Acantholytic Squamous Cell Carcinoma and Salivary Duct Carcinoma Ex-pleomorphic Adenoma of the Submandibular Gland: A Report of Two Extremely Rare Cases with an Immunohistochemical Analysis.

Carcinoma ex pleomorphic adenoma (CXPA) is a malignant tumor of the salivary gland that arises from pleomorphic adenoma (PA). Squamous cell carcinoma (SCC) is extremely rare in the salivary glands. We report two cases of acantholytic SCC (ASCC) ex PA. Case 1 involved a 72-year-old female, and case 2 involved a 67-year-old male. Histologically, both cases involved PA, and salivary duct carcinoma (SDC) components, which were positive for androgen receptor (AR) and gross cystic disease fluid protein (GCDFP)-15 but negative for HER2, were seen in the intracapsular regions. The invasive components consisted of ASCC, which were positive for cytokeratin 5/6 and p63 but negative for AR and GCDFP-15. The SDC and ASCC components were positive for the epidermal growth factor receptor. In both cases, the cytoplasmic localization or decreased expression of E-cadherin was observed in the ASCC. In the early phase, CXPA might emerge as SDC, and it might change into SCC as it invades beyond the capsule due to changes in microenvironment. Also, the aberrant expression of E-cadherin is related to acantholysis in SCC.

Carcinoma ex basal cell adenoma of the parotid gland: A report of an extremely rare case.

Malignant non-basaloid tumors that arise from basal cell adenoma (BCA) are extremely rare. The patient was a 72-year-old Japanese male, who had noticed swelling of the left parotid region 21 years ago. A superficial lobectomy was performed. About 60% of the tumor was made up of cribriform and trabecular tissue composed of basaloid cells, which exhibited mild atypia and nuclear expression of ß-catenin. This portion of the tumor was considered to be a BCA. In the other part of the tumor, the proliferation of large eosinophilic atypical cells, most of which formed intraductal structures, was observed. These tumor cells displayed cellular atypia, and some of them formed Roman bridge structures or contributed to intracapsular invasion. Immunohistochemically, these cells were positive for cytokeratin 7, gross cystic disease fluid proten-15 (GCDFP-15), androgen receptor (AR), and mammaglobin (MMG) and exhibited a high Ki-67 labeling index. So, this portion of the tumor was considered to be a salivary duct carcinoma (SDC). The tumor's final diagnosis was SDC ex BCA (intracapsular type), which is extremely rare. GCDFP-15, AR, MMG, and Ki-67 are useful immunohistochemical markers for diagnosing SDC ex BCA.

Salivary duct carcinoma with rhabdoid features: a salivary counterpart of pleomorphic lobular carcinoma of the breast.

AIM: To analyse the clinicopathological features and immunohistochemical characteristics of nine cases of salivary duct carcinoma (SDC) with rhabdoid features (SDCRF), representing a new, extremely rare type of salivary gland malignancy. METHODS AND RESULTS: We analysed 2511 cases of salivary gland tumour, clinicopathologically and immunohistochemically. The incidence of SDCRF was 0.4%. Eight patients were male. The age of patients ranged from 36 years to 85 years (mean, 61 years). SDC arose from the parotid glands and submandibular gland in six and three cases, respectively. Seven cases appeared as a carcinoma component of carcinoma ex pleomorphic adenoma cases. Six patients died of disease. Histologically, diffuse proliferations of non-coherent large ovoid or polygonal carcinoma cells with eosinophilic cytoplasm and eccentric nuclei were observed in all cases; such cytological characteristics were defined as 'rhabdoid features'. Immunohistochemically, all cases were positive for cytokeratin, gross cystic disease fluid protein-15, androgen receptor, and SMARCB1, seven cases were positive for HER2, and two cases were positive for epidermal growth factor receptor. However, all cases were negative for vimentin and myoepithelial markers. Eight cases showed no or aberrant expression of E-cadherin and ß-catenin. The results suggest that SDCRF is an extremely rare subtype of SDC, and not a sarcomatoid variant of SDC. SDCRF is histologically unique, and is positive for SDC markers but negative for vimentin, unlike rhabdoid-type carcinomas arising from other organs. CONCLUSIONS: The morphogenesis of SDCRF is related to no or aberrant expression of cell-cell adhesion molecules. Therefore, SDCRF could be a salivary counterpart to pleomorphic lobular breast carcinoma.

