Targeting therapy for hepatocellular carcinoma by delivering microRNAs as exosomal cargo

Exosomes,the smallest extracellular vesicles,have gained significant attention as key mediators in intercellular communication,influencing both physiological and pathological processes,particularly in cancer progression.A recent review article by Wang et al was published in a timely manner to stimulate future research and facilitate practical developments for targeted treatment of hepatocellular carcinoma using exosomes,with a focus on the origin from which exosomes derive.If information about the mechanisms for delivering exosomes to specific cells is incorporated,the concept of targeted therapy for hepatocellular carcinoma using exosomes could be more comprehensively understood.

Shear wave velocity is a useful marker for managing nonalcoholic steatohepatitis

AIM:To investigate whether a noninvasive measurement of tissue strain has a potential usefulness for management of nonalcoholic steatohepatitis(NASH).METHODS:In total 26 patients,23 NASHs and 3 normal controls were enrolled in this study.NASH was staged based on Brunt criterion.At a region of interest(ROI),a shear wave was evoked by implementing an acoustic radiation force impulse(ARFI),and the propagation velocity was quantif ied.RESULTS:Shear wave velocity(SWV) could be reproducibly quantified at all ROIs in all subjects except for 4 NASH cases,in which a reliable SWV value was not calculated at several ROIs.An average SWV of 1.34 ± 0.26 m/s in fibrous stage 0-1 was significantly slower than 2.20 ± 0.74 m/s and 2.90 ± 1.01 m/s in stages 3 and 4,respectively,but was not significantly different from 1.79 ± 0.78 m/s in stage 2.When a cutoff value was set at 1.47 m/s,receiver operating characteristic analysis showed significance to dissociate stages 3 and 4 from stage 0-1(P=0.0092) with sensitivity,specificity and area under curve of 100%,75% and 94.2%,respectively.In addition,the correlation between SWV and hyaluronic acid was significant(P<0.0001),while a tendency toward negative correlation was observed with serum albumin(P=0.053).CONCLUSION:The clinical implementation of ARFI provides noninvasive repeated evaluations of liver stiffness at an arbitrary position,which has the potential to shed new light on NASH management.

Active treatments are a rational approach for hepatocellular carcinoma in elderly patients

AIM: To determine whether an active intervention is beneficial for the survival of elderly patients with hepa-tocellular carcinoma (HCC). METHODS: The survival of 740 patients who received various treatments for HCC between 1983 and 2011 was compared among different age groups using Cox regression analysis. Therapeutic options were principal-ly selected according to the clinical practice guidelines for HCC from the Japanese Society of Hepatology. The treatment most likely to achieve regional control capa-bility was chosen, as far as possible, in the following order: resection, radiofrequency ablation, percutaneous ethanol injection, transcatheter arterial chemoembo-lization, transarterial oily chemoembolization, hepatic arterial infusion chemotherapy, systemic chemotherapy including molecular targeting, or best supportive care.Each treatment was used alone, or in combination, with a clinical goal of striking the best balance be-tween functional hepatic reserve and the volume of the targeted area, irrespective of their age. The percent survival to life expectancy was calculated based on a Japanese national population survey. RESULTS: The median ages of the subjects during each 5-year period from 1986 were 61, 64, 67, 68 and 71 years and increased significantly with time (P<0.0001). The Child-Pugh score was comparable among younger (59 years of age or younger), middle-aged (60-79 years of age), and older (80 years of age or older) groups (P=0.34), whereas the tumor-node-metastasis stage tended to be more advanced in the younger group (P=0.060). Advanced disease was significantly more frequent in the younger group compared with the middle-aged group (P=0.010), whereas there was no difference between the middle-aged and elderly groups (P=0.75). The median sur-vival times were 2593, 2011, 1643, 1278 and 1195 d for 49 years of age or younger, 50-59 years of age, 60-69 years of age, 70-79 years of age, or 80 years of age or older age groups, respectively, whereas the me-dian percent survival to life expectancy were 13.9%, 21.9%, 24.7%, 25.7% and 37.6% for each group, respectively. The impact of age on actual survival time was significant (P=0.020) with a hazard ratio of 1.021, suggesting that a 10-year-older patient has a 1.23-fold higher risk for death, and the overall survival was the worst in the oldest group. On the other hand, when the survival benefit was evaluated on the basis of per-cent survival to life expectancy, age was again found to be a significant explanatory factor (P=0.022); how-ever, the oldest group showed the best survival among the five different age groups. The youngest group revealed the worst outcomes in this analysis, and the hazard ratio of the oldest against the youngest was 0.35 for death. The survival trends did not differ substan-tially between the survival time and percent survival tolife expectancy, when survival was compared overall or among various therapeutic interventions. CONCLUSION: These results suggest that a thera-peutic approach for HCC should not be restricted due to patient age.

