Yohei Mano

Neutrophil–lymphocyte ratio reflects hepatocellular carcinoma recurrence after liver transplantation via inflammatory microenvironment

BACKGROUND & AIMS Although the Milan criteria (MC) have been used to select liver transplantation candidates among patients with hepatocellular carcinoma (HCC), many patients exceeding the MC have shown good prognosis. Preoperative neutrophil-lymphocyte ratio (NLR) is a predictor of patient prognosis, but its mechanism has never been clarified. METHODS We assessed outcomes in 158 patients who had undergone living-donor liver transplantation (LDLT) for HCC. Recurrence-free survival (RFS) was determined in patients with high (≥ 4) and low (<4) NLR. Levels of expression of vascular endothelial growth factor (VEGF), interleukin (IL)-8, IL-17, CD68, and CD163 were measured. RESULTS The 5-year RFS rate was significantly lower in patients with high (n=26) than with low (n=132) NLR (30.3% vs. 89.0%, p<0.0001), in patients with high (n=15) than with low (n=79) NLR who met the MC (73.6% vs. 100%, p=0.0008) and in patients with high (n=11) than with low (n=53) NLR who exceeded the MC (0% vs. 76.1%, p=0.0002). Tumor expression of VEGF, IL8, IL-17, CD68, and CD163 was similar in the high and low NLR groups, but serum and peritumoral IL-17 levels were significantly higher in the high-NLR group (p=0.01 each). The density of peritumoral CD163 correlated with the density of peritumoral IL-17-producing cells (p=0.04) and was significantly higher in the high-NLR group (p=0.005). CONCLUSIONS NLR predicts outcomes after LDLT for HCC via the inflammatory tumor microenvironment. Combined with the MC, NLR may be a new criterion for LDLT candidates with HCC.

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.

Role of tumor-associated macrophages in the progression of hepatocellular carcinoma

Recent studies have shown that the tumor microenvironment plays an important role in cancer progression. Tumor-associated macrophages (TAMs), in particular, have been found to be associated with tumor progression. Macrophages have multiple biological roles, including antigen presentation, target cell cytotoxicity, removal of foreign bodies, tissue remodeling, regulation of inflammation, induction of immunity, thrombosis, and endocytosis. Recent immunological studies have identified two distinct states of polarized macrophage activation: the classically activated (M1) and the alternatively activated (M2) macrophage phenotypes. Bacterial moieties such as lipopolysaccharides and the Th1 cytokine interferon-γ polarize macrophages toward the M1 phenotype. The M2 polarization was discovered as a response to the Th2 cytokine interleukin-4. In general, M2 macrophages exert immunoregulatory activity, participate in polarized Th2 responses, and aid tumor progression. TAMs have recently been found to play an important role in hepatocellular carcinoma (HCC) progression. Based on the properties of TAMs, obtained from pathological examination of resected specimens, we have identified new therapeutic approaches, involving the targeting of TAMs with adjuvant therapy after hepatic resection for HCC. This review discusses the roles of TAM in HCC progression and the possibility of new therapies targeting TAMs.

Tumor-associated macrophage promotes tumor progression via STAT3 signaling in hepatocellular carcinoma.

Objective: Signal transducer and activator of transcription 3 (STAT3) is activated in hepatocellular carcinoma (HCC), and tumor-associated macrophage plays an important role in tumor progression. Therefore, we examined STAT3 activation, cytokine expression and infiltration of tumor-associated macrophages in resected HCCs as well as the alteration of cell growth and migration by cytokine stimulation in HCC cell lines. Methods: Immunohistochemical staining of phosphorylated STAT3 (pSTAT3), CD163, interleukin (IL)-6, Ki-67 and Bcl-XL was performed for 101 cases of resected HCC, and correlations between pSTAT3 staining and clinicopathological findings were analyzed. In HCC cell lines (PLC/PRF/5 and Huh7), cell proliferation and migration by IL-6 stimulation and S3I-201 (STAT3 inhibitor) treatment were analyzed. Results: In HCC specimens, the pSTAT3-positive group showed high levels of α-fetoprotein (p = 0.0276), large tumor size (p = 0.0092), frequent intrahepatic metastasis (p = 0.0214), high Ki-67 (p = 0.0002) and Bcl-XL (p = 0.0001), poor prognosis (p = 0.0234), and high recurrence rate (p = 0.0003). CD163-positive cells were frequently observed in the pSTAT3-positive group (p = 0.0013). In two HCC cell lines, IL-6 stimulation promoted cell proliferation and migration via the STAT3 phosphorylation, and S3I-201 inhibited this activation. Conclusions: STAT3 activation was correlated with aggressive behavior of HCC and may be mediated via tumor-associated macrophage. We expect that STAT3 signaling and tumor-associated macrophages can be attractive therapeutic targets in HCC patients.

