ISNO consensus guidelines for practical adaptation of the WHO 2016 classification of adult diffuse gliomas
Keywords: Consensus guidelines, diagnosis, diffuse glioma, management
The year 2016 has seen a major change in the diagnostic criteria for adult diffuse gliomas. For the first time, an integrated histo-molecular approach was suggested for diagnosis. Although this modification was widely welcomed, it has generated challenges as to the practical applicability of the new guidelines, especially in a resource limited setting like India. This article discusses the practical considerations of implementing the recommendations in the World Health Organization (WHO) 2016 classification of adult diffuse gliomas and provides practical guidelines suitable for our country.
World Health Organization (WHO) 2016 classification of diffuse gliomas
The World Health Organization (WHO) 2016 classification of central nervous system (CNS) tumors represents a paradigm shift from the previous classifications, as, for the first time, the definition of diffuse gliomas is based on both histological and molecular alterations. The changes in classification directly impact neuropathological assessment and work-up of brain tumor samples; diagnostic lexicon and terminology; and ultimately patient management, with the pivotal aim of providing higher diagnostic accuracy and prognostic precision.
The three clinically relevant biomarkers identified in recent years include 1p/19q codeletion, mutations of isocitrate dehydrogenase (IDH 1 and 2) genes, and alpha thalassemia/mental retardation syndrome X-linked gene (ATRX) gene. These have now been used to create a molecular diagnostic algorithm, which is found to be superior to the traditional histological entities and hence incorporated into the WHO 2016 classification. With this, there has been a major reorganization in the classification of diffuse gliomas by the WHO, and they are now classified under the common header of “diffuse astrocytic and oligodendroglial tumors.” Within this category, diffuse astrocytoma (grade II) is designated as IDH-mutant or IDH-wild type with gemistocytic astrocytoma being IDH-mutant. Anaplastic astrocytoma (grade III) is either IDH-mutant or IDH-wild type. Oligodendroglioma (grade II) and anaplastic oligodendroglioma (grade III) are defined by IDH mutation and 1p/19q codeletion. The samples, where relevant molecular testing cannot be done or are partially performed, are classified according to histomorphology alone with the suffix “NOS” (Not Otherwise Specified) This has immediate clinical relevance in the diagnosis of diffuse gliomas with overlapping morphological features (mixed oligoastrocytoma). With the updated classification, it is now incumbent upon the neuropathologist to molecularly dissect the tumor and genetically identify its molecular signature (astrocytoma or oligodendroglioma), thereby limiting the use of the term “oligoastrocytoma, NOS” in clinical practice.
Glioblastoma, WHO grade IV, is now also classified according to IDH status as glioblastoma; IDH-mutant and glioblastoma; IDH-wild type. The histological variants of glioblastoma; IDH-wild type, include gliosarcoma, giant cell glioblastoma, and epithelioid glioblastoma (associated with the BRAF mutation). Again, an NOS designation can be applied in cases of insufficient molecular testing. The glioma variants that have been deleted from the WHO 2016 classification are the protoplasmic and fibrillary astrocytoma, and the tumor entity deleted is gliomatosis cerebri. Furthermore, the newly introduced tumor entity is the diffuse midline glioma, histone H3 K27M-mutant.
Although the neuro-oncology community welcomed the updated novel classification as a step in the right direction, such fundamental and conceptual modification often evokes critical comments. The implementation of molecular diagnostic testing as recommended by the updated 2016 WHO classification poses considerable technical and practical challenges, more so, in a large and diverse country like India with widespread socio-economic disparities, variable health-care infrastructure, and prevalent non-uniformity of care, thereby calling for a structured, tiered, and tailored approach, which utilizes the available economical tests to reach a meaningful diagnosis in every case.
Approach to a patient with glioma: Clinical evaluation and surgical management
A detailed neurological history will throw light on the dynamics of the glioma. A rapidly growing glioma will have a short clinical profile, and a slower growing glioma will have a longer duration history. A detailed physical examination helps in ruling out differentials like metastasis and other medically manageable conditions. Karnofsky and neurologic performance scores, neurocognitive assessment, and overall patient functional scores help in assessing response to treatment. A dedicated brain tumor imaging study should be considered as far as possible, which includes a standardized magnetic resonance imaging (MRI) with T2-weighted, fluid-attenuated inversion recovery (FLAIR) and plain, and postcontrast T1-weighted images. Wherever feasible, additional MRI sequences of MR spectroscopy, MR perfusion, diffusion/apparent diffusion coefficient (ADC) maps, tractography, etc., may be performed on an individual case-to-case basis, which may aid in the accurate interpretation, diagnosis, and in deciding appropriate therapeutic strategy.
