Novel RNA oligonucleotide improves liver function and inhibits liver carcinogenesis in vivo.

UNLABELLED: Hepatocellular carcinoma (HCC) occurs predominantly in patients with liver cirrhosis. Here we show an innovative RNA-based targeted approach to enhance endogenous albumin production while reducing liver tumor burden. We designed short-activating RNAs (saRNA) to enhance expression of C/EBPα (CCAAT/enhancer-binding protein-α), a transcriptional regulator and activator of albumin gene expression. Increased levels of both C/EBPα and albumin mRNA in addition to a 3-fold increase in albumin secretion and 50% decrease in cell proliferation was observed in C/EBPα-saRNA transfected HepG2 cells. Intravenous injection of C/EBPα-saRNA in a cirrhotic rat model with multifocal liver tumors increased circulating serum albumin by over 30%, showing evidence of improved liver function. Tumor burden decreased by 80% (P = 0.003) with a 40% reduction in a marker of preneoplastic transformation. Since C/EBPα has known antiproliferative activities by way of retinoblastoma, p21, and cyclins, we used messenger RNA (mRNA) expression liver cancer-specific microarray in C/EBPα-saRNA-transfected HepG2 cells to confirm down-regulation of genes strongly enriched for negative regulation of apoptosis, angiogenesis, and metastasis. Up-regulated genes were enriched for tumor suppressors and positive regulators of cell differentiation. A quantitative polymerase chain reaction (PCR) and western blot analysis of C/EBPα-saRNA-transfected cells suggested that in addition to the known antiproliferative targets of C/EBPα, we also observed suppression of interleukin (IL)6R, c-Myc, and reduced STAT3 phosphorylation. CONCLUSION: A novel injectable saRNA-oligonucleotide that enhances C/EBPα expression successfully reduces tumor burden and simultaneously improves liver function in a clinically relevant liver cirrhosis/HCC model.

MTL-CEBPA, a Small Activating RNA Therapeutic Upregulating C/EBP-α, in Patients with Advanced Liver Cancer: A First-in-Human, Multicenter, Open-Label, Phase I Trial.

PURPOSE: Transcription factor C/EBP-α (CCAAT/enhancer-binding protein alpha) acts as a master regulator of hepatic and myeloid functions and multiple oncogenic processes. MTL-CEBPA is a first-in-class small activating RNA oligonucleotide drug that upregulates C/EBP-α. PATIENTS AND METHODS: We conducted a phase I, open-label, dose-escalation trial of MTL-CEBPA in adults with advanced hepatocellular carcinoma (HCC) with cirrhosis, or resulting from nonalcoholic steatohepatitis or with liver metastases. Patients received intravenous MTL-CEBPA once a week for 3 weeks followed by a rest period of 1 week per treatment cycle in the dose-escalation phase (3+3 design). RESULTS: Thirty-eight participants have been treated across six dose levels (28-160 mg/m2) and three dosing schedules. Thirty-four patients were evaluable for safety endpoints at 28 days. MTL-CEBPA treatment-related adverse events were not associated with dose, and no maximum dose was reached across the three schedules evaluated. Grade 3 treatment-related adverse events occurred in nine (24%) patients. In 24 patients with HCC evaluable for efficacy, an objective tumor response was achieved in one patient [4%; partial response (PR) for over 2 years] and stable disease (SD) in 12 (50%). After discontinuation of MTL-CEBPA, seven patients were treated with tyrosine kinase inhibitors (TKIs); three patients had a complete response with one further PR and two with SD. CONCLUSIONS: MTL-CEBPA is the first saRNA in clinical trials and demonstrates an acceptable safety profile and potential synergistic efficacy with TKIs in HCC. These encouraging phase I data validate targeting of C/EBP-α and have prompted MTL-CEBPA + sorafenib combination studies in HCC.

Pilot study for a new bipolar radiofrequency ablation/aspirator device in the management of primary and secondary liver cancers.

