Hepatocellular carcinoma (HCC) is a challenging cancer with high mortality rates, limited predictability, and a lack of effective prognostic indicators. The relationship between small nucleolar RNAs (snoRNAs) and HCC is poorly understood. Based on the literature data, snoRNA studies were primarily focused on viral-related causes of HCC, such as Hepatitis B or C viruses (HBV or HCV). According to these studies, we selected four snoRNAs (snoRA12, snoRA47, snoRA80E, and snoRD126) for exploration in the context of non-viral-related causes, including non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver diseases (NAFLD), and alcohol steatohepatitis. The primary goal of this study was to gain a deeper understanding of how snoRNA expression affects patient outcomes and whether it can serve as a prognostic tool for non-viral HCC. We conducted a study on tissue samples from 35 HCC patients who had undergone resection at Pilsen University Hospital. SnoRA12, snoRA47, snoRA80E, and snoRD126 were studied by quantitative real-time PCR (qRT-PCR) in tumor and non-tumor adjacent tissue (NTAT) samples. Kaplan-Meier analysis was performed to assess the association of snoRNAs expression levels with patient outcomes: time to recurrence (TTR), disease-free survival (DFS) and overall survival (OS). In tumor tissues, snoRA12, snoRA47 and snoRA80E were upregulated, while snoRD-126 was downregulated compared to NTAT. Low expression of snoRA47 and snoRD126 in patients was associated with longer TTR and DFS. The individual expression of snoRA12 and snoRA80E did not show associations with TTR and DFS. However, a combination of medium expression of snoRD126 and snoRA80E was associated with longer TTR and DFS, while high and low expressions of the combined snoRA126 and snoRA80E showed no significant association with TTR, DFS, and OS. Conversely, a combination of high expression of snoRA12 and snoRD126 was associated with shorter TTR. In conclusion, the results indicate that snoRA47 and snoRD126 exhibit good prognostic power specifically for non-viral related HCC. Both snoRA47 and snoRD126 showed favorable prognostication in single and combined analysis when assessing patient outcomes. Also, in combination analysis, snoRA80E and snoRA12 showed favorable prognosis, but not alone.
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The prognostic significance of mast cells and different phenotypes of macrophages in the microenvironment of hepatocellular carcinoma (HCC) following resection is unclear. We aimed in this study to assess the local distribution of infiltrating macrophages and mast cells of specific phenotypes in tissues of HCC and to evaluate their prognostic values for survival of post-surgical patients. METHODS: The clinicopathological and follow-up data of 70 patients with HCC, who underwent curative resection of tumor from 1997 to 2019, were collected. The infiltration of CD68+ and CD163+ macrophages and CD117+ mast cells was assessed immunohistochemically in representative resected specimens of HCC and adjacent tissues. The area fraction (AF) of positively stained cells was estimated automatically using QuPath image analysis software in several regions, such as tumor center (TC), inner margin (IM), outer margin (OM), and peritumor (PT) area. The prognostic significance of immune cells, individually and in associations, for time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS) was evaluated using Kaplan-Meier and Cox regression analyses. RESULTS: High AF of CD68+ macrophages in TC and IM and high AF of mast cells in IM and PT area were associated with a longer DFS. High AF of CD163+ macrophages in PT area correlated with a shorter DFS. Patients from CD163TChigh & CD68TClow group had a shorter DFS compared to all the rest of the groups, and cases with CD163IMlow & CD68IMhigh demonstrated significantly longer DFS compared to low AF of both markers. Patients from CD68IMhigh & CD163PTlow group, CD117IMhigh & CD163PTlow group, and CD117PThigh & CD163PTlow group had a significantly longer DFS compared to all other combinations of respective cells. CONCLUSIONS: The individual prognostic impact of CD68+ and CD163+ macrophages and mast cells in the microenvironment of HCC after resection depends on their abundance and location, whereas the cumulative impact is built upon combination of different cell phenotypes within and between regions.
