Benchmark

All IHC was performed in an integrated laboratory (Northern Ireland Molecular Pathology Laboratory) that has UK Clinical Pathology Accreditation [39 (link)]. Sections were cut from the whole face or TMA blocks for H&E staining and IHC. The initial H&E section was used to assess TMA quality and appropriate tumour, lymphoid, or stromal content for subsequent IHC localizations and analysis. Sections for IHC were cut at 4 microns on a rotary microtome, dried at 37°C overnight, and then used for IHC that was performed on automated immunostainers (Ventana BenchMark or Leica Bond-Max, Milton Keynes, UK). See Supp Table 2 for primary antibody, pretreatment, and biomarker detection details all of which were optimised prior to application to TMA sections. Subsequently, all sections were visualized with diaminobenzidine, counterstained with haematoxylin, and then dehydrated and tape-mounted using a Sakura autostainer. All slides were scanned on an Aperio AT2 Digital scanner at x40.

Histologic diagnosis of PDOX tumors and matched patient samples was assessed by hematoxylin and eosin stained section by a board-certified neuropathologist (B.A.O.) according to the criteria specified in the WHO Classification of Tumours of the Central Nervous System [23 (link)]. Immunohistochemistry was performed on 4-µm-thick formalin-fixed paraffin embedded sections using automated Ventana Benchmark or Leica Bond III machines with appropriate secondary reagents. Specific antibody clones used are listed in Supplementary Table S1, Online Resource. Dual-color FISH was performed on 4 µm paraffin embedded tissue sections. Probes were derived from BAC clones (BACPAC Resources, Oakland, CA) and labeled with either AlexaFluor-488 or AlexaFluor-555 fluorochromes (Supplementary Table S2, Online Resource). Briefly, probes were co-denatured with the target cells on a slide moat at 90 °C for 12 min. The slides were incubated overnight at 37 °C on a slide moat and then washed in 4 M Urea/2xSSC at 25 °C for 1 min. Nuclei were counterstained with DAPI (200 ng/ml; Vector Labs) for viewing on an Olympus BX51 fluorescence microscope equipped with a 100 watts mercury lamp; FITC, Rhodamine, and DAPI filters; 100× PlanApo (1.40) oil objective; and a Jai CV digital camera. Images were captured and processed using the Cytovision v7.3 software from Leica Biosystems (Richmond, IL).

Immunohistochemical staining was performed on a Benchmark immunohistochemistry staining system (Bond; Leica, Wetzlar, Germany) with BOND polymer refine detection solution for DAB, using anti‐MGMT (1 : 800, abcam, Cambridge, UK, ab39253) primary antibody as substrate as previously described [45 (link)]. Images were acquired using an Olympus BX46 microscope (Shinjuku City, Tokyo, Japan) as previously described. MGMT immunoreactivity was scored semi‐quantitatively by multiplying the proportion of MGMT positive cells (%) and the staining intensity (0 = none; 1 = weak; 2 = intermediate; and 3 = strong). Statistical comparison was performed using paired Wilcoxon test.