 mutation; sequencing methods can also be employed. (3) Some commented that TP53 status should be evaluated in all IDH-mutant DGs. Based on the evidence, it was concluded that this is not necessary if whole-arm 1p/19q codeletion is known to be present. All comments were taken into consideration and the recommendation remained as initially stated.
Recommendation 4
Confirmation of whole-arm 1p/19q codeletion in an IDH-mutant DG is essential to render the diagnosis of oligodendroglioma, IDH-mutant and 1p/19q codeleted. Landmark genomic analyses have firmly established that whole-arm 1p/19q codeletion, the product of an unbalanced translocation involving chromosomes 1 and 19, is entirely restricted to gliomas that also harbor mutations in either IDH1 or IDH2.4,6 Therefore, assessment of 1p/19q codeletion status is only relevant in IDH-mutant DGs. 1p/19q testing is not recommended in IDH-WT gliomas, as false-positive test results (see below) may complicate effective diagnostic categorization and the results have no bearing on patient outcome or treatment.105,106
As described above, 1p/19q codeletion arises with near mutual exclusivity with respect to inactivating alterations of TP53 and ATRX.4,6 Multiple studies have concluded that loss of nuclear ATRX expression by IHC is strongly predictive of a 1p/19q noncodeleted status.74,76,82 Confirmed TP53 mutation or p53 overexpression is similarly inversely associated with 1p/19q codeletion.74,75 Loss of ATRX expression or TP53 mutation has been noted in a small minority of IDH-mutant gliomas that also exhibit 1p/19q codeletion (2%–8%).4,6,74,76,82 The extent to which these cases reflect false-positive testing for 1p/19q codeletion is unclear. Of note, The Cancer Genome Atlas analysis, which employed cytogenomic arrays to confirm whole-arm 1p/19q codeletion, revealed low rates of coinciding ATRX or TP53 mutations (2%–3%),6 suggesting that under strict testing criteria, the mutual exclusivity between ATRX/TP53 mutations and 1p/19q codeletion is near absolute. Nevertheless, the small degree of overlap between these biomarkers may justify 1p/19q codeletion assessment in some IDH-mutant gliomas with established ATRX loss or TP53 mutation/p53 overexpression if oligodendroglioma is suspected based on histopathologic features. As mentioned above, rare cases of carefully documented “dual genotype” IDH-mutant DGs have demonstrated the co-occurrence of ATRX loss, TP53 mutation, and whole-arm 1p/19q codeletion.95–97 Nonetheless, for most cases, 1p/19q testing is no longer needed if an IDH-mutant DG demonstrates ATRX loss and TP53 mutation, as highlighted in cIMPACT-NOW update 2.98
1p/19q codeletion is optimally assessed by cytogenomic array or NGS methodologies capable of definitively demonstrating deletion of whole chromosomal arms of 1p and 19q.35,107 Other approaches, such as fluorescence in situ hybridization (FISH) and PCR-based testing, have also been effectively applied to 1p/19q analysis4,74,76,82 and have lower cost and quicker turnaround time than their genomic counterparts. DNA methylation profiling can identify a methylome highly characteristic of oligodendroglioma and has the added benefit of providing copy number alterations that can directly inform the status of 1p/19q codeletion.12
FISH is the most widely used test and has the advantage of assessing a limited sample of tissue (2 slides; a minimum of 50 tumor nuclei required per slide), which is often the case in small biopsy samples. One must consider the possibility of false-positive results, attributable to partial arm deletions that are detected by FISH, yet do not represent whole-arm deletions associated with the unbalanced translocation. False-positive test results on 1p/19q testing by FISH are also noted in IDH-WT GBMs, because they are genomically unstable,105,106 underlying our recommendation that 1p/19q assessment be exclusively restricted to IDH-mutant gliomas.
Public Comment Response to Recommendation 4
There were 89 respondents, of whom 76 (85.4%) agreed or agreed with modifications, 10 (11.2%) disagreed, and 3 (3.7%) were neutral. Thirty comments were received. One comment suggested that the term “codeletional status” used in the prior version of the recommendation be removed and replaced with “codeletion” for simplicity. The text was modified in response to this recommendation. Multiple comments suggested that 1p/19q testing be performed more broadly, on all DGs or on all IDH-mutant DGs. The panel did not reach these conclusions based on review of the evidence, and the detailed reasoning is explained in the preceding paragraph. A related series of comments was made regarding the importance of p53 IHC or TP53 mutational analysis in the classification of DGs. One suggested using the phrase “ATRX loss with or without TP53 mutations” instead of “ATRX loss or TP53 mutations.” While it was acknowledged that IHC for p53 has limitations, and that TP53 mutations may rarely co-occur with 1p/19q codeletion in IDH-mutant gliomas (see above), the evidence suggests that TP53 mutations are one of the defining genetic characteristics of IDH-mutant astrocytoma (see preceding sections) and its inclusion is justified in the diagnostic workup of IDH-mutant DGs. Multiple comments argued for the formal incorporation of histopathological criteria in addition to or in lieu of biomarkers for the classification of oligodendroglioma. Based on our review of the evidence, the designation of glioma subclasses based on IDH mutation and 1p/19q codeletion is more robust and clinically meaningful than histopathology alone.4,6,74,76,82 There is also considerable evidence showing that the interpretation of histopathologic features varies considerably, even among experienced pathologists. Multiple comments suggested that pediatric gliomas be explicitly excluded from this recommendation. The sentiment underlying this suggestion is correct, given that oligodendroglioma, IDH-mutant and 1p/19q codeleted is rare in children. However, because IDH mutations occur predominantly in adults and our recommendation for 1p/19q codeletion assessment is only for IDH-mutant DGs, testing in pediatric gliomas is essentially ruled out in most cases. Finally, multiple comments urged specific recommendations regarding the most appropriate testing modalities for 1p/19q assessment. This point was addressed in the preceding paragraphs.
Conditional Recommendation: 5
CDKN2A/B homozygous deletion testing should be performed on IDH-mutant astrocytomas.
The quality of evidence to support this recommendation was assessed as moderate.
Quality Summary
The evidence base informing this recommendation comprises 2 prospective cohort studies77,78 and 9 retrospective cohort studies.41,84,85,99,108–112 The included studies were assessed as intermediate,77 intermediate-low,78 low,41,84,85,99,110–112 and very low quality108,109 based on risk of bias in selection,‡‡ performance,78,99,108,112 detection,§§ and reporting‖‖ domains. In addition, 2 of the studies reported statistical analyses that were underpowered,109,112 and 1 did not report on sources of funding.109 Although the aggregate risk of bias across the evidence base was very serious, the evidence was upgraded based on a strong association between CDKN2A/B deletion and diffuse glioma WHO classification. Refer to the SDC for the quality assessment of included retrospective cohort studies and the GRADE Quality of Evidence Assessment. See Table 5 for a summary of the mutational status of CDKN2A/B across all DG subtypes.