The primary aim of surgery in gliomas generally is to perform a maximal safe resection of the tumor, to relieve raised intracranial pressure, and to obtain tissue for diagnosis and molecular studies., In cases, wherein, radical resection is not possible due to a deep seated, diffuse midline, or an eloquent area location, a biopsy with or without stereotactic guidance may be considered. This will help to obtain an integrated diagnosis, which will enable counseling of patients regarding prognostication.
Clinically relevant molecular markers of diffuse gliomas
Isocitrate dehydrogenase (IDH)
Isocitrate dehydrogenase (IDH) 1 and 2 mutations are seen mainly in diffuse grade II and III gliomas and to a lesser extent, in glioblastoma. IDH is an essential enzyme in the citric acid cycle, which catalyzes the oxidative decarboxylation of isocitrate to alpha ketaglutarate. Of the IDH mutations found in gliomas, the vast majority are in IDH1, the commonest being IDH1 (R132H). IDH 2 mutation frequently leads to alteration in arginine at position 172. These IDH mutations are thought to be important initial players in gliomagenesis. The IDH mutations are seen in 100% of oligodendroglioma and 70%–80% of WHO grade II and III astrocytomas. Only 8%–10% of glioblastomas are IDH-mutant. The IDH-mutant gliomas have a better prognosis than their wild type counterparts irrespective of their histological grade. Immunohistochemistry (IHC) is the most prevalent method across the world and a very robust method to demonstrate IDH1 (R132H) mutant protein [which showed 100% concurrence with sequencing for IDH1 (R132H)]. However, other IDH1 and IDH 2 mutations can be tested by DNA sequencing only. Thus, all cases of diffuse glioma should be first evaluated by IHC for IDH1 (R132H), and only those that are negative by IHC can be subjected to deoxyribonucleic acid (DNA) sequencing for detection of other IDH1 and 2 mutations. However, direct evaluation for all IDH1 and 2 mutations by DNA sequencing without the initial IHC can also be done, where feasible.
Unbalanced translocation between the short arm of chromosome 1 and long arm of chromosome 19 with subsequent loss of the derivative chromosome, del (1;19) (p10; q10), is seen in oligodendrogliomas. Codeletion of these chromosome arms is a specific molecular alteration, now considered essential for the diagnosis of oligodendroglioma. This alteration is associated with improved survival and also serves as a predictor of sensitivity to chemotherapy. Hence, 1p/19q codeletion is used as a potential diagnostic, prognostic, and predictive marker in oligodendroglial tumors. The genes on 1p and 19 that are thought to be responsible for the effect of 1p/19q codeletion are FUBP1 and CIC, respectively.
Over the years, 1p19q codeletion has been evaluated by two major methods of polymerase chain reaction (PCR) based loss of heterozygosity, fluorescent in situ hybridization (FISH), and also by different next generation sequencing (NGS) platforms. However, the currently preferred technique for assessing 1p/19q codeletion is the FISH technique owing to the ease of availability of commercial target probes and feasibility to perform the test on formalin-fixed paraffin embedded (FFPE) tissue sections.
ATRX (Alpha Thalassemia Mental Retardation X-linked gene) mutation
ATRX is involved in chromatin remodeling, histone regulation, nucleosome assembly, and in maintenance of telomeres. Mutations in this gene, located at Xq210.1 lead to loss of function. The ATRX mutation is reliably detected by IHC where a tumor with the mutation is immunonegative for ATRX protein, and positivity is seen in native glial/microglial cells, neurons, inflammatory cells, and endothelial cells. Mutations in ATRX have a strong correlation with tumors of astrocytic phenotype, that harbor TP53 and IDH mutations., ATRX mutation is not seen in any oligodendroglial tumors and is mutually exclusive with 1p/19q codeletion. It is presumed that ATRX mutation follows IDH mutation during astrocytoma tumorigenesis.
