A multimodal atlas of hepatocellular carcinoma reveals convergent evolutionary paths and 'bad apple' effect on clinical trajectory.

BACKGROUND & AIMS: Hepatocellular Carcinoma (HCC) is a highly fatal cancer characterized by high intra-tumor heterogeneity (ITH). A panoramic understanding of its tumor evolution, in relation to its clinical trajectory, may provide novel prognostic and treatment strategies. METHODS: Through the Asia-Pacific Hepatocellular Carcinoma (AHCC) trials group (NCT03267641), we recruited one of the largest prospective cohorts of HCC with over 600 whole genome and transcriptome samples from 123 treatment-naïve patients. RESULTS: Using a multi-region sampling approach, we revealed seven convergent genetic evolutionary paths governed by the early driver mutations, late copy number variations and viral integrations, which stratify patient clinical trajectories after surgical resection. Furthermore, such evolutionary paths shaped the molecular profiles, leading to distinct transcriptomic subtypes. Most significantly, although we found the coexistence of multiple transcriptomic subtypes within certain tumors, patient prognosis was best predicted by the most aggressive cell fraction of the tumor, rather than by overall degree of transcriptomic ITH level - a phenomenon we termed the 'bad apple' effect. Finally, we found that characteristics throughout early and late tumor evolution provide significant and complementary prognostic power in predicting patient survival. CONCLUSIONS: Taken together, our study generated a comprehensive landscape of evolutionary history for HCC and provided a rich multi-omics resource for understanding tumor heterogeneity and clinical trajectories. CLINICAL TRIAL NUMBER: NCT03267641 (Observational cohort) IMPACT AND IMPLICATIONS: This prospective study, utilizing comprehensive multi-sector, multi-omics sequencing and clinical data from surgically resected HCC, reveals critical insights into the role of tumor evolution and intra-tumor heterogeneity (ITH) in determining the prognosis of Hepatocellular Carcinoma (HCC). These findings are invaluable for oncology researchers and clinicians, as they underscore the influence of distinct evolutionary paths and the 'bad apple' effect, where the most aggressive tumor fraction dictates disease progression. These insights not only enhance prognostic accuracy post-surgical resection but also pave the way for developing personalized therapies tailored to specific tumor evolutionary and transcriptomic profiles. The co-existence of multiple sub-types within the same tumor prompts a re-appraisal of the utilities of depending on single samples to represent the entire tumor and suggests the need for clinical molecular imaging. This research thus marks a significant step forward in the clinical understanding and management of HCC, underscoring the importance of integrating tumor evolutionary dynamics and multi-omics biomarkers into therapeutic decision-making.

A genomic enhancer signature associates with hepatocellular carcinoma prognosis.

Background & Aims: Lifestyle and environmental-related exposures are important risk factors for hepatocellular carcinoma (HCC), suggesting that epigenetic dysregulation significantly underpins HCC. We profiled 30 surgically resected tumours and the matched adjacent normal tissues to understand the aberrant epigenetic events associated with HCC. Methods: We identified tumour differential enhancers and the associated genes by analysing H3K27 acetylation (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) and Hi-C/HiChIP data from the resected tumour samples of 30 patients with early-stage HCC. This epigenome dataset was analysed with previously reported genome and transcriptome data of the overlapping group of patients from the same cohort. We performed patient-specific differential expression testing using multiregion sequencing data to identify genes that undergo both enhancer and gene expression changes. Based on the genes selected, we identified two patient groups and performed a recurrence-free survival analysis. Results: We observed large-scale changes in the enhancer distribution between HCC tumours and the adjacent normal samples. Many of the gain-in-tumour enhancers showed corresponding upregulation of the associated genes and vice versa, but much of the enhancer and gene expression changes were patient-specific. A subset of the upregulated genes was activated in a subgroup of patients' tumours. Recurrence-free survival analysis revealed that the patients with a more robust upregulation of those genes showed a worse prognosis. Conclusions: We report the genomic enhancer signature associated with differential prognosis in HCC. Findings that cohere with oncofoetal reprogramming in HCC were underpinned by genome-wide enhancer rewiring. Our results present the epigenetic changes in HCC that offer the rational selection of epigenetic-driven gene targets for therapeutic intervention or disease prognostication in HCC. Impact and Implications: Lifestyle and environmental-related exposures are the important risk factors of hepatocellular carcinoma (HCC), suggesting that tumour-associated epigenetic dysregulations may significantly underpin HCC. We profiled tumour tissues and their matched normal from 30 patients with early-stage HCC to study the dysregulated epigenetic changes associated with HCC. By also analysing the patients' RNA-seq and clinical data, we found the signature genes - with epigenetic and transcriptomic dysregulation - associated with worse prognosis. Our findings suggest that systemic approaches are needed to consider the surrounding cellular environmental and epigenetic changes in HCC tumours.

