Eiji Tsujita

Laparoscopic hepatectomy for hepatocellular carcinoma

S. Maehara Background: No reports exist on the role of laparoscopic hepatectomy in the short- and long-term outcomes of patients with hepatocellular carcinoma (HCC). We present our results from using laparoscopic hepatectomy for HCC and discuss the importance of this procedure. Methods: To investigate the role of laparoscopic hepatectomy in the short- and long-term outcomes, 17 patients with HCC who underwent laparoscopic hepatectomy (laparoscopic hepatectomy group) were compared with 38 patients who underwent conventional open hepatectomy (open hepatectomy group) during the same period. Results: No differences in operation time, blood loss, rate of blood transfusion, or incidence of postoperative complications were found between the two groups. The postoperative hospital stay for the laparoscopic hepatectomy group was significantly shorter than for the open hepatectomy group. With long-term prognosis, no difference was found in survival rate and disease-free survival rate between the two groups. No recurrence was found in the stump of the remaining liver after laparoscopic hepatectomy. Conclusions: Laparoscopic hepatectomy has resulted in a better short-term outcome after surgery than conventional open hepatectomy. The long-term prognosis in the laparoscopic hepatectomy group was similar to that in the open hepatectomy group. Therefore, laparoscopic hepatectomy can be a new alternative for treatment of cirrhotic patients with HCC when patients are strictly selected.

Preoperative neutrophil-to-lymphocyte ratio is a predictor of survival after hepatectomy for hepatocellular carcinoma: a retrospective analysis.

Objective: To clarify the prognostic value of the preoperative blood neutrophil-to-lymphocyte ratio (NLR) in patients undergoing hepatectomy for hepatocellular carcinoma (HCC). Background: Although a high NLR has been reported to be a predictor of poor survival in patients with various cancers, it has not been extensively examined in patients with HCC. Methods: This retrospective study enrolled 958 patients who underwent hepatectomy without preoperative therapy for HCC from 1996 to 2009. Clinicopathological parameters, including NLR, were evaluated to identify predictors of overall and recurrence-free survival after hepatectomy. Univariate and multivariate analyses were performed, using the Cox proportional hazards model. The best cutoff was determined with time-dependent receiver operating characteristic curve. To determine the mechanism of NLR elevation, immunohistological examination using CD163 staining was performed in 150 patients. Results: Univariate and multivariate analyses showed that NLR was an independent prognostic factor in overall and recurrence-free survival. The best cutoff of NLR was 2.81, and 238 of 958 patients (24.8%) had NLR of more than 2.81. The 5-year survival rate after hepatectomy was 72.9% in patients with NLR less than 2.81 and 51.5% in those with NLR 2.81 or more (P < 0.0001). CD163-positive cell counts were significantly higher in tumors in the group with NLR 2.81 or more than in the group with NLR less than 2.81 (P = 0.0004). Conclusions: Our results show that NLR is an independent predictor of survival after hepatectomy in patients with HCC. Accumulation of tumor-associated macrophages in the tumor is associated with a high NLR.

In vivo gene transfer into muscle via electro-sonoporation

Among the nonviral techniques for gene transfer in vivo, electroporation is simple, potent, inexpensive, and safe. To upregulate the expression levels of the transferred gene, we investigated the applicability of in vivo electro-sonoporation, which consists of a combination of electric pulse and ultrasound, for gene transfer using plasmid DNA encoding luciferase and mouse interleukin-12 (mIL-12). The quadriceps muscles of mice were injected with plasmid DNA, then sonoporated for 5 min, and electroporated by a pair of electrode plates at the middle of the duration of sonoporation. Three days later, mice that had undergone electro-sonoporation demonstrated twofold higher luciferase activity and low tissue damage in quadriceps muscle compared to mice having undergone electroporation alone. Serum mIL-12 levels in mice that had undergone electro-sonoporation (peaking at 25.5 ng/ml) were twofold higher after gene transfer than were those in mice having undergone electroporation alone (peaking at 14.3 ng/ml), and maintained high serum level of 13.9 ng/ml at 28 days after gene transfer. The efficacy of gene transfer via electro-sonoporation was superior when the plasmid DNA solution was 0.85% NaCl compared to albumin microbubble echo-contrast material. These results demonstrated that gene transfer into muscle via electro-sonoporation could provide a new potent nonviral technique for gene transfer in vivo.

