|Year : 2021 | Volume
| Issue : 3 | Page : 166-174
Approach to integrating molecular markers for assessment of pediatric gliomas
Swati Mahajan, MC Sharma, Chitra Sarkar, Vaishali Suri
Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
|Date of Web Publication||02-Nov-2021|
Dr. Vaishali Suri
Professor, Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi
Source of Support: None, Conflict of Interest: None
Recent research has promoted elucidation of the diverse biological processes that occur in pediatric central nervous system (CNS) tumors. These molecular data are leading to new guidelines for the diagnosis, offering prognostic implications and novel molecular targeted therapies. The consortium to inform molecular and practical approaches to CNS tumor taxonomy-NOW have made practical recommendations using recent advances in CNS tumor classification, particularly in molecular discernment of these neoplasms as morphology-based classification of tumors is being replaced by molecular-based classification. Here, we review the specific molecular drivers that help to define the entities that fall under the umbrella of pediatric gliomas and how to effectively test them in cost-effective manner. We discuss briefly the proposed risk-based stratification system that considers both clinical and molecular parameters to aid clinicians in making treatment decisions and the availability of an increasing array of molecular-directed therapies.
Keywords: Glioma, high-grade glioma, low-grade glioma, molecular, pediatric
|How to cite this article:|
Mahajan S, Sharma M C, Sarkar C, Suri V. Approach to integrating molecular markers for assessment of pediatric gliomas. Int J Neurooncol 2021;4, Suppl S1:166-74
| Introduction|| |
Gliomas are the most common primary central nervous system (CNS) tumors in the pediatric population, with an extremely broad range of clinical behavior, histology, molecular biology, and outcome. Majority of pediatric gliomas are low grade, slow-growing lesions classified as Grade I or II by the World Health Organization (WHO) classification of CNS tumors and account for 25%–30% of pediatric CNS tumors. The 10-year overall survival (OS) of these tumors is over 95%, but the10-year progression-free survival (PFS) is only approximately 50%., Although complete surgical resection is the most favorable predictor of survival, half of these patients still require adjuvant therapy. A significant fraction of pediatric gliomas develop over a short period and progress rapidly and are, therefore, classified as WHO Grade III or IV pediatric high-grade gliomas (pHGGs). They comprise 8%–12% of all pediatric brain tumors and unlike histologically similar lesions in adults, where large proportion of tumors develop from the preexisting lesion and tend to be restricted to the cerebral hemispheres, pHGGs are predominantly de novo and can occur throughout the CNS. Approximately one-half of pHGGs occur in the midline location (brainstem), in particular, the ventral pons as diffuse intrinsic pontine gliomas (DIPGs) (80%) and thalamus (13%). Despite all therapeutic efforts, they remain largely incurable, with 2-year survival rates for children with hemispheric high-grade gliomas being 10%–30% and diffuse midline HGG being <10%.,,,
Recently, next-generation genomics have shed light on the broad molecular heterogeneity of pediatric gliomas. Molecular genetic analysis has identified key genes and signaling pathways that serve to drive tumor proliferation, which are different from those observed in adults. Since the 2016 WHO classification of CNS tumors, molecular profiling has become indispensable resulting in the morphology-based classification of tumors being replaced by molecular-based classification with significant clinical correlations in terms of age at presentation, anatomical location, and consequently improving treatment approaches and prognosis.,, Consortium to inform molecular and practical approaches to CNS tumor taxonomy-NOW (cIMPACT-NOW) is making practical recommendations based on recent advances in CNS tumor classifications, particularly those based on molecular discernment of these neoplasms., In this review, we provide an insight into the key molecular alterations that have expanded our knowledge in pediatric gliomas. Further, we aim to encapsulate the diagnostic, prognostic, and predictive molecular biomarkers and consolidate various economical platforms that can be used for routine assessment in resource-constrained countries.
