Circulating cell-free DNA fragmentation is a stepwise and conserved process linked to apoptosis.

BACKGROUND: Circulating cell-free DNA (cfDNA) is a pool of short DNA fragments mainly released from apoptotic hematopoietic cells. Nevertheless, the precise physiological process governing the DNA fragmentation and molecular profile of cfDNA remains obscure. To dissect the DNA fragmentation process, we use a human leukemia cell line HL60 undergoing apoptosis to analyze the size distribution of DNA fragments by shallow whole-genome sequencing (sWGS). Meanwhile, we also scrutinize the size profile of plasma cfDNA in 901 healthy human subjects and 38 dogs, as well as 438 patients with six common cancer types by sWGS. RESULTS: Distinct size distribution profiles were observed in the HL60 cell pellet and supernatant, suggesting fragmentation is a stepwise process. Meanwhile, C-end preference was seen in both intracellular and extracellular cfDNA fragments. Moreover, the cfDNA profiles are characteristic and conserved across mammals. Compared with healthy subjects, distinct cfDNA profiles with a higher proportion of short fragments and lower C-end preference were found in cancer patients. CONCLUSIONS: Our study provides new insight into fragmentomics of circulating cfDNA processing, which will be useful for early diagnosis of cancer and surveillance during cancer progression.

Identification and validation of a fatty acid metabolism-related lncRNA signature as a predictor for prognosis and immunotherapy in patients with liver cancer.

BACKGROUND: Fatty acid (FA) metabolism is considered the emerging cause of tumor development and metastasis, driving poor prognosis. Long non-coding RNAs (lncRNAs) are closely related to cancer progression and play important roles in FA metabolism. Thus, the discovery of FA metabolism-related lncRNA signatures to predict outcome and immunotherapy response is critical in improving the survival of patients with hepatocellular carcinoma (HCC). METHODS: FA metabolism scores and a FA metabolism-related lncRNA signature were constructed using a single-sample gene set enrichment analysis based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. "ConsensusClusterPlus" was used to screen molecular subtypes. Chi-squared test and Fisher's exact test were applied to explore the relationship between clinical, genomic mutation characteristics and subtypes. Transcription factor (TF) activity scores, cellular distributions, immune cell infiltration, and immunotherapy response were employed to investigate the functions of FA metabolism-related lncRNA signatures. FA metabolism microarray and western blot were performed to detect the biological function of candidate lncRNAs. RESULTS: A total of 70 lncRNAs that highly correlated with FA metabolism scores in two cohorts were used to construct two distinct clusters. Patients in cluster 2 had lower FA metabolism scores and worse survival than those in cluster 1. Patients in cluster 2 exhibited a high frequency of DNA damage, gene mutations, oncogenic signaling such as epithelial-to-mesenchymal transition, and a high degree of immune cell infiltration. Moreover, the lncRNA signature could predict the effects of immunotherapy in patients with HCC. Furthermore, three lncRNAs (SNHG1, LINC00261, and SNHG7) were identified that were highly correlated with FA metabolism. Additionally, SNHG1 and SNHG7 were found to regulate various FA metabolism-related genes and ferroptosis-related genes in vitro experiments. GSEA analysis revealed that SNHG1 and SNHG7 promote fatty acid beta-oxidation. SNHG1 and SNHG7 silencing dramatically reduced lipid droplets in HCC cells. Many immune-infiltration genes and TFs were overexpressed in HCC tissues with SNHG1 and SNHG7 high expression. CONCLUSIONS: A novel molecular model of FA metabolism-related lncRNAs was developed, which has significantly prognostic potential in HCC diagnosis and aids in clinical decision making.

Prognostic value of preoperative peripheral monocyte count in patients with hepatocellular carcinoma after liver transplantation.

