Carol Walker

Extent of MGMT promoter methylation correlates with outcome in glioblastomas given temozolomide and radiotherapy

Background:Epigenetic silencing of O6-methylguanine-DNA-methyltransferase (MGMT) by promoter methylation is associated with improved survival in glioblastomas treated with alkylating agents. In this study, we investigated MGMT promoter methylation in glioblastomas treated with temozolomide and radiotherapy in a single UK treatment centre.Methods:Quantitative methylation data at individual CpG sites were obtained by pyrosequencing for 109 glioblastomas.Results:Median overall survival (OS) was 12.4 months with 2-year survival of 17.9%. Pyrosequencing data were reproducible with archival samples yielding data for all glioblastomas. Variation in methylation patterns of discrete CpG sites and intratumoral methylation heterogeneity were observed. A total of 58 out of 109 glioblastomas showed average methylation >non-neoplastic brain in at least one clinical sample; 86% had homogeneous methylation status in multiple samples. Methylation was an independent prognostic factor associated with prolonged progression-free survival (PFS) and OS. Cases with methylation more than 35% had the longest survival (median PFS 19.2; OS 26.2 months, 2-year survival of 59.7%). Significant differences in PFS were seen between those with intermediate or high methylation and unmethylated cases, whereas cases with low, intermediate or high methylation all showed significantly different OS.Conclusions:These data indicate that MGMT methylation is prognostically significant in glioblastomas given chemoradiotherapy in the routine clinic; furthermore, the extent of methylation may be used to provide additional prognostic stratification.

Oestrogen receptor expression in the normal and pre-cancerous breast

As oestrogen is associated with most of the epidemiological risk factors for breast cancer, the number and distribution of oestrogen receptor positive (ER+) cells could have a bearing on the development of the disease. ER+ cells were thus studied in the normal breast and in the spectrum of in situ proliferations which range from non‐atypical hyperplasia to in situ carcinoma and are associated with different levels of risk for developing breast cancer. In the normal pre‐menopausal breast, ER+ cells comprised the minority and were distributed singly, being surrounded by oestrogen receptor negative (ER−) cells. ER+ cells showed a statistically significant increase with age, reaching a plateau after the menopause, and the increase was associated with a tendency for positive cells to become contiguous in patches of variable size. A small proportion of lobules showing involutional change comprised over 90 per cent ER+ cells. The significance of this feature is not clear but no evidence was found that it was pre‐cancerous. The percentage of ER+ cells was slightly increased in hyperplasia of usual type (non‐atypical hyperplasia, HUT) and the relationship to age was maintained. The staining pattern was variable; in some lesions ER+ cells were surrounded by ER− cells whereas in others there were contiguous groups of positive cells sometimes accounting for more than 90 per cent of cells in the lesion. In contrast, all cases of atypical ductal hyperplasia (ADH), lobular in situ neoplasia (LIN) and ductal carcinoma in situ (DCIS) exhibited positivity of contiguous cells accounting for the majority in the lesions. Furthermore, the relationship between ER+ cell numbers and age was lost in these lesions, indicating autonomy of ER expression or of proliferation of cells expressing the receptor. It is hypothesized that this dysregulation of receptor expression or of ER+ cell numbers at the ADH stage may be the precursor of abnormal expression of cyclins and other cell cycle control proteins which have been shown first to appear in DCIS. Copyright

Neutrophil infiltration into human gliomas

Abstract Human gliomas were analysed for the infiltration of neutrophils using immunohistochemistry by staining sections for CD15-positive and myeloperoxidase-positive cells. Over 70% of all glioma samples analysed (n = 105) had significant neutrophil infiltration, but there was a marked and significant correlation between tumour grade and the extent of the neutrophil infiltration. In the low grade tumours only 40–50% had significant infiltration, while in glioblastoma multiforme over 85% of the samples analysed had significant infiltration. Numbers of neutrophils infiltrating glioblastoma multiforme tumours were also greater than in the other tumour groups. Circulating white blood cell counts were elevated above the normal range in all glioma patients, but this elevation was entirely due to increased numbers of circulating neutrophils. Again, the highest numbers of circulating neutrophils were seen in the glioblastoma multiforme patients. These experiments indicate that glioma-derived factors may directly or indirectly affect the number of circulating neutrophils and influence their infiltration into the tumours.

Cerebral blood volume, genotype and chemosensitivity in oligodendroglial tumours.

