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Analysis of circulating cell-free DNA (cfDNA) of colorectal adenoma (AD) and cancer (CRC) patients provides a minimally invasive approach that is able to explore genetic alterations. It is unknown whether there are specific genetic variants that could explain the high prevalence of CRC in Hungary. Whole-exome sequencing (WES) was performed on colon tissues (27 AD, 51 CRC) and matched cfDNAs (17 AD, 33 CRC); furthermore, targeted panel sequencing was performed on a subset of cfDNA samples. The most frequently mutated genes were APC, KRAS, and FBN3 in AD, while APC, TP53, TTN, and KRAS were the most frequently mutated in CRC tissue. Variants in KRAS codons 12 (AD: 8/27, CRC: 11/51 (0.216)) and 13 (CRC: 3/51 (0.06)) were the most frequent in our sample set, with G12V (5/27) dominance in ADs and G12D (5/51 (0.098)) in CRCs. In terms of the cfDNA WES results, tumor somatic variants were found in 6/33 of CRC cases. Panel sequencing revealed somatic variants in 8 out of the 12 enrolled patients, identifying 12/20 tumor somatic variants falling on its targeted regions, while WES recovered only 20% in the respective regions in cfDNA of the same patients. In liquid biopsy analyses, WES is less efficient compared to the targeted panel sequencing with a higher coverage depth that can hold a relevant clinical potential to be applied in everyday practice in the future.

Up-regulation of the long non-coding RNA LINC00152 can contribute to cancer development, proliferation and invasion, including colorectal cancer, however, its mechanism of action in colorectal carcinogenesis and progression is only insufficiently understood. In this work we correlated LINC00152 expression with promoter DNA methylation changes in colorectal tissues along the normal-adenoma-carcinoma sequence and studied the effects of LINC00152 silencing on the cell cycle regulation and on the whole transcriptome in colon carcinoma cells using cell and molecular biology techniques. LINC00152 was significantly up-regulated in adenoma and colorectal cancer (p

Absztrakt: A colorectalis carcinoma (CRC) az egyik leggyakrabban előforduló daganatos megbetegedés világszerte. A sporadikus vastagbélrák incidenciája ötvenéves kor alatt alacsonyabb, majd az életkor előrehaladtával nő, továbbá jellegzetes klinikai, lokalizáció szerinti és molekuláris eltérést mutathat a nők és a férfiak között. Epidemiológiai és molekuláris biológiai kutatások eredményei szerint az ösztradiol (E2) által szabályozott jelútrendszer meghatározó szerepet játszik a CRC kialakulásában és prognosztikájában, döntően a vastagbélhámban domináns ösztrogénreceptor-bétán (ERβ) keresztül. Az ösztradiol emésztőrendszeri hatásai igen sokrétűek, az ép és tumoros vastagbélhámsejtekre gyakorolt hatását in vitro és in vivo vizsgálatok egyaránt igazolták. Az ösztrogénreceptor-alfával (ERα) ellentétben az ERβ aktivációja a sejtosztódást gátolja és az apoptózist fokozza, a béta-receptor kifejeződése ugyanakkor mind az élettani öregedés, mind a vastagbél kórállapotaiban megváltozhat. Az ösztradiol ERβ által közvetített daganatellenes hatása a sejtproliferáció gátlása, az apoptózis serkentése, az áttétképzés gátlása és gyulladáscsökkentő hatása révén valósulhat meg. Sejtkultúra- és állatkísérletes kutatások eredményei alapján az ösztrogénreceptor-bétára szelektíven ható receptormodulátorok (szelektív ösztrogénreceptor-modulátor [SERM]) és a fitoösztrogének új, hozzáadott kezelési lehetőséget jelenthetnek az idült gyulladással és a kóros sejtproliferációval jellemezhető colorectalis megbetegedésekben. Orv Hetil. 2020; 161(14): 532–543.

Folic acid (FA) is a synthetic form of vitamin B9, generally used as a nutritional supplement and an adjunctive medication in cancer therapy. FA is involved in genetic and epigenetic regulation; therefore, it has a dual modulatory role in established neoplasms. We aimed to investigate the effect of short-term (72 h) FA supplementation on colorectal cancer; hence, HT-29 and SW480 cells were exposed to different FA concentrations (0, 100, 10,000 ng/mL). HT-29 cell proliferation and viability levels elevated after 100 ng/mL but decreased for 10,000 ng/mL FA. Additionally, a significant (p ≤ 0.05) improvement of genomic stability was detected in HT-29 cells with micronucleus scoring and comet assay. Conversely, the FA treatment did not alter these parameters in SW480 samples. RRBS results highlighted that DNA methylation changes were bidirectional in both cells, mainly affecting carcinogenesis-related pathways. Based on the microarray analysis, promoter methylation status was in accordance with FA-induced expression alterations of 27 genes. Our study demonstrates that the FA effect was highly dependent on the cell type, which can be attributed to the distinct molecular background and the different expression of proliferation- and DNA-repair-associated genes (YWHAZ, HES1, STAT3, CCL2). Moreover, new aspects of FA-regulated DNA methylation and consecutive gene expression were revealed.