Effects of thrombopoietin on growth of hepatocellular carcinoma: Is thrombopoietin therapy for liver disease safe or not?

AIM: Liver cirrhosis (LC) is the end stage of chronic liver disease. No definitive pharmacological treatment is currently available. We previously reported that thrombopoietin (TPO) promoted liver regeneration and improved liver cirrhosis by increasing platelet count. TPO is therefore considered to be a therapeutic agent for LC; however, it is unclear whether TPO has proliferative effects on hepatocellular carcinoma (HCC), which arises frequently in cirrhotic livers. In this study, we examined the effects of TPO on growth of HCC. METHODS: Expression of the TPO receptor, myeloproliferative leukemia virus oncogene (MPL) was examined in various liver tumor cell lines and liver cell types. In an in vitro study, the effects of TPO on signal transduction, cell proliferation, migration and invasion were examined in Huh7 cells, in which MPL is highly expressed. In an in vivo study, we subcutaneously transplanted Huh7 cells into nude mice that were divided into a TPO-treated group and a control group, and the tumor volume of each group was measured. RESULTS: MPL was expressed strongly in hepatocytes but not in other cell types. Among liver tumor cell lines, Huh7 showed the highest expression of MPL. In Huh7, the addition of TPO activated Akt phosphorylation but not cell proliferation, migration or invasion. In the mouse experiment, there was no significant difference in tumor volume between the two groups. CONCLUSION: TPO had no proliferative effect on HCC in vitro or in vivo, and could therefore be useful in the treatment of liver cirrhosis.

Platelet-derived adenosine 5'-triphosphate suppresses activation of human hepatic stellate cell: In vitro study.

AIM: Activated hepatic stellate cells (HSC) play a critical role in liver fibrosis. Suppressing abnormal function of HSC or reversion from activated to quiescent form is a hopeful treatment for liver cirrhosis. The interaction between platelets and HSC remains unknown although platelets go through hepatic sinusoids surrounded by HSC. This study aimed at clarifying the hypothesis that platelets control activation of HSC. METHODS: We used human platelets, platelet extracts, and primary or immortalized human HSC. We examined the effect of platelets on the activation, DNA synthesis, type I collagen production, and fibrosis-relating gene expressions of HSC. We investigated what suppressed activation of HSC within platelets and examined the mechanism of controlling activation in vitro. RESULTS: Platelets and platelet extracts suppressed activation of HSC. Platelets decreased type I collagen production without affecting DNA synthesis. Platelets increased the expression of matrix metallopeptidase 1. As platelet extracts co-cultured with an enzyme of degrading adenosine 5'-triphosphate (ATP) suppressed activation, we detected adenine nucleotides within platelets or on their surfaces and confirmed the degradation of adenine nucleotides by HSC and the production of adenosine. Adenosine and platelets increased the intracellular cyclic adenosine 5'-monophosphate (cAMP), which is important in quiescent HSC. A great amount of adenosine and ATP also suppressed activation of HSC. CONCLUSION: Activation of human HSC is suppressed by human platelets or platelet-derived ATP via adenosine-cAMP signaling pathway in vitro. Therefore, platelets have the possibility to be used in the treatment of liver cirrhosis.

Platelets prevent acute hepatitis induced by anti-fas antibody.

