Biomarkers as prognostic and predictive factors in patients with hepatocellular carcinoma undergoing radiological oncological interventions.

BACKGROUND: Hepatocellular carcinoma is the most common malignant liver tumor in adults and thermal ablation and transarterial embolization are important methods of therapy. Thermal ablation can be used in early stages. Methods based on the transarterial approach, especially transarterial chemoembolization, play an important role in intermediate stage diseases. The success of procedures depends not only on the biological nature and the size of the tumor, on the technical design of the procedure and on the patient's response to treatment, but also on the molecular changes associated with these procedures. In addition to classic predictive and prognostic factors including age, patient comorbidities, Child-Pugh score, tumor characteristics, presence of large surrounding vessels, and portal vein thrombosis, molecular prognostic and predictive factors (serum biomarkers) are often mentioned in studies. Currently, only a-fetoprotein is routinely used as a prognostic biomarker; however, there are studies referring to new serum biomarkers that can potentially help to classical markers and imaging methods to determine the cancer prognosis and predict the success of therapy. These biomarkers most often include g-glutamyltranspeptidase, des- g-carboxyprothrombin, some types of microRNAs, inflammatory and hypoxic substances, whose serum levels are changed by the intervention therapies. Evaluation of these molecules could lead to the optimization of the medical intervention (choice of therapy method, timing of treatment) or change the management of patient follow-up after interventions. Although several biomarkers have shown promising results, most serum biomarkers still require validation in phase III studies. PURPOSE: The aim of this work is to present a comprehensive overview of classical and molecular biomarkers that could potentially help in the prognostic stratification of patients and better predict the success and effect of radiological intervention methods.

Chemoembolization for Treatment of Hepatocellular Carcinoma - National Registry-Based Analysis.

BACKGROUND: To evaluate survival benefit in patient undergoing transarterial chemoembolization (TACE) for hepatocellular carcinoma (HCC) by national registry data analysis and comparison of regions with unequal usage of interventional radiology procedures. MATERIALS AND METHODS: A total of 4,343 patients with primary diagnosis of HCC between 2010-2016 were extracted from the databases of The Czech National Cancer Registry. The analysis was supported by data from the National Registry of Paid Health Services and the Death Records Database. Primary treatment option was categorized as liver resection, ablation, TACE and chemotherapy. The regional data analysis provided information of interventional radiology procedures frequency for primary treatment of HCC. The 14 main regions were symmetrically divided to group with well-developed interventional radiology service and low-developed interventional radiology service according the frequency of stage adjusted interventional radiology procedure usage (< 15%, > 15%). Kaplan-Meier and Cox regression were used for survival and hazard ratios (HR) analyses. RESULTS: Only 1,730 patients had assessed any primary treatment option, 16.5 % (285) were treated by TACE. Median of survival were significantly different in regions with well and low developed interventional radiology service for whole study population (13.2 vs. 6.5 months, p < 0.001), patients treated in regions with well-developed interventional radiology service had lower risk of death during treatment (HR 0.73; 0.66-0.81). The patient treated by TACE had median of survival 15.8 months (13.5-18.1), while the survival was not significantly different in region groups. CONCLUSION: The usage of anticancer therapies based on interventional radiology procedures is a huge factor influences the survival of HCC patient according population-based data. Studies gathering data from cancer register databases can provide further information on treatment effectiveness. This work was created at Masaryk University in the project „Oncological radiological interventions and their benefit in complex oncological treatment, comparison of dedicated oncological treatment results data of the Czech republic II“ (MUNI/A/1574/2018), supported by Ministry of Education, Youth and Sports. This publication was additionally suported by Ministry of Health grant No. 15-32484A. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 1. 3. 2019 Accepted: 4. 3. 2019.

Dynamic Changes in Circulating MicroRNA Levels in Liver Cancer Patients Undergoing Thermal Ablation and Transarterial Chemoembolization.