BackgroundHypomethylation of long interspersed nuclear element 1 (LINE-1) is characteristic of various cancer types, including colorectal cancer (CRC). Malfunction of several factors or alteration of methyl-donor molecules’ (folic acid and S-adenosylmethionine) availability can contribute to DNA methylation changes. Detection of epigenetic alterations in liquid biopsies can assist in the early recognition of CRC. Following the investigations of a Hungarian colon tissue sample set, our goal was to examine the LINE-1 methylation of blood samples along the colorectal adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore the possible underlying mechanisms of global DNA hypomethylation formation on a multi-level aspect.MethodsLINE-1 methylation of colon tissue (n = 183) and plasma (n = 48) samples of healthy controls and patients with colorectal tumours were examined with bisulfite pyrosequencing. To investigate mRNA expression, microarray analysis results were reanalysed in silico (n = 60).Immunohistochemistry staining was used to validate DNA methyltransferases (DNMTs)and folate receptor beta(FOLR2)expression along with the determination of methyl-donor molecules’ in situ level (n = 40).ResultsSignificantly decreased LINE-1 methylation level was observed in line with cancer progression both in tissue (adenoma: 72.7 ± 4.8%, and CRC: 69.7 ± 7.6% vs. normal: 77.5 ± 1.7%,p ≤ 0.01) and liquid biopsies (adenoma: 80.0 ± 1.7%, and CRC: 79.8 ± 1.3% vs. normal: 82.0 ± 2.0%,p ≤ 0.01). However, no significant changes were recognized in inflammatory bowel disease cases. According to in silico analysis of microarray data, altered mRNA levels of several DNA methylation-related enzymes were detected in tumours vs. healthy biopsies, namely one-carbon metabolism-related genes—which met our analysing criteria—showed upregulation, whileFOLR2was downregulated. Using immunohistochemistry,DNMTs,andFOLR2expression were confirmed. Moreover, significantly diminished folic acid and S-adenosylmethionine levels were observed in parallel with decreasing 5-methylcytosine staining in tumours compared to normal adjacent to tumour tissues (p ≤ 0.05).ConclusionOur results suggest that LINE-1 hypomethylation may have a distinguishing value in precancerous stages compared to healthy samples in liquid biopsies. Furthermore, the reduction of global DNA methylation level could be linked to reduced methyl-donor availability with the contribution of decreasedFOLR2expression.

Abstract: The incidence and mortality of colorectal cancer (CRC) are considerably high in Central European countries, it is the second most common cancer in both men and women in Hungary with 10,000 newly registered patients per year. These data indicate the necessity of new screening methods that are more comfortable for patients, hence the compliance can be increased. Cell-free DNA (cfDNA) level in blood is elevated in certain physiological conditions, such as pregnancy or high physical activity. Furthermore, cfDNA concentration alterations can also be detected in some pathological processes; increased cfDNA amount was observed in autoimmune and inflammatory diseases, as well as in various cancers including CRC. Numerous studies about origin, function, and mechanism of cfDNA can be found in the scientific literature. In this review, we aimed to describe the quantitative and qualitative changes of cfDNA, to present its functions, and to provide an overview of the available diagnostic applications for CRC. CfDNA can be released to the circulatory system via apoptosis, necrosis or by direct secretions by living cells. In cancer patients, cfDNA can originate from healthy and cancer cells, hence genetic (e.g. mutations in APC, KRAS, BRAF) and epigenetic (e.g. methylation in SEPT9, SFRP1) alterations of tumor cells can be examined in cfDNA fraction. Several high-throughput, sensitive and even automated methods are available providing opportunity to perform standardized sample preparation and to analyse biomarker candidates quantitatively. These enhancements can help to develop alternative screening methods that can be easily integrated into the clinical practice and can contribute to early cancer detection. Orv Hetil. 2019; 160(30): 1167–1177.

Abstract: Vitamin B9, also known as folate, can be found in natural and synthetic forms, mostly in vegetables or folic acid containing food supplements. By participating in the proper cell development and division, its presence is indispensable for certain basic metabolic processes. The decreased folate level of the body, mainly caused by environmental and hereditary factors as well as aging, can lead to genetic, epigenetic and metabolic changes. It can be related to the development of megaloblastic anemia, various cardiovascular diseases (such as atherosclerosis, stroke) obstetrical complications (such as abruption of the placentae, spontaneous abortion, preterm delivery, neural tube defect), neuropsychiatric diseases (such as Alzheimer’s disease, Parkinson’s disease, depression) and tumors. The vitamin has a preventive effect in all the above-mentioned diseases, however, in the case of tumor existence, its therapeutic use requires great care, as it may promote the progression of certain precancerous lesions. Food fortification with folic acid is currently being carried out in more than 60 countries in order to ensure a minimum vitamin B9 requirement for the population and therefore to prevent the development of the diseases that are connected to folic acid deficiency. Due to its assumable role in carcinogenesis, an initial concern had taken place when fortification was implemented (1998), however, the present statistical data do not confirm such adverse health effects. On the other hand, several beneficial properties can be connected to the vitamin, that can be the reason why more and more countries are considering to join this program. Besides the fact that folic acid is a widely used food supplement, it is also applied in oncological medicine (leucovorin) to increase the effectiveness of certain chemotherapeutical drugs (e.g. methotrexate, 5-fluorouracil). Orv Hetil. 2019; 160(28): 1087–1096.