Prognostic value of peripheral monocyte, as a member of inflammatory cells, was widely being investigated. The aim of this study was to evaluate the prognostic value of preoperative peripheral blood monocyte count for hepatocellular carcinoma (HCC) patients who underwent liver transplantation (LT) and the relationship between monocyte count and tumor-related characteristics. We retrospectively analyzed the clinical data of 101 HCC patients after LT. Preoperative monocyte count and demographic, clinical, and pathologic data were analyzed. The optimal cutoff value of monocyte count was 456/mm(3), with the sensitivity and specificity of 69.4 and 61.5 %, respectively. Elevated preoperative peripheral blood monocyte count was significantly associated with large tumor size. The 1-, 3-, and 5-year disease-free survival (DFS) (80.9, 70.1, and 53.3 % vs 55.1, 38.7, and 38.7 %, P = 0.007) and overall survival (OS) rates (95.7, 76.6, and 64.8 % vs 72.2, 44.1, and 36.1 %, P = 0.002) of HCC patients in the peripheral blood monocyte count ≤456/mm(3) group were higher than those in the peripheral blood monocyte count >456/mm(3) group. In conclusion, elevated preoperative peripheral blood monocyte count was significantly associated with advanced tumor stage and it can be considered as a prognostic factor for HCC patients after LT.

Case report: Intrahepatic cholangiectasis with Clonorchis sinensis infection.

Clonorchis sinensis infections persist globally among humans. These pathogens mainly inhabit the intrahepatic biliary system. Most individuals with clonorchiasis exhibit mild symptoms. The absence of distinctive symptoms often results in delayed diagnosis and treatment, potentially leading to chronic infection. We herein report a case of a 29-year-old female presented with a year-long history of abdominal distention and dyspepsia. Imaging revealed intrahepatic bile duct dilatation, intrahepatic bile duct cyst, and associated deposits. One month post-cystectomy, the patient developed massive ascites and a significant increase in eosinophil count. After treatment, multiple worms were observed in the drainage tube. Morphological and DNA metagenomic analyses confirmed the presence of C. sinensis. Clinical manifestations of C. sinensis vary widely. Imaging serves as a valuable diagnostic tool in endemic areas, especially in detecting intrahepatic duct dilation where the flukes reside. In addition to intrahepatic bile duct dilation, abnormal echoes within the bile duct and the presence of floating objects in the gallbladder significantly aid in diagnosis. Clinicians may encounter these parasitic diseases unexpectedly, underscoring the importance of understating such cases in routine practice and contributing to our broader understanding of managing similar cases in clinical settings.

CDCA8 induced by NF-YA promotes hepatocellular carcinoma progression by regulating the MEK/ERK pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most lethal malignant tumors. Cell division cycle associated 8 (CDCA8) is an important multifactorial regulator in cancers. However, its up and downstream targets and effects in HCC are still unclear. METHODS: A comprehensive bioinformatics analysis was performed using The Cancer Genome Atlas dataset (TCGA) to explore novel core oncogenes. We quantified CDCA8 levels in HCC tumors using qRT-PCR. HCC cell's proliferative, migratory, and invasive abilities were detected using a Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, clone formation, and a Transwell assay. An orthotopic tumor model and tail vein model were constructed to determine the effects of CDCA8 inhibition in vivo. The mechanism underlying CDCA8 was investigated using RNA sequencing. The prognostic value of CDCA8 was assessed with immunohistochemical staining of the tissue microarrays. RESULTS: CDCA8 was identified as a novel oncogene during HCC development. The high expression of CDCA8 was an independent predictor for worse HCC outcomes both in publicly available datasets and in our cohort. We found that CDCA8 knockdown inhibited HCC cell proliferation, colony formation, and migration by suppressing the MEK/ERK pathway in vitro. Moreover, CDCA8 deficiency significantly inhibited tumorigenesis and metastasis. Next-generation sequencing and laboratory validation showed that CDCA8 silencing inhibited the expression of TPM3, NECAP2, and USP13. Furthermore, NA-YA overexpression upregulated the expression of CDCA8. CDCA8 knockdown could attenuate NF-YA-mediated cell invasion in vitro. The expression of NF-YA alone or in combined with CDCA8 were validated as significant independent risk factors for patient survival. CONCLUSION: Our findings revealed that the expression of CDCA8 alone or in combined with NF-YA contributed to cancer progression, and could serve as novel potential therapeutic targets for HCC patients.

Overexpression of SRD5A3 in Hepatocellular Carcinoma and Its Molecular Mechanism: A Study of Bioinformatics Exploration Analysis with Experimental Verification.

Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and more prevalent among males than females. However, the biological role of enzyme 5α-reductase (SRD5A3), which plays a critical role in the androgen receptor signaling pathway during HCC development, remains poorly understood. Methods: ONCOMINE, GEPIA, UALCAN, and Kaplan-Meier Plotter were used to analyze the expression and prognostic value of SRD5A3 in HCC. STRING and Metascape were applied to analyze potential target and molecular pathways underlying SRD5A3 in HCC. A real-time quantitative reverse transcription-polymerase chain reaction was used to validate the downstream target expression of SRD5A3. Results: The expression of SRD5A3 was significantly overexpressed in HCC tissues compared with normal tissues, while the expression of SRD5A1 and SRD5A2 were downregulated in multiple public datasets. It may be that the low methylation of the SRD5A3 promoter leads to its overexpression. The level of SRD5A3 tended to be higher expressed in clinical samples with advanced stage and positive node metastasis. Furthermore, the patients with higher SRD5A3 were remarkably associated with poorer overall survival and disease-free survival in the TCGA data. In addition, the increased mRNA expression of SRD5A3 could predict poorer overall survival in Kaplan-Meier Plotter database including different patient cohorts. Moreover, HCC patients with higher level of SRD5A3 had significantly shorter recurrence-free survival, progression-free survival, and disease-specific survival. Furthermore, enrichment analysis demonstrated that multiple processes, such as steroid hormone biosynthesis, lipid biosynthetic process, and androgen metabolic process, were affected by SRD5A1-3 alterations. In vitro experiments showed that the expression of SRD5A3 was increased in HCC tissues than that in adjacent tissues. SRD5A3 silencing promoted the expression of DOLK in two HCC cell lines. Conclusions: This study identified SRD5A3/DOLK as a novel axis to regulate HCC development.

Inhibition of NEK7 Suppressed Hepatocellular Carcinoma Progression by Mediating Cancer Cell Pyroptosis.

NIMA-related kinase 7 (NEK7) is a serine/threonine kinase involved in cell cycle progression via mitotic spindle formation and cytokinesis. It has been related to multiple cancers, including breast cancer, hepatocellular cancer, lung cancer, and colorectal cancer. Moreover, NEK7 regulated the NLRP3 inflammasome to activate Caspase-1, resulting in cell pyroptosis. In the present study, we investigated whether NEK7 is involved in cell pyroptosis of hepatocellular carcinoma (HCC). Interestingly, we found that NEK7 was significantly related to expression of pyroptosis marker GSDMD in HCC. We found that NEK7 expression was significantly correlated with GSDMD expression in bioinformatics analysis, and NEK7 expression was significantly co-expressed with GSDMD in our HCC specimens. Cell viability, migration, and invasion capacity of HCC cell lines were inhibited, and the tumor growth in the xenograft mouse model was also suppressed following knockdown of NEK7 expression. Mechanistic studies revealed that knockdown of NEK7 in HCC cells significantly upregulated the expression of pyroptosis markers such as NLRP3, Caspase-1, and GSDMD. Coculture of HCC cells stimulated hepatic stellate cell activation by increasing p-ERK1/2 and α-SMA. Knockdown of NEK7 impaired the stimulation of HCC cells. Therefore, downregulation of NEK7 inhibited cancer-stromal interaction by triggering cancer cell pyroptosis. Taken together, this study highlights the functional role of NEK7-regulated pyroptosis in tumor progression and cancer-stromal interaction of HCC, suggesting NEK7 as a potential target for a new therapeutic strategy of HCC treatment.

Prognostic Value of Preoperative Serum Leucine Aminopeptidases in Hepatocellular Carcinoma Patients Who Underwent Liver Transplantation.

