TU-100 Antagonizes the M2 Polarization Phenotype of Macrophages in the Tumor Microenvironment by Suppressing the TLR4/NF-B/STAT3 Axis.

BACKGROUND/AIM: Macrophages are the most abundant immune cells in the tumor stroma, and their polarization states within the tumor microenvironment (TME) exert critical roles in tumorigenesis. TU-100 (Daikenchuto) is a commonly prescribed Japanese herbal medicine that has shown anti-cancer effects by regulating cancer-associated fibroblasts (CAFs) in the TME. However, its effects on tumor-associated macrophages (TAMs) remain unclear. MATERIALS AND METHODS: TAMs were generated by macrophage exposure to tumor-conditioned medium (CM), and their polarization states were evaluated after TU-100 treatment. The underlying mechanism was further studied. RESULTS: TU-100 exhibited little cytotoxicity over a range of doses in M0 macrophages and TAMs. However, it could antagonize the M2-like polarization of macrophages evoked by tumor-CM exposure. These effects might be caused by the inhibition of TLR4/NF-B/STAT3 signaling in the M2-like phenotype of macrophages. Interestingly, TU-100 antagonized the malignancy promoting effects of M2 macrophages on hepatocellular carcinoma cell lines in vitro. Mechanistically, the administration of TU-100 restrained the high expression of MMP-2, COX-2, and VEGF in TAMs. CONCLUSION: TU-100 may alleviate the progression of cancer by regulating the M2 polarization of macrophages within the TME, suggesting a viable therapeutic approach.

Frailty Can Predict Prognosis After Hepatectomy in Patients With Colorectal Liver Metastasis.

BACKGROUND/AIM: The aim of this study was to investigate frailty as a prognostic factor in patients with colorectal liver metastasis undergoing hepatectomy. PATIENTS AND METHODS: Eighty-seven patients who underwent hepatectomy at our institution were enrolled. Frailty was defined as a score of ≥4 on a clinical frailty scale. Patients were divided into frailty (n=29) and non-frailty (n=58) groups. RESULTS: Overall and cancer-specific survival rates were significantly worse in the frailty group compared with the non-frailty group, and multivariate analysis revealed frailty as an independent prognostic factor. Disease-free survival tended to be worse in the frailty group. Fifty-eight patients relapsed after the first hepatectomy. Twenty-one of 58 recurrent patients were allocated to the frailty group. After recurrence, chemotherapy was significantly more frequently performed in the non-frailty group compared with the frailty group. CONCLUSION: Frailty can predict the prognosis of patients with colorectal liver metastasis undergoing hepatectomy.

Bile Metabolites and Risk of Carcinogenesis in Patients With Pancreaticobiliary Maljunction: A Pilot Study.

BACKGROUND/AIM: Pancreaticobiliary maljunction (PBM), a disease with reflux of pancreatic and bile juice in the pancreaticobiliary tract, is a high-risk factor for biliary tract cancer. The aim of this study was to investigate the mechanism of carcinogenesis in PBM using a metabolomics analysis of bile sampled during surgery. PATIENTS AND METHODS: Three patients with PBM without biliary tract cancer, four patients with extrahepatic bile duct cancer (EHBC), and three controls with benign disease were enrolled. Metabolomics analysis of bile samples was performed using capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry to discriminate the amino acid and lipidomic profiles. RESULTS: The principal component analysis in the capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry revealed similar metabolites in patients with PBM and those with EHBC; furthermore, there was a clear difference between patients with PBM or EHBC compared to controls. The amino acid profiles revealed the following 20 potential carcinogenic candidates for PBM: isoleucine, phenylalanine, tyrosine, leucine, tryptophan, arginine, lysine, valine, asparagine, methionine, aspartic acid, serine, threonine, histidine, glutamine, alanine, proline, glutamic acid, and pyruvic acid. The lipidomic profiles revealed the following 11 carcinogenic candidates: lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidyl glycerol, lysophosphatidyl glycerol, triacylglycerol, diacylglycerol, ceramide, sphyngomyeline, fatty acid, hyperforin, and vitamin D. Among these characteristic metabolites, the branched-chain amino acids, methionine and lysophosphatidylcholine are known to be related to carcinogenesis. CONCLUSION: The bile metabolites were extremely similar in patients with PBM and those with EHBC. Furthermore, amino acid and lipid metabolism was markedly different in patients with PBM or EHBC compared to healthy controls.

