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Molecular Analysis for Gliomas - CAM 337

Description
Glioma refers to tumors resulting from metaplastic transformation of glial tissue of the nervous system. Tumors have historically been classified by the retained histologic features of the three types of glial cells: astrocytes, oligodendrocytes, and ependymal cells. Tumors of each type can vary widely in aggressiveness, response to treatment, and prognosis.

Molecular genetic features were added to histopathologic appearance in the current WHO classification to yield more biologically homogeneous and narrowly defined diagnostic entities for greater diagnostic accuracy, improved patient management, more accurate determinations of prognosis, and better treatment response.

REGULATORY STATUS
No diagnostic tests have been specifically approved for use in detecting mutations in gliomas as of July 19, 2019 (FDA, 2019). Additionally, many labs have developed specific tests that they must validate and perform in house. These laboratory-developed tests (LDTs) are regulated by the Centers for Medicare & Medicaid Services (CMS) as high-complexity tests under the Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88). As an LDT, the U.S. Food and Drug Administration has not approved or cleared this test; however, FDA clearance or approval is not currently required for clinical use.

Policy 
Application of coverage criteria is dependent upon an individual’s benefit coverage at the time of the request. 

  1. For the prognosis of gliomas, the follow tests is considered MEDICALLY NECESSARY:  
    1. Array-based genomic copy number testing, fluorescence in situ hybridization (FISH), or polymerase chain reaction (PCR) testing for the co-deletion of 1p and 19q
    2. ATRX mutation testing via EITHER immunohistochemistry OR gene sequencing
    3. BRAF fusion and mutation testing, including BRAF V600E common variant
    4. IDH1 and IDH2 testing
    5. MGMT promoter methylation testing
    6. Genetic sequencing of TERT
  2. For the prognosis of diffuse midline gliomas, the following tests is considered MEDICALLY NECESSARY:
    1. H3-3A (formerly H3F3A) and HIST1H3B gene sequencing
    2. H3-3A (formerly H3F3A) testing using a K27M histone antibody
  3. For the prognosis of ependymomas, ZFTA fusion testing using either RNA sequencing analysis (RNA-Seq) or FISH is considered MEDICALLY NECESSARY.
  4. ATRX mutation co-testing using BOTH immunohistochemistry and gene sequencing is NOT MEDDICALLY NECESSARY.

NOTE: For 5 or more gene tests being run on the same platform, such as multi-gene panel next generation sequencing, please refer to CAM 235.

Rationale 
According to the American Cancer Society, an estimated 25,050 adults in the United States will be diagnosed with malignant tumors of the brain and spinal cord in 2022. In children, brain and spinal cord tumors are the second most common cancers and account for about 1 out of 4 children’s cancers (American Cancer Society, 2022). As of 2022, an estimated 18,280 people will die from brain and spinal cord tumors (American Cancer Society, 2022).

Studies over the past two decades have clarified the genetic basis of tumorigenesis in the common, and some rarer, brain tumor entities (Louis et al., 2016), and identified clinically relevant molecular genetic characterizations that complement standard histologic analysis providing additional diagnostic and prognostic information to improve diagnostic accuracy, influence treatment selection, and improve survival. Molecular and/or genetic characterization do not replace standard histologic assessment, but rather serve as a complimentary approach (NCCN, 2022). 

Isocitrate dehydrogenase (IDH1/2) mutations
IDH 1 and 2 are metabolic enzymes that oxidize isocitrate to alpha-ketoglutarate, and are important in the mitigation of cellular oxidative damage (Horbinski, 2013b). Mutations in genes encoding these enzymes leads to the aberrant production of D-2 hydroxyglutarate (Dang et al., 2009), an oncometabolite that causes epigenetic modifications in affected cells (Horbinski, 2013b).

IDH mutations are a defining feature of WHO grade II and III astrocytomas and oligodendrogliomas (Louis et al., 2016). Their presence distinguishes lower grade gliomas from primary glioblastomas, which are IDH wild type. IDH mutations are commonly associated with O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and are also associated with a relatively favorable prognosis (Brat et al., 2015; Eckel-Passow et al., 2015).

O-6-methylguanine-DNA methyltransferase (MGMT) methylation
MGMT is a DNA repair enzyme that reverses the DNA damage caused by alkylating agents, resulting in tumor resistance to temozolomide and nitrosourea-based chemotherapy. Methylation of the MGMT promoter silences MGMT, making the tumor more sensitive to treatment with alkylating agents (Esteller et al., 2000; Gusyatiner & Hegi, 2018).

MGMT promoter methylation is strongly associated with IDH mutation and genome-wide epigenetic change (Eckel-Passow et al., 2015); it is also associated with longer survival in patients with glioblastoma who receive alkylating agents (Hegi et al., 2005; Zhao et al., 2016). MGMT promoter methylation is particularly useful in treatment decisions for elderly patients with high grade gliomas (Malmstrom et al., 2012; Wick et al., 2012; Wick et al., 2014).

