Yoshiyuki Sawai

Gd-EOB-DTPA-enhanced magnetic resonance images of hepatocellular carcinoma: correlation with histological grading and portal blood flow

Objective:To retrospectively investigate enhancement patterns of hepatocellular carcinoma (HCC) and dysplastic nodule (DN) in the hepatobiliary phase of gadolinium-ethoxybenzyl-diethylenetriamine (Gd-EOB-DTPA)-enhanced MRI in relation to histological grading and portal blood flow.Methods:Sixty-nine consecutive patients with 83 histologically proven HCCs and DNs were studied. To assess Gd-EOB-DTPA uptake, we calculated the EOB enhancement ratio, which is the ratio of the relative intensity of tumorous lesion to surrounding nontumorous area on hepatobiliary phase images (post-contrast EOB ratio) to that on unenhanced images (pre-contrast EOB ratio). Portal blood flow was evaluated by CT during arterial portography.Results:Post-contrast EOB ratios significantly decreased as the degree of differentiation declined in DNs (1.00 ± 0.14) and well, moderately and poorly differentiated HCCs (0.79 ± 0.19, 0.60 ± 0.27, 0.49 ± 0.10 respectively). Gd-EOB-DTPA uptake, assessed by EOB enhancement ratios, deceased slightly in DNs and still more in HCCs, while there was no statistical difference in the decrease between different histological grades of HCC. Reductions in portal blood flow were observed less frequently than decreases in Gd-EOB-DTPA uptake in DNs and well-differentiated HCCs.Conclusions:Reduced Gd-EOB-DTPA uptake might be an early event of hepatocarcinogenesis, preceding portal blood flow reduction. The hepatobiliary phase of Gd-EOB-DTPA-enhanced MRI may help estimate histological grading, although difficulties exist in differentiating HCCs from DNs.

Expression and prognostic role of RhoA GTPases in hepatocellular carcinoma

The Rho sub-family of proteins is involved in regulating the organization of the cytoskeleton and in cell motility. Our aim is to clarify the clinical significance of Rho protein in hepatocellular carcinomas (HCC) and to determine the relationship between the level of expression and patient outcome following hepatectomy. The expression of RhoA protein in HCC and corresponding non-tumor tissues of 26 patients who underwent surgical resection was analyzed by immunoblotting. The expression level of each case was calculated as tumor/non-tumor (T/N) ratios. High expression (T/N≥1) of RhoA protein in HCC compared to the paired non-tumor tissues was recognized in 18 patients (69.2%) of 26 samples. The activity of RhoA is also increased in HCC with high expression of RhoA. The high expression of RhoA protein did not correlate with various clinical parameters. However, the disease-free survival rates of the RhoA-high expression group (T/N≥1) were significantly lower than those of the RhoA-low expression group (T/N<1) (P<0.05). The high expression of RhoA protein in HCC plays an important role in intrahepatic recurrence of patients who underwent a hepatectomy for HCC, and RhoA is a useful marker for predicting early recurrence in an early-stage HCC.

Serum Mac‐2 binding protein levels as a novel diagnostic biomarker for prediction of disease severity and nonalcoholic steatohepatitis

Mac‐2 binding protein (Mac‐2bp) is one of the major fucosylated glycoproteins, which we identified with glycol‐proteomic analyses. We previously reported that fucosylated glycoproteins are secreted into bile, but scarcely secreted into sera in normal liver and hypothesized that the fucosylation‐based sorting machinery would be disrupted in ballooning hepatocytes due to the loss of cellular polarity. In the present study, we investigated the availability of Mac‐2bp for differential diagnosis of nonalcoholic steatohepatitis (NASH) from nonalcoholic fatty liver disease (NAFLD) as a biomarker.

