Ondrej Vycital

HOTAIR long non-coding RNA is a negative prognostic factor not only in primary tumors, but also in the blood of colorectal cancer patients

Colorectal cancer (CRC) is one of the main causes of death of neoplasia. Demand for predictive and prognostic markers to reverse this trend is increasing. Long non-coding RNA HOTAIR (Homeobox Transcript Antisense Intergenic RNA) overexpression in tumors was previously associated with poor prognosis and higher mortality in different carcinomas. We analyzed HOTAIR expression levels in tumor and blood of incident sporadic CRC patients in relation to their overall survival with the aim to evaluate surrogate prognostic marker for CRC. Tissue donor group consisted of 73 CRC patients sampled for tumor and normal tissue. Blood donor group was represented by 84 CRC patients compared with 40 healthy controls. Patients were characterized for tumor-node-metastasis stage, tumor grade, microsatellite instability and tumor penetration by stromal cells. HOTAIR levels were assessed by real-time quantitative PCR. CRC patients had higher HOTAIR expression in blood than healthy controls (P = 0.0001), whereas there was no difference in HOTAIR levels between tumor and adjacent mucosa of CRC patients. HOTAIR levels positively correlated between blood and tumor (R = 0.43, P = 0.03). High HOTAIR levels in tumors were associated with higher mortality of patients [Coxs proportional hazard, hazard ratio = 4.4, 95% confidence interval: 1.0-19.2, P = 0.046]. The hazard ratio was even higher when blood HOTAIR levels were taken into account (hazard ratio = 5.9, 95% confidence interval: 1.3-26.1, P = 0.019). Upregulated HOTAIR relative expression in primary tumors and in blood of CRC patients is associated with unfavorable prognosis. Our data suggest that HOTAIR blood levels may serve as potential surrogate prognostic marker in sporadic CRC.

Immunohistochemical Detection of Cancer Stem Cell Related Markers CD44 and CD133 in Metastatic Colorectal Cancer Patients

Aim. The goal of this study was to semiquantitatively detect presence of cancer stem cells markers CD44 and CD133 in immunohistochemically stained paired samples of colorectal cancer (CRC) and colorectal liver metastases (CLM). Level of staining intensity was compared to clinical and pathological characteristics of tumors with the aim to identify impact of CD44 or CD133 expression on tumor behavior. Patients and Methods. Formalin fixed paraffin embedded samples from 94 patients with colorectal tumor and liver metastases were collected at Sikls Department of Pathology. Samples were stained by antibodies against CD44 and CD133. Presence and intensity of staining was assessed semiquantitatively by three trained researchers. Results. Patients with higher level of CD133 staining in CRC had longer disease free interval (Cox-Mantel P = 0.0244), whereas we found no relation between CD44 expression and overall survival or disease free interval. CD133 expression in CRC and CLM differed based on CRC grading; in case of CD44 we found differences in staining intensity in individual stages of tumor lymph node invasion. Conclusion. Effect of cancer stem cell markers on prognosis of colorectal cancer can vary depending on pathological classification of tumor, and we have shown that CD133, generally considered to be a negative marker, can bear also clinically positive prognostic information in group of patients with colorectal liver metastases.

The association of miR-126-3p, miR-126-5p and miR-664-3p expression profiles with outcomes of patients with metastatic colorectal cancer treated with bevacizumab

MicroRNAs regulate the expression of genes involved in several important cancer-related processes including cell adhesion, proliferation, and tumour angiogenesis. Bevacizumab is routinely used in the treatment of patients with metastatic colorectal cancer, but, so far, no reliable biomarker predicting response to bevacizumab has been established. The aim of our retrospective study was to evaluate the association of miR-126-3p, miR-126-5p and miR-664-3p tumour expression levels with outcomes of patients with metastatic colorectal cancer treated with bevacizumab. The study included 63 patients. For the assessment of microRNA expression, gene-specific TaqMan assays were used. The median progression-free survival and overall survival for patients with low tumour expression of miR-126-3p were 8.8 and 20.6 months versus 13.5 months and median overall survival was not reached for patients with high expression (p = 0.0064 and p = 0.0027), respectively. The median progression-free survival and overall survival for patients with low tumour expression of miR-126-5p were 9.0 and 22.2 months versus 12.0 and 23.4 months for patients with high expression (p = 0.2113 and 0.6858), respectively. The median progression-free survival and overall survival for patients with low tumour expression of miR-664-3p were 9.1 and 22.5 months versus 8.8 and 23.4 months for patients with high expression (p = 0.2542 and p = 0.1922), respectively. The multivariable Cox proportional hazards model revealed that miR-126-3p expression was significantly associated with progression-free survival (hazard ratio = 0.28, p = 0.0053) and also with overall survival (hazard ratio = 0.18, p = 0.0046). In conclusion, the results of this study suggest that the expression of miR-126-3p in the tumour tissue was associated with outcome of metastatic colorectal cancer patients treated with bevacizumab.

