Wolfgang Feiden

Low values of 5‐hydroxymethylcytosine (5hmC), the “sixth base,” are associated with anaplasia in human brain tumors

5‐Methylcytosine (5mC) in genomic DNA has important epigenetic functions in embryonic development and tumor biology. 5‐Hydroxymethylcytosine (5hmC) is generated from 5mC by the action of the TET (Ten‐Eleven‐Translocation) enzymes and may be an intermediate to further oxidation and finally demethylation of 5mC. We have used immunohistochemistry (IHC) and isotope‐based liquid chromatography mass spectrometry (LC‐MS) to investigate the presence and distribution of 5hmC in human brain and brain tumors. In the normal adult brain, IHC identified 61.5% 5hmC positive cells in the cortex and 32.4% 5hmC in white matter (WM) areas. In tumors, positive staining of cells ranged from 1.1% in glioblastomas (GBMs) (WHO Grade IV) to 8.9% in Grade I gliomas (pilocytic astrocytomas). In the normal adult human brain, LC‐MS also showed highest values in cortical areas (1.17% 5hmC/dG [deoxyguanosine]), in the cerebral WM we measured around 0.70% 5hmC/dG. 5hmC levels were related to tumor differentiation, ranging from lowest values of 0.078% 5hmC/dG in GBMs (WHO Grade IV) to 0.24% 5hmC/dG in WHO Grade II diffuse astrocytomas. 5hmC measurements were unrelated to 5mC values. We find that the number of 5hmC positive cells and the amount of 5hmC/dG in the genome that has been proposed to be related to pluripotency and lineage commitment in embryonic stem cells is also associated with brain tumor differentiation and anaplasia.

Evaluation of l-3-[123I]iodo-α-methyltyrosine SPET and [18F]fluorodeoxyglucose PET in the detection and grading of recurrences in patients pretreated for gliomas at follow-up: a comparative study with stereotactic biopsy

Abstract. Based on the results of stereotactic biopsy, we evaluated in a prospective fashion the efficiency of l-3-[123I]iodo-α-methyltyrosine-single-photon emission tomography (SPET) and [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET) in the detection and grading of recurrences in patients previously treated for gliomas. The patient population comprised 30 individuals, nine with astrocytomas of grade II, ten with astrocytomas of grade IV, three with oligoastrocytomas of grade II, six with oligodendrogliomas of grade II and two with anaplastic oligodendrogliomas of grade III) suspected of recurrence and scheduled for further treatment. IMT SPET data were acquired using either by dual-or a triple-headed SPET camera, Multispect 2/3. FDG uptake was measured with an ECAT ART PET camera. Two independent observers classified PET and SPET images as positive or negative for tumour tissue. Uptake of FDG and IMT was evaluated visually and, in the case of IMT, also quantitatively by calculating the ratios between tracer accumulation in the lesion and the unaffected contralateral regions of reference using the region of interest (ROI) technique. The PET and SPET results were compared with the histopathological findings obtained either by stereotactic biopsy or in one case by open surgery. Glucose metabolism and amino acid uptake of recurrences of brain tumours as assessed by FDG-PET and IMT-SPET correlated highly with the histopathological findings. Based on the histopathological data, FDG-PET and IMT-SPET findings confirmed recurrence in all cases of high-grade gliomas (IV). A difference could be demonstrated in low-grade (II–III) tumour recurrences. True-positive IMT-SPET results were found in 86% of grade III and 75% of grade II recurrences, whereas FDG-PET yielded a sensitivity of 71% in tumours of grade III and 50% in those of grade II. With respect to the grade of malignancy of brain tumours at recurrence, IMT-SPET, in contrast to FDG-PET, does not permit adequate in vivo grading of non-mixed brain tumours of astrocytic or oligodendroglial origin. However, in this study FDG-PET did not permit discrimination between upgrading of low-grade oligoastrocytomas (II) into anaplastic oligodendrogliomas (III) and upgrading into glioblastomas (IV) The results of this study indicate that FDG-PET and IMT-SPET are equivalent to stereotactic biopsy in their ability to identify high-grade tumours at recurrence. IMT-SPET proved to be superior to FDG-PET in confirming low-grade recurrences. In the case of suspected progression of the grade of malignancy in ordinary gliomas, FDG-PET correlated significantly with the histopathological grading, whereas IMT-SPET did not. However, tumour grading by FDG-PET has a limitation in mixed brain tumours in that it is not possible to discriminate between progression of the oligo- versus the astrocytic tumour entity. In this case histopathological evaluation of the tumour grade remains necessary.

