Plasma cell-free RNA profiling distinguishes cancers from pre-malignant conditions in solid and hematologic malignancies.

Cell-free RNA (cfRNA) in plasma reflects phenotypic alterations of both localized sites of cancer and the systemic host response. Here we report that cfRNA sequencing enables the discovery of messenger RNA (mRNA) biomarkers in plasma with the tissue of origin-specific to cancer types and precancerous conditions in both solid and hematologic malignancies. To explore the diagnostic potential of total cfRNA from blood, we sequenced plasma samples of eight hepatocellular carcinoma (HCC) and ten multiple myeloma (MM) patients, 12 patients of their respective precancerous conditions, and 20 non-cancer (NC) donors. We identified distinct gene sets and built classification models using Random Forest and linear discriminant analysis algorithms that could distinguish cancer patients from premalignant conditions and NC individuals with high accuracy. Plasma cfRNA biomarkers of HCC are liver-specific genes and biomarkers of MM are highly expressed in the bone marrow compared to other tissues and are related to cell cycle processes. The cfRNA level of these biomarkers displayed a gradual transition from noncancerous states through precancerous conditions and cancer. Sequencing data were cross-validated by quantitative reverse transcription PCR and cfRNA biomarkers were validated in an independent sample set (20 HCC, 9 MM, and 10 NC) with AUC greater than 0.86. cfRNA results observed in precancerous conditions require further validation. This work demonstrates a proof of principle for using mRNA transcripts in plasma with a small panel of genes to distinguish between cancers, noncancerous states, and precancerous conditions.

In vitro expansion of cirrhosis derived liver epithelial cells with defined small molecules.

BACKGROUND & AIMS: Mature hepatocytes have limited expansion capability in culture and rapidly loose key functions. Recently however, tissue culture conditions have been developed that permit rodent hepatocytes to proliferate and transform into progenitor-like cells with ductal characteristics in vitro. Analogous cells expressing both hepatic and duct markers can be found in human cirrhotic liver in vivo and may represent an expandable population. METHODS: An in vitro culture system to expand epithelial cells from human end stage liver disease organs was developed by inhibiting the canonical TGF-ß, Hedgehog and BMP pathways. RESULTS: Human cirrhotic liver epithelial cells became highly proliferative in vitro. Both gene expression and DNA methylation site analyses revealed that cirrhosis derived epithelial liver cells were intermediate between normal hepatocytes and cholangiocytes. Mouse hepatocytes could be expanded under the same conditions and retained the ability to re-differentiate into hepatocytes upon transplantation. In contrast, human cirrhotic liver derived cells had only low re-differentiation capacity. CONCLUSIONS: Epithelial cells of intermediate ductal-hepatocytic phenotype can be isolated from human cirrhotic livers and expanded in vitro. Unlike their murine counterparts they have limited liver repopulation potential.

AAV integration in human hepatocytes.

Recombinant adeno-associated viral (rAAV) vectors are considered promising tools for gene therapy directed at the liver. Whereas rAAV is thought to be an episomal vector, its single-stranded DNA genome is prone to intra- and inter-molecular recombination leading to rearrangements and integration into the host cell genome. Here, we ascertained the integration frequency of rAAV in human hepatocytes transduced either ex vivo or in vivo and subsequently expanded in a mouse model of xenogeneic liver regeneration. Chromosomal rAAV integration events and vector integrity were determined using the capture-PacBio sequencing approach, a long-read next-generation sequencing method that has not previously been used for this purpose. Chromosomal integrations were found at a surprisingly high frequency of 1%-3% both in vitro and in vivo. Importantly, most of the inserted rAAV sequences were heavily rearranged and were accompanied by deletions of the host genomic sequence at the integration site.

Liver Injury Increases the Incidence of HCC following AAV Gene Therapy in Mice.