| Molecular Landscape of Pediatric High-Grade Gliomas|| |
The key discovery that best illustrates the unique biology of pHGG's was the identification of somatic histone mutations. Specific and mutually exclusive mutations in the genes encoding the histone H3.3 (H3F3A) and H3.1 (HIST1H3B, HIST1H3C) variants along with BRAF V600E mark distinct subgroups of disease in children and young adults. Nearly half of tumors harbor numerous overlapping putative drivers or other epigenomic characteristics; yet they do not form well-validated biological and clinical subgroups. A small proportion of children (mostly adolescents) with IDH1 mutations represent the lower tail of age distribution of an otherwise adult subgroup. pHGGs have broadly been classified as:
Diffuse midline glioma, H3-K27M-altered
Diffuse midline gliomas, H3-K27M mutant was recognized as a new diagnostic entity in the updated 2016 WHO classification of CNS tumors unifying DIPGs and gliomas from the thalamus and spinal cord harboring a histone H3-K27M mutation., Recently, studies identified a few cases of midline glial tumors that lacked H3-K27M mutation, but showed H3K27me3 loss along with either enhancer of zest homologs inhibitory protein (EZHIP) overexpression or epidermal growth factor receptor (EGFR) mutation, thus extending the spectrum of DMG's beyond H3-K27M mutation.,, Thus, loss of H3K27me3 can now be regarded as a unifying feature of midline gliomas. The logical approach will be to integrate these alterations into a novel tumor type “DMG, H3K27-altered,” with the three molecular classes (DMG-H3K27M, DMG-EZHIP, and DMG-EGFR) as subtypes. H3K27M-mutant tumors remain by far the most common subtype, with the remaining two subclasses representing rare exceptions.
Diffuse midline glioma, H3-K27M mutant
Majority of high-grade infiltrative astrocytomas arising within midline structures (thalamus, brain stem, and spinal cord) in pediatric patients are characterized by somatic driver mutations in H3F3A or HIST1H3B/C, encoding for histone 3 variants H3.3 and H3.1, respectively.,, The H3-K27M-mutant protein inhibits polycomb repressive complex 2 (PRC2) activity via sequestration of its catalytic subunit enhancer of zest 2 (EZH2), resulting in globally decreased H3-K27 trimethylation (H3K27me3).,,, Overall incidence of H3-K27M mutations in DMGs is estimated to be 80% in pediatric and 38.2%–72% in adult patients with worse OS (median OS 11–15 months) when compared with wild type, independent of patient age and histological diagnosis.,, Immunohistochemistry (IHC) is the most prevalent method worldwide and a very robust method to demonstrate H3-K27M mutation or loss of mutant protein (H3K27me3). Studies have shown 100% concurrence with sequencing for H3-K27M mutation.
Diffuse midline glioma, enhancer of zest homologs inhibitory protein overexpression
A small subset of DMG harbor H3K27me3 loss but lack histone gene (H3) mutations; however they express the H3-K27M-mimic (EZHIP/CXorf67)., EZHIP expression is mutually exclusive to H3-K27M mutation for driving the massive loss of H3K27me3. This genomic alteration is seen in older children as compared to H3-K27M mutant cases. OS of these patients is similar to H3.1-K27M patients (median OS of 15.9 vs. 15 months, respectively). The EZHIP expression is reliably detected by IHC as positive staining of the tumor nuclei. This IHC has a reported sensitivity and specificity of more than 95%.
Diffuse midline glioma, Epidermal growth factor receptor mutant
Primary bilateral thalamic gliomas, represent an exceedingly rare subset with the incidence of 0.84%–5.2% among all intracranial tumors; however, their actual incidence in children has not been established yet. These cases are not amenable to surgical resection and have a uniformly poor outcome despite radiation and conventional cytotoxic chemotherapy., These gliomas harbor frequent mutations in the EGFR oncogene in the absence of accompanying EGFR amplification (85%), with only rare histone H3 mutation (9%), and a distinct genome-wide DNA methylation profile compared to all other glioma subtypes. In addition, inactivating mutations of the TP53 gene are present in 62% of cases and H3K27me3 protein loss is seen in 89% of cases., These mutations are found predominantly in infants and older children (7 to 16 years) and are associated with similar poor outcomes as H3-K27M mutant cases (median OS-11 months). The presence or absence of the EGFR mutation can be detected by Sanger sequencing.
