Acquired resistance or tolerance? - in search of mechanisms underlying changes in the resistance profile of Candida albicans and Candida parapsilosis as a result of exposure to methotrexate.

The increasing prevalence of fungal strains showing acquired resistance and multidrug resistance is an increasing therapeutic problem, especially in patients with a severely weakened immune system and undergoing chemotherapy. What is also extremely disturbing is the similarity of the resistance mechanisms of fungal cells and other eukaryotic cells, including human cells, which may contribute to the development of cross-resistance in fungi in response to substances used in e.g. anticancer treatment. An example of such a drug is methotrexate, which is pumped out of eukaryotic cells by ABC transmembrane transporters - in fungi, used to remove azoles from fungal cells. For this reason, the aim of the study was to analyze the expression levels of genes: ERG11, MDR1 and CDR1, potentially responsible for the occurrence of cross-resistance in Candida albicans and Candida parapsilosis as a result of fungal exposure to methotrexate (MTX). In vitro exposure of C. albicans and C. parapsilosis strains to methotrexate showed a high increase in resistance to fluconazole and a partial increase in resistance to voriconazole. Analysis of the expression of resistance genes showed varied responses of the tested strains depending on the species. In the case of C. albicans, an increase in the expression of the MDR1 gene was observed, and a decrease in ERG11 and CDR1. However, for C. parapsilosis there was an increase in the expression of the CDR1 gene and a decrease in ERG11 and MDR1. We noted the relationship between the level of resistance to voriconazole and the level of ERG11 gene expression in C. albicans. This indicates that this type of relationship is different for each species. Our research confirms that the mechanisms by which fungi acquire resistance and develop cross-resistance are highly complex and most likely involve several pathways simultaneously. The emergence of multidrug resistance may be related to the possibility of developing tolerance to antimycotics by fungi.

Cytokine and LL-37 gene expression levels in Bartonella spp. seropositive and seronegative patients of a rheumatology clinic.

PURPOSE: The variation in the immune response to Bartonella spp. infection in humans remains unclear. The present study compares the expression of selected interleukins, cytokines and cathelicidin (LL-37) in rheumatology clinic patients suffering from musculoskeletal symptoms with healthy blood donors. The patients had previously been tested for the presence of Bartonella henselae antibodies. METHODS: Gene expression of LL-37, interleukin (IL)-2, IL-4, IL-6, IL-12, interferon-(IFN)-γ, and tumor necrosis factor (TNF-α)-α was determined in blood samples using quantitative Polymerase Chain Reaction (qPCR). Statistical analysis was prepared with STATISTICA. RESULTS: Statistically significant differences in the mRNA levels of the tested cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-12; p<0.0001) were observed between the healthy controls and patients; however, no difference was observed for LL37 mRNA (p â€‹= â€‹0.1974). No significant differences in mRNA expression were observed between IgG in anti-Bartonella seropositive and seronegative individuals (p>0.05). The only significant differences between the Bartonella spp. DNA positive and negative patients, indicated by PCR, were observed for TNF-α and IL-12 mRNA (p â€‹= â€‹0.0045 and p â€‹= â€‹0.0255, respectively). CONCLUSION: A broadly similar immune response to the tested cytokines was observed among the participants irrespective of anti-Bartonella spp. IgG seropositivity. However, the Bartonella DNA-positive participants demonstrated significantly lower expression of IL-12 and TNF-α mRNA; this may indicate that these bacteria have a suppressive influence on the immune system.

Evaluation of Changes to the Oral Microbiome Based on 16S rRNA Sequencing among Children Treated for Cancer.