Synthetic vitamin B9, also known as folic acid (FA), is an extensively used nutritional supplement as well as an adjunctive medication in cancer therapy. However, there is increasing evidence that FA can promote the progression of established colorectal cancers (CRC); therefore, great care is required in the case of its application. In order to gain knowledge about the underlying mechanisms, we analyzed the genomic and the epigenomic effect of different FA supplies on two CRC cell lines with distinct molecular backgrounds. HT-29 and SW480 cells were kept in FA-free media (0 ng/mL) or treated with 100 ng/mL and 10000 ng/mL FA for 72 hours. Firstly, cell proliferation and cell viability alterations were determined with Supforhodamine B and AlamarBlue assays; then, cell cycle analysis was performed using fluorescence-activated cell sorting (FACS). Global DNA methylation level was investigated with the pyrosequencing of long interspersed nuclear element 1 (LINE-1) retrotransposons. Micronucleus scoring performed on DAPI- and anti-γ-H2AX-stained slides, as well as Comet assay, were used to detect the impact of FA on genome integrity. Finally, we analyzed gene expression alterations with Human Transcriptome Array (HTA) 2.0. Our data revealed that 100 ng/mL FA induced significant (p≤0.05) elevation (HT-29100: 128.4±24.9%) of HT-29 cell proliferation compared to the other two circumstances (HT-290: 101.3±13.5%; HT-2910000: 86.1±20.8%). The tendency of cell viability was analogous to the results detected during cell proliferation analyses (HT-290: 91.6±13.3%; HT-29100: 115.8±30.9%; HT-2910000: 64.1±20.2%). The genomic stability of FA-supplemented HT-29 samples was improved in a significant manner (p≤0.05), based on the results of micronucleus scoring (HT-290: 0.56±0.05%; HT-29100: 0.17±0.05%; HT-2910000: 0.25±0.09%) and Comet assay (HT-290: 37.4±3.5%; HT-29100: 31.2±3.4%; HT-2910000: 20.1±3.6%). However, in SW480 cells, remarkable alterations were not detected concerning these parameters. Fundamental differences were observed between the two cell lines in the case of cell cycle (HT-29: G0/1 phase dominance; SW480: S phase dominance) and global DNA methylation analysis (HT-29: 59.1±1.0; SW480: 49.0±0.2), but exposing them to different FA doses did not lead to significant changes. Gene expression alterations were more diverse, as genes involved in carcinogenesis were either up- (HES1, SLC7A11) or downregulated (CCL2) for FA supplementation. We concluded that the effect of FA was considerably influenced by the cell type and the applied FA concentration. Thereby, translating our in vitro results to patient care, we would emphasize the importance of genetic and epigenetic investigations coupled with the choice of proper FA dose upon CRC diagnosis to achieve the best disease outcome. Citation Format: Sára Zsigrai, Alexandra Kalmár, Barbara Kinga Barták, Zsófia Brigitta Nagy, Krisztina Andrea Szigeti, Gábor Valcz, Titanilla Dankó, Anna Sebestyén, Gábor Barna, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Folic acid has a cell type- and dose-dependent effect on the genome and the epigenome of colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3730.

Reduction of global DNA methylation is a characteristic epigenetic alteration of various cancer types, including colorectal cancer. Abnormality of several factors, such as DNA methyltransferases (DNMT), demethylases, or deviation in methyl-donor (folate and S-adenosylmethionine) availability can contribute to the development of genome-wide hypomethylation. Detection of epigenetic changes as global DNA hypomethylation in cell-free DNA fraction obtained from blood samples can expand the opportunities for the early recognition of colorectal cancer. One of our main goals was the investigation of global DNA methylation patterns in tissue biopsies (n=183) and cell-free DNA fraction of blood samples (n=48) along the colorectal normal-adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore possible underlying mechanisms of genome-wide hypomethylation formation in 12 colorectal tumor tissue sections, containing transition zones. Using LINE-1 pyrosequencing, significantly reduced global DNA methylation level was detected in line with cancer progression in tissue specimens (normal: 77.5±1.7%, adenoma: 72.7±4.8%, carcinoma: 69.7±7.6%, p≤0.0001) and in liquid biopsies as well (normal: 82.0±2.0%, adenoma: 80.0±1.7%, carcinoma: 79.8±1.3%, p≤0.01). However, no significant methylation changes were found in inflammatory bowel disease cases. Analyzing microarray data in silico, altered mRNA expression of certain methylation-, and one-carbon metabolism-related genes were detected in tumorous specimens vs. healthy biopsies, from which DNMT1 was upregulated, and folate receptor 2 (FOLR2) was downregulated. DNMT and FOLR2 expression were validated by immunohistochemistry. Furthermore, significantly reduced folic acid and S-adenosylmethionine content were observed in parallel with diminishing 5-methylcytosine levels in adenoma and carcinoma sections compared to normal adjacent to tumor tissue areas by immunolabeling (p≤0.05). Our results suggest that intraindividual monitoring of genome-wide hypomethylation may assist in the recognition of adenoma formation, cancer progression, or remission as well. Moreover, lower global DNA methylation level could be connected to decreased methyl-donor availability with the contribution of reduced FOLR2 expression. Citation Format: Krisztina Andrea Szigeti, Alexandra Kalmár, Gábor Valcz, Barbara Kinga Barták, Zsófia Nagy, Sára Zsigrai, Ildikó Felletár, Árpád V Patai, Tamás Micsik, Márton Papp, Eszter Márkus, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Global DNA hypomethylation can be linked to decreased methyl-donor content in colorectal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3745.