Different roles of s100p overexpression in intrahepatic cholangiocarcinoma : Carcinogenesis of perihilar type and aggressive behavior of peripheral type

S100P is expressed in several kinds of malignant tumors. Intracellular S100P interacts with ezrin, and extracellular S100P activates the receptor for advanced glycation endproducts. However, little is known about the biological significance of S100P and related proteins in cholangiocarcinoma. Biliary intraepithelial neoplasia (BilIN) is a precursor lesion of hilar or perihilar cholangiocarcinoma. We examined S100P, ezrin, and the receptor for advanced glycation end product expression in 39 BilIN and 110 intrahepatic cholangiocarcinoma (ICC) cases, and analyzed its relationship with clinicopathologic factors and outcomes. S100P expression increased from reactive epithelium to low-grade BilIN to high-grade BilIN. S100P and ezrin expression rates in perihilar-type ICC were higher than those in peripheral-type ICC (P<0.0001, P=0.0008, respectively). S100P nuclear expression in peripheral-type ICC significantly correlated with vascular invasion (P=0.0209), lymphatic invasion (P=0.0003), and lymph node metastasis (P=0.003). S100P and ezrin expression was significantly correlated. S100P-positive and ezrin-positive cases indicate shorter survival in survival analysis of the peripheral type (P=0.001, P=0.0728, respectively). Our results suggest that S100P-ezrin signaling has different roles of carcinogenesis of perihilar ICC and an aggressive course of peripheral ICC.

Impact of tumor size, number of tumors and neutrophil-to-lymphocyte ratio in liver transplantation for recurrent hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is primarily treated with hepatic resection and/or locoregional therapy. When HCC recurs and further treatment is no longer possible owing to poor liver function, liver transplantation (LT) or living‐donor LT (LDLT) is considered. The aim of this study was to clarify risk factors for tumor recurrence after LDLT in patients with recurrent HCC.

Kinetics of anti-blood type isoagglutinin titers and B lymphocytes in ABO-incompatible living donor liver transplantation with rituximab and plasma exchange.

Background. A novel immunosuppression protocol using rituximab and plasma exchange treatment was developed for ABO-incompatible living donor liver transplantation (ABO-I LDLT). The aim of this study was to investigate the kinetics of anti-blood type isoagglutinin titers and the number of blood B lymphocytes in ABO-I LDLT with the new protocol and their impact on the outcomes after ABO-I LDLT. Methods. Fifteen patients underwent ABO-I LDLT plus splenectomy with the new protocol between November 2005 and December 2010, and their data were retrospectively analyzed. Results. CD19-positive lymphocytes in the blood rapidly disappeared after rituximab treatment and began to recover approximately 6 months later. Anti-blood type isoagglutinin titers were lowered by pretransplant plasma exchange (23∼212→21∼28). Although the anti-donor blood type isoagglutinin titers remained consistently low after transplantation in comparison to the pretreatment levels, they persisted long after LDLT, whereas posttransplant biopsy specimens showed sustained A/B antigens on the graft livers. ABO-I hepatitis C virus-positive patients were prone to acceleration of hepatitis C viremia and cytomegalovirus antigenemia in comparison to the control patients. Conclusions. Although the new protocol for ABO-I LDLT yielded great success with 100% graft survival, the acceptable anti-blood type isoagglutinin titers just before LDLT, and its application to hepatitis C-positive patients must be determined.