CDKN2A lies adjacent to CDKN2B in a region of chromosome 9 that is frequently mutated and deleted in a wide variety of human cancers. CDKN2A/B deletion has been shown by multiple investigations to be an adverse prognostic factor in IDH-mutant astrocytomas.¶¶ Subsequent analyses of multiple cohorts of IDH-mutant astrocytomas showed that homozygous deletion of CDKN2A/B was the most relevant adverse prognostic indicator.110 Patients with histologic grade 3 IDH-mutant astrocytomas that harbored homozygous deletion of CDKN2A/B had shorter survivals than patients with histologic grade 4 tumors that did not have CDKN2A/B homozygous deletion. Because homozygous deletion of CDKN2A/B can be observed in other types of primary brain tumors that have highly variable clinical outcomes, including IDH-WT GBM, pleomorphic xanthoastrocytoma (PXA), and anaplastic astrocytoma with piloid features, this recommendation pertains specifically to IDH-mutant astrocytomas.
In addition to studies captured in our systematic review, other studies1,113 have shown similar associations between CDKN2A/B homozygous deletion and shorter survival among patients with IDH-mutant astrocytomas. Such findings culminated in the cIMPACT-NOW recommendation that CDKN2A/B homozygous deletion could be used as a grade 4 criterion in a histologic grade 2 or 3 IDH-mutant astrocytoma11 and led to its inclusion as a grade 4 criterion within the WHO, 5th edition.7 While not a part of the current recommendations, CDKN2A/B homozygous deletion has also been shown to be a marker of poor prognosis in oligodendroglioma, IDH-mutant and 1p/19q codeleted, which may be relevant to future recommendations or grading schemes for these tumors.111
Public Comment Response to Recommendation 5
Of 86 total respondents to this recommendation, 66 (76.7%) agreed or agreed with modification, 8 (9.3%) disagreed, and 12 (14.0%) were neutral. Twenty-three written comments were received. Some respondents expressed a desire to strengthen the language of the recommendation from “may be performed” to “should be performed.” In response to this suggestion, the recommendation was strengthened to “should be performed” based on review of the evidence. Others requested advice regarding methods, desired reconciliation with subsequent cIMPACT-NOW 5 statements, or underscored the importance of the distinction between heterozygous and homozygous loss of CDKN2A/B. The evidence suggests that only homozygous deletion of CDKN2A/B is strongly and reproducibly associated with decreased survival that is independent of tumor grade. These comments were taken into consideration and specific concerns were addressed and clarified in the preceding paragraphs.
Strong Recommendation: 6
MGMT promoter methylation testing should be performed on all GBM IDH-WT. The quality of evidence to support this recommendation was assessed as moderate.
Quality Summary
This recommendation was informed by 2 meta-analyses,114,115 3 genome sequencing studies,5,21,22 and 5 retrospective cohort studies.86,108,116–118 The included meta-analyses were assessed as high115 and high-intermediate114 quality. Neither of these 2 studies reported on using publication status as a study selection inclusion criteria,114,115 and 1 did not report on conflict of interest or sources of funding.114 The genome sequencing studies were all assessed as intermediate-low quality based on retrospective acquisition of samples in all of them,5,21,22 plus individual moderate risk of reporting21 and detection bias.5,21 Finally, the retrospective cohort studies were assessed as low86,116,117 and very low quality108,118 based on risk of bias. The aggregate risk of bias of the evidence base was serious and the evidence was not further upgraded or downgraded for any domain. Refer to the SDC for the quality assessment of genome sequencing studies, retrospective cohort studies, meta-analyses, and the GRADE Quality of Evidence Assessment. See Table 6 for a summary of the promoter methylation status of MGMT in GBMs.
The MGMT protein, encoded by the MGMT gene, binds to DNA and repairs mutations that occur during DNA replication. Through these actions, it is a key mediator of resistance to alkylating chemotherapy in the treatment of DGs. Randomized studies have shown that the clinical benefit of adding temozolomide to radiotherapy is predominantly among patients with MGMT promoter methylated GBM (“predictive marker”).119,120 Temozolomide monotherapy in newly diagnosed GBM is only effective in patients with tumors that have MGMT promoter methylation.121,122 In GBM patients treated with combined temozolomide chemoirradiation, MGMT promoter methylation is an established prognostic parameter. In a large randomized trial on temozolomide dose intensification, MGMT promoter methylation was associated with improved overall survival (21.2 versus 14 months; hazard ratio, 1.74; P < .001).123 Clinical benefit of treatment with temozolomide or lomustine at the time of tumor progression after initial therapy is particularly observed in patients with methylated MGMT promoter at the time of first diagnosis.124–126 Assessment of MGMT promoter methylation can be useful for making treatment decisions for patients when either radiotherapy or chemotherapy is considered contraindicated, and in patients with recurrent tumors for whom second line alkylating chemotherapy is unlikely to be successful.