Hepatic Stellate Cells Contribute to the Tumor Malignancy of Hepatocellular Carcinoma Through the IL-6 Pathway.

BACKGROUND/AIM: The hepatic stellate cells (HSCs) have relationship to cancer progression. The aim of this study is to investigate the effect of HSCs and the role of IL-6/Stat3 pathway on hepatocellular carcinoma (HCC) progression. MATERIALS AND METHODS: HCCs were co-cultured with HSCs. The viability and migration ability of cancer cells were detected. Epithelial-mesenchymal transition (EMT) marker (E-cadherin), stem cell marker (CD44) and p-signal transducer and activator of transcription 3 (p-STAT3) of cancer cells were evaluated. Finally, interleukin-6 (IL-6) neutralization was performed. RESULTS: Co-culture of HCCs with HSCs increased cancer cell viability and migration ability. EMT and stemness of cancer cells increased with HSCs. Following IL-6 neutralization, phospho-STAT3 activation, cancer cell viability and migration, as well as EMT, and stemness of cancer cells decreased. CONCLUSION: HSCs promoted HCC progression through the IL-6/STAT3 pathway.

Carbohydrate Antigen 19-9 Is a Prognostic Factor Which Correlates With HDAC1 and HIF-1α for Intrahepatic Cholangiocarcinoma.

BACKGROUND/AIM: Carbohydrate antigen 19-9 (CA19-9) is a poor prognostic marker in intrahepatic cholangiocarcinoma (IHCC). Previous studies have shown a link between hypoxia and CA19-9 in cancer, and we have previously demonstrated a correlation between HDAC1 and HIF-1α in IHCC. Here, we evaluated the expression and correlation of CA19-9 with HIF-1 and HDAC in IHCC. PATIENTS AND METHODS: This study included 62 patients with IHCC who underwent primary hepatectomy at our department. Clinicopathological characteristics were examined. Immunohistochemical expression of HIF-1 and HDAC1 in specimens was quantitatively evaluated. RESULTS: Patients with high preoperative serum CA19-9 levels showed clinicopathological characteristics associated with tumour progression. High CA19-9 levels were associated with worse overall and recurrence-free survival. Univariate and multivariate analysis detected high CA19-9 levels as an independent poor prognostic factor for IHCC. Serum CA19-9 was significantly correlated with both HIF-1α and HDAC1 expression. CONCLUSION: High serum CA19-9 level is a poor prognostic factor for overall survival in IHCC and correlates with HIF-1α and HDAC1 expression.

Impact of Bevacizumab on Liver Damage After Massive Hepatectomy in Rats.