Efficacy of a polyurethane foam/spheroid artificial liver by using human hepatoblastoma cell line (Hep G2)

We investigated the availability of human hepatoblastoma cell line (Hep G2), compared with human primary hepatocytes (HH) and porcine primary hepatocytes (PH), as a cell source for the hybrid artificial liver support system (HALSS) by using polyurethane foam (PUF). All three kinds of hepatocytes spontaneously formed spherical multicellular aggregates (spheroids) of 100–200 μm diameter in the pores of PUF within 3 days of culture. In a PUF stationary culture, Hep G2 spheroids recovered the ammonia removal activity that was lost in monolayer culture, although the removal for each unit cell number was about one tenth that of HH spheroids and about one eighth of PH spheroids. The synthesis activities of albumin and fibrinogen of each unit cell number of Hep G2 were also upregulated by PUF spheroid culture, and were about twice as high as in monolayer culture. The albumin secretion activity of Hep G2 spheroids was almost the same as that of PH spheroids. HH scarcely secreted these proteins in this experiment, probably because they were cultured in a serum-free medium. In the PUF module in a circulation culture, HH had high ammonia removal and low synthesis activities similar to stationary culture. Hep G2 proliferated to a high cell density, such as about 4.8 × 107 cells/cm3-module at 10 days of culture. Although Hep G2 spheroids had low ammonia removal activity in each cell, the removal rate in the PUF module was almost the same as for PH at 7 days of culture because of the high cell density culture by cell proliferation. The albumin secretion rate by Hep G2 in the PUF module also increased with cell proliferation and was about 10 times higher than the initial rate for PH at 7 days of culture. These results suggest that Hep G2 is a potential cell source for the PUF-HALSS.

Suppressed MKP-1 Is an Independent Predictor of Outcome in Patients with Hepatocellular Carcinoma

Objective: An increase in the activity of mitogen-activated protein kinases (MAPKs) has been correlated with a more malignant phenotype in several tumor models in vivo. This study was designed to clarify the expression of MKP-1 in surgically resected hepatocellular carcinoma (HCC). Methods: We reviewed the cases of 77 patients who had undergone initial liver resection for HCC without preoperative treatment. Immunohistochemical analysis of MKP-1 was performed on paraffin-embedded tissues. The correlation between MKP-1 expression and clinical outcome was investigated. Results: Tumor cells were immunohistochemically stained for MKP-1 expression, and the same levels as in normal hepatocytes were detected in 66 (85%) of 77 HCC patients, being decreased in 11 (15%) HCCs. Decreased MKP-1 expression significantly correlated with serum α-fetoprotein levels and tumor size (p < 0.05). The disease-free survival rates in MKP-1-negative and -positive patients were 0 and 31.0% at 5 years, respectively (p < 0.01). The survival rates after a surgical resection in MKP-1-negative and -positive patients were 18.2 and 65.5% at 5 years, respectively (p < 0.01). Conclusions: The MKP-1 expression in HCC was an independent prognostic factor for outcome in HCC patients. In the future, it will be useful to explore whether the phosphatase expression might account for the response to HCC treatments targeting at MAPK activation.

Polyurethane foam/spheroid culture system using human hepatoblastoma cell line (Hep G2) as a possible new hybrid artificial liver.

The risk of xenozoonosis infections poses the greatest obstacle against the clinical application of hybrid artificial liver support system (HALSS). Primary human hepatocytes are an ideal source for HALSS, but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, we used human hepatocytes with replication capacity (fetal hepatocytes, Hep G2, and Huh 7) in a polyurethane foam (PUF)/spheroid culture system in vitro, and analyzed liver functions such as ammonia removal and albumin synthesis capacity; results were compared to those of porcine hepatocytes. Human fetal hepatocytes, Hep G2, and Huh 7 formed spheroids spontaneously within 24 h in a PUF/spheroid culture system; ammonia removal activity (μmol/106 nuclei/h) was upregulated, as was albumin synthesis activity (μg/106 nuclei/day). In particular, Hep G2 spheroids demonstrated high ammonia removal and albumin synthesis activities: 85% of the ammonia removal activity and 171.7% of the albumin synthesis activity of porcine hepatocytes in the monolayer culture. These results indicate the possibility of the development of a multicapillary PUF (MC-PUF) packed-bed culture system of hepatocyte spheroids as a HALSS using Hep G2.

Outcome of repeat hepatectomy in patients with hepatocellular carcinoma aged 75 years and older.

BACKGROUND We sought to evaluate the influence of age on the outcome of repeat hepatectomies in patients > or = 75 years with recurrent hepatocellular carcinoma (HCC). METHODS We studied 121 curative repeat hepatectomies retrospectively. Among the 121 patients, 100, 20, and 1 received second, third, and fourth hepatectomies, respectively. The short-term surgical results of a younger group (<75 years; n = 88) and those of an elderly group (> or = 75 years; n = 33) were compared. The long-term prognosis of the patients who underwent second hepatectomies was also compared between a younger group (<75 years; n = 77) and an elderly group (> or = 75 years; n = 23). RESULTS The patients in the elderly group displayed more comorbid conditions pre-operatively, including hypertension and cardiovascular diseases, than the younger group (P < .05); however, there was no significant difference in the incidence of postoperative complications or the duration of postoperative hospital stay. The long-term prognosis in the elderly group was almost identical to that in the younger group. The 3-year overall survival rates for the younger group and the elderly group were 83 vs 73% (P = .51). Disease-free, 3-year survival rates for the younger group and the elderly group were 35% vs 38% (P = .88). CONCLUSION Our findings suggest that advanced age by itself does not have an adverse effect on operative outcomes, including postoperative complications and long-term prognosis. Repeat hepatectomy may, therefore, be justified for recurrent HCC in selected elderly patients.