TP53 is an essential regulator of the cell cycle, forming a part of the tumor suppressor gene family. The TP53 gene provides instructions for making a protein called tumor protein p53 (or p53) that acts as a tumor suppressor. TP53 mutations are found to be almost mutually exclusive with 1p/19q codeletion in gliomas and correlate strongly with an astrocytic morphology. Approximately, 50% of astrocytomas harbor TP53 mutations, in contrast to just 10% of oligodendrogliomas. As TP53 mutations show a close relationship to ATRX mutations and IDH mutations, co-existence of these three mutations can be viewed as a molecular signature of astrocytomas.
On IHC, p53 protein accumulation is detected as positive staining of the nuclei and serves as a surrogate marker for detection of the mutation. Null mutations will not be detected by IHC as the protein is absent. In addition, immunopositivity for p53, while highly concordant with Tp53 mutation, cannot always be taken as concrete proof of mutation. DNA sequencing is the gold standard to detect the presence of a TP53 mutation in a particular sample.
O6-methylguanine DNA methyltransferase (MGMT) promoter methylation
The MGMT gene located at 10q26 encodes for the DNA repair enzyme, O6-methylguanine DNA methyltransferase, which is responsible for removing the alkyl adducts from the O 6 position of guanine. The enzyme ensures rapid repair of DNA, making a cell more resistant to alkylating or methylating chemotherapeutic agents. Epigenetic silencing of this gene by promoter methylation has been found to be an independent prognostic marker regardless of the type of treatment. Although some studies have suggested that the prognostic power of MGMT promoter methylation is derived from its close association with the IDH mutation, other studies have observed that the methylation status of the MGMT promoter is a predictive marker of the response to chemotherapy in IDH-wild type tumors as well., MGMT promoter methylation is commonly assessed by methylation specific PCR (conventional or real-time) and pyrosequencing.
Telomerase reverse transcriptase (TERT) promoter mutation
In recent years, although telomerase reverse transcriptase (TERT) promoter mutations have been actively studied as mutations that are quite prevalent in gliomas as well as being significantly associated with survival and prognosis, the nature of this association depends in a large part upon the presence of other molecular alterations., The significance of TERT promoter mutations in IDH-wild type glioblastoma remains controversial. However, the analysis of other molecular markers alongside TERT promoter mutations may better define prognostic subgroups in gliomas. The TERT promoter mutations are detected by Sanger sequencing. A chromatogram showing TERT promoter mutation 228 C > T is depicted in [Figure 2].
Epidermal growth factor receptor (EGFR) gene mutation
One of the most common mutations that occur in IDH-wild type glioblastomas is the gain of 7p, harboring the epidermal growth factor receptor (EGFR) gene. Approximately, 40% of glioblastomas carry this mutation. The EGFR gene codes for a cell surface tyrosine kinase receptor that initiates the PI3K and MAPK pathways, which contribute to cell survival and proliferation. The most common EGFR gene aberration encountered in glioblastoma is its amplification. A proportion of the EGFR amplified tumors carry the EGFR vIII mutation, which plays a significant role in gliomagenesis, increasing the survival of the mutant cells as well as adjacent cells through paracrine activity. The EGFR amplification can be assessed by the FISH technique. The presence or absence of the EGFR mutation can be detected by IHC using antibodies to EGFR vIII, or by PCR sequencing.
BRAFV600E mutation has been described frequently in circumscribed gliomas like pleomorphic xanthoastrocytoma (PXA) and less often in diffuse adult gliomas. Approximately, 50% of cases of epithelioid glioblastoma harbor this mutation. The survival implication of this mutation is not well established to date with the few case reports suggesting a variable prognosis. BRAFV600E mutation can be detected using IHC.
Histone (H3 K27M) mutation
Histone protein H3 is one of the four proteins essential for the formation of octamer complex around which the DNA is wrapped to form a nucleosome. Diffuse midline gliomas often harbor mutation in the H3 protein. These are infiltrative midline gliomas characterized by K27M mutation in either H3F3A or HIST1H3B/C genes. Mutation occurring in either H3.1 or H3.3 protein leads to H3 K27M mutation, which prevents the methylation/acetylation of lysine at position 27. Trimethylated H3K27 (H3 K27Me3) is essential for tumor suppression and differentiation. Thus, H3 K27M-mutant diffuse midline gliomas show a decrease in H3 K27Me3, which confers very poor prognosis to these tumors. H3 K27M-mutant protein can be detected by IHC. The mutation, while commonly presents in diffuse midline gliomas, may also be present in gliomas in other locations. However, the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy– Not Official WHO (cIMPACT-NOW) working group has clarified that only those diffuse gliomas located in the midline and positive for H3 K27M are to be designated as “diffuse midline glioma, H3 K27M-mutant.”