Effect of double platinum agents, combination of miriplatintransarterial oily chemoembolization and cisplatinhepatic arterial infusion chemotherapy, in patients with hepatocellular carcinoma: Report of two cases

Hepatocellular carcinoma(HCC) is one of the most common cancers and the third highest cause of cancerassociated mortality worldwide. The treatment of HCC is complicated by its variable biological behavior and the frequent coexistence of chronic liver disease, particularly cirrhosis. To date, multiple treatment modalities have been developed according to the stage of the tumor and the hepatic functional reserve, including transarterial treatments such as transarterial chemoembolization, transarterial oily chemoembolization(TOCE), and hepatic arterial infusion chemotherapy(HAIC). We conducted a phase I and II study of the combination therapy with double platinum agents, miriplatin and cisplatin, and confirmed its safety and efficacy. Here, we describe two cases of unresectable HCC who were successfully treated by miriplatin-TOCE/cisplatin-HAIC combination therapy, resulting in complete responses with no significant adverse events. This report will provide that the combination therapy can be the therapeutic option for HCC patients in the advanced stage.

Rational arrangement of measuring shear wave speed in the liver

BACKGROUND Shear wave speed has been widely applied to quantify a degree of liver fibrosis. However, there is no standardized procedure, which makes it difficult to utilize the speed universally. AIM To provide procedural standardization of shear wave speed measurement. METHODS Point shear wave elastography (pSWE) was measured in 781 patients, and twodimensional shear wave elastography (2dSWE) was measured on the same day in 18 cases. Regions-of-interest were placed at 12 sites, and the median and robust coefficient-of-variation (CVR) were calculated. A residual sum-of-square (Σdi2) was computed for bootstrap values of 1000 iterations in 18 cases with each assumption of 1 to 12 measurements. The proportion of the Σdi2 (%Σdi2) was calculated as the ratio of Σdi2 to pSWE after converting it based on the correlation between pSWE and 2dSWE. RESULTS The CVR showed a significantly broader distribution in the left lobe (P < 0.0001),and the smallest CVR in the right anterior segment that covered 95% cases was 40.4%. pSWE was significantly higher in the left lobe than in the right lobe (1.63 ± 0.78 m/s vs 1.61 ± 0.78 m/s, P = 0.0004), and the difference between the lobes became further discrete when the subjects were limited to the cases with a CVR less than 40.4% in any segment (1.76 ± 0.80 m/s vs 1.70 ± 0.82 m/s, P < 0.0001). The highest values of the CVR in every 0.1 m/s interval were plotted in convex upward along pSWE and peaked at 1.93 m/s. pSWE and 2dSWE were significantly correlated (P < 0.0001, r = 0.95). In 216000 resamples from 18 cases, the %Σdi2 of 12 sites was 8.0% and gradually increased as the acquisition sites decreased to reach a significant difference with a %Σdi2 of 7 sites (P = 0.027). CONCLUSION These data suggest that shear wave speed should be measured at 8 or more sites of spreading in both lobes.

