Consecutive adult patients diagnosed with anaplastic glioma and referred to the Neuro-oncology Multidisciplinary Tumour Board at the Northern Sydney Cancer Centre were entered into a prospective Anaplastic Glioma Database, approved by Institutional Ethics Review Board. All methods were performed in accordance with guidelines and regulations as per the Northern Sydney Local Health District Human Research Ethics Committee .
Eligible patients for this study were those patients in the Anaplastic Glioma Database with an IDH-mutated tumour with extension into the insular region, and managed with IMRT between January 2008 and June 2019. In the database, histopathological classification was initially recorded as anaplastic astrocytoma (AA), anaplastic oligoastrocytoma (AOA) or anaplastic oligodendroglioma (AOD) based on WHO 2007 Classification . Molecular factors such as 1p19q codeletion, IDH1/2 mutation (evaluated by immunohistochemical and pyrosequencing techniques) and ATRX mutation were recorded where available. The availability of these results varied over the years as new molecular pathology techniques were sequentially introduced into clinical practice .
Patients had histopathology subsequently updated as per the WHO 2016 Classification ; and included those with recently diagnosed WHO Grade III Pathology or patients with known WHO Grade II Pathology from prior diagnosis but had recent radiological or metabolic progression consistent with anaplastic change. The latter required imaging criteria of progressive MRI T1 radiological abnormality within a six-month period, in addition to either new gadolinium enhancement on MRI or FDG uptake in absence of enhancement. These progressive IDH mutated low grade tumours were presumed to have an equivalent natural history to anaplastic glioma  and were included in the study.
Gadolinium-enhanced 3T MRI was the principal diagnostic procedure at initial diagnosis and RT planning. Tumour was delineated on T1 and T2Flair sequences. Presence of contrast enhancement was recorded as absent, patchy (<10 mm) or diffuse regions (>10 mm) .
Nuclear medicine imaging with combined FET and FDG-PET was commenced in 2011 and subsequently performed routinely on all patients with IDH mutation. The FET studies were acquired on a Siemens Biograph mCT PET/CT scanner with extended axial field of view and time of flight (ToF) imaging capability; with 20 minute dynamic and 10 minute static acquisitions following a 3 minute FET infusion period.
An insular tumour for this study was defined as having MRI evidence of infiltration into the gyri of the insular cortex. The insular tumours were then classified radiologically as per the Berger-Sanai Classification  with involvement of regions classifying the tumours as occupying Zones I-IV with a further category (Zone V) classifying giant tumours that occupy all zones. This classification reflects the geographical site of tumour within the insular region, related superior and inferior to the line of the Sylvian fissure; and anterior/posterior to a perpendicular plane crossing the foramen of Monro. Patients were described to the zones involved as well as the major zone involved.
Patients were referred from a number of neurosurgical centres with varying approaches to the extent of surgical intervention and timing of IMRT for anaplastic glioma. Data was recorded regarding the number of salvage craniotomies, the time from initial surgery to radiation therapy and the extent of gross residual disease on MRI T1 and T2Flair sequences prior to IMRT .
Radiation Therapy Management
IMRT was utilised for all patients and management was through one clinician. The target volume defined by preoperative and postoperative MRI and importantly any historical MRI imaging, especially for patients with progressive disease after prior surveillance was imported for assistance with target volume determination. FDG-PET and FET-PET were utilised where available for target volume determination .
Prior to 2011, the tumour was defined as a gross tumour volume (GTV) for each imaging modality and separate T1, T2Flair, FDG-PET and FET-PET GTVs were delineated. These were combined to produce a final GTV, expanded by 10 mm to a clinical target volume (CTV) and then a further 3 mm to a planning target volume (PTV). This volume received a dose of 59.4Gy in 33 fractions and delivered over a period of 6-7 weeks.