BACKGROUND AND AIM: Platelets provide many functions in the body, especially to the liver. The purpose of this study is to investigate the effect of thrombocytosis with acute hepatitis induced by anti-Fas antibody and its mechanism. METHODS: Acute hepatitis was induced by administration of anti-Fas antibody in normal and thrombocytotic C57BL6J mice. For thrombocytosis, thrombopoietin; PEG-rHuMGDF was injected 5 days before and just prior to administration of anti-Fas antibody. To investigate the mechanisms, hepatocyte cell line (AML12) and sinusoidal endothelial cell line (M1) were induced apoptosis by staurosporine. They were cultured with platelets or thrombopoietin. Examination items were as follows: platelet number, alanine aminotransferase (ALT), histological findings, TUNEL (TdT-mediated dUTP-biotin Nick End Labeling) staining, and the expression of proteins associated with apoptosis in vivo and in vitro. RESULTS: Platelets were significantly increased in the thrombocytotic group (P < 0.01). Serum ALT levels were significantly reduced by thrombocytosis at 6, 24 and 72 h after the administration (P < 0.05). In histological findings, hemorrhagic necrosis was observed in the normal group, but not observed in the thrombocytotic group. TUNEL-positive hepatocytes were reduced and the expression of cleaved caspase-3 was significantly decreased in the thrombocytotic group. The phosphorylation of Akt, the increment of Bcl-xL and the decrease of cleaved caspase-3 were observed in AML12 cells cultured with platelets, but were not observed cultured with thrombopoietin. Platelets and thrombopoietin had no anti-apoptotic effect on M1 cells. CONCLUSION: Increase of platelets has a preventative effect against acute hepatitis induced by the anti-Fas antibody. It is suggested that platelets have a direct protective effect against apoptosis of hepatocytes.

Platelets prevent acute liver damage after extended hepatectomy in pigs.

BACKGROUND/PURPOSE: Platelets develop tissue repair and promote liver regeneration. We investigated whether platelets prevented acute liver damage after extended hepatectomy in pigs. METHODS: Thrombocytosis was induced by the following two methods; afterwards 80% hepatectomy was performed in pigs. In the first method, the pigs received administration of thrombopoietin [TPO (+) group], and they were compared with a control group [TPO (-) group]. In the second method, the pigs received a splenectomy [Sp (+) group], and theywere compared with another control group [Sp (-) group]. Platelet counts, biochemical examination of blood, and histopathological findings of the residual liver were examined. RESULTS: Serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and total bilirubin (T-Bil) levels were significantly decreased in the thrombocytotic groups compared with the control groups in the early period after hepatectomy. In the histopathological findings, hemorrhagic necrosis with a bile plug was observed in the control groups, but this phenomenon was not observed in the thrombocytotic groups. On transmission electron microscopy, the sinusoidal endothelial lining was destroyed and detached into the sinusoidal space with enlargement of Disse's spaces in the thrombocytotic groups, but these findings were not observed in the control groups. CONCLUSION: An increased number of platelets prevents acute liver damage after extended hepatectomy.

Activation of human liver sinusoidal endothelial cell by human platelets induces hepatocyte proliferation.

BACKGROUND & AIMS: We previously reported that platelets promote hepatocyte proliferation. In this study, we focused on the role of platelets in liver sinusoidal endothelial cells (LSECs) in addition to their role in hepatocyte in liver regeneration. METHODS: Immortalized human LSECs (TMNK-1) were used. The LSECs were co-cultured with human platelets, and the proliferation of LSECs and the excretion of growth factors and interleukin-6 (IL-6) were subsequently measured. The main factor from platelets which induced the excretion of IL-6 from LSECs was determined using inhibitors of each component contained in the platelets. The need for direct contact between platelets and LSECs was investigated using cell culture inserts. The proliferation of human primary hepatocytes was measured after the addition of the supernatant of LSECs cultured with or without platelets. RESULTS: The number of LSECs cocultured with platelets significantly increased. Excretion of IL-6 and vascular endothelial growth factor (VEGF) increased in LSECs with platelets. JTE-013, a specific antagonist for sphingosine 1-phosphate (S1P) 2 receptors, inhibited the excretion of IL-6 from LSECs after the addition of platelets. When the platelets and LSECs were separated by the cell culture insert, the excretion of IL-6 from LSECs was decreased. DNA synthesis was significantly increased in human primary hepatocytes cultured with the supernatant of LSECs with platelets. CONCLUSIONS: Platelets promote LSEC proliferation and induce IL-6 and VEGF production. Direct contact between the platelets and LSECs and S1P, that are contained in platelets, were involved in the excretion of IL-6 from LSECs. IL-6 from LSECs induced proliferation of parenchymal hepatocytes.

Autonomic regulation of liver regeneration after partial hepatectomy in mice.