IntroductionThe biological factors responsible for differential chemoresponsiveness in oligodendroglial tumours with or without the −1p/−19q genotype are unknown, but tumour vascularity may contribute. We aimed to determine whether dynamic susceptibility contrast (DSC) magnetic resonance imaging (MRI) could distinguish molecular subtypes of oligodendroglial tumour, and examined the relationship between relative cerebral blood volume (rCBV) and outcome following procarbazine, lomustine and vincristine (PCV) chemotherapy.MethodsPretherapy rCBV was calculated and inter- and intraobserver variability assessed. Allelic imbalance in 1p36, 19q13, 17p13, 10p12–15, and 10q22–26 and p53 mutation (exons 5–8) were determined. rCBV was compared with genotype and clinicopathological characteristics (n=37) and outcome following PCV chemotherapy (n=33).Results1p/19q loss was seen in 6/9 grade II oligodendrogliomas, 6/14 grade II oligoastrocytomas, 4/4 grade III oligodendrogliomas, and 3/10 grade III oligoastrocytomas. rCBV measurements had good inter- and intraobserver variability, but did not distinguish histology subtype or grade. Tumours with 1p/19q loss had higher rCBV values (Student’s t-test P=0.001). Receiver operating characteristic analysis revealed a cut-off of 1.59 for identifying genotype (sensitivity 92%, specificity 76%). Tumours with high and low rCBV showed response to chemotherapy. The −1p/−19q genotype, but not rCBV, was strongly associated with response, progression-free and overall survival following PCV chemotherapy. Tumours with high rCBV and intact 1p/19q were associated with shorter progression-free and overall patient survival than those with intact 1p/19q and low rCBV or high rCBV and 1p/19q loss.ConclusionrCBV identifies oligodendroglial tumours with 1p/19q loss, but does not predict chemosensitivity. The prognostic significance of rCBV may differ in oligodendroglial tumours with or without the −1p/−19q genotype.

Expression of ADAMTS-8, a secreted protease with antiangiogenic properties, is downregulated in brain tumours.

Angiogenesis and extracellular matrix degradation are key events in tumour progression, and factors regulating stromal–epithelial interactions and matrix composition are potential targets for the development of novel anti-invasive/antiangiogenic therapies. Here, we examine the expression of ADAMTS-8, a secreted protease with antiangiogenic properties, in brain tissues. Using quantitative RT–polymerase chain reaction (PCR), high, equivalent expression of ADAMTS-8 was found in normal whole brain, cerebral cortex, frontal lobe, cerebellum and meninges. ADAMTS-8 expression in 34 brain tumours (including 22 high-grade gliomas) and four glioma cell lines indicated at least two-fold reduction in mRNA compared to normal whole brain in all neoplastic tissues, and no detectable expression in 14 out of 34 (41%) tumours or four out of four (100%) cell lines. In contrast, differential expression of TSP1 and VEGF was seen in nine out of 15 (60%) and seven out of 13 (54%) tumours, with no relationship in the expression of these genes. Immunohistochemistry and Western analysis indicated downregulation of ADAMTS-8 protein in >77% tumours. Methylation-specific PCR analysis of ADAMTS-8 indicated promoter hypermethylation in one out of 24 brain tumours (a metastasis) and three out of four glioma cell lines suggesting an alternative mechanism of downregulation. These data suggest a role for ADAMTS-8 in brain tumorigenesis, warranting further investigation into its role in regulation of tumour angiogenesis and local invasion.

Molecular pathology and clinical characteristics of oligodendroglial neoplasms

To evaluate the role of molecular genetics in the routine clinic, we investigated allelic imbalance at 1p36, 19q13, 17p13, 10p12‐15, and 10q22‐26 and p53 mutation in 100 oligodendroglial neoplasms diagnosed at a single treatment center between 2000 and 2003. The ‐1p/‐19q genotype, seen in 64, 34, 77, and 30% of OII, OAII, OIII, and OAIII respectively, was inversely related to p53 mutation and 17p13 loss. Genotype was unrelated to tumor location and could not distinguish high‐grade tumors that presented de novo from those that progressed from a previous lower grade malignancy. Presentation with seizures was more common in cases with the ‐1p/‐19q genotype, and these remained stable for longer before treatment. In longitudinal samples, 74% retained their initial histological differentiation, whereas 29% showed new genetic alterations, the ‐1p/‐19q genotype being acquired in three cases. Loss of 1p36 and 19q13, 17p13, chromosome 10, and p53 mutation were significantly associated with survival from presentation in Kaplan–Meier analysis (p < 0.01), and loss of 1p36 and 19q13 and loss of 17p13 retained significance in multivariate analysis. In this recently diagnosed unselected series, clinical differences in tumors with and without the ‐1p/‐19q genotype support a genetic approach to aid diagnosis and prognostication for oligodendroglial neoplasms. Ann Neurol 2005;57:855–865