A child's mouth is the gateway to many species of bacteria. Changes in the oral microbiome may affect the health of the entire body. The aim of the study was to evaluate the changes in the oral microbiome of childhood cancer survivors. Saliva samples before and after anti-cancer treatment were collected from 20 patients aged 6-18 years, diagnosed de novo with cancer in 2018-2019 (7 girls and 13 boys, 7.5-19 years old at the second time point). Bacterial DNA was extracted, and the microbial community profiles were assessed by 16S rRNA sequencing. The relative abundances of Cellulosilyticum and Tannerella genera were found to significantly change throughout therapy (p = 0.043 and p = 0.036, respectively). However, no differences in the alpha-diversity were observed (p = 0.817). The unsupervised classification revealed two clusters of patients: the first with significant changes in Campylobacter and Fusobacterium abundance, and the other with change in Neisseria. These two groups of patients differed in median age (10.25 vs. 16.16 years; p = 0.004) and the length of anti-cancer therapy (19 vs. 4 months; p = 0.003), but not cancer type or antibiotic treatment.

MiR-21, miR-34a, miR-125b, miR-181d and miR-648 levels inversely correlate with MGMT and TP53 expression in primary glioblastoma patients.

INTRODUCTION: TP53 and MGMT alterations play a crucial role in glioblastoma (GB) pathogenesis. TP53 and MGMT function is affected by several pathologic mechanisms, such as point mutations or promoter methylation, which are well characterized. Expression of both genes can be regulated by other mechanisms as well, e.g., microRNAs (miRNAs). Moreover, cross-talk among various pathologic processes may occur, further affecting MGMT and TP53 functionality. MATERIAL AND METHODS: In 49 GB patients, we analyzed the possible associations between TP53 and its miRNA regulators miR-125b, miR-21, and miR-34a, as well as MGMT and its miRNA regulators miR-181d and miR-648. We evaluated the possible influence of mutational and methylation status on the pre-identified associations. RESULTS: In patients with immunohistochemistry-detected TP53 overexpression, expression levels of miR-34a and TP53 were negatively correlated (r = -0.56, p = 0.0195), and in patients with TP53 mutations, expression levels of TP53 and miR-21 were negatively correlated (r = -0.67, p = 0.0330). In patients with MGMT methylation, expression levels of MGMT were negatively correlated with miR-648 and miR-125b expression levels (r = -0.61, p = 0.0269 and r = -0.34, p = 0.0727, respectively). CONCLUSIONS: Our findings demonstrate that selected miRNAs are significantly correlated with MGMT and TP53 levels, but the extent of this correlation differs regarding the TP53 and MGMT mutational and promoter methylation status.

Dysregulation of microRNAs in triple-negative breast cancer.

OBJECTIVES: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited treatment options and poor prognosis. TNBC is usually diagnosed at a relatively young age and is characterized by high risk of developing metastases. Some epigenetic regulation of gene expression is associated with TNBC. Expression of microRNAs (miRNAs) can serve as a potential tool for identifying critical biomarkers in TNBC. The aim of our study is to examine expression of selected miRNAs in TNBC and to assess the relationship between miRNA expression and clinicopathological factors. MATERIAL AND METHODS: Expression levels of 19 selected miRNAs were compared between cancerous and normal breast tissues by use of qPCR method. We have evaluated the relationship between the expression level of miRNAs and clinicopathological factors such as: age, tumor size and lymph node status. RESULTS: We found that in TNBC tissues, when compared with normal breast tissues, the expression of miR-190a, miR- 136-5p and miR-126-5p was significantly reduced (p = 0.0041, p = 0.0007, p = 0.0007, respectively) whereas expression of miR-135b-5p and miR-182-5p was significantly increased (p = 0.0194, p = 0.0041, respectively). We found a linear trend for tumor size and expression of miR-126-5p (p = 0.0296) and miR-135b-5p (p = 0.0241). CONCLUSIONS: Our study confirms that miRNA expression profile is dysregulated in TNBC patients compared to healthy controls. MiR-190a, miR-136-5p, miR-126-5p, miR-135b-5p and miR-182-5p may be associated with development and progression of TNBC.

Genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia cell line.

