Immunophenotypes and Tumor Immune Microenvironment in Hepatocellular Carcinoma With Macrotrabecular Massive and Vessels Encapsulating Tumor Clusters.

BACKGROUND/AIM: Recently, vessels encapsulating tumor clusters (VETC) pattern and macrotrabecular massive (MTM) pattern of hepatocellular carcinoma (HCC) have been reported as aggressive histological types. These histological patterns showed an immunosuppressive tumor immune microenvironment (TIME). Since there have been no reports on the differences of these two subtypes simultaneously, this study examined the immunophenotypes and TIME of MTM-HCC and VETC-HCC immunohistochemically. PATIENTS AND METHODS: Seventy-four cases of previously diagnosed HCC, including 32 MTM-HCCs, 21 VETC-HCCs, and 21 conventional HCCs, were enrolled in immunohistochemical analysis. We conducted immunohistochemical analysis. RESULTS: We found that MTM-HCC showed less frequent expression of HepPar-1, which is one of the most common hepatocytic markers. In MTM-HCC, the frequency of high expression levels of Keratin19, carbonic anhydrase (CA) IX, and PD-L1 was higher compared to VETC-HCC and conventional HCC. PD-L1 expression was found in 34.4% of MTM-HCC, 0% of VETC-HCC, and 19.0% of conventional HCC. The rate of PD-L1 expression in MTM-HCC was significantly higher than the others (p=0.0015). PD-L1 expression was significantly associated with epithelial cell adhesion molecules and CA IX expression, which are representative markers of tumor stemness and hypoxic conditions, respectively. The CD8 infiltration in VETC-HCC was significantly lower than that in conventional HCC. CONCLUSION: MTM-HCC had different immunophenotypes and TIMEs compared to HCC with the VETC pattern. Although both had immunosuppressive TIME, the elements forming TIME were quite different. To enhance the immune checkpoint inhibitor efficacy, changing TIME from a suppressive to an active form is essential.

Clinicopathological characteristics and molecular analysis of lymphocyte-rich hepatocellular carcinoma.

Lymphocyte-rich hepatocellular carcinoma (LR-HCC), a newly proposed subtype of HCC, is characterized with abundant lymphocyte infiltration in the tumor. LR-HCC has a relatively good prognosis and is quite rare (<1% of all HCC). We examined LR-HCC clinicopathological and molecular characteristics by analyzing 451 surgically resected HCC cases without any prior treatment history at our hospital between 2012 and 2021. Clinicopathological features of LR-HCC and other HCCs (non-LR-HCC) were compared. Neoplastic and nonneoplastic hepatocytes from LR-HCC (n = 4) were collected with a laser microdissection system; RNA was extracted, followed by microarray analysis to examine lymphocytic infiltration-related molecular targets. Immunohistochemical staining of identified molecular target was performed in LR-HCC and non-LR-HCC. CD3, CD20, and CD8 immunostaining was also performed in LR-HCCs. There were 28 cases of LR-HCC (6%). No statistically significant differences were found in clinicopathological features, except for gross type, between LR-HCC and non-LR-HCC cases. The LR-HCC 5-year survival rate was >90%. Microarray analysis revealed high CCL20 expression in LR-HCC cases; immunohistochemical study showed significantly higher CCL20 expression in LR-HCC (P < 0.01) than in non-LR-HCC. CCR6, the only CCL20 receptor, was observed in infiltrating lymphocytes and HCC cells in LR-HCC. There were significantly more CD3-positive cells than CD20-positive cells (P < 0.0001) in tumor-infiltrating lymphocytes, most of which were CD8-positive T cells. In conclusion, there were no significant differences in clinicopathological characteristics between LR-HCC and non-LR-HCC, except for gross and LR microscopic features. CCL20 expression in LR-HCC may contribute to infiltration of large numbers of CD8-positive lymphocytes.

The Sympathetic Nervous System Promotes Hepatic Lymphangiogenesis, which Is Protective Against Liver Fibrosis.