BACKGROUND: Leucine aminopeptidases (LAPs) have been reported to be involved in tumor cell proliferation, invasion and angiogenesis. However, the relationship between serum leucine aminopeptidases and prognosis of hepatocellular carcinoma (HCC) patients who underwent liver transplantation (LT) was not yet reported. We aimed to evaluate the prognostic value of preoperative serum leucine aminopeptidases in these patients. METHODS: Clinical data of 106 HCC patients who underwent LT were retrospectively analyzed. The sex ratio, age, HBV infection, Child-Pugh stage, preoperative tumor therapy, AFP, the largest tumor size, tumor number, Edmondson grading, macro- and micro-vascular invasion of patients with different serum LAP level and compositions of patients who met the criteria of Milan, UCSF or Hangzhou were compared using the chi-square test. The Kaplan-Meier method was performed in survival analysis and the log rank test was used in survival comparison. RESULTS: Serum LAPs were correlated with alpha-fetoprotein (AFP), the largest tumor size, tumor number and macro-vascular invasion. Patients with serum LAPs > 87 U/L showed significantly poorer disease-free survival (DFS) and overall survival (OS) than those with serum LAPs ≤ 87 U/L. Univariate analysis indicated that serum LAPs, AFP, the largest tumor size, tumor number, and macro- and micro-vascular invasion were all associated with DFS and OS. Multivariate analysis showed that serum LAPs, macro-vascular invasion and the largest tumor size were independently correlated with DFS and OS. Serum LAPs could also distinguish prognosis between patients with different status of AFP, the largest tumor size, tumor number, and macro- and micro-vascular invasion, as well as patients within and beyond selection criteria, such as Milan, University of California, San Francisco and Hangzhou criteria. CONCLUSION: Elevated preoperative serum LAPs were associated with advanced tumor stage and aggressive biological behavior, and thus a poor outcome, which could be a prognostic marker for HCC patients who underwent LT.

Noninvasive Detection of Hepatocellular Carcinoma with Circulating Tumor DNA Features and α-Fetoprotein.

Liver cancer is the fifth-most common cancer worldwide, with the third-highest rate of cancer-related mortality. Hepatocellular carcinoma (HCC) is the leading pathologic subtype, contributing 85% to 90% of cases of primary liver cancer. Most HCC patients are diagnosed at an advanced stage at which treatment is not curative. This study assessed the performance of a newly developed blood-based assay that utilizes genomic features and protein markers for the early detection of HCC. Two cancer-associated hallmarks, copy-number aberrations (CNA) and fragment size (FS), were characterized by shallow whole-genome sequencing of cell-free DNA and utilized to differentiate cancer patients from healthy subjects. As a clinically implemented biomarker of HCC, plasma α-fetoprotein (AFP) was also used with the genomic surrogates to optimize the detection of HCCs. The sensitivity of AFP ≥20.0 µg/L in detecting HCC was 57.9%. The combined genomic classifier CNA + FS via cell-free DNA shallow whole-genome sequencing identified nearly half of AFP-negative HCC patients (43.8%). By integrating CNA, FS as well as AFP (HCCseek), 75.0% sensitivity was achieved at 98.0% specificity, resulting in 92.6% accuracy, with 58.6% sensitivity in stage I HCC. The quantitative output of HCCseek was correlated with the severity of the disease (tumor size, stage, and recurrence-free survival). In summary, this study describes an efficient, noninvasive, and cost-effective method to detect HCC.

CFDA-SE Combined with MACSiBeads™ Particles to Evaluate the Inhibitory Effect of Treg Cells in vitro.

OBJECTIVE: To explore the application of carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and MACSiBeads™ Particles within in vitro suppression assays of regulatory T (Treg) cells. METHODS: CD4+CD25+ Treg cells and CD8+ T cells were sorted using magnetically activated cell sorting. CD8+ T cells were subjected to CFDA-SE staining to determine their optimal staining concentration. MACSi-Beads™ Particles, a component of the Treg expansion kit, were used as stimulators in suppression assays. Five experimental groups were set based on the condition of MACSiBeads™ Particles, CFDA-SE staining and cell composition of co-culture system, which were stimulated CD8+ cells without CFDA-SE staining, unstimulated CD8+ cells with CFDA-SE staining, stimulated CD8+ cells with CFDA-SE staining, co-cultured with Treg cells at a ratio of 1:0.25, 1:0.125 and 1:0, respectively. Flow cytometry was performed using the BD FACS Canto™ and flow data was analyzed using FCS Express 4 Plus software. RESULTS: CFSE fluorescence intensity correlated with cell type and culture time. The final CFDA-SE staining concentration was 0.5µM. Within in vitro suppression assays, Treg cells showed a significant inhibitory effect on the proliferation of CD8+ T cells increasing as its concentration increased. CONCLUSION: CFDA-SE combined with MACSiBeads™ Particles can be used to evaluate the in vitro inhibition effect of Treg cells.

lncRNAs as potential molecular biomarkers in the clinicopathology and prognosis of cholangiocarcinoma: a systematic review and meta-analysis.