Codeletion of chromosomes 1p and 19q
The codeletion of 1p and 19q represents an unbalanced translocation (1;19)(q10;p10) leading to the whole arm deletion of chromosome 1p and chromosome 19q (Jenkins et al., 2006). Codeletion of 1p and 19q is a defining feature of oligodendroglial tumors, is strongly associated with oligodendroglial histology, and helps to confirm the oligodendroglial character of tumors with equivocal or mixed histologic features (Brat et al., 2015; Burger et al., 2001; Eckel-Passow et al., 2015). Combined loss involving chromosomes 1p and 19q is significantly associated with both favorable therapeutic response and longer recurrence-free survival after chemotherapy (Cairncross et al., 1998).

Alpha-thalassemia/mental retardation syndrome X-linked (ATRX) mutations
Mutations in the chromatin regulator gene, ATRX, enable alternative lengthening of telomeres (Abedalthagafi et al., 2013). ATRX is a switch/sucrose helicase that assists with H3.3 chromatin deposition in telomeric regions. Disruption of this gene leads to the alternative lengthening of telomeres stated above and is thought to represent an early event in gliomagenesis (Batchelor, 2020).

ATRX mutations in glioma are strongly associated with IDH and TP53 mutations and are nearly always mutually exclusive with 1p19q codeletion (Reuss et al., 2015). ATRX deficiency, coupled with IDH mutation, is typical of astrocytoma (Brat et al., 2015).

Tumor protein 53 (TP53) mutation
TP53 is essential for regulating cell division and preventing tumor formation (Parikh et al., 2014). Missense mutations in the TP53 gene are present in the clear majority of IDH-mutant astrocytomas (Brat et al., 2015). Immunopositivity for mutant p53 is not entirely sensitive or specific for a TP53 mutation, however, and loss of ATRX expression may be a more reliable marker of astrocytic differentiation (Louis et al., 2020).

Telomerase reverse transcriptase (TERT) mutations
TERT encodes the catalytic active site of telomerase, the enzyme responsible for maintaining telomere length in dividing cells. TERT mutations in the noncoding promoter region cause increased expression of the TERT protein and are one of the major mechanisms of telomerase activation in gliomas (Arita et al., 2013). TERT mutations are strongly associated with 1p19q codeletion and are found in most glioblastomas. A TERT mutation in combination with an IDH mutation and 1p19q codeletion is characteristic of oligodendroglioma. The absence of a TERT mutation, coupled with an IDH mutation, designates astrocytoma (Eckel-Passow et al., 2015). In terms of survival, mutation in the TERT promoter is generally unfavorable in the absence of IDH mutation and favorable in the presence of IDH mutation and 1p/19q codeletion. TERT promoter mutation is associated with an older age of the patient at presentation, regardless of whether IDH mutation is present (Eckel-Passow et al., 2015).

Histone (H3FA) mutations
A lysine to methionine substitution in the H3F3A gene (H3K27M) is the most common histone mutation in brain tumors and inhibits the trimethylation of H3.3 histone (Sturm et al., 2012), arresting cells in a primitive state refractory to differentiation induction (Weinberg et al., 2017). G34R/G34V mutations in the H3F3A gene are more common in cortical gliomas in children (Schwartzentruber et al., 2012). H3FA mutations can be useful in the diagnosis of infiltrative glioma (Sturm et al., 2012). The H3K27M mutation is an adverse prognostic marker in children and adults (Meyronet et al., 2017). The G34 mutation does not appear to have any prognostic significance once the diagnosis of a glioblastoma has been established (Sturm et al., 2012).

A similar mutation to H3K27M may also occur in the HIST1H3B/C gene, which encodes the histone H3.1 variant. However, the mutation at HIST1H3B/C is about one third as common as H3F3A and often confers a better prognosis than its H3F3A counterpart (Louis et al., 2020).

B-Raf proto-oncogene (BRAF) mutations
BRAF is a serine-threonine protein kinase involved in cell survival, proliferation, and differentiation (Davies et al., 2002). Activating mutations in BRAF, most often V600E, have been discovered in most pediatric and some adult gliomas (Chappe et al., 2013; Horbinski, 2013a), including approximately 80% of pleomorphic xanthoastrocytomas, 20% of gangliogliomas, 10% of pilocytic astrocytomas, and occasionally diffuse gliomas (Chi et al., 2013). Tandem duplication of chromosome 7q34 resulting in an activating fusion of the BRAF and KIAA1549 genes occur in 60-80% of pilocytic astrocytoma (Jones et al., 2008).