Liver is the largest lymph-producing organ. In cirrhotic patients, lymph production significantly increases concomitant with lymphangiogenesis. The aim of this study was to determine the mechanism of lymphangiogenesis in liver and its implication in liver fibrosis. Liver biopsies from portal hypertensive patients with portal-sinusoidal vascular disease (n = 22) and liver cirrhosis (n = 5) were evaluated for lymphangiogenesis and compared with controls (n = 9 and n = 6, respectively). For mechanistic studies, rats with partial portal vein ligation (PPVL) and bile duct ligation (BDL) were used. A gene profile data set (GSE77627), including 14 histologically normal liver, 18 idiopathic noncirrhotic portal hypertension, and 22 cirrhotic patients, was analyzed. Lymphangiogenesis was significantly increased in livers from patients with portal-sinusoidal vascular disease, cirrhotic patients, as well as PPVL and BDL rats. Importantly, Schwann cells of sympathetic nerves highly expressed vascular endothelial growth factor-C in PPVL rats. Vascular endothelial growth factor-C neutralizing antibody or sympathetic denervation significantly decreased lymphangiogenesis in livers of PPVL and BDL rats, which resulted in progression of liver fibrosis. Liver specimens from cirrhotic patients showed a positive correlation between sympathetic nerve/Schwann cell-positive areas and lymphatic vessel numbers, which was supported by gene set analysis from patients with noncirrhotic portal hypertension and cirrhotic patients. Sympathetic nerves promote hepatic lymphangiogenesis in noncirrhotic and cirrhotic livers. Increased hepatic lymphangiogenesis can be protective against liver fibrosis.

Endotheliopathy of liver sinusoidal endothelial cells in liver disease.

Liver is the largest solid organ in the abdominal cavity, with sinusoid occupying about half of its volume. Under liver disease, hemodynamics in the liver tissue dynamically change, resulting in injury to liver sinusoidal endothelial cells (LSECs). We discuss the injury of LSECs in liver diseases in this article. Generally, in noninflamed tissues, vascular endothelial cells maintain quiescence of circulating leukocytes, and unnecessary blood clotting is inhibited by multiple antithrombotic factors produced by the endothelial cells. In the setting of inflammation, injured endothelial cells lose these functions, defined as inflammatory endotheliopathy. In chronic hepatitis C, inflammatory endotheliopathy in LSECs contributes to platelet accumulation in the liver tissue, and the improvement of thrombocytopenia by splenectomy is attenuated in cases with severe hepatic inflammation. In COVID-19, LSEC endotheliopathy induced by interleukin (IL)-6 trans-signaling promotes neutrophil accumulation and platelet microthrombosis in the liver sinusoids, resulting in liver injury. IL-6 trans-signaling promotes the expression of intercellular adhesion molecule-1, chemokine (C-X-C motif) ligand (CXCL1), and CXCL2, which are the neutrophil chemotactic mediators, and P-selectin, E-selectin, and von Willebrand factor, which are involved in platelet adhesion to endothelial cells, in LSECs. Restoring LSECs function is important for ameliorating liver injury. Prevention of endotheliopathy is a potential therapeutic strategy in liver disease.

Transcription factor activating enhancer-binding protein 2B expression correlates with invasiveness and prognosis of extramammary Paget's disease.