BACKGROUND: In the US, the thermal ablation workload for cancer involving the liver is predicted to more than double in the next 5 years, emphasising the need to develop and improve the current technology. STUDY DESIGN: A multicentre nonrandomised prospective clinical trial (NCT00514930) was undertaken, to assess the efficacy and safety of a new bipolar radiofrequency ablation/aspirator device, in the treatment of primary and secondary cancers of the liver. RESULTS: A total of 34 lesions in 16 patients were ablated at laparotomy and followed up at 4 weeks. The mean diameter of lesion before ablation was 3.2+/-2.22 (range 1-10) cm, the mean volume aspirated during ablation was 9.25+/-7.3 (range 0-25) ml and the mean operative time was 145.95+/-40.7 (range 60-215) min. There was one major complication of a pleural effusion, which required drainage. The mean length of stay was 8+/-3.2 (range 3-14) days. In 11 patients, the ablated tumour was resected. On histological assessment, there was no evidence of viable cancer at the tumour edge. On follow-up computed tomography, the ablation zone fully encompassed the targeted tumour and there were no local complications related to ablation. CONCLUSION: Initial analysis of the data from this small cohort, with only a short-term follow-up, shows this device to be safe and effective.

Autologous infusion of expanded mobilized adult bone marrow-derived CD34+ cells into patients with alcoholic liver cirrhosis.

OBJECTIVES: Recent advances in regenerative medicine, including hematopoietic stem cell (HSC) transplantation, have brought hope for patients with severe alcoholic liver cirrhosis (ALC). The aim of this study was to assess the safety and efficacy of administering autologous expanded mobilized adult progenitor CD34+ cells into the hepatic artery of ALC patients and the potential improvement in the liver function. METHODS: Nine patients with biopsy-proven ALC, who had abstained from alcohol for at least 6 months, were recruited into the study. Following granulocyte colony-stimulating factor (G-CSF) mobilization and leukapheresis, the autologous CD34+ cells were expanded in vitro and injected into the hepatic artery. All patients were monitored for side effects, toxicities, and changes in the clinical, hematological, and biochemical parameters. RESULTS: On average, a five-fold expansion in cell number was achieved in vitro, with a mean total nucleated cell count (TNCC) of 2.3 x 10(8) pre infusion. All patients tolerated the procedure well, and there were no treatment-related side effects or toxicities observed. There were significant decreases in serum bilirubin (P < 0.05) 4, 8, and 12 wk post infusion. The levels of alanine transaminase (ALT) and aspartate transaminase (AST) showed improvement through the study period and were significant (P < 0.05) 1 wk post infusion. The Child-Pugh score improved in 7 out of 9 patients, while 5 patients had improvement in ascites on imaging. CONCLUSION: It is safe to mobilize, expand, and reinfuse autologous CD34+ cells in patients with ALC. The clinical and biochemical improvement in the study group is encouraging and warrants further clinical trials.

High frequency of fetal cells within a primitive stem cell population in maternal blood.

BACKGROUND: During pregnancy, fetal cells enter the maternal bloodstream resulting in fetal cell microchimerism. The fetal cells persist in the mother for decades and colonize a variety of maternal organs. They are associated with maternal autoimmune diseases and may also participate in tissue repair. The identity of the microchimeric cells is not certain but they must be able to persist long-term and have potential for multitissue differentiation. METHODS AND RESULTS: Here we tested the hypothesis that the fetal microchimeric cells are primitive stem cells, represented by CD34+ adherent cells, which have a wide potential for differentiation. We isolated these stem cells from the blood of pregnant females (n = 25) and detected fetal cells of the correct gender, using fluorescence in situ hybridization, in a high proportion (71% male fetuses and 90% female fetuses; false positive rate 11%, false negative rate 29%) of cases. By RT-PCR, we demonstrated that the cells express Oct-4, Nanog and Rex-1. No fetal cells were detected in the mononuclear or total CD34+ cell populations but high frequencies (mean 11.8%) of fetal cells were detected in the adherent CD34+ cell population. CONCLUSIONS: These results identify adherent CD34+ stem cells as candidate fetal microchimeric cells, which are capable of sustaining the fetal cell population in the long term and have the ability to colonize multiple tissues and organs.

Repeat hepatic resection using a radiofrequency-assisted technique.

