Michal Bienkowski

Glioma cells showing IDH1 mutation cannot be propagated in standard cell culture conditions.

Background:It has recently been reported by several sources that original (i.e., present in vivo) glioma cell phenotypes or genotypes cannot be maintained in vitro. For example, glioblastoma cell lines presenting EGFR amplification cannot be established.Methods and results:IDH1 sequencing and loss of heterozygosity analysis was performed for 15 surgery samples of astrocytoma and early and late passages of cells derived from those and for 11 archival samples. We were not able to culture tumour cells presenting IDH1 mutations originating from currently proceeded 10 tumours; the same results were observed in 7 samples of archival material.Conclusion:The IDH1 mutation is expected to be almost mutually exclusive with EGFR amplification, so glioma cells with IDH1 mutations seem to represent a new group of tumour cells, which cannot be readily analysed in vitro because of their elimination. The reasons for this intriguing phenomenon should be investigated since its understanding can help to define a new therapeutic approach based on simulating in vivo conditions, responsible for tumour cells elimination in vitro. Moreover, a new model for culturing glioma cells in vitro should be designed since the current one does not provide conditions corresponding to in vivo growth.

Prognostic role of tumour-infiltrating inflammatory cells in brain tumours: literature review.

PURPOSE OF REVIEW Both primary and metastatic brain tumours pose a significant and unmet clinical need. Immune cells infiltrating the tumour have been shown to affect the clinical course of various extracranial tumour types, but there is little knowledge on the role of tumour-infiltrating immune cells in brain tumours. Thus, the aim of this review was to recapitulate the reports on immune infiltrates in brain tumours and their prognostic significance. RECENT FINDINGS Immune infiltrates composed of various lymphocyte subsets and microglia/macrophages are frequently observed in brain tumours; however, their density and prognostic role seem to differ between tumour types. Central nervous system (CNS) metastases, particularly of melanoma, lung cancer and renal cell cancer, commonly show high amounts of tumour-infiltrating lymphocytes and tumour-infiltrating lymphocytes density strongly correlate with patients overall survival times in patients with CNS metastases. In gliomas and primary CNS lymphomas, some studies also suggest a prognostic role of immune cell infiltration; however, methodological issues such as low sample size and retrospective study designs with heterogeneous patient populations preclude definite conclusions. Meningiomas typically harbour inflammatory infiltrates, but their correlation with the clinical course is unclear because of the lack of studies correlating immune cell infiltrates with outcome parameters. SUMMARY The available literature suggests a relevant role of immune infiltrates in the clinical course of some brain tumour types; however, further studies are required to better understand the interaction of the immune system and CNS neoplasms and to explore therapeutic opportunities with immunotherapies such as vaccines or immune checkpoint modulators.

Screening for EGFR Amplifications with a Novel Method and Their Significance for the Outcome of Glioblastoma Patients

Glioblastoma is a highly aggressive tumour of the central nervous system, characterised by poor prognosis irrespective of the applied treatment. The aim of our study was to analyse whether the molecular markers of glioblastoma (i.e. TP53 and IDH1 mutations, CDKN2A deletion, EGFR amplification, chromosome 7 polysomy and EGFRvIII expression) could be associated with distinct prognosis and/or response to the therapy. Moreover, we describe a method which allows for a reliable, as well as time- and cost-effective, screening for EGFR amplification and chromosome 7 polysomy with quantitative Real-Time PCR at DNA level. In the clinical data, only the patient’s age had prognostic significance (continuous: HR = 1.04; p<0.01). At the molecular level, EGFRvIII expression was associated with a better prognosis (HR = 0.37; p = 0.04). Intriguingly, EGFR amplification was associated with a worse outcome in younger patients (HR = 3.75; p<0.01) and in patients treated with radiotherapy (HR = 2.71; p = 0.03). We did not observe any difference between the patients with the amplification treated with radiotherapy and the patients without such a treatment. Next, EGFR amplification was related to a better prognosis in combination with the homozygous CDKN2A deletion (HR = 0.12; p = 0.01), but to a poorer prognosis in combination with chromosome 7 polysomy (HR = 14.88; p = 0.01). Importantly, the results emphasise the necessity to distinguish both mechanisms of the increased EGFR gene copy number (amplification and polysomy). To conclude, although the data presented here require validation in different groups of patients, they strongly advocate the consideration of the patient’s tumour molecular characteristics in the selection of the therapy.