Diffuse hemispheric glioma, H3G34-mutant
Mutations on H3F3A that substitutes glycine to arginine or valine at position 34 (G34R/V) have been identified in approximately 20% of the pHGG located in the cerebral hemispheres., They occur predominantly within the age range of 11–30 years (median 15 years). Patients harboring tumor with this mutation tend to have longer OS (median survival of 20 months) than DMG. A considerable number of H3.3G34R/V mutated tumors have co-mutations in TP53 and in chaperone genes ATRX and DAXX.,
Histologically, these tumors display two distinct patterns, “Glioblastoma multiforme (GBM)-like” and “Primitive neuroectodermal tumor (PNET)-like.” GBM like tumors show immunoreactivity for glial fibrillary acidic protein (GFAP). In contrast, PNET-like tumors show features of an undifferentiated embryonal tumor with limited to absent immunoreactivity for GFAP. Both histological variants lack expression of Olig2 protein. Strong nuclear accumulation of p53 protein and complete nuclear negativity for antibodies against ATRX protein are seen in >90% of cases., Although high specificity of H3G34R/V mutation-specific antibodies has been claimed (>95%), occasional false-negative cases have been observed raising a word of caution in their use as surrogates for the detection of G34 mutations.,,,
Pediatric high-grade glioma-IDH mutated
Missense mutations in either the IDH1 or IDH2 genes represent <5% of pHGGs. IDH-mutant gliomas are generally seen in adolescent patients and are associated with a more favorable prognosis compared to wild-type tumors. IHC for IDH1-R132H protein is the most cost-effective diagnostic aid, and positive immunoreactivity confirms an IDH1-mutant diffuse glioma.
Diffuse pediatric type high-grade glioma, H3 and IDH wildtype
Genome-wide DNA methylation profiling delineated three biological subtypes of H3-/IDH-wt pediatric GBM which show a variety of genomic and epigenetic profiles., These tumors dominantly show either MYCN upregulation, platelet-derived growth factor receptor (PDGFRA) amplification, or frequent amplification of EGFR/TERT mutation. Further investigations of these heterogeneous subgroups are needed to improve integrated molecular diagnostics and patient stratification for more tailored treatment based on molecular targets.
Pleomorphic xanthoastrocytoma like/low-grade glioma like
These tumors are predominantly cortical, have histological, genetic and epigenetic similarities to pleomorphic xanthoastrocytoma and low-grade gliomas (LGGs) with median OS of 63 months.,,
Hypermutant glioblastoma in patients with constitutional mismatch repair deficiency
An autosomal recessive childhood cancer syndrome is caused by biallelic mutations in the mismatch repair pathway (bMMMR). It is associated with both extracranial and malignant brain tumors in children and/or young adults. The average age at diagnosis is 12 years with 5-year OS survival of 30%.
Recently, infantile gliomas have been proposed to be subclassified into hemispheric receptor tyrosine kinase (RTK)-driven, hemispheric RAS/MAPK-driven gliomas or midline RAS/MAPK-driven gliomas with distinct demographic profiles and latter having the worst clinical outcome.
| Molecular Landscape of Pediatric Low-Grade Gliomas|| |
Pediatric low-grade gliomas (pLGG's) include glial, glio-neuronal and neuronal tumors: pilocytic astrocytoma (PA), dysembryoplastic neuroepithelial tumor (DNET), ganglioglioma (GG), pleomorphic xanthoastrocytoma (PXA), subependymal giant cell astrocytoma, diffuse astrocytoma (DA), diffuse oligodendroglial tumors, and other rare tumors. The majority of pLGG are driven by a single genetic event resulting in up-regulation of the RAS/mitogen-activated protein kinase (MAPK) pathway.,, In addition, rarer alterations affecting RAS/MAPK signaling, including those involving FGFR1/2/3, NTRK2, RAF1, ALK, and ROS1 as well as non-RAS/MAPK alterations, such as MYB, MYBL1, IDH1, H3F3A mutations and CDKN2A/B homozygyous deletion have been identified in a small numbers of cases. An overview of the most common aberrations in pLGG is as follows:
It is the most frequent molecular alteration in pLGG (30%–40%) and is significantly enriched in PA (70%) and in tumors arising in the posterior fossa/cerebellum. The presence of KIAA1549-BRAF fusion aid in tumor diagnosis as it is not found in adult-type diffuse glioma and, with rare exceptions, confirms a pLGG diagnosis., Tumors with this fusion are often amendable to complete surgical resection, have excellent OS and rarely progress.