The expression of the MGMT protein is inhibited by methylation of specific CpG sites in the MGMT promoter and laboratory testing is therefore focused on these regions.116,127 Despite the overall correlation between MGMT promoter methylation and clinical outcome, test characteristics remain suboptimal. MGMT promoter methylation results are affected by the choice of CpG islands and promoter regions that are tested, as well as the platform employed. Techniques used to assess MGMT promoter methylation status include qualitative or quantitative methylation-specific PCR,128 pyrosequencing,129 multiplex ligation-dependent probe amplification,130 genome-wide methylation analysis,116 and IHC.131 With any of these available assays, the determination of the optimal technical cut off between methylated and unmethylated remains a challenge.132 In 1 study that compared 5 techniques for measuring MGMT promoter methylation on a series of 100 GBMs, the percentage of tumors with promoter methylation varied between 30% and 60%, a difference that was attributed to both variations between techniques and between laboratories.123,133,134
A major obstacle for current studies on the interpretation and optimization of these techniques is the lack of randomized trials with an untreated control arm that would allow assessment of the predictive value of MGMT promoter methylation. Current studies therefore assess the prognostic significance of various MGMT promoter methylation tests in GBM patients being treated with chemoirradiation with temozolomide134–136 or the concordance of results between various techniques.129 Individual detection techniques show only modest correlation when compared with one another.137,138 MGMT protein expression by IHC was found unreliable in 1 series due to large interobserver variation, yet another study concluded that IHC results correlated with methylation-specific PCR.131,139,140 Also, in some studies MGMT promoter status was found to vary between paired samples obtained at the time of first diagnosis and progression.141 With a quantitative methylation-specific PCR technique, analysis within cohorts has revealed a clinically relevant gray zone between MGMT promoter clearly methylated and unmethylated tumors. Establishing an appropriate threshold within this test gray zone has substantial implications for patients in clinical trials in which temozolomide is excluded from therapy based on MGMT methylation status.142
Public Comment Response to Recommendation 6
Of 86 respondents, 66 (76.7%) agreed or agreed with modifications, 8 (9.3%) disagreed, and 12 (14.0%) were neutral. Twenty-five comments were received. Comments emphasized that patients with GBM that had an unmethylated MGMT promoter may also benefit from the addition of temozolomide to radiotherapy, assumed the impact was mainly prognostic and stated that the need for testing depended on clinical decision-making and treatment planning. While most of the statement remained unchanged, the panel clarified that the recommendation applies to all GBM IDH-WT.
Conditional Recommendation: 7
For IDH-mutant DGs, MGMT promoter methylation testing may not be necessary. The quality of evidence to support this recommendation was assessed as low.
Quality Summary
The evidence base informing this recommendation comprises 1 genome sequencing study5 and 4 low-quality retrospective cohort studies.31,36,40,143 The genome sequencing study5 was assessed as intermediate-low quality based on a serious risk of selection bias and a moderate risk of detection bias. All retrospective cohort studies were limited by critical risk of selection bias.31,36,40,143 Individual studies were further limited by risk of bias in performance,31,40 reporting,31,36,40,143 and detection domains,36,143 as well as underpowered statistical analyses in 2,31,143 and a lack of reporting funding sources in 1.40 The aggregate risk of bias across these studies was very serious but evidence was not further downgraded for any domain. Refer to the SDC for the quality assessment of included genome sequencing studies, retrospective cohort studies, and the GRADE Quality of Evidence Assessment.
In the section above, data supporting MGMT promoter methylation testing in GBM, IDH-WT were presented. The clinical relevance of testing for MGMT promoter methylation in IDH-mutant DGs is not as firmly established. IDH mutations lead directly to global DNA hypermethylation and the establishment of the CpG island methylator phenotype, as demonstrated experimentally and in numerous investigations of clinical specimens.5,144,145 Therefore, the prevalence of MGMT promoter methylation in IDH-mutant DGs is very high.40,144,146,147
Data supporting this recommendation from our systematic review documents the tight correlation between IDH-mutant DGs and MGMT promoter methylation.31,36,40,143,148,149 Using bisulfite treatment, pyrosequencing, and focusing on 16 selected CpG sites, Mulholland et al found that 127 of 129 gliomas (98.5%) with IDH mutations were MGMT methylated, irrespective of pathologic group.143 A strong association between IDH mutation and MGMT promoter methylation has also been demonstrated by others.31,40 Thus, the high correlation between IDH-mutation and MGMT promoter methylation suggests that testing for MGMT promoter methylation in IDH-mutant DGs may not be necessary.
Public Comment Response to Recommendation 7
There were 89 respondents, of whom 65 (73.0%) agreed or agreed with modifications, 15 (16.9%) disagreed, and 9 (10.1%) were neutral. There were 17 written comments, many of which stated that MGMT testing decisions should be left to the treating oncologist and questioned whether it is worthwhile to issue a recommendation for every test that should not be performed. We have carefully taken these comments into consideration and have kept the recommendation as is, because MGMT promoter methylation testing is one of the most frequently requested tests for DGs, and we did not find sufficient evidence for reflexively ordering it in the group of IDH-mutant DGs. The strong association between IDH mutation and MGMT promoter methylation supports a role for IDH mutation (when present) resulting in MGMT promoter methylation, with the limitations of predominantly retrospective evidence. The recommendation is listed as conditional, which leaves room for individual and institutional treatment decisions.
Conditional Recommendation: 8
TERT promoter mutation testing may be used to provide further support for the diagnosis of oligodendroglioma and IDH-WT GBM. The quality of evidence to support this recommendation was assessed as low.
Quality Summary
This recommendation was informed by 2 genome sequencing studies5,6 and 11 retrospective cohort studies.## The genome sequencing studies5,6 were assessed as intermediate-low quality while the retrospective cohort studies were assessed as low*** and very low quality.4,18,34,152 Included studies suffered from risk of bias in selection,††† performance,4,18,34,150 reporting,‡‡‡ and detection§§§ domains. The aggregate risk of bias across these studies was very serious but evidence was not further downgraded for any domain. Refer to the SDC for the quality assessment of included genome sequencing studies, retrospective cohort studies, and the GRADE Quality of Evidence Assessment. See Table 7 for a summary of the mutational status of the TERT promoter across all DG subtypes.
Representative Studies Reporting on TERT Promoter, EGFR, Chromosome 7, and Chromosome 10 Status Across Diffuse Glioma (DG) Subtypes

As discussed for ATRX (Recommendation 2), cancerous proliferation requires escape from cellular senescence. In oligodendrogliomas, IDH-mutant and 1p/19q codeleted, and IDH-WT GBM this predominantly occurs via point mutations in the TERT promoter, which leads to enhanced TERT expression. Point mutations occur in 2 hotspots located 124 base pairs (bp) and 146 bp upstream of the translation start site and are referred to as C228T and C250T.34,151 Testing for promoter mutation is performed by DNA sequence analysis, either by Sanger methods, pyrosequencing, PCR, or targeted NGS.25,34,84,150,154,155
In contrast to oligodendrogliomas and IDH-WT GBM, IDH-mutant astrocytomas primarily use the alternative lengthening of telomeres pathway to escape senescence, which is driven by inactivation of ATRX. Of note, TERT promoter mutations and ATRX loss/mutations are mutually exclusive,4,6,150 consistent with our understanding that these 2 genetic mechanisms confer equivalent selective growth advantages. Thus, in the setting of IDH-mutation, TERT promoter mutations can provide additional support for the diagnosis of oligodendroglioma, IDH-mutant and 1p/19q codeleted, WHO grade 2 or 3. It can also provide diagnostic support in cases in which immunostaining for ATRX is equivocal or was not performed. By the same logic, TERT promoter sequencing may not be necessary in IDH-mutant gliomas with sufficient evidence of ATRX loss. Similarly, if there is laboratory evidence that clearly establishes the diagnosis of oligodendroglioma (IDH-mutant and 1p/19q codeleted), then TERT promoter testing may not be necessary.
