Summary: imaging, histology, and molecular testing

Diagnosis combines MRI with histopathology and molecular testing in an integrated, layered report, as endorsed by WHO CNS5. Molecular assays used for pLGG include immunohistochemistry for BRAF p.V600E, fluorescence in situ hybridization, RNA-based multiplex fusion panels (NanoString), next-generation sequencing panels, and DNA methylation profiling. Consensus reviews state that testing must detect structural variants and fusions, copy-number alterations, and single nucleotide variants. In a German population-based cohort of more than 1,200 children, multi-omic integration refined the diagnosis through a DNA methylation class in 50% and detected diagnostic or therapeutically relevant alterations in 47%. In the United States, FoundationOne CDx was approved as a companion diagnostic for OJEMDA in January 2025. Biopsy may be omitted for visual pathway gliomas with characteristic MRI findings, particularly in children with NF1; 115 of 1,031 children (11.2%) in HIT-LGG-1996 and 68 of 497 patients (13.7%) in the SIOP-LGG 2004 randomized trial had no histological verification.

Integrated and layered diagnosis under WHO CNS5

“Such reports feature an integrated diagnosis at the top, followed by layers that display histological, molecular, and other key types of information (Table 4).” (opens the source at this quote in a new tab)

“Another key concept in the 2021 WHO CNS tumor classification is the recommendation to utilize integrated and layered diagnoses including tumor site, a combination of a tissue-based histologic diagnosis and molecular diagnosis, histopathological classification, CNS tumor grade, and molecular information.” (opens the source at this quote in a new tab)

“Given this, a layered diagnostic approach is recommended where both the histologic classification and molecular findings are reported in an integrated diagnosis” (opens the source at this quote in a new tab)

Molecular testing methods: immunohistochemistry, FISH, RNA fusion panels, sequencing, and methylation profiling

“With respect to pLGG, IHC has been faithfully utilized in the detection of BRAF p.V600E” (opens the source at this quote in a new tab)
“FISH is widely available and can be used on FFPE material but is relatively labor-intensive, and can only test for a single alteration at a time.” (opens the source at this quote in a new tab)
“NanoString panels can be used to screen for the common fusions such as those involving BRAF (including both the canonical KIAA1549-BRAF and the non-canonical fusions described above) and FGFR1-TACC1” (opens the source at this quote in a new tab)
“Sequencing based approaches have the benefit of simultaneous detection of most clinically relevant alterations in a single test from which diagnostic, prognostic and therapy decisions can be made.” (opens the source at this quote in a new tab)
“However, the utility of the methylation classifier may be less robust in pLGG, possibly due to frequent inclusion of normal tissue in these tumors” (opens the source at this quote in a new tab)

Consensus recommendations on the scope of molecular testing

“At a minimum, assays need to be designed to detect genetic alterations that are relevant to pLGG. An integrated approach that is sufficient to detect structural variants/copy-number alterations, single nucleotide variants, and expressed fusion proteins to help inform histopathological interpretation is essential.” (opens the source at this quote in a new tab)

“Therefore, comprehensive molecular profiling using platforms capable of detecting SNVs, copy-number changes, and structural variants (including gene fusions and internal tandem duplications [ITDs]) is integral to contemporary practice” (opens the source at this quote in a new tab)

Multi-omic neuropathology in a population-based pediatric cohort

“We show that the multi-omic integration increased diagnostic accuracy in a substantial proportion of patients through annotation to a refining DNA methylation class (50%), detection of diagnostic or therapeutically relevant genetic alterations (47%) or identification of cancer predisposition syndromes (10%).” (opens the source at this quote in a new tab)
“For the most commonly affected gene BRAF (272/1,034), fusion events were observed in 158 of 237 DNA methylation-defined infratentorial (124/160), midline (28/51) or cortical LGG (6/26), whereas V600E mutations were further observed in GG (7/13) and PXA (17/23).” (opens the source at this quote in a new tab)

United States practice: routine genomic profiling and companion diagnostic

“Comprehensive genomic profiling of newly diagnosed or recurrent/progressive pLGG is standard practice within pediatric neuro-oncology programs across the United States, either utilizing CLIA/College of American Pathologists, or CAP, accredited hospital laboratories or third-party commercial vendors.” (opens the source at this quote in a new tab)
“In January 2025, the FDA approved FoundationOne CDx to be used as a companion diagnostic for OJEMDA.” (opens the source at this quote in a new tab)