Reduced expression of ELAVL4 in male meningioma patients

Meningioma is a frequently occurring tumor of the central nervous system. Among many genetic alternations, the loss of the short arm of chromosome 1 is the second most frequent chromosomal abnormality observed in these tumors. Here, we focused on the previously described and well-established minimal deletion regions of chromosome 1. In accordance with the Knudson suppressor theory, we designed an analysis of putative suppressor genes localized in the described minimal deletion regions. The purpose was to determine the molecular background of the gender-specific occurrence of meningiomas. A total of 149 samples were examined for loss of heterozygosity (LOH). In addition, 57 tumor samples were analyzed using real-time polymerase chain reaction. We examined the association between the expression of selected genes and patient age, gender, tumor grade and presence of 1p loss. Furthermore, we performed an analysis of the most stable internal control for real-time analysis in meningiomas. LOH analysis revealed gender-specific discrepancies in the frequency of 1p aberrations. Moreover, statistical correlation between the gene expression level and gender was significant for the ELAVL4 gene as we found it to be lower in males than in females. We conclude that meningiomas present different features depending on patient gender. We suggest that ELAVL4 can be involved in the pathogenesis of meningiomas in male patients.

The Failure in the Stabilization of Glioblastoma-Derived Cell Lines: Spontaneous In Vitro Senescence as the Main Culprit

Cell line analysis is an important element of cancer research. Despite the progress in glioblastoma cell culturing, the cells isolated from the majority of specimens cannot be propagated infinitely in vitro. The aim of this study was to identify the processes responsible for the stabilization failure. Therefore, we analyzed 56 primary GB cultures, 7 of which were stabilized. Our results indicate that senescence is primarily responsible for the glioblastoma cell line stabilization failure, while mitotic catastrophe and apoptosis play a minor role. Moreover, a new technical approach allowed for a more profound analysis of the senescent cells in primary cultures, including the distinction between tumor and normal cells. In addition, we observed that glioblastoma cells in primary cultures have a varied potential to undergo spontaneous in vitro senescence, which is often higher than that of the normal cells infiltrating the tumor. Thus, this is the first report of GB cells in primary cell cultures (including both monolayer and spheroid conditions) rapidly and spontaneously becoming senescent. Intriguingly, our data also suggest that nearly half of GB cell lines have a combination of TP53 mutation and CDKN2A homozygous deletion, which are considered as mutually exclusive in glioblastoma. Moreover, recognition of the mechanisms of senescence and mitotic catastrophe in glioblastoma cells may be a step towards a potential new therapeutic approach.

BRAF inhibitors in BRAF-V600 mutated primary neuroepithelial brain tumors

Introduction: Primary neuroepithelial brain tumors encompass a wide variety of glial and glioneuronal neoplasms. Malignant tumors, tumors located in surgically inaccessible locations (e.g., eloquent brain areas, deep structures, brain stem) and recurrent or progressive tumors pose considerable treatment challenges and are candidates for novel therapeutics based on molecular insights. Small kinase inhibitors of v-RAF murine sarcoma viral oncogene homologue B1 (BRAF) have shown considerable antineoplastic activity in some tumor types harboring activating BRAF-V600 mutations (e.g., melanoma) and promising data are emerging on BRAF inhibitor therapy of mutation-bearing primary brain tumors. Areas covered: This review summarizes the available data on BRAF-V600 point mutations and the antineoplastic activity and toxicity profiles of BRAF inhibitors in neuroepithelial brain tumors including diffuse gliomas (glioblastomas, astrocytomas, oligodendrogliomas), pilocytic astrocytomas, pleomorphic xanthoastrocytomas and gangliogliomas. Expert opinion: Activating BRAF-V600 mutations are recurrently found in several glial and glioneuronal brain tumors and the available data indicate that BRAF inhibitors are active and well-tolerated in such tumors. Thus, BRAF inhibitors represent a novel and promising therapeutic opportunity that may alter the disease course of molecularly selected CNS neoplasms in a clinically meaningful way. However, so far the evidence is anecdotal and prospective clinical studies should be conducted.

