Harry R Haynes

EGFR and EGFRvIII analysis in glioblastoma as therapeutic biomarkers

Abstract Introduction. EGFR and EGFRvIII analysis is of current interest because of new EGFRvIII vaccine trials opened in the UK. EGFR activation promotes cellular proliferation via activation of MAPK and PI3K-Akt pathways. EGFRvIII is the most common variant resulting from an in-frame deletion of 801bp, leading to constitutively active EGFR. Method. 51 glioblastoma samples from a cohort of 50 patients were tested for EGFR amplification by FISH and immunohistochemistry and EGFRvIII expression by reverse-transcriptase PCR (RT-PCR), and immunohistochemistry. EGFR and EGFRvIII expression was compared with Overall Survival in the cohort. Results. Overall 22/51 samples (43%) were positive for EGFR, 16/51 (31%) were positive for EGFRvIII and 13/51 (25%) were positive for both. 9/51 cases (18%) were positive for EGFR alone, and 3/51 (6%) were positive for EGFRvIII alone. Of the EGFR positive cases, 22/51 (43%) were positive by FISH, 24/51 (47%) were positive by IHC and 2/51 (4%) were discrepant between methods (positive by IHC but non-amplified by FISH). Of the EGFRvIII positive cases, 16/51 (31%) were positive by RT-PCR, 17/51 (33%) were positive by IHC and 1/51 (2%) sample was discrepant (positive by IHC but not by RT-PCR). Neither EGFRvIII or EGFR are predictive of overall survival in this cohort. Conclusion. In our cohort, 25/51 (49%) of GBM showed EGFR alterations, including 16/51 (31%) with EGFRvIII. There was high concordance between IHC and FISH (96%) and IHC and RT-PCR (98%) as diagnostic methods. Neither EGFR or EGFRvIII is predictive of overall survival in this cohort. These results are key for selecting patients for novel individualised anti-EGFR therapies.

IDH mutation analysis in gliomas as a diagnostic and prognostic biomarker

Abstract Introduction. There is a high rate of IDH1/2 mutations in low grade gliomas and in high grade gliomas deriving from them. IDH analysis of gliomas is a novel method of classification and an independent prognostic marker. We compared antibody and sequencing methods for the detection of IDH mutations. Method. 88 samples from 74 patients were identified. For immunohistochemistry: sections were stained with anti-IDH1R132H antibody. For sequencing: DNA was extracted from fresh, frozen tissue. Results. 28% (20/71) of cases were positive for the R132H IDH1 mutation by antibody. An IDH1 mutation was detected by molecular genetics in 37% (21/57) of cases and no IDH2 mutations were detected. 24% (5/21) had rare IDH1 mutations not detected by immunohistochemistry. Where sufficient tissue was available, immunohistochemistry and DNA analysis were fully concordant for the p.Arg132His mutation. Both Grade II gliomas and anaplastic astrocytomas showed a statistically different distribution of IDH1 mutation load compared to GBMs (p < 0.0001; p = 0.0021 respectively). Conclusion. A rationalised combined approach involving R132H antibody testing and sequencing of negative cases would be ideal for the detection of IDH1 mutations - antibody testing is cheaper than sequencing but sequencing demonstrates rare IDH1 mutations not detected by immunohistochemistry.

Convection enhanced delivery of panobinostat (LBH589)-loaded pluronic nano-micelles prolongs survival in the F98 rat glioma model

Background The pan-histone deacetylase inhibitor panobinostat is a potential therapy for malignant glioma, but it is water insoluble and does not cross the blood–brain barrier when administered systemically. In this article, we describe the in vitro and in vivo efficacy of a novel water-soluble nano-micellar formulation of panobinostat designed for administration by convection enhanced delivery (CED). Materials and methods The in vitro efficacy of panobinostat-loaded nano-micelles against rat F98, human U87-MG and M059K glioma cells and against patient-derived glioma stem cells was measured using a cell viability assay. Nano-micelle distribution in rat brain was analyzed following acute CED using rhodamine-labeled nano-micelles, and toxicity was assayed using immunofluorescent microscopy and synaptophysin enzyme-linked immunosorbent assay. We compared the survival of the bioluminescent syngenic F98/Fischer344 rat glioblastoma model treated by acute CED of panobinostat-loaded nano-micelles with that of untreated and vehicle-only-treated controls. Results Nano-micellar panobinostat is cytotoxic to rat and human glioma cells in vitro in a dose-dependent manner following short-time exposure to drug. Fluorescent rhodamine-labelled nano-micelles distribute with a volume of infusion/volume of distribution (Vi/Vd) ratio of four and five respectively after administration by CED. Administration was not associated with any toxicity when compared to controls. CED of panobinostat-loaded nano-micelles was associated with significantly improved survival when compared to controls (n=8 per group; log-rank test, P<0.001). One hundred percent of treated animals survived the 60-day experimental period and had tumour response on post-mortem histological examination. Conclusion CED of nano-micellar panobinostat represents a potential novel therapeutic option for malignant glioma and warrants translation into the clinic.