Practical considerations for implementation of World Health Organization (WHO) 2016 classification for diagnosis of diffuse glioma in a resource limited setup
The erstwhile WHO 2007 classification based on histomorphology alone allowed for providing a definitive diagnosis in diffuse gliomas within a few days after surgery. However, molecular testing to provide an integrated diagnosis, as per updated 2016 WHO classification would prolong the time to a conclusive diagnosis. The calculation of increased costs of incorporating integrated diagnosis into routine clinical practice is a complex and onerous task that depends to a large extent upon the number of molecular markers tested, assays/methods used for such molecular testing, total number of samples tested, and of course, the health-care setting. The cost of these tests is generally not affordable by patients from the lower economic strata. These are some of the major concerns to be considered in a resource constraint setting that exists not only in our country but also in other developing nations. In India, whilst IHC for IDH1 (R132H), ATRX, and P53 is available in most of the diagnostic histopathology laboratories, FISH and DNA sequencing techniques are offered mostly in major diagnostic/research centers. In keeping with all the above issues, there is an absolute need to design a practical, economical, working algorithm for the integrated diagnosis of diffuse gliomas, suitable for a resource constraint environment.
The Indian Society of Neuro-oncology (ISNO) guidelines reflect an adaptation of the WHO 2016 classification and recommendations, to suit a resource constraint setting. Given the logistic and financial constraints in our country, the ISNO recommends a more practical and pragmatic approach of using ATRX IHC in addition to IDH mutation for classification of adult diffuse gliomas. This has also been further clarified by the cIMPACT-NOW, which suggests that diffuse astrocytic tumors that are WHO grade II or grade III and that have IDH mutation can be diagnosed as diffuse astrocytoma, IDH-mutant or anaplastic astrocytoma, IDH-mutant (respectively), if there is definite loss of ATRX nuclear expression and/or strong, diffuse p53 immunopositivity, without the need for 1p/19q testing. The guidelines are framed for adult diffuse gliomas (grades II and III) as well as glioblastoma (grade IV) and its variants and provide details of the required histological and molecular work-up. The guidelines emphasize on the uniform neuropathology reporting of adult diffuse gliomas and the associated treatment protocols in our country that would benefit therapy decision and follow-up of patients. The ISNO guidelines for medulloblastoma have been widely adopted by the Indian clinicians and pathologists. Hopefully, the ISNO guidelines for gliomas also find their place in the routine clinical practice in the country.
Histology [Figure 1]
The initial screen is histological review, where the diffuse infiltrative growth pattern should be confirmed and only then the further steps can be followed. Once this is confirmed, the phenotype of the tumor should be identified, i.e., astrocytoma, oligodendroglioma, and oligoastrocytoma (ambiguous morphological pattern). These tumors are then graded (as II or III) based on the traditional histological criteria of cellularity and mitosis. Subsequent to histology, IHC is carried out.
IHC [Figure 2]
The sequence of IHC markers to be tested is as follows:
In summary, following histology and IHC with two markers: IDH1 (R132H) and ATRX, one can derive four subgroups in diffuse glioma: Astrocytoma-IDH-mutant, astrocytoma- NOS, oligoastrocytoma-NOS, and oligodendroglioma-NOS.
FISH [Figure 2]
DNA Sequencing [Figure 2]
The simplified algorithm for the approach to the classification of diffuse glioma in a resource limited setup is depicted in [Figure 3].
Histology [Figure 1]
The entry point for diagnosis of glioblastoma, as in grade II and grade III diffuse gliomas, is histopathology. On the basis of histomorphology, the variant of glioblastoma is determined. Certain variants may demand testing for a specific molecular marker, for example, epitheloid glioblastoma phenotype warrants a search for BRAFV600E mutation.
