Sakiko Sanada

Clinicopathologic analysis of combined hepatocellular-cholangiocarcinoma according to the latest WHO classification.

Combined hepatocellular-cholangiocarcinoma comprises <1% of all liver carcinomas. The histogenesis of combined hepatocellular-cholangiocarcinoma has remained unclear for many years. However, recent advances in hepatic progenitor cell (HPC) investigations have provided new insights. The concept that combined hepatocellular-cholangiocarcinoma originates from HPCs is adopted in the chapter “combined hepatocellular-cholangiocarcinoma” of the latest World Health Organization (WHO) classification. In this study, we conducted clinicopathologic analysis of combined hepatocellular-cholangiocarcinoma according to the latest WHO classification. Fifty-four cases were included in this study. Pathologic diagnosis was made according to the WHO classification. When a tumor contained plural histologic patterns, predominant histologic pattern (≥50%) was defined. Minor histologic patterns were also appended. Immunohistochemical staining with biliary markers (CK7, CK19, and EMA), hepatocyte paraffin (HepPar)-1, HPC markers (CD56, c-kit, CD133, and EpCAM), and vimentin was performed. Forty-five and 50 patients were analyzed for progression-free survival and overall survival, respectively. Ten, 1, 32, and 11 cases were diagnosed as: combined hepatocellular-cholangiocarcinoma, classical type; combined hepatocellular-cholangiocarcinoma, stem cell features, typical subtype; combined hepatocellular-cholangiocarcinoma, stem cell features, intermediate cell subtype; and combined hepatocellular-cholangiocarcinoma, stem cell features, cholangiolocellular type, respectively. Combined hepatocellular-cholangiocarcinomas usually have high expression of biliary markers. CD56, c-kit, and EpCAM were expressed to various degrees in all combined hepatocellular-cholangiocarcinomas apart from the hepatocellular carcinoma component of combined hepatocellular-cholangiocarcinoma, classical type. The expression of CD133 and vimentin was observed only in combined hepatocellular-cholangiocarcinoma, stem cell features of intermediate cell subtype and cholangiolocellular subtype. The expression of CD133, EpCAM, and vimentin was significantly high in combined hepatocellular-cholangiocarcinoma, subtypes with stem cell features, especially cholangiolocellular subtype. Minor histologic patterns were significantly frequent in combined hepatocellular-cholangiocarcinoma, subtypes with stem cell features, compared with combined hepatocellular-cholangiocarcinoma, classical type. There was no significant difference in clinical outcome between each subtype. Combined hepatocellular-cholangiocarcinoma has wide histologic diversity and shows immunophenotypic expression of not only biliary markers but also HPC markers to various degrees, suggesting that the histogenesis of combined hepatocellular-cholangiocarcinoma could be strongly associated with HPCs. Our results pathologically validate the latest WHO classification of combined hepatocellular-cholangiocarcinoma. However, the complex mixture of histologic subtypes has presented a challenge to the classification of combined hepatocellular-cholangiocarcinoma. Further study should be conducted using a large cohort to support this classification.

Aldehyde Dehydrogenase 1 Identifies Cells with Cancer Stem Cell-Like Properties in a Human Renal Cell Carcinoma Cell Line

Cancer stem cells (CSC) or cancer stem cell-like cells (CSC-LCs) have been identified in many malignant tumors. CSCs are proposed to be related with drug resistance, tumor recurrence, and metastasis and are considered as a new target for cancer treatment; however, there are only a few reports on CSCs or CSC-LCs in renal cell carcinoma (RCC). Different approaches have been reported for CSC identification, but there are no universal markers for CSC. We used two different approaches, the traditional side population (SP) approach, and the enzymatic (aldehyde dehydrogenase 1 (ALDH1)) approach to identify CSC-LC population in two RCC cell lines, ACHN and KRC/Y. We found that ACHN and KRC/Y contain 1.4% and 1.7% SP cells, respectively. ACHN SP cells showed a higher sphere forming ability, drug resistance, and a slightly higher tumorigenic ability in NOD/SCID mice than Non-SP (NSP) cells, suggesting that cells with CSC-LC properties are included in ACHN SP cells. KRC/Y SP and NSP cells showed no difference in such properties. ALDH1 activity analysis revealed that ACHN SP cells expressed a higher level of activity than NSP cells (