Expression of ephrin-B1 in hepatocellular carcinoma: possible involvement in neovascularization

BACKGROUND/AIMS Hepatocellular carcinoma (HCC) is, in general, a hypervascular tumor. Ephrin/Eph molecules have recently been reported as possible regulators of angiogenesis. We aimed to clarify the role of ephrin-Bs (B1-B3) in the progression of HCC. METHODS Ephrin-Bs transcripts in 26 HCC and their corresponding non-tumor liver tissues were analyzed by the quantitative reverse transcription-polymerase chain reaction. We established ephirn-B1 overexpressing cell in a human HCC cell line, PLC/PRF/5 cell, and their in vivo growth monitored after subcutaneous injection into nude mice. Neovascularization in the inoculated tumors was evaluated by the immunohistochemical staining of CD31. The migration and proliferation of human umbilical vein endothelial cells (HUVECs) in response to soluble ephrin-B1-Fc was examined. RESULTS The expression of the ephrin-B1 transcript but not -B2 and -B3 transcripts was significantly higher in HCC tissues than in non-tumor tissues (P<0.05). The ephrin-B1 overexpressing cells developed tumors more rapidly than controls in vivo (P<0.05), although in vitro cell growth was not affected. The tumor vessel number significantly increased in the ephrin-B1 overexpressing tumors (P<0.0001). In addition, in vitro studies revealed that ephrin-B1 induced migration and proliferation of HUVECs. CONCLUSIONS Ephrin-B1 may be involved in in vivo tumor progression by promoting neovascularization in HCC.

Regorafenib as Second-Line Systemic Therapy May Change the Treatment Strategy and Management Paradigm for Hepatocellular Carcinoma

At the European Society of Medical Oncology World Congress of Gastrointestinal Cancer held in Barcelona, Spain, on 30th June 2016, positive outcomes were reported by the Study of Regorafenib after Sorafenib in Patients with Hepatocellular Carcinoma (RESORCE) trial, which investigated the efficacy of regorafenib as second-line therapy after sorafenib failure [1]. In this clinical trial, the group who received regorafenib achieved a survival benefit of approximately 2.8 months compared to the placebo group. Overall survival (OS) was 10.6 months in the regorafenib arm compared with 7.8 months in the placebo arm, with a hazard ratio (HR) of 0.62 (95% confidence interval [CI]: 0.50–0.78; p<0.001). These are groundbreaking results. The positive outcome achieved by this second-line systemic therapy is a major development, especially after the numerous reports of failures in clinical studies of first-and second-line systemic therapeutic agents (table ​(table1).1). Regorafenib therapy is expected to significantly prolong life expectancy by approximately 2.8 months in patients with hepatocellular carcinoma (HCC) who develop progressive disease (PD) during sorafenib therapy. This development will certainly lead to drastic changes in the treatment strategy and management paradigm for HCC. Table 1 Phase III Clinical Trials of Japanese Participation for HCC Design of the RESORCE Trial The RESORCE trial enrolled 573 patients with advanced HCC corresponding to Barcelona Clinic Liver Cancer (BCLC) stage B or C who were unresponsive to sorafenib. The patients were divided into placebo and regorafenib arms at a 1:2 ratio for the daily administration of placebo and oral regorafenib (160 mg), respectively, for three weeks on and one week off (four weeks/cycle) (fig. ​(fig.1).1). Geographic region, performance status on the Eastern Cooperative Oncology Group scale, α-fetoprotein level (≥400 or <400 ng/mL), macrovascular invasion, and extrahepatic disease were used as allocation factors. This study excluded patients who were intolerant of sorafenib and who discontinued the treatment because of side effects. It enrolled only those patients who discontinued sorafenib because of evidence of PD on imaging studies. In addition, patients were included only if they had received ≥400 mg sorafenib for at least 20 of 28 days immediately prior to radiologically detected PD. In other words, this trial was designed (1) to ensure regorafenib tolerance among patients, and to reduce the occurrence of the drug-specific skin symptoms because the compound is structurally similar to sorafenib [2,3] (fig. ​(fig.2)2) and (2) to reduce the effect of post-trial treatment on OS in both the placebo and treatment arms by using a homogeneous group of patients who developed PD due to sorafenib failure. Fig. 1 Design of the RESORCE Trial. ECOG PS=Eastern Cooperative Oncology Group Performance Status; RECIST=Response Evaluation Criteria in Solid Tumors. Fig. 2 Chemical structure of Regorafenib is very similar to that of Sorafenib. In general, post-progression survival (PPS) is defined as the time interval between the diagnosis of PD after primary treatment and the patients death, and OS is the sum of PPS and progression-free survival (PFS). Therefore, even significant differences in PFS can be canceled out because PPS is prolonged. Indeed, OS showed a stronger correlation with PPS than with PFS in a clinical trial of sorafenib [4]. Because HCC responds extremely well to locoregional therapy, it is often used as post-trial treatment even in cases in which locoregional therapy is no longer applicable and molecular targeted agents are subsequently administered in accordance with the protocol, provided that the patients general condition is stable. This rarely happens with other types of cancer and is therefore essentially unique to HCC, owing to the availability of powerful locoregional therapies such as intra-arterial infusion chemotherapy [5, 6, 7], transcatheter arterial chemoembolization (TACE) [8, 9], and radiofrequency ablation [10, 11, 12]. These post-trial treatments are capable of canceling out any difference in the primary endpoint OS by prolonging PPS [13]. Indeed, previous clinical trials of second-line agents other than regorafenib have always included patients intolerant to sorafenib, which may have increased the influence of post-trial treatment and thus contributed to their negative outcomes. Patients unresponsive to sorafenib are those who develop PD during sorafenib therapy and are likely to have relatively poor hepatic function and overall general condition. By contrast, patients intolerant to sorafenib are those who discontinue the treatment because of side effects; these patients are in relatively stable conditions because of negligible amounts of internalized sorafenib, and a lack of HCC progression. Because of their clinical stability, patients intolerant to sorafenib are inevitably treated by locoregional therapy or various other post-trial treatments, including the re-administration of sorafenib, regardless of whether they received an actual second-line agent or placebo during the trial. With this in mind, clinical trials of second-line agents should enroll only patients who are unresponsive to sorafenib [14]. The RESORCE trial was the first clinical study to reflect this point in the trial design (fig. ​(fig.1).1). The benefit of excluding patients intolerant to sorafenib was demonstrated in the subanalysis of a previous phase II study of axitinib, which generated an excellent HR and a significant study outcome [15, 16]. The second noteworthy point in the design of the RESORCE trial is that the allocation factors of macrovascular invasion and extrahepatic disease were treated as independent stratification factors. In general, the designs of previous clinical trials of molecular targeted agents involved allocation factors specifying “vascular invasion and/or extrahepatic spread” or “neither.” However, because vascular invasion is an extremely poor prognostic factor for HCC, assigning vascular invasion to the same category as extrahepatic spread may have influenced the outcome of these clinical trials. For example, when the treatment group contains more patients with vascular invasion but the placebo group includes more patients with extrahepatic spread, such sampling bias will put the treatment group at a significant disadvantage. In fact, such allocation imbalance apparently contributed to a negative outcome in a clinical trial of brivanib as second-line therapy [17] (table ​(table22). Table 2 Imbalance between Brivanib and Placebo Arm in BRISK-PS Trial The design of the RESORCE trial is excellent because it reflects what was learned from the negative outcomes of past trials and the reasons for those outcomes.