Thorough examination of genetic heterogeneity of cell lines is uncommon. In order to address this issue, the present study analyzed the genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia (T-ALL) cell line. For this purpose, traditional techniques such as fluorescence in situ hybridization and immunocytochemistry were used, in addition to more advanced techniques, including cell sorting, Sanger sequencing and massive parallel sequencing. The results indicated that the RPMI-8402 cell line consists of several genetically different cell subpopulations. Furthermore, massive parallel sequencing of RPMI-8402 provided insight into the evolution of T-ALL carcinogenesis, since this cell line exhibited the genetic heterogeneity typical of T-ALL. Therefore, the use of cell lines for drug testing in future studies may aid the progress of anticancer drug research.

PIN3 duplication may be partially responsible for TP53 haploinsufficiency.

BACKGROUND: Previously 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. METHODS: 250 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. RESULTS: Haplotype 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. CONCLUSION: TP53 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.

TP53 promoter methylation in primary glioblastoma: relationship with TP53 mRNA and protein expression and mutation status.

Reduced expression of TP53 by promoter methylation has been reported in several neoplasms. It remains unclear whether TP53 promoter methylation is associated with reduced transcriptional and protein expression in glioblastoma (GB). The aim of our work was to study the impact of TP53 methylation and mutations on TP53 mRNA level and protein expression in 42 molecularly characterized primary GB tumors. We also evaluate the impact of all molecular alterations on the overall patient survival. The frequency of TP53 promoter methylation was found in 21.4%. To the best of our knowledge, this is the first report showing such high frequency of TP53 promoter methylation in primary GB. There was no relation between TP53 promoter methylation and TP53 mRNA level (p=0.5722) and between TP53 promoter methylation and TP53 protein expression (p=0.2045). No significant associations were found between TP53 mRNA expression and mutation of TP53 gene (p=0.9076). However, significant association between TP53 mutation and TP53 protein expression was found (p=0.0016). Our data suggest that in primary GB TP53 promoter methylation does not play a role in silencing of TP53 transcriptional and protein expression and is probably regulated by other genetic and epigenetic mechanisms associated with genes involved in the TP53 pathway.

Astrocytoma-associated antigens - IL13Rα2, Fra-1, and EphA2 as potential markers to monitor the status of tumour-derived cell cultures in vitro.

BACKGROUND: The molecular heterogeneity of high-grade astrocytomas underlies the difficulties in the development of representative and valuable in vitro experimental models for their studies. The purpose of our study was to estimate the value of astrocytoma-associated antigens (AAAs) - IL13Rα2, Fra-1, EphA2 - and the most common molecular aberrations typical for astrocytomas as potential markers to screen the status of tumour-derived cell cultures in vitro. METHODS: The tumour-derived cell cultures were established from high-grade astrocytomas. The expression analyses of the tested genes were performed via semi-quantitative real-time PCR and subsequently verified by immunohistochemical and immunocytochemical technique. The analyses of molecular aberrations at DNA level included gene dosage status evaluation based on real-time PCR, sequencing analysis, and loss of heterozygosity (LOH) assay. RESULTS: The expression analyses based on semi-quantitative real-time PCR showed that in the final stage of culture the expression level of all tested AAAs was significantly higher or at least comparable to that of primary tumours; however, two expression patterns were observed during cell culture establishment. Analysis at the single cell level via immunocytochemistry also demonstrated an increase of the level of tested proteins and/or selection of tumour cell populations strongly positive for AAAs vs. other cell types including admixed non-tumoural cells. Confrontation of AAA expression data with the results of molecular analyses at DNA level seems to support the latter, revealing that the expression pattern of astrocytoma-associated antigens in tumour-derived cells in subsequent stages of culture is convergent with changes in the molecular profile of examined cell populations. CONCLUSIONS: The consistency of the obtained results seems to support the use of the selected AAAs, in particular IL13Rα2 and Fra-1, as tools facilitating the establishment of tumour-derived cultures. However, the intratumoural heterogeneity of high-grade astrocytomas may require further detailed characterisation of the molecular profile of a tumour in order to evaluate the value of the experimental model in relation to the individual context of particular studies.

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.

Molecular alterations in meningiomas: association with clinical data.