BACKGROUND: Extramammary Paget's disease (EMPD) is a slowly advancing malignancy that sometimes progresses to the invasion of the dermis, systemic metastases, and death. Although there have been reports that dermal invasion is associated with poor prognosis, no molecular markers of this invasion have been identified thus far. The aim of this study was to identify key molecules for predicting the risk of EMPD dermis invasion. METHOD: We performed microarray screening for three cases of in-situ EMPDs, three cases of invasive EMPDs, and three cases of normal epidermis. We identified a molecule that exhibited a stepwise increase in expression. Further, we analyzed 47 cases of EMPD using immunohistochemical staining (IHC) and examined the correlated clinicopathological findings, including prognosis. RESULT: We examined molecules that showed stepwise differences with invasion. We focused on transcription factor activating enhancer-binding protein 2 B (TFAP2B). Of the 47 EMPD patients, 38 (80.9 %) and 9 (19.1 %) had low and high TFAP2B expression, respectively. TFAP2B expression was significantly correlated with invasion into the dermis, mass formation, and preoperative lymph node metastasis (p = 0.001, 0.042, and 0.033, respectively). The cumulative postoperative recurrence-free rate in the TFAP2B-high expression group was significantly lower than that in the TFAP2B-low expression group (P < 0.001). In univariate analysis of recurrence-free survival, TFAP2B expression was found to be a significant factor (p = 0.006). CONCLUSION: The expression of TFAP2B, which was comprehensively found by microarray screening, may correlate with the invasiveness of EMPD and may be an unfavorable prognostic factor.

Prognostic impact of vessels encapsulating tumor clusters and macrotrabecular patterns in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) shows a high mortality rate. A macrotrabecular (MT) pattern and vessels encapsulating tumor clusters (VETC) pattern have been reported as aggressive histological patterns in HCC. However, their cut-off values have been contentious. METHOD: Nine hundred eighty-five cases of previously diagnosed HCC were enrolled. The percentage areas of the MT and/or VETC pattern with ≥ 5% at every 10% increment were assessed. Clinicopathological analysis including patients' prognosis was conducted. RESULT: One hundred fifty-eight and eighty-four cases were accompanied by 5-49% and ≥ 50% MT components, respectively. Two hundred six and twenty-nine cases had 5-49% and ≥ 50% VETC components, respectively. Cases with these histological patterns in common had aggressive characteristics and worse prognosis compared to cases with none of these patterns. The presence of 5-49% VETC pattern was independent worse prognostic factor in overall survival (P = 0.046). HCCs with the MT pattern and the VETC pattern were significantly accompanied by the VETC pattern and the MT pattern (P < 0.001), respectively. CONCLUSION: As even 5% of the MT pattern and/or VETC pattern affected the prognosis of patients with HCC, the amount of these pattern should be described in pathological reports. This information could be useful in expecting patients' prognosis and providing proper post-operative treatments.

Utility of sonic hedgehog and keratin 8/18 immunohistochemistry for detecting ballooned hepatocytes.

AIMS: Ballooned hepatocytes represent liver cell degeneration and are histological hallmarks in the diagnosis of non-alcoholic steatohepatitis, a severe form of non-alcoholic fatty liver disease. However, the identification of ballooned hepatocytes is often difficult, especially in the clinical setting of patients with other chronic liver diseases. In this study, we investigated the utility of immunostaining for positive sonic hedgehog (SHh) protein and negative Keratin 8/18 (K8/18) expression on ballooned hepatocytes. METHODS AND RESULTS: Immunohistochemistry for SHh and K8/18 was evaluated independently by two experienced liver pathologists in non-tumorous liver tissue from 100 cases of resected hepatocellular carcinoma of various aetiology. The degree of hepatocyte ballooning was scored as follows: 0, none; 1, few; 2, many ballooned hepatocytes. These evaluations were performed using routine haematoxylin and eosin (H&E) staining, followed by immunostaining for SHh or K8/18. Using SHh or K8/18 immunostaining combined with H&E staining, the score of ballooned hepatocytes was upgraded in 20 and 19 cases, and downgraded in none and 2 cases, respectively. The percentage of observed agreement for ballooned hepatocytes scoring was 85% and 92%, and the weighted kappa value was 0.806 and 0.893 with SHh or K8/18 immunohistochemistry. Considering the immunohistochemistry results, background liver disease diagnosis was changed in 15 out of 100 cases (15%) evaluated. CONCLUSIONS: SHh and K8/18 immunohistochemistry are useful in detecting ballooned hepatocytes, regardless of background liver disease, and improving pathological diagnosis accuracy.

Covid-19 and Liver Injury: Role of Inflammatory Endotheliopathy, Platelet Dysfunction, and Thrombosis.