Diagnosis without biopsy: visual pathway gliomas and NF1

“Initial surgical intervention achieved complete resection in 34.8% of patients, 204 tumors underwent biopsy only (19.8%), and 115 children received a diagnosis of tumor on the basis of neuroradiologic findings (11.2%), with 65 being NF-1.” (opens the source at this quote in a new tab)
“For VP gliomas, MRI criteria combined with clinical information, such as NF-1 status, allowed diagnosis of the presence of an LGG and justified dispensing with a biopsy.” (opens the source at this quote in a new tab)
“although outside the VPs, LGG histology should be confirmed” (opens the source at this quote in a new tab)

“Children with chiasmatic-hypothalamic tumours may be eligible without histological diagnosis, if neuroradiologic findings meet unequivocal criteria for the presence of a low grade glioma.” (opens the source at this quote in a new tab)
“Sixty-eight patients (13.7%) did not have verification biopsy of the LGG, due to unequivocal neuroradiological appearances.” (opens the source at this quote in a new tab)

“In addition, chiasmatic-hypothalamic tumors intrinsic to the optic pathway were eligible without pathologic confirmation.” (opens the source at this quote in a new tab)

“Historically, NF1-pLGG are not biopsied due to their precarious location and the lack of clinical utility of the additional information obtained.” (opens the source at this quote in a new tab)
“Therefore, obtaining a biopsy from, at minimum, patients deemed higher risk, may prove valuable in identifying patients that require refined and/or novel treatments and distinguishing them from NF1-HGG, particularly in adults.” (opens the source at this quote in a new tab)

Molecular testing of midline tumors that are not routinely biopsied

“Of importance, the mutation was common in midline tumors, including optic pathway, brainstem, and spinal cord tumors.” (opens the source at this quote in a new tab)
“For deeply located tumors, such as hypothalamic/chiasmatic LGGs, the need for biopsy before treatment decisions are made for these children is still debated.” (opens the source at this quote in a new tab)

Imaging: MRI standards and response assessment

“RAPNO recommends that baseline brain MRI (or baseline spine MRI depending upon the primary lesion location), be performed pre-operatively and 24–72 h post-operatively, and that the post-operative scan should be utilized as the new baseline for response to systemic (and RT) therapy assessments.” (opens the source at this quote in a new tab)
“RAPNO specifically recommends utilizing T2/FLAIR images to measure a tumor and in assessing radiographic response, as contrast enhancement in pLGG is an unreliable measure of tumor response or progression.” (opens the source at this quote in a new tab)

“When a symptomatic NF1-OPG is suspected, MRI of the brain with dedicated high-resolution sequences of the optic nerves and chiasm is the imaging technique of choice to evaluate the extent of OPG involvement and to monitor for radiographic progression or response.” (opens the source at this quote in a new tab)

European guideline: observation versus treatment after diagnosis

“Following incomplete resection observation is recommended, as long as there is no radiologic tumor growth and the patient does not suffer from significant, tumor-related symptoms. This also applies to patients with a diagnosis of LGG on the basis of radiological criteria. By contrast, clinical worsening and / or radiologic progression are an indication to treatment with either chemo- or radiotherapy.” (opens the source at this quote in a new tab)

Tiered molecular testing pipeline and turnaround time per tier

“Our tiered testing pipeline included three sequential stages, each increasing in cost and TAT as they progress. Tier 1 consisted of IHC. Tier 2 included FISH or an RNA-based targeted gene fusion panel (NanoString nCounter).” (opens the source at this quote in a new tab)
“LGG samples underwent Tier 1 IHC testing to assess BRAF p.V600E and IDH1 p.R132H (the latter if over age 10 years).” (opens the source at this quote in a new tab)
“TAT was defined as the time it takes from the acquisition of the tumor sample until a clinical report is generated in business days. The TAT per test is 2 days, 7 days, and 14 days for Tier 1, Tier 2, and Tier 3, respectively.” (opens the source at this quote in a new tab)

Testing yield in pediatric low-grade glioma and the proportion receiving a targeted agent

“Using our tiered approach, 95% (78 of 82) of LGGs were successfully molecularly characterized.” (opens the source at this quote in a new tab)
“Within the LGG cohort, 95.1% (78 of 82) of patients had a targetable alteration detected using the tiered approach. Interestingly, only 23% (18 of 78) of those received a targeted agent at some point during therapy.” (opens the source at this quote in a new tab)
“Of the remaining 60 patients with a targetable alteration, 30 underwent a curative total resection and were subsequently monitored.” (opens the source at this quote in a new tab)

Yield of a tiered molecular characterization pipeline in pediatric glioma

“Overall, the tiered approach successfully identified a molecular alteration in 96% (95 of 99) of gliomas diagnosed over a 2-year period.” (opens the source at this quote in a new tab)
“Within the LGG cohort, 95.1% (78 of 82) of patients had a targetable alteration detected using the tiered approach.” (opens the source at this quote in a new tab)