The main function of the single-carbon metabolic cycle is maintaining nucleotide pool and DNA methylation which is highly influenced by the chemotherapy protocols. This study aimed to evaluate the ultrashort effect of reduced folate combined anticancer treatment on peripheral blood parameters of colorectal cancer (CRC) patients. Post-operative CRC patients were treated with chemotherapy using the combination of oxaliplatin, 5-FU, leucovorin, and capecitabine. Blood samples were taken until the beginning of the treatment and immediately after the therapy. Plasma fractions were separated and the levels of S-adenosylmethionine (SAM), S-adenosyl homocysteine (SAH) and the simultaneous quantification of nucleotides (adenine, cytosine, thymine, guanine and uracil) were detected by HPLC-MS/MS. Homocysteine (HCY) was also determined from plasma specimens. Cell-free DNA (cfDNA) was isolated from plasma and DNA methylation was analyzed by LINE-1 bisulfite pyrosequencing. Moreover, LINE-1 methylation statuses were also examined from PBMC cells that were separated from whole blood by a density gradient centrifugation. In each patient, the HCY level of plasma was decreased by an average of 17% after oxaliplatin treatment with the combination of capecitabine (XELOX) and also with 5-FU + leucovorin (FOLFOX) agents. Our study also confirmed that cancer patients have an elevated cfDNA level (avg. 20ng/plasma mL); however, its concentration decreased significantly (p=0.02) on average by 50% after treatment compared to the baseline. The same tendency was observed in the case of SAM after XELOX and FOLFOX adjuvant therapy in plasma samples (p=0.03). Moreover, mostly elevated SAM/SAH ratios were observed in parallel with reduced SAH level after the therapy. Significant (p=0.0001) mean LINE-1 hypomethylation was detected in PBMC (81.84%) compared to cfDNA of plasma (73.76%), furthermore, slightly DNA hypermethylation was noticed in CpG1 position of LINE-1 after chemotherapy in both mononuclear cells and circulating DNA. Lower level of plasma nucleotides was identified in case of adenine, cytosine and uracil after treatment, while thymine and guanine were below the detection limit. The present study demonstrates that the DNA-damaging chemotherapy results in a detectable decrease in cfDNA immediately after the treatment, that was supported by the decrease in the level of the nucleotides measured in the plasma fraction. The methionine cycle is responsible for the DNA methylation maintenance, and though the amount of members of this pathway was reduced, only a moderately increased DNA methylation could be detected even in such a short period time. Co-administration of high dose leucovorin as a folate derivative and a methyl donor with further chemotherapeutic agents may actively contribute to genome alterations. Citation Format: Zsófia Brigitta Nagy, Barbara Kinga Barták, Tamás Fodor, Gellért Balázs Karvaly, Sára Zsigrai, Krisztina Andrea Szigeti, Alexandra Kalmár, Magdolna Dank, István Takács, Béla Molnár. Folic acid combined chemotherapy has an immediate effect on C1 methionine cycle and consequential DNA methylation in liquid biopsy samples of colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3741.

Background and aims: The early detection of colorectal cancer (CRC) recurrence and the monitoring of therapeutic response are crucial steps in the determination and modification of treatment strategies. The currently used gold standard tumor markers such as CEA and CA 19-9 and the different imaging techniques have several limitations in many cases. Therefore, we aimed to establish a liquid biopsy-based approach for tracking tumor dynamics in post-operative non-metastatic (n=32) and metastatic (n=23) CRC patients. Methods: Blood samples were collected from each patient before and during chemotherapy, and finally, patients were classified according to disease outcome. Longitudinal investigations of the total amount, global and local (SFRP2 and SDC2 genes) DNA methylation pattern of cell-free DNA (cfDNA) fraction were performed. The plasma concentration of homocysteine was also determined, as it is one of the main components of the DNA methylation process, and its level defines the methylation potential. We examined how the parameters mentioned above were affected depending on the different therapy responses. Results: The average cfDNA amount was significantly higher (p Conclusion: Our study offers the possibility to monitor the therapeutic response during chemotherapy with a minimally-invasive blood-based method that combines the analysis of cfDNA, its global and local DNA methylation pattern, and homocysteine level. Citation Format: Barbara Kinga Bartak, Tamás Fodor, Alexandra Kalmár, Zsófia Brigitta Nagy, Sára Zsigrai, Krisztina Andrea Szigeti, William Kothalawala, Gábor Valcz, Péter Igaz, István Takács, Magdolna Dank, Béla Molnár. Monitoring chemotherapy response status of colorectal cancer patients using liquid biopsy samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3738.

Global DNA hypomethylation is a characteristic feature of colorectal carcinoma (CRC). The tumor inhibitory effect of S-adenosylmethionine (SAM) methyl donor has been described in certain cancers including CRC. However, the molecular impact of SAM treatment on CRC cell lines with distinct genetic features has not been evaluated comprehensively. HT-29 and SW480 cells were treated with 0.5 and 1 mmol/L SAM for 48 h followed by cell proliferation measurements, whole-genome transcriptome and methylome analyses, DNA stability assessments and exome sequencing. SAM reduced cell number and increased senescence by causing S phase arrest, besides, multiple EMT-related genes (e.g., TGFB1) were downregulated in both cell lines. Alteration in the global DNA methylation level was not observed, but certain methylation changes in gene promoters were detected. SAM-induced &gamma, H2AX elevation could be associated with activated DNA repair pathway showing upregulated gene expression (e.g., HUS1). Remarkable genomic stability elevation, namely, decreased micronucleus number and comet tail length was observed only in SW480 after treatment. SAM has the potential to induce senescence, DNA repair, genome stability and to reduce CRC progression. However, the different therapeutic responses of HT-29 and SW480 to SAM emphasize the importance of the molecular characterization of CRC cases prior to methyl donor supplementation.