Advanced MRI in the management of adult gliomas

Gliomas are a heterogeneous group that account for ∼86% of primary brain neoplasms, and include astrocytic and oligodendroglial tumours, as well as a variety of less common histopathological subtypes. Magnetic resonance imaging has become the accepted mode of imaging for the clinical management of these tumours. MRI features bear close resemblance to the histopathology grading and prognosis of these tumours. Currently, conventional MRI is used to aid diagnosis, plan neurosurgical approaches, and monitor response to therapy and disease progression. More recent developments in the field of MRI include MR spectroscopy, perfusion MRI, diffusion-weighted imaging, intraoperative MRI and functional MRI. These newer techniques have been adopted with varying success in the management of adult gliomas. This review focuses on the application of advanced MR imaging in the clinical management of adult gliomas.

Mutation in the PTEN/MMAC1 gene in archival low grade and high grade gliomas

SummaryThe PTEN gene, located on 10q23.3, has recently been described as a candidate tumour suppressor gene that may be important in the development of advanced cancers, including gliomas. We have investigated mutation in the PTEN gene by direct sequence analysis of PCR products amplified from samples microdissected from 19 low grade (WHO Grade I and II) and 27 high grade (WHO grade III and IV) archival, formalin-fixed, paraffin-embedded gliomas. Eleven genetic variants in ten tumours have been identified. Eight of these are DNA sequence changes that could affect the encoded protein and were present in 0/2 pilocytic astrocytomas, 0/2 oligoastrocytomas, 0/1 oligodendroglioma, 0/14 astrocytomas, 3/13 (23%) anaplastic astrocytomas and 5/14 (36%) glioblastomas. PTEN mutations were found exclusively in high grade gliomas; this finding was statistically significant. Only two of the PTEN genetic variants have been reported in other studies; two of the genetic changes are in codons in which mutations have not been found previously. The results of this study indicate that mutation in the PTEN gene is present only in histologically more aggressive gliomas, may be associated with the transition from low histological grade to anaplasia, but is absent from the majority of high grade gliomas.

Expression of the BCL-2 protein in normal and dysplastic bronchial epithelium and in lung carcinomas

Although expression of the bcl-2 protein has been investigated in a number of non-haematological malignancies, little is known of its distribution in premalignant lesions. Expression of bcl-2 was investigated immunohistochemically in archival biopsies of normal (n = 8) and dysplastic bronchial epithelium (n = 56) and in 31 bronchial resection margins and their corresponding carcinomas. All dysplasias had lost the prominent basal staining pattern seen in histologically normal epithelium. Two were negative and six had occasional basal positive cells. In 37 cases up to 66% of the epithelial cells throughout the full epithelial thickness were bcl-2 positive with weak to moderate staining intensity. In 11 cases, all severe dysplasias, strong expression was observed in > 90% of the epithelial cells. Four patterns of bcl-2 expression in dysplasias were identified and an increasingly aberrant pattern of bcl-2 expression correlated with an increasing grade of dysplasia (Spearmans rank correlation, P < or = 0.0001). Sixty-five per cent of the carcinomas contained bcl-2-positive cells. Patients with non-small-cell lung carcinomas (n = 27) in which > 50% of the tumour cells were bcl-2 positive showed a survival advantage compared with those with 0-25% bcl-2-positive cells (P = 0.02). No correlation was found between p53 expression (Walker et al., 1994) and bcl-2 expression in dysplasias or carcinomas.

Apparent diffusion coefficients in oligodendroglial tumors characterized by genotype.

To investigate whether oligodendroglial tumors with or without 1p/19q loss differ in their diffusion‐weighted imaging characteristics. Oligodendroglial tumors with or without 1p/19q loss differ in their therapeutic responsiveness and prognosis, and recent reports also suggest that these tumors may differ in their magnetic resonance characteristics and blood volume.