Liver injury, characterized predominantly by elevated aspartate aminotransferase and alanine aminotransferase, is a common feature of coronavirus disease 2019 (COVID-19) symptoms caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Additionally, SARS-CoV-2 infection is associated with acute-on-chronic liver failure in patients with cirrhosis and has a notably elevated mortality in patients with alcohol-related liver disease compared to other etiologies. Direct viral infection of the liver with SARS-CoV-2 remains controversial, and alternative pathophysiologic explanations for its hepatic effects are an area of active investigation. In this review, we discuss the effects of SARS-CoV-2 and the inflammatory environment it creates on endothelial cells and platelets more generally and then with a hepatic focus. In doing this, we present vascular inflammation and thrombosis as a potential mechanism of liver injury and liver-related complications in COVID-19.

Expression and significance of laminin receptor in squamous cell carcinoma of the tongue.

BACKGROUND: Laminin receptor is a non-integrin cell-surface receptor that binds laminin present on the basement membrane. It has been reported to be associated with infiltration and metastasis of various malignant tumors. However, no studies regarding tongue cancer have been reported. This study aimed to clarify the role of laminin receptor in squamous cell carcinoma of the tongue. METHODS: We performed immunohistochemical staining of specimens from 66 patients with squamous cell carcinoma of the tongue and assessed laminin receptor expression and clinicopathological factors. As epithelial-mesenchymal transition has been shown to be associated with infiltration and metastasis of malignant tumors, staining for E-cadherin, vimentin, and N-cadherin were also performed. RESULTS: Of 20 patients with postoperative recurrence, 14 exhibited high laminin receptor expression (p = 0.0025). Kaplan-Meier analysis revealed a significantly shorter time to postoperative recurrence for the high laminin receptor expression group than that for the low laminin receptor expression group (p = 0.0008). Based on multivariate analyses for postoperative recurrence, high laminin receptor expression was associated with poor prognosis (high expression vs. low expression; HR =3.19, 95% CI =0.92-11.08; p = 0.0682). There was a correlation between laminin receptor and N-cadherin (p = 0.0089) but not between laminin receptor and E-cadherin (p = 0.369) or vimentin (p = 0.4221). CONCLUSION: These results suggest that high laminin receptor expression is a useful prognostic factor for postoperative recurrence and may be a target for molecular therapy to treat squamous cell carcinoma of the tongue.

Clinicopathological Analysis of Non-B Non-C Hepatocellular Carcinoma Focusing on Cellular Proliferation.

BACKGROUND/AIM: Non-B non-C hepatocellular carcinomas (NBNC-HCCs) are larger than hepatitis virus-related HCCs. We conducted a clinicopathological study of patients who underwent curative NBNC-HCC resection, including proliferative activity assessments, such as nuclear grade and Ki-67 labelling index (LI). MATERIALS AND METHODS: Histopathological findings of 197 patients were examined, including 56 NBNC-HCCs, 45 hepatitis B virus (HBV)-related HCCs (HBV-HCC), and 96 hepatitis C virus (HCV)-related HCCs (HCV-HCC). RESULTS: NBNC-HCCs were significantly larger than HCV-HCCs, but not significantly different from HBV-HCCs. Mitotic counts, nuclear grade, and Ki-67 LI of NBNC-HCCs were not significantly different from those of HCV-HCCs, but were significantly lower than those of HBV-HCCs. Recurrence-free survival was significantly better in the NBNC-HCC group than in the HBV-HCC group in cases with mild liver fibrosis. CONCLUSION: NBNC-HCCs were significantly larger in diameter, but their nuclear grade or Ki-67 LI were not significantly different from those of other HCCs, suggesting that they do not have a higher proliferative activity.

Pathological findings of liver steatosis that is difficult to evaluate with ultrasound.