From 2011 with the commencement of routine IDH mutation testing and the recognition of a potential favourable subgroup of patients with long-term survival, a new protocol was commenced for patients with IDH mutation . This incorporated an IMRT integrated boost with two dose levels 59.4Gy and 54Gy defined both by MRI and PET with FDG or FET tracers. The high dose region (GTV59.4) encompassed any areas of gadolinium enhancement, T1 density or FDG uptake. The lower dose region (GTV54) included this volume, as well as region of T2Flair and FET uptake outside of this volume. Patients with wild-type IDH tumours on both immunohistochemistry and subsequent protein sequencing were managed with a single-phase treatment to 59.4Gy as per the EORTC CATNON Protocol [15,20].
Systemic Therapy Management
The protocol for systemic therapy evolved over the duration of study with the addition of adjuvant temozolomide commencing in early 2012 following release of long-term results of the RTOG and EORTC studies showing improved median survival for patients with oligodendroglial tumours receiving adjuvant chemotherapy [21,22]. This was extrapolated to the cohort of patients with IDH mutation rather than just those oligodendroglial tumours with 1p19q co-deletion .
Patients were evaluated at one month post-IMRT with MRI and then commenced a surveillance programme with 3 monthly MRI for years 1-2, then 4 monthly in year 3 followed by 6 monthly until year 5 .
MRI Tumour Volume Calculation
Measurements were performed by the lead author, an advanced trainee radiation oncologist. The volume of residual tumour pre-RT was calculated in cm3 by contouring the complete volume on MRI fused to the simulation CT scan in the treatment planning system. Surgical cavity was excluded in selection of the maximum dimension in any plane. This was conducted on T1 and T2Flair image sequences (Figure 1). Any uncertainty over volume delineation was clarified with a neuroradiologist .
At set time points for study evaluation, three months (month+3) and twelve months (month+12) following completion of RT, the same procedure was undertaken on these images fused into the radiation therapy treatment planning system (Figure 2).
Additionally, the presence of contrast enhancement was recorded from T1 with gadolinium sequences and categorised as absent, patchy (<10 mm) or diffuse regions (>10 mm). Where measurable, the volume of the largest contrast enhancing area was calculated at each time point.
The primary endpoint was change in the MRI tumour volume as measured on both T1 and T2Flair sequences performed at 12 months post-IMRT completion. A secondary endpoint was the similar procedure at 3 months post-RT completion. The volumetric endpoint was calculated as a median reduction for the total group; as well as the median of the volume reductions experienced by each patient.
Relapse-free survival (as measured from date of commencement of RT to date of relapse or last follow-up) was analysed as a measure of treatment efficacy. Relapse was defined using RANO Criteria post 2012 . For patients within 18 months of completion of IMRT, in which uncertainty existed whether the MRI changes were related to tumour or treatment effect, sequential MRIs were utilised to confirm diagnosis but date of relapse was taken from initial MRI if subsequently confirmed. The relapse site was categorised in relation to the IMRT defined GTV (CTV59.4) and defined as: Infield (>50% within CTV59.4/54); Marginal (>50% within 20 mm of CTV59.4/54); Distant (>50% beyond 20 mm from CTV59.4/54); or Combined (synchronous sites of relapse). Additionally, involvement of ventricular system was reported if a distant relapse was evident.
No detailed acute or late toxicity criteria were assessed for this study; however a measurement of longer performance status and impact of therapy were explored using functional outcome measures obtained at month +12 and month +60 post-IMRT. These were change in Eastern Co-operative Oncology Group Scale (ECOG) as well as change in Employment Status from baseline.
All patients had clinical and MRI volume data entered on an Excel database at Northern Sydney Cancer Centre and updated for outcome events. Kaplan-Meier estimates of survival distribution were used to calculate relapse-free survival. Volume reductions were calculated at each time point from baseline as both the central tendency measured as median score; and the dispersion of data by the interquartile range (q1-3). Log-rank test was used to investigate associations between the volume reduction and potential predictive factors. All reported p-values are two-tailed. Statistical significance was defined as p<0.05 in all cases. IBM SPSS Statistics Version 23 was used for statistical analysis .