IHC [Figure 2]
FISH and DNA sequencing [Figure 2]
Thus, a definitive integrated diagnosis of glioblastoma can be arrived at with IHC and FISH in a majority of the cases without the need for DNA sequencing.
The simplified algorithm for the approach to the classification of glioblastoma in a resource limited setup is depicted in [Figure 4].
Impact of IDH mutation in clinical practice
As discussed, molecular classification of histologically-defined diffuse gliomas currently divides them into IDH-mutant and IDH-wild type tumors. IDH-mutant gliomas are associated with better outcomes than IDH-wild type gliomas. Although the generally followed standard of care in higher grade diffuse gliomas is maximal safe resection followed by adjuvant treatment with radiotherapy (RT) and chemotherapy, management of lower grade diffuse gliomas may be observational or conservative following surgery. However, some practitioners embrace the more aggressive course of management of lower grade diffuse gliomas similar to higher grade diffuse gliomas. In this scenario, IDH mutation status in the patient may aid in the decision-making on the management of the patient.
IDH-wild type low/lower grade gliomas
The absence of IDH mutation in grade II/III gliomas marks a distinct IDH-wild type subgroup, which when compared to IDH-mutant gliomas, has been shown to be associated with relatively poor prognosis than is expected for low/lower grade gliomas.
Suggested ISNO guidelines:
IDH-mutant WHO grade II glioma
IDH-mutant adult diffuse WHO grade II low-grade gliomas are relatively slow growing tumors. It is important to characterize their lineage (astrocytoma vs. oligodendroglioma) as the median survival in grade II oligodendroglioma is >15 years and grade II astrocytoma is 7-10 years.
World Health Organization (WHO) grade III anaplastic gliomas with IDH mutation and 1p19q codeletion (anaplastic oligodendrogliomas)
This group of tumors do generally well with median survivals reported to be more than 10 years with postoperative adjuvant chemo-radiation post-surgery. It is recommended to give radiation therapy and chemotherapy, either in the form of RT + PCV or with TMZ followed by adjuvant 12 cycles of monthly TMZ, as per the standard Stupp regimen. The ongoing phase III intergroup study of RT with concomitant and adjuvant TMZ versus RT with adjuvant PCV chemotherapy in patients with 1p/19q codeleted anaplastic glioma or low-grade glioma (CODEL study) is currently ongoing and should eventually answer the question of the relative efficacy of TMZ versus PCV chemotherapy. Radiation doses typically have been 59.4 Gy/33 fractions but in view of good long-term survivals in these patients, there is emerging interest in reducing the RT doses to 54–55.8 Gy/30–31 fractions.
World Health Organization grade III anaplastic gliomas with IDH mutation and 1p19q non-codeletion (anaplastic astrocytomas)
This group has a median survival of 3.5 years. The interim results from the Phase III trial on concurrent and adjuvant TMZ chemotherapy in 1p/19q non-codeleted anaplastic glioma (CATNON trial) was associated with a significant survival benefit at 5 years to be 56% in the RT + TMZ arm vs. 44% with RT alone, and hence, is the recommendation in these patients.
IDH-wild type glioblastoma
IDH-wild type glioblastoma, also known as primary glioblastoma, is the most aggressive of all diffuse gliomas. The recommended treatment in IDH-wild type glioblastoma is maximal safe resection followed by radiotherapy with 59.4 Gy/33 fractions and chemotherapy with TMZ, followed by cyclical TMZ, unless contraindicated.
IDH-mutant glioblastomas (about 10% of cases) correspond to what has been ascribed to secondary glioblastoma with a longer history/history of prior lower grade diffuse glioma, and arises in relatively younger patients. Evidence demonstrates that survival of IDH-mutated GBM to be more favorable than that for non-mutated grade III astrocytoma, thus showing the strong prognostic value of this finding. However, the concurrence of both mutated-IDH and methylated MGMT promoter has stronger prognostic value than either one of these genetic aberrations alone and is associated with improved survival irrespective of the treatment administered.
