Circulating cell-free DNA species affect the risk of hepatocellular carcinoma in treated chronic hepatitis B patients.

Hepatocellular carcinoma (HCC) may still develop in chronic hepatitis B (CHB) patients even under effective long-term oral antiviral therapy, but its pathogenesis in the setting of long-standing inhibition of viral replication has not been completely elucidated. We investigated whether species of circulating cell-free DNA (cfDNA) may be involved in the process of hepatocarcinogenesis in treated CHB patients. Serum samples were obtained from HBeAg-negative CHB patients with (HCC cases, n = 37) or without HCC development during the first 5 years of oral antiviral therapy (controls, n = 74). HCC cases and controls were matched 1:2 for age, sex and platelets. Determination of different circulating cfDNA species (before HCC diagnosis in HCC cases) including total cfDNA quantity, levels of Alu repeat DNA and RNase P coding DNA, copies of mitochondrial DNA and levels of 5-methyl-2'-deoxycytidine as an indicator of DNA methylation was performed. HCC cases compared with controls had higher median levels of Alu247 (123 vs 69 genomic equivalent, p = .042) and RNase P coding DNA (68 vs 15 genomic equivalent, p < .001). In contrast, median cfDNA concentration, Alu115 levels, Alu247/Alu115 ratio as an index of DNA integrity and mitochondrial DNA copies did not differ significantly between HCC cases and controls. Receiver operating characteristic curve analysis showed that levels RNase P coding DNA offered good prediction of subsequent HCC development (c-statistic: 0.80, p < .001). In conclusion, serum levels of RNase P coding DNA are increased years before HCC diagnosis and could be potentially helpful in the prediction of the HCC risk in treated HBeAg-negative CHB patients.

Significance of Circulating Cell-Free DNA Biomarkers in HBeAg-Negative Chronic Hepatitis B Virus Infection and Their Changes after Treatment Initiation.

BACKGROUND: Chronic hepatitis B virus (HBV) infection is a common chronic liver disease that is closely associated with increased morbidity and mortality. Circulating cell-free DNA (cf-DNA) and global DNA methylation, expressed as circulating levels of 5-methyl-2'-deoxycytidine, are increasingly used to monitor chronic inflammatory diseases of several etiologies. This study attempts to investigate the serum levels of circulating cf-DNA and 5-methyl-2'-deoxycytidine in HBeAg-negative patients with chronic infection (carriers) and chronic hepatitis B (CHB), as well as their changes after treatment initiation in CHB. METHODS: Serum samples from a total of 61 HBeAg-negative patients (30 carriers and 31 CHB patients) were included in order to quantify the levels of circulating cf-DNA and 5-methyl-2'-deoxycytidine. In addition, serum samples from 17 CHB patients in complete virological and biochemical remission after initiation of treatment with a nucleos(t)ide analogue were included. RESULTS: Circulating cf-DNA concentration was significantly increased after the initiation of treatment (15 vs. 10 ng/mL, p = 0.022). There was a trend in higher mean levels of circulating 5-methyl-2'-deoxycytidine in carriers compared to CHB patients (211.02 vs. 175.66 ng/mL, p = 0.089), as well as a trend in increasing 5-methyl-2'-deoxycytidine levels after treatment initiation in CHB patients compared to pre-treatment levels (215 vs. 173 ng/mL, p = 0.079). CONCLUSIONS: Both circulating levels of cf-DNA and 5-methyl-2'-deoxycytidine might be useful biomarkers in order to monitor liver disease activity and response to antiviral treatment in HBeAg-negative chronic HBV patients, but further studies are essential in order to validate these intriguing findings.

Hematological Abnormalities in COVID-19 Disease: Association With Type I Interferon Pathway Activation and Disease Outcomes.

Increased expression of interferon (IFN)-stimulated genes (ISGs) in peripheral blood, has been previously reported in viral infections, as well as in autoimmune disorders, in association with reduced leukocyte and platelet counts. Though cytopenias are common in patients with COVID-19 disease and predict severe outcomes, the underlying mechanisms have not been fully elucidated. In the current study, we aimed to determine the prevalence of hematological abnormalities in the setting of active COVID-19 infection and to explore whether they associate with disease outcomes and activation of type I IFN pathway. One-hundred-twenty-three consecutive SARS-CoV2 infected patients were included in the study. Clinical and laboratory parameters were recorded for all study participants. In 114 patients, total RNA was extracted from whole peripheral blood and subjected to real time PCR. The relative expression of three interferon stimulated genes (ISGs; IFIT1, MX-1, and IFI44) was determined and a type I IFN score reflecting peripheral type I IFN activity was calculated. The rates of anemia, leukopenia, and thrombocytopenia were 28.5, 14.6, and 24.4%, respectively. Among leukocytopenias, eosinopenia, and lymphopenia were the most prominent abnormalities being found in 56.9 and 43.1%, respectively. Of interest, patients with either eosinopenia and/or thrombocytopenia but no other hematological abnormalities displayed significantly increased peripheral type I IFN scores compared to their counterparts with normal/high eosinophil and platelet counts. While eosinopenia along with lymphopenia were found to be associated with increased risk for intubation and severe/critical disease, such an association was not detected between other hematological abnormalities or increased type I IFN scores. In conclusion, hematological abnormalities are commonly detected among patients with COVID-19 infection in association with severe disease outcomes and activation of the type I IFN pathway.