BACKGROUND: The aim of this study was to evaluate the impact of pretreatment with bevacizumab on liver damage in a rat model of massive hepatectomy (Hx) model, as a surrogate model of massive Hx for liver metastasis from colorectal cancer. MATERIALS AND METHODS: Male Wister rats (n=24) were separated into the following two groups: 90% Hx and 90% Hx plus bevacizumab group. Bevacizumab (5 mg/kg) was injected intraperitoneally 7 days before Hx. Samples of blood and remnant liver tissue were obtained 24 hours after hepatectomy and the following parameters were evaluated: Biochemical analysis; liver regeneration rate; survival rate; and real-time polymerase chain reaction for interleukin-1 beta (Il1b), tumor necrosis factor alpha (Tnfa), matrix metalloproteinase (Mmp) 2 and Mmp9 mRNA. In addition, samples of whole liver tissue were obtained immediately before Hx and real-time polymerase chain reaction was performed for X-box binding protein 1 (Xbp1), activating transcription factor 6 (Atf6), C/EBP homologous protein (Chop), glucose-regulated protein 78 (Grp78) and heat-shock protein 70 (Hsp70), as markers of endoplasmic reticulum stress response. RESULTS: The levels of transaminases 24 hours after Hx were significantly reduced in the group pretreated with bevacizumab compared to that not pretreated (p<0.05). The liver regeneration rate at 24 hours after Hx was significantly increased in the group pretreated with bevacizumab compared with the group which underwent Hx alone (p<0.05). The survival rate for the group pretreated with bevacizumab tended to be higher than that of the Hx-only group, 72 hours after Hx (p=0.09). The expressions of Il1b, Mmp2 and Mmp9 mRNA 24 hours after Hx in the group pretreated with bevacizumab tended to be lower than that of rats which underwent Hx alone (p=0.11, 0.09 and 0.15, respectively). The expression of Xbp1, Chop, Grp78 and Hsp70 mRNA immediately before Hx in the group pretreated with bevacizumab were significantly higher than the 90% Hx group (p<0.05). CONCLUSION: Bevacizumab pretreatment had protective effects on liver injury after massive hepatectomy in rats, apparently via the induction of the endoplasmic reticulum stress response, i.e. the so-called unfolded protein response.

Clinical Impact of FOLFOXIRI Aiming for Conversion Surgery in Unresectable Multiple Colorectal Liver Metastasis.

BACKGROUND/AIM: We evaluated the clinical impact of FOLFOXIRI regimen aiming for conversion surgery in patients with unresectable multiple colorectal liver metastasis (CRLM). PATIENTS AND METHODS: A total of 42 patients with unresectable multiple CRLM who received chemotherapy with molecular agents were included in the analysis. The clinical results of FOLFOXIRI with other regimens were compared. RESULTS: The total conversion rate of 42 unresectable CRLM was 48.1%, and conversion cases had a better prognosis. Clinicopathological characteristics of conversion cases were more frequent in FOLFOXIRI induction, liver limited disease and maximum diameter × number (MDN) over 70. FOLFOXIRI achieved a higher conversion rate compared to other regimens (72.2% vs. 37.5%, p=0.0334), and significantly reduced the medication period until conversion surgery (median 5.8 courses) with a higher tumour necrotic rate. Consequently, the overall survival of conversion cases with FOLFOXIRI was better than that with other regimens (p=0.0055). CONCLUSION: FOLFOXIRI plus molecular agents might provide a higher probability of conversion surgery with a prognostic benefit.

The Outcome of Sorafenib Therapy on Unresectable Hepatocellular Carcinoma: Experience of Conversion and Salvage Hepatectomy.

BACKGROUND/AIM: We report the outcomes of sorafenib therapy for advanced hepatocellular carcinoma (HCC) in our Department. PATIENTS AND METHODS: Thirty-eight patients with unresectable HCC who were administrated sorafenib from 2009 to 2015 were investigated retrospectively. RESULTS: The 1-year overall survival rate was 59.3%. The macroscopic vascular invasion and response rate were independent prognostic factors of survival. Surgical resection after sorafenib achieved long-term survival in two cases. Case 1: A patient with locally unresectable HCC showed significant response induced by sorafenib, which allowed complete surgical resection. This tumor tested positive for FGF4. Case 2: A patient with a history of hepatectomy for HCC had multiple distant metastases. Most lesions were reduced in size after sorafenib therapy and new lesions in the remnant liver and residual lung metastases were resected. The sorafenib-resistant lesions were negative for FGF4. CONCLUSION: Sorafenib combined with surgical resection is a feasible option in advanced HCC patients, if sorafenib has been effective.

Effect of Adjuvant Gemcitabine Combined with Low-dose 5-Fluorouracil and Cisplatin Chemotherapy for Advanced Biliary Carcinoma.

