Petr Hrabal

Cytogenetic analyses in 81 patients with brain gliomas: correlation with clinical outcome and morphological data

Specific gene mutations, loss of heterozygosity, deletions and/or amplifications of entire chromosomal regions and gene silencing have been described in gliomas. 82 samples from 81 patients were investigated to detect the deletion of TP53, RB1, CDKN2A genes, deletion of 1p36 and 19q13.3 region, amplification of EGFR gene, trisomy of chromosome 7 and monosomy of chromosome 10 in glial cells. Dual-colour interphase fluorescence in situ hybridization (I-FISH) with locus-specific and/or chromosome enumeration DNA probes were used for cytogenetic analyses. In the study, molecular cytogenetic analyses were successfully performed in 74 patients (91.3%) and were uninformative in 7 only (8.7%). The cytogenetic analyses were correlated with morphological data and clinical outcome. I-FISH was the essential part of diagnostics. In comparison with the clinical data, the patients’ age seems to be a factor more important for the overall survival, rather than cytogenetic findings in glial tumours. The combined deletion of 1p36 and 19q13.3 chromosomal regions predicts longer overall survival for patients with oligodendroglial tumours.

Co-expression of the homologous proteases fibroblast activation protein and dipeptidyl peptidase-IV in the adult human Langerhans islets.

Abstract Fibroblast activation protein (FAP, seprase, EC 3.4.21.B28) and dipeptidyl peptidase-IV (DPP-IV, CD26, EC 3.4.14.5) are homologous serine proteases implicated in the modulation of the bioavailability and thus the function of a number of biologically active peptides. In spite of their generally nonoverlapping expression patterns, DPP-IV and FAP are co-expressed and probably co-regulated in certain cell types suggesting that for some biological processes their functional synergy is essential. By an in situ enzymatic activity assay, we show an abundant DPP-IV-like enzymatic activity sensitive to a highly specific DPP-IV inhibitor sitagliptin and corresponding DPP-IV immunoreactivity in the adult human islets of Langerhans. Moreover, the homologous protease FAP was present in the human endocrine pancreas and was co-expressed with DPP-IV. DPP-IV and FAP were found in the pancreatic alpha cells as determined by the co-localization with glucagon immunoreactivity. In summary, we show abundant enzymatic activity of the canonical DPP-IV (CD26) in Langerhans islets in the natural tissue context and demonstrate for the first time the co-expression of FAP and DPP-IV in pancreatic alpha cells in adult humans. Given their ability to proteolytically modify several biologically active peptides, both proteases have the potential to modulate the paracrine signaling in the human Langerhans islets.

Genetic and epigenetic characterization of low-grade gliomas reveals frequent methylation of the MLH3 gene.

Diffuse astrocytomas and oligodendrogliomas (WHO grade II) are the most common histological subtypes of low‐grade gliomas (LGGs). Several molecular and epigenetic markers have been identified that predict tumor progression. Our aim was in detail to investigate the genetic and epigenetic background of LGGs and to identify new markers that might play a role in tumor behavior. Twenty‐three patients with oligodendroglioma or oligoastrocytoma (LGO) and 22 patients with diffuse astrocytoma (LGA) were investigated using several molecular‐cytogenetic and molecular methods to assess their copy number variations, mutational status and level of promoter methylation. The most frequent findings were a 1p/19q codeletion in 83% of LGO and copy‐neutral loss of heterozygosity (CN‐LOH) of 17p in 72% of LGA. Somatic mutations in the isocitrate dehydrogenase 1 or 2 (IDH1/IDH2) genes were detected in 96% of LGO and 91% of LGA. The O‐6‐methylguanine‐DNA‐methyltransferase (MGMT) promoter was methylated in 83% of LGO and 59% of LGA. MutL homolog 3 (MLH3) promoter methylation was observed in 61% of LGO and 27% of LGA. Methylation of the MGMT promoter, 1p/19q codeletion, mutated IDH1, and CN‐LOH of 17p were the most frequent genetic aberrations in LGGs. The findings were more diverse in LGA than in LGO. To the best of our knowledge, this is the first time description of methylation of the MLH3 gene promoter in LGGs. Further studies are required to determine the role of the methylated MLH3 promoter and the other aberrations detected.

Recognition of anaplastic foci within low-grade gliomas using MR spectroscopy.

