Martin Pesta

Relevance of miR-21 and miR-143 expression in tissue samples of colorectal carcinoma and its liver metastases

MicroRNAs, which are endogenously expressed regulatory noncoding RNAs, have an altered expression in colorectal cancer. The aim of our study was to assess the relationship of miR-21 and miR-143 expression to the prognostic/clinicopathological features of colorectal carcinoma (CRC) and colorectal liver metastases (CLM). The estimation was performed in 46 paired (tumor and control) tissue samples of CRC. Further, we studied 30 tissue samples of CLM. MiR-21 and miR-143 expressions were quantified by using the quantitative reverse transcription polymerase chain reaction method. Relation of miR-21 and miR-143 expression to disease-free interval (DFI) (Wilcoxon; P = 0.0026 and P = 0.0191, respectively) was recorded. There was shorter DFI in patients with a higher expression of miR-21 and, surprisingly, also in patients with a higher expression of miR-143, which is a putative tumor suppressor. There was a higher expression of miR-21 and lower expression of miR-143 in CRC tissue in comparison with adjacent normal colon tissue (P < 0.0001; P < 0.0001, respectively). Similarly, we observed a higher expression of miR-21 and a lower expression of miR-143 in CLM in comparison with normal colon tissue (P < 0.0001; P < 0.0001, respectively). Our results support the hypothesis about oncogenic function of miR-21 and show its relation to DFI. The role of miR-143 in carcinogenesis seems to be more complex.

Clinical Relevance of the Expression of mRNA of MMP-7, MMP-9, TIMP-1, TIMP-2 and CEA Tissue Samples from Colorectal Liver Metastases

Background: Nowadays we know that survival rates do not differ between repeated and single liver resections for colorectal liver metastases (CLM). To be able to determine patients prone to early recurrence, the use of different markers with a better prognostic value than the routinely employed tumor markers is required. Aim of Study: The aim of our study was to assess mRNA expression of MMP-7, MMP-9, TIMP-1, TIMP-2 and CEA in tissue samples from CLM and their relationship to disease-free interval (DFI) and overall survival (OS). Patients and Methods: The liver tumor biopsies were obtained from 40 patients suffering from CLM treated with radical surgery. mRNA expression levels of CEA, MMPs and TIMPs and a housekeeping gene (GAPDH) were quantified using RT-PCR. Results: The increased expression of CEA, MMP-9 and TIMP-1 in CLM was associated with a short DFI and a high tendency to early CLM recurrence. Statistical analysis confirmed CEA, MMP-9 and TIMP-1 expression as prognostic factors of survival. Conclusion: This study demonstrated the importance of CEA, MMP-9 and TIMP-1 in the prognostication of DFI and OS.

May CTC technologies promote better cancer management

In the case of cancer, death is usually not due to the primary tumor itself but due to dissemination. Analysis of the circulating tumor cells (CTCs), i.e., cells responsible for a formation of metastases, should provide information useful for the management of cancer patients, fulfilling the objectives of predictive, preventive, and personalized medicine (PPPM). Despite promising results, the decisions on stage of disease and how to guide the adjuvant treatment still do not include results of CTC assessment. We want to describe two major reasons why the recent diagnostic value of CTC analysis is not sufficient for clinical use. The first reason arises from the biological nature of the tumor itself and the second reason is associated with an interdisciplinary status of CTC diagnostics in the sense that it is neither a theme purely for pathologists nor for haemato-oncologists nor clinical biochemists. We anticipate that there are at least three areas where CTCs can be useful for clinical practice. The first is monitoring of treatment efficacy of cancer patients. The second is a molecular characterization of captured CTCs for targeted treatment, and the third is a cultivation of captured CTCs for drug sensitivity testing. All of these approaches allow researchers recognize and respond to changes of phenotype of cancer cells during disease progression and introduce PPPM into clinical practice.

Testing for oncogenic molecular aberrations in cell-free DNA-based liquid biopsies in the clinic: Are we there yet?

The optimal choice of cancer therapy depends upon analysis of the tumor genome for druggable molecular alterations. The spatial and temporal intratumor heterogeneity of cancers creates substantial challenges, as molecular profile depends on time and site of tumor tissue collection. To capture the entire molecular profile, multiple biopsies from primary and metastatic sites at different time points would be required, which is not feasible for ethical or economic reasons. Molecular analysis of circulating cell-free DNA offers a novel, minimally invasive method that can be performed at multiple time-points and plausibly better represents the prevailing molecular profile of the cancer. Molecular analysis of this cell-free DNA offers multiple clinically useful applications, such as identification of molecular targets for cancer therapy, monitoring of tumor molecular profile in real time, detection of emerging molecular aberrations associated with resistance to particular therapy, determination of cancer prognosis and diagnosis of cancer recurrence or progression.