BACKGROUND: Repeat hepatic resection for recurrent primary or secondary liver cancer is performed due to advances in resection techniques and evidence of survival benefit. This paper presents the safety and efficacy of repeat radiofrequency-assisted hepatic resection to highlight the utility of the technique. METHODS: 264 consecutive hepatic resections performed on 218 patients were identified. The subset of patients with recurrent disease (n = 24) suitable for repeat hepatic resection had their records reviewed. RESULTS: Including initial (n = 24), second (n = 24) and third hepatic resection (n = 6), a total of 54 hepatic resections were performed in 24 patients. Non-anatomical resection in the form of metastasectomy was the most common procedure. There were no post-operative deaths. Four patients (17%) had complications after their second resection and 1 (17%) after the third resection. There were no cases of bile leak or liver failure. The proportion of repeat hepatic resection for recurrent disease was high: 50% of recurrences were suitable for further resection after initial resection and 43% after second resection. CONCLUSION: Radiofrequency-assisted repeat hepatic resection is a safe procedure and may increase the proportion of patients who can be considered for a curative repeat hepatic resection.

Radiofrequency assisted liver resection--a novel technique.

Surgical resection remains the curative procedure for liver tumors, but even with improvements in method it is still a major operation with significant morbidity and mortality in experts' hands, and a long learning curve for those surgeons who undertake it. Recently radiofrequency ablation has gained some credibility as an alternative method of dealing with liver tumors deemed unresectable. A novel technique of liver resection assisted by the application of radiofrequency is described here. A patient with colorectal liver metastases underwent a segment II/III liver resection with this technique. Following laparotomy, the tumor was identified with intraoperative ultrasound and a 'cooled-tipped' radiofrequency probe was used to ablate liver parenchyma 2cm away from the edge of the tumor. To achieve full thickness of radiofrequency ablation, several insertions were applied. The effect of radiofrequency on liver parenchyma was monitored with an intraoperative ultrasound by micro-bubbles generated by radiofrequency ablation. The length of the resection was 45 min with a blood loss of 30mL. The patient was discharged on the 6th postoperative day without complications. In this report we indicate how the use of radiofrequency ablation can be combined with standard surgical resection of liver cancers to provide a quick, and relatively bloodless operation that is likely to reduce morbidity and mortality and is easy for its practitioners to learn.

Hepatectomy using intraoperative ultrasound-guided radiofrequency ablation.

Radiofrequency (RF) ablation is emerging as a new therapeutic method for management of hepatic tumors. Here we report a new technique for hepatectomy using ultrasound-guided RF, which renders liver resection a less complex surgical procedure. Under intraoperative ultrasound (IOUS) guidance, a Cool-Tip RF probe is inserted into the liver parenchyma surrounding the tumor. A zone of coagulative desiccation is created around the tumor ensuring a 1-cm resection margin. Then, using a scalpel, the liver parenchyma is divided, and the tumor is removed with minimal blood loss. RF works by the conversion of RF waves into heat. Coagulative desiccation occurs and results in sealing of blood and biliary vessels. This is a new technique for liver resection that enables the surgeon to operate in a virtually bloodless field without the use of ischemia, sutures, or ties. It also spares the need for intraoperative blood transfusion and postoperative intensive care unit facilities, and it reduces the length of in patient stay.

A perspective on non-catalytic Src homology (SH) adaptor signalling proteins.

Intracellular adaptor signalling proteins are members of a large family of mediators crucial for signal transduction pathways. Structurally, these molecules contain one Src Homology 2 (SH2) domain and one or more Src Homology 3 (SH3) domain(s); with either a catalytic subunit, or with other non-catalytic modular subunits. Cells depend on these regulatory signalling molecules to transmit information to the nucleus from both external and internal cues including growth factors, cytokines and steroids. Although there is a vast library of adaptor signalling proteins expressed ubiquitously in cells, the vital role these SH containing proteins play in regulating cellular signalling lacks the recognition they deserve. Their target selection method via the SH domains is simple yet highly effective. The SH3 domain(s) interact with proteins that contain proline-rich motifs, whereas the SH2 domain only binds to proteins containing phosphotyrosine residues. This unique characteristic physically enables proteins from a diverse range of networks to assemble for amplification of a signalling event. The biological consequence generated from these adaptor signalling proteins in a constantly changing microenvironment have profound regulatory effect on cell fate decision particularly when this is involved in the progression of a diseased state.

