Assessment of molecular markers demonstrates concordance between samples acquired via stereotactic biopsy and open craniotomy in both anaplastic astrocytomas and glioblastomas.

The classification, treatment and prognosis of high-grade gliomas has been shown to correlate with the expression of molecular markers (e.g. MGMT promotor methylation and IDH1 mutations). Acquisition of tumor samples may be obtained via stereotactic biopsy or open craniotomy. Between the years 2009 and 2013, 22 patients initially diagnosed with HGGs via stereotactic biopsy, that ultimately underwent open craniotomy for resection of their tumor were prospectively included in an institutional glioma database. MGMT promotor analysis was performed using methylation-specific (MS)-PCR and IDH1R132H mutation analysis was performed using immunohistochemistry. Three patients (13.7%) exhibited IDH1R132H mutations in samples obtained via stereotactic biopsy. Tissue derived from stereotaxic biopsy was demonstrated to have MGMT promotor methylation in ten patients (45.5%), while a non-methylated MGMT promotor was demonstrated in ten patients (45.5%); inconclusive results were obtained for the remaining two patients (9%) within our cohort. The initial histologic grading, IDH1R132H mutation and MGMT promotor methylation results were confirmed using samples obtained during open craniotomy in all but one patient; here inconclusive MGMT promotor analysis was obtained in contrast to that which was obtained via stereotactic biopsy. Tumor samples acquired via stereotactic biopsy provide accurate information with regard to clinically relevant molecular markers that have been shown to impact patient care decisions. The profile of markers analyzed in our cohort was nearly concordant between those samples obtained via stereotactic biopsy or open craniotomy thereby suggesting that clinical decisions may be based on the molecular profile of the tumor samples obtained via stereotactic biopsy.

Rapid colorimetric determination of reduced and oxidized glutathione using an end point coupled enzymatic assay.

A simple and rapid colorimetric coupled enzymatic assay for the determination of glutathione is described. The proposed method is based on the specific reaction catalyzed by γ-glutamyltransferase, which transfers the γ-glutamyl moiety from glutahione to an acceptor, with the formation of the γ-glutamyl derivative of the acceptor and cysteinylglycine. The latter dipeptide is a substrate of leucyl aminopeptidase, which hydrolyzes cysteinylglycine to glycine and cysteine that can be easily measured spectrophotometrically. The proposed method was used to measure the content of glutathione in acid extracts of bovine lens, to follow the NADPH-dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) catalyzed by the enzyme glutathione reductase and to determine the glutathione content in human astrocytoma ADF cells subjected to oxidative stress. The results obtained showed that the method can be suitably used for the determination of GSH and GSSG in different biological samples and to monitor tissue or cell redox status under different conditions. It is also applicable for following reactions involving GSH and/or GSSG.

Proliferative and metabolic markers in incompletely excised pediatric pilocytic astrocytomas--an assessment of 3 new variables in predicting clinical outcome.

Although pilocytic astrocytoma (PA) is the most common brain tumor diagnosed in children, few prognostic variables have been delineated that stratify the risk of clinical progression in patients with this tumor. In this study, the MIB-1 labeling index was compared with 2 other immunohistochemical markers of cell proliferation, phospho-histone H3 (PHH3) and mini-chromosomal maintenance protein 2 (MCM2) in 80 incompletely resected PAs to see which was best able to identify patients at risk for tumor progression. 0(6)-Methylguanine-DNA methyltransferase (MGMT) protein expression, which has been predictive of progression-free survival (PFS) in high-grade gliomas in children, was also evaluated in these cases. The mean follow-up period was 7.81 ± 3.9 years, and 42.8% of tumors have shown progression at the time of censoring. A MIB-1 labeling index ≥2.0 was associated with shortened PFS as a grouped variable by log-ranked analysis (P = .03) and by Cox regression analysis as a continuous variable (P = .007). None of the other potential biomarkers was significantly predictive of PFS. Although the amount of MCM2 staining correlated with the MIB-1 labeling index (P < .001), MCM2 reactivity was not independently associated with outcome. We conclude that MIB-1 labeling remains the best predictor of PFS in pediatric PAs.

Assessment of 1p/19q status by fluorescence in situ hybridization assay: A comparative study in oligodendroglial, mixed oligoastrocytic and astrocytic tumors.