Molecular profile of 5-fluorouracil pathway genes in colorectal carcinoma

BackgroundThis study addresses involvement of major 5-fluorouracil (5-FU) pathway genes in the prognosis of colorectal carcinoma patients.MethodsTesting set and two validation sets comprising paired tumor and adjacent mucosa tissue samples from 151 patients were used for transcript profiling of 15 5-FU pathway genes by quantitative real-time PCR and DNA methylation profiling by high resolution melting analysis. Intratumoral molecular profiles were correlated with clinical data of patients. Protein levels of two most relevant candidate markers were assessed by immunoblotting.ResultsDownregulation of DPYD and upregulation of PPAT, UMPS, RRM2, and SLC29A1 transcripts were found in tumors compared to adjacent mucosa in testing and validation sets of patients. Low RRM2 transcript level significantly associated with poor response to the first-line palliative 5-FU-based chemotherapy in the testing set and with poor disease-free interval of patients in the validation set irrespective of 5-FU treatment. UPP2 was strongly methylated while its transcript absent in both tumors and adjacent mucosa. DPYS methylation level was significantly higher in tumor tissues compared to adjacent mucosa samples. Low intratumoral level of UPB1 methylation was prognostic for poor disease-free interval of the patients (P = 0.0002). The rest of the studied 5-FU genes were not methylated in tumors or adjacent mucosa.ConclusionsThe observed overexpression of several 5-FU activating genes and DPYD downregulation deduce that chemotherapy naïve colorectal tumors share favorable gene expression profile for 5-FU therapy. Low RRM2 transcript and UPB1 methylation levels present separate poor prognosis factors for colorectal carcinoma patients and should be further investigated.

The focus on sample quality: Influence of colon tissue collection on reliability of qPCR data

Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling. The collection procedures of the biological material were implemented in two hospitals during colon cancer surgery in order to identify the impact of the collection method on the experimental outcome. Our study shows that the pre-analytical sample handling has a significant effect on the quality of RNA and on the variability of qPCR data. PAXgene collection mode proved to be more easily implemented in the operating room and moreover the quality of RNA obtained from human colon tissues by this method is superior to the one obtained by snap freezing.

Tumor Infiltrating Lymphocytes as Prognostic Factor of Early Recurrence and Poor Prognosis of Colorectal Cancer After Radical Surgical Treatment

Sixty percent of patients with colorectal cancer (CRC) are afflicted with distant metastases (liver or lung metastatic process) or a local relapse of malignancy (Bird et al., 2006). The possibilities of surgical and oncological treatment of this disease offer us a large spectrum of treatments including the combination of surgical procedures and consecutive oncological treatments. In the case of radical surgical therapy we can consider the curative access. The main medical problem of CRC is the high rate of recurrences after radically performed surgical therapy. The operability of recurrence is only about 30% in the case of local relapse and 20% in the case of distant metastases (Coleman et al., 2008; Kobayashi et al., 2007). The second dominant problem is the early recurrence of CRC after radical surgical treatment, when the patients undergo a difficult and exhausting procedure with a high risk of perioperative complications without any significant differences in overall survival against modern palliative therapy (Van den Eynde & Hendlisz, 2009). Contemporary clinical and histopathological prognostic factors (staging, grading, etc.) used for the detection of patients with a high risk of relapse and a short overall survival rate and for the indication of adjuvant oncological treatment after radical surgery are not sufficient. Tumor infiltrating lymphocytes (TIL) were described as a good prognostic factor for patients with a high risk of relapse. They are critical indicators of efficient antitumor immunological response. Their number, type and morphology of TIL cells determine resulting tumor prognosis (Atreya & Neurath, 2008; Galon et al., 2006). They could be connected also with the suppression of micrometastatical disease after radical surgery (Gajewski et al., 2006; Pages et al., 2005). We can recognize either the type of immune cells or distinguish their morphological aspects (infiltration of any part of tumor or surrounding of tumor or tributary lymph nodes) (Talmadge et al., 2007).