Liver-targeted hydrodynamic gene therapy: Recent advances in the technique

One of the major research focuses in the field of gene therapy is the development of clinically applicable, safe, and effective gene-delivery methods. Since the first case of human gene therapy was performed in 1990, a number of gene-delivery methods have been developed, evaluated for efficacy and safety, and modified for human application. To date, viral-vectormediated deliveries have shown effective therapeutic results. However, the risk of lethal immune response and carcinogenesis have been reported, and it is still controversial to be applied as a standard therapeutic option. On the other hand, delivery methods for nonviral vector systems have been developed, extensively studied, and utilized in in vivo gene-transfer studies. Compared to viral-vector mediated gene transfer, nonviral systems have less risk of biological reactions. However, the lower gene-transfer efficiency was a critical hurdle for applying them to human gene therapy. Among a number of nonviral vector systems, our studies focus on hydrodynamic gene delivery to utilize physical force to deliver naked DNA into the cells in the living animals. This method achieves a high gene-transfer level by DNA solution injections into the tail vein of rodents, especially in the liver. With the development of genome editing methods, in vivo gene-transfer therapy using this method is currently the focus in this research field. This review explains the method principle, efficiency, safety, and procedural modifications to achieve a high level of reproducibility in large-animal models.

Identification of cellular genes showing differential expression associated with hepatitis B virus infection

AIM: To investigate the impact of hepatitis B virus (HBV) infection on cellular gene expression, by conducting both in vitro and in vivo studies. METHODS: Knockdown of HBV was targeted by stable expression of short hairpin RNA (shRNA) in huH-1 cells. Cellular gene expression was compared using a human 30K cDNA microarray in the cells and quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR) (qRT-PCR) in the cells, hepatocellular carcinoma (HCC) and surrounding non-cancerous liver tissues (SL). RESULTS: The expressions of HBsAg and HBx protein were markedly suppressed in the cells and in HBx transgenic mouse liver, respectively, after introduction of shRNA. Of the 30K genes studied, 135 and 103 genes were identified as being down- and up-regulated, respectively, by at least twofold in the knockdown cells. Functional annotation revealed that 85 and 62 genes were classified into four up-regulated and five down-regulated functional categories, respectively. When gene expression levels were compared between HCC and SL, eight candidate genes that were confirmed to be up- or down-regulated in the knockdown cells by both microarray and qRT-PCR analyses were not expressed as expected from HBV reduction in HCC, but had similar expression patterns in HBV- and hepatitis C virus-associated cases. In contrast, among the eight genes, only APM2 was constantly repressed in HBV non-associated tissues irrespective of HCC or SL. CONCLUSION: The signature of cellular gene expression should provide new information regarding the pathophysiological mechanisms of persistent hepatitis and hepatocarcinogenesis that are associated with HBV infection.

Factors predicting aggressiveness of non-hypervascular hepatic nodules detected on hepatobiliary phase of gadolinium ethoxybenzyl diethylene-triamine-pentaacetic-acid magnetic resonance imaging