The presence of a BRAF fusion is reliable evidence that the tumor is a pilocytic astrocytoma and predicts a better clinical outcome (Hawkins et al., 2011). A BRAF mutation is more complicated, as it can occur in a variety of tumors and requires integration with histology. Tumors with BRAF mutations may respond to BRAF inhibitors; however, in pediatric gliomas, BRAF V600E indicates poor prognosis when treated with current adjuvant therapy, especially in combination with a CDKN2A mutation (Lassaletta et al., 2017).

v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA, p65, NFKB3) fusion
Fusion between the C11orf95 and RELA genes defines approximately 70 percent of all childhood supratentorial ependymomas (Louis et al., 2020). These fusions are associated with increased NF-kappa-B (NFkB) signaling and poor outcome (Malgulwar et al., 2018). Normally, NFkB is an inactive transcription factor in the cytoplasm. When its inhibitor degrades, it activates transcription of certain genes, RELA among them (NCBI, 2011). New research supports the hypothesis that the status of RELA fusion and p53 overexpression are significantly associated with the prognosis of supratentorial extraventricular ependymomas (Wang et al., 2019).

New Tests
Assessment of gliomas is incredibly difficult, and new methods of molecular analyses for gliomas are consistently being developed. For example, Miller et al. (2019) devised a liquid-biopsy based method to evaluate cerebrospinal fluid from 42 (of 85) patients. The genomic profile developed from the cerebrospinal fluid (CSF) samples closely matched established profiles, such as the characteristic 1p/19q codeletion and IDH1/2 mutations. The authors stated that the ability to monitor the glioma genome in real time could be useful in management of this condition (Miller et al., 2019). Other researchers report that “A cerebrospinal fluid ct-DNA liquid biopsy approach may virtually support all the stages of glioma management, from facilitating molecular diagnosis when surgery is not feasible, to monitoring tumor response, identifying early recurrence, tracking longitudinal genomic evolution, providing a new molecular characterization at recurrence and allowing patient selection for targeted therapies” (Simonelli et al., 2020).

Clinical Utility and Validity
Nikiforova et al. (2016) validated GlioSeq, a commercial next generation sequencing (NGS) panel of 30 genes, in 54 patients with CNS tumors against fluorescence in-situ hybridization (FISH), Sanger sequencing, and reverse transcription polymerase chain reaction (PCR). GlioSeq correctly identified 71/71 (100%) genetic alterations known to be present by conventional techniques. The assay sensitivity was 3%-5% for mutant alleles of single nucleotide variants (SNVs), and 1%-5% for gene fusions. Likewise, Zacher et al. (2017) developed an NGS panel of 20 genes that allowed for molecular classification of 121 gliomas. The researchers conclude that gene panel NGS is a promising diagnostic technique that may facilitate integrated histological and molecular glioma classification.

Ramkissoon et al. (2017) used OncoPanel and OncoCopy to identify targetable alterations in tumors for the establishment of best practices in routine clinical pediatric oncology. They analyzed 117 samples by OncoPanel and 146 by OncoCopy; further, 60 tumors were subjected to both methodologies. OncoPanel revealed clinically relevant alterations in 56% of patients (44 cancer mutations and 20 rearrangements), including BRAF alterations that directed the use of targeted inhibitors. Rearrangements in MYB-QKI, MYBL1, BRAF, and FGFR1 were also detected. Furthermore, while copy number profiles differed across histologies, the combined use of OncoPanel and OncoCopy identified subgroup-specific alterations in 89% (17/19) of medulloblastomas.

Ryall et al. (2016) evaluated the prognostic impact of H3K27M and MAPK pathway aberrations in 64 gliomas (44 low grade, 22 high grade). Tumors are designated as low-grade if the cells are well differentiated, are less aggressive overall, and suggest a better prognosis for the patient. Five low grade gliomas contained the H3F3A/HIST1H3B K27M (H3K27M) mutation, and 11 high grade gliomas contained the H3K27M mutation. Survival analysis evaluated the median survival at 9.12 years for wildtype H3 patients compared to 1.02 years for patients with the H3K27M mutation. MAPK pathway mutations (through BRAF or FGFR1 mutation) were associated with long-term survival in absence of H3K27M mutations. Further, H3K27M status and high-grade histology were found to be the most significant independent predictors of poor overall survival with hazard ratios of 6.945 and 7.721 respectively. MAPK pathway activation was a predictor of “favourable patient outcome,” but dependent on other factors (Ryall et al., 2016).

Houdova Megova et al. (2017) evaluated the prognostic value of the IDH1/2 mutation in glioblastomas. A total of 37 IDH mutations were examined and studied. The authors found that IDH1 mutations were positively associated with MGMT methylation (odds ratio [OR]: 3.08), 1p/19q co-loss (OR: 8.85), and negatively associated with EGFR amplification. IDH-mutant patients had an overall survival of 25 months compared to only 9 months for IDH-wildtype gliomas (Houdova Megova et al., 2017).

