Quantitative analysis of 2-hydroxyglutarate as a controversial oncometabolite in malignant gliomas.

There is a great effort to connect the accumulation of 2-hydroxyglutarate (2-HG) oncometabolite with cellular onco-epigenetic status and subsequently predict the prognoses of glioma patients. In this observational study, the concentrations of D- and L- 2-HG were determined in 57 tumor tissue samples of glioma patients (n=57) WHO grade I through IV (astrocytoma, oligodendroglioma, secondary glioblastoma, and glioblastoma multiforme) in vitro. Also, genetic mutation status on isocitrate dehydrogenase 1 and 2 (IDH 1/2) was determined from these samples. The objective of this study was to confirm or to reject the hypothesis of the direct correlation of 2-HG concentration in tumor tissue and the results from IDH 1/IDH 2 point mutation analyses. The concentrations of 2-HG were quantified using high sensitive HPLC and Q-TOF HRMS spectrometer setup. Concurrently, the genetic mutation analyses of both IDH 1 (cytosolic) and IDH 2 (mitochondrial) were performed by the isolation of tumor tissue DNA, PCR amplification, and subsequent Sanger forward sequencing. Our results indicate that there is no definite correlation between the two as we identified cases of glioma tumors with significantly increased concentration of one or both L- and D- 2-HG but no IDH 1/2 mutations (44% 2-HG positive cases).

Detection of driver mutations in FFPE samples from patients with verified malignant melanoma.

Malignant melanoma is an oncological disease characterized by etiologic heterogeneity and it has increasing incidence and mortality in the Slovak Republic. While it is treated surgically in combination with chemotherapy, targeted therapy, and immunotherapy, malignant melanomas can ulcerate and are susceptible to infections. These are highly aggressive cancers with metastasis, and recent studies have shown the presence of mutations in RAC1, PPP6C and STK19 genes in melanoma patients. Mutations in these genes are driver mutations; important in oncogenesis and providing selective advantage to tumor cells. The aim of our study is to establish a method to detect driver mutations in formalin-fixed, paraffin embedded (FFPE) tissue DNA. We applied Sanger sequencing to detect driver somatic mutations in RAC1, PPP6C, STK19 and BRAF genes in patients with malignant melanoma. Confirmation of BRAF V600E mutation was obtained by allele-specific PCR. The BRAF V600E mutation was present in 15 of 113 patients (13.2%) and the driver mutation in 7 of 113 patients (6.2 %). Our results demonstrate that Sanger sequencing analysis detects mutations in FFPE clinical samples. The identification of these somatic driver mutations in samples with verified malignant melanomas enabled development of a molecular classification of melanomas, and our study provides evidence of diversity of novel driver mutations implicated in malignant melanoma pathogenesis. These findings could have very important implications for targeted therapy.

Epigenetics: an alternative pathway in GISTs tumorigenesis.

Many diseases have different pathological backgrounds responsible for abnormal cell behavior and exhibiting altered function and signal transduction. This is especially true for tumors and although changes affecting DNA sequence, irreversible mutations and chromosomal aberrations in gastrointestinal stromal tumors (GISTs) have been widely studied, the importance of reversible epigenetic changes increasingly recognized in many cancers has received insufficient attention in these tumors. Epigenetic mechanisms are part of normal development and gene expression under normal conditions, but malfunction of these processes leads to malignant transformation by disturbing both intra- and intercellular communication. GISTs are a specific group of gastrointestinal tract tumors resistant to conventional chemotherapy and radiotherapy. Although they account for only 1% to 2% of tumors, they are among the most widespread gastrointestinal mesenchymal tumors. DNA hyper/hypomethylation overexpression/underexpression of miRNAs or abnormal histone modification may provide an alternative to the genetic modifications responsible for GIST pathology, response to treatment, prognosis and overall survival. This review summarizes the known epigenetic mechanisms involved in GIST pathogenesis; including onset, progression, and GISTs resistance. Reversible epigenetic changes are a novel and appropriate approach to halt the spread of metastases and the emergence of resistance in GIST treatment, and these changes depend on the type of epigenetic alternation, including inhibitors of histone acetyltranferase and deacetylase and DNA methyltransferases.