This was a retrospective observational study performed at a tertiary referral center conforming to the institutional research protocols. We searched our hospital's database retrospectively for the gliomas involving various midline structures of the brain, including the septal region, hypothalamus, thalamus brainstem, cerebellum, middle cerebellar peduncles, and pineal region from June 2016 to April 2020.
Patient cohort
The cases included in the study had, i) MRI suggestive of DMG, ii) who had not received any therapy or had not undergone biopsy or surgical resection before the MRI examination, and
iii) for whom the histopathology and immunohistochemistry (IHC) reports were available. All the patients underwent stereotactic biopsy or surgical resection within one month of the MRI. The exclusion criteria were: 1. patients with cerebral hemispheric mass lesions; 2. patients with spinal cord lesions; 3. those subjects where imaging or HPE was suggestive of WHO grade I pilocytic astrocytomas or tumor subtypes other than glioma; 4. inconclusive biopsy; 5. biopsy-proven diffuse midline gliomas with no MRI study; and 6. immunohistochemistry for H3K27M mutation status unavailable. A total of 123 consecutive cases fulfilled the inclusion criteria and were finally enrolled in the study, of which 61 patients had mutant DMGs (age (years) = 24.13+13.13), and 62 patients had WT DMGs (age = 35.79+18.74) (in Online Resource 1).
Histopathology and Immunohistochemistry
Ninety-seven of 123 patients underwent surgical resection, and 26 patients underwent stereotactic biopsy. All the tumors were reviewed by the Neuropathologist and were histologically categorized as phenotypic low-grade diffuse astrocytomas (grade II), anaplastic astrocytomas (grade III), glioblastomas (grade IV) and diffuse midline gliomas (grade IV). For IHC, formalin- fixed paraffin-embedded sections (4 µm) from the blocks were collected on Silane coated slides. IHC was performed using the Ventana Benchmark automated staining system (Ventana Benchmark-XT). Briefly, the sections were subjected to antigen retrieval followed by incubation with primary and then secondary antibody. Counterstaining was done with hematoxylin. The antibodies used were as follows: H3K27me3 (Millipore, 07-449; 1:100) (H3.3K27Mme3, Malaysia, RM192, 1:100); Anti-mIDH1 R132H (dilution 1:50, internal clone H06, Dianova, Hamburg, Germany); Sigma polyclonal anti ATRX antibody in 1:100 dilutions; and DAKO P53 antibody in 1:200 dilutions, D07 clone. Appropriate positive or negative controls were included in each batch of staining. The neuroradiologists were blinded to the pathology results of the cases
Image acquisition
MRI was performed on the 1.5 (Aera 1.5 T, Siemens Medical Systems, Erlangen, Germany) or 3.0 (Achieva 3T, Philips Medical Systems, Best, Netherlands) Tesla MR Scanners using 32 channel head coil as per standard operating procedure with/without sedation. MRI protocol included the following sequences: T2-weighted Turbo spin-echo axial and coronal (TR/TE: 3000-4900/80-99 ms), T1-weighted spin-echo axial (TR/TE: 500-600/10 ms), fluid- attenuated inversion recovery (FLAIR) (TR/TE: 9000-11000/ 87-125ms, IR delay- 2500-28003 ms) and SWI (susceptibility-weighted imaging)/ Venobold (TR/TE: 31-49/7.2-30 ms), prior to contrast administration with 512 x 512 matrix size, 5mm slice thickness and 1 mm inter-slice gap. Post-contrast 3D T1 MPRAGE (Magnetization prepared rapid gradient echo) sequence was acquired after intravenous administration of gadolinium-based contrast agent (Gadovist (Gadobutrol)- 0.1 mmol/kg): (256 x 256) matrix size, 1 x 1 x1mm3 in-plane resolution, TR/TE=6.7/3 ms (3T) and 2200/2.6 ms (1.5T). Intratumoral susceptibility signal (ITSS) scoring was also done on the Venobold and SWI images.
Qualitative imaging features
All the MRI scans were reviewed on picture archiving and communication system (PACS) by two independent neuroradiologists. Our analysis adopted the broad range of imaging attributes
as laid out in the VASARI dataset for a detailed characterization of the lesions on conventional MRI. The following parameters were evaluated: tumor location, side of lesion center, involvement of eloquent brain, enhancement quality, proportion contrast-enhancing tumor (CET), proportion non-contrast enhancing tumor (NCET), proportion necrosis, cysts, multifocal or multicentric or gliomatosis pattern, T1/FLAIR ratio, thickness of enhancing margin (maximum thickness), definition of the enhancing margin, definition of the non-enhancing margin, proportion of edema, hemorrhage, pial invasion, ependymal extension, cortical involvement, deep white matter invasion, NCET crosses midline, CET crosses midline and presence of satellites lesions as per the definition [12]. Few parameters like overall tumor margin and presence of exophytic component, hydrocephalus, and mass effect were also evaluated other than the VASARI feature set.
ITSS scoring was also performed as specified by Park et al. [15]. ITSS was defined as low signal tubular or dot-like structures with or without conglomeration within the tumor in high- resolution SWI. ITSS was divided into 4 grades: grade 0 (No ITSS); grade 1 (1-5 dot-like or tubular ITSS); grade 2 (6-10 dot-like or tubular ITSS), and grade 3 (> 11 dot-like or tubular ITSS).
Intergroup analysis of various features was done between overall mutant and WT DMGs irrespective of tumor grade and location. Furthermore, subgroup analysis for thalamic, brainstem, and grade IV mutant and WT DMGs was also performed.
Statistical analysis
Data was collated offline in a Microsoft Excel 2007 spreadsheet in a de-identified manner. The analysis was conducted using R software version 3.5.2. Interval scale data were presented as means and standard deviations, and nominal scale data are presented as frequencies and percentages. Between-group analysis of interval scale data was conducted using the non- parametric Mann-Whitney U test. Normality of within-group data was observed qualitatively using histograms, and for conformity of analysis, non-parametric methods were chosen. Between-group analysis of nominal scale data was conducted using a Chi-square test with or without Yate's correction as appropriate. A P-value of < 0.05 was considered statistically significant.