Although new ultrasound (US) methods able to quantitatively assess liver fat content have been recently developed, B-mode US is still the major method for detecting liver steatosis during medical checkups. However, some pathological cases yield false-positive or false-negative liver steatosis results using B-mode US. In addition, histologically, the degree of fat deposits and the size of fat droplets in the liver can affect the sensitivity and specificity of the diagnosis of liver steatosis using B-mode US. As B-mode US evaluation of fatty liver relies on operator expertise, the operator should be aware that there are some cases of liver steatosis that are difficult to evaluate with B-mode US. Here, we describe the pathological findings of liver steatosis that is difficult to evaluate with US.

Pathological characteristics of liver sinusoidal thrombosis in COVID-19 patients: A series of 43 cases.

AIM: Coronavirus disease (COVID-19) is characterized by pneumonia with secondary damage to multiple organs including the liver. Liver injury (elevated alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) often correlates with disease severity in COVID-19 patients. The aim of this study is to identify pathological microthrombi in COVID-19 patient livers by correlating their morphology with liver injury, and examine hyperfibrinogenemia and von Willebrand factor (vWF) as mechanisms of their formation. METHODS: Forty-three post-mortem liver biopsy samples from COVID-19 patients were obtained from Papa Giovanni XXIII Hospital in Bergamo, Italy. Three morphological features of microthrombosis (sinusoidal erythrocyte aggregation [SEA], platelet microthrombi [PMT], and fibrous thrombi) were evaluated. RESULTS: We found liver sinusoidal microthrombosis in 23 COVID-19 patients (53%) was associated with a higher serum ALT and AST level compared to those without (ALT: 10-fold, p = 0.04; AST: 11-fold, p = 0.08). Of 43 livers, PMT and SEA were observed in 14 (33%) and 19 (44%) cases, respectively. Fibrous thrombi were not observed. Platelet microthrombi were associated with increased ALT (p < 0.01), whereas SEA was not (p = 0.73). In COVID-19 livers, strong vWF staining in liver sinusoidal endothelial cells was associated with significantly increased platelet adhesion (1.7-fold, p = 0.0016), compared to those with weak sinusoidal vWF (2-fold, p < 0.0001). Sinusoidal erythrocyte aggregation in 19 (83%) liver samples was mainly seen in zone 2. Livers with SEA had significantly higher fibrinogen (1.6-fold, p = 0.031) compared to those without SEA in COVID-19 patients. CONCLUSIONS: Liver PMT is a pathologically important thrombosis associated with liver injury in COVID-19, while SEA is a unique morphological feature of COVID-19 patient livers. Sinusoidal vWF and hyperfibrinogenemia could contribute to PMT and SEA formation.

Liver injury in COVID-19 and IL-6 trans-signaling-induced endotheliopathy.

BACKGROUND AND AIMS: COVID-19 is associated with liver injury and elevated interleukin-6 (IL-6). We hypothesized that IL-6 trans-signaling in liver sinusoidal endothelial cells (LSECs) leads to endotheliopathy (a proinflammatory and procoagulant state) and liver injury in COVID-19. METHODS: Coagulopathy, endotheliopathy, and alanine aminotransferase (ALT) were retrospectively analyzed in a subset (n = 68), followed by a larger cohort (n = 3,780) of patients with COVID-19. Liver histology from 43 patients with COVID-19 was analyzed for endotheliopathy and its relationship to liver injury. Primary human LSECs were used to establish the IL-6 trans-signaling mechanism. RESULTS: Factor VIII, fibrinogen, D-dimer, von Willebrand factor (vWF) activity/antigen (biomarkers of coagulopathy/endotheliopathy) were significantly elevated in patients with COVID-19 and liver injury (elevated ALT). IL-6 positively correlated with vWF antigen (p = 0.02), factor VIII activity (p = 0.02), and D-dimer (p <0.0001). On liver histology, patients with COVID-19 and elevated ALT had significantly increased vWF and platelet staining, supporting a link between liver injury, coagulopathy, and endotheliopathy. Intralobular neutrophils positively correlated with platelet (p <0.0001) and vWF (p <0.01) staining, and IL-6 levels positively correlated with vWF staining (p <0.01). IL-6 trans-signaling leads to increased expression of procoagulant (factor VIII, vWF) and proinflammatory factors, increased cell surface vWF (p <0.01), and increased platelet attachment in LSECs. These effects were blocked by soluble glycoprotein 130 (IL-6 trans-signaling inhibitor), the JAK inhibitor ruxolitinib, and STAT1/3 small-interfering RNA knockdown. Hepatocyte fibrinogen expression was increased by the supernatant of LSECs subjected to IL-6 trans-signaling. CONCLUSION: IL-6 trans-signaling drives the coagulopathy and hepatic endotheliopathy associated with COVID-19 and could be a possible mechanism behind liver injury in these patients. LAY SUMMARY: Patients with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection often have liver injury, but why this occurs remains unknown. High levels of interleukin-6 (IL-6) and its circulating receptor, which form a complex to induce inflammatory signals, have been observed in patients with COVID-19. This paper demonstrates that the IL-6 signaling complex causes harmful changes to liver sinusoidal endothelial cells and may promote blood clotting and contribute to liver injury.