TERT promoter mutations were initially noted in the majority of histologically defined GBMs.156,157 Subsequently, IDH mutations and TERT promoter mutations were shown to be mutually exclusive in GBMs, with those GBMs harboring TERT promoter mutations associated with shorter survivals compared with those with IDH mutations.34,151,155 Further comprehensive investigations demonstrated that those adult DGs that harbored TERT promoter mutations but did not have IDH mutations, were tightly associated with genetic signatures of IDH-WT GBM and short overall patient survivals.4,5,158 TERT promoter mutations are not specific for IDH-WT GBM, because they have been documented in other forms of CNS neoplasia in addition to oligodendroglioma, IDH-mutant and 1p/19q codeleted.156,158,159 However, in the proper clinical, radiologic and neuropathologic setting, TERT promoter mutations may be used to support the diagnosis of IDH-WT GBM.
Public Comment Response to Recommendation 8
There were 88 respondents, of which 69 (73.9%) agreed or agreed with modifications, 9 (17.1%) disagreed, and 10 (10.2%) were neutral. There were 15 comments, including several that reiterated the importance of interpreting TERT promoter mutations in the context of IDH-status, as well as its established relationships with ATRX mutations and 1p/19q codeletions, as discussed above. The recommendation was unchanged.
Strong Recommendation: 9
For histologic grade 2 to 3 DGs that are IDH-WT, testing should be performed for whole chromosome 7 gain/whole chromosome 10 loss, EGFR amplification, and TERT promoter mutation to establish the molecular diagnosis of IDH-WT GBM, grade 4. The quality of evidence to support this recommendation was assessed as moderate.
Quality Summary
The evidence base informing this recommendation includes studies evaluating testing of chromosome 7, chromosome 10, EGFR, and TERT promoter. One genome sequencing study21 and 1 retrospective cohort study108 comprise the evidence base for chromosome 7, 1 retrospective cohort study was included for chromosome 10,108 2 genome sequencing studies,21,22 and 8 retrospective cohort studies18,31,49,108,160–163 for EGFR, and 1 genome sequencing study5 and 8 retrospective cohort studies for TERT.4,18,23,49,150–153 Included studies were assessed as intermediate-low through very low quality and suffered from risk of bias in selection,‖‖‖ performance,4,18,31,108,150,163 reporting,¶¶¶ and detection### domains. Quality of evidence was assessed for each target individually and overall for the recommendation. For both chromosome 7 and EGFR testing, the aggregate risk of bias for studies included in the evidence base was serious and evidence was not further downgraded, resulting in moderate quality of evidence. For chromosome 10 the risk of bias of the 1 included study was very serious, thus carrying a low quality of evidence. Finally, the quality of evidence for TERT was also low based on a very serious aggregate risk of bias and no further downgrading of quality. The overall recommendation was assessed as moderate. Refer to the SDC for the quality assessment of included genome sequencing studies, retrospective cohort studies, and the GRADE Quality of Evidence Assessment. See Table 8 for a summary of the mutational status of EGFR, TERT promoter, chromosome 7, and chromosome 10 across all DG subtypes.
Representative Studies Reporting on Histone H3 Mutation Frequency Across Diffuse Glioma (DG) Subtypes

A substantial subset of IDH-WT diffuse astrocytic gliomas in adults are considered grade 2 or 3 based on histologic criteria (no microvascular proliferation or necrosis), yet have an aggressive clinical course, with overall survival times similar to patients with IDH-WT GBM. Numerous studies have attempted to identify molecular genetic biomarkers that reliably identify histologic grade 2 and 3 tumors that behave most aggressively. The strongest evidence indicates that the following markers identify IDH-WT diffuse astrocytic gliomas with grade 4 clinical behavior: (1) whole chromosome 7 gain together with whole chromosome 10 loss (+7/−10); (2) EGFR amplification; or (3) TERT promoter mutation. Studies that have assessed the frequency of +7/−10, EGFR amplification and TERT promoter mutations have demonstrated higher percentages of these alterations in histologically defined GBMs as compared to histologic grade 2 or 3 DGs, lending support to the association between these markers and the molecular signature of IDH-WT GBM.****
IDH-WT diffuse astrocytic gliomas of histologic grade 2 or 3 that carry EGFR amplification are associated with significantly shorter patient survival and have outcomes similar to patients with histologically defined IDH-WT GBM.5,6,18,158,160 EGFR amplification has excellent specificity for gliomas with aggressive behavior and is not present in other glioma subtypes that display a more indolent clinical course. It is typically accompanied by gain of chromosome 7 and loss of chromosome 10 in adult DGs, as well as in a smaller subset of pediatric high-grade gliomas.158,164
Similarly, those histologic IDH-WT diffuse astrocytic gliomas of histologic grade 2 or 3 with the +7/−10 signature are associated with short patient survival, similar to histologically defined IDH-WT GBM.5,158,165 The +7/−10 signature also has excellent specificity for DGs, with the rare exception of PXAs.158 Most investigations have focused on the prognostic role of whole gains of chromosome 7 and whole losses of chromosome 10 (+7/−10), because these are most frequent; the prognostic association of other, far less common combinations of imbalances, such as +7q/−10q, +7/−10q or +7q/−10, await further evaluation.
TERT promoter mutation in IDH-WT diffuse astrocytic gliomas of histologic grade 2 or 3 is associated with significantly shorter patient survival.4,18,23,160 However, other types of IDH-WT glial neoplasms without WHO grade 4 histology or aggressive behavior also occasionally harbor TERT promoter mutations.156,158,159 Therefore, TERT promoter mutations can be considered a marker for grade 4 behavior if the clinical, radiologic and histopathologic features are those of diffusely infiltrative astrocytic glioma. Further, more recent investigations have concluded that a subset of histologic grade 2 IDH-WT DGs that harbor only TERT promoter mutations (no other genetic alterations involving canonical IDH-WT GBM pathways) have longer clinical outcomes than expected for a grade 4 neoplasm.166,167 The combination of TERT promoter mutation with other markers, such as EGFR amplification and +7/−10 adds specificity as a marker of grade 4 behavior.158
cIMPACT-NOW update 3 has recommended that IDH-WT DGs of histologic grade 2 or 3 that have +7/−10, EGFR amplification or TERT promoter mutation should be considered grade 4 and more recently, these molecular alterations have been added as criteria for the diagnosis of GBM, IDH-WT, WHO grade 4 by the WHO 5th edition.7,10 Clinical implications for the use of these biomarkers to establish the molecular diagnosis of IDH-WT GBM, grade 4 include the use of combined chemotherapy and radiotherapy and expanded clinical trial inclusion.
