Aurora B Kinase Inhibition by AZD1152 Concomitant with Tumor Treating Fields Is Effective in the Treatment of Cultures from Primary and Recurrent Glioblastomas.

Tumor Treating Fields (TTFields) were incorporated into the treatment of glioblastoma, the most malignant brain tumor, after showing an effect on progression-free and overall survival in a phase III clinical trial. The combination of TTFields and an antimitotic drug might further improve this approach. Here, we tested the combination of TTFields with AZD1152, an Aurora B kinase inhibitor, in primary cultures of newly diagnosed (ndGBM) and recurrent glioblastoma (rGBM). AZD1152 concentration was titrated for each cell line and 5-30 nM were used alone or in addition to TTFields (1.6 V/cm RMS; 200 kHz) applied for 72 h using the inovitro™ system. Cell morphological changes were visualized by conventional and confocal laser microscopy. The cytotoxic effects were determined by cell viability assays. Primary cultures of ndGBM and rGBM varied in p53 mutational status; ploidy; EGFR expression and MGMT-promoter methylation status. Nevertheless; in all primary cultures; a significant cytotoxic effect was found following TTFields treatment alone and in all but one, a significant effect after treatment with AZD1152 alone was also observed. Moreover, in all primary cultures the combined treatment had the most pronounced cytotoxic effect in parallel with morphological changes. The combined treatment of TTFields and AZD1152 led to a significant reduction in the number of ndGBM and rGBM cells compared to each treatment alone. Further evaluation of this approach, which has to be considered as a proof of concept, is warranted, before entering into early clinical trials.

Intratumoral heterogeneity and TERT promoter mutations in progressive/higher-grade meningiomas.

BACKGROUND: Recent studies have reported mutations in the telomerase reverse transcriptase promoter (TERTp) in meningiomas. We sought to determine the frequency, clonality and clinical significance of telomere gene alterations in a cohort of patients with progressive/higher-grade meningiomas. METHODS: We characterized 64 temporally- and regionally-distinct specimens from 26 WHO grade III meningioma patients. On initial diagnoses, the meningiomas spanned all WHO grades (3 grade I, 13 grade II and 10 grade III). The tumor samples were screened for TERTp and ATRX/DAXX mutations, and TERT rearrangements. Additionally, TERTp was sequenced in a separate cohort of 19 patients with radiation-associated meningiomas. We examined the impact of mutational status on patients' progression and overall survival. RESULTS: Somatic TERTp mutations were detected in six patients (6/26 = 23%). Regional intratumoral heterogeneity in TERTp mutation status was noted. In 4 patients, TERTp mutations were detected in recurrent specimens but not in the available specimens of the first surgery. Additionally, a TERT gene fusion (LPCAT1-TERT) was found in one sample. In contrary, none of the investigated samples harbored an ATRX or DAXX mutation. In the cohort of radiation-induced meningiomas, TERTp mutation was detected in two patients (10.5%). Importantly, we found that patients with emergence of TERTp mutations had a substantially shorter OS than their TERTp wild-type counterparts (2.7 years, 95% CI 0.9 - 4.5 years versus 10.8 years, 95% CI 7.8 -12.8 years, p=0.003). CONCLUSIONS: In progressive/higher-grade meningiomas,TERTp mutations are associated with poor survival, supporting a model in which selection of this alteration is a harbinger of aggressive tumor development. In addition, we observe spatial intratumoral heterogeneity of TERTp mutation status, consistent with this model of late emergence in tumor evolution. Thus, early detection of TERTp mutations may define patients with more aggressive meningiomas. Stratification for TERT alterations should be adopted in future clinical trials of progressive/higher-grade meningiomas.

IDH mutations as an early and consistent marker in low-grade astrocytomas WHO grade II and their consecutive secondary high-grade gliomas.

This study investigated the prognostic and predictive significance of IDH1 and IDH2 mutations in low-grade astrocytomas (LGA). The presence and consistency of IDH mutations during the progression of LGA to secondary high-grade gliomas (sHGG) were detected. Samples of patients with LGA and sHGG were investigated. The genomic regions around IDH1 codon 132 and IDH2 codon 172 were PCR amplified and directly sequenced. Furthermore, the MGMT promoter status was provided using the methylation-specific PCR. Our population comprised 71 patients with a total of 45 pairs of LGA and their consecutive sHGG. Median follow-up was 9.6 years. IDH mutations were found in 36/45 LGA (80%) and their sHGG without changes in the mutation status. A total of 71 patients with LGA were analyzed according to clinical and molecular tumor-related factors: 56/71 patients (78.8%) had an IDH mutation without significant influence on the progression-free or overall survival (OS), and 22/71 (31%) of the patients received postoperative radiotherapy (RT) after diagnosis of LGA. Patients with early RT but without IDH mutations had the shortest survival. Our study shows that IDH mutation status is stable during the progression course of LGA to sHGG. The presence of IDH mutations fails to demonstrate a significant influence on survival in the multivariate analysis of LGA patients. Early RT appears to be beneficial only LGA patients with IDH-mutations.

Isolation and enrichment of urologic tumor cells in blood samples by a semi-automated CD45 depletion autoMACS protocol.

The purpose of this study was to demonstrate the efficacy of an enrichment protocol for the detection of circulating carcinoma cells in the bloodstream of patients with various urologic cancers. Using 16-ml peripheral blood samples (BS) the mononuclear cells were isolated by density gradient centrifugation. The CD45 leukocyte depletion method based on a previous study was slightly modified and semi-automated by an immunomagnetic cell separation unit (autoMACS). Enriched tumor cells were analyzed on a single slide by cytokeratin (CK) immunocytochemistry. The number of recovered DU-145 prostate cancer cells in various spiking experiments was 70-88%. By the optimized tumor cell enrichment protocol 186 BS originated from 128 patients with different urologic cancers (60 prostate carcinoma, 34 bladder cancers, 24 renal cell carcinoma and 10 other tumors) were investigated before, during and after tumor surgery. In 59 BS from 52 patients on average 5 tumor cells were detected in each BS containing tumor cells. The median number of identified tumor cells was 8 cells per BS and patient. Tumor cells were found for the 3 tumor types with representative BS numbers in 29-39% of the investigated BS and in 38-53% of the affected patients. The detection rates increased in the order prostate carcinoma < renal cell carcinoma < bladder cancer. Surprisingly, in four bladder tumor cases with identified disseminated tumor cells in BS, the histopathological examination of the transurethral resection of bladder tumor specimens showed no evidence for tumor cells in situ but the affected patients had clinically known and histologically defined tumor residue or a bladder tumor recurrence during the follow-up. The semi-automated CD45 autoMACS depletion protocol for the enrichment and the detection of disseminated tumor cells in the peripheral bloodstream allows to study up to 20 BS per working day prospectively by one technician. The improved sensitivity and specificity might be of importance when applying the protocol to BS in future clinical studies.