BACKGROUND: Cholangiocarcinoma (CCA) is the second most common fatal primary hepatobiliary malignant carcinoma, characterized by early invasion and extremely poor outcomes. It is therefore necessary to identify a novel biomarker to better diagnose CAA and predict its prognosis. Recently, emerging evidence has revealed that some lncRNAs play an important role in the tumorigenesis and progression of CAA. In order to support this search for novel diagnostic and prognostic biomarkers for CAA, we conducted a meta-analysis to analyze the published association between lncRNA expression and its clinical value in CAA. METHODS: Eligible studies were pooled and analyzed according to our inclusion and exclusion criteria after a comprehensive literature search. Stata 14.0 software was used to analyze the data from relevant studies and to construct a forest plot. Different effect sizes were selected for the meta-analysis. RESULTS: In total, 24 publications were included in this meta-analysis. After review of their full-text, 16 articles studied the association between lncRNAs and clinicopathological characteristics, 2 discussing diagnosis and 16 discussing prognosis. Our results showed that overexpression of CCAT1 was significantly correlated with tumor stage (I + II vs III + IV) (OR, 4.99; 95% CI 2.77-8.99; P<0.001) and lymph node metastasis in CCA (OR, 4.75; 95% CI 2.65-8.52; P<0.001). Furthermore, elevated CCAT lncRNA family expression predicted a shorter overall survival (HR, 2.09; 95% CI 1.17-3.00; P<0.001), especially CCAT2. Upregulation of CCAT2 was also obviously associated with tumor stage in CCA (OR, 5.29; 95% CI 2.64-10.58; P=0.001). CONCLUSION: This is the first meta-analysis to assess the relationship between expression of lncRNAs and the clinical values of patients with CCA. lncRNAs can function as potential molecular biomarkers of the clinicopathology and prognosis of CCA.

Severe sclerosing cholangitis after Langerhans cell histiocytosis treated by liver transplantation: An adult case report.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare hematopoietic disorder of unknown pathogenesis. LCH diseases may occur in a single organ or multisystem organ. The patients with multisystem involvement usually have a poor prognosis. Liver involvement in multisystem LCH results in severe complications, such as obvious sclerosing cholangitis (SC) with jaundice. METHODS: We reported a 31-year-old man developed severe SC due to multisystem LCH and was successfully treated by liver transplantation (LT). In addition, we firstly used tacrolimus and mycofenolate mofetil as immunosuppressants to treat LCH after LT. RESULTS: We performed the immunosuppressants to deal with the LCH after LT, now the patient is currently well with normal liver function and no evidence of recurrence of LCH for 4 and a half years follow-up. CONCLUSION: LT should be recommended as an effective treatment for these adults with severe SC due to multisystem LCH. Finally, using tacrolimus and mycofenolate mofetil as immunosuppressants to treat LCH might be favorable to prevent LCH recurrence.

Active immunization in patients transplanted for hepatitis B virus related liver diseases: A prospective study.

INTRODUCTION: Prophylactic administration of hepatitis B immunoglobulin (HBIG) and nucleos(t)ide analogues (NAs) is the standard treatment for controlling hepatitis B virus (HBV) recurrence after liver transplantation (LT). Since lifelong use of HBIG is expensive and inconvenient and the antibodies level in anti-hepatitis B surface (HBs) is not sustainable and stable, an alternative strategy is to produce anti-HBs antibodies by active immunization. Our present study aimed to prospectively investigate the efficacy and safety of procedural HBV vaccination in transplanted patients. METHODS: Recipients who had undergone LT for hepatitis B related liver diseases more than one year before, with no evidence of HBV recurrence or rejection and normal liver function were enrolled. All subjects received the hepatitis B vaccine (40 µg) by intramuscular injection at months 0, 1, 2, 6 and 12 after enrollment with continuous administration of NAs. The liver function and anti-HBs titers were measured before each vaccination and HBIG (400U) was administrated intramuscularly when anti-HBs titer was lower than 30 IU/L during the course. The results of routine blood tests, liver function, concentration of immunosuppressant, and HBV-DNA copies were monitored during the research. After completion of the vaccination procedure, recipients were regarded as responders if their anti-HBs greater than 30 IU/L were maintained for up to six months without using HBIG and vaccine. RESULTS: Twenty-seven patients were enrolled in this study and the average anti-HBs titer before vaccination was 19.86±14.80 IU/L. The average anti-HBs titer of the nine responders at the end of the follow-up was 57.14±22.75 IU/L, giving an overall response rate of 33.3% (9/27). There were no reports of reactivation of HBV, rejection, severe anaphylaxis or other adverse events. Responders and non-responders showed their significant difference in anti-HBs titers after the fourth vaccination (P<0.01). Moreover, the majority of non-responders (11/18, 63.64%) had high LY/EO rates (lymphocyte number/eosinophil number>15) while most responders (8/9, 88.89%) had low LY/EO rates at the beginning of vaccination (P = 0.019). CONCLUSIONS: Active immunization is an effective, cost-saving, and safe method for the prevention of HBV reactivation in patients transplanted for hepatitis B virus related liver diseases. The LY/EO rate may be a valuable indicator in selecting potential recipients for vaccination.