Abstract Background. The recognition of anaplastic foci within low-grade gliomas is of extreme importance in patients under follow-up for Grade II gliomas. We present the algorithm of MR spectroscopy (MRS)-guided brain biopsy and its correlation with tumour histology. Methods. Twenty-seven patients harbouring suspected Grade II/III glioma were examined on our 3T MR. 2D PRESS-CSI metabolite images of Choline/Creatine, Creatine/N-acetylaspartate and Choline/N-acetylaspartate were calculated and exported to the DICOM format. According to these maps, a stereobiopsy was performed at the point of maximum Choline/Creatine ratio prior to tumour resection. In the case of enhancing tumour, a subsequent biopsy was performed from the point of enhancement. Comparisons were made between the histology of the biopsied specimens and the resected tumours. Results. Eleven tumours were diagnosed as high-grade and sixteen as low-grade lesions. The correlation between main spectroscopic ratios (Cho/Cr and Cho/NAA) was strongly positive at the points of maximum Cho/Cr. Similar results were obtained at the points of contrast enhancement. Comparison of histological parameters of biopsy samples at the points of maximum Cho/Cr and histological examination of the completely resected tumours gives a strong correlation of tumour grade, number of mitoses and Ki-67 expression. The diagnostic accuracy of MRS-guided biopsy was 84%. The absolute value of Cho/NAA was higher in high-grade compared to that of low-grade lesions. The value of Cho/NAA ratio of 0.9 using MRS produced a sensitivity and specificity of 78% in the differentiation between low-grade and high-grade lesions. Combining MRS with structural MR, the sensitivity increased to 86% and the specificity to 80%. Conclusions. Strong correlation was demonstrated between Cho/Cr and Ch/NAA ratios. Strong correlation was demonstrated between histological parameters of biopsy samples taken using Cho/Cr ratio and those from total tumour examination. Diagnostic accuracy of MRS-guided biopsy was 84%. Sensitivity and specificity of MRS combined with structural MR reaches 86% and 80%.

Increased tissue and circulating levels of dipeptidyl peptidase-IV enzymatic activity in patients with pancreatic ductal adenocarcinoma

BACKGROUND/OBJECTIVES Pancreatic ductal adenocarcinoma (PDAC) is frequently heralded by an impairment of glucose homeostasis. Dipeptidyl peptidase-IV (DPP-IV) and fibroblast activation protein alpha (FAP) are aminopeptidases that regulate several bioactive peptides involved in glucoregulation, and are frequently dysregulated in cancer. The present study analyzes blood plasma levels and the quantity and localization of DPP-IV and FAP in PDAC tissues. METHODS DPP-IV and FAP concentration and enzymatic activity were evaluated in the plasma from 93 PDAC, 39 type 2 diabetes mellitus (T2DM) and 29 control subjects, and in matched paired non-tumorous and tumor tissues from 48 PDAC patients. The localization of DPP-IV and FAP was determined using immunohistochemistry and catalytic histochemistry. RESULTS The enzymatic activity and concentration of DPP-IV was higher in PDAC tumor tissues compared to non-tumorous pancreas. DPP-IV was expressed in cancer cells and in the fibrotic stroma by activated (myo)fibroblasts including DPP-IV(+)FAP(+) cells. FAP was expressed in stromal cells and in some cancer cells and its expression was increased in the tumors. Plasmatic DPP-IV enzymatic activity, and in particular the ratio between DPP-IV enzymatic activity and concentration in PDAC with recent onset DM was higher compared to T2DM. In contrast, the plasmatic FAP enzymatic activity was lower in PDAC compared to T2DM and controls and rose after tumor removal. CONCLUSIONS DPP-IV-like enzymatic activity is upregulated in PDAC tissues. PDAC patients with recent onset diabetes or prediabetes have increased plasmatic DPP-IV enzymatic activity. These changes may contribute to the frequently observed association of PDAC and recent onset impairment of glucoregulation.

Early detection of pancreatic cancer: impact of high-resolution imaging methods and biomarkers.

High-resolution imaging methods (HRIMs) and biomarkers present the second step of pancreatic cancer (PC) diagnostics in at-risk individuals. These include patients with positive risk factors, early symptoms, nonresponders to the initial antidiabetic therapy, patients older than 50 years of age with new-onset unstable diabetes requiring insulin as well as patients with long-term insulin-non-dependent diabetes and recent (up to 6 months) failure of antidiabetic therapy. The procedures should be started without delay and the co-operation between the primary and tertiary medical centers is highly desirable. An early indication of HRIMs and biomarkers is a prerequisite for the diagnosis of a resectable PC. This publication reviews the recent contribution of HRIMs and biomarkers toward an early diagnosis of PC.