Colorectal cancer (CRC) is one of the most common types of cancers worldwide. The incidence of sporadic CRC is lower in individuals below 50 years and increases with age, furthermore, it shows typical clinical, macroscopic and molecular differences between females and males. According to the results of epidemiological and molecular biology studies, the estradiol-regulating signaling pathway plays an important role in the development and prognosis of CRC, predominantly through estrogen receptor beta (ERβ), which is dominant in the colonic epithelium. Estradiol has multiple gastrointestinal effects, which were confirmed byAbsztrakt: A colorectalis carcinoma (CRC) az egyik leggyakrabban előforduló daganatos megbetegedés világszerte. A sporadikus vastagbélrák incidenciája ötvenéves kor alatt alacsonyabb, majd az életkor előrehaladtával nő, továbbá jellegzetes klinikai, lokalizáció szerinti és molekuláris eltérést mutathat a nők és a férfiak között. Epidemiológiai és molekuláris biológiai kutatások eredményei szerint az ösztradiol (E2) által szabályozott jelútrendszer meghatározó szerepet játszik a CRC kialakulásában és prognosztikájában, döntően a vastagbélhámban domináns ösztrogénreceptor-bétán (ERβ) keresztül. Az ösztradiol emésztőrendszeri hatásai igen sokrétűek, az ép és tumoros vastagbélhámsejtekre gyakorolt hatását

The incidence and mortality of colorectal cancer (CRC) are considerably high in Central European countries, it is the second most common cancer in both men and women in Hungary with 10,000 newly registered patients per year. These data indicate the necessity of new screening methods that are more comfortable for patients, hence the compliance can be increased. Cell-free DNA (cfDNA) level in blood is elevated in certain physiological conditions, such as pregnancy or high physical activity. Furthermore, cfDNA concentration alterations can also be detected in some pathological processes; increased cfDNA amount was observed in autoimmune and inflammatory diseases, as well as in various cancers including CRC. Numerous studies about origin, function, and mechanism of cfDNA can be found in the scientific literature. In this review, we aimed to describe the quantitative and qualitative changes of cfDNA, to present its functions, and to provide an overview of the available diagnostic applications for CRC. CfDNA can be released to the circulatory systemAbsztrakt: A vastagbélrák (CRC) incidenciája és mortalitása is kiemelkedően magas a közép-európai országokban, hazánkban a második leggyakoribb daganattípus mind a férfiak, mind a nők körében. Az évente újonnan regisztrált betegek száma 10 000 köré tehető. Ezek az adatok jelzik, hogy szükséges olyan szűrőmódszerek kifejlesztése, amelyek a betegek számára kevéssé megterhelőek, ezáltal növelhető a vizsgálatokon történő részvétel. A vérben található, sejten kívüli szabad DNS (skDNS) szintje bizonyos fiziológiás állapotokban megnő, többek között terhesség vagy erőteljes fizikai aktivitás esetén. Az skDNS koncentrációja azonban egyes kórállapotokban, például autoimmun és gyulladásos megbetegedésekben, valamint különböző daganattípusokban, többek között vastagbélrákban is emelkedett értéket mutat. Az skDNS eredetére, funkciójára és hatásmechanizmusára vonatkozóan számos tanulmány található a szakirodalomban. Jelen összefoglaló közleményünk célja a szabad DNS mennyiségi és minőségi változásainak ismertetése, funkcióinak áttekintése, valamint diagnosztikus alkalmazási lehetőségeinek bemutatása a vastagbélrák korai észlelésének szempontjából. A szabad-DNS-molekulák többféle módon kerülhetnek a keringésbe, az apoptózis és nekrózis mellett az élő sejtek által történő direkt szekréció is lehetséges. Daganat kialakulása esetén az egészséges és a rákos sejtek egyaránt képesek DNS-t kibocsátani a perifériás vérbe, így a tumorsejtekben bekövetkező genetikai (például mutáció

Vitamin BAbsztrakt: A B

Cell-free DNA (cfDNA) level increases in several physiological conditions, such as pregnancy, aging or high physical activity. Furthermore, alterations of cfDNA concentration and genetic or epigenetic (e.g. DNA methylation) patterns can also be observed in pathological processes, as inflammatory diseases or different cancer types including colorectal cancer (CRC). However, studies about quality changes of cfDNA aimed to analyze its fragment length profile and DNA methylation pattern are lacking. We aimed to define the quantity and quality changes of cfDNA, including concentration, fragment length distribution and global DNA methylation pattern during high physical exercise, and in colorectal adenoma and cancer patients. Moreover, we aimed to investigate the treatment response in plasma of metastatic CRC (mCRC) patients analyzing the above-mentioned parameters and the DNA methylation level of SFRP2 gene during therapy. Plasma fraction was separated from blood samples of 6 healthy athletes before, during and after physical training, and from healthy (n=16), adenoma (n=13), IBD (n=19), and CRC (n=16) patients. Moreover, plasma longitudinal assessment was performed in the case of 20 mCRC patients treated with chemotherapy. CfDNA level was quantified with Qubit 1.0 (Thermo Fisher Scientific), fragment length distribution was examined by 2100 Bioanalyzer instrument (Agilent), and global DNA methylation level was measured with bisulfite pyrosequencing of long interspersed nuclear element-1 (LINE-1) (Qiagen) in all samples. Besides, DNA methylation status of SFRP2 gene was determined with droplet digital PCR (Bio-Rad) in mCRC plasma samples. Increased cfDNA amount was observed during physical exercise (198.5±90ng), in comparison with control phase (86.3±40.6ng), and then in restitution period, lower cfDNA level (155.6±119.3ng) was detected. Elevated cfDNA amounts have been found in plasma of adenoma (72.1±37.5ng), IBD (78.0±50.9ng) and CRC (84.5±70.1ng) patients compared to controls (36.2±11.3ng). CfDNA fragment length distribution showed different patterns in each sample group, longer fragments (200-600bp) enriched in AD and CRC samples in addition to short ( Citation Format: Barbara K. Bartak, Krisztina Andrea Szigeti, Alexandra Kalmar, Orsolya Galamb, Zsófia Brigitta Nagy, Sara Zsigrai, Zsolt Tulassay, Peter Igaz, Magdolna Dank, Bela Molnar. Analyses of cell-free DNA alterations in physiological conditions and colon diseases for screening, diagnostics and therapy follow-up [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1276.