Alcohol-induced Hsp90 acetylation is a novel driver of liver sinusoidal endothelial dysfunction and alcohol-related liver disease.

BACKGROUND & AIMS: Liver sinusoidal endothelial cell (LSEC) dysfunction has been reported in alcohol-related liver disease, yet it is not known whether LSECs metabolize alcohol. Thus, we investigated this, as well as the mechanisms of alcohol-induced LSEC dysfunction and a potential therapeutic approach for alcohol-induced liver injury. METHODS: Primary human, rat and mouse LSECs were used. Histone deacetylase 6 (HDAC6) was overexpressed specifically in liver ECs via adeno-associated virus (AAV)-mediated gene delivery to decrease heat shock protein 90 (Hsp90) acetylation in ethanol-fed mice. RESULTS: LSECs expressed CYP2E1 and alcohol dehydrogenase 1 (ADH1) and metabolized alcohol. Ethanol induced CYP2E1 in LSECs, but not ADH1. Alcohol metabolism by CYP2E1 increased Hsp90 acetylation and decreased its interaction with endothelial nitric oxide synthase (eNOS) leading to a decrease in nitric oxide (NO) production. A non-acetylation mutant of Hsp90 increased its interaction with eNOS and NO production, whereas a hyperacetylation mutant decreased NO production. These results indicate that Hsp90 acetylation is responsible for decreases in its interaction with eNOS and eNOS-derived NO production. AAV8-driven HDAC6 overexpression specifically in liver ECs deacetylated Hsp90, restored Hsp90's interaction with eNOS and ameliorated alcohol-induced liver injury in mice. CONCLUSION: Restoring LSEC function is important for ameliorating alcohol-induced liver injury. To this end, blocking acetylation of Hsp90 specifically in LSECs via AAV-mediated gene delivery has the potential to be a new therapeutic strategy. LAY SUMMARY: Alcohol metabolism in liver sinusoidal endothelial cells (LSECs) and the mechanism of alcohol-induced LSEC dysfunction are largely unknown. Herein, we demonstrate that LSECs can metabolize alcohol. We also uncover a mechanism by which alcohol induces LSEC dysfunction and liver injury, and we identify a potential therapeutic strategy to prevent this.

Programmed Death-Ligand 1 and Programmed Death-Ligand 2 Expression Can Affect Prognosis in Extramammary Paget's Disease.

BACKGROUND: Extramammary Paget's disease (EMPD) is a type of carcinoma that usually progresses slowly but may cause metastasis and subsequent death of patients. We investigated the relationship between the expression of programmed death-ligand 1 (PD-L1)/programmed death-ligand 2 (PD-L2) and stromal CD8+ tumor-infiltrating lymphocytes (TILs) in EMPD and clinicopathological findings, including prognosis. MATERIALS AND METHODS: We examined 47 cases of EMPD and performed immunohistochemical staining of formalin-fixed paraffin-embedded full-face sections. RESULTS: PD-L1 expression in tumor cells was observed in 13 cases (27.7%) while PD-L2 expression was observed in 21 cases (44.7%). The cumulative postoperative recurrence-free rate in the group with positivity for PD-L1 and/or PD-L2 with a low CD8+ TIL count was significantly lower than that of the corresponding group with a high CD8+ TIL count and of the PD-L1- and PD-L2-negative group (p=0.026). CONCLUSION: The expression of PD-L1/PD-L2 in tumor cells was shown to be a factor for poor prognosis.