Johnson et al. (2017) performed comprehensive genomic profiling of 282 pediatric gliomas: 157 high-grade and 125 low-grade. The investigators used a 315 gene panel and calculated the tumor mutational burden (TMB). In low grade gliomas, BRAF was the most frequent mutation found (48%), followed by FGFR missense (17.6%), NF1 loss of function (8.8%), and TP53 (5.6%). Rearrangements were found in 35% of low-grade gliomas. In high-grade gliomas, TP53 was the most frequent mutation found (49%), followed by H3F3A (37.6%), ATRX (24.2%), NF1 (22.2%), and PDGFRA (21.7%). H3F3A mutations were found to be the K28M variant. Approximately 6% of the high-grade gliomas were found have a TMB of > 20 mutations/Mb (“hypermutated”) (Johnson et al., 2017).

Back et al. (2020) studied the pattern of failure in anaplastic glioma (AG) patients with an IDH1/2 mutation. A total of 156 patients participated in the study, with data collected from 2008 to 2014; the median follow-up time was 5.1 years. Of all 156 patients, 75% were found to have an IDH1 or IDH2 mutation. The authors concluded that “patients with IDH-mutated AG have improved outcomes”; however, this population also had a greater number of distant relapses approximately two years after intensity-modulated radiation therapy compared to individuals with IDH wild type mutations (Back et al., 2020).

Ji et al. (2021) studied the clinical utility of comprehensive genomic profiling to detect CNS tumors in children and young adults using the OncoKids next-generation sequencing panel, chromosomal microarray analysis, and germline testing. NGS was performed on 222 samples and CMA was performed on 146 of the 222 samples. The OncoKids NGS panel identified diagnostic biomarkers in 138/222 samples (62%), prognostic information in 49/222 cases (22%), and targetable genomic alterations in 41/222 samples (18%). Additionally, CMA revealed prognostic copy number alterations (CNA) in 101/146 cases (69%). Further, germline cancer predisposition testing was performed in 57 of 212 patients which identified 20 patients which a confirmed germline pathogenic/likely pathogenic variant of genes TP53, NF1, SMARCB1, NF2, MSH6, PMS2, and a patient with Klinefelter syndrome. Overall, the authors conclude that there is "significant clinical utility of integrating genomic profiling into routine clinical testing for pediatric and young adult patients with CNS tumors" (Ji et al., 2021).

Muralidharan et al. (2021) studied the diagnostic utility of a novel digital droplet PCR (ddPCR) assay for detection of two TERT promoter mutations (C228T and C250T) and monitoring of gliomas. In comparison with the gold-standard tumor tissue-based detection of TERT mutations, the ddPCR assay had an overall sensitivity of 62.5% and a specificity of 90%. Longitudinal monitoring of five patients demonstrated that the peripheral TERT mutant allele frequency reflects the clinical course of the disease. TERT mutant alleles decreased after surgical intervention and pharmacotherapy but increased with tumor progression. The authors conclude that the ddPCR assay has feasibility in "detecting circulating cfDNA TERT promoter mutations in patients with glioma with clinically relevant sensitivity and specificity" (Muralidharan et al., 2021).

Cabezas-Camarero et al. (2021) studied the levels of soluble PD-L1 (sPD-L1) in patients with gliomas according to histologic grade and IDH mutation status, evaluating its predicted role and dynamic changes in sPD-L1. Plasma samples were obtained prior to and after radiotherapy/chemotherapy and were evaluated using ELISA. The authors compared 12 healthy controls with 57 patients with grade II to IV gliomas. They found that sPD-L1 levels were numerically higher in glioma patients as compared to the healthy control group. The authors found that elevated sPD-L1 levels pre- and post-treatment associated with a worse prognosis in IDH-MUT gliomas. Dynamics of SPDL1 and other immune-biomarkers should still be explored in gliomas (Cabezas-Camarero et al., 2021).

National Comprehensive Cancer Network (NCCN)
The NCCN published Clinical Practice Guidelines in Oncology (2022) for Central Nervous System Cancers which recommend:

IDH1 and IDH2 mutation

Recommendation: IDH1 mutation testing is required for the workup of glioma.

“The most common IDH1 mutation (R132H) is reliably screened by mutation specific immunohistochemistry, which is recommended for all glioma patients. If the R132H immunostain result is negative, in the appropriate clinical context, sequencing of IDH1 and IDH2 is highly recommended to detect less common IDH1 and IDH2 mutations. Prior to age 55 years, sequencing of IDH1 and IDH2 is required if the R132H immunostain result is negative. Standard sequencing methods include Sanger sequencing, pyrosequencing, and next-generation sequencing, and should be performed on formalin fixed, paraffin embedded tissue (NCCN, 2022).”