Adeno-associated virus (AAV) integrates into host genomes at low frequency, but when integration occurs in oncogenic hotspots it can cause hepatocellular carcinoma (HCC). Given the possibility of recombinant AAV (rAAV) integration leading to HCC, common causes of liver inflammation like non-alcoholic fatty liver disease (NAFLD) may increase the risk of rAAV-induced HCC. A rAAV targeting the oncogenic mouse Rian locus was used, and as expected led to HCC in all mice infected as neonates, likely due to growth-related hepatocyte proliferation in young mice. Mice infected with rAAV as adults did not develop HCC unless they were fed a diet leading to NAFLD, with increased inflammation and hepatocyte proliferation. Female mice were less susceptible to rAAV-induced HCC, and male mice with NAFLD treated with estrogen exhibited less inflammation and immune exhaustion associated with oncogenesis compared to those without estrogen. Adult NAFLD mice infected with a non-targeted control rAAV also developed HCC, though only half as frequently as those exposed to the Rian targeted rAAV. This study shows that adult mice exposed to rAAV gene therapy in the context of chronic liver disease developed HCC at high frequency, and thus warrants further study in humans given the high prevalence of NAFLD in the population.

Multidisciplinary Team Management of Hepatocellular Carcinoma Is Standard of Care.

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality, but unlike other leading causes of cancer death, HCC is increasing in mortality and burden of management. Management of HCC is unique because it usually arises in a diseased liver, which itself may be a driver of mortality. Multidisciplinary teams (MDTs) for the management of complex diseases are becoming more common, but are especially needed in the management of patients with HCC. Liver cancer MDTs are used in most centers providing comprehensive care for patients with HCC, and should be considered the standard of care for these patients.

Correction to: Yttrium-90 Radioembolization for BCLC Stage C Hepatocellular Carcinoma Comparing Child-Pugh A Versus B7 Patients: Are the Outcomes Equivalent?

The name of one of the co-authors was slightly misspelled. Kristian Enestvedt is listed currently as "Kristian K. Enestvedt" and should be listed instead as "C. Kristian Enestvedt."

Battle Royale: Systemic Versus Locoregional Therapy for Unresectable Hepatocellular Carcinoma.

OBJECTIVE: This commentary discusses research examining comparisons of locoregional therapy with systemic therapy for unresectable hepatocellular carcinoma (HCC). CONCLUSION: Comparison of patient outcomes after locoregional or systemic therapy for HCC is confounded by selection bias, wherein patients chosen for locoregional therapy usually have better underlying liver function that may contribute to the observed longer survival.

Long-Term Correction of Diabetes in Mice by In Vivo Reprogramming of Pancreatic Ducts.

Direct lineage reprogramming can convert readily available cells in the body into desired cell types for cell replacement therapy. This is usually achieved through forced activation or repression of lineage-defining factors or pathways. In particular, reprogramming toward the pancreatic ß cell fate has been of great interest in the search for new diabetes therapies. It has been suggested that cells from various endodermal lineages can be converted to ß-like cells. However, it is unclear how closely induced cells resemble endogenous pancreatic ß cells and whether different cell types have the same reprogramming potential. Here, we report in vivo reprogramming of pancreatic ductal cells through intra-ductal delivery of an adenoviral vector expressing the transcription factors Pdx1, Neurog3, and Mafa. Induced ß-like cells are mono-hormonal, express genes essential for ß cell function, and correct hyperglycemia in both chemically and genetically induced diabetes models. Compared with intrahepatic ducts and hepatocytes treated with the same vector, pancreatic ducts demonstrated more rapid activation of ß cell transcripts and repression of donor cell markers. This approach could be readily adapted to humans through a commonly performed procedure, endoscopic retrograde cholangiopancreatography (ERCP), and provides potential for cell replacement therapy in type 1 diabetes patients.

Safety and Efficacy of Accelerated Hypofractionation and Stereotactic Body Radiation Therapy for Hepatocellular Carcinoma Patients With Varying Degrees of Hepatic Impairment.