Multi-region sampling with paired sample sequencing analyses reveals sub-groups of patients with novel patient-specific dysregulation in Hepatocellular Carcinoma.

BACKGROUND: Conventional differential expression (DE) testing compares the grouped mean value of tumour samples to the grouped mean value of the normal samples, and may miss out dysregulated genes in small subgroup of patients. This is especially so for highly heterogeneous cancer like Hepatocellular Carcinoma (HCC). METHODS: Using multi-region sampled RNA-seq data of 90 patients, we performed patient-specific differential expression testing, together with the patients' matched adjacent normal samples. RESULTS: Comparing the results from conventional DE analysis and patient-specific DE analyses, we show that the conventional DE analysis omits some genes due to high inter-individual variability present in both tumour and normal tissues. Dysregulated genes shared in small subgroup of patients were useful in stratifying patients, and presented differential prognosis. We also showed that the target genes of some of the current targeted agents used in HCC exhibited highly individualistic dysregulation pattern, which may explain the poor response rate. DISCUSSION/CONCLUSION: Our results highlight the importance of identifying patient-specific DE genes, with its potential to provide clinically valuable insights into patient subgroups for applications in precision medicine.

Dynamic phenotypic heterogeneity and the evolution of multiple RNA subtypes in hepatocellular carcinoma: the PLANET study.

Intra-tumor heterogeneity (ITH) is a key challenge in cancer treatment, but previous studies have focused mainly on the genomic alterations without exploring phenotypic (transcriptomic and immune) heterogeneity. Using one of the largest prospective surgical cohorts for hepatocellular carcinoma (HCC) with multi-region sampling, we sequenced whole genomes and paired transcriptomes from 67 HCC patients (331 samples). We found that while genomic ITH was rather constant across stages, phenotypic ITH had a very different trajectory and quickly diversified in stage II patients. Most strikingly, 30% of patients were found to contain more than one transcriptomic subtype within a single tumor. Such phenotypic ITH was found to be much more informative in predicting patient survival than genomic ITH and explains the poor efficacy of single-target systemic therapies in HCC. Taken together, we not only revealed an unprecedentedly dynamic landscape of phenotypic heterogeneity in HCC, but also highlighted the importance of studying phenotypic evolution across cancer types.

Current role of selective internal radiation with yttrium-90 in liver tumors.

An expert panel met to review the evidence for selective internal radiation therapy (SIRT) using yttrium-90 microspheres in hepatocellular carcinoma and metastases from colorectal cancer and neuroendocrine tumors. There is now convincing evidence for the safety and efficacy of SIRT in these situations albeit mostly from retrospective cohort studies. There are a number of ongoing prospective randomized controlled clinical trials investigating the role of SIRT in liver tumors; however, data from these trials are still several years away (although the SIRFLOX study has been recently published). In this evolving environment, published evidence and the authors' experience were used to summarize the current and potential role of SIRT in the management of hepatocellular carcinoma of intermediate or advanced stage and in liver-dominant metastatic colorectal cancer and metastatic neuroendocrine tumors.

Living donor hepatectomy in a pregnant woman.

Liver transplantation with a live donor is an effective way to expand the donor pool. Restrictive selection of living donors may assure donor safety but limit the utility of this resource. A 12-month-old recipient with biliary atresia was rapidly deteriorating with hepatic encephalopathy, massive ascites and coagulopathy. Her mother, the only possible living donor, expressed a strong desire to donate part of liver to her baby, although she was found to be pregnant. The donor hepatectomy was then undertaken at 18 weeks of gestation. A left lateral segmentectomy was performed. Her postoperative course was uneventful and she was discharged 7 days after the operation. She gave birth to a healthy term baby without any complications 5 months later. Both recipient and her younger brother are well 12 months after the operation. Despite the limited experience reported herein, pregnancy may no longer be considered an absolute contraindication for live liver donation.

Characterization of liver enzymes on living related liver transplantation patients with acute rejection.