MGMT promoter methylation:

Recommendation: MGMT promoter methylation is an essential part of molecular diagnostics for all high-grade gliomas (grade III and IV). The NCCN also notes that “MGMT promoter methylation is strongly associated with IDH mutations and other genome-wide epigenetic changes (G-CIMP phenotype)” (NCCN, 2022).

“There are multiple ways to test for MGMT promoter methylation, including methylation-specific PCR, methylation-specific high-resolution melting, pyrosequencing, and droplet-digital PCR” (NCCN, 2022).

“MGMT promoter methylation testing is particularly useful in treatment decisions for older adult patients with high-grade gliomas (grades 3–4). ” (NCCN, 2022).

Codeletion of 1p and 19q:
Recommendation: 1p19q testing is an essential part of molecular diagnostics for oligodendroglioma.

“The codeletion of 1p and 19q is detectable by array-based genomic copy number testing, fluorescence in situ hybridization (FISH), or polymerase chain reaction (PCR) … IDH-mutated gliomas that do NOT show loss of ATRX (for example, by IHC) should be strongly considered for 1p/19q testing, even if not clearly oligodendroglial by histology. Conversely, IDH1 wild-type gliomas do not contain true whole-arm 1p/19q codeletion. Therefore, 1p/19q testing is unnecessary if a glioma is definitely IDH-wild-type, and a glioma should not be regarded as 1p19q codeleted without an accompanying IDH mutation, regardless of the test results (NCCN, 2022).”

ATRX mutation:
“Recommendation: ATRX mutation testing is required for the workup of glioma.”

“ATRX mutations can be detected by IHC for wild-type ATRX (loss of wild type expression) and/or sequencing. ATRX mutations in glioma are strongly associated with IDH mutations and are nearly always mutually exclusive with 1p/19q codeletion. ATRX deficiency, coupled with IDH mutation, is typical of astrocytoma. A lack of ATRX immunostaining in glioblastoma should trigger IDH1/2 sequencing if IDH1 R132H immunostaining is negative, due to frequent co-occurrence of ATRX and IDH mutations (NCCN, 2022).”

TERT mutation:
“Recommendation: TERT mutation testing is required for the workup of gliomas. TERT mutation can be detected by sequencing of the promoter region (NCCN, 2022).”

“TERT promoter mutations are nearly always present in 1p/19q codeleted oligodendroglioma and are found in most glioblastomas. TERT promoter mutation, in combination with IDH mutation and 1p/19q codeletion, is characteristic of oligodendroglioma. Absence of TERT promoter mutation, coupled with the presence of mutant IDH, designates astrocytoma. (NCCN, 2022).”

H3F3A and HIST1H3B mutation br /> “Recommendation: H3-3A and HIST1H3B mutation testing is recommended in the appropriate clinical context.”

“Although a K27M histone antibody is available, it is not 100% specific and interpretation can be difficult for non-experts. Therefore, screening by H3F3A and HIST1H3B sequencing is a viable alternative and preferred approach, especially since it will also detect mutations in G34 (NCCN, 2022).”

“Histone mutations most commonly occur in pediatric midline gliomas (e.g., diffuse intrinsic pontine gliomas [DIPG]), although midline gliomas in adults can also contain histone modifications. Their presence can be considered solid evidence of an infiltrative glioma, which is often helpful in small biopsies of midline lesions that may not be fully diagnostic with light microscopy or do not fully resemble infiltrative gliomas. The K27M gliomas typically do not have MGMT promoter methylation, and the mutation is an adverse prognostic marker in children and adults. The G34 mutation does not appear to have any prognostic significance once the diagnosis of a glioblastoma has been established (NCCN, 2022).”

“Diffuse midline gliomas should be screened for H3F3A mutations, specifically the H3K27M mutation. While sequencing is the “gold standard,” H3K27M-specific IHC, paired with HeK27 trimethylation immunostaining, is a reasonable alternative, especially when tissue is scarce” (NCCN, 2022).

BRAF mutation:
“Recommendation: BRAF fusion and/or mutation testing is recommended in the appropriate clinical context.”

“BRAF V600E is best detected by sequencing, and BRAF fusions can be detected with RNA-Seq or other PCR-based breakpoint methods that capture the main 16-9, 15-9, and 16-11 breakpoints between BRAF and its main fusion partner, KIAA1549. FISH is too unreliable to detect BRAF fusions” (NCCN, 2022).

“The presence of a BRAF fusion is reliable evidence that the tumor is a pilocytic astrocytoma, provided the histology is compatible. BRAF V600E is more complicated, as it can occur in a variety of tumors over all four WHO grades and requires integration with histology. Tumors with BRAF fusions tend to be indolent, with occasional recurrence but only rare progression to lethality. BRAF V600E tumors show a much greater range of outcomes and need to be considered in context with other mutations and clinicopathologic findings (eg, CDKN2A/B deletion) (NCCN, 2022).”