PURPOSE: To report the toxicities and outcomes for stereotactic body radiation therapy (SBRT) and accelerated hypofractionated radiation therapy (AHRT) in patients with Child-Pugh (CP) class A, B, or C and albumin-bilirubin (ALBI) score 1, 2, or 3 hepatocellular carcinoma. METHODS AND MATERIALS: We retrospectively reviewed the data from 146 patients with hepatocellular carcinoma who had undergone SBRT (50 Gy in 5 fractions) or AHRT (45 Gy in 18 fractions). The primary endpoint was liver toxicity, defined as an increase in the CP score of ≥2 within 6 months of radiation therapy. The secondary endpoints of ALBI change, overall survival, and local control were also calculated. RESULTS: The median follow-up was 23 months (range 1-59). Most received SBRT (72%), and 28% received AHRT. Of all 146 patients, 45 (31%) had a CP score elevation of ≥2 within 6 months of radiation therapy (RT) (27 patients [28%] with baseline CP-A/B7 and 18 [35%] with baseline CP-B8/B9/C cirrhosis; P = .45). On multivariate analysis, neither baseline CP nor ALBI score was predictive of toxicity. No patient with a decline in liver functionality of CP ≥2 within 6 months of RT returned to baseline at later time points. Eleven grade 4 toxicities were observed. The mean change in the raw ALBI score at ∼6 months was similar for all baseline ALBI groups. Twenty-two patients underwent orthotopic liver transplantation after RT, 13 of whom had baseline CP-B8/B9/C liver functionality. For all patients, the 1- and 2-year treated-lesion local control was greater for SBRT than for AHRT (2-year 94% vs 65%, P < .0001). CONCLUSIONS: The tolerability of SBRT or AHRT as measured by a CP score decline of ≥2 within 6 months of RT was similar across baseline liver functionality groups. Compared with AHRT, SBRT was associated with superior local control. Because the true tolerability of limited-volume RT for patients with CP-B or CP-C cirrhosis is unknown, prospective trials validating its safety and efficacy are warranted.

Symptom Distress in Patients With Hepatocellular Carcinoma Toward the End of Life.

PURPOSE/OBJECTIVES: To describe the presence, frequency, severity, and distress of symptoms in outpatients with advanced hepatocellular carcinoma toward the end of life, and the variability in psychological and physical symptom distress between and within patients over time. 
. DESIGN: A prospective, longitudinal, descriptive design. 
. SETTING: Outpatient clinics at two healthcare institutions. 
. SAMPLE: 18 patients (15 men and 3 women) with hepatocellular carcinoma and a mean age of 63.3 years (range = 54-81 years).
. METHODS: Data were collected monthly for six months. Patients completed the Memorial Symptom Assessment Scale, which reports a total score, and three subscales that provide global distress, psychological distress, and physical distress scores.
. MAIN RESEARCH VARIABLES: Global, psychological, and physical distress.
. FINDINGS: Patients reported lack of energy and pain as the most frequent and distressing symptoms. Problems with sexual interest or activity was the fourth most present symptom after drowsiness. Global Distress Index mean scores had notable variability between and within patients over time. During data collection, six patients died. None were referred to palliative care.
. CONCLUSIONS: Gaining knowledge about symptom distress and prevalent symptoms experienced by patients with advanced hepatocellular carcinoma is critical for designing symptom management strategies that are comprehensive and tailored to patients to optimize their quality of life as they approach death.
. IMPLICATIONS FOR NURSING: Nurses play a vital role in advocating for, initiating, and providing comprehensive holistic care based on individual patient needs by facilitating discussions about apparent and less apparent distressing symptoms, including those related to sexuality.

Malignant infiltration of the liver presenting as acute liver failure.

There have been few reports of acute liver failure (ALF), with encephalopathy and coagulopathy, caused by infiltration of the liver by malignant cells. We describe a case series of 27 patients with ALF caused by malignancy. We examined a large, multicenter ALF registry (1910 patients; mean age, 47.1 ± 13.9 y) and found only 27 cases (1.4%) of ALF attributed to malignancy. Twenty cases (74%) presented with abdominal pain and 11 presented with ascites. The most common malignancies included lymphoma or leukemia (33%), breast cancer, (30%), and colon cancer (7%); 90% of the patients with lymphoma or leukemia had no history of cancer, compared with 25% of patients with breast cancer. Overall, 44% of the patients had evidence of liver masses on imaging. Diagnosis was confirmed by biopsy in 15 cases (55%) and by autopsy for 6 cases. Twenty-four patients (89%) died within 3 weeks of ALF.