Prognostic Significance of the Mitotic Index Using the Mitosis Marker Anti-Phosphohistone H3 in Meningiomas

Mitotic activity is one of the most reliable prognostic factors in meningiomas. The identification of mitotic figures (MFs) and the areas of highest mitotic activity in H&E-stained slides is a tedious and subjective task. Therefore, we compared the results from immunostaining for the mitosis-specific antibody anti-phosphohistone H3 (PHH3 mitotic index [MI]) with standard MF counts (H&E MI) and the Ki-67 labeling index (LI). The relationship between these proliferation indices and prognosis was investigated in a retrospective series of 265 meningiomas. The PHH3 staining method yielded greater sensitivity in the detection of MFs and facilitated MF counting. Mitotic thresholds of H&E MI of 4 or more per 10 high-power fields (HPF) and PHH3 MI of 6 or more per 10 HPF were found as the most appropriate prognostic cutoff values for the prediction of recurrence-free survival. All 3 proliferation indices were univariately associated with recurrences and deaths. In contrast with the Ki-67 LI, H&E MI and PHH3 MI also remained as independent predictors in the multivariate Cox hazards modeling (P = .0007 and P = .0004, respectively).

Loss of Alkaline Phosphatase Activity in Meningiomas: A Rapid Histochemical Technique Indicating Progression-associated Deletion of a Putative Tumor Suppressor Gene on the Distal Part of the Short Arm of Chromosome 1

Apart from defined histomorphologic features, increased Ki-67 indices and various numeric and structural chromosome aberrations, meningiomas of the intermediate (WHO grade II, atypical meningioma) and anaplastic type (WHO grade III) are cytogenetically distinguished from common-type meningiomas (WHO grade I) by frequent loss of the distal part of the short arm of one chromosome 1 (1p-), which formerly proved to be an independent predictor of shorter recurrence-free intervals. Histochemically, loss of alkaline phosphatase activity (ALPL, liver/bone/kidney type, EC 3.1.3.1) was another frequent, specific finding in meningiomas with signs of dedifferentiation. In a prospective study including 66 meningiomas, all common-type meningiomas except one case (18/19) were reactive for ALPL, whereas 75% (30/39) of intermediate type and all anaplastic meningiomas (8/8) showed loss of enzyme activity in large areas of the tumor. Exclusively, the ALPL negative phenotype was associated with 1p loss (15/19). Our data suggest that ALPL, which is coded as a single copy gene on chromosome 1p36.1-p34, is a useful marker enzyme for the loss of a putative regulatory (tumor suppressor) gene on chromosome 1p, or that ALPL itself represents a new tumor suppressor gene homozygously inactivated in meningiomas.

Clinical Value of Iodine-123-Alpha-Methyl-l-Tyrosine Single-Photon Emission Tomography in the Differential Diagnosis of Recurrent Brain Tumor in Patients Pretreated for Glioma at Follow-Up

PURPOSE To assess the clinical potential of iodine-123-alpha-methyl-L-tyrosine (IMT) and single-photon emission tomography (SPET) in the differential diagnosis of recurrences in patients pretreated for gliomas at follow-up. PATIENTS AND METHODS Seventy-eight patients were examined after primary therapy over 36 months. Histopathologic diagnoses of all patients was known at first treatment; magnetic resonance and/or computed tomography examination was performed in addition to IMT-SPET. Cerebral SPET images were acquired 20 minutes after intravenous application of 190 +/- 10 MBq of IMT. SPET images were classified as positive or negative for recurrent tumor visually and by calculating the ratios between tracer accumulation in the lesion and the unaffected contralateral regions of reference using region of interest. Final diagnoses were based on prospective clinicopathologic findings obtained independently of IMT-SPET. RESULTS IMT-SPET detected all high-grade recurrent gliomas (grade 4; sensitivity, 100%). A difference could be demonstrated in grade 2 and 3 recurrences (sensitivity, 84% and 92%, respectively). Moreover, benign posttherapeutic lesions (postoperative scars, radiation necrosis) were correctly diagnosed as negative for tumor recurrence. In general, IMT uptake in grade 2 (1.45 +/- 0.24) was significantly lower than that in grades 3 (1.70 +/- 0.41) and 4 (1.88 +/- 0.32). However, it was difficult to evaluate tumor grade only from the IMT accumulation in individual cases. CONCLUSION IMT-SPET seems highly useful for detecting and delineating recurrent gliomas and differentiating between benign posttherapeutic lesions and malignant tumor tissue. It may be a valuable clinical tool to diagnose recurrences in patients pretreated for gliomas at follow-up. However, it showed limitations in determining histologic tumor grade.

