Mateusz Banaszczyk

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.

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.

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.

EGFR Activation Leads to Cell Death Independent of PI3K/AKT/mTOR in an AD293 Cell Line

The Epidermal Growth Factor Receptor (EGFR) and its mutations contribute in various ways to tumorigenesis and biology of human cancers. They are associated with tumor proliferation, progression, drug resistance and the process of apoptosis. There are also reports that overexpression and activation of wild-type EGFR may lead to cell apoptosis. To study this phenomenon, we overexpressed in an AD293 cell line two most frequently observed forms of the EGFR receptor: wild-type and the constitutively active mutant–EGFR variant III (EGFRvIII). Then, we compared the effect of EGF stimulation on cell viability and downstream EGFR signaling. AD293 cells overexpressing wild-type EGFR, despite a significant proliferation increase in serum supplemented medium, underwent apoptosis after EGF stimulation in serum free conditions. EGFRvIII expressing cells, however, were unaffected by either serum starvation or EGF treatment. The effect of EGF was completely neutralized by tyrosine kinase inhibitors (TKIs), indicating the specificity of this observation. Moreover, apoptosis was not prevented by inhibiting EGFR downstream proteins (PI3K, AKT and mTOR). Here we showed another EGFR function, dependent on environmental factors, which could be employed in therapy and drug design. We also proposed a new tool for EGFR inhibitor analysis.

Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes.

Glioblastoma is the most common and malignant brain tumor, characterized by high cellular heterogeneity. About 50% of glioblastomas are positive for EGFR amplification, half of which express accompanying EGFR mutation, encoding truncated and constitutively active receptor termed EGFRvIII. Currently, no cell models suitable for development of EGFRvIII-targeting drugs exist, while the available ones lack the intratumoral heterogeneity or extrachromosomal nature of EGFRvIII. The reports regarding the biology of EGFRvIII expressed in the stable cell lines are often contradictory in observations and conclusions. In the present study, we use DK-MG cell line carrying endogenous non-modified EGFRvIII amplicons and derive a sub-line that is near depleted of amplicons, whilst remaining identical on the chromosomal level. By direct comparison of the two lines, we demonstrate positive effects of EGFRvIII on cell invasiveness and populational growth as a result of elevated cell survival but not proliferation rate. Investigation of the PI3K/Akt indicated no differences between the lines, whilst NFκB pathway was over-active in the line strongly expressing EGFRvIII, finding further supported by the effects of NFκB pathway specific inhibitors. Taken together, these results confirm the important role of EGFRvIII in intrinsic and extrinsic regulation of tumor behavior. Moreover, the proposed models are stable, making them suitable for research purposes as well as drug development process utilizing high throughput approach.

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)

Different mutational characteristics of TSG in cell lines and surgical specimens

One of the most crucial concerns of cancer research pertains to the differences between the neoplastic cells in tumor specimens in vivo and their counterparts in cell lines. The huge amount of results deposited in cancer genetic databases allows to address this issue from a wider perspective. Our analysis of the Sanger Institute Catalog Of Somatic Mutations In Cancer (COSMIC) database v61 showed a lower percentage of homozygous mutations in a group of tumor suppressor genes in surgical samples (in vivo) in comparison to their frequency in cell lines (in vitro). Similarly, the mutations resulting in the lack of protein (e.g., nonsense mutations or whole gene deletions) of several tumor suppressor genes (TSGs) were more frequently observed in vitro than in vivo. In this article, we suggest two potential explanations of these data. Firstly, TSG heterozygous mutations resulting in the modified protein (e.g., missense mutations) may be gradually (when the specific molecular context is achieved) changed to homozygous mutations resulting in the lack of protein during carcinogenesis. Secondly, among different independent pathways of tumorigenesis, those leading to homozygous nonsense mutations are characteristic for cells which are more efficiently stabilized in vitro. To conclude, these observations may be interesting for researchers working with cell line in vitro models illustrating the extent to which they reflect the tumors in vivo.

PIN3 duplication may be partially responsible for TP53 haploinsufficiency

BackgroundPreviously we have suggested that cancer cells develop a mechanism(s) which allows for either: silencing of the wild-type TP53 transcription, degradation of the wild-type TP53 mRNA, or selective overproduction of the mutated TP53 mRNA, which is the subject of this article. Sequencing of TP53 on the respective cDNA and DNA templates from tumor samples were found to give discordant results. DNA analysis showed a pattern of heterozygous mutations, whereas the analysis of cDNA demonstrated the mutated template only. We hypothesized that different TP53 gene expression levels of each allele may be caused by the polymorphism within intron 3 (PIN3). The aim of this study was to test if one of the polymorphic variants of PIN3 (A1 or A2) in the heterozygotes is associated with a higher TP53 expression, and therefore, responsible for the haploinsufficiency phenomenon.Methods250 tumor samples were tested. To analyze the involvement of PIN3 polymorphic variant (A1 or A2) on TP53 mRNA expression regulation, bacterial subcloning combined with sequencing analyses, dual luciferase reporter assays and bioinformatic analysis were performed.ResultsHaplotype analysis showed the predominance of the mutated template during the cDNA sequencing in all samples showing a heterozygous TP53 mutation and PIN3 heterozygosity. Out of 30 samples (from the total of 250 tested samples) which carried TP53 mutations and had a bias in allelic expression 6 were heterozygous for the A1/A2 polymorphism, and all 6 (p = 0.04) samples carried the mutation within the PIN3 longer allele (A2). Reporter assays revealed higher luciferase activity in cells transfected with the plasmid containing A2 construct than A1 and control. A2/A1 ratio ranged from 1.16 for AD293 cell line (p = 0.019) to 1.59 for SW962 cell line (p = 0.0019). Moreover, bioinformatic analyses showed that PIN3 duplication stabilized secondary DNA structures – G-quadruplexes.ConclusionTP53 alleles are not equivalent for their impact on the regulation of expression of TP53 mRNA. Therefore, in PIN3-heterozygous cases a single TP53 mutation of the longer allele might sufficiently destabilize its function. Secondary DNA structures such as quadruplexes can also play a role in PIN3-dependent TP53 haploinsufficiency.