Serum Fucosylated Haptoglobin as a Novel Diagnostic Biomarker for Predicting Hepatocyte Ballooning and Nonalcoholic Steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is a growing medical problem around the world. NAFLD patients with nonalcoholic steatohepatitis (NASH) can develop cirrhosis and hepatocellular carcinoma. The ability to distinguish NASH from simple steatosis would be of great clinical significance. Ballooning hepatocytes are characteristic of typical pathological NASH; here, the polarized secretion of proteins is disrupted due to destruction of the cytoskeleton. We previously reported that fucosylated glycoproteins are secreted into bile, but not into sera in normal liver. Therefore, we hypothesized that the fucosylation-based sorting machinery would be disrupted in ballooning hepatocytes, and serum fucosylated glycoproteins would increase in NASH patients. To confirm our hypothesis, we evaluated serum fucosylated haptoglobin (Fuc-Hpt) levels in biopsy-proven NAFLD patients (n = 126) using a lectin-antibody ELISA kit. Fuc-Hpt levels were significantly increased in NASH patients compared with non-NASH (NAFLD patients without NASH) patients. Interestingly, Fuc-Hpt levels showed a significant stepwise increase with increasing hepatocyte ballooning scores. Multiple logistic regression analysis showed that Fuc-Hpt levels were independent and significant determinants of the presence of ballooning hepatocytes. Moreover, Fuc-Hpt levels were useful in monitoring liver fibrosis staging. Next, to investigate the significance of serum Fuc-Hpt in a larger population, we measured Fuc-Hpt levels in ultrasound-diagnosed NAFLD subjects (n = 870) who received a medical health checkup. To evaluate NAFLD disease severity, we used the FIB-4 index (based on age, serum AST and ALT levels, and platelet counts). Fuc-Hpt levels increased stepwise with increasing FIB-4 index. Conclusion Measurement of serum Fuc-Hpt levels can distinguish NASH from non-NASH patients, and predict the presence of ballooning hepatocytes in NAFLD patients with sufficient accuracy. These results support the potential usefulness of measuring Fuc-Hpt levels in clinical practice.