Assessing CpG island methylator phenotype, 1p/19q codeletion, and MGMT promoter methylation from epigenome-wide data in the biomarker cohort of the NOA-04 trial

BACKGROUND Molecular biomarkers including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation, 1p/19q codeletion, and O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter methylation may improve prognostication and guide treatment decisions for patients with World Health Organization (WHO) anaplastic gliomas. At present, each marker is individually tested by distinct assays. Illumina Infinium HumanMethylation450 BeadChip arrays (HM450) enable the determination of large-scale methylation profiles and genome-wide DNA copy number changes. Algorithms have been developed to detect the glioma CpG island methylator phenotype (G-CIMP) associated with IDH1/2 mutation, 1p/19q codeletion, and MGMT promoter methylation using a single assay. METHODS Here, we retrospectively investigated the diagnostic and prognostic performance of these algorithms in comparison to individual marker testing and patient outcome in the biomarker cohort (n = 115 patients) of the NOA-04 trial. RESULTS Concordance for IDH and 1p/19q status was very high: In 92% of samples, the HM450 and reference data agreed. In discordant samples, survival analysis by Kaplan-Meier and Cox regression analyses suggested a more accurate assessment of biological phenotype by the HM450 analysis. The HM450-derived MGMT-STP27 model to calculate MGMT promoter methylation probability revealed this aberration in a significantly higher fraction of samples than conventional methylation-specific PCR, with 87 of 91 G-CIMP tumors predicted as MGMT promoter-methylated. Pyrosequencing of discordant samples confirmed the HM450 assessment in 14 of 17 cases. CONCLUSIONS G-CIMP and 1p/19q codeletion are reliably detectable by HM450 analysis and are associated with prognosis in the NOA-04 trial. For MGMT, HM450 suggests promoter methylation in the vast majority of G-CIMP tumors, which is supported by pyrosequencing.

PHF3-specific antibody responses in over 60% of patients with glioblastoma multiforme

Glioblastoma multiforme (GBM), a malignant astrocytic tumour, represents the most frequent tumour of the human brain. Nevertheless, its molecular pathology is not well understood. We utilized the immune system, which contributes to cancer protection, to help identify new GBM-related genes. By screening a human GBM cDNA library with autologous patient serum (SEREX-approach), we isolated a gene termed PHF3 (PHD finger protein 3). The gene product of PHF3 is immunogenic in GBM as tested in an allogenic patient serum screening demonstrating antibodies in 24 of 39 (61.53%) sera, whereas none of the 14 healthy persons had antibodies against PHF3. While previous SEREX studies revealed allogenic antibody responses up to 40%, our results for PHF3 represent the highest reported rate for a specific antibody response. We show that GBM patients with an antibody response against PHF3 show significant better survival than patients without PHF3-specific antibodies. Because the amino acid sequence of PHF3 contains a PHD finger (also termed LAP motif), a TFIIS homology, a proline rich region and nuclear localization signals, it supposedly functions as a transcription factor. A polyclonal antibody generated against PHF3 shows nuclear expression in most investigated formalin-fixed, paraffin embedded tissues. In GBM, PHF3 expression is concentrated in cells surrounding necroses.

Matrix-Metallo-Proteinases and their tissue inhibitors in radiation-induced lung injury