Limited importance of the dominant-negative effect of TP53 missense mutations

BackgroundHeterozygosity of TP53 missense mutations is related to the phenomenon of the dominant-negative effect (DNE). To estimate the importance of the DNE of TP53 mutations, we analysed the percentage of cancer cases showing a single heterozygous mutation of TP53 and searched for a cell line with a single heterozygous mutation of this gene. This approach was based on the knowledge that genes with evident DNE, such as EGFR and IDH1, represent nearly 100% of single heterozygous mutations in tumour specimens and cell lines.MethodsGenetic analyses (LOH and sequencing) performed for early and late passages of several cell lines originally described as showing single heterozygous TP53 mutations (H-318, G-16, PF-382, MOLT-13, ST-486 and LS-123). Statistical analysis of IARC TP53 and SANGER databases. Genetic analyses of N-RAS, FBXW7, PTEN and STR markers to test cross-contamination and cell line identity. Cell cloning, fluorescence-activated cell sorting and SSCP performed for the PF-382 cell line.ResultsA database study revealed TP53 single heterozygous mutations in 35% of in vivo (surgical and biopsy) samples and only 10% of cultured cells (in vitro), although those numbers appeared to be overestimated. We deem that published in vivo TP53 mutation analyses are not as rigorous as studies in vitro, and we did not find any cell line showing a stable, single heterozygous mutation. G16, PF-382 and MOLT-13 cells harboured single heterozygous mutations temporarily. ST-486, H-318 and LS-123 cell lines were misclassified. Specific mutations, such as R175H, R273H, R273L or R273P, which are reported in the literature to exert a DNE, showed the lowest percentage of single heterozygous mutations in vitro (about 5%).ConclusionWe suggest that the currently reported percentage of TP53 single heterozygous mutations in tumour samples and cancer cell lines is overestimated. Thus, the magnitude of the DNE of TP53 mutations is questionable. This scepticism is supported by database investigations showing that retention of the wild-type allele occurs with the same frequency as either nonsense or missense TP53 mutations.

Validation of nuclear STAT6 immunostaining as a diagnostic marker of meningeal solitary fibrous tumor (SFT)/hemangiopericytoma

INTRODUCTION NAB2-STAT6 gene fusion is a molecular characteristic of solitary fibrous tumors (SFT) and hemangiopericytoma, underscoring their definition as one diagnostic entity. NAB2-STAT6 fusion is associated with nuclear relocation of STAT6 protein that can be detected by immunohistochemistry. We evaluated the diagnostic value of STAT6 expression in meningeal tumors. METHODS 77 meningeal tumors (17/77 (22.0%) SFT/hemangiopericytoma, 11/77 meningothelial meningioma, 10/77 atypical meningioma 8/77 chordoid meningioma, 9/77 fibroblastic meningioma, 10/77 transitional meningioma, 3/77 rhabdoid meningioma and 9/77 anaplastic meningioma) were included. STAT6 immunohistochemistry was performed on FFPE specimens using a fully automated slide-staining system and anti-STAT6 antibody SC-20:sc621. Two independent observers analyzed all specimens blinded to histological diagnoses, and a third observer was consulted in case of discordancy. RESULTS STAT6 immunohistochemistry yielded an exclusively nuclear immunostaining signal. 16/17 (94%) SFT/hemangiopericytoma specimens presented with clear-cut, wide-spread, and moderate to strong staining in tumor cell nuclei and were rated as STAT6-positive. In only 1 SFT case with weak and focal nuclear STAT6 immunostaining signal, STAT6 expression was rated discordant (observer 1: STAT6-negative, observers 2 and 3: STAT6-positive). All non-SFT/hemangiopericytoma cases were unanimously rated as STAT6-negative. In 76/77 (98.7%) cases the evaluation of STAT6 immunostaining results was in agreement among observers. CONCLUSION STAT6 immunohistochemistry is a robust method to verify diagnosis of SFT/hemangiopericytoma and should therefore be included in the diagnostic work-up of meningeal tumors. In singular cases, weak and focal STAT6 expression may lead to false-negative evaluation and may prompt further molecular work-up.
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Glioblastoma specimens with TP53 mutations do not show EGFRvIII amplification