Fluorescence in situ hybridization (FISH) analysis is widely used to detect BRAF fusion. Though it is sensitive for its detection, results are often difficult to interpret because the KIAA1549 and BRAF genes lie in close proximity on chromosome 7q34, and it is occasionally difficult to distinguish a fusion signal from a normal signal. Moreover, analysis may also be complicated by amplification of the 7q34 region in some tumors without gene fusion. It also does not give information regarding the fusion partner. The reverse transcription-polymerase chain reaction (RT-PCR) is increasingly being implemented over or alongside FISH in a diagnostic setting with sensitivity and specificity of 97% and 91%, and also gives additional information as to the type of fusion variant.
This mutation has been reported in approximately 40%–80% of PXAs, 18% to 33% of GGs, 20%–25% of DNETs and 9% of PAs, and in rare cases of low-grade diffuse gliomas in children., It has been reported in ~10% of glioblastomas, especially the epithelioid variant. As a group, pLGGs with BRAF V600E have worse OS and PFS compared to other pLGGs., Further, BRAFV600E pLGG, with co-occurring CDKN2A deletions are significantly more likely to transform into HGG; an event that may occur 10–20 years after the initial diagnosis.
Though Sanger sequencing is the gold standard, it may miss mutations with <10% to 20% mutant alleles because of its lower analytic sensitivity. A Food and Drug Administration approved V600E-specific mouse monoclonal antibody (VE1) allows the identification of mutant tumors in small biopsy or specimens, with scattered tumor cells intermingled with abundant nonneoplastic cells, with a sensitivity and specificity of >95%.,
Fibroblast growth factor receptor 1(FGFR1) alterations
It is the second most commonly altered gene in pLGGs. FGFR1 aberrations include FGFR1 mutations, FGFR1-TACC1 fusions, and FGFR1-TKD duplications. Becker et al., in their description of FGFR1 mutations in PA, noted that mutated tumors had a worse prognosis than their wild-type counterparts. The mutations are detected by either IHC (Phospho-ERK antibody CS-4376 (1:200) optimized in Ventana machine) or Sanger sequencing whereas evaluation of fusions and duplications require either nanostring assay (reported sensitivity and specificity of >95%) or targeted next-generation sequencing (NGS), where feasible.
KRAS, PTPN11 mutations, and CRAF fusions
A small subset (1%–5%) of pLGGs harbor these alterations, the identification of which may offer access to targeted therapies.,,
ALK, ROS1 and NTRK alterations
These mutations have also been identified in pLGG, though rarely. The targeted agents for these are already developed, approved and are available, due to their higher prevalence in other adult malignancies.
Non-RAS/MAPK pathway alterations
MYB and MYBL1 alterations
Rearrangements of MYB or MYBL1 genes have been substantially detected in specific clinicopathological subgroups of IDH-wt/H3-wt diffuse gliomas from a large pediatric-based cohort, thereby strictly indicating the need of instilling their diagnostic testing. These alterations are detected more frequently in young children (median age, 5 years) and are significantly enriched for the cerebral hemispheres. MYB alterations have been identified to be histologically restricted to angiocentric gliomas (87%) and pediatric DAs (41%). MYBL1 alterations are rare and are detected in DAs. The clinical course is generally indolent and Chiang et al., reported that the 10-year OS is 90%, and 10-year PFS is 95%. MYB/MYBL1 alterations can be demonstrated by IHC, FISH, or quantitative RT-PCR in routine practice.,,
| Diagnostic, Prognostic, and Therapeutic Implications|| |
The field of neuro-oncology has made headway in uncovering the key oncogenic drivers in pediatric gliomas. This not only has diagnostic implications but also prognostic and predictive value that continues to rapidly evolve with important implications for the standard of care and for the clinical management of these patients.
Approach to integrating molecular markers for diagnosis
Given the range of molecular alterations and their overlap amongst different tumor histologies, devising a simple testing recommendation for pediatric gliomas can be difficult. There can be two primary approaches (i) sequential testing of the most common alterations in a tier-based approach; and, (ii) upfront NGS panels. Given the logistic and financial constraints, the use of sequential testing is largely recommended as majority of pediatric gliomas harbor a single molecular driver only.