AIM: To establish a prognostic formula that distinguishes non-hypervascular hepatic nodules(NHNs) with higher aggressiveness from less hazardous one. METHODS: Seventy-three NHNs were detected in gadolinium ethoxybenzyl diethylene-triamine-pentaaceticacid magnetic resonance imaging(Gd-EOB-DTPA-MRI) study and confirmed to change 2 mm or more in size and/or to gain hypervascularity. All images were interpreted independently by an experienced, board-certified abdominal radiologist and hepatologist; both knew thatthe patients were at risk for hepatocellular carcinoma development but were blinded to the clinical information. A formula predicting NHN destiny was developed using a generalized estimating equation model with thirteen explanatory variables: age, gender, background liver diseases, Child-Pugh class, NHN diameter, T1-weighted imaging/T2-weighted imaging detectability, fat deposition, lower signal intensity in arterial phase, lower signal intensity in equilibrium phase, α-fetoprotein, des-γ-carboxy prothrombin, α-fetoprotein-L3, and coexistence of classical hepatocellular carcinoma. The accuracy of the formula was validated in bootstrap samples that were created by resampling of 1000 iterations. RESULTS: During a median follow-up period of 504 d, 73 NHNs with a median diameter of 9 mm(interquartile range: 8-12 mm) grew or shrank by 68.5%(fifty nodules) or 20.5%(fifteen nodules), respectively, whereas hypervascularity developed in 38.4%(twenty eight nodules). In the fifteen shrank nodules, twelve nodules disappeared, while 11.0%(eight nodules) were stable in size but acquired vascularity. A generalized estimating equation analysis selected five explanatories from the thirteen variables as significant factors to predict NHN progression. The estimated regression coefficients were 0.36 for age, 6.51 for lower signal intensity in arterial phase, 8.70 or 6.03 for positivity of hepatitis B virus or hepatitis C virus, 9.37 for des-γ-carboxy prothrombin, and-4.05 for fat deposition. A formula incorporating the five coefficients revealed sensitivity, specificity, and accuracy of 88.0%, 86.7%, and 87.7% in the formulating cohort, whereas these of 87.2% ± 5.7%, 83.8% ± 13.6%, and 87.3% ± 4.5% in the bootstrap samples. CONCLUSION: These data suggest that the formula helps Gd-EOB-DTPA-MRI detect a trend toward hepatocyte transformation by predicting NHN destiny.

Methotrexate-related lymphoproliferative disorders in the liver: Case presentation and mini-review

BACKGROUND Immunosuppression is effective in treating a number of diseases,but adverse effects such as bone marrow suppression,infection,and oncogenesis are of concern.Methotrexate is a key immunosuppressant used to treat rheumatoid arthritis.Although it is effective for many patients,various side effects have been reported,one of the most serious being methotrexate-related lymphoproliferative disorder.While this may occur in various organs,liver involvement is rare.Information on these liver lesions,including clinical characteristics,course,and imaging studies,has not been summarized to date.CASE SUMMARY We present a case of 70-year-old woman presented with a 2-wk history of fever and abdominal pain.She had had rheumatoid arthritis for 5 years and was being treated with medication including methotrexate.Contrast-enhanced computed tomography revealed multiple low density tumors in the liver and the histological analyses showed significant proliferation of lymphocytes in masses that were positive on immunohistochemical staining for CD3,CD4,CD8,and CD79a but negative for CD20 and CD56.Staining for Epstein-Barr virus-encoded RNA was negative.And based on these findings,the liver tumors were diagnosed as Methotrexate-related lymphoproliferative disorders.A timedependent disappearance of the liver tumors after stopping methotrexate supported the diagnoses.CONCLUSION The information obtained from our case and a review of 9 additional cases reported thus far assist physicians who may face the challenge of diagnosing and managing this disorder.

Multicentric occurrence of hepatocellular carcinoma with nonalcoholic steatohepatitis

AIM:To reveal the manner of hepatocellular carcinoma (HCC) development in patients with nonalcoholic steatohepatitis(NASH) focusing on multicentric occurrence (MO) of HCC.METHODS:We compared clinicopathological characteristics between patients with and without MO of HCC arising from NASH background.The clinical features were implicated with reference to the literature available.RESULTS:MO of HCC was identified with histological proof in 4 out of 12 patients with NASH-related HCC(2 males and 2 females).One patient had synchronous MO;an advanced HCC,two well-differentiated HCCs and a dysplastic nodule,followed by the development of metachronous MO of HCC.The other three patients had multiple advanced HCCs accompanied by a well-differentiated HCC or a dysplastic nodule.Of these three patients,one had synchronous MO,one had metachronous MO and the other had both synchronous and metachronous MO.There were no obvious differences between the patients with or without MO in terms of liver function tests,tumor markers and anatomical extent of HCC.On the other hand,all four patients with MO of HCC were older than 70 years old and had the comorbidities of obesity,type 2 diabetes mellitus(T2DM),hypertension and cirrhosis.Although these conditions were not limited to MO of HCC,all the conditions were met in only one of eight patients without MO of HCC.Thus,concurrence of these conditions may be a predisposing situation to synchronous MO of HCC.In particular,old age,T2DM and cirrhosis were suggested to be prerequisite for MO because these factors were depicted in common among two other cases with MO of HCC under NASH in the literature.CONCLUSION:The putative predisposing factors and necessary preconditions for synchronous MO of HCC in NASH were suggested in this study.Further investigations are required to clarify the accurate prevalence and predictors of MO to establish better strategies for treatment and prevention leading to the prognostic improvement in NASH.