ZFTA fusion:
“Testing for ZFTA and YAP1 fusions is recommended in the appropriate clinical context. ... Ependymomas arising in the supratentorium often contain activating fusions of ZFTA. This leads to increased NF-kappa-B signaling and more aggressive behavior. This event is more common in children than in adults, and occurs only in the supratentorium, not the posterior fossa or spine (NCCN,2022).”

“The most common ZFTA fusion partner is RELA. This can be detected with RNA sequencing or a break-apart FISH probe set. … Detection of RELA fusion is not required for the diagnosis of ependymoma, as this entity is still diagnosed by light microscopy (NCCN,2022).”

“RELA fusion-positive ependymomas are now a distinct entity in the WHO classification of CAN tumors, as this subset of ependymomas tends to be far more aggressive than other supratentorial ependymomas (NCCN, 2022).”

Miscellaneous
Other markers have been suggested for various uses in evaluating gliomas. For example, other markers for subtyping grade II – III gliomas include assessment of PTEN, TP53, NOTCH1, CIC, FUBP1, EGFR, chromosome 7 gain, and chromosome 10 loss. However, these markers are not currently widely accepted as markers for gliomas.

Finally, the NCCN states there are no identified targeted agents with demonstrated efficacy in glioblastoma (NCCN, 2022).

National Institute for Health and Care Excellence (NICE)
NICE recommends the following molecular markers for investigation of gliomas: IDH1/2 mutations, ATRX mutations, 1p/19q co-deletion, histone H3.3 K27M, BRAF mutation, and MGMT promoter methylation (for prognosis). NICE also notes that testing IDH wild type gliomas for TERT promoter mutations may be considered (NICE, 2021).

European Society for Medical Oncology (ESMO)
The ESMO has published clinical practice guidelines for the diagnosis, treatment, and follow-up of high-grade gliomas. They state that MGMT promoter methylation status, IDH1/2 mutation status, and 1p/19q codeletions are “commonly determined” for assessment of gliomas (ESMO, 2014).

World Health Organization (WHO)
In 2016 and 2021, the WHO published guidelines on the classification of central nervous system tumors. These WHO guidelines, for the first time, incorporated molecular testing in the diagnosis of gliomas and medulloblastomas. The following key points were given by the WHO regarding molecular testing:

  • “IDH1 R132H, which accounts for approximately 90% of IDH mutations, can be detected immunohistochemically. If this testing is negative, sequencing of IDH1 and IDH2 is necessary to ensure that no other IDH mutations are present.”
  • Given the importance of IDH mutational status in the diagnosis or gliomas, at a minimum, it will be important that most institutions have the capacity to both stain tumor specimens for IDH1 R132H by immunohistochemistry and, ideally, sequence those tumors that are negative for both IDH1 and IDH2 mutations” (Wen & Huse, 2016).
  • “Because of the growing importance of molecular information in CNS tumor classification, diagnoses and diagnostic reports need to combine different data types into a single, ‘integrated’ diagnosis. To display the full range of diagnostic information available, the use of layered (or tiered) diagnostic reports is strongly encouraged. Such reports feature an integrated diagnosis at the top, followed by layers that display histological, molecular, and other key types of information” (Louis et al., 2021).
  • Certain tumors (Diffuse astrocytoma, MYB- or MYBL1-altered; Angiocentric glioma; Polymorphous low-grade neuroepithelial tumor of the young; and Diffuse low-grade glioma, MAPK pathway-altered) “require molecular characterization and the integration of histopathological and molecular information in a tiered diagnostic format as molecular work-up helps to characterize the lesion as one type or the other” (Louis et al., 2021).
  • For other tumors such as Myxopapillary ependymoma and Subependymoma, “although these can be identified with methylome studies, molecular classification does not provide added clinicopathological utility for these 2 tumors” (Louis et al., 2021).
  • “Several molecular biomarkers are also associated with classification and grading of meningiomas, including SMARCE1 (clear cell subtype), BAP1 (rhabdoid and papillary subtypes), and KLF4/TRAF7 (secretory subtype) mutations, TERT promoter mutation and/or homozygous deletion of CDKN2A/B62, H3K27me3 loss of nuclear expression (potentially worse prognosis), and methylome profiling (prognostic subtyping)” (Louis et al., 2021).