BACKGROUND: Hepatic cancer patients who cannot undergo surgical resection of tumour are candidates for methods of interventional radiology - transarterial chemoembolization (TACE) or thermal ablative (TA) therapy. Both methods are causing characteristic changes in liver tissue (inflammatory immune response, hypoxia, elevated temperature, tissue destruction) which are accompanied with systemic secretion of cytokines or microRNAs (miRNAs). The aim of our study was to investigate whether the level of circulating miRNAs related to hypoxia (miR-21 and miR-210), liver injury (miR-122) and epithelial-mesenchymal transition (miR-200a) could reflect systemic effect of these intervention techniques. MATERIALS AND METHODS: Study consisted of 10 primary hepatocellular carcinoma patients treated with TACE and 10 patients with liver metastases of colorectal cancer treated with TA. Thermal ablation was performed using the radiofrequency or microwave generator (RITA, Microsulis, AngioDynamics,Inc), for TACE drug eluting beads (DCBeads, Biocompatibles Ltd.) were used. Tumours were evaluated using RECIST (Response Evaluation Criteria in Solid Tumours), mRECIST (modified RECIST) criterion and volumetry. For all patients we determined concentrations of miRNA in blood plasma samples from four time points (before intervention, immediately after intervention, 24 hours after intervention, 1 week after intervention) using TaqMan® Assays and quantitative real time polymerase chain reaction method. RESULTS: After both intervention techniques we observed changes in circulating miRNA levels. In TA cases we observed significant increase of miR-122 and miR-200a concentrations immediately after intervention, on the contrary in TACE we observed increase in miRNA concentration at time point 24 hours after intervention (miR-21, miR-210, miR-122, miR-200a). Increased concentration of circulating miRNA was followed by subsequent decline to initial level. These changes were consistent with presumed biological effect of TA and TACE on tumour tissue. CONCLUSION: Data of this pilot study show potential usage of circulating miRNA for monitoring of systemic effect of thermal ablative and intraarterial therapies. This work was created at Masaryk University as part of the project MUNI/A/1574/2018 and it was supported by Czech Ministry of Health grants No. 15-32484A, 16-31765A and 16-31314A. The authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 1. 3. 2019 Accepted: 4. 3. 2019.

Modeling cancer driver events in vitro using barrier bypass-clonal expansion assays and massively parallel sequencing.

The information on candidate cancer driver alterations available from public databases is often descriptive and of limited mechanistic insight, which poses difficulties for reliable distinction between true driver and passenger events. To address this challenge, we performed in-depth analysis of whole-exome sequencing data from cell lines generated by a barrier bypass-clonal expansion (BBCE) protocol. The employed strategy is based on carcinogen-driven immortalization of primary mouse embryonic fibroblasts and recapitulates early steps of cell transformation. Among the mutated genes were almost 200 COSMIC Cancer Gene Census genes, many of which were recurrently affected in the set of 25 immortalized cell lines. The alterations affected pathways regulating DNA damage response and repair, transcription and chromatin structure, cell cycle and cell death, as well as developmental pathways. The functional impact of the mutations was strongly supported by the manifestation of several known cancer hotspot mutations among the identified alterations. We identified a new set of genes encoding subunits of the BAF chromatin remodeling complex that exhibited Ras-mediated dependence on PRC2 histone methyltransferase activity, a finding that is similar to what has been observed for other BAF subunits in cancer cells. Among the affected BAF complex subunits, we determined Smarcd2 and Smarcc1 as putative driver candidates not yet fully identified by large-scale cancer genome sequencing projects. In addition, Ep400 displayed characteristics of a driver gene in that it showed a mutually exclusive mutation pattern when compared with mutations in the Trrap subunit of the TIP60 complex, both in the cell line panel and in a human tumor data set. We propose that the information generated by deep sequencing of the BBCE cell lines coupled with phenotypic analysis of the mutant cells can yield mechanistic insights into driver events relevant to human cancer development.

Base changes in tumour DNA have the power to reveal the causes and evolution of cancer.

Next-generation sequencing (NGS) technology has demonstrated that the cancer genomes are peppered with mutations. Although most somatic tumour mutations are unlikely to have any role in the cancer process per se, the spectra of DNA sequence changes in tumour mutation catalogues have the potential to identify the mutagens, and to reveal the mutagenic processes responsible for human cancer. Very recently, a novel approach for data mining of the vast compilations of tumour NGS data succeeded in separating and precisely defining at least 30 distinct patterns of sequence change hidden in mutation databases. At least half of these mutational signatures can be readily assigned to known human carcinogenic exposures or endogenous mechanisms of mutagenesis. A quantum leap in our knowledge of mutagenesis in human cancers has resulted, stimulating a flurry of research activity. We trace here the major findings leading first to the hypothesis that carcinogenic insults leave characteristic imprints on the DNA sequence of tumours, and culminating in empirical evidence from NGS data that well-defined carcinogen mutational signatures are indeed present in tumour genomic DNA from a variety of cancer types. The notion that tumour DNAs can divulge environmental sources of mutation is now a well-accepted fact. This approach to cancer aetiology has also incriminated various endogenous, enzyme-driven processes that increase the somatic mutation load in sporadic cancers. The tasks now confronting the field of molecular epidemiology are to assign mutagenic processes to orphan and newly discovered tumour mutation patterns, and to determine whether avoidable cancer risk factors influence signatures produced by endogenous enzymatic mechanisms. Innovative research with experimental models and exploitation of the geographical heterogeneity in cancer incidence can address these challenges.