Prognostic Value of Tumor Proportion Score in Salivary Gland Carcinoma.

OBJECTIVE: Limited information is available regarding the role of programmed death ligand 1 (PD-L1) expression and CD8+ tumor-infiltrating lymphocyte (TIL) density in the tumor immune microenvironment (TIM) of patients with salivary gland carcinoma (SGC). This study aimed to assess the association between the prognosis of SGC patients and the probability of PD-L1 expression in tumor and/or immune cells using the tumor proportion score (TPS), mononuclear immune cell density score (MIDS), combined positive score (CPS), and CD8+ TIL density in the TIM. STUDY DESIGN: Retrospective cohort study. METHODS: We retrospectively reviewed 73 SGC patients treated with definitive surgery between 2000 and 2015. Immunohistochemical analysis was used to assess TPS, MIDS, CPS, and CD8+ TIL density, followed by prognostic evaluation of these immune-related parameters. RESULTS: Histological grade was associated with TPS, MIDS, and CPS based on PD-L1 expression, and these scores exhibited a significant association with CD8+ TIL density. Patients with positive TPS had an unfavorable disease-free survival and overall survival. Multivariate analyses indicated that the TPS was a significant and independent prognostic factor. CONCLUSION: Our results suggest that TPS might be a useful prognostic biomarker in SGC patients receiving definitive surgery. Laryngoscope, 131:E1481-E1488, 2021.

The lymphatic system in alcohol-associated liver disease.

The lymphatic system plays vital roles in interstitial fluid balance and immune cell surveillance. The effect of alcohol on the lymphatic system is poorly understood. This review article explores the role of the lymphatic system in the pathogenesis of alcohol-related disease including alcoholic liver disease (ALD) and the therapeutic potential of targeting hepatic lymphatics for the treatment of ALD.

Usefulness of Tumor Tissue Biopsy for Predicting the Biological Behavior of Hepatocellular Carcinoma.

BACKGROUND/AIM: Assessment of the biological behavior of tumors is important for choosing an appropriate cancer therapy. In hepatocellular carcinoma (HCC), the biological behaviour can be assessed by tumor morphology and molecular biology. This study investigated the usefulness of tumor tissue biopsy for predicting the biological behavior of HCC. PATIENTS AND METHODS: We studied 43 patients who underwent hepatectomy and preoperative liver tumor biopsy for HCC. We performed clinicopathological and immunohistochemical (IHC) analyses. The expression of the following molecules was examined: regulator of G-protein signaling 5 (RGS5), glypican-3 (GPC3), keratin 19 (K19), epithelial cell adhesion molecule (EpCAM), protein induced by vitamin K absence or antagonist-II (PIVKA-II), ß-Catenin, and p53. RESULTS: There was an overall 83.7% agreement regarding tumor differentiation between the preoperative biopsy specimens and the resected specimens. The accuracy of IHC analysis was more than 70% for all molecules between the preoperative biopsy specimens and the resected specimens. The RGS5-positive biopsy cases had higher serum α-fetoprotein levels (p=0.04), a higher rate of moderately or poorly differentiated tumors (p=0.02) and portal vein invasion (p=0.0003) than the RGS5-negative biopsy cases. The GPC3-positive biopsy cases were younger (p=0.04), had higher serum PIVKA-II levels (p=0.01), and a higher rate of portal vein invasion (p=0.03) than the GPC3-negative biopsy cases. The PIVKA-II-positive biopsy cases had significantly higher serum PIVKA-II levels than the PIVKA-II-negative biopsy cases (p=0.02). The other molecular markers showed no significantly different clinical findings between the positive and negative cases. CONCLUSION: In HCC, there was a high agreement rate of both the histopathological and IHC findings between preoperative biopsy specimens and resected specimens. In the biopsy specimens of HCC, RGS5 and GPC3 expression were useful molecular makers for predicting portal vein invasion. Liver tumor biopsy is useful for predicting the biological behavior of HCC through histopathological and immunohistochemical findings.