BACKGROUND/AIM: The aim of this retrospective study was to clarify the effectiveness of chemotherapy with gemcitabine combined with low-dose 5-fluorouracil and cisplatin (GFP) for advanced biliary carcinoma after hepatectomy. PATIENTS AND METHODS: Sixty-two patients had biliary carcinoma with lymph node metastasis, intrahepatic metastasis or positive surgical margins, including intrahepatic cholangiocarcinoma (IHC, n=25), hilar cholangiocarcinoma (HC, n=14), and gallbladder cancer (GBC, n=23). Twenty-eight patients (IHC; n=9, HC; n=8, GBC; n=11) received adjuvant GFP chemotherapy. RESULTS: We found no significant difference in clinicopathological factors in patients treated with or without adjuvant GFP chemotherapy. Overall, survival in the adjuvant GFP group was significantly better than that in the non-adjuvant GFP group (3-year survival: 61.9% vs. 8.8%, p<0.001), as was relapse-free survival. CONCLUSION: Adjuvant GFP chemotherapy after hepatectomy may be a promising option for improving surgical outcomes in patients with advanced biliary carcinoma.

Peripheral Tr1 and Foxp3+ Treg as Markers of Recurrent Malignancies in Patients with Hepato-Biliary Pancreatic Cancers.

BACKGROUND/AIM: Recently CD4+CD49b+ LAG3+regulatory T (Tr1) cells are reported to be IL-10 driven, have strong regulatory activities. Thus, this study aimed to investigate whether Treg and Tr1 cells participate in immunological status against cancer. PATIENTS AND METHODS: Peripheral blood was withdrawn from patients (n=78), and healthy volunteers as controls (n=23). The peripheral blood mononuclear cells were subjected to FACScan analysis after labeling with anti-CD4, -CD25, - Foxp3, -CD49b and -LAG3 antibodies. Resected specimens were stained for IL-10. Patients' clinical course and clinicopathological factors were compared. RESULTS: Tr1 was significantly higher in patients with cancer (p=0.02). Among patients who underwent R0 resection, those with early recurrence had a significantly higher pre-/post-operative Tr1 ratio (p<0.05). Median pre-/post-operative ratios of Foxp3Tregs and Tr1s predicted early recurrence with 85.6% sensitivity and 93.3% specificity. Disease-free survival was significantly lower in patients with high IL-10 expression in resected specimens. CONCLUSION: Peripheral Tregs and Tr1 indicate immunological state against cancer.

Accurate Estimation of Functional Liver Volume Using Gd-EOB-DTPA MRI Compared to MDCT/99mTc-SPECT Fusion Imaging.

BACKGROUND/AIM: We assessed the utility of dynamic magnetic resonance imaging (MRI) with gadoxetate-ethoxybenzyl-diethylenetriamine penta-aceticpenta-acetic acid (Gd-EOB-DTPA) (EOB-MRI) for estimating functional liver volume compared to 99mTc-galactosyl albumin single-photon-emission computed tomography (99mTc-GSA SPECT). PATIENTS AND METHODS: Regional functional liver volume (left lateral, medial, right anterior, right posterior) of 58 hepatectomized patients was assessed using EOB-MRI and 99mTc-GSA SPECT, and compared to the actual liver volume with MDCT-3D volumetry. RESULTS: 99mTc-GSA SPECT found a significantly lower functional volume of the left lateral section than the actual volume found by MDCT-3D volumetry (p=0.003) and EOB-MRI (p<0.001). Functional liver volume of right anterior section found with 99mTc-GSA SPECT was significantly higher than that found by MDCT-3D volumetry (p=0.04), despite no differences in asialoglycoprotein receptor 1 (ASGR1) or ATP-dependent organic anion transporting polypeptide 1 (OATP) expression between the left lateral and right anterior sections. CONCLUSION: 99mTc-GSA SPECT might underestimate the function of the left lobe and overestimate that of the right lobe. Therefore, EOB-MRI could be better for estimating the true regional functional liver reserve.

CD44 Expression Is a Prognostic Factor in Patients with Intrahepatic Cholangiocarcinoma After Surgical Resection.