BACKGROUND/AIMS: The autonomic vagus nerve is thought to play an essential role in liver regeneration since hepatic vagotomy delays hepatic DNA synthesis. However, how the parasympathetic vagus nerve is involved in liver regeneration remains obscure. Kupffer cells are located in liver sinusoids adjacent to hepatocytes and might regulate liver regeneration by releasing interleukin-6 (IL-6). The present study examines the role of the vagus nerve and how Kupffer cells are involved in parasympathetic nerve-mediated liver regeneration in mice. METHODS: We performed surgical vagotomy of the hepatic branch and then partial hepatectomy (PH); some mice received acetylcholine (ACh) agonist/antagonist before PH. We then evaluated liver regeneration and signal transducer and activator of transcription-3 (STAT3) activation. We also investigated whether ACh stimulates IL-6 release from Kupffer cells. RESULTS: Surgical vagotomy impaired liver regeneration. STAT3, which is activated by IL-6 after hepatectomy and plays a pivotal role in liver regeneration, was less activated in vagotomized mice after PH. Post-PH STAT3 activation was recovered by administering vagotomized mice with an ACh agonist. Furthermore, ACh stimulated IL-6 release in Kupffer cells in vitro. CONCLUSION: The parasympathetic system (vagus nerve) contributes to liver regeneration after hepatectomy by stimulating IL-6 release from Kupffer cells followed by STAT3 activation in hepatocytes.

Role of platelets on liver regeneration after 90% hepatectomy in mice.

BACKGROUND/AIMS: Mortality after 90% partial hepatectomy in mice was associated with severe acute liver failure. Recently, we revealed that platelets have a strong promotional effect on hepatic regeneration. In the present study, we investigated the effect of thrombocytosis on liver regeneration after 90% hepatectomy in mice. METHODS: For thrombocytosis induction PEG-rHuMGDF was injected 5 days before operation. Hepatectomy, sparing only the caudate lobe, was performed in normal and thrombocytotic BALB/c mice. Survival rate, platelet number, liver weight/body weight ratio, proliferating cell nuclear antigen, serum parameters, signal transduction and overexpressed genes were examined. RESULTS: Platelet number was significantly higher in thrombocytotic group. All mice in normal group died within 30 h after hepatectomy. Survival rate in thrombocytotic group was 6/11 at 30 h and 3/11 one week after hepatectomy. Activation of Akt and STAT3 signaling pathways in thrombocytotic group was observed earlier and recognized to be stronger compared to normal group. Cell cycle, signaling pathways, metabolism and transport genes were significantly overexpressed in thrombocytotic group up to 24h after hepatectomy. CONCLUSIONS: Under the thrombocytotic condition, liver regeneration occurred even in 90% hepatectomized mice. Platelets contribute to cell cycle progression and metabolic pathways in addition to preventing acute liver failure.

Platelet dynamics in the early phase of postischemic liver in vivo.

BACKGROUND: In liver surgery, ischemia/reperfusion injury occasionally leads to liver failure by activating Kupffer cells (KCs) and leukocytes. However, few reports have demonstrated a relationship between KCs and platelets in vivo. This study investigated the relationship between these cells using intravital microscopy. MATERIALS AND METHODS: Male Wistar rats were divided into two groups: (1) KC+ group, receiving 1 mL saline; and (2) KC- group, intravenously injected with liposome-encapsulated dichloromethylene disphosphonate for elimination of KCs. At 48 h after administration, 20 min of total normothermic hepatic ischemia was induced. Rhodamine-6G-labeled platelets and sinusoidal alterations were monitored using intravital microscopy up to 120 min after reperfusion. P-selectin, accumulated leukocytes and morphological damage, and alanine aminotransferase were evaluated. RESULTS: In the KC+ group, numbers of adherent platelets increased significantly within 30 min after reperfusion. Endothelial cells of sinusoids in which KCs were mainly located were destroyed and the sinusoids were significantly constricted after reperfusion. Conversely, in the KC- group, adherent platelets in sinusoids were suppressed, and sinusoidal perfusion, endothelial cell damage and serum alanine aminotransferase levels were significantly improved. P-selectin on sinusoidal endothelial cells was not observed up to 120 min after reperfusion in either group. CONCLUSIONS: Adherent platelets appear to reflect activation of KCs and lead to leukocyte accumulation, resulting in sinusoidal perfusion disturbance and liver failure. Evaluation of adherent platelets in the microcirculation offers an important marker of hepatic injury.