Diffuse midline gliomas
- All midline gliomas should initially be assessed with IHC using anti-H3-K27M and H3K27me3 antibodies. For H3-K27M mutation-specific antibody, intense nuclear staining (3+) in more than 80% of tumor cells and negative staining in normal cellular components such as endothelial cells, inflammatory cells, and entrapped neurons is taken as positive.,,, Pale staining cells interpreted as 1+/2+ or granular cytoplasmic staining is considered nonspecific. For H3 K27me3, nonneoplastic cellular components should be nuclear positive, and the tumor nuclei should be negative. IHC for H3K27me3, should only be used in conjunction with H3-K27M IHC, since loss of H3K27me3 expression is by itself not specific for H3-K27M mutation., DMGs that show H3-K27M immunopositivity and H3K27me3 loss are designated as DMG, H3-K27M mutant
- H3 wild-type cases showing H3K27me3 loss should be assessed for EZHIP expression by IHC and if positive should be designated as DMG, H3wildtype with EZHIP overexpression. Strong nuclear staining in >80% tumor cells is interpreted as positive for EZHIP,,
- The H3 wild-type case showing H3K27me3 loss but negative EZHIP expression can be reported as either DMG, NOS or can be assessed for EGFR mutation by DNA sequencing if resources permit. If EGFR mutation is detected the tumor should be designated as DMG, EGFR mutant.
Where feasible, the H3-wild type DMG lacking the above alterations could be considered for testing with other molecular markers such as BRAFV600E, Mismatch repair genes, MYCN, RTK's, etc., as this might identify potential prognostic and therapeutic markers. Considering the limitations in resource-constrained settings, DNA sequencing to identify rare molecular alterations in pHGGs is optional and not mandatory.
Hemispheric high-grade gliomas
- All hemispheric HGGs must be initially subjected to IHC for OLIG2, ATRX, and P53. OLIG2 nuclear positivity in at least 1% of the tumor cells is considered positive. ATRX mutation is considered where tumor cells are immunonegative for ATRX protein, and positivity is seen in native glial/microglial cells, neurons, inflammatory cells, and endothelial cells. On IHC, p53 protein accumulation is detected as strong positive staining of the nuclei and serves as a surrogate marker for the detection of the mutation
- Cases immunonegative for OLIG2 and ATRX and/or immunopositive for p53 expression should be assessed for H3G34R and H3G34V mutant protein-specific antibodies. G34R/V mutation is considered positive if >5% tumor cells show nuclear staining associated with the negativity of the control endothelial cell nuclei. H3G34R/V positive tumors should be entitled as Diffuse hemispheric glioma, H3G34-Mutant
- In H3G34R/V negative cases, IDH1 R132H IHC should be performed to segregate glioma into IDH1 (R132H) mutant and wildtype subgroups. For IDH1, combined cytoplasmic and nuclear staining in >10% of tumor cells should be interpreted as immunopositive
- Testing IDH1 R132H wildtype cases for other rare molecular markers can be considered wherever feasible in accordance with the age of the patient (infant or child). Else, the tumor may be designated as diffuse pediatric-type HGG/infant-type Hemispheric glioma, H3wt, NOS.
The simplified algorithm for the approach to the classification of pediatric HGGs in a resource-limited setup is depicted in [Figure 1].
|Figure 1: Molecular testing decision tree for pediatric high grade gliomas|
Click here to view
Pediatric low-grade gliomas
- Molecular characterization of majority of pLGGs can be performed by simple and robust tests to detect BRAF fusions (FISH or RT PCR) and BRAF V600E mutation (IHC). KIAA1549-BRAF assay requires minimum cut-off value of 10%–15% fusion-positive tumor cells., BRAF V600E immunoreactivity is characterized by intense cytoplasmic stain, with somewhat granular appearance. Focal/weak immunostaining should be interpreted as nonspecific and negative
- BRAF V600E mutant gliomas like PXA or PAs with atypical features should be assessed for CDKN2A deletion (FISH)
- IHC for IDHR132H, ATRX, P53, H3-K27M, and H3G34R/V mutations should be performed in cases with DA or diffuse oligodendroglial like histology
- Based upon histologic features alone, or if molecular analysis fails to discover one of the enlisted genetic alterations, the default diagnosis would be “DA, NOS/NEC” or “Oligodendroglioma, NOS/NEC.”