Distinctive clinicopathological features and KRAS and IDH1/2 mutation status of cholangiolocellular carcinoma.

AIM: Cholangiolocellular carcinoma (CLC) is classified as a subtype of combined hepatocellular cholangiocarcinoma with stem-cell features (CHC-SC) in the latest World Health Organization classification. This subclassification of CHC-SCs is controversial and the relevance of such classification is unclear. METHODS: We analyzed a series of CHC-SCs and intrahepatic cholangiocarcinoma (iCCA) to clarify the clinicopathological features and mutational status of each tumor. RESULTS: Background liver disease, fibrosis stage, microvascular invasion, nodal metastasis, and IDH1/2 mutation status were associated with their histology. Compared with the intermediate cell subtype of CHC-SC (CHCs-SC-int), CLCs were less frequently associated with chronic viral hepatitis, and showed lower levels of serum alpha-fetoprotein. Compared with iCCAs, CLCs showed lower levels of serum carbohydrate antigen 19-9 (CA19-9) and a lower frequency of expression of S100P. Patients with iCCA showed worse overall survival than those with CLC or CHC-SC-int. In patients with iCCA, CLC, or CHC-SC-int, a histology of iCCA, microvascular invasion, and serum CA19-9 value of >100 U/mL were significant poor prognostic factors for overall survival in univariate analysis. Multivariate analysis showed that a high serum CA19-9 value was an independent poor prognostic factor for overall survival. CONCLUSIONS: Patients with CLC are likely to have a different etiology and mutational background from those with CHC-SC-int. Their clinicopathological manifestations are also different from those with classic iCCA. Our results suggest that CLC might be a distinct entity among primary liver carcinomas.

Antiproliferative Effect of Lenvatinib on Human Liver Cancer Cell Lines In Vitro and In Vivo.

BACKGROUND/AIM: Lenvatinib is a potent inhibitor of receptor tyrosine kinases, targeting vascular endothelial growth factor receptors (VEGFR1-3), fibroblast growth factor receptors (FGFR1-4), KIT, and RET. Here, we investigated the antiproliferative effects of lenvatinib in liver cancer cells in vitro and in vivo. MATERIALS AND METHODS: Eleven hepatocellular carcinoma cell lines and two combined hepatocellular/cholangiocarcinoma cell lines were treated with 0-30 µM lenvatinib. Cell growth, apoptosis and the expression of FGFR1-4, FGF19, fibroblast growth factor receptor substrate (FRS)2α and RET were examined. Two HCC cell lines were subcutaneously implanted on nude mice and mice were treated with 3, 10, 30 mg/kg/day of lenvatinib or vehicle for 14 consecutive days. Tumor volume was measured every 3 days. Mice were sacrificed on day 15 and tumors were processed for histological examination. Blood vessels, microvessel density, necrosis, and apoptosis were also examined. RESULTS: Lenvatinib dose- and time-dependently inhibited growth of all cell lines; however, sensitivity to lenvatinib varied. Apoptosis was not observed in any cell line, and expression of FGFR1, -2, -3 and -4, FGF19, FRS2α, and RET were observed in these cell lines. Cell lines with high expression of these factors showed higher response to lenvatinib. In mice, lenvatinib dose-dependently suppressed tumor growth. Blood vessels and microvessel density were significantly reduced and the rate of necrosis was significantly increased by lenvatinib; apoptosis was not observed. CONCLUSION: Antiproliferative effects of lenvatinib on liver cancer cells were observed in vitro and in vivo. Lenvatinib may suppress tumor formation by inhibiting angiogenesis, and via an additional direct antiproliferative effect in some liver cancer cells.