A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma

Low-passage, serum-free cell lines cultured from patient tumour tissue are the gold-standard for preclinical studies and cellular investigations of glioblastoma (GBM) biology, yet entrenched, poorly-representative cell line models are still widely used, compromising the significance of much GBM research. We submit that greater adoption of these critical resources will be promoted by the provision of a suitably-sized, meaningfully-described reference collection along with appropriate tools for working with them. Consequently, we present a curated panel of 12 readily-usable, genetically-diverse, tumourigenic, patient-derived, low-passage, serum-free cell lines representing the spectrum of molecular subtypes of IDH-wildtype GBM along with their detailed phenotypic characterisation plus a bespoke set of lentiviral plasmids for bioluminescent/fluorescent labelling, gene expression and CRISPR/Cas9-mediated gene inactivation. The cell lines and all accompanying data are readily-accessible via a single website, Q-Cell (qimrberghofer.edu.au/q-cell/) and all plasmids are available from Addgene. These resources should prove valuable to investigators seeking readily-usable, well-characterised, clinically-relevant, gold-standard models of GBM.


HISTORY
Frozen section: Right frontal/temporal lesion. History of expressive dysphasia. MACROSCOPIC 1. The specimen jar is labelled Right frontal parietal tumour and consists of soft gelatinous tissue measuring 10mm in extent. Frozen section remanent (1A); remainder of the tissue blocked in toto in (1B).

2.
The specimen jar is labelled Right frontal parietal tumour and consists of two grey pink soft pieces of tissue, the larger measuring 40×25×15mm and the smaller measuring 12×7×5mm. Reserved tissue blocked in (2A -2F).

MICROSCOPIC Right frontal/parietal tumour:
The cerebral cortex tumour is a primary glial tumour showing the histomorphology of a glioblastoma multiforme. The tumour presents has a mass lesion with a relatively circumscribed margin. The tumour is not a metastatic malignancy as was suggested in the frozen section report.

MACROSCOPIC
1. The container is labelled R Frontal lesion. The specimen consists of pale areas of tissue measuring 10×8×2mm for smear and frozen section.
Frozen section diagnosis: Necrosis with adjacent high grade glioma.

2.
The second specimen is labelled Right frontal tumour. The specimen consists of a piece of brain measuring 60×55×50mm. The gyri look flattened and there are some excrescences on the surface. The cut surface looks largely necrotic and there are some clots within the brain parenchyma. There is also expansion of the white matter adjacent to the necrotic margin focally with thinning of the overlying grey matter and blurring of the white grey junction.

MICROSCOPIC
Paraffin sections confirm the frozen section diagnosis of a high grade glioma. The tumour exhibits features of a Glioblastoma multiforme. Areas with small cell morphology are present and there is prominent serpiginous palisaded necrosis and areas of vascular endothelial proliferation.

SUMMARY
Right frontal lobe resection: Glioblastoma multiforme: WHO grade IV.

4.
The specimen container is labelled brain tumour: The specimen consists of a single piece of irregularly shaped haemorrhagic soft tissue measuring 33×27×14mm. The external surface is inked blue. The specimen is entirely embedded as serial transverse sections (4A to 4D). Sectioning reveals a heterogeneous cut surface with firm cream areas interspersed with gelatinous areas.

5.
The specimen container is labelled right temporal lobe: the specimen consists of two portions of tissue. The specimen weights 17.7g. One appears to represent a portion of external brain tissue with vascular channels, gyri and sulci. This part of the specimen measures 42×42mm by up to 23mm. The apposing surface is irregularly roughened and haemorrhagic. There is also a second small piece of tissue in the specimen container measuring 20×8×5mm. The smaller piece inked bisected, all embedded (5A), the larger piece of tissue is photographed, inked blue and divided into serial transverse sections and all embedded from one side to the other as per the photograph. What is possible false cavity margin is inked black (5B to 5L). (5E, 5F, 5I & 5G) each contain piece bisected. Refer to photograph for blocking information. Specimen has been entirely embedded.

6.
The specimen container is labelled abnormal bone: The specimen consists of a single fragment of bone measuring 10x5x5mm. Specimen is inked and blocked in toto (6A).

-4. Brain tumour:
There is a high grade glial tumour composed of anaplastic, highly atypical round and spindly glial cells. Tumour cells show remarkable nuclear enlargement, nuclear hyperchromasia and nuclear pleomorphism together with increased mitotic rate, multi-nucleated tumour cells with bizarre nuclei and also karhyorectic cells. There is extensive coagulative tumour necrosis in areas with characteristic palisading necrosis. There is microvascular proliferation with glomeruloid structures. Tumour cells show positive reaction for GFAP and mild focal positivity for cytokeratin. The features are in keeping with glioblastoma multiforme (WHO Grade IV).

Right temporal lobe:
the specimen is composed of brain tissue infiltrated by a Grade 2 diffused astrocytoma which shows transition to anaplastic astrocytoma (WHO Grade 3) and glioblastoma (WHO Grade 4). The later features suggest differentiation of a Grade 2 astrocytoma.