BACKGROUND: ATP-binding cassette (ABC) transporters translocate various substances across cellular membranes. Their deregulation may cause cancer drug resistance or perturbations in the supply of building blocks for cancer cells and modify patients' prognosis. This study investigated protein expression and cellular localization of the previously suggested putative prognostic biomarkers - ABCB2/TAP1, ABCC7/CFTR, ABCC8/SUR1, and ABCD4 in patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: Protein expression and localization were assessed by immunohistochemistry in formalin-fixed paraffin-embedded primary tumor tissue blocks of 61 PDAC patients and associated with clinical data and the survival of patients. RESULTS: No CFTR protein expression was observed in PDAC, while TAP1 and ABCC8 were expressed predominantly in the cytoplasm of tumor cells. Most samples (81 %) had detectable both membranous and cytoplasmic ABCD4 staining and 42 % had ABCD4 expressed in the apical orientation. Negative membranous ABCD4 staining was significantly more frequent in advanced stage III or IV tumors (p = 0.022). Small or medium counts of individual ABCC8-positive cells in the stroma surrounding tumor tubules were also more often found in stage III or IV (p = 0.044). Patients with moderate or strong ABCC8 cytoplasmic staining intensity in tumor cells had a 3.5-fold higher risk of disease progression than those with weak staining (p = 0.002). CONCLUSIONS: The study shows for the first time that the cytoplasmic ABCC8 protein expression has prognostic value in PDAC.
- MeSH
- ABC transportéry genetika MeSH
- adenokarcinom * patologie MeSH
- duktální karcinom pankreatu * patologie MeSH
- lidé MeSH
- nádorové biomarkery metabolismus MeSH
- nádory slinivky břišní * diagnóza patologie MeSH
- prognóza MeSH
- progrese nemoci MeSH
- receptory sulfonylurey * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: Analysis of somatic variant profiles in retrospectively collected pairs of primary tumors and synchronous liver metastases from surgically treated patients with colorectal carcinomas. Mutational profiles were compared between groups of patients stratified by response to chemotherapy and survival. PATIENTS AND METHODS: The study used whole-exome sequencing of tumor sample pairs from 20 patients diagnosed and treated at a single center. The Cancer Genome Atlas COAD-READ data set (n = 380) was used for validation in silico, where possible. RESULTS: The most frequently altered oncodrivers were APC (55% in primaries and 60% in metastases), TP53 (50/45), TRIP11 (30/5), FAT4 (20/25), and KRAS (15/25). Harboring variants with a high or moderate predicted functional effect in KRAS in primary tumors was significantly associated with poor relapse-free survival in both our sample set and the validation data set. We found a number of additional prognostic associations, including mutational load, alterations in individual genes, oncodriver pathways, and single base substitution (SBS) signatures in primary tissues, which were not confirmed by validation. Altered ATM, DNAH11, and MUC5AC, or a higher share of SBS24 signature in metastases seemed to represent poor prognostic factors, but because of a lack of suitable validation data sets, these results must be treated with extreme caution. No gene or profile was significantly associated with response to chemotherapy. CONCLUSION: Taken together, we report subtle differences in exome mutational profiles between paired primary tumors and synchronous liver metastases and a distinct prognostic relevance of KRAS in primary tumors. Although the general scarcity of primary tumor-synchronous metastasis sample pairs with high-quality clinical data makes robust validation difficult, this study provides potentially valuable data for utilization in precision oncology and could serve as a springboard for larger studies.