BACKGROUND/AIMS: To determine the biochemical data that reliably predict allograft injury from acute rejection (AR) in patients with living related liver transplantation (LRLT), liver function test and histopathological characteristics of AR were compared and analyzed retrospectively. METHODOLOGY: From Aug. 1994 to Nov. 2000, 101 cases received orthotopic liver transplantation (OLT), which included 53 patients with LRLT in our series. Completed liver functions including aspartate transferase (AST), alanine transferase (ALT), bilirubin total/direct (Bil.T/D), alkaline phosphatase (ALP) and gamma glutamyl transpeptidase (GGT) were collected with peak level when AR was diagnosed by liver biopsy. The best data of the same patients when disease free, were compared and analyzed with non-parametric Wilcoxon signed ranks test and Mann-Whitney test. All of the ARs were reversed with steroid pulse therapy, and two cases converted to FK506. No steroid-resistant rejection or chronic rejection was found in our series. RESULTS: In the patients with LRLT, 17 episodes in 13 patients with AR were found. The incidence of histological analysis proved AR was 12.9% (13/101) in OLT and 24.5% (13/53) in LRLT respectively. Among the liver function tests, AST (p<0.0001), ALT (p<0.0001), Bil.T (p=0.001), Bil.D (p=0.001), GGT (p<0.0001), and INR (p=0.034) were the significant predictors respectively in the patients with AR episode. Once liver enzymes had elevated, the AST/ALT ratio <1.0 showed a more significant difference in AR than in those of the no rejection group (p<0.0001). ALP showed significant difference in our series. The severity of histological change was not correlated to the degree of liver enzymes elevation. CONCLUSIONS: Complete liver function tests especially AST, ALT, Bil.T/D, GGT and the ratio of AST/ALT are very sensitive tests in a group of patients receiving LRLT with AR. The severity of AR is based on the histopathologic change but is not related to the degree of liver enzymes elevation itself. Meanwhile, the outcome of acute rejection in living related liver transplantation is quite good.

Stress response to hepatectomy in patients with a healthy or a diseased liver.

Partial hepatectomy is a major upper abdominal operation associated with certain stress to the patient. Successful adaptation to such stress is a prerequisite for survival. Donor hepatectomy with maximal safety is a principal concern during living donor liver transplantation. The purpose of the study was to compare the stress response by assessing cytokines and the acute-phase response induced by hepatectomy in patients with a healthy liver and those with a diseased liver. Fourteen patients undergoing partial right hepatectomy were enrolled in this study. Seven of them were donors for living related liver transplantation (group 1, or GI); the other seven were patients with hepatocellular carcinoma due to chronic hepatitis B (Child's class A) (GII). Blood samples for interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFalpha), and C-reactive protein (CRP) assays were collected before the operation, at the beginning and end of the operation, and 24 and 48 hours after the operation. The data were analyzed and compared in the same group using the Friedman test and between groups using the Mann-Whitney U-test. A value of p < 0.05 was regarded as significant. Results showed that resection of the liver in patients with both healthy and disease livers leads to significant increases in IL-6 and CPR but not TNFalpha. Significantly lower levels of IL-6 before and after operation in GI patients compared to those in GII patients suggests that GI patients adapted to surgical stress more easily than did the GII patients.

Right lobe living donor liver transplantation-addressing the middle hepatic vein controversy.

OBJECTIVE: To describe our approach in the decision-making for taking the middle hepatic vein with the graft or leaving it with the remnant liver in right lobe live donor liver transplantation. SUMMARY BACKGROUND DATA: Right lobe living donor liver transplantation has been successfully performed. However, the extent of donor hepatectomy is still a subject of debate and the main considerations in the decision making are graft functional adequacy and donor safety. METHODS: An algorithm based on donor-recipient body weight ratio, right lobe-to-recipient standard liver volume estimate, and donor hepatic venous anatomy was used to decide the extent of donor hepatectomy. This algorithm was applied in 25 living donor liver transplant operations performed between January 1999 and January 2002. In grafts taken without the middle hepatic vein, anterior segment tributaries draining into it were not reconstructed. Outcomes between right lobe liver transplants with (Group I) and without (Group II) the middle hepatic vein were compared. RESULTS: Ten grafts included the middle hepatic vein and 15 did not. The mean graft to recipient standard liver volume ratio was 58% and 64% in Groups I and II, respectively, and the difference was not statistically significant. Donors from both groups had comparable recovery, with 2 complications, 1 from each group, requiring a percutaneous drainage procedure. The recipient outcomes were, likewise, comparable and there was 1 case of structural outflow obstruction in Group I, which required venoangioplasty and stenting. There were 2 recipient mortalities, 1 due to a biliary complication and the other to recurrent hepatitis C. Another patient required retransplantation for secondary biliary cirrhosis. The overall actuarial graft and patient survival rates are 84% and 96%, respectively, at a median follow-up of 16 months. CONCLUSION: Based on certain preoperative criteria, a right lobe graft can be taken with or without the middle hepatic vein with equally successful outcomes in both the donors and recipients. The decision, therefore, of the extent of right lobe donor hepatectomy should be tailored to the particular conditions of each case.