BACKGROUND: Due to overlapping histomorphological features, difference in clinical behavior and treatment response, establishing potential molecular markers to facilitate diagnosis of various genetic subtypes of diffuse gliomas is essential. AIM: To analyze 1p/19q status in diffuse gliomas and correlate it with epidermal growth factor receptor (EGFR) and p53 protein expression. MATERIALS AND METHODS: 1p/19q status in 43 cases was evaluated by fluorescence in situ hybridization assay. Glial fibrillary acidic protein (GFAP), EGFR and p53 were assessed by immunohistochemistry. RESULTS: Glial fibrillary acidic protein immunopositivity was observed in oligodendrogliomas within minigemistocytes and gliofibrillary oligodendrocytes as perinuclear homogenous blobs. It also highlighted the intermingled reactive astrocytes. Astrocytomas and the astrocytic component of oligoastrocytomas showed a diffuse fibrillary type of staining. 1p and/or 19q loss was seen in 65% (13/20) of oligodendrogliomas and 66.6% (5/9) of mixed oligoastrocytomas. There was one case each of pediatric oligodendroglioma and mixed oligoastrocytoma, none of which showed 1p/19q loss. None of the astrocytomas including two pediatric cases showed this alteration (P < 0.05). p53 was expressed in 57.1% of astrocytomas (8/14), 33% of mixed oligoastrocytomas (3/9) and 10% of oligodendrogliomas (2/20). Majority of oligodendrogliomas (85%; 17/20) and oligodendroglial areas in mixed oligoastrocytomas (77.7%; 7/9) showed a membranous lace-like immunopositivity with EGFR. In contrast, all astrocytomas (Grade II and III) were EGFR negative. CONCLUSION: Loss of 1p/19q is strongly associated with oligodendroglial phenotype, while astrocytic tumors are more likely to show p53 over-expression. p53 expression and 1p/19q status appear to be mutually exclusive.

Pitfalls in the assessment of MGMT expression and in its correlation with survival in diffuse astrocytomas: proposal of a feasible immunohistochemical approach.

Immunohistochemical studies showed that O(6)-methylguanine-DNA methyltransferase (MGMT) protein expression is negatively associated with survival in glioblastomas treated with alkylating agents in accordance with previous results of methylation-specific PCR. Implementation of this data in routine clinical diagnostics is limited due to often inappropriate study designs, e.g. pooling of tumor entities, WHO grades or primary and secondary glioblastomas, disregard concerning the infiltration zone or various epidemiological factors. The aim of our study was to evaluate MGMT expression and its prognostic value taking into consideration the aforementioned deficiencies. For this, 162 astrocytic tumors WHO II-IV (36 diffuse astrocytomas WHO II, 51 anaplastic astrocytomas, 75 primary glioblastomas) as well as 25 glioblastoma infiltration zones and 19 glioblastoma relapses were analyzed for immunohistochemical MGMT protein expression using tissue microarray technique. Expression of MGMT significantly decreased from WHO grade II (25.6%) to glioblastoma (16.8%, p = 0.01) with lowest levels in grade III tumors (10.2%, II/III p < 0.0001). Significant negative associations of MGMT and survival were detected for WHO grade II and IV (p = 0.003 and 0.013). The optimal cut-off value of MGMT positive nuclei in primary glioblastomas discriminating patients with significantly different survival rates was at 15% (Log-Rank p = 0.0002). Individual relapse tumors showed changes of MGMT expression to a varying degree. The infiltration zone demonstrated a significant increase of MGMT (p < 0.0001). We conclude that immunohistochemical MGMT assessment has potential as a powerful diagnostic tool but analysis should only be performed in a grade dependent manner, before radio-/chemotherapy and with special attention to the infiltration zone of diffuse astrocytomas.

Assessment and prognostic significance of mitotic index using the mitosis marker phospho-histone H3 in low and intermediate-grade infiltrating astrocytomas.