- MeSH
- individualizovaná medicína MeSH
- kolorektální nádory * farmakoterapie genetika MeSH
- lidé MeSH
- lokální recidiva nádoru MeSH
- nádory jater * farmakoterapie genetika MeSH
- prognóza MeSH
- protoonkogenní proteiny p21(ras) genetika MeSH
- retrospektivní studie MeSH
- sekvenování exomu MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Anastomotický leak je v kolorektální chirurgii závažnou komplikací. Problematikou poruch hojení střevní anastomózy se zabývá řada experimentálních prací zejména ve smyslu aplikovaného výzkumu. Design zvířecích modelů je přitom různorodý a výsledky jednotlivých prací jsou těžko porovnatelné. Tato práce pojednává souhrnně o hlavních bodech problematiky plánování zvířecích modelů střevních anastomóz, které jsou jednotlivě rozebrány. Hlavní částí textu je popis modelu defektní střevní anastomózy prasete. Anastomóza je v modelu konstruována s lokalizovanou protruzí sliznice. Zvířata jsou pooperačně sledována 3 týdny, monitorovány jsou jak klinický stav, tak změny vitálních hodnot, laboratorních parametrů, je prováděno v definovaných bodech CT vyšetření. Na konci observačního období jsou odebrány vzorky, hodnocen makroskopický nález v dutině břišní, skórování adhezí, známky leaku či poruch pasáže. Histologicky jsou preparáty hodnoceny jak standardními metodami analyzujícími vaskularitu, zánětlivou infiltraci a podíl kolagenu, tak metodami vyvinutými de novo pro potřeby experimentu, jako je analýza integrity střevní stěny v místě slizniční protruze. Experimentálního modelu si ceníme pro možnost systematické a podrobné analýzy stavu zhojení anastomózy v kombinaci s podrobným observačním protokolem, jež vytvářejí klinicky relevantní výsledky.
Anastomotic leak is a serious complication in colorectal surgery. The problem of intestinal anastomosis healing disorders is dealt with by a number of experimental studies, especially in applied research. The design of animal models is diverse and the results of individual studies are difficult to compare. This paper summarizes the main issues of planning animal models of intestinal anastomoses, which are discussed individually. The main part of the text is a description of the defective intestinal anastomosis model of the pig. The anastomosis is constructed in the model with a localized mucosal protrusion. The animals are monitored for 3 weeks postoperatively, the clinical condition and changes in vital values and laboratory parameters are monitored, and CT examinations are performed at defined points. At the end of the observation period, samples are taken, macroscopic findings in the abdominal cavity are evaluated, adhesions are scored, and signs of leakage or passage disorders are assessed. The preparations are evaluated histologically both by standard methods analyzing vascularity, inflammatory infiltration and the proportion of collagen, and by methods developed de novo for the needs of the experiment, such as the analysis of the integrity of the intestinal wall at the site of the mucosal protrusion. We value the experimental model for the possibility of a systematic and detailed analysis of the healing state of the anastomosis in combination with a detailed observation protocol, which produces clinically relevant results.
BACKGROUND: Gastrointestinal anastomoses are performed in many patients every year. The pathogenesis of aberrant anastomotic healing and the causes of intestinal leakage are not fully understood. The present study gathered and critically evaluated histological quantitative data to deepen current knowledge of anastomotic healing in the small and large intestine and its complications and outline the options for further experimental in vivo research in large porcine animal models. METHODS: Three groups of porcine intestinal anastomoses were compared: small intestine without defect (SI; n = 7), small intestine with an additional defect (SID; n = 8), and large intestine (LI; n = 7). Multilevel sampling (2112 micrographs) and stereological methods were used for histological quantification of proliferation (Ki-67 immunohistochemistry), neutrophil infiltration (myeloperoxidase staining), vascularity (von Willebrand factor) and type I and type III collagen formation (picrosirius red in polarized light) within the region of anastomosis compared to the region outside of anastomosis. RESULTS: Quantitative histological evaluation revealed the following results. i) Proliferation, vascularity, and collagen, but not neutrophils, were more highly expressed within the anastomosis than outside of the anastomosis region. ii) Porcine large and small intestine were not interchangeable based on histological evaluation of surgical experiments. The presence or absence of an additional experimental defect strongly affected healing, but the healing seemed complete after 21 days. iii) The microscopic structure of small intestine segments was more affected by their proximity to the anastomosis than the structure of large intestine segments. CONCLUSIONS: Histological quantification was more laborious than the previously used semiquantitative scoring system evaluating the healing rate of intestinal anastomoses, but it provided detailed maps of biological processes within individual intestine layers. The primary data collected in the study are open and available for power sample analyses to calculate the minimum numbers of samples justified in future experiments on porcine intestines. The porcine intestine is a promising animal model with translational potential for human surgery.