Isocitrate Dehydrogenase-1 Mutations as Prognostic Biomarker in Glioblastoma Multiforme Patients in West Bohemia

Introduction. Glioblastoma multiforme (GBM) is the most malignant primary brain tumor in adults. Recent whole-genome studies revealed novel GBM prognostic biomarkers such as mutations in metabolic enzyme IDH—isocitrate dehydrogenases (IDH1 and IDH2). The distinctive mutation IDH1 R132H was uncovered to be a strong prognostic biomarker for glioma patients. We investigated the prognostic role of IDH1 R132H mutation in GBM patients in West Bohemia. Methods. The IDH1 R132H mutation was assessed by the RT-PCR in the tumor samples from 45 GBM patients treated in the Faculty Hospital in Pilsen and was correlated with the progression free and overall survival. Results. The IDH1 R132H mutation was identified in 20 from 44 GBM tumor samples (45.4%). The majority of mutated tumors were secondary GBMs (16 in 18, 89.9%). Low frequency of IDH1 mutations was observed in primary GBMs (4 in 26, 15.3%). Patients with IDH R132H mutation had longer PFS, 136 versus 51 days (P < 0.021, Wilcoxon), and OS, 270 versus 130 days (P < 0.024, Wilcoxon test). Summary. The prognostic value of IDH1 R132H mutation in GBM patients was verified. Patients with mutation had significantly longer PFS and OS than patients with wild-type IDH1 and suffered more likely from secondary GBMs.

Asymptomatic Abdominal Aortic Aneurysms Show Histological Signs of Progression: A Quantitative Histochemical Analysis

Objective: Abdominal aortic aneurysm (AAA) is a serious disease due to its covert nature, relatively high prevalence and fatal prognosis in the case of rupture. To obtain new insights into AAA pathogenesis, we examined the relationships between histopathology, multiplex in vitro immunoassay data, diameter and symptomatology. Methods: In a prospective, non-randomised study, we evaluated samples from 6 normal infrarenal aortae and 65 AAA patients (65 walls, 55 thrombi). The AAA patients were either asymptomatic (n = 44), symptomatic (n = 7) or with ruptured AAA (n = 14). The AAA diameter was classified as small (<5 cm, n = 18), medium (5–7 cm, n = 26) and large (>7 cm, n = 21). We quantified the histopathology of the AAA wall and the adjacent thrombus. We assessed the expression of proteins in the same samples. Results: Asymptomatic AAAs had walls with more abundant inflammatory infiltrates, lower amounts of PAI-1, a higher number of tPA-positive elements, a tendency towards decreased collagen content, whereas the adjacent thrombi had a greater concentration of VCAM-1 and MMP-2 when compared with symptomatic AAAs. Compared with the aneurysmatic aorta, the normal aorta contained less collagen and more elastin, actin, desmin and PAI-1-positive elements; in addition, it was more vascular. Medium-sized AAAs were the most actin and vimentin rich, and large AAAs were the most vascular. Conclusion: Our results show that asymptomatic AAA walls often have more potentially deleterious histopathological alterations than symptomatic AAA walls. This result indicates that a progression from an asymptomatic AAA to rupture can be expected and screening patients who are at risk of rupture could be beneficial.

Association between endothelial NO synthase polymorphisms and arterial properties in the general population.

OBJECTIVE Nitric oxide plays an important role in vascular biology. Several single nucleotide polymorphisms (SNP) in the endothelial nitric oxide gene (NOS3) have been previously associated with arterial hypertension. We investigated whether these SNPs might be associated with arterial phenotypes in the Czech general population. METHODS We genotyped three NOS3 SNPs in 426 subjects not treated for arterial hypertension (mean age, 49.1 years; 55.9% women). Arterial properties were measured using applanation tonometry. In multivariate-adjusted analyses, we assessed the gene effects of rs3918226 (-665 C>T), rs1799983 (glu298asp G>T) and rs2070744 (786 T>C) on augmentation index (AIx), central augmentation pressure (AP) and aortic pulse wave velocity (PWV). RESULTS Carriers of rs3918226 mutated T allele had marginally higher AIx (145.3 ± 2.5 vs. 140.2 ± 1.1%; P = 0.064) and significantly higher AP (12.7 ± 0.7 vs. 11.1 ± 0.3 mm Hg; P = 0.033). These associations were independent of potential confounding factors. Aortic PWV was not different in the two rs39182226 genotypes groups (P = 0.35). In single gene analyses, we did not observe any association between measured phenotypes and rs1799983 or rs2070744 (P ≥ 0.11). In haplotype analysis, we observed trend for higher PWV in haplotypes containing rs3918226 mutated T allele compared with other allelic combination (P ≤ 0.079). CONCLUSION Mutated T allele of rs3918226 polymorphism in NOS3 gene was associated with parameters reflecting central arterial stiffness and wave reflection. We hypothesize that genetic modulation of intermediate arterial phenotypes might lead to higher blood pressure.