Distinguishing between grade II and grade III diffuse astrocytomas is important both for prognosis and for treatment decision-making. However, current methods for distinguishing between grades based on proliferative potential are suboptimal, making identification of clear cutoffs difficult. In this study, we compared the results from immunohistochemical staining for phospho-histone H3 (pHH3), a specific marker of cells undergoing mitosis, with standard mitotic counts (number of mitoses/10 high-power fields) and MIB-1 labeling index values for assessing proliferative activity. We tested the relationship between pHH3 staining and tumor grade and prognosis in a retrospective series of grade II and III infiltrating astrocytomas from a single institution. The pHH3 index (per 1000 cells), MIB-1 index (per 1000 cells), and number of mitoses per 10 high-power fields were determined for each of 103 cases of grade II and III diffuse astrocytomas from patients with clinical follow-up. pHH3 staining was found to be a simple and reliable method for identifying mitotic figures, allowing a true mitotic index to be determined. The pHH3 mitotic index was significantly associated both with the standard mitotic count and with the MIB-1 index. Univariate analyses revealed that all 3 measurements of proliferation were significantly associated with survival. However, the pHH3 mitotic index accounted for a larger proportion of variability in survival than standard mitotic count or MIB-1/Ki-67 labeling index. After adjusting for age, extent of resection, and performance score, the pHH3 mitotic index remained an independent predictor of survival. Thus, pHH3 staining provides a simple and reliable method for quantifying proliferative potential and for the stratification of patients with diffuse astrocytomas into typical grade II and III groups. These results also suggest that pHH3 staining may be a useful method in other neoplasms in which accurate determination of proliferation potential is relevant to tumor grading or clinical treatment decision-making.

Positron emission tomographic assessment of cerebral hemocirculation and glucose metabolism in malignant glioma following treatment with intracarotid recombinant human tumor necrosis factor-alpha.

Cerebral hemocirculation and glucose metabolism in a malignant astrocytoma were repeatedly quantified before and after intracarotid injection of recombinant human tumor necrosis factor-alpha (rH-TNF) using positron emission tomography (PET). The patient received an intracarotid injection of a 3 x 10(4) U/m2 dose of rH-TNF three times over a two week period. PET was performed prior to and 24 hr after the first injection, and two weeks after the third injection. Prior to the first rH-TNF treatment, two lesions demonstrating high perfusion and hypermetabolism of glucose were noted in the right frontal and temporal regions. The frontal hypermetabolic lesion showed decreases in hemocirculation and metabolism 24 hr after the first injection and then increases beyond the pre-treatment level two weeks after the third treatment, whereas the temporal lesion remained unchanged during the follow-up period. No appreciable changes were noted in the adjacent cortex where rH-TNF was perfused, with the exception of a transient decrease in regional blood volume. Magnetic resonance images of the tumor showed no changes as a result of treatment with intracarotid rH-TNF. Intracarotid rH-TNF preferentially affects tumor tissue as opposed to normal cortex.

MR assessment of radiation-induced blood-brain barrier permeability changes in rat glioma model.

PURPOSE: To assess the potential of a T1-weighted, gadolinium-enhanced MR technique for quantifying radiation-induced changes of blood-brain barrier permeability in a model of stereotactically implanted intracerebral gliomas in rats. METHODS: We calculated the gadolinium blood-to-tissue transport coefficient for gadopentetate dimeglumine from signal intensities in sequential MR images in nine control animals that were not irradiated and in five and three animals that had received 2500 cGy and 1500 cGy whole-brain irradiation, respectively, at 2 days before imaging. RESULTS: The average blood-to-tissue transport coefficient values were 9.76 mL.kg-1.min-1 in the control group, 23.41 mL.kg-1.min-1 in the 2500 cGy group, and 25.63 mL.kg-1.min-1 in the 1500-cGy group. Blood-to-tissue transport coefficients were significantly higher after irradiation, indicating increased radiation-induced blood-brain barrier permeability. Similar increased blood-brain barrier leakiness in brain tumors after high-dose irradiation has been shown by previous nuclear medicine studies using quantitative autoradiography. CONCLUSION: Contrast-enhanced dynamic MR of brain gliomas is a sensitive method to document radiation-induced blood-brain barrier breakdown. Quantitative gadolinium-enhanced MR may become a useful tool for the management of patients with brain tumors undergoing radiation therapy.

Increased energy expenditure in a patient with diencephalic syndrome.

Total energy expenditure, measured in an infant with diencephalic syndrome with doubly labeled water, was 30% to 50% higher than that reported for other healthy infants whose energy expenditure was measured by the same technique and 13% higher than the patient's energy intake. This result suggests that the weight loss and cachexia of the diencephalic syndrome are the result of abnormally increased energy expenditure.