Watanabe et al. described TP53 mutation and epidermal growth factor receptor (EGFR) amplification as being mutually exclusive (1). In 2003, however, Okada et al. (2) showed that peripheral or local EGFR amplification occurs in four out of six cases presenting with a TP53 mutation. They suggested that EGFR amplification occurs commonly in glioblastomas (GBMs) with TP53 mutations, although this amplification occurs in a very small proportion of cells (less than 1%). In 2008, Yoshimoto et al. (3) described a highly sensitive technique for detecting the EGFRvIII variant by means of specific RT-PCR. EGFRvIII, characterized by the deletion of 267 amino acids in the extracellular domain, is the most common EGFR variant in GBMs. Moreover, this mutation leads to the constitutive activation of the receptor and enhanced tumorigenic potential (4). EGFRvIII is generally observed in 30% of unselected GBMs and 60% of GBMs showing EGFR amplification, indicating that there is a close association between these two EGFR alterations (3,5,6). Moreover, to date, EGFRvIII has been detected almost exclusively in cases showing concurrent wild-type EGFR amplification (5,7,8). We decided to verify whether EGFRvIII amplification occurs frequently in a low percentage of glioblastoma cells with TP53 mutations. The presence of concurrent TP53 mutations and EGFRvIII would confirm the findings of Okada et al. (2). To this end, we searched for EGFRvIII by means of primer-specific RT-PCR in 80 samples of GBM. Twentythree cases showed TP53 mutations. Our data revealed no variant III in the 23 cases with TP53 mutations. Considering the fact that the sensitivity of the EGFRvIII RT-PCR technique is higher than that of the anti-EGFRvIII immunohistochemical assay (3), we conclude that none of the GBMs with a TP53 mutation had the EGFRvIII variant. It is possible that there are cases with TP53 mutations that have wild-type EGFR amplification, but GBMs with TP53 mutations do not present with amplification of variant III. EGFRvIII was detected in 60% of glioblastomas showing EGFR amplification (5). Okada et al. (2) showed concurrent EGFR amplification and TP53 mutation; however, they did not include information about the distribution of EGFR amplification (vIII type vs. EGFR wild type) in samples presenting with TP53 mutations. We recognize that the group of GBMs analyzed here showed a lower percentage of cases with EGFRvIII than that reported in the majority of articles to date (20% in our study vs. 30% in the majority of publications)

KINFix--A formalin-free non-commercial fixative optimized for histological, immunohistochemical and molecular analyses of neurosurgical tissue specimens.

An optimal fixative should ideally combine the advantages of formalin fixation and freezing, allowing for good preservation of histology and molecular components, easy handling and storage, lack of toxicity, and low costs. Most of these criteria are fulfilled by ethanol-based solutions, and due to our good experience with the commercial RCL2 fixative, reflected by our published single-center trial, we initiated a multicenter ring trial. However, during its course, RCL2 was discontinued on the market. Therefore, we created our own agent, KINFix, composed of the same main constituents as RCL2, and employed it in our laboratory with similar results. Here we present our evaluation of the three fixatives formalin, RCL2, and KINFix from the perspective of histopathology as well as nucleic acid and protein analyses in comparison to fresh frozen tissues together with the multicenter ring trial data for RCL2. We observe that RCL2 and KINFix offer comparable histomorphology and superior template for molecular analyses than formalin. Moreover, KINFix as freely available fixative might overcome some of the difficulties related to the commercial agents. Therefore, we conclude that KINFix might be an attractive complement to formalin in tissue processing and advocate its use in neuropathological practice.