Gravity assistance enables liver stiffness measurements to detect liver fibrosis under congestive circumstances

BACKGROUND As survival has been prolonged owing to surgical and medical improvements,liver failure has become a prognostic determinant in patients with congestive heart diseases.Congestive hepatopathy,an abnormal state of the liver as a result of congestion,insidiously proceed toward end-stage liver disease without effective biomarkers evaluating pathological progression.Regular measurements of shear wave elastography cannot qualify liver fibrosis,which is a prognosticator in any type of chronic liver disease,in cases of congestion because congestion makes the liver stiff without fibrosis.We hypothesized that the effects of congestion and fibrosis on liver stiffness can be dissociated by inducing architectural deformation of the liver to expose structural rigidity.To establish a strategy measuring liver stiffness as a reflection of architectural rigidity under congestion.METHODS Two-dimensional shear wave elastography(2dSWE)was measured in the supine(Sp)and left decubitus(Ld)positions in 298 consecutive cases as they were subjected to an ultrasound study for various liver diseases.Regions of interest were placed at twelve sites,and the median and robust coefficient of variation were calculated.Numerical data were compared using the Mann-Whitney U or Kruskal-Wallis test followed by Dunn's post-hoc multiple comparisons.The inferior vena cava(IVC)diameters at different body positions were compared using the Wilcoxon matched pairs signed rank test.The number of cases with cardiothoracic ratios greater than or not greater than 50%was compared using Fisher’s exact test.A correlation of 2dSWE between different body positions was evaluated by calculating Spearman correlation coefficients.RESULTS The IVC diameter was significantly reduced in Ld in subjects with higher 2dSWE values in Ld(LdSWE)than in Sp(SpSWE)(P=0.007,(average±SD)13.9±3.6 vs 13.1±3.4 mm)but not in those with lower LdSWE values(P=0.32,13.3±3.5 vs 13.0±3.5 mm).In 81 subjects,SpSWE was increased or decreased in Ld beyond the magnitude of robust coefficient of variation,which suggests that body postural changes induced an alteration of liver stiffness significantly larger than the technical dispersion.Among these subjects,all 37 with normal SpSWE had a higher LdSWE than SpSWE(Normal-to-Hard,SpSWE-LdSWE(Δ2dSWE):(minimum-maximum)-0.74--0.08 m/sec),whereas in 44 residual subjects with abnormal SpSWE,LdSWE was higher in 27 subjects(Hard-to-Hard,-0.74--0.05 m/sec)and lower in 17 subjects(Hard-to-Soft,0.04-0.52 m/sec)than SpSWE.SpSWE was significantly correlated withΔ2dSWE only in Hard-to-Soft(P<0.0001).Δ2dSWE was larger in each lobe than in the entire liver.When Hard-to-Hard and Hard-to-Soft values were examined for each lobe,fibrosis-4 or platelet counts were significantly higher or lower only for Hard-to-Soft vs Normal-to-Hard cases.CONCLUSION Gravity alters the hepatic architecture during body postural changes,causing outflow blockage in hepatic veins.A rigid liver is resistant to structural deformation.Stiff-liver softening in the Ld position suggests a fibrous liver.