Background: Colorectal cancer (CRC) has high incidence in Eastern-Europe, especially in Hungary. In addition to environmental factors, the genetic and epigenetic background of this phenomenon still remains unknown. Aims: We aimed to perform whole exome sequencing along the colorectal adenoma-carcinoma sequence on tissue samples in order to identify characteristic germline and somatic variants. Materials & methods: Genomic DNA was isolated from 111 colonic tissue samples including 20 adenomas (AD) with matched normal adjacent tissue (NAT) pairs, 18 CRC with matched NAT pairs, 3 normal (N) young donors, 1 CRC and 1 AD and from 2 colorectal cancer cell lines (HT29, SW480), furthermore, 20 buffy coat (from 10 healthy, 5 adenoma and CRC patients). Exome enrichment was done with the Nextera DNA Exome Kit and sequencing was performed with the NextSeq 500/550 High Output v2 kit using the NextSeq 500 Instrument. Raw data analysis and demultiplexing was completed on BaseSpace Sequence Hub. Germline and somatic variants were determined by Genome Analysis Toolkit (GATK) and MuTect2 algorithms by using the default filter settings and were confirmed by ddPCR. Results: The total number of detected variants increased along the adenoma-carcinoma sequence and NAT samples showed higher number of variants compared to healthy tissues. On the basis of the somatic variant analysis, certain genes with the most frequent CRC-associated pathogenic mutations could be detected in relatively higher [e.g. APC (89% of AD samples, 74% of CRC samples)], with relatively lower [e.g. KRAS (47% of ADs, 33% CRC), TP53 (26% of ADs, 14% of CRCs), or with average frequencies [PIK3CA (4.8% of AD samples, 11% of CRC samples), NRAS (5% of ADs, 4.7% of CRCs)]. One adenoma sample (4.7%) exerted BRAF mutation. On the basis of the COSMIC database, the expected variant profile of the CRC cell lines could be confirmed (HT-29: BRAF, TP53; SW480: KRAS, TP53). Among the germline variants, MTHFR C677T (rs1801133) were detected in 4 N (40% CT) 8 AD (37% CT, 5% TT) 9 CRC samples (37% CT, 11% TT), and MTHFR A1298C (rs1801131) were detected in 7 N (50% AC, 20% CC) 12 AD (47% AC, 16% CC) and 10 CRC samples (42% AC, 11% CC) representing a relatively higher frequency in the Hungarian population compared to the European and worldwide statistics. Conclusion: Identification of CRC-associated pathogen variant patterns in the Hungarian population revealed a higher tumor mutation burden already in pre-malignant colorectal adenoma samples, as well as in CRC. Alterations in the frequencies of CRC-associated somatic variants compared to the European averages might contribute to the high CRC incidence observed in the Hungarian population. The germline variants of MTHFR gene were observed with a relatively higher frequency in our cohort having an effect on the DNA methylation regulation system that might be a link between the genetic and epigenetic alterations of the CRC development. Citation Format: Alexandra Kalmar, Orsolya Galamb, Barnabas Wichmann, Barbara B Bartak, Zsofia B Nagy, Sara Zsigrai, Krisztina Szigeti, Orsolya Pipek, Anna Medgyes-Horváth, Istvan Csabai, Zsolt Tulassay, Peter Igaz, Bela Molnar. Differences in the frequency of CRC-associated pathogen variants in colorectal adenomas and cancers from the Hungarian population [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3623.

