Izabela Zawlik

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.

Regulation of PrPC expression: nerve growth factor (NGF) activates the prion gene promoter through the MEK1 pathway in PC12 cells.

A high expression of PrP(C) in cells is one factor that increases the risk of conversion to the misfolded, disease-associated form (PrP(Sc)) characteristic of transmissible spongiform encephalopathies. Thus, developing a method to control the level of PrP(C) expression in cells could be one way to delay or prevent the onset of clinical signs of these diseases. In this study the mechanisms controlling the expression of the Prnp gene in PC12 cells and in rat brain were examined. We observed a slight activation of a cloned fragment of the human PRNP gene promoter using the luciferase reporter system in PC12 cells stimulated with nerve growth factor (NGF). The activating effect of NGF was enhanced by mitogen-activated protein kinase (MEK1) and suppressed by myristylated serine/threonine kinase (myrAKT). These results suggest that MEK1 is a positive activator of the PRNP promoter that inhibits the AKT pathway. Independent experiments suggested that high expression of PrP(C) in the brain depends on the rate of translation and/or the efficiency of PrP(C) stabilization. We also investigated the epigenic status of the Prnp promoter. We observed no increase of PrP(C) or Prnp mRNA levels in PC12 cells after treatment with the DNA-demethylating agent. The Prnp promoter did not display methylation either in NGF-treated and untreated PC12 cells, or in the rat brain. These results improve the understanding of the regulation of the Prnp gene promoter, a DNA regulatory element controlling the expression of PrP(C), a protein involved in several neurological diseases.

Earlier Onset of Alzheimer's Disease: Risk Polymorphisms Within PRNP, PRND, CYP46, and APOE Genes

We studied eight polymorphisms within APOE, PRNP, PRND, and CYP46 genes in 213 Polish late-onset patients with Alzheimers disease (AD) and 171 non-demented elderly controls. A latent classification approach, grade-of-membership analysis, was taken to identify three extreme pure type risk sets defined by the probabilities of being affected with AD and for genotypes found at the examined genes. Sets I and II represented high intrinsic risk, having a higher density of various genotypes compared to set III, at low intrinsic risk. A gradient of onset age depending on membership in the risk sets was also observed. Logistic regression analysis showed that the highest risk for AD was found for individuals who co-inherited APOE epsilon4 allele, PRNP codon 129 homozygosity, PRND codon 174 Thr allele, and CYP46 rs754203 g allele. AD can be influenced by genetic profiles leading to appearance of the disease, composed of genes which separately evoke a little or unnoticeable effect. Moreover, there may be multiple sufficient risk sets for AD. Looking at multiple genes together rather than analyzing them individually, may improve identification of risk alleles.

Apoptosis-, proliferation, immune function-, and drug resistance- related genes in ER positive, HER2 positive and triple negative breast cancer.

The aim of our study was to examine an association between gene expression assessed using a 23-gene microarray and receptor status of breast cancer samples categorized as ER positive, HER2 positive and triple negative subtypes. The ER positive cohort was subsequently divided into Luminal A, Luminal B HER2 negative and Luminal B HER2 positive subtypes. Core- needle biopsies were collected from 78 female patients with inoperable locally advanced breast cancer or resectable tumors suitable for downstaging, before any treatment. Expressions of 23 genes were determined by means of TagMan Low Density Arrays. Analysis of variance was used to select genes with discriminatory potential between receptor subtypes. We introduced a correction for false discovery rates (presented as q values) due to testing multiple hypothesis. Pairwise post-hoc comparisons of receptor subtypes were performed using Tukey s HSD test. Five genes out of a 23-gene microarray differed significantly in relation to breast cancer receptor-based subtypes. Among these five genes, we identified: BCL2 (p=0.0002, q=0.0009), MKI67 (p=0.0037, q=0.0064), IGF1R (p=0.0040, q=0.0064), FOXC1 (p=0.0113, q=0.0135) and IRF1 (p=0.0435, q=0.0416) as ones showing ER positive, HER2 positive and triple negative -subtype specific expression profiles. When incorporating Luminal A, Luminal B HER2 negative, Luminal B HER2 positive subtypes into analysis, four genes: BCL2 (p=0.0006, q=0.0034), MKI67 (p=0.0078, q=0.0198), FOXC1 (p=0.0102, q=0.0198) and IGF1R (p=0.0174, q=0.0254) were selected. Elevated levels of IGF1R and BCL2 were significantly linked with Luminal A subtype. Triple negative breast cancer subtype was associated with higher expression of IRF1, FOXC1 and MKI67. In HER2 positive cohort lower expression of all five analyzed genes was noted.