Ethics and rationale of living-donor liver transplantation in Asia.

Living-donor liver transplantation took root in Asia as a natural result of circumstances, because the supply of organs from the cadaveric pool remained scarce over the years. In contrast to Western countries, the majority of organs for liver transplantation in Asia come from live donations. As the expertise of the transplant teams grows, patient outcomes improve, and public awareness increases, the option of live donation of the liver is increasingly chosen. Although no live liver donor death has yet been reported from Asia, the risk is not eliminated and remains a major consideration in the potential donor's decision to donate. The low morbidity and mortality rate could be attributed to the extensive experience of surgeons in liver surgery, because surgical liver disease is highly prevalent in Asia. Although the donor risk is estimated to be low, live organ donation should be absolutely voluntary, with consent given on the basis of unbiased information and chosen only when the option for obtaining a cadaveric graft is practically nil. It is only under these conditions that living-donor liver transplantation should be perpetuated. Although the disease-donation-transplantation process involves a complex interplay of psychosocial and family dynamics, the potential candidate's perception will necessarily depend on the surgeon's explanation. The ethical soundness of the practice of living-donor liver transplantation rests primarily on the ones who deliver the service.

Evaluation of living liver donors.

Preoperative evaluation of donors for living-donor liver transplantation aims to select a suitable donor with optimal graft quality and to ensure donor safety. There are minor variations in the donor selection process among different centers, but the safety of the donor remains central to the entire process. The potential donors are evaluated in a stepwise manner including medical, physical, laboratory, psychosocial, and imaging assessment to disqualify unsuitable donors as early as possible in the evaluation process. The main goal of the imaging study is to provide an accurate picture of liver vascular anatomy and liver volume measurement for surgical guidance or for exclusion of unsuitable donors. All imaging studies can now be obtained using noninvasive modalities, thereby decreasing the risk associated with the donor evaluation process. This article describes the donor selection practice in our center including the details of the imaging evaluation.

Right anterior sector drainage in right-lobe live-donor liver transplantation.

The necessity of providing venous drainage for the right anterior sector of a right-lobe graft in adult-to-adult right-lobe live-donor liver transplantation has been controversial. Inclusion of the middle hepatic vein in the right-lobe graft to ensure better early graft function is also under debate. This report summarizes the views of five Asian centers on the necessity of providing venous drainage to the right anterior sector in a right-lobe graft as presented at the Asian Living Donor Transplantation Symposium 2002. All five centers recognize the importance of adequate drainage of the right anterior sector, but they adopt different approaches in including the middle hepatic vein in the graft. Tokyo University uses an occlusion test of the right hepatic artery and middle hepatic vein to define whether the right anterior sector is dusky or regurgitation of blood flow is present in the right anterior portal vein before the decision is made for middle hepatic vein reconstruction. The Asan Medical Center uses hydrostatically dilated saphenous venous graft to anastomose prominent segment V and VIII hepatic vein branches to the inferior vena cava. The University of Hong Kong Medical Centre includes the middle hepatic vein in every graft and anastomoses it to the recipient's middle or left hepatic vein. Kyoto University uses venous jump graft for anastomosing prominent middle hepatic vein branches to the inferior vena cava for recipients receiving a small-for-size graft or graft with dominant middle hepatic vein drainage. The Chang Gung Memorial Hospital adopts a flexible approach in inclusion of the middle hepatic vein in the graft depending on the donor size and the hepatic venous configuration of the right-lobe graft. In summary, the criteria for inclusion and reconstruction of the middle hepatic vein vary. Further analysis is needed to confirm the importance of adequate drainage of the right anterior sector in right-lobe live-donor liver transplantation.

Hepatitis B core antibody-positive grafts: recipient's risk.