Usefulness of the Multimodality Fusion Imaging for the Diagnosis and Treatment of Hepatocellular Carcinoma

A multimodality fusion imaging system has been introduced for the clinical practice of diagnosis and treatment of hepatocellular carcinoma (HCC), especially for loco-regional treatment. An ultrasonography (US) fusion imaging system can provide a side-by-side display of real-time US images and any cross-sectional images of multiplanar reconstruction of CT or MRI that synchronize real-time US. The US fusion imaging system enables us to perform radiofrequency ablation (RFA) for HCCs difficult to detect on conventional US safely. Besides, we can evaluate the treatment effects of RFA easily at the bedside by combining the contrast-enhanced US and the US fusion imaging system. Fusion images of pre- and post-RFA CT have been utilized for the assessment of the treatment effects of RFA. Although the treatment effects of RFA have been conventionally evaluated, comparing pre- and post-RFA CT side-by-side, the evaluation tends to be inaccurate. On CT fusion images, the tumor and the ablation zone are overlaid and we can grasp the positional relation easily, leading to quantitative and more accurate evaluation. The multimodality fusion imaging system has become quite an important tool for loco-regional treatment of HCC because of its usefulness for both the guidance during the RFA procedure and the evaluation of its treatment effects.

Ultrasonography fusion imaging system increases the chance of radiofrequency ablation for hepatocellular carcinoma with poor conspicuity on conventional ultrasonography.

Objectives: To investigate the usefulness of the ultrasonography (US) fusion imaging system for radiofrequency ablation (RFA) for hepatocellular carcinoma (HCC). Methods: Since the US fusion imaging system became available in 2010, we have conducted RFA with this system in all cases. The characteristics of 75 patients with 120 HCCs and 89 patients with 123 HCCs who underwent RFA before the introduction of this system (period A) and after it (period B), respectively, were retrospectively compared. Results: Significant difference in the characteristics of the patients and HCCs between the two periods was found only in the proportion of HCCs with poor conspicuity on grayscale US treated with RFA (1.7%, 2/120 for period A vs. 15.4%, 19/123 for period B, p < 0.01). Among the 19 HCCs with poor conspicuity on grayscale US for period B, 5 and 9 HCCs were identified on grayscale US and contrast-enhanced US, respectively, by the use of the US fusion imaging system, whereas the 5 remaining undetectable HCCs were treated by using the system in conjunction with reference images displayed side-by-side with grayscale US. Conclusion: Since the introduction of the US fusion imaging system, it has become possible to perform RFA for HCCs with poor conspicuity on grayscale US.

Clinicopathological characteristics of serum amyloid A-positive hepatocellular neoplasms/nodules arising in alcoholic cirrhosis

To characterize serum amyloid A (SAA)‐positive hepatocellular neoplasms/nodules arising in alcoholic cirrhosis, which are detected as hypervascular hepatocellular nodules resembling hepatocellular carcinoma on imaging.

Hypervascular hepatocellular carcinoma : Combined dynamic MDCT and SPIO-enhanced MRI versus combined CTHA and CTAP

Aim:  Recently, many diagnostic modalities have been developed for the detection of hepatocellular carcinoma (HCC). Of these, a less invasive and more accurate diagnostic procedure is desirable. This study was undertaken to compare combined dynamic multidetector row helical computerized tomography (MDCT) and superparamagnetic iron oxide (SPIO)‐enhanced magnetic resonance imaging (MRI) with combined CT hepatic arteriography (CTHA) and CT during arterial portography (CTAP) for the detection of hypervascular HCC.