Tumor-infiltrating lymphocytes and hepatocellular carcinoma: pathology and clinical management

The presence of tumor-infiltrating lymphocytes (TILs) in hepatocellular carcinoma (HCC) is relatively rare. The prognosis of patients with HCC and marked TILs is better than that of patients with HCC without TILs. TILs in HCC tissues are mainly T cells, and previous reports suggested that TILs might be important antitumor effector cells. TILs have been extensively analyzed, and subpopulations of CD3+, CD4+, and CD8+ T cells are often present in HCC. Some studies have reported that the percentage of CD8+ T cells, which might have cytotoxic activity, is decreased in tumors with TILs, as compared with noncancerous tissues. Although the antitumor effects of TILs seem to be impaired in HCCs, the underlying mechanism has remained unclear until quite recently. Pathological and in vitro studies have now shown that regulatory T cells play important roles in the deterioration of the antitumor effects of TILs. The aim of this review is to introduce recent pathological findings for TILs in HCC and to evaluate new therapeutic strategies in this field.

Histologic classification of microscopic portal venous invasion to predict prognosis in hepatocellular carcinoma

Portal venous invasion is one of the most important prognostic factors after surgical resection of hepatocellular carcinoma. Microscopic portal venous invasion can be evaluated histologically. We examined 280 hepatocellular carcinomas with microscopic portal venous invasion (n = 125) or without it (n = 155) for 3 characteristics: the number of invaded portal vessels, the maximum number of invading carcinoma cells, and the farthest distance from the tumor. Univariate analysis of overall and disease-free survival revealed that the number of invaded portal vessels and the number of invading carcinoma cells were poor prognostic factors. Therefore, we classified patients with microscopic portal venous invasion into 2 groups: a high-microscopic portal venous invasion group, in which there were multiple invaded portal venous vessels (≥2) and more than 50 invading carcinoma cells (n = 57), and a low-microscopic portal venous invasion group, in which microscopic portal venous invasion was observed but with invasion of only a single portal venous vessel or fewer than 50 invading carcinoma cells (n = 68). The high-microscopic portal venous invasion group showed significantly higher α-fetoprotein levels, larger tumor size, and higher frequencies of poorly differentiated histology, capsule infiltration, and intrahepatic metastasis compared with the low-microscopic portal venous invasion group (P = .0496, P < .0001, P = .0431, P = .0180, and P = .0012, respectively). The high-microscopic portal venous invasion group showed poorer overall survival and disease-free survival rates than the low-microscopic portal venous invasion group (P = .0004 and P = .0003), and the high-microscopic portal venous invasion group was an independent prognostic factor for disease-free survival (P = .0259). We proposed a new definition for classifying microscopic portal venous invasion and documented the necessity of definite histologic evaluation of it.

p62+ Hyaline Inclusions in Intrahepatic Cholangiocarcinoma Associated With Viral Hepatitis or Alcoholic Liver Disease

Mallory bodies (MBs) and hyaline globules (HGs) are recognized as hepatocellular cytoplasmic inclusions in liver diseases. We reviewed 123 intrahepatic cholangiocarcinomas (ICCs) and encountered 16 cases (13.0%) in which cancer cells had MB-type inclusions and/or HG-type inclusions, both of which are positive for p62 and ubiquitin. The HG type was present in all 16 cases, and 5 cases contained the MB type. Of 16 patients, 12 had chronic liver disease that was related to alcoholic abuse in 4, hepatitis B surface antigen-positive in 3, and hepatitis C virus antibody-positive in 8. Viral infection and liver cirrhosis were more common in ICCs with p62+ inclusions (P = .0004 and P = .0199, respectively). Of 16 ICCs, 15 with hyaline inclusions had a peripheral tumor location (P = .0052). On ultrastructural examination, the MB type had an electron-dense fibrillar appearance, while the HG type appeared as rounded masses of granular materials. Our results suggest that intracytoplasmic hyaline bodies occasionally can be found in cholangiocarcinoma with chronic liver disease related to viral hepatitis or alcoholic intake.