Building the multidisciplinary team for management of patients with hepatocellular carcinoma.

Optimal care of the patient with hepatocellular carcinoma (HCC) necessitates the involvement of multiple providers. Because the patient with HCC often carries 2 conditions with competing mortality risks (cancer and underlying cirrhosis), no single provider is equipped to deal with all of these patients' needs adequately. Multidisciplinary teams (MDTs) have evolved to facilitate care coordination, reassessments of clinical course, and nimble changes in treatment plans required for this complex group of patients. Providers or sites that elect to manage patients with HCC thus are increasingly aware of the need to build their own MDT or communicate with an established one. The availability of new communication technologies, such as teleconferencing or teleconsultation, offers the possibility of MDT expansion into underserved or rural areas, as well as areas such as correctional facilities. Although the availability of resources for HCC patient care varies from site to site, construction of an MDT is possible in a wide spectrum of clinical practices, and this article suggests a blueprint for assembly of such collaboration. Research strategies are needed to explain how MDTs improve clinical outcomes so that MDTs themselves can be improved.

Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes.

Adult liver progenitor cells are biliary-like epithelial cells that emerge only under injury conditions in the periportal region of the liver. They exhibit phenotypes of both hepatocytes and bile ducts. However, their origin and their significance to injury repair remain unclear. Here, we used a chimeric lineage tracing system to demonstrate that hepatocytes contribute to the progenitor pool. RNA-sequencing, ultrastructural analysis, and in vitro progenitor assays revealed that hepatocyte-derived progenitors were distinct from their biliary-derived counterparts. In vivo lineage tracing and serial transplantation assays showed that hepatocyte-derived proliferative ducts retained a memory of their origin and differentiated back into hepatocytes upon cessation of injury. Similarly, human hepatocytes in chimeric mice also gave rise to biliary progenitors in vivo. We conclude that human and mouse hepatocytes can undergo reversible ductal metaplasia in response to injury, expand as ducts, and subsequently contribute to restoration of the hepatocyte mass.

Waiting time predicts survival after liver transplantation for hepatocellular carcinoma: a cohort study using the United Network for Organ Sharing registry.

Recipients of liver transplantation (LT) for hepatocellular carcinoma (HCC) have an 8% to 20% risk of HCC recurrence. Single-center studies suggest that a period of waiting after HCC therapy may facilitate the selection of patients at low risk for post-LT HCC recurrence and mortality. We evaluated whether a longer waiting time after Model for End-Stage Liver Disease (MELD) prioritization for HCC predicts longer post-LT survival. From the United Network for Organ Sharing registry, we selected 2 groups registered for LT between March 2005 and March 2009: (1) HCC patients receiving MELD prioritization and (2) non-HCC patients. Patients were stratified by their MELD status at LT (a marker of time on the wait list after HCC MELD prioritization) and were followed from LT until death or censoring through October 2012. By comparing post-LT survival to intention-to-treat (ITT) survival from registration, we assessed predictors of post-LT survival and estimated the benefit of LT. The median MELD scores at LT were 22 (HCC) and 24 (non-HCC). A higher MELD score at LT was independently associated with lower post-LT mortality in the HCC group [hazard ratio (HR) = 0.84, 95% confidence interval (CI) = 0.73-0.98] and higher post-LT mortality in the non-HCC group (HR = 1.20, 95% CI = 1.15-1.25). Compared with the HCC group, the non-HCC group had lower post-LT mortality [relative risk (RR) = 0.85, log-rank P < 0.01] but higher ITT mortality (RR = 1.25, log-rank P < 0.01) because of a 33 percentage point lower probability of undergoing LT. In conclusion, a longer waiting time before LT for HCC predicted longer post-LT survival in a national transplant registry. Delaying LT for HCC may reduce disparities in ITT survival and access to LT among different indications and thereby improve system utility and organ allocation equity for the overall pool of LT candidates.