The aim of our study was to evaluate the frequency of deletions on chromosomes 1, 9, 10, 14, 18 and 22 in 75 benign and 15 atypical meningiomas and correlate them with clinical findings. Paired normal and tumor DNA samples were analyzed for loss of heterozygosity (LOH), using 24 microsatellite markers and PCR techniques. Statistical analysis showed that deletions on chromosomes 14 and 18 were significantly associated with tumor grade of meningiomas (p = 0.048 and p = 0.03, respectively). In addition, we found a marginally increased frequency of LOH on chromosome 9 in atypical meningiomas (p = 0.06). Interestingly, LOH on chromosome 14 was significantly associated with tumor size (p = 0.049), as the risk of developing a tumor of more than 4 cm in diameter was 6-times the risk of developing tumor with diameter below 4 cm. The most frequent genetic abnormality in meningiomas is 22 LOH, which seems to be confirmed by the present study in which high frequency of such abnormality was observed (67%). We found associations between chromosome 22 status and histological subtype. LOH on chromosome 22 was more frequent in fibrous meningiomas than in the meningothelial variant (p = 0.001). Besides that, we found a relationship between 22 LOH status and tumor localization: the frequency of LOH in skull base-localized tumors was significantly lower compared to parasagittal meningiomas (p = 0.0004). Our results indicated that allelic loss on chromosomes 9, 10, 14, 18 and 22 may be associated with meningioma pathogenesis and progression.

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

BACKGROUND: Heterozygosity 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. METHODS: Genetic 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. RESULTS: A 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%). CONCLUSION: We 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.

Detection of P53 mutations in different cancer types is improved by cDNA sequencing.

Recently published data show discrepancies between P53 cDNA and DNA sequencing results in glioblastoma, colorectal cancer and pleomorphic xanthoastrocytoma. We hypothesized that similar discrepancies are observed in other types of human cancers. Using DNA and cDNA direct sequencing, we analyzed 40 cases of invasive breast duct carcinoma, 23 cases of acute myeloblastic leukaemia, 12 cases of astrocytoma and 40 cases of soft tissue sarcoma for P53 mutations. Additionally, we used real-time quantitative PCR to estimate the normalized relative P53 expression. In the comparative study, the P53 mutation was detected more frequently when using cDNA sequencing than DNA sequencing in all of the cancer types. Furthermore, several samples presented missense P53 mutations, with visible wild-type nucleotide on the DNA sequence. In contrast, elimination of the wild-type allele or selective overproduction of the mutated allele was observed on the cDNA sequence. P53 expression was not significantly different between the cases with or without P53 mutations. These results indicate that cDNA sequencing improves the detection of P53 mutations in these cancers. We suggest that the true incidence of P53 mutations in these cancers is underestimated at the DNA level, and evaluation of the alteration should be carried out using cDNA analysis.

Prevalence of mutated TP53 on cDNA (but not on DNA template) in pleomorphic xanthoastrocytoma with positive TP53 immunohistochemistry.

Pleomorphic xanthoastrocytoma (PXA) is a tumor of astrocytic lineage that may have anaplastic features; such a phenotype usually correlates with a less favorable outcome. The molecular profile of PXA differs from other astrocytic tumors, but the molecular mechanisms of its formation and progression are still undefined. We analyzed a loss of heterozygosity and mutations of the SMARCB1 (also known as SNF5 or INI1) and TP53 genes in four cases of PXA. In just one case a TP53 mutation (Cys238Tyr) was readily detectable at the mRNA level, but it was almost undetectable during DNA sequencing. This case was also the only one exhibiting anaplastic features and TP53 overexpression as defined by immunohistochemistry. This observation supports our earlier findings on discrepancies in TP53 status in glioblastomas. We suggest that the incidence of TP53 mutations in pleomorphic xanthoastrocytoma may be underestimated and that molecular approaches should be used for greater diagnostic precision.

cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer.

BACKGROUND: Recently published data showed discrepancies between P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. METHODS: To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. RESULTS: We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. CONCLUSION: In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis.

Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors.