Background: The balance of dNTP pool is needed for normal DNA synthesis. Nucleotide depletion can cause DNA damage thus it could lead to tumor development. The supplementation of substrates that are involved in DNA synthesis and methylation reactions could be a preventive step against oncogenic processes. Furthermore, tumor origin and heterogeneity could influence the cell response depend on experimental models (human SW480 versus animal-derived MCA38 colon adenocarcinoma). Aims: Our aims were to investigate the effect of elevated dNTP pool and modified DNA fragments on tumor growth, furthermore to detect the influence of different types of folates on tumor formation. Methods: C57BL/6 (n=35) mice with implanted MCA38 mouse colorectal adenocarcinoma cells were divided into 7 groups. Mice were injected subcutaneously on every 2nd days over 4 weeks in the doses of 10ug/animal with the following materials: dNTP (Group1) and methylated (d5mCTP) deoxynucleotide solution (Group2). In Group 3-5, mice were injected with artificially methylated and unmethylated spleen-derived DNA in different doses. Group 6 was supplemented with 20mg/kg folic acid orally, while saline (0.9% NaCl) was used as a vehicle in control group. In parallel, human-derived SW480 colon cancer cells (5 × 106) were injected subcutaneously on SCID mice (n=15). Xenograft mice were divided into 3 groups. 20mg/kg folic acid (Group A) and 20mg/kg L-methylfolate (Group B) were supplemented orally 5 times a week, as well as control mice were treated with saline. The length and width of the implanted tumors were measured with sliding calipers two times weekly by the same observer. Results: In the animal-derived (MCA38) tumor model, slower tumor developing tendency was observed in the dNTP supplementation group; d5mCTP supplementation moderately decreased tumor growth. Non-methylated DNA has also a concentration-dependent growth decreasing effect in MCA38 tumor development. However, folate supplementation has a different effect on tumor development based on the origin of the implanted colon tissue. Slower tumor developing tendency could be detected in the case of C57BL/6-C38 tumor model compared to controls, while folate supplementation stimulated the growth and proliferation of tumor cells in SW480-derived xenograft model (p Conclusion: Our study described the effect of differently methylated DNA fragments and precursor molecules on the growth of tumor transplants. According to our results, tumor volume decrease was observed by providing sufficient DNA components to normal synthesis. Based on our results folate treatment has a different effect on cancer development regarding the tumor origin. Folate has a key role in nucleotide synthesis and also in DNA methylation therefore the selection of the appropriate dosing time is crucial. Citation Format: Zsófia Brigitta Nagy, Barbara Kinga Barták, Sára Zsigrai, Alexandra Kalmár, Krisztina Szigeti, Peter Igaz, Bela Molnár. Effect of DNA fragments and nucleotid supplementation on tumor development in mice model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2761.

Global DNA hypomethylation can be observed along with the aging of normal cells, and it is also related to tumor initiation and progression. S-adenosylmethionine (SAM) is a universal methyl donor molecule, used as a dietary supplement. SAM is involved in DNA methylation processes, thereby it may have a favorable effect on gene expression of cancer-associated genes through epigenetic modifications, but may also influence DNA folding during repair processes. Our aim was to analyze the effect of SAM treatment on global and promoter-specific DNA methylation level, gene expression, DNA integrity, cell cycle and the proliferation of two different colorectal cancer cell lines (HT-29, SW480). HT-29 and SW480 cells were treated with SAM in different concentrations (0, 0.5, 1 mmol/l) for 48 hours. Global DNA methylation status was analyzed by bisulfite pyrosequencing of long interspersed nuclear element-1 (LINE-1) retrotransposons. Promoter-specific DNA methylation alterations were determined by Reduced Representation Bisulfite Sequencing (RRBS) method. Gene expression changes were detected using Human Transcriptome Array 2.0 (HTA 2.0). DNA integrity analysis was performed with γH2AX ELISA, immunostaining and Comet Assay. Flow cytometry measurement and Sulforhodamine B (SRB) assay were assessed for cell cycle and proliferation determination. Global and promoter-specific DNA methylation alterations, as well as decreased expression (p< 0.05) of genes, that are involved in epithelial-mesenchymal transition were observed after SAM treatment. Increased phosphorylation of H2AX (74.9, 166.5, 200.6 pM) and decreased micronucleus number (1.47, 0.76, 0.45% of cells) were referred to the activation of reparative processes, that was supported by the changes of comet tail lengths. Proportion of cells was decreased in the G0/G1 (48.4, 28.5, 20.4%) phase; however, it was increased in both S (45.7, 61.7, 67.0%) and G2/M (6.0, 10.7, 12.5%) phases. Significant (p< 0.05) decrease of cell proliferation (99.5, 78.0, 70.6%) was also detected with SRB assay. SAM is able to alter the DNA methylation pattern of tumor cells and can induce DNA repair. Activation of these processes can lead to cell cycle arrest, decreased proliferation, and inhibition of epithelial-mesenchymal transition. Tumor cells could be targeted by SAM through different pathways; therefore, it may enhance the effect of chemotherapeutic agents. Citation Format: Sára Zsigrai, Alexandra Kalmár, Krisztina Andrea Szigeti, Zsófia Brigitta Nagy, Barbara K. Barták, Gábor Valcz, Orsolya Galamb, Titanilla Dankó, Zsolt Tulassay, Péter Igaz, Béla Molnár. Alteration of DNA methylation, DNA repair and epithelial-mesenchymal transition in colorectal cancer cell lines by S-adenosylmethionine treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 164.