Plasma D-dimer level as a mortality predictor in patients with advanced or recurrent colorectal cancer.

Objective: Plasma D-dimer levels are elevated in patients with a variety of solid tumors. Recently, it has been reported that the level before curative surgery is a prognostic factor for colorectal cancer (CRC). We investigated whether the plasma D-dimer level before systemic chemotherapies is a predictor for advanced or recurrent unresectable CRC. Methods: This study included 42 patients treated with systemic chemotherapies for advanced or recurrent unresectable CRC. Variables including clinicopathological factors, plasma D-dimer levels and the modified Glasgow Prognostic Factor Score (mGPS) were evaluated. Results: The plasma D-dimer level was closely related to the mGPS. Survival was shorter for patients with plasma D-dimer levels >5 µg/ml than for those with lower levels. Compared with an mGPS of 0 or 1, an mGPS of 2 was predictive of poor prognosis (p < 0.0001). Old age, advanced stage, plasma D-dimer level and mGPS were significantly associated with mortality, but plasma D-dimer level was the only independent risk factor in multivariate analysis, and was significant related to the clinical response to chemotherapy (p < 0.05). Conclusions: Survival was significantly shorter in patients with elevated plasma D-dimer levels having advanced or recurrent CRC. The plasma D-dimer level before systemic chemotherapies was an independent mortality predictor.

Mass preparation of primary porcine hepatocytes and the design of a hybrid artificial liver module using spheroid culture for a clinical trial.

To isolate a large number of porcine hepatocytes, we originally developed a mass preparation method that combined the usual collagenase perfusion method of a whole liver with a collagenase redigestion method of tissue fragments after liver perfusion. Using a pig of 10kg, collagenase perfusion only resulted in a yield of 63 ± 78 x 108 total cells with a viability of 69.2 ± 25.3 %, but our combined method had a yield of 167 ± 31 x 108 total cells with a viability of 87.9 ± 4.4 % (mean ± SD). Also, the combined method was applied to two pigs of 10kg body weight at the same time, and isolated 387 ± 89 x 108 hepatocytes with a viability of 87.1 ± 6.9 % and a purity of 93.6 ± 2.8 % in 11 experiments. We designed a large multi-capillary polyurethane foam (MC-PUF) packed-bed module containing 1 x 1010 porcine hepatocytes on a clinical trial scale. The porcine hepatocytes in the module formed spherical multicellular aggregates (spheroids) of 200 – 500 μm diameter. Most hepatocytes forming spheroids were viable judged by fluorescein diacetate and ethidium bromide staining. The activities of ammonia removal, albumin secretion and oxygen consumption of the large MC-PUF module were the same as for a small MC-PUF module containing 2 x 108 porcine hepatocytes, and were maintained for at least 9 days of culture. These results show that a large MC-PUF module is successfully scaled up 50 times. In conclusion, we succeeded in developing a mass preparation method of porcine hepatocytes and a large hybrid artificial liver module on a clinical trial scale.

Prospective randomized controlled trial investigating the type of sutures used during hepatectomy

AIM To determine whether absorbable sutures or non-absorbable sutures are better in preventing surgical site infection (SSI), in this paper we discuss the results of a randomized clinical trial which examined the type of sutures used during hepatectomy. METHODS All hepatic resections performed from January 2007 to November 2008 at the Department of Surgery at Iizuka Hospital in Japan were included in this study. There were 125 patients randomly assigned to an absorbable sutures (Vicryl) group or non-absorbable sutures (Silk) group. RESULTS SSI was observed in 13.6% (17/125) patients participating in this study, 11.3% in the Vicryl group and 15.8% in the Silk group. Incisional SSI including superficial and deep SSI, was observed in 8% of the Vicryl group and 9.5% of the Silk group. Organ/space SSI was observed in 3.2% of the Vicryl group and 6.0% of the Silk group. There were no significant differences, but among the patients with SSI, the period for recovery was significantly shorter for the Vicryl group compared to the Silk group. CONCLUSION The incidence of SSI in patients receiving absorbable sutures and silk sutures is not significantly different in this randomized controlled study; however, the period for recovery in patients with SSI was significantly shorter for absorbable sutures.