Impact of MGMT promoter methylation status in glioblastoma
MGMT promoter methylation as a prognostic and predictive factor
Several landmark papers have proven the prognostic and predictive value of MGMT promoter methylation in glioblastoma with a methylated MGMT promoter being associated with significantly longer survival than an unmethylated phenotype.,,,,,,,,,,,,,,,,,,,,,,, This finding has triggered a clinical trial with dose dense TMZ in glioblastoma, which failed to show any significant survival benefit. Furthermore, trials testing alternate chemotherapeutic agents in glioblastoma with unmethylated MGMT promoter have shown no survival advantage when compared to standard TMZ therapy, thus suggesting that the standard adjuvant TMZ treatment (days 1 through 5 of a 28-day cycle, 150–200 mg/m 2) should be employed in clinical practice. Hence, RT + TMZ followed by adjuvant TMZ remains the standard of care for both methylated and unmethylated tumors.
MGMT promoter methylation and pseudoprogression
Pseudoprogression is a transient MRI pattern mimicking tumor progression but not necessarily accompanied by clinical deterioration. The incidence of pseudoprogression in glioma patients, especially glioblastoma treated with concurrent TMZ and RT is 20%–35%, which is often seen around 3–6 months after of completion of chemo-RT. In clinical practice, it is important to review all clinico-radiological features in a multi-disciplinary tumor board meeting to discriminate pseudoprogression from an early progression. MGMT promoter methylation status has been frequently associated with pseudoprogression and found to have a 3.5 fold greater possibility of having pseudoprogression than early progression  and can, therefore, serve as a very useful complementary tool to help in this evaluation.
MGMT promoter methylation status in elderly patients with glioblastoma
Older adults with glioblastoma have generally a worse prognosis as compared to younger patients. Treatment decisions in older adults can be additionally complicated by factors such as comorbid disease and increased susceptibility to side effects. Older adults also tend to receive less aggressive therapy, which may influence outcomes. MGMT promoter methylation status predicted an improved OS and PFS, as well as quality of life, even in patients aged 70 years or older, who had a poor performance status. Phase III studies have shown the effectiveness of shorter courses (40 Gy in 15 fractions) of RT as compared with standard RT or TMZ alone in elderly patients with glioblastoma with MGMT promoter methylation and their recommendations can be adopted in clinical practice based on the performance status of the patient.
Suggested ISNO guidelines for elderly patients with glioblastoma
Hence, we recommend the assessment of MGMT promoter methylation status in glioblastoma, which may aid in personalization of treatment, especially in the elderly.
MGMT promoter methylation status at recurrence
There is no accepted standard of care for management of recurrent glioblastoma, and treatment recommendations can vary from patient to patient. Modalities include repeat surgery or re-irradiation in case of relapse or with second line systemic therapies such as lomustine and bevacizumab, depending on the decision in a tumor board meeting.
Emerging evidence from phase II studies suggests that the addition of bevacizumab to lomustine might improve OS as compared with monotherapies, and patients with methylated MGMT promoter have a longer median overall survival than those with unmethylated MGMT promoter (13.5 months versus 8.0 months).,
Impact of histone mutations in midline glioma
The evaluation of H3 K27M mutation may aid in the diagnosis in clinical situations where there is a clinico-radiological dilemma in midline gliomas, especially between a low grade pilocytic astrocytoma and glioblastoma. H3 K27M-mutant tumors are to be treated with aggressive adjuvant protocols (RT + TMZ). Various therapeutic strategies including peptide vaccines and targeted agents against histone mutations in these groups of patients are currently being investigated in clinical trials and may alter outcomes in the near future.
BRAF gene alteration in epithelioid glioblastoma
As discussed earlier, BRAF V600E mutations, are present in about 50% of epithelioid glioblastoma, a rare aggressive variant and its clinical implication is not yet clear. However, novel BRAF inhibitors such as vemurafenib and dabrafenib are currently being tested in clinical trials and may further optimize clinical outcomes in the future.
The ISNO guidelines were framed by an expert panel comprising neuropathologists, neurosurgeons, and radiation/medical oncologists, to suit a resource limited setting. Our guidelines allow the multidisciplinary treating team to arrive at a definitive diagnosis with the minimum number of molecular tests possible and also help in deciding an appropriate management strategy in individual cases. The guidelines will be updated as required.
The authors acknowledge the contribution of Dr. Raees Tonse, in the preparation of the manuscript and the neuro-oncology team, NIMHANS for their technical support. We thank Mr. K. Manjunath for preparing the photomontages.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]