BACKGROUND: Although features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed. METHODS: Following methods were used to compare glioblastoma cells and GFAP+NNP (NHA): exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells EGFR amplification analysis, LOH/MSI analysis, and P53 nucleotide sequence analysis were performed. RESULTS: In vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP). Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8), as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum. CONCLUSION: Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present in vitro multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.

High incidence of MGMT promoter methylation in primary glioblastomas without correlation with TP53 gene mutations.

O(6)-methylguanine DNA methyltransferase (MGMT) reduces cytotoxicity of therapeutic or environmental alkylating agents. MGMT promoter methylation has been associated with TP53 G: C to A:T transition mutations in various types of cancers, and with poor prognosis in patients who did not receive chemotherapy. Mutations of TP53 are more frequent in secondary than in primary glioblastoma, thus the expected MGMT promoter methylation was low in primary glioblastoma. Glioblastoma patients with MGMT promoter methylation showed better response to chemotherapy based on alkylating agents and longer survival than patients without MGMT methylation. We examined 32 primary glioblastomas, treated with radiotherapy and surgery, for TP53 mutation by direct sequencing and MGMT promoter methylation by methylation-specific PCR. MGMT promoter methylation and TP53 mutations were detected in 72% and 31% of primary glioblastoma, respectively. Although not statistically significant, the frequency of TP53 G:C to A:T mutations were higher in cases with (26%) than without (11%) MGMT promoter methylation (p=0.376). MGMT promoter methylation had no impact on patient survival. Our data indicate that MGMT promoter methylation occurs frequently in primary glioblastoma, but does not lead to G:C to A:T TP53 mutations, has no independent prognostic value and is not a predictive marker unless glioblastoma patients are treated with chemotherapy.

Diverse molecular pattern in a bihemispheric glioblastoma (butterfly glioma) in a 16-year-old boy.

Glioblastoma multiforme (GBM), the most common malignant brain tumor of adults, is relatively rare in children. In a GBM affecting a 16-year-old boy, the tumor spread across the corpus callosum (butterfly glioma). This type of bilateral hemispheric growth has previously been thought to result from spread along the white matter tracts. Two samples obtained from opposite sides of the same tumor were analyzed comprehensively for loss of heterozygosity (LOH) and microsatellite instability (MSI). Amplification of EGFR and MDM2 was studied by means of multiplex polymerase chain reaction. Exons 5, 6, 7, and 8 of TP53 were screened for mutations by sequencing. In neither specimen were molecular alterations found in the EGFR, MDM2, or TP53 genes. The specimen obtained from the right hemisphere exhibited a high level of MSI and LOH in chromosome arms 5q, 9p, and 13q. The specimen from the left hemisphere exhibited LOH in chromosome arms 3p, 5q, 9p, 9q, 10p, 10q, and 13q. Here we propose four plausible hypothetical scenarios underlying the tumorigenesis of this GBM.

KCTD11 expression in medulloblastoma is lower than in adult cerebellum and higher than in neural stem cells.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood, and the most frequent associated genetic alteration is loss of heterozygosity on chromosome region 7p13. Two genes mapping to this region, KCTD11 (alias REN) and HIC1, have been proposed as involved in MB pathogenesis. We used real-time polymerase chain reaction in 20 tissue samples of primary MB to examine the transcriptional level of the two genes, with reference to two types of controls: adult cerebellum and fetal neural stem cells. A significant reduction of KCTD11 expression relative to adult normal cerebellum was detected in 14 of 20 (70%) of MB samples. Neural stem cells had even lower levels of KCTD11 expression than did MB. HIC1 gene expression was low ( approximately 100 times lower than KCTD11 expression) in MB, and low also in both adult cerebellum and neural stem cells. Hypermethylation of the 5'UTR or the central region of HIC1 (or both) was detected in a significant number of MB samples, as well as in cerebellum and neural stem cells. Our data suggest that KCTD11 may play an important role in MB tumorigenesis, but do not support the role of HIC1 in this tumor development. We argue that recognition of the gene or genes important in MB tumorigenesis depends in part on defining an appropriate control.