Efficient in vivo microRNA targeting of liver metastasis.

Targeting oncogenic microRNAs (miRNAs) is emerging as a promising strategy for cancer therapy. In this study, we provide proof of principle for the safety and efficacy of miRNA targeting against metastatic tumors. We tested the impact of targeting miR-182, a pro-metastatic miRNA frequently overexpressed in melanoma, the in vitro silencing of which represses invasion and induces apoptosis. Specifically, we assessed the effect of anti-miR-182 oligonucleotides synthesized with 2' sugar modifications and a phosphorothioate backbone in a mouse model of melanoma liver metastasis. Luciferase imaging showed that mice treated with anti-miR-182 had a lower burden of liver metastases compared with control. We confirmed that miR-182 levels were effectively downregulated in the tumors of anti-miR-treated mice compared with tumors of control-treated mice, both in the liver and in the spleen. This effect was accompanied by an upregulation of multiple miR-182 direct targets. Transcriptional profiling of tumors treated with anti-miR-182 or with control oligonucleotides revealed an enrichment of genes controlling survival, adhesion and migration modulated in response to anti-miR-182 treatment. These data indicate that in vivo administration of anti-miRs allows for efficient miRNA targeting and concomitant upregulation of miRNA-controlled genes. Our results demonstrate that the use of anti-miR-182 is a promising therapeutic strategy for metastatic melanoma and provide a solid basis for testing similar strategies in human metastatic tumors.

Large-scale analysis of ion channel gene expression in the mouse heart during perinatal development.

The immature and mature heart differ from each other in terms of excitability, action potential properties, contractility, and relaxation. This includes upregulation of repolarizing K(+) currents, an enhanced inward rectifier K(+) (Kir) current, and changes in Ca(2+), Na(+), and Cl(-) currents. At the molecular level, the developmental regulation of ion channels is scantily described. Using a large-scale real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay, we performed a comprehensive analysis of ion channel transcript expression during perinatal development in the embryonic (embryonic day 17.5), neonatal (postnatal days 1-2), and adult Swiss-Webster mouse hearts. These data are compared with publicly available microarray data sets (Cardiogenomics project). Developmental mRNA expression for several transcripts was consistent with the published literature. For example, transcripts such as Kir2.1, Kir3.1, Nav1.5, Cav1.2, etc. were upregulated after birth, whereas others [e.g., Ca(2+)-activated K(+) (KCa)2.3 and minK] were downregulated. Cl(-) channel transcripts were expressed at higher levels in immature heart, particularly those that are activated by intracellular Ca(2+). Defining alterations in the ion channel transcriptome during perinatal development will lead to a much improved understanding of the electrophysiological alterations occurring in the heart after birth. Our study may have important repercussions in understanding the mechanisms and consequences of electrophysiological alterations in infants and may pave the way for better understanding of clinically relevant events such as congenital abnormalities, cardiomyopathies, heart failure, arrhythmias, cardiac drug therapy, and the sudden infant death syndrome.

Genetic programs of epithelial cell plasticity directed by transforming growth factor-beta.

Epithelial-mesenchymal transitions (EMTs) are an essential manifestation of epithelial cell plasticity during morphogenesis, wound healing, and tumor progression. Transforming growth factor-beta (TGF-beta) modulates epithelial plasticity in these physiological contexts by inducing EMT. Here we report a transcriptome screen of genetic programs of TGF-beta-induced EMT in human keratinocytes and propose functional roles for extracellular response kinase (ERK) mitogen-activated protein kinase signaling in cell motility and disruption of adherens junctions. We used DNA arrays of 16,580 human cDNAs to identify 728 known genes regulated by TGF-beta within 4 hours after treatment. TGF-beta-stimulated ERK signaling mediated regulation of 80 target genes not previously associated with this pathway. This subset is enriched for genes with defined roles in cell-matrix interactions, cell motility, and endocytosis. ERK-independent genetic programs underlying the onset of EMT involve key pathways and regulators of epithelial dedifferentiation, undifferentiated transitional and mesenchymal progenitor phenotypes, and mediators of cytoskeletal reorganization. The gene expression profiling approach delineates complex context-dependent signaling pathways and transcriptional events that determine epithelial cell plasticity controlled by TGF-beta. Investigation of the identified pathways and genes will advance the understanding of molecular mechanisms that underlie tumor invasiveness and metastasis.