Backgrounds: Global DNA hypomethylation is characteristic in various cancer types including colorectal cancer. Alterations of DNA methylation related enzymes expression and decreased level of methyl-donor molecules (folic acid (FA), S-adenosylmethionine (SAM)) can lead to aberrant DNA methylation pattern and elevated homocysteine level. Aims: Our aim was to examine global DNA methylation changes during aging and colorectal normal-adenoma-carcinoma sequence and in inflammatory bowel disease in tissue and liquid biopsy samples for diagnostic purposes. Moreover, we aimed to explore the reasons of global hypomethylation on gene expression level and methyl-donor molecule content. Methods: Bisulfite treatment was performed on DNA isolated from 30 normal (N), 10 adenoma (Ad), 10 colorectal carcinoma (CRC), 10 colitis ulcerosa (UC) tissue samples and on 11 N, 10 Ad, 15 CRC, 12 UC plasma specimens. 30 N samples contained different age groups derived from under 20 to 70 years old healthy controls for examination of aging process. LINE-1 PCR product was generated and pyrosequenced. Whole genome expression level of 60 biopsy samples was evaluated by HTA 2.0 RNA microarraychip (Affymetrix). In situ tissue appearance of 5-methylcytosine, FA, SAM, homocysteine, and expression of DNA methyltransferases (DNMTs) were analyzed by immunohistochemistry staining (IHC). Results: According to LINE-1 bisulfite sequencing results, DNA methylation was 72.6±1% in samples of healthy controls under 50 years old and 71.6±1.8% in specimens of patients over 50 years old. Significant DNA hypomethylation was found in CRC (62.9±8.7%; p Conclusion: Significant decrease in DNA methylation level was found in tissue and liquid biopsy samples of colorectal normal-adenoma-carcinoma sequence, but not in UC specimens. Our results suggest that determination of global DNA hypomethylation could have prognostic and diagnostic value as well, and reduction of DNA methylation level could be linked to decreased FA and SAM availability and not to methylation related ezymes activity. Citation Format: Krisztina A. Szigeti, Orsolya Galamb, Alexandra Kalmár, Gábor Valcz, Sára Zsigrai, Barbara K. Barták, Zsófia B. Nagy, Zsolt Tulassay, Péter Igaz, Béla Molnár. Global DNA hypomethylation can be caused by decreased methyl-donor content in tissue and liquid biopsy samples in colorectal cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 155.

Introduction In addition to expression level analysis, gene splicing variant determination is also technically available now on microarrays and by RNA sequencing. In this study colorectal cancer related gene expression, gene splicing analysis performed with Kegg pathway and gene ontology evaluation with protein interaction network evaluation. Material and methods Biopsy samples after collection were immediately put in stabilisation reagent and stored at −80°C from healthy colonic normal (n=20) and malignant biopsy specimen (n=20). Human Transcriptome Microarrays (HTA 2.0) washed, scanned and analysed. Gene +Exon SST-RMA summarization was performed by Affymetrix TAC 4.0 software. In silico analysis was performed from the GEO database (GSE73360), in which 37 CRC biopsies from 27 patients, and 19 NAT biopsies (3–6 cm away from the tumour) were collected. Differently expressed genes from normal versus cancer pairwise comparison were determined and three transcript set variants were first defined. Transcripts with significant (>abs 2) fold changes and with exon-splicing events were in transcript set 1. Low level fold changes ( abs 2) but without any exon-splicing event. For KEGG and GO analysis DAVID while for key control gene determinations STRING ppi network was applied. Results and discussions GEO in silico confirmation was applied before pathway analysis. For transcript set 1 five clusters of genes were determined, which involved into the most common pathways such as RNA transport (NUP43, 107 and 205) and Cell cycle (MCM3, 5 and 7). For transcript set 2 one clusters of genes were determined which involved pathways were Epstein-Barr virus infection (CD39, IRAK1 and POLR3H), Ribosome biogenesis in eukaryotes (GAR1, IMP4 and RAN), Purine and Pyrimidine metabolism (POLD2 and POLR1B) pathways. For transcript set 3 four clusters of genes were determined Cell cycle (CCNA2, CCNB1, RBL1 and TFDP1) and 3 in PPAR signalling pathway (ACSL4, MMP1 and SCD2). Conclusion Different splicing transcript variants, in addition to their expression level alterations may play important role in CRC cancer pathways. Splicing events with or without fold changes participate in additional signal transduction processes in CRC development like nucleotide metabolism.

Introduction MiRNAs has a critical relevance in regulation during tumorigenesis. The expression profiles of miRNAs alter along tumour progression moreover; these miRNA may spread into tumour macro- and microenvironment. Extracellular miRNAs are stable and its expression is less characterised in plasma. Altered and overlapped miRNA profiles between tissue and plasma are less explored. The present study was designed to characterise the tissue and circulating miRNA profile through colorectal adenoma-carcinoma sequence in human specimens and also in mice peripheral blood samples. Furthermore, the purpose of our study was to determine the origin of detected miRNAs in tumor-adherent C38/C57BL/6 and non-adherent CBAJ mice tumour models. Material and methods To achieve that goal, human peripheral blood and biopsy of normal (N), tubular (AT), tubulovillous adenoma (ATV) and colorectal cancer (CRC) were selected and plasma were also collected two times a week over 45 days from C57BL/6 C38, CBAJ mice. MiRNAs were isolated and Affymetrix GeneChip miRNA array analysis was performed for screening of the altered miRNA profile. RT-qPCR method was used for validation. Results and discussions In the case of human samples out of 1733 detectable miRNAs, 306 miRNAs were expressed in normal, 334 in adenoma and 321 in CRC. Characteristic miRNA expression alteration was observed in comparison of AD vs. CRC (miR-149*, miR-3196, miR-4687) in plasma. In the case of N vs. CRC, overexpression of miR-612, miR-1296, miR-933, miR-937 and miR-1207 was validated by RT-PCR (p We identified high plasma levels of 94 miRNAs in healthy animals and 176 miRNAs in late tumour stages. Based on CBAJ-C38 mice model experiment where the injected tumorous cells could not adhered miR-676 found to be a host originated while miR-92a was a tumor-derived miRNA. MiR-676 and miR-92a shown significant overexpression (388x and 37x p Conclusion Characteristic tissue miRNA patterns were determined during neoplastic development. Moreover, circulating miRNAs alteration could observe in animal models and in the human circulatory system. Cancer-associated miRNAs in the circulation may originate from the immunologic system or from other metastatic regions far from the primer tumour location.