Abnormal bone:
There is compact cortical bone with no evidence of tumour invasion. SUMMARY 1 -6. Brain tumour: Glioblastoma multiforme (WHO Grade IV).

MN1 -Primary Glioblastoma
Pathology report: HISTORY Two request forms and two frozen sections forms.
The first request form reads: Frozen section L frontal lobe lesion. Presents with expressive dysphasia. The surgeon gives a history of colorectal carcinoma.
There is another request form which reads: Formal histology left frontal lobe lesion. Presents with expressive dysphasia. MACROSCOPIC 1. The first frozen section reads: Container labelled Brain tumour. The specimen consists of a piece of cream to pink soft tissue measuring 5×4×3mm. Smear x1 and remaining tissue for frozen section. (1A) frozen section remnant.
Frozen section diagnosis: Glioma, favour high grade.

2.
There is a second frozen section form which reads: Container labelled Brain lesion number 2. The specimen consists of three to four pieces of cream to pink tissue measuring 2-3mm each. Smear x 1 and one piece of frozen section. (2A) frozen section remnant of specimen 2; (2B) remainder of the tissue in specimen 2.

Frozen section diagnosis: Glioma.
3. The specimen is labelled Brain lesion and consists of fragments of brain tissue measuring 13×12×10mm in aggregate. Wrapped and blocked in toto in (3A).
4. The specimen in labelled Brain tumour deep margin and consists of four fragments of pale tissue, in aggregate measuring 8x7x3mm. Wrapped and blocked in toto in (4A).

2. & 3. Brain tumour:
The paraffin sections confirm the frozen section diagnosis of high-grade diffusely infiltrating glioma. The tumour cells appear astocytic and are relatively monomorphous with focal perivascular orientation of tumour cells. There evidence of palisaded necrosis and frequent mitoses are present in some areas. Vascular endothelial proliferation is also present. The features are consistent with a glioblastoma.

SUMMARY
Left frontal lobe brain tumour: Glioblastoma (WHO grade IV).

HISTORY
Frozen section. Left temporal lobe tumour.

MACROSCOPIC
1. The specimen is labelled Brain tumour. The specimen consists of two fragments of cream to pink tissue measuring in aggregate 5×4×2mm. Two smears are prepared and one rep, section per piece for frozen section. Block (1A) frozen section remnants; (1B) remaining tissue. Specimen blocked in toto.

2.
The specimen is labelled Brain tumour. The specimen consists of multiple fragments of cream to maroon tissue measuring in aggregate 20×10×5mm. The largest fragment measures 12×3×3mm. Blocks (2A) and (2B) specimen wrapped and blocked in toto.

MICROSCOPIC
1. Brain tumour: the paraffin sections confirm the frozen section diagnosis of a high grade glioma. There are large areas of coagulative necrosis which shows pseudo-palisading in one of the two fragments. There is crowding of pleomorphic glial cells with hyperchromatic irregular nuclei. There is focal endothelial proliferation.
2. Brain tumour: some of the fragments show diathermy artefact which limits interpretation, however the appearances are similar to those in Specimen 1 with crowding of pleomorphic glial cells with hyperchromatic irregular nuclei. There is a proliferation of blood vessels of small to medium diameter, some of which show endothelial proliferation. There are focal areas of haemorrhage and calcification, as well as areas of coagulative necrosis.
COMMENT: the appearances in both specimens are consistent with Glioblastoma multiforme, WHO Grade IV.

2.
The specimen is labelled Brain tumour and consists of multiple pieces of light tan tissue measuring 18×12×4mm in aggregate. (2A) specimen blocked in total. MICROSCOPIC 1. and 2. Brain tumour and brain tumour: The sections show a densely cellular proliferation composed of medium-sized to large cells in a background of smaller oligodendrocyte-like cells and fibrillary stroma. There is vascular hyperplasia and areas of necrosis. There are frequent foci of vascular hyperplasia. Mitoses are frequent. There are some areas of degenerative tissue, but no real tumour necrosis. SUMMARY 1. and 2. Brain tumour and brain tumour: the lesion is best regarded as a diffuse infiltrative malignant glioma.
Further studies (p53 and FISH) are pending to allow a better categorisation.

SUPPLEMENTARY REPORT
Thank you for the opportunity to review this case. In some areas this tumour demonstrates oligodendroglioma features with perinuclear halo formation, an arborescent vascular pattern and punctuate calcifications. However, other areas demonstrate astrocytic features with background fibrillar tumour cell processes, perivascular orientation and scattered bizarre giant cells. Frequent mitoses are present and there is evidence of vascular proliferation. Focal necrosis is identified at the edge of one biopsy fragment. Immunohistochemical staining for p53 is normal with only focal positive immunoreactivity.

SUMMARY
Left temporal brain biopsy: Glioblastoma multiforme with oligodendroglioma component; 1p/19q deletions not present; WHO grade IV.