MicroRNA-181a* Targets Nanog in a Subpopulation of CD34(+) Cells Isolated From Peripheral Blood.

Exploiting the properties of stem cells by microRNA (miRNA) profiling offers an attractive approach to identify new regulators of stem cell fate. Although numerous miRNA have been screened from hematopoietic stem cells (HSC), the targets corresponding to many of these miRNA have not yet been fully elucidated. By miRNA profiling in a subpopulation of CD34+ cells isolated from peripheral blood, we have identified eight clusters of miRNA that were differentially expressed. Further analysis of one of the clusters by bioinformatics revealed that a miRNA, miR-181a*, which is highly expressed in the adherent CD34+ cells, affects the expression levels of Nanog, a stem cell surrogate marker. We show specifically by reporter assay and mutational analysis that miR-181a* targets a seedless 3' compensatory site in the 3'UTR of Nanog and affects gene expression. We demonstrate that inhibiting miR-181a* upregulates the Nanog expression level, in addition to an increase in alkaline phosphatase activity. Our studies suggest that miR-181a* may be important in controlling the expression level of Nanog in a subpopulation of CD34+ cells.

Characterization and clinical application of human CD34+ stem/progenitor cell populations mobilized into the blood by granulocyte colony-stimulating factor.

A phase I study was performed to determine the safety and tolerability of injecting autologous CD34(+) cells into five patients with liver insufficiency. The study was based on the hypothesis that the CD34(+) cell population in granulocyte colony-stimulating factor (G-CSF)-mobilized blood contains a subpopulation of cells with the potential for regenerating damaged tissue. We separated a candidate CD34(+) stem cell population from the majority of the CD34(+) cells (99%) by adherence to tissue culture plastic. The adherent and nonadherent CD34(+) cells were distinct in morphology, immunophenotype, and gene expression profile. Reverse transcription-polymerase chain reaction-based gene expression analysis indicated that the adherent CD34(+) cells had the potential to express determinants consistent with liver, pancreas, heart, muscle, and nerve cell differentiation as well as hematopoiesis. Overall, the characteristics of the adherent CD34(+) cells identify them as a separate putative stem/progenitor cell population. In culture, they produced a population of cells exhibiting diverse morphologies and expressing genes corresponding to multiple tissue types. Encouraged by this evidence that the CD34(+) cell population contains cells with the potential to form hepatocyte-like cells, we gave G-CSF to five patients with liver insufficiency to mobilize their stem cells for collection by leukapheresis. Between 1 x 10(6) and 2 x 10(8) CD34(+) cells were injected into the portal vein (three patients) or hepatic artery (two patients). No complications or specific side effects related to the procedure were observed. Three of the five patients showed improvement in serum bilirubin and four of five in serum albumin. These observations warrant further clinical trials.

New technique for liver resection using heat coagulative necrosis.

OBJECTIVE: To assess a new bloodless technique using radiofrequency energy for segmental liver resection of hepatic tumors. SUMMARY BACKGROUND DATA: Liver resection remains a formidable surgical procedure; safe performance requires a high level of training and skill. Intraoperative blood loss during liver resection remains a major concern because it is associated with a higher rate of postoperative complications and shorter long-term survival. METHODS: From January 2000 to June 2001, 15 patients with various hepatic tumors were operated on using radiofrequency energy to remove the tumor in its entirety. Radiofrequency energy was applied along the margins of the tumor to create "zones of necrosis" before resection with a scalpel. RESULTS: No blood transfusions were required. The mean blood loss during resection was 30 +/- 10 mL. No mortality or morbidity was observed. The median postoperative stay was 8 days (range 5-9). No liver recurrence was detected in patients undergoing resection with this technique during follow-up periods ranging from 2 to 20 months. CONCLUSIONS: Segmental and wedge liver resection assisted by radiofrequency is safe. This novel technique offers a new method for transfusion-free resection.