Ribosomal protein S19 gene mutations in patients with diamond-blackfan anemia and identification of ribosomal protein S19 pseudogenes.

Diamond-Blackfan anemia (DBA) is a rare congenital pure red cell hypoplasia characterized by a selective defect of erythropoiesis with a normochromic macrocytic anemia and reticulocytopenia often accompanied by various congenital anomalies. The critical region responsible for the pathogenesis of DBA has been mapped in some patients to chromosome 19q13.2 (P Gustavsson, E Garelli, N Draptchinskaia, et al. Am. J. Hum. Genet. 63:1388-1395, 1998) and the gene encoding ribosomal protein S19 (RPS19) is believed to be the candidate gene. Here we present molecular analysis of the RPS19 gene in DBA patients from the Czech National DBA Registry. We found that the RPS19 gene was mutated in 25% (5/20) of DBA patients (insertion, deletion, and point mutations, but no nonsense or splice site mutations). Point mutations were localized to hot spots defined by Willig (TN Willig, N Draptchinskaia, I Dianzani, et al. Blood 94:4294-4306, 1999). Moreover, we describe two processed RPS19 pseudogenes, which were not expressed. Possible models of the DBA pathogenesis in the view of RPS19 mutations are discussed.

An antisense transcript to SMAD5 expressed in fetal and tumor tissues.

SMAD5, a transducer of TGF-beta/BMP inhibitory signals and a tumor suppressor candidate, localizes to the region of invariant loss in human myeloid neoplasms, on chromosome 5q31.1. Recent evidence indicates a gene-dosage effect along the TGF-beta/BMP signaling pathways. We have identified a novel transcript designated DAMS, whose 3' exonic sequences contain in part an alternate 5' exon of SMAD5, in the antisense orientation. Expressed sequenced tags (ESTs) for DAMS are found in fetal tissues (heart, adrenal glands, and total fetus) and pancreatic tumor cDNA libraries. In contrast to SMAD5, DAMS expression is not readily detectable in adult and fetal tissues. Semiquantitative PCR suggests that the stoichiometry between SMAD5 and DAMS transcripts ranges between 15 and 120 in normal and malignant hematopoietic cells. The findings raise the possibility that DAMS may be a fail-safe mechanism for precise regulation of SMAD5 transcript levels that may be critical in maintaining normal homeostasis.

Autosomal dominant distal renal tubular acidosis is associated in three families with heterozygosity for the R589H mutation in the AE1 (band 3) Cl-/HCO3- exchanger.

Distal renal tubular acidosis (dRTA) is characterized by defective urinary acidification by the distal nephron. Cl-/HCO3- exchange mediated by the AE1 anion exchanger in the basolateral membrane of type A intercalated cells is thought to be an essential component of lumenal H+ secretion by collecting duct intercalated cells. We evaluated the AE1 gene as a possible candidate gene for familial dRTA. We found in three unrelated families with autosomal dominant dRTA that all clinically affected individuals were heterozygous for a single missense mutation encoding the mutant AE1 polypeptide R589H. Patient red cells showed approximately 20% reduction in sulfate influx of normal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid sensitivity and pH dependence. Recombinant kidney AE1 R589H expressed in Xenopus oocytes showed 20-50% reduction in Cl-/Cl- and Cl-/HCO3- exchange, but did not display a dominant negative phenotype for anion transport when coexpressed with wild-type AE1. One apparently unaffected individual for whom acid-loading data were unavailable also was heterozygous for the mutation. Thus, in contrast to previously described heterozygous loss-of-function mutations in AE1 associated with red cell abnormalities and apparently normal renal acidification, the heterozygous hypomorphic AE1 mutation R589H is associated with dominant dRTA and normal red cells.

Smad5, a tumor suppressor candidate at 5q31.1, is hemizygously lost and not mutated in the retained allele in human leukemia cell line HL60.

Deletions of the long arm of chromosome 5 with common overlapping segment 5q31.1 are among the most frequent cytogenetic aberrations in myelodysplastic syndromes and acute myeloid leukemias (MDS/AML). We have constructed a YAC-based physical map of the 5q31.1 critical locus and localized the transcriptional transactivator Smad5 adjacent to loci showing consistent loss of heterozygosity in these disorders. Smad5 plays a key role along the bone morphogenetic protein-4 (BMP-4) inhibitory signalling pathway inducing embryonic hematopoiesis. Smad5 homologs Smad2 and DPC4 have recently been linked to human cancer. FISH analysis of AML-M2 cell line HL60 and of four MDS/AML patients revealed consistent hemizygous loss of the Smad5 locus. In HL60 cells, a translocation event within 5q31.1 associated with loss of adjacent material leads to disruption of the critical locus with partial retention of the 5q31.1 genomic sequences on a marker chromosome. RT-PCR sequencing analysis of the HL60 Smad5 remaining allele ruled out the functional inactivation of the gene analogous to that occurring in the Smad5 homologs DPC4 and Smad2 in cases of pancreatic and colorectal cancers. Mutational analysis of Smad5 in MDS/AML cases is in progress.