BACKGROUND/AIM: Cancer stem cells (CSC) plays an important role in various kinds of cancers. The aim of this study was to clarify the role of CD44 expression in intrahepatic cholangiocarcinoma (IHCC) as a marker of CSCs. MATERIALS AND METHODS: Thirty-five patients with IHCC patients who underwent hepatectomy were evaluated. CD44 expression was determined immunohistochemically. The patients were divided into a CD44-positive group (n=22) or CD44-negative group (n=13). Clinicopathological variables including prognosis were compared between the two groups. RESULTS: The CD44-positive group had a worse prognosis than the CD44-negative group (5-year survival: 19.3% vs. 55.5%, respectively, p=0.016), although no difference in the background variables was observed. In multivariate analysis, CD44-positivity was identified as an independent prognostic factor (hazard ratio=3.676, p=0.034). CONCLUSION: These data suggest that CD44-positivity might be a candidate CSC marker in IHCC and a prognostic indicator.

miR-223 and Stathmin-1 Expression in Non-tumor Liver Tissue of Patients with Hepatocellular Carcinoma.

AIM: Overexpression of stathmin (STMN1) has been reported for several tumor entities. STMN1 expression correlated with the detection of mutant p53, also suggesting loss-of-function-dependent mechanisms for its accumulation in hepatocellular carcinoma (HCC) cells. On the other hand, miR-223 has been identified as one of the most down-regulated miRNAs in HCC, and its expression was shown to be negatively correlated with STMN1 expression. The aim of this study was to investigate the clinical significance of STMN1 and miRNA-223 expression. PATIENTS AND METHODS: Fifty-six consecutive patients with HCC who underwent curative hepatectomy as initial treatment were enrolled. They were divided into two groups based on the STMN1 expression level: high (n=36) and low (n=20) gene-expression groups. We compared the clinicopathological factors between the groups with high and low expression in non-tumor tissues. Thirty out of 56 patients were also divided into groups with high (n=15) and low (n=15) miR-223 expression and compared the clinicopathological factors between the two groups. RESULTS: There were no significant differences in patient background between high and low STMN1 expression groups. The incidence of multicentric (MC) recurrence in the high-expression group was significantly higher than in the low-expression group and high STMN1 expression was shown to be an independent risk factor for MC recurrence. There were also no significant differences in patient background between high and low miR-223 expression groups; however, the disease-free survival rate in the group with low expression was significantly worse. Furthermore, MC-related miRNAs identified by miRNA microarray clearly clustered patients into MC recurrence and non-recurrence groups. CONCLUSION: Both gene and miRNA expression profiles in non-tumor liver tissues could predict the risk for MC recurrence. Such molecular information may be useful in enabling better decision making for patients with HCC.

Maximum Diameter and Number of Tumors as a New Prognostic Indicator of Colorectal Liver Metastases.

BACKGROUND: Surgical resection is currently considered the only potentially curative option as a treatment strategy of colorectal liver metastases (CRLM). However, the criteria for selection of resectable CRLM are not clear. The aim of this study was to confirm a new prognostic indicator of CRLM after hepatic resection. PATIENTS AND METHODS: One hundred thirty nine patients who underwent initial surgical resection from 1994 to 2015 were investigated retrospectively. Prognostic factors of overall survival including the product of maximum diameter and number of metastases (MDN) were analyzed. RESULTS: Primary tumor differentiation, vessel invasion, lymph node (LN) metastasis, non-optimally resectable metastases, H score, grade of liver metastases, resection with non-curative intent and MDN were found to be prognostic factors of overall survival (OS). In multivariate analyses of clinicopathological features associated with OS, MDN and non-curative intent were independent prognostic factors. Patients with MDN ≥30 had shown significantly poorer prognosis than patients with MDN <30 in OS and relapse-free survival (RFS). CONCLUSION: MDN ≥30 is an independent prognostic factor of survival in patients with CRLM and optimal surgical criterion of hepatectomy for CRLM.

Elevated Preoperative Serum CEA Level Is Associated with Poor Prognosis in Patients with Hepatocellular Carcinoma Through the Epithelial-Mesenchymal Transition.