Public Comment Response to Recommendation 9
Of 89 respondents, 77 (86.5%) agreed or agreed with modifications, 6 (6.7%) disagreed, and 6 (6.7%) were neutral. Twenty written comments were received, many expressing concern about the use of “grade IV astrocytic glioma.” As “astrocytic glioma” is not currently an official WHO class, respondents requested that the statement be changed to “molecular diagnosis of GBM.” Additional comments requested that the recommendation include ‘and' instead of ‘or' for the target testing to reduce turnaround time. These suggestions were incorporated into the final recommendation.
Strong Recommendation: 10
H3 K27M testing must be performed in DGs that involve the midline in the appropriate clinical and pathologic setting. The quality of evidence to support this recommendation was assessed as moderate.
Quality Summary
This recommendation was informed by 2 retrospective cohort studies.88,168 Both retrospective studies were limited by risk of bias in selection, performance, reporting, and detection domains.88,168 In addition, 1 study reported underpowered statistical analyses.168 The aggregate risk of bias for the evidence base was very serious and the quality of evidence was upgraded based on a strong association between H3 K27M testing and DG diagnosis in the WHO classification. Refer to the SDC for the quality assessment of included retrospective cohort studies and the GRADE Quality of Evidence Assessment. See Table 8 for a summary of the mutational status of histone H3 in GBM, midline glioma, and pediatric primitive neuroectodermal tumors of the CNS.
Diffuse midline glioma with histone H3 K27M mutation was a newly recognized diagnostic entity in the 4th edition update of the WHO Classification of Tumours of the Central Nervous System.1 The diagnosis relies on the histologic identification of diffusely infiltrating glioma within midline CNS structures such as spinal cord, brainstem, or thalamus and the identification of K27M mutation in either H3F3A or HIST1H3B/C using anti-H3 K27M IHC or by sequencing methods.90,169 By histology, tumors can show a large degree of histopathologic and cytopathologic variability, ranging from a low-density infiltrate to an undifferentiated, high-grade neoplasm. 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 4.88,168 While the number of studies investigating this particular mutation is small, the implications for diagnosis, prognosis, and treatment are critically important. This underlies the strong recommendation that H3 K27M testing must be performed in DGs that involve the midline in the appropriate clinical and pathologic setting. 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.91
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.88,168,170–172 IHC can be particularly useful in small biopsy samples, when tissue is limited. Several anti-H3 K27M antibodies are commercially available for on-site laboratory development, and reference laboratories also offer testing. Of note, the staining pattern of a positive test is nuclear, and therefore staining of cytoplasmic and/or cellular processes should not be interpreted as positive. Confirmatory molecular sequencing may be of utility in equivocal cases. 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.173 A smaller subset of diffuse midline gliomas that show loss of H3 K27me3 do not have H3 K27M mutations, but rather have EGFR mutations (see Good Practice Statement 1) or overexpression of EZHIP.172,174,175
Public Comment Response to Recommendation 10
There were 88 respondents, of whom 85 (96.6%) agreed or agreed with modifications and 3 (3.4%) disagreed. There were 24 written comments. Originally, the statement included that the recommendation applied to “the appropriate clinical and pathologic setting.” Some commented that this phrase needed to be further explained. There were also suggestions that the statement should recommend that the H3 K27M test phrasing of “should” be performed rather than “must” be performed. Others indicated that the methods of testing (eg, IHC versus sequencing) should be described. These comments were taken into consideration. While the recommendation remained the same, these comments have been addressed in the preceding paragraphs.
Conditional Recommendation: 11
H3 G34 testing may be performed in pediatric and young adults with IDH-WT DGs. The quality of evidence to support this recommendation was assessed as low.
Quality Summary
The evidence base informing this recommendation comprises 2 low-quality retrospective cohort studies.3,171 In addition to suffering from critical risk of selection bias, both studies were limited by a moderate risk of reporting bias3,171 and one was also limited by risk of performance and detection bias.3 The aggregate risk of bias for the studies was very serious and the evidence was not further downgraded for any domain. Refer to the SDC for the quality assessment of included retrospective cohort studies and the GRADE Quality of Evidence Assessment. See Table 8 for a summary of the mutational status of histone H3 in GBM, midline glioma, and pediatric embryonal tumors in the CNS.
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.3,171,176,177 G34R/V mutant DGs occur in a somewhat older age group in comparison with K27M cases (median age, 19 years for G34R/V mutation; median age, 6 for K27M mutation) and tend to involve the cerebral hemispheres rather than midline locations.176 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). “Diffuse hemispheric glioma H3 G34-mutant” was recently introduced as a distinct entity in the WHO 5th edition, underscoring the importance of testing for this molecular alteration.7 Of note, these DGs with G34R/V mutations should not be lumped together with other IDH-WT gliomas, as they carry a disease-defining genetic alteration that directs aggressive behavior corresponding to a WHO grade 4 neoplasm regardless of histologic appearance.3,171 Testing by sequencing or with mutation-specific (H3 G34R) antibodies is recommended for nonmidline DGs in pediatric and young adult populations.
Public Comment Response to Recommendation 11
There were 89 respondents, of whom 75 (83.2%) agreed or agreed with modification, 4 (4.5%) disagreed, and 11 (12.4%) were neutral. There were several written comments, including 1 that suggested that the age of the patient should be considered but that older adults might also benefit from testing in some cases. Others indicated that the methods of testing (ie, IHC versus sequencing) should be described. These comments were taken into consideration. While the recommendation remained the same, the comments were addressed in the text above.
Conditional Recommendation: 12
BRAF mutation testing (V600) may be performed in DGs that are IDH-WT and H3-WT. The quality of evidence to support this recommendation was assessed as low.
Quality Summary
The evidence base informing this recommendation consists of 1 intermediate quality prospective cohort study77 and 4 retrospective cohort studies.18,178–180 Two included retrospective studies were assessed as very low quality based on serious or critical risk of bias across more than 1 domain.18,178 The other 2 retrospective studies were also assessed as very low quality and this was based on critical risk of selection bias, moderate risk of bias in other domains, underpowered statistical analyses, and a lack of funding source reporting.179,180 The aggregate risk of bias for the studies was very serious but evidence was not further downgraded for any domain. Refer to the SDC for the quality assessment of retrospective cohort studies and the GRADE Quality of Evidence Assessment. See Table 9 for a summary of the mutational status of BRAF, MYB/MYBL1, and FGFR1 across DG subtypes.