Investigation of single and synergic effects of NLRC5 and PD-L1 variants on the risk of colorectal cancer

Constitutive activation of interferon signaling pathways has been reported in colorectal cancer (CRC), leading to a strong CD8+ T cell response through stimulation of NLRC5 expression. Primed CD8+ T cell expansion, however, may be negatively regulated by PD-L1 expression. Additionally, aberrant PD-L1 expression enables cancer cells to escape the immune attack. Our study aimed to select potential regulatory variants in the NLRC5 and PD-L1 genes by using several online in silico tools, such as UCSC browser, HaploReg, Regulome DB, Gtex Portal, microRNA and transcription factor binding site prediction tools and to investigate their influence on CRC risk in a Czech cohort of 1424 CRC patients and 1114 healthy controls. Logistic regression analysis adjusted for age and gender reported a moderate association between rectal cancer risk and two NLRC5 SNPs, rs1684575 T>G (OR: 1.60, 95% CI: 1.13–2.27, recessive model) and rs3751710 (OR: 0.70, 95% CI: 0.51–0.96, dominant model). Given that a combination of genetic variants, rather than a single polymorphism, may explain better the genetic etiology of CRC, we studied the interplay between the variants within NLRC5, PD-L1 and the previously genotyped IFNGR1 and IFNGR2 variants, to evaluate their involvement in the risk of CRC development. Overall we obtained 18 pair-wise interactions within and between the NLRC5 ad PD-L1 genes and 6 more when IFNGR variants were added. Thirteen out of the 24 interactions were below the threshold for the FDR calculated and controlled at an arbitrary level q*<0.10. Furthermore, the interaction IFNGR2 rs1059293 C>T—NLRC5 rs289747 G>A (P<0.0001) remained statistically significant even after Bonferroni correction. Our data suggest that not only a single genetic variant but also an interaction between two or more variants within genes involved in immune regulation may play important roles in the onset of CRC, providing therefore novel biological information, which could eventually improve CRC risk management but also PD-1-based immunotherapy in CRC.

lncRNAs in Non-Malignant Tissue Have Prognostic Value in Colorectal Cancer

Although colorectal cancer (CRC) is the third most frequent cause of cancer related death in Europe, clinically relevant biomarkers for therapy guidance and prognosis are insufficiently reliable. Long non-coding RNAs (lncRNAs) are RNAs over 200 nucleotides long that are not translated into proteins but can influence biological processes. There is emerging evidence for their involvement in solid cancer as oncogenes, tumour suppressors or regulators of cell proliferation and metastasis development. The goal of this study was to evaluate the prognostic effect of selected lncRNAs in a retrospective study on CRC patients from the Czech Republic. We used a quantitative PCR approach to measure the expression in paired non-malignant and tumour tissue samples of CRC patients of nine lncRNAs previously shown to be involved in cancer progression—ANRIL, CCAT1, GAS5, linc-ROR, MALAT1, MIR155HG, PCAT1, SPRY4-IT1 and TUG1. Associations between expression and expression ratios and clinical characteristics and survival were assessed by using univariable Cox proportional hazards models, Kaplan-Meier estimations with the Gehan-Wilcoxon test, the Mann-Whitney U test, the Kruskal-Wallis test and Spearman’s correlations. A comparison of expression in tumour tissue (TT) and non-malignant mucosa tissue (MT) showed significant upregulation of CCAT1 and linc-ROR in TT (p < 0.001 and p = 0.001, respectively) and downregulation of ANRIL, MIR155HG and MALAT1 (p = 0.001, p = 0.010, p = 0.001, respectively). Linc-ROR was significantly associated with the presence of synchronous metastases (p = 0.033). For individual tissue types, lower MIR155HG expression in TT was correlated with both shorter overall survival (p = 0.008) and shorter disease-free survival (p = 0.040). In MT, expression ratios of CCAT1/ANRIL and CCAT1/MIR155HG were associated with overall survival (p = 0.005 and p = 0.006, respectively). Our results revealed that changes in expression of lncRNAs between MT and TT hold potential to be used as prognostic biomarkers in CRC patients. Moreover, the ratios of CCAT1 to ANRIL and MIR155HG in MT also exhibit potential for prognosis assessment without tumour sampling. Our results also indicate that cancer progression is associated with detrimental system-wide changes in patient tissue, which might govern patient survival even after successful elimination of tumour or cancerous cells.