- MeSH
- anastomóza chirurgická metody MeSH
- hojení ran * MeSH
- lidé MeSH
- prasata MeSH
- střeva MeSH
- tenké střevo * MeSH
- tlusté střevo MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Hepatocellular carcinoma (HCC) is a fatal disease characterized by early genetic alterations in telomerase reverse transcriptase promoter (TERTp) and β-catenin (CTNNB1) genes and immune cell activation in the tumor microenvironment. As a novel approach, we wanted to assess patient survival influenced by combined presence of mutations and densities of CD8+ cytotoxic T cells. METHODS: Tissue samples were obtained from 67 HCC patients who had undergone resection. We analysed CD8+ T cells density, TERTp mutations, rs2853669 polymorphism, and CTNNB1 mutations. These variables were evaluated for time to recurrence (TTR) and disease free survival (DFS). RESULTS: TERTp mutations were found in 75.8% and CTNNB1 mutations in 35.6% of the patients. TERTp mutations were not associated with survival but polymorphism rs2853669 in TERTp was associated with improved TTR and DFS. CTNNB1 mutations were associated with improving TTR. High density of CD8+ T-lymphocytes in tumor center and invasive margin correlated with longer TTR and DFS. Combined genetic and immune factors further improved survival showing higher predictive values. E.g., combining CTNNB1 mutations and high density of CD8+ T-lymphocytes in tumor center yielded HRs of 0.12 (0.03-0.52), p = 0.005 for TTR and 0.25 (0.09-0.74), p = 0.01 for DFS. CONCLUSION: The results outline a novel integrative approach for prognostication through combining independent predictive factors from genetic and immune cell profiles. However, larger studies are needed to explore multiple cell types in the tumor microenvironment.
- MeSH
- beta-katenin genetika MeSH
- CD8-pozitivní T-lymfocyty patologie MeSH
- hepatocelulární karcinom * genetika patologie chirurgie MeSH
- jednonukleotidový polymorfismus MeSH
- lidé MeSH
- mutace MeSH
- nádorové mikroprostředí genetika MeSH
- nádory jater * genetika patologie chirurgie MeSH
- počet buněk MeSH
- telomerasa * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The use of biologically derived vessels as small-diameter vascular grafts in vascular diseases is currently intensely studied. Vessel decellularization provides a biocompatible scaffold with very low immunogenicity that avoids immunosuppression after transplantation. Good scaffold preservation is important as it facilitates successful cell repopulation. In addition, mechanical characteristics have to be carefully evaluated when the graft is intended to be used as an artery due to the high pressures the vessel is subjected to. Here, we present a new and fast decellularization protocol for porcine carotid arteries, followed by investigation of the quality of obtained vessel scaffolds in terms of maintenance of important extracellular matrix components, mechanical resistance, and compatibility with human endothelial cells. Our results evidence that our decellularization protocol minimally alters both the presence of scaffold proteins and their mechanical behavior and human endothelial cells could adhere to the scaffold in vitro. We conclude that if a suitable protocol is used, a high-quality decellularized arterial scaffold of non-human origin can be promptly obtained, having a great potential to be recellularized and used as an arterial graft in transplantation medicine.