Genotoxicity of industrial effluents, river waters, and their fractions using the Ames test and in vitro cytogenetic assay.

Total and fractionated organic extracts from industrial effluents and Labe river water were tested for mutagenicity using the Ames test with TA98 strain and its YG derivatives (YG1021, YG1024 and YG1041) and cytogenetic analysis with human peripheral lymphocytes in vitro. The bacterial mutagenicity results showed a dose-dependent increase in numbers of TA98 revertants (10(5)) with effluent extracts and lower, but still significant, increase (10(3)) with river water extracts 6 km downstream. The further increase of YG revertants indicates the possible presence of nitroarenes and aromatic amines in the tested samples. In fractionated samples the significant mutagenicity was detected in two-polar acidic and polar basic-fractions, but the numbers of revertants in all effluent fractions were about one order of magnitude lower compared with total extracts results. The cytogenetic effect in human peripheral lymphocytes in vitro was not significant. The increase in chromosomal aberrations was not clearly dose-dependent and may reflect more the combination of toxic and clastogenic effects.

Influence of optical fibers on the spectrum of transmitted light-emitting-diode radiation.

Propagation of Ga(x)In(1-x)As(y)P (1-y) light-emitting-diode radiation through an optical fiber was found to affect its spectral curve very strongly, and the origin of this effect was analyzed. Various kinds of fibers were found to induce interferencelike modulations superimposed on the smooth spontaneous spectral curve of the diodes. We were able to show that when a monomode polarization-preserving fiber is used, the modulations are mostly due to intermodal interference.

Consistent loss of the D5S89 locus mapping telomeric to the interleukin gene cluster and centromeric to EGR-1 in patients with 5q- chromosome.

Interstitial deletions of the long arm of chromosome 5 are common in a number of disorders of leukemic and preleukemic myeloid disorders. Although the limits of these deletions vary among patients, a region of cytogenetic overlap that includes band 5q31 is deleted consistently, suggesting loss of 5q31 loci critical for normal myeloid differentiation and leukemogenesis. An anonymous genomic DNA segment D5S89, previously mapped to 5q21-31, detects consistent loss of alleles in cases showing the 5q- chromosome at presentation or relapse. Analysis of a panel of natural-deletion somatic-cell hybrids in conjunction with irradiation hybrids containing fragments of human chromosome 5q shows that the D5S89 locus is telomeric to the interleukin (IL) genes (IL-3, IL-4, IL-5, IL-9, and granulocyte-macrophage colony-stimulating factor [GM-CSF]) and interferon response factor-1 (IRF-1) gene and centromeric to the early response transcription factor (early growth response gene-1 [EGR-1]) on 5q31. To further define the principal region of loss, we have isolated and characterized yeast artificial chromosomes (YACs) spanning D5S89. The presence of several CpG islands within the 300-kb YAC is suggestive of multiple transcription units. However, IL-4, IL-5, IRF-1, IL-3, GM-CSF, and EGR-1 genes were not detected in the YAC clone spanning D5S89, implying that none of these genes are in the vicinity of the D5S89 marker. Further characterization of these YACs should facilitate the isolation of novel candidate genes that may play a role in the evolution of the abnormal phenotype associated with 5q- chromosome.

Factor VIII gene deletions in haemophilia A patients in Czechoslovakia.

Genomic DNA from 90 Czechoslovak haemophilia A patients from 81 pedigrees was analysed by Southern blotting and hybridization with factor VIII cDNA probes. Three partial deletions of the factor VIII gene were identified and characterized: a 4.8 kilobase (kb) deletion eliminating exon 10 in one patient with severe haemophilia A without inhibitor, a 6.1 kb deletion eliminating the 3' part of intron 13 and the 5' part of exon 14 in two related severe haemophiliacs, but only one of them produced inhibitor, and a 4.6 kb deletion eliminating the 3' part of intron 13 and the 5' part of exon 14 in a severe haemophiliac with high-titre inhibitor. Besides these three deletions, three different restriction site variants without apparent loss of DNA sequence were found.