Elimination of wild-type P53 mRNA in glioblastomas showing heterozygous mutations of P53

We screened 50 glioblastomas for P53 mutations. Five glioblastomas showed heterozygous mutations, while three were putatively heterozygous. Six of these eight glioblastomas showed elimination of wild-type P53 mRNA. These results strongly suggest that some sort of mechanism(s) favouring mutated over wild-type P53 mRNA exists in glioblastoma cells with heterozygous mutations of this gene.

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

BackgroundRecently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers.MethodsTo this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry.ResultsWe 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.ConclusionIn 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.

Association of microRNA-93, 190, 200b and Receptor Status in Core Biopsies from Stage III Breast Cancer Patients

Oncologists now favor more personalized treatment strategies in breast cancer patients. Gene expression analysis has been widely used, but less is known about epigenetic factors, for example, microRNAs (miRNAs). The aim of this study was to determine the relationship between selected miRNAs and receptor status in core biopsies sampled before preoperative chemotherapy in stage III locally advanced breast cancer (LABC) patients. In 37 LABC core biopsies, three miRNAs per sample were analyzed: hsa-miR-93-5p, hsa-miR-190a, and hsa-miR-200b-3p, and hsa-miR-103a-3p as an endogenous control (TaqMan(®) RT-PCR; Applied Biosystems). Receptor status was determined by a dedicated pathologist. The Mann-Whitney U, Shapiro-Wilk, and Levenes tests were used to compare related samples. Levels of miRNA-93 differed significantly in core biopsies of LABC patients with different expressions of ER (estrogen receptor) and PR (progesterone receptor). Higher levels of miRNA-93 were found in ER-negative (p=0.0027) and PR-negative patients (p=0.0185). Levels of miRNA-190 and 200b did not differ significantly in core biopsies of LABC patients who expressed ER and PR differently (p=0.7727, p=0.9434, p=0.6213, and p=0.1717). Levels of miRNA-93, 190, and 200b were not significantly different in core biopsies of LABC patients with different HER2 (human epidermal growth factor 2) expressions (p=0.8013, p=0.2609, and p=0.3222). The assessment of core biopsy miRNA profiles and receptor-based subtypes may identify new signaling pathways for improved breast cancer classification.

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.

Immune checkpoints in aggressive breast cancer subtypes.

Immune checkpoints are molecules referred to inhibitory pathways in the immune system that play a pivotal role in prevention of autoimmunity and oncogenesis. The aim of the study was to evaluate expression levels of selected immune checkpoints- PD-1 (programmed cell death protein 1), and PD-L1 (programmed cell death 1 ligand 1) in breast cancer patients, suitable for breast conservation and sentinel node biopsy and determine their associations with clinicopathological factors.Expression of the genes coding for PD-1 and PD-L1 was analyzed in formalin-fixed paraffin-embedded specimens using real-time PCR. mRNA expression levels were determined using beta actin (ACTB) as an endogenous control. There was a trend towards significance between higher PD-1 and PD-L1 levels in triple negative breast cancers (p=0.1). Higher PD-L1 expression was also found in aggressive breast cancer subtypes e.g. triple negative and HER2 (human epidermal growth factor receptor 2) -positive as compared with subtypes with better prognosis such as luminal A and luminal BHER2-negative (p=0.05). There was a trend towards significance in higher PD-1 levels in triple negative and HER-2 positive breast cancers (p=0.1). A statistically significant difference was found between PD-L1 expression and tumor grade (p=0.01). Elevated PD-L1 levels were noted in G3 tumors. Immunogenicity appears to be gaining importance in triple negative and HER2-positive molecular subtypes of breast cancer, and the results in this study provide a basis for further investigation into the role of immune checkpoints in breast cancer.

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.