- Publikační typ
- časopisecké články MeSH
Úvod: Při využití prasete jako experimentálního zvířete je často nezbytné zajištění permanentního žilního vstupu, který umožní nejen opakované krevní odběry. Cílem práce bylo vyhodnotit průchodnost a komplikace implantabilních intravenózních port katétrů u prasat zařazených do různých chirurgických experimentů. Metody: Port katétry byly implantovány cestou zevní jugulární žíly celkem 211 prasatům zařazeným do 7 různých experimentů. Retrospektivně byly hodnoceny všechny zaznamenané komplikace. Výsledky: U 157 zvířat (74,4 %) nebyly zaznamenány žádné komplikace. Nejméně závažnou komplikací byl otok (12 zvířat, 5,7 %) a serom v okolí portu (3 zvířata, 1,4 %). Přechodné potíže s aspirací krve byly zaznamenány u 13 zvířat (6,2 %). Nejzávažnější komplikace, kvůli kterým nemohl být port katétr využíván k aspiraci ani aplikaci, nastaly u 26 zvířat (12,3 %). Jednalo se o absces v okolí portu (12 zvířat), nekrózu kožního krytu (2 zvířata), částečnou dehiscenci rány (2 zvířata) a ztrátu funkce port katétru z nespecifikovaných důvodů (10 zvířat). U žádného ze zvířat nemusel být port katétr explantován a žádné zvíře nemuselo být z důvodů komplikací port katétru vyřazeno z experimentu. Cestou port katétru lze bezpečně aplikovat i jodovou kontrastní látku při CT vyšetření. Závěr: I přes zaznamenané komplikace je implantabilní port katétr vhodný k zajištění permanentního intravenózního vstupu u prasat v rámci chirurgických experimentů. Tato metodika umožňuje minimalizovat stres zvířat v pooperačním období a redukovat jejich počet.
Introduction: Permanent intravenous access is usually required in pigs used for surgical experiments, not only to enable repeated blood sample collections. The aim of this study was to evaluate the patency and complications of vascular access ports (VAP) implanted in pigs included in different surgical experiments. Methods: VAPs were implanted via the external jugular vein in a total of 211 pigs from 7 different experiments. All observed complications were retrospectively evaluated. Results: No complications were observed in 157 animals (74.4%). Complications of the least severity were edema or seroma around the port which were observed in 12 (5.7%) and 3 (1.4%) animals, respectively. Temporary problems with aspiration of blood via the port occurred in 13 animals (6.2%). The most severe complications which prevented the use of the VAP for aspiration and application were recorded in 26 animals (12.3%). These complications included: abscess formation around the port (12 animals), skin necrosis over the port (2 animals), partial wound dehiscence (2 animals) and loss of the VAP function due to an unspecified cause (10 animals). Removal of the VAP was not needed in any of the animals and none of the animals had to be excluded from the experiment due to the complications. The VAP can also be used for safe administration of iodine contrast agent during CT examination. Conclusion: Despite the observed complications the VAP is suitable as permanent intravenous access in pigs used for surgical experiments. This method helps to minimize the stress of the animals in the postoperative period and to reduce the number of experimental animals.
- MeSH
- modely u zvířat MeSH
- prasata MeSH
- zaváděcí katétry * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
In this retrospective study on 67 patients with hepatocellular carcinoma (HCC), after tumor resection, we evaluated the significance of CD3+ and CD8+ T-lymphocytes and CD20+ B-lymphocytes in tumor and non-tumor liver for time to recurrence (TTR), disease-free survival (DFS) and overall survival. After immunohistochemical staining, the density of nucleated lymphocyte profiles (QA) was estimated stereologically in the tumor center (TC), inner margin (inn M), outer margin (out M), peritumor and non-tumor liver. In TC, intermediate and high QA of CD8+ cells predicted longer TTR, whereas CD3+ and CD20+ were predictive only at high QA. DFS was predicted by high QA of CD3+, CD8+ and CD20+ cells in TC. The inn M harbored smaller QA of CD3+, CD8+ and CD20+ lymphocytes than out M. In contrast to out M, high T-cells' QA and intermediate and high B-cell QA in inn M predicted longer TTR and DFS. High inn M/out M QA ratios of CD3+ and CD20+ cells were associated with longer TTR and DFS, whereas high inn M/out M QA ratio of CD8+ was predictive only for DFS. Patients with intermediate-high QA of combined CD8+ and CD20+ cells in inn M showed longer TTR and DFS, compared to CD8+-high or CD20+-high alone. Our findings highlight overall heterogeneity of the tumor invasive margin, the importance of inn M, and the predictive role of B-cells.
- Publikační typ
- časopisecké články MeSH