Diagnostic and Prognostic Value of microRNA-21 in Colorectal Cancer: An Original Study and Individual Participant Data Meta-analysis

Background: We aimed to systematically summarize the diagnostic and prognostic value of circulating/tissue miR21 in patients with colorectal cancer. Methods: An original study was conducted to explore the potential value of circulating miR21 in colorectal cancer diagnosis and tissue miR21 in colorectal cancer prognosis. PUBMED and EMBASE were searched (to August, 2013) to identify eligible studies. To explore the diagnostic performance of circulating miR21, meta-analysis methods were used to pool sensitivity, specificity, positive and negative likelihood ratio, diagnostic OR and to construct a summary ROC curve. For prognostic meta-analysis, study-specific HRs of tissue miR21 for survival were summarized. Subgroup and sensitivity analyses were applied to explore heterogeneity. Results: Finally, 14 studies (including our study) were included in the meta-analyses. The pooled sensitivity, specificity, and AUC of circulating miR21 were 0.76 [95% confidence interval (CI), 0.59–0.88], 0.81 (95% CI, 0.76–0.85), and 0.81 (95% CI, 0.78–0.85) in diagnosing colorectal cancer. Patients with higher expression of tissue miR21 had significant inferior overall survival (OS; pooled HR, 1.56; 95% CI, 1.16–2.11) and disease-free survival (DFS; pooled HR, 1.35; 95% CI, 1.08–1.69). The individual participant data (IPD) meta-analysis demonstrated that tissue miR21 level was independently associated with worse colorectal cancer OS (HR, 1.69; 95% CI, 1.07–2.67; P = 0.023), whereas this association seems to be confined to males (P = 0.007) but not for females (P = 0.845). Conclusions: Circulating miR21 level has potential value for colorectal cancer early detection, whereas high tissue miR21 level is associated with adverse colorectal cancer prognosis. Impact: miR21 is a promising biomarker for early detection and prognosis of colorectal cancer. Cancer Epidemiol Biomarkers Prev; 23(12); 2783–92. ©2014 AACR.

Tissue microRNAs as predictive markers for gastric cancer patients undergoing palliative chemotherapy

MicroRNAs have the potential to become valuable predictive markers for gastric cancer. Samples of biopsy tissue, routinely taken from gastric cancer patients undergoing palliative chemotherapy, constitute suitable material for microRNA profiling with the aim of predicting the effect of chemotherapy. Our study group consisted of 54 patients, all of whom underwent palliative chemotherapy based on 5-fluorouracil (5-FU) or 5-FU in combination with platinum derivatives between 2000 and 2013. The expression of 29 selected microRNAs and genes BRCA1, ERCC1, RRM1 and TS, in gastric cancer tissue macrodissected from FFPE tissue samples, was measured by quantitative RT-PCR. The relationship between gene expression levels and time to progression (TTP) and overall survival (OS) was analysed. From the set of the 29 microRNAs of interest, we found high expression of miR-150, miR-342-3p, miR-181b, miR-221, miR-224 and low levels of miR-520h relate to shorter TTP. High levels of miR-150, miR-192, miR-224, miR-375 and miR-342-3p related to shorter OS. In routinely available FFPE tissue samples, we found 6 miRNAs with a relation to TTP, which may serve as predictors of the effectiveness of palliative treatment in gastric cancer patients. These miRNAs could also help in deciding whether to indicate palliative chemotherapy.

Morphological analysis of embryonic cerebellar grafts in SCA2 mice.

SCA2 transgenic mice are thought to be a useful model of human spinocerebellar ataxia type 2. There is no effective therapy for cerebellar degenerative disorders, therefore neurotransplantation could offer hope. The aim of this work was to assess the survival and morphology of embryonic cerebellar grafts transplanted into the cerebellum of adult SCA2 mice. Four month-old homozygous SCA2 and negative control mice were treated with bilateral intracerebellar injections of an enhanced green fluorescent protein-positive embryonic cerebellar cell suspension. Graft survival and morphology were examined three months later. Graft-derived Purkinje cells and the presence of astrocytes in the graft were detected immunohistochemically. Nissl and hematoxylin-eosin techniques were used to visualize the histological structure of the graft and surrounding host tissue. Grafts survived in all experimental mice; no differences in graft structure, between SCA2 homozygous and negative mice, were found. The grafts contained numerous Purkinje cells but long distance graft-to-host axonal connections to the deep cerebellar nuclei were rarely seen. Relatively few astrocytes were found in the center of the graft. No signs of inflammation or tissue destruction were seen in the area around the grafts. Despite good graft survival and the presence of graft-derived Purkinje cells, the structure of the graft did not seem to promise any significant specific functional effects. We have shown that the graft is available for long-term experiments. Nevertheless, it would be beneficial to search for ways of enhancement of connections between the graft and host.