Purpose: Remodeling of extracellular matrix (ECM) after lung damage depends on collagen degrading Matrix-Metallo-Proteinases (MMP) and their endogenous inhibitors (Tissue-Inhibitors of Metallo-Proteinases, TIMP). Transforming growth factor (TGF)-β1 has been implicated in the pathogenesis of radiation-induced lung fibrosis upon its effects on fibroblast proliferation and collagen synthesis. Lung cancer patients have often elevated TGF-β1 plasma levels as a result of increased TGF-β1 expression in their tumours. On this background, we investigated the effect of irradiation on the MMP/TIMP system in the lung tissue of normal and transgenic TGF-β1 mice, in which TGF-β1 is overexpressed in the liver resulting in high TGF-β1 plasma levels. Material and methods: Transgenic (TG) and wild-type (WT) mice underwent thoracic irradiation with 12 Gy or sham-irradiation. For each study group (TG 12 Gy; TG 0 Gy; WT 12 Gy; WT 0 Gy) 8 mice were sacrificed at 4 and 8 weeks after (sham-) irradiation. The TGF-β1, TIMP-1/-2/-3 expression in the lung tissue was quantified by Western blot; the MMP-2 and MMP-9 activity was analysed by zymography. The cellular origin of the MMP and TIMP was localised by immunohistochemistry. Results: Irradiation had no influence on the TIMP-1/-2/-3, but increased significantly the MMP-2 /-9 expression. In the lung tissue of TG mice the TIMP-1/-2/-3 expression was elevated, the MMP-9 activity was decreased. The immunhistochemical study showed that parenchymal and inflammatory cells express these MMP/TIMP. Conclusion: Our results provide evidence that the overexpression of MMP-2 and MMP-9 is involved in the inflammatory response of radiation-induced lung injury. MMP-2 and MMP-9 are known to degrade collagen IV of basement membranes, therefore affecting the structural integrity of lung tissue. In contrast, in lung tissue of TG mice the TIMP-1/-2/-3 expression was up-regulated and the MMP-9 activity was diminished, thereby decreasing possibly the ECM degradation leading to lung fibrosis.

Diagnosis of primary cerebral lymphoma with particular reference to CT-guided stereotactic biopsy

In establishing the histological diagnosis of primary cerebral lymphoma, stereotactic brain tumour biopsy is the method of choice as the mainstay of therapy is radiation and chemotherapy. This study describes the histopathology and diagnostic immunohistochemistry of 54 primary brain lymphomas in a mainly non-AIDS population. The stereotactic biopsies were performed using the Leksell CT stereotactic frame and a spiral needle which procured about 10-mm-long tissue cylinders. Usually, three successive biopsy cylinders were taken along the target trajectory. Histological examination revealed the prevalence of high-grade non-Hodgkins lymphoma of the polymorphous centroblastic type. The series did not include any low-grade lymphomas or T-cell lymphomas. L-26 immunohistochemistry resulted in a positive staining of the blasts, thus confirming the B-cell origin of primary brain lymphomas. Small reactive T-lymphocytes and monohistiocytic cells were also found within and at the periphery of the lymphomas and in areas of degeneration. In the biopsies of nine patients, who had shown significant reduction of the lesions on the CT scans, after corticosteroid medication, regressive tissue changes were predominant and consisted of T-lymphocytes, macrophages, and occasionally bizarre reactive astrocytes.

Correspondence of tumor localization with tumor recurrence and cytogenetic progression in meningiomas.

OBJECTIVEMeningiomas are mostly benign tumors that originate from the coverings of the brain and spinal cord. Cytogenetically, they reveal a normal karyotype or, typically, monosomy of chromosome 22. Progression of meningiomas is associated with a non-random pattern of secondary losses of other autosomes. Deletion of the short arm of one chromosome 1 is a decisive step to anaplastic growth in meningiomas. METHODSStatistical analyses were performed for the karyotypes of 661 meningiomas with respect to localization, progression, and recurrence of the tumor. A mathematical mixture model estimates typical pathogenetic routes in terms of the accumulation of somatic chromosome changes in tumor cells. The model generates a genetic progression score (GPS) that estimates the prognosis as related to the cytogenetic properties of a given tumor. RESULTSIn 53 patients, one or several recurrences were documented over the period of observation. This corresponds to a total rate of recurrence of 8.0% after macroscopically complete tumor extirpation. Higher GPS values were shown to be strongly correlated with tumor recurrence (P = 2.9 × 10−7). High-risk tumors, both in terms of histology and cytogenetics, are localized much more frequently at the brain surface than at the cranial base (P = 1.2 × 10−5 for World Health Organization grade and P = 3.3 × 10−12 for GPS categorization). CONCLUSIONThe tendency of cranial base meningiomas to recur seems to depend on surgical rather than biological reasons. As a quantitative measure, the GPS allows for a more precise assessment of the prognosis of meningiomas than the established categorical cytogenetic markers.