Prognostic value of combined preoperative fibrinogen and neutrophil-lymphocyte ratio in patients with hepatocellular carcinoma after liver transplantation.

OBJECTIVES: Elevated plasma fibrinogen (Fib) correlated with patient's prognosis in several solid tumors. However, few studies have illuminated the relationship between preoperative Fib and prognosis of HCC after liver transplantation. We aimed to clarify the prognostic value of Fib and whether the prognostic accuracy can be enhanced by the combination of Fib and neutrophil-lymphocyte ratio (NLR). RESULTS: Fib was correlated with Child-pugh stage, alpha-fetoprotein (AFP), size of largest tumor, macro- and micro-vascular invasion. Univariate analysis showed preoperative Fib, AFP, NLR, size of largest tumor, tumor number, macro- and micro- vascular invasion were significantly associated with disease-free survival (DFS) and overall survival (OS) in HCC patients with liver transplantation. After multivariate analysis, only Fib and macro-vascular invasion were independently correlated with DFS and OS. Survival analysis showed that preoperative Fib > 2.345 g/L predicted poor prognosis of patients HCC after liver transplantation. Preoperative Fib showed prognostic value in various subgroups of HCC. Furthermore, the predictive range was expanded by the combination of Fib and NLR. MATERIALS AND METHODS: Data were collected retrospectively from 130 HCC patients who underwent liver transplantation. Preoperative Fib, NLR and clinicopathologic variables were analyzed. The survival analysis was performed by the Kaplan-Meier method, and compared by the log-rank test. Univariate and multivariate analyses were performed to identify the prognostic factors for DFS and OS. CONCLUSIONS: Preoperative Fib is an independent effective predictor of prognosis for HCC patients, higher levels of Fib predict poorer outcomes and the combination of Fib and NLR enlarges the prognostic accuracy of testing.

Elevated Preoperative Serum Gamma-glutamyltranspeptidase Predicts Poor Prognosis for Hepatocellular Carcinoma after Liver Transplantation.

Gamma-glutamyltransferase (γ-GGT) is a membrane-bound enzyme that is involved in biotransformation, nucleic acid metabolism, and tumourigenesis. Elevated serum γ-GGT levels are related to an increased cancer risk and worse prognosis in many cancers. In the present study, we evaluated the prognostic value of preoperative serum γ-GGT in patients with hepatocellular carcinoma (HCC) who underwent liver transplantation (LT). A total of 130 HCC patients after LT were included in the study. The optimal cut-off value of γ-GGT was 128U/L by receiver operating characteristic analysis, with a sensitivity and specificity of 60.0% and 72.9%, respectively. Elevated preoperative serum γ-GGT was significantly associated with high alpha-fetoprotein (AFP), large tumor size, and macro- and micro-vascular invasion. The 1-, 3-, 5-year disease-free survival (DFS) and overall survival (OS) rates of HCC patients in the γ-GGT > 128U/L group were poorer than those in the γ-GGT ≤ 128U/L group. Stratification analysis revealed that γ-GGT exhibited a greater predictive value for DFS and OS in HCC patients beyond the Milan criteria and no macro-vascular invasion. In conclusion, elevated preoperative serum γ-GGT was significantly associated with advanced tumor stage and aggressive tumor behaviors, and serum γ-GGT can be considered as a prognostic factor for HCC patients after LT, especially for patients beyond the Milan criteria or without macro-vascular invasion.