A possible testing strategy highlighting the most probable molecular alterations in pLGG is depicted in [Figure 2].
|Figure 2: Molecular testing decision tree for pediatric low grade gliomas|
Click here to view
Prognostic significance: Distinct biological risk group stratification
A comprehensive risk-based classification has been proposed which integrates molecular profiling data with clinical information such as demographics and outcomes. In a recent study, molecular-based risk stratification for pLGGs was proposed by Ryall et al. pLGG harboring gene fusions or germline NF1 mutations comprise the low-risk group with an OS of 98%. These tumors require conservative management as therapy may carry higher long-term morbidity than the tumor itself. The intermediate-risk group of pLGG includes tumors with BRAF V600E mutation without CDKN2A deletion, FGFR1 single nucleotide variation (SNV), IDH1 p. R132H, or MET mutations. Intermediate-risk tumors had a 10-year PFS and OS of 35% and 90%, respectively. These patients may, therefore, require multiple treatment courses and longer-term follow-up. High-risk pLGG include those with H3.3 K27M or BRAF V600E mutation with CDKN2A deletion. These tumors need aggressive treatment and the introduction of novel, targeted agents. They invariably progress (10-year PFS of 0%) and these patients often succumb to their disease (10-year OS of 41%). Integrated genetic and epigenetic characterization allowed delineation of pHGGs into a high-risk group based upon K27M mutation and/or amplification of PDGFRA, MYCN, etc., and an intermediate group enriched for G34R/V and IDH1 mutations. Low-risk group comprise PXA/LGG like tumors harboring BRAF V600E mutation frequently co-segregating with CDKN2A deletion. Lastly, there are nonH3-, non-IDH1, non-BRAF mutated tumors with remarkably stable genome profiles, for whom prognosis remains elusive.,
The role of precision diagnostics and therapies in neuro-oncology is rapidly evolving with several clinical trials utilizing the specific molecular alterations of pediatric brain tumors for novel therapeutic strategies. Molecular targeted clinical trials in BRAFV600E mutated LGGs and/or relapsed/refractory HGG (Trametinib, MEK inhibitor in combination with Dabrafenib/Vemurafenib, BRAF inhibitor), recurrent or refractory LGGs (Selumetinib MEK1/2 inhibitor), new diagnosed DIPG/recurrent/refractory H3K27M gliomas (Panobinostat, HDAC inhibitors or GDC-0084 targeting PI3K/Akt/mTOR), IDH1/2 mutant Gliomas (BGB-290 + TMZ targeting PARP) have uncovered new therapeutic options to improve patient OS and reduce long-term side effects.,,,, Molecular inhibitors of FGFR like AZD4547 (NCT02824133) have also been developed and are in clinical trials for an array of malignancies. Treatment with targeted kinase inhibitors (EGFR inhibitors) in bithalamic gliomas manifested encouraging results. Majority (75%) of H3G34R/V mutant tumors show MGMT promoter methylation, which is rare in other pHGGs subgroups, implying a likely high efficacy of temozolomide-based therapy (or other alkylating agents).
| Conclusion|| |
The identification of driver alterations in pediatric gliomas has highlighted their biological and clinicopathological heterogeneity and opened new therapeutic avenues. Keeping pace with recent advances, molecular markers will now be incorporated as diagnostic and prognostic indicators in the updated WHO 2021 classification. However, it is not possible for laboratories in a developing country to utilize high throughput expensive techniques on all cases. We have here reviewed common molecular alterations found in pediatric gliomas and provided the guidelines for using cost-effective and robust molecular genetic tests in routine clinical practice for an accurate diagnosis. Rich and useful data will be obtained with careful morphologic analysis, judicious use of immunohistochemical stains, FISH and Sanger sequencing technologies, which are accessible at most institutions and will give adequate classification of most pediatric gliomas. This complex molecular data will be essential for patient stratification in future clinical trials and will further help to develop new efficient targeted therapies for these tumors.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]