The Impact of Immune Interaction on the Metastatic Infiltration of Colorectal Carcinoma to Lymph Nodes

Background/Aim: Tumour-infiltrating lymphocytes (TILs) and Granzyme B play crucial roles in immune reactions against colorectal carcinoma (CRCa). The inhibitor of Granzyme B is Serpin B9. The aim of this study was to evaluate the effect of immunohistological parameters of TILs on the prognosis of CRCa and presence of lymph node metastasis. Patients and Methods: A total of 152 patients who underwent surgery for CRCa were analyzed, including 63 patients in cohort stage II, according to the Union for International Cancer Control (UICC), and 89 patients in cohort UICC stage III. The TIL pattern was classified as peritumoural (PTL), intratumoural (ITL), intrastromal (ISL) or Crohn-like, and immunohistological staining of TIL and cancer cells was also performed. Results: A significantly higher density of CD8+ and CD4+ TILs was observed in the UICC II group, and significantly higher densities of CD4+ TILs were observed in the UICC II group in all distinguished patterns. In the same cohort, higher numbers of CD57+ cells and FoxP3+ TILs and Granzyme B levels were observed. In cohort UICC III, there was a higher density of ISL, PTL CD8+, CD25+ TILs and cancer cells showed staining for Serpin B9. CD57, Granzyme B and CD8 were demonstrated as positive prognostic factors of overall survival, and CD57 and CD4+ TILs were demonstrated as positive prognostic factors of recurrence. Conclusion: TILs and CD57 are promising prognostic factors of CRCa. The association of Serpin B9 with lymph node metastasis reveals a potential mechanism for tumour resistance to immune reaction.

Liver Parenchyma Regeneration in Connection with Extended Surgical Procedure - Experiment on Large Animal

Liver surgery underwent enormous evolution after development and introduction of new technical skills in surgical praxis. Nevertheless many patients with primary or secondary liver malignancies are not indicated to radical surgical therapy that could reach complete remission of malignant disease because the frontiers of liver surgery are limited today by the functional reserves of remnant parenchyma. The main argument to non surgical treatment is increased risk of acute liver failure after extended liver resection, where retained liver parenchyma is to small to sustain the liver functions (Abdalla, 2001). Portal vein embolization (PVE) can multiply the future liver remnant volume (FLRV) in spite of affection of only one of liver lobes by malignant diseases (Makuuchi, 1984, Makuuchi, 1990, Harada, 1997). This procedure was performed firstly in 1984 by Makuuchi (Abdalla, 2001, Makuuchi, 1984, Makuuchi, 1990). PVE of portal branch of with malignancy afflicted liver lobe initiates compensatory hypertrophy of contralateral non-occluded lobe. The occluded lobe underlies atrophy. The compensatory hypertrophy is supposed to be stimulated by increased flow of portal blood, that contains hepatotrophic substances (Kusaka, 2004, Azoulay, 2000). Liver resection after PVE is performed only in 63-96% of patients (Kokudo, 200, Stefano, 2005, Lagasse, 2000). The main reason for this resolution is unsuccessful hypertrophy of FLRV or progression of malignancy. Liver resection after PVE is performed only in 63-96% of patients (Azoulay, 200, Kokudo, 2001, Stefano, 2005). The main reason for this resolution are unsuccessful hypertrophy of FLRV or progression of malignancy.