Representative Studies Reporting on BRAF, MYB/MYBL1, and FGFR1 Status Across Diffuse Glioma (DG) Subtypes

BRAF V600E mutation is a driver mutation that is present in a wide variety of neoplasms, including melanoma, hairy cell leukemia, papillary thyroid carcinoma, colorectal carcinoma, lung carcinoma, Langerhans cell histiocytosis, Erdheim Chester disease, papillary craniopharyngioma, and ameloblastoma. It is present in a minority of DGs, but prevalence is enriched in epithelioid GBMs178–180 and other IDH- and H3-WT DGs.2,18 Of note, BRAF V600E mutation is distinct from the KIAA1549::BRAF fusion and other types of BRAF fusion, which are especially frequent in cerebellar pilocytic astrocytomas.
In addition to these studies, the recommendation was influenced by the availability of targeted therapy181 and the occurrence of BRAF V600E within a variety of brain tumors other than DGs, where the mutation is more prevalent; these include PXAs, ganglioglioma (GG), and a minority of pilocytic astrocytomas.2,182 Although BRAF V600E mutation occurs in a higher proportion of these generally circumscribed neuroepithelial tumors, PXA, GG, and pilocytic astrocytoma fell outside of the scope and search criteria used to develop these guidelines. However, histologic overlap between these entities can be considerable; given the presence of targeted therapy, a liberal approach to testing was favored in borderline or ambiguous cases where the distinction between a diffuse and circumscribed neoplasm could not be made with certainty.
This recommendation 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.”10 Evidence for BRAF V600E testing in the setting of an IDH-WT GBM with typical histologic features or with canonical genetic alterations in older adults is currently lacking.
While many of the markers considered in this guideline pertain principally to DGs in adults, it should be noted that a small number of pediatric DGs with BRAF V600E mutation have been identified,2 and that BRAF V600E testing should be considered in the context of IDH-WT and H3-WT DGs in children.9 This is particularly important given relatively common histologic overlap between circumscribed and DGs in children.
Public Comment Response to Recommendation 12
Of 89 respondents to this recommendation, 75 (84.3%) agreed or agreed with modifications, 10 (11.2%) disagreed, and 4 (4.5%) were neutral. Of 24 written comments, 7 solicited advice as to the best method of testing, and 5 emphasized the importance of clinical and pathologic correlation. The remaining 12 comments expressed diverse thoughts, both supportive and skeptical, including concern about the difference between the strength of the recommendation and the level of evidence (2 comments). These helpful comments were considered and the initial recommendation was streamlined to encompass all IDH-WT and H3-WT DGs (within the appropriate clinicopathologic context) instead of epithelioid GBM and “other” IDH-WT and H3-WT DGs. We also included a more specific description of the appropriate clinicopathologic context for testing in the text above.
Conditional Recommendation: 13
MYB/MYBL1 and FGFR1 testing may be performed in children and young adults with DGs that are histologic grade 2 to 3 and are IDH-WT and histone H3-WT. The quality of evidence to support this recommendation was assessed as low.
Quality Summary
The evidence base informing this recommendation includes three retrospective cohort studies evaluating MYB/MYBL1 testing18,183,184 and 1 retrospective cohort study evaluating FGFR1 testing.2 The 4 studies were assessed as low183,184 and very low quality2,18 based on risk of bias in selection,2,18,183,184 performance,18 reporting,2,18,183,184 and detection2,183,184 domains. Quality of evidence was assessed for each target individually and then overall for the entire statement. The aggregate risk of bias for MYB/MYBL1 studies was very serious but evidence was not further downgraded for any domain. The risk of bias for the one FGFR1 study was very serious. As only one study was identified, quality of evidence is dependent only on the risk of bias. The quality of evidence for the entire recommendation was assessed as low. Refer to the SDC for the quality assessment of included retrospective cohort studies and the GRADE Quality of Evidence Assessment. See Table 9 for a summary of the mutational status of BRAF, MYB/MYBL1, and FGFR1 across DG subtypes.
Recent studies have identified subsets of DGs in children and young adults that lack alterations in IDH1, IDH2, or histone H3 genes. In addition to BRAF, mentioned above, genes that are recurrently altered in this group include MYB proto-oncogene (MYB), MYB proto-oncogene like 1 (MYBL1), and FGFR1. These tumors are technically IDH-WT and H3-WT, but in retrospective studies they seem to exhibit an indolent clinical course, especially when compared with most DGs in adults.18 Genetic mechanisms of activation of these drivers are heterogeneous, which may affect the availability of specific assays for detection. These include duplications, truncations, amplifications, and rearrangements resulting in fusion genes (MYB and MYBL1) and internal tandem duplications of tyrosine kinase domains and single nucleotide variants (FGFR1). The prevalence of these alterations is difficult to assert with certainty, because most of the early data were from small retrospective studies. Integrated molecular and clinical analyses of a large cohort of pediatric low-grade gliomas provide insight into the spectrum and frequency of these alterations.185 Specific genetic alterations seem to correlate with glioma subtypes, with MYBL1 rearrangements being reported in up to 50% pediatric diffuse astrocytoma,184 MYB amplifications/copy number gains in 2 of 14 (14.3%) pediatric diffuse astrocytoma183 and 33 of 166 (19.9%) adult IDH-WT lower grade gliomas.18 Conversely, in one study FGFR1 alterations were frequent in rare low-grade neoplasms with ‘oligodendroglial' phenotype affecting children and young adults, 39 of 42 (92.9%).2 In addition, MYB::QKI fusions are found in nearly all angiocentric glioma (WHO grade 1), a low-grade DG often associated with epilepsy.186 Given the diagnostic and prognostic implications for these genetic drivers in gliomas of children and young adults, testing for them may be advisable depending on laboratory capabilities, but the quality of evidence is lower than that supporting testing for other more commonly altered genes (eg, IDH1/2 and BRAF).
Public Comment Response to Recommendation 13
There were 88 respondents, of whom 66 (75.0%) agreed or agreed with modifications, 8 (9.1%) disagreed, and 14 (15.9%) were neutral. There were 19 written comments, including some that suggested that evidence for the recommendation was not strong and the targets are not actionable; questions about testing methods; requests to clarify what is meant by “in the appropriate clinical setting” and inclusion of other genetic drivers (ie, panels). These comments were taken into consideration and the recommendation was revised or addressed by the other recommendations (eg, testing for BRAF).