The transmission of hepatitis B virus infection through hepatitis B core antibody (anti-HBc)-positive liver grafts in hepatitis B surface antigen (HBsAg)-negative recipients has been established. The mandatory use of immunosuppression in transplant patients favors reactivation of latent virus that may be present in grafts from HBsAg-negative anti-HBc-positive donors. With the persistent organ donor scarcity, the use of these grafts cannot be avoided, especially in urgent cases and in areas where the prevalence of the hepatitis B virus is high, as in Asia. The recognition of posttransplant de novo hepatitis B from core antibody-positive liver donors has, therefore, led to modifications in graft allocation policies and the introduction of strategies for prophylaxis. The risk of developing this type of new-onset hepatitis B virus infection in liver transplant recipients and the various approaches to minimize this risk are reviewed. The peculiar implications of using core antibody-positive grafts in the context of living donor liver transplantation in Asia are discussed.

Split liver transplantation.

Greater experience and improved outcomes in liver transplantation have necessarily led to longer waiting lists against a constantly limited donor pool. Split liver transplantation has been conceived as a means to increase the supply of liver grafts. The bipartition of a whole liver provides grafts for two recipients in a complex operation with equally complex manpower and logistical demands. The in situ technique of splitting offers advantages over the ex vivo technique, although after the time-dependent learning curve is overcome, they may theoretically be used interchangeably with acceptable outcomes. Aside from surgical expertise, donor characteristics and recipient pre-transplant status are risk factors for survival. This review will address the salient aspects of split liver transplantation, summarize the world experience with this procedure and describe the preliminary attempts in Asia.

Prevention of de novo hepatitis B virus infection in living donor liver transplantation using hepatitis B core antibody positive donors.

Exclusion of liver grafts from hepatitis B core antibody (anti-HBc) positive donors to prevent de novo hepatitis B virus (HBV) infection after liver transplantation is not feasible in areas highly endemic for HBV virus like Taiwan, where approximately 80% of adults are anti-HBc(+). The efficacy of lamivudine monotherapy to prevent de novo HBV infection after living donor liver transplantation (LDLT) using grafts from anti-HBc(+) donors remains to be elucidated. From June 1994 to August 2000, LDLT was performed in 42 recipients. Twenty-four of the 42 donors were anti-HBc(+) (57%). Pre-transplant HBV vaccination was given to all recipients irrespective of anti-HBc status at monthly intervals for 3 months. Until December 1997, eight recipients received liver grafts from anti-HBc(+) donors without prophylaxis. Since January 1998, prophylaxis with lamivudine monotherapy was given to 16 recipients receiving liver grafts from anti-HBc(+) donors. De novo HBV infection occurred in three of the eight recipients (37.5%) who did not receive prophylaxis, while none of the 16 recipients given lamivudine developed de novo HBV infection after a mean follow-up of 25 months. Two of the three recipients with de novo HBV infection were anti-HBs(-) and one recipient was anti-HBs(+). Lamivudine was well tolerated, and no side effects were noted. These results suggest that lamivudine monotherapy for recipients receiving anti-HBc(+) liver grafts is a simple, relatively inexpensive and effective prophylactic regimen for prevention of de novo HBV infection. The additive protection provided by vaccine-induced or natural immunity is uncertain.

The effect of hypernatremia on liver allografts in rats.

UNLABELLED: Hypernatremia in the donor organ is one of the most dangerous risk factors that may cause primary graft loss after orthotopic liver transplantation (OLT). However, the viability of donor grafts from acute hypernatremic donors, which is likely to occur during resuscitation of trauma patients with hypertonic saline solution, has not been studied precisely. In the present study, we sought to evaluate whether the hypernatremia, per se, induced by hypertonic saline solution, affects the outcome of liver transplantation in the normal rat. Thirty minutes after the induction of hypernatremia (>160 mEq/L), the livers of nine Wistar rats were removed under ether anesthesia. Six livers were immediately transplanted into normal Wistar rats, whereas the other three were preserved in 4 degrees C University of Wisconsin solution for 6 h before transplantation in the recipients. Liver function variables of the donor rats at graft procurement and of the recipients at Day 7 after OLT were compared with a control group. The water content of the graft at procurement and the survival of the recipients at 7 days after OLT were, likewise, compared with the untreated control group. Results showed that there were no significant differences in the liver function tests of the donors and recipients, as well as in the water content of the grafts, between groups. All the rats survived the observation period of 7 days. This study showed that acute hypernatremia induced by the infusion of 10% saline solution before graft procurement in a nonbrain-dead donor rat model did not lead to a deterioration of liver graft viability after OLT. IMPLICATIONS: Hypernatremia in cadaveric donors may be detrimental to the graft in clinical liver transplantation, but acute donor hypernatremia induced by an IV infusion of 10% saline solution before graft procurement in nonbrain-dead rats did not affect the survival of the recipient rats in an experimental liver transplantation model.