European Association of Neuro-Oncology (EANO)
In 2021, the EANO published guidelines regarding diagnosis and management of adult patients with diffuse gliomas. The following recommendations were made on molecular testing:

  • “Patients with relevant germline variants or suspected hereditary cancer syndromes should receive genetic counselling and might subsequently be referred for molecular genetic testing.
  • Immunohistochemistry for mutant IDH1 R132H protein and nuclear expression of ATRX should be performed routinely in the diagnostic assessment of diffuse gliomas.
  • If immunohistochemistry for IDH1 R132H is negative, sequencing of IDH1 codon 132 and IDH2 codon 172 should be conducted in all WHO grade 2 and 3 diffuse astrocytic and oligodendroglial gliomas as well as in all glioblastomas of patients aged < 55 years to enable integrated diagnoses according to the WHO classification and to guide treatment decisions.
  • 1p/19q codeletion status should be determined in all IDH-mutant gliomas with retained nuclear expression of ATRX.
  • MGMT promoter methylation status should be determined in glioblastoma, notably in elderly or frail patients, to aid in decision-making for the use of temozolomide.
  • CDKN2A/B homozygous deletions should be explored in IDH-mutant astrocytomas.
  • Combined chromosome 7 gain and chromosome 10 loss (+7/–10 signature), EGFR amplification and TERT promoter mutation should be tested in IDH-wild-type diffuse gliomas lacking microvascular proliferation and necrosis as histological features of WHO grade 4 to allow for a diagnosis of IDH-wild-type glioblastoma” (Weller et al., 2021).

In addition, EANO published a table to summarize the molecular markers used for the diagnosis and management of gliomas.

Table 1: Molecular Markers for the Diagnosis and Management of Gliomas (Weller et al., 2021)

Molecular Marker

Diagnostic Roles

IDH1 R132 or IDH2 R172 mutation

“Distinguishes diffuse gliomas with IDH mutation from IDH-wild-type glioblastomas and other IDH-wild-type gliomas

1p/19q codeletion

Distinguishes oligodendroglioma, IDH-mutant and 1p/19q-codeleted from astrocytoma, IDH-mutant

Loss of nuclear ATRX

Loss of nuclear ATRX in an IDH-mutant glioma is diagnostic for astrocytic lineage tumours

Histone H3 K27M mutation

Defining molecular feature of diffuse midline glioma, H3 K27M-mutant

Histone H3.3 G34R/V mutation

Defining molecular feature of diffuse hemispheric glioma, H3.3 G34-mutant

MGMT promoter methylation

None, but is a predictive biomarker of benefit from alkylating chemotherapy in patients with IDH-wild-type glioblastoma

Homozygous deletion of CDKN2A/CDKN2B

A marker of poor outcome and WHO grade 4 disease in IDH-mutant astrocytomas

EGFR amplification

EGFR amplification occurs in ~ 40–50% of glioblastoma, IDH wild type

 Molecular marker of glioblastoma, IDH wild type, WHO grade 4

TERT promotor mutation

TERT promoter mutation occurs in ~ 70% of glioblastoma, IDH wild type and > 95% of oligodendroglioma, IDH-mutant and 1p/19q-codeleted

Molecular marker of glioblastoma, IDH wild type, WHO grade 4

+7/–10 cytogenetic signature

Molecular marker of glioblastoma, IDH wild type, WHO grade 4

BRAFV600E mutation

Rare in adult diffuse gliomas but amenable to pharmacological intervention” (Weller et al., 2021).

College of American Pathologists
In 2022, The College of American Pathologists in Collaboration with the American Association of Neuropathologists, Association for Molecular Pathology, and Society for Neuro-Oncology published guidelines that suggests these following recommendations for molecular biomarker testing for the diagnosis of diffuse gliomas (Brat et al., 2022):

  • “IDH mutational testing must be performed on all DGs.”
  • “ATRX status should be assessed in all IDH-mutant DGs unless they show 1p/19q codeletion.”
  • “TP53 status should be assessed in all IDH-mutant DGs unless they show 1p/19q codeletion.”
  • “1p/19q codeletion must be assessed in IDH-mutant DGs unless they show ATRX loss or TP53 mutations.”
  • “CDKN2A/B homozygous deletion testing should be performed on IDH-mutant astrocytoma’s.”
  • “MGMT promoter methylation testing should be performed on all GBM, IDH-WT.” 
  • “For IDH-mutant DGs, MGMT promoter methylation testing may not be necessary.”
  • “TERT promoter mutation testing may be used to provide further support for the diagnosis of oligodendroglioma and IDH-WT GBM.”
  • “For histologic grade 2-3 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 GBM, IDH-WT, grade 4.”
  • “H3 K27M testing must be performed in DGs that involve the midline in the appropriate clinical and pathologic setting.” 
  • “H3 G34 testing may be performed in pediatric and young adult patients with IDH-WT DGs.”
  • “BRAF mutation testing (V600) may be performed in DGs that are IDH-WT and H3-WT.”
  • “MYB/MYBL1 AND FGFR1 testing may be performed in children and young adults with DGs that are histologic grade 2-3 and are IDH-WT and H3-WT.”