Current Understanding of Circulating Tumor Cells - Potential Value in Malignancies of the Central Nervous System.

Detection of circulating tumor cells (CTCs) in the blood via so-called “liquid biopsies” carries enormous clinical potential in malignancies of the central nervous system (CNS) because of the potential to follow disease evolution with a blood test, without the need for repeat neurosurgical procedures with their inherent risk of patient morbidity. To date, studies in non-CNS malignancies, particularly in breast cancer, show increasing reproducibility of detection methods for these rare tumor cells in the circulation. However, no method has yet received full recommendation to use in clinical practice, in part because of lack of a sufficient evidence base regarding clinical utility. In CNS malignancies, one of the main challenges is finding a suitable biomarker for identification of these cells, because automated systems, such as the widely used Cell Search system, are reliant on markers, such as the epithelial cell adhesion molecule, which are not present in CNS tumors. This review examines methods for CTC enrichment and detection, and reviews the progress in non-CNS tumors and the potential for using this technique in human brain tumors.

Diagnosis of primary cerebral lymphomas: possible value of PCR testing in equivocal cases requiring rebiopsy

Abstract Introduction. Rebiopsy rates as high as 12% have been reported in previous studies of Primary Central Nervous System Lymphoma (PCNSL). This can lead to secondary operations, increasing risks of morbidity to the patient and costs for the NHS. Polymerase Chain Reaction (PCR) testing for clonality in haematological malignancies has been applied to cases of lymphoma outwith the central nervous system (CNS), but is less commonly used in the diagnosis of CNS lymphomas. Clonality in B- and T-cell populations may indicate the presence of malignancy. We aimed to identify factors to reduce the rebiopsy rate in PCNSL. Methods. We examined a cohort of 102 suspected cerebral lymphoma cases biopsied at Frenchay Hospital, Bristol over a 10-year period (2000–2010). Clinical data, including age, sex, location, pre-biopsy steroid use, the need for rebiopsy and histological diagnosis, were collected. We retrospectively reviewed rebiopsied cases and they subsequently underwent PCR testing for clonality. Results. Overall, 96/102 (94%) cases achieved a histological diagnosis after one or more biopsies. 81/96 (84%) of these were lymphomas involving the brain and 15/96 (16%) were spinal lymphomas. The majority of these were B-cell lymphomas (95/96 (99%)), with one case of peripheral T-cell lymphoma (1/96 (1%)). Due to insufficient histological evidence of PCNSL after the first biopsy, 9/102 (9%) of cases had required rebiopsy. In 7/9 (78%) of these cases, we undertook PCR testing for clonality on tissue from the first biopsy. We found 3/7 (43%) cases were monoclonal for B or T populations, raising the possibility of PCNSL. Conclusions. We recommend that all CNS lymphoproliferative lesions be assessed by haematopathologists, with the inclusion of PCR testing particularly in equivocal cases. This would reduce the number of patients going for rebiopsy and reduce the patient morbidity and costs for the NHS.

The transcription factor PPARalpha is overexpressed and is associated with a favourable prognosis in IDH-wildtype primary glioblastoma

PPARα agonists are in current clinical use as hypolipidaemic agents and show significant antineoplastic effects in human glioblastoma models. To date however, the expression of PPARα in large‐scale glioblastoma datasets has not been examined. We aimed to investigate the expression of the transcription factor PPARα in primary glioblastoma, the relationship between PPARα expression and patients’ clinicopathological features and other molecular markers associated with gliomagenesis.

Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays

Aims Histopathological tissue samples are being increasingly used as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control central nervous system (CNS) formalin-fixed paraffin embedded (FFPE) tissue-derived RNA for gene expression analyses. Methods Total RNA was extracted from control (temporal lobe resection tissue) and glioblastoma FFPE tissue samples. RNA purity (260/280 ratios) was determined and RNA integrity number (RIN) analysis was performed. RNA was subsequently used for RT-qPCR for two reference genes, 18S and GAPDH. Results Reference gene expression was equivalent between control and glioblastoma tissue when using RNA extracted from FFPE tissue, which has key implications for biological normalisation for CNS gene expression studies. There was a significant difference between the mean RIN values of control and glioblastoma FFPE tissue. There was no significant correlation between 260/280 or RIN values versus total RNA yield. The age of the tissue blocks did not influence RNA yield, fragmentation or purity. There was no significant correlation between RIN or 260/280 ratios and mean qPCR cycle threshold for either reference gene. Conclusions This study showed that routinely available CNS FFPE tissue is suitable for RNA extraction and downstream gene expression studies, even after 60 months of storage. Substantial RNA fragmentation associated with glioblastoma and control FFPE tissue blocks did not preclude downstream RT-qPCR gene expression analyses. Cross validation with both archival and prospectively collated FFPE specimens is required to further demonstrate that CNS tissue blocks can be used in novel translational molecular biomarker studies.

Bone marrow transplantation stimulates neural repair in Friedreich's ataxia mice

Friedreichs ataxia is an incurable inherited neurological disease caused by frataxin deficiency. Here, we report the neuroreparative effects of myeloablative allogeneic bone marrow transplantation in a humanized murine model of the disease.

Isocitrate dehydrogenase mutation analysis in gliomas as a diagnostic and prognostic biomarker

Abstract Background There is a high rate of isocitrate dehydrogenase (IDH) 1 and 2 mutations in low grade gliomas and in high grade gliomas derived from them. IDH analysis of gliomas is a novel adjunct to traditional classification and an independent prognostic marker. We compared antibody and sequencing methods for the detection of IDH mutations. Methods 88 samples from 74 patients were identified: 16 patients had WHO grade II gliomas, 30 had WHO grade III gliomas, and 28 had WHO grade IV glioblastoma multiforme (GBM). 31 samples had insufficient material available for DNA extraction. For immunohistochemistry, sections were stained with anti-IDH1R132H antibody. For sequencing, DNA was extracted from fresh, frozen tissue. Findings 20 (28%) of 72 patients were positive for the R132H IDH1 mutation by antibody. An IDH1 mutation was detected by molecular genetics in 21 (37%) of 57 patients, and no IDH2 mutations were detected (6% expected from previous studies). 5 (24%) of 21 patients had rare IDH1 mutations not detected by immunohistochemistry (7% expected). Three of these patients displayed the p.Arg132Cys mutation (two anaplastic astrocytomas, one fibrillary astrocytoma) and two displayed p.Arg132Gly (one anaplastic astrocytoma, one anaplastic oligoastrocytoma). Where sufficient tissue was available, immunohistochemistry and DNA analysis were fully concordant for the p.Arg132His mutation. We found a high rate of IDH1 mutations in lower grade lesions (WHO grade II and III) (54% [25/46]) and a low rate in GBMs (7% [2/28]). Both grade II gliomas and anaplastic astrocytomas showed a statistically different distribution of IDH1 mutation load compared with GBMs (p Interpretation A rationalised combined approach involving R132H antibody testing and sequencing of negative cases would be ideal for the detection of IDH1 mutations. Antibody testing is cheaper than sequencing but sequencing demonstrates rare IDH1 mutations not detected by immunohistochemistry. Funding Brain Tumour Bank Frenchay, British Neuropathological Society, and Brain Tumour Action.

A case of chronic asymptomatic central pontine myelinolysis with histological evidence of remyelination

Central pontine myelinolysis (CPM) is a neurological demyelinating disease of the pons. Although usually associated with rapid correction of hyponatremia, CPM may occur despite normonatremia, is often associated with chronic alcoholism and may be asymptomatic. Histological confirmation of asymptomatic CPM is rare. We describe an unusual post-mortem case of extensive but asymptomatic CPM in a chronic alcoholic patient with normonatremia. The affected part of the pons contained thinly myelinated axons with appearances supporting remyelination. We suggest that remyelination may account for the subclinical nature of this patients CPM.