BACKGROUND: Serum carcinoembryonic antigen (CEA) is used as an indicator of tumor progression in a variety of carcinomas. A subset of patients with hepatocellular carcinoma (HCC) exhibit increased serum CEA level, but the significance of this is unclear. In this study, we investigated the prognosis of patients with HCC with increased serum CEA, and explored the correlations with expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and epithelial-mesenchymal transition (EMT) and tumor angiogenesis. MATERIALS AND METHODS: One hundred and twenty-three patients with HCC who underwent radical resection were divided into two groups according to a cut-off value of 5.0 ng/ml for serum CEA: high (n=24) and normal (n=99) groups. We compared the clinicopathological factors with serum CEA levels and its correlations with CEACAM1 expression, EMT-related factors and microvessel density (MVD) of tumor tissues by immunohistochemistry. RESULTS: In the high CEA group, the disease-free survival (DFS) rate was significantly worse than in the normal CEA group. Multivariate analysis revealed that a high CEA level was an independent factor predictive of recurrence. Furthermore, increased serum CEA levels were positively correlated with CEACAM1 expression. Moreover, CEACAM1 expression was positively correlated with expression of EMT-related factors and MVD of tumor tissues. CONCLUSION: Increased serum CEA level reflected CEACAM1 expression and was an independent factor predictive of recurrence in HCC through EMT and tumor angiogenesis.

Loss of SFRP1 Expression Is Associated with Poor Prognosis in Hepatocellular Carcinoma.

BACKGROUND: Secreted frizzled-related protein-1 (SFRP1) is a well-known inhibitor of the wingless type (WNT)-ß-catenin signaling pathway and its inactivation plays an important role in the development and progression of various types of cancer. However, the clinical significance of SFRP1 expression in patients with hepatocellular carcinoma (HCC) remains unknown. MATERIALS AND METHODS: A total of 63 patients with HCC who underwent hepatectomy at our Institution were enrolled in this study. A quantitative real-time polymerase chain reaction (RT-PCR) was performed to determine the SFRP1 mRNA expression level in both the tumorous and non-tumorous tissues of HCC. The patients were divided into low and high gene-expression groups based on the SFRP1 gene expression level in their tumor tissues. We analyzed the differences in clinicopathological characteristics between these two groups of patients. RESULT: The expression level of SFRP1 was significantly lower in tumor tissue than in non-tumor tissue (p<0.0001). Significant correlations were observed between a high expression of SFRP1 in tumor tissue and older than 65 years (p=0.030), tumor size less than 5 cm (p=0.011); and no vascular invasion (p=0.004). Patients with high SFRP1 expression in tumor tissue had a significantly better overall survival rate (p=0.040). However, the SFRP1 expression level was not defined as an independent risk factor for patient survival based on results of multivariate analysis. CONCLUSION: SFRP1 may play a role in the development and progression of HCC. Therefore, more studies are required to investigate a potential role of SFRP1 in HCC prognosis.

High NEK2 Expression Is a Predictor of Tumor Recurrence in Hepatocellular Carcinoma Patients After Hepatectomy.

BACKGROUND/AIM: Better prognosis of cancer including hepatocellular carcinoma (HCC) remains unsatisfactory due to recurrence and chemoresistance. In this respect it is important to identify molecular targets specific to the disease in order to design effective therapeutic strategies. In the present study, we investigated the prognostic role of Never-in-mitosis-A-related kinase 2 (NEK2) in HCC. MATERIALS AND METHODS: Fifty HCC patients who underwent hepatectomy were enrolled in the study. NEK2 gene and protein expression was examined by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry, respectively. RESULTS: Higher expression of NEK2 was detected in HCC tumoral compared to adjacent non-tumor tissues (p<0.001), and protein expression was also relatively high in tumor than corresponding non-tumor tissues. Furthermore, high NEK2 expression was positively correlated with hepatic venous invasion (p=0.047), des-gammacarboxy prothrombin (p=0.003), and alpha-fetoprotein (AFP) (p=0.024). Patients with high NEK2 expression had significantly poor recurrence-free survival (p=0.042) and early recurrence. CONCLUSION: Overall, these results suggest that NEK2 could be a promising biomarker for HCC recurrence.