Epstein-Barr virus (EBV) related acute liver failure: a case series from the US Acute Liver Failure Study Group.

PURPOSE: Acute liver failure (ALF) is a rare clinical syndrome associated with a high case fatality rate. Asymptomatic primary infection with Epstein-Barr virus (EBV) is common in the general population while acute hepatitis and jaundice are much less common and ALF has been rarely reported. We reviewed the presenting features as well as clinical outcomes amongst consecutive adults with EBV-related ALF. METHODS: Amongst the 1,887 adult ALF patients enrolled into the US ALF Study Group from January 1998 to February 2012, there were four patients (0.21 %) with EBV-related ALF. Diagnostic criteria for acute EBV infection included compatible serologies and/or the detection of EBV-encoded RNA (EBER) in liver tissue. RESULTS: Median patient age was 30 years (range 18-44); 75 % were male, and only 25 % were immunosuppressed. The median presenting ALT was 504 IU/mL (range 156-4,920), median Alk P was 431 (range 136-1,009), and median bilirubin was 17 mg/dL (range 13-22.1). Liver biopsy findings ranged from cholestasis to submassive necrosis with EBER + staining in two of the three samples tested. Although all of the patients were treated with an antiviral agent, two died of ALF, one underwent liver transplantation (LT) and one survived with supportive care and is well at 5 years. A review of the literature identified four additional LT recipients with favorable long-term outcomes. CONCLUSION: Primary EBV infection accounts for <1 % of consecutive adult ALF cases but is associated with a high case fatality rate. LT is associated with favorable short- and long-term outcomes.

Impact of locoregional therapy and alpha-fetoprotein on outcomes in transplantation for liver cancer: a UNOS Region 6 pooled analysis.

Liver transplantation (LT) provides optimal long-term disease-free survival for hepatocellular carcinoma (HCC). High pre-LT alpha-fetoprotein (AFP) has been associated with HCC recurrence, but it is unclear whether a drop in AFP or locoregional therapy impacts survival/recurrence after LT. LT-recipients transplanted for HCC in three centers (UNOS Region 6) were reviewed (2006-2009) for demographics, tumor characteristics, locoregional therapy, AFP, recurrence, and survival. Among 211 LT recipients (mean age 56.4 yr, 83% male, mean MELD 12.2), 94% met Milan criteria and 61% received locoregional therapy. Mean disease-free survival (DFS) was 1549.7 d, and 84% are currently alive. Factors affecting DFS included recurrence (RR, 0.074; 95% CI, 0.038-0.14), normal peak AFP (29.6, 95% CI, 2.96-296.3), peak AFP >400 (RR, 0.15; 95% CI, 0.03-0.73) and AFP at LT >400 (RR, 15.5; 95% CI, 2.4-100.5). Twenty-one patients had recurrence and were more likely beyond Milan criteria (5/23(21%) vs. 8/220 (4%), p = 0.0038), with peak AFP >400 and AFP at LT >400 (p = 0.001). Locoregional therapy did not affect mean DFS (1458.0 vs. 1603.8 d, p = 0.05) or recurrence (12.5% vs. 6%). Predictors of recurrence were similar to previous studies, including high AFP and tumor outside Milan criteria. While locoregional therapy itself did not affect DFS/recurrence, a decrease in AFP pre-transplant appears to positively influence outcomes in those who received locoregional therapy.

Portal Biliopathy Causing Recurrent Biliary Obstruction and Hemobilia.

A 63-year-old man with extrahepatic portal vein thrombosis presented with biliary obstruction and hemobilia after a liver biopsy. Balloon sweep of the common bile duct removed clotted blood, and cholangiogram showed a common bile duct narrowing, treated with biliary stenting. A percutaneous biliary catheter was later required for recurrent biliary obstruction and hemobilia, and repeat cholangiogram confirmed portal biliopathy-a large peri-biliary varix was compressing the common bile duct, causing biliary obstruction and intermittent portal hypertensive hemobilia. A transjugular intrahepatic portosystemic shunt was inserted, followed by embolization of the peri-biliary varix. Delayed diagnosis of portal biliopathy may lead to significant patient morbidity.