Table of Terminology

Term Definition
AG Anaplastic glioma
ATRX Alpha-Thalassemia/Mental Retardation Syndrome X-Linked
BRAF B-Raf proto-oncogene
CLIA ’88 Clinical Laboratory Improvement Amendments Of 1988
CMS Centers for Medicare & Medicaid Services
CNA Copy number alterations
CNS Central nervous system
CSF Cerebrospinal fluid
ddPCR Digital droplet polymerase chain reaction
DIPG Diffuse intrinsic pontine gliomas
EANO European Association of Neuro-Oncology
ELISA Enzyme-linked immunoassay
ESMO European Society for Medical Oncology
FISH Fluorescence in situ hybridization
H3-3A H3.3 histone A
H3F3A Previous gene name of H3.3 histone A
H3FA H3 clustered histone 1
IDH1 Isocitrate Dehydrogenase 1
IDH2 Isocitrate Dehydrogenase 2
LDTs Laboratory-Developed Tests
MGMT O-6-Methylguanine-Dna Methyltransferase
NCCN National Comprehensive Cancer Network
NICE National Institute for Health and Care Excellence
NFkB Nuclear factor kappa B
NGS Next-generation sequencing
NICE National Institute for Health and Care Excellence
NFkB Nuclear factor kappa B
NGS Next-generation sequencing
NICE National Institute for Health and Care Excellence
PCR Polymerase chain reaction
RELA RELA proto-oncogene, NF-kB subunit
RNA-Seq Ribonucleic acid sequencing analysis
SNVs Single nucleotide variants
sPD-L1 Soluble programmed cell death ligand 1
TERT Telomerase reverse transcriptase
TMB Tumor mutational burden
TP53 Tumor protein 53
WHO World Health Organization
YAP1 Yes1 associated transcriptional regulator
ZFTA Zinc finger translocation associated

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Coding Section

Codes Number Description
CPT  81120 (effective 1/1/2018) 

IDH1 (isocitrate dehydrogenase 1 [NADP+], soluble) (e.g., glioma), common variants (e.g., R132H, R132C) 
  81121 (effective 1/1/2018) 

IDH2 (isocitrate dehydrogenase 2 [NADP+], mitochondrial) (e.g., glioma), common variants (e.g., R140W, R172M) 
  81210 

BRAF (B-Raf proto-oncogene, serine/threonine kinase) (e.g., colon cancer, melanoma), gene analysis, V600 variant(s); 
  81287

MGMT (0-6-methylguanine-DNA methyltransferase) (e.g., glioblastoma multiforme), methylation analysis
  81345 

TERT (telomerase reverse transcriptase) (e.g., thyroid carcinoma, glioblastoma multiforme) gene analysis, targeted sequence analysis (e.g., promoter region)
  81479 

Unlisted molecular pathology procedure 

H3F3A gene sequencing 

RELA Testing 

H3F3A testing using a K27M histone antibody 
  88374

Morphometric analysis, in situ hybridization (quantitative or semi-quantitative), using computer-assisted technology, per specimen; each multiplex probe stain procedure  
  88377 

Morphometric analysis, in situ hybridization (quantitative or semi-quantitative), manual, per specimen; each multiplex probe stain procedure 
  C71.0-C71.9

Malignant neoplasm of brain code range
ICD-10-PCS (effective (10/01/15)  

Not applicable. ICD-10-PCS codes are only used for inpatient services. There are no ICD procedure codes for laboratory tests.
Type of Service    
Place of Service    

Procedure and diagnosis codes on Medical Policy documents are included only as a general reference tool for each policy. They may not be all-inclusive.

This medical policy was developed through consideration of peer-reviewed medical literature generally recognized by the relevant medical community, U.S. FDA approval status, nationally accepted standards of medical practice and accepted standards of medical practice in this community, Blue Cross Blue Shield Association technology assessment program (TEC) and other nonaffiliated technology evaluation centers, reference to federal regulations, other plan medical policies, and accredited national guidelines.

"Current Procedural Terminology © American Medical Association. All Rights Reserved" 

History From 2015 Forward     

11/02/2022 Annual review, updating policy to allow coverage criteria 2b and 3 as medically necessary. Adding table of terminology. Updating rational and references.

10/15/2021 

Annual review, adding coverage statement for H3F3A testing using K27M. Also updating policy number background, rationale and references. 

10/01/2020 

Annual review, adding coverage criteria for HIST1H3B; otherwise, no change to policy intent. 

10/17/2019 

Annual review, no change to policy intent. Reformatting for clarity. 

11/15/2018 

Annual review. Updating policy to include medical necessity for multiple mutations and deletions. Also updating coding. 

12/7/2017 

Updating policy with 2018 coding. No other changes. 

11/09/2017 

Annual review, providing medical necessity criteria for testing for prognosis of malignant gliomas.  Updating background, description, regulatory status, rationale, references and appendixes. 

04/25/2017 

Updated category to Laboratory. No other changes.

11/07/2016 

Annual review,adding Inflammadry and Liquid Biopsy as investigational. No other changes to policy intent. 

02/10/2016 

Annual review, no change to policy intent. 

02/09/2015

NEW POLICY
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