GOOD PRACTICE STATEMENTS
During the period of the systematic literature review and drafting of recommendations, several clinically relevant investigations were published on the topic of biomarker testing for DG subtypes not originally covered by the Patient/population, Intervention, Comparator, and Outcome frameworks. To include guidance on these subtypes, we performed a focused literature review and included Good Practice Statements based on this second review. A description of the methods used to formulate these statements is included in the SDC.
The targeted search included alterations of ALK receptor tyrosine kinase (ALK), EGFR, ROS proto-oncogene 1 (ROS1), neurotrophic tyrosine receptor kinase (NTRK), MET, PDGFRA, and MYCN proto-oncogene (MYCN) within bithalamic gliomas, infantile-type hemisphere gliomas, or diffuse pediatric-type high-grade gliomas. The panel viewed the statements below as important, but there was not a formal rating for the certainty of evidence. The decision to form these statements was based on (1) emerging evidence with limited number of studies and samples and (2) anticipated updates to the WHO occurring at the time that this manuscript was being written. The panel believed that the tests would provide prognostic or treatment-related information for clinicians to consider. Future updates to the guideline will address these tests using a formal rating of the evidence and will include a grade for the strength of recommendation. Refer to Table 10 for the list of Good Practice Statements.
Statement 1
EGFR mutation testing may be performed on DGs that involve the midline.
A small subset of diffusely infiltrating midline gliomas that harbor EGFR mutations have a distinct methylation profile.174,187 While most of these DGs with EGFR mutations are histone H3-WT, some contain H3 K27M mutations. Like H3 K27M mutant gliomas involving the midline, EGFR-mutant DGs in this location that are histone H3-WT typically show loss of H3 K27me3, which can be detected by IHC. Small in-frame insertions/duplications of the tyrosine kinase domain and point mutations of the extracellular domain are detected by NGS and are potential therapeutic targets.
Statement 2
Testing for alterations in ALK, ROS1, MET, and NTRK genes may be performed on cerebral hemispheric DGs of infancy that are WT for IDH and histone H3.
DGs that occur in infancy are usually molecularly distinct from those of older childhood and adults. These large cerebral hemispheric tumors are typically wild type for IDH and histone H3 and often harbor fusions (rarely other activating alterations) involving ROS1, ALK, MET, or the NTRK family.188,189 The identification of such fusions or other activating alterations could lead to targeted therapy. Fusions can be identified by NGS and by FISH if appropriate probes are available.
Statement 3
Testing for DNA methylation class or for alterations in PDGFRA, EGFR, MYCN may be performed in pediatric high-grade DGs that are WT for IDH and histone H3.
Among pediatric diffuse high-grade gliomas that are IDH-WT and histone H3-WT, 3 molecular subclasses can be identified by DNA methylation profiling that have differing molecular genetic alterations and clinical outcomes. One class is generally characterized by PDGFRA amplifications or mutations; a second class enriched for EGFR amplifications is associated with the longest survival; and the third often harbors MYCN amplifications and is associated with the shortest survival.164,190
FUTURE CONSIDERATIONS
The recommendations provided are based on evidence available at the time of literature review. It is expected that additional studies and new technologies will emerge that will need to be incorporated into future guidelines. In the category of IDH-WT diffuse astrocytic gliomas of adults, it has been recommended that histologic grade 2 and 3 DGs should be tested for chromosome 7 gain/whole chromosome 10 loss, EGFR amplification, and TERT promoter mutation to establish the molecular diagnosis of GBM, grade 4. It might be expected that additional biomarkers or combinations of biomarkers will be identified and validated in histologic grade 2 and 3 DGs that are also capable of predicting grade 4 clinical behavior, including alterations in PDGFRA, FGFR3, MET, TP53, NF1, PTEN, among others.10 For IDH-mutant astrocytomas of histologic grade 2 and 3, future studies could uncover and validate additional markers that can more optimally stratify risk or predict grade 4 clinical behavior in addition to CDKN2A/B homozygous deletion, such as PDGFRA, MYCN, or CDK4 amplifications, mutations in CDKN2A/B or RB1, among others.11,65,85 CDKN2A/B homozygous deletion have recently been shown to be a marker of poor prognosis in oligodendroglioma, IDH-mutant and 1p/19q codeleted, which along with other biomarkers, may be relevant to future recommendations or grading schemes.111 Similarly, it is likely that genetic or epigenetic markers will be uncovered and validated that are capable of stratifying risk among patients with DGs driven by histone H3, mitogen-activated protein kinase pathway or MYB/MYBL1 genes.9,191
The laboratory findings of tumor mutational burden (TMB), mismatch repair (MMR) loss and microsatellite instability are of interest in multiple forms of cancer, because the findings from these tests predict response to immunotherapies, including immune checkpoint inhibitors in some cancer types.192–194 Initial randomized clinical trials of checkpoint inhibitors in newly diagnosed and recurrent GBM have failed to demonstrate improved survival.195 Guidelines for testing of tumor mutational burden, mismatch repair, or microsatellite instability in DGs will be developed if and when clinical care warrants them.
Whole-genome DNA methylation profiling represents a robust and reproducible method for precisely segregating tumor types that have similar genetic alterations, epigenetic signatures, and clinical behaviors.5,12 IDH-WT GBM, IDH-mutant astrocytomas, oligodendrogliomas, IDH-mutant and 1p/19q codeleted, and histone H3-mutant gliomas cluster tightly within their own class with little overlap based on DNA methylation profiling.5,6,22 Other DGs, such as those that occur in children and are driven by mitogen-activated protein kinase pathway or MYB/MYBL1 genomic alteration also cluster together, indicating a high degree of specificity for each signature.164,190 While the literature indicates this method is superior for classification purposes and could have a role in future classification and grading, it currently lacks widespread clinical implementation due to regulatory challenges, prevailing practice patterns, and uncertainties in reimbursement. Currently, DNA methylation profiling is an attractive alternative for identifying DG classes that will likely be implemented into clinical practice soon.
LIMITATIONS
We were unable to answer KQs 1b—What are the acceptable techniques/methods for mutation testing of DGs and what are the expected turnaround times for individual assays? While we collected the various methods of testing, none performed poorly in our assessment. Each have varying strengths and weaknesses. For the latter question, no studies from the literature search reported turnaround time data and so, no recommendation was made. It is up to each laboratory to determine the best methodology and acceptable turnaround times. Although the individual DG entities have prognostic characteristics that have been well defined in the literature,2–7 most of the studies that comprised the evidence base for the recommendations correlated the reported outcomes to DG entities and not to survival outcomes. Additional studies that investigate outcomes related to biomarkers and DG subtypes would increase certainty of the evidence base and lead to stronger recommendations.
CONCLUSIONS
The 13 evidence-based recommendations and 3 good practice statements that are provided for biomarker testing of DGs, together with their explanations and justifications, are intended to guide practice and improve the clinical care of patients with these diseases. For more than a century, the diagnosis of DG was based primarily on histologic appearance, yet recent clinically relevant molecular genetic discoveries have forced a re-evaluation of diagnostic definitions and criteria. We are now firmly within the molecular era in which integrated diagnoses are formulated by directly incorporating molecular biomarker test results. The complexity of establishing an integrated diagnosis has increased and pace of change has been rapid, warranting the recommendations that we have provided. Neuropathologists, molecular pathologists, and treating physicians will continue to work closely together to provide optimal clinical care by using molecular biomarkers for diagnostic and treatment purposes.
DISCLAIMER
The CAP developed the Pathology and Laboratory Quality Center for Evidence-based Guidelines as a forum to create and maintain laboratory practice guidelines (LPGs). Guidelines are intended to assist physicians and patients in clinical decision-making, and to identify questions and settings for further research. With the rapid flow of scientific information, new evidence may emerge between the time an LPG is developed and when it is published or read. LPGs are not continually updated and may not reflect the most recent evidence. LPGs address only the topics specifically identified therein and are not applicable to other interventions, diseases, or stages of diseases. Furthermore, guidelines cannot account for individual variation among patients and cannot be considered inclusive of all proper methods of care or exclusive of other treatments. It is the responsibility of the treating physician or other health care provider, relying on independent experience and knowledge, to determine the best course of treatment for the patient. Accordingly, adherence to any LPG is voluntary, with the ultimate determination regarding its application to be made by the physician considering each patient's individual circumstances and preferences. The CAP and its collaborators make no warranty, express or implied, regarding LPGs and specifically excludes any warranties of merchantability and fitness for a particular use or purpose. The CAP and its collaborators assume no responsibility for any injury or damage to persons or property arising out of or related to any use of this statement or for any errors or omissions.
We thank the following collaborating medical societies and their staff involved in the development of this guideline: American Association of Neuropathologists, Association for Molecular Pathology, Society for Neuro-Oncology. We thank the American Society of Clinical Oncology for identifying representatives to serve on the expert panel. We also thank the following advisory panel members representing these organizations for their guidance throughout the development of the guideline and for their thoughtful review of this work: Tracy T. Batchelor, MD; David W. Ellison, MD, PhD; Matthew Foster, MD; Azra H. Ligon, PhD; Keith L. Ligon, MD, PhD; Morgan Mosky, patient advocate; Joanna J. Phillips, MD, PhD; Gordana Raca, MD, PhD; Sharleen Rapp, BS, MB(ASCP)cm; Michelle Stoffel, MD, PhD; and Mary Kay Washington, MD, PhD.
References
APPENDIX
Disclosed Interests and Activities From July 2017 to April 2021
Daniel J. Brat, MD, PhD
Position of Influence: Society for Neuro-Oncology, American Association of Neuropathologists
Julia A. Bridge, MD
Research Grant: Cepheid
Consulting fees or Advisory Board: Bayer, Bristol Myers Squibb/National Comprehensive Cancer Network, Epizyme, Merck, Moffitt Cancer Center, Translational Genomics Research Institute, VENTANA/Roche
Position of Influence: United States & Canadian Academy of Pathology
Howard Colman, MD, PhD
Consulting fees or Advisory Board: AbbVie, Adastra Pharmaceuticals, Bayer, Best Doctors, CytRx Corporation, F. Hoffman-La Roche, Forma Therapeutics, Foundation Medicine, Genentech, Insys Therapeutics, Karyopharm Therapeutics, NewLink Genetics, Novocure, Omniox, Orbus Therapeutics, OXiGENE, Private Health, Upsher-Smith
Speakers' bureau/lecture fees/honoraria: Merck
Institutional contracts/clinical trials: AbbVie, Array BioPharma, Bayer, BeiGene, Bristol Myers Squibb, DNAtrix, Forma Therapeutics, Global Coalition for Adaptive Research, Kadmon Holdings, Karyopharm Therapeutics, Merck, NewLink Genetics, Nuvation Bio, Orbus, Plexxikon
Meera R. Hameed, MD
Consulting fees or Advisory Board: Novartis
Eyas M. Hattab, MD, MBA
Consulting fees or Advisory Board: Arbor Pharmaceuticals, Cell Marque, E.R. Squibb & Sons, Bristol Myers Squibb, Ventana Medical System/Roche Diagnostics
Speakers' bureau/lecture fees/honoraria: Arbor Pharmaceuticals, Roche Diagnostics
Position of Influence: College of American Pathologists, United States & Canadian Academy of Pathology
Other: International Collaboration on Cancer Reporting member
Jason T. Huse, MD, PhD
Research Grants: Taiho Oncology, Inc.
Martin J. van den Bent, MD, PhD
Consulting fees or Advisory Board: AbbVie, Agios, Bayer, Boehringer Ingelheim, Bristol Myers Squibb, CarThera, Celgene, Celldex Therapeutics, Genenta Science, Karyopharm Therapeutics, Nerviano Medical Sciences, VAXIMM
Speakers' bureau/lecture fees/honoraria: Merck Sharp & Dohme Corp
Research Grants: AbbVie
No Disclosures to Report
Arie Perry, MD; Brent T. Harris, MD, PhD; Michelle Hawks Yount, MS; Fausto J. Rodriguez, MD; Robert B. Jenkins, MD, PhD; William C. McDonald, MD; Kenneth Aldape, MD; Peter Canoll, MD, PhD; Dolores H. Lopez-Terrada, MD, PhD; Lesley H. Souter, PhD; Carol Colasacco, MLIS, SCT(ASCP); Nicole E. Thomas, MPH, CT(ASCP)cm
Author notes
Supplemental digital content is available for this article at https://meridian.allenpress.com/aplm in the May 2022 table of contents.
Competing Interests
Authors' disclosures of potential conflicts of interest and author contributions are found in the Appendix at the end of this article.