The Significance of Circulating Cell-Free DNA Markers in Chronic Hepatitis B Patients with Hepatocellular Carcinoma.

(1) Background: Hepatocellular carcinoma (HCC) is the most serious complication of chronic hepatitis B (CHB). Recently, the detection of circulating cell-free (cf) DNA and nucleosomes has found numerous applications in oncology. This study aimed to examine the levels of serum cfDNA markers and nucleosomes in CHB patients with and without HCC and assess their potential association with HCC patients' survival. (2) Methods: Nineteen patients with CHB and HCC and 38 matched patients with CHB without cancer development during 5 years of antiviral therapy were included. Stored serum samples were analyzed for cfDNA species, including the cfDNA concentration and levels of Alu115, Alu247, and nucleosomes. DNA integrity was expressed as the Alu247/Alu115 ratio. (3) Results: Compared to controls, HCC patients had higher median Alu247 levels (64.2 vs. 23.2 genomic equivalent, p = 0.004) and DNA integrity (1.0 vs. 0.7, p < 0.001) and a trend for a higher median cfDNA concentration (36.0 vs. 19.5 ng/mL, p = 0.064). Increased DNA integrity (Alu247/Alu115 > 1) was associated with an increased risk of death during the first year after HCC diagnosis (p = 0.016). (4) Conclusions: Levels of Alu247 and DNA integrity in serum cfDNA are elevated in CHB patients with HCC, whereas increased DNA integrity seems to be associated with a worse short-term prognosis in this setting.

Metabolic inflammation as an instigator of fibrosis during non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive storage of fatty acids in the form of triglycerides in hepatocytes. It is most prevalent in western countries and includes a wide range of clinical and histopathological findings, namely from simple steatosis to steatohepatitis and fibrosis, which may lead to cirrhosis and hepatocellular cancer. The key event for the transition from steatosis to fibrosis is the activation of quiescent hepatic stellate cells (qHSC) and their differentiation to myofibroblasts. Pattern recognition receptors (PRRs), expressed by a plethora of immune cells, serve as essential components of the innate immune system whose function is to stimulate phagocytosis and mediate inflammation upon binding to them of various molecules released from damaged, apoptotic and necrotic cells. The activation of PRRs on hepatocytes, Kupffer cells, the resident macrophages of the liver, and other immune cells results in the production of proinflammatory cytokines and chemokines, as well as profibrotic factors in the liver microenvironment leading to qHSC activation and subsequent fibrogenesis. Thus, elucidation of the inflammatory pathways associated with the pathogenesis and progression of NAFLD may lead to a better understanding of its pathophysiology and new therapeutic approaches.

Circulating tumor cells in colorectal cancer: detection methods and clinical significance.

Colorectal cancer is one of the most frequently diagnosed malignancies in both men and women. Although curative resection is the major treatment option, approximately half of all patients eventually develop distant metastases. Thus, the need for early detection of occult metastases has led to extensive investigation with regard to the detection of disseminated tumor cells in biological fluids, including peripheral blood or bone marrow of cancer patients. In this review, we summarize the methods currently implemented for disseminated tumor cell detection in colorectal cancer. In addition, we discuss the pitfalls of each method and the future perspectives in the development of an easily applied, quick and inexpensive method which will enable the reliable detection of circulating tumor cells with optimal sensitivity and specificity.

Detection of circulating tumor cells in breast cancer patients using multiplex reverse transcription-polymerase chain reaction and specific primers for MGB, PTHRP and KRT19 correlation with clinicopathological features.

AIM: The aim of this study was to correlate the clinicopathological features of breast cancer patients with the positive detection of parathyroid hormone-related protein (PTHRP), cytokeratin protein 19 (KRT19) and mammaglobin (MGB) using a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay developed to detect circulating tumor cells (CTCs) in peripheral blood of patients with breast cancer. PATIENTS AND METHODS: Peripheral blood samples were collected from 54 breast cancer patients and 20 healthy blood donors. Subsequently, the samples were processed for RNA extraction and analyzed for the expression of PTHRP, KRT19 and MGB using specific primers and multiplex RT-PCR. RESULTS: The positive detection rates in breast cancer patients for PTHRP, KRT19 and MGB were 68.5%, 63% and 22.2% and for healthy donors 10%, 0% and 10%, respectively. The statistical analysis revealed that PTHRP- and KRT19-positive detections correlated with the diagnosis of breast cancer while the combined positive detections of PTHRP-plus-KRT19 correlated with the presence of distant metastasis, especially with bone metastasis. Moreover, positive detections of KRT19 correlated with high proliferation rate of breast cancer tumors. MGB-positive detections did not add any diagnostic advantage in such analysis. CONCLUSION: Multiplex-PCR based detection of CTCs using PTHRP and KRT19 primers can provide useful information for the disease.

Multiplex RT-PCR-based detections of CEA, CK20 and EGFR in colorectal cancer patients.

AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting circulating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors' clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo). RESULTS: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P < 0.001) and preoperative serum levels of CEA (P = 0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented significant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts. CONCLUSION: These data suggest that multiplex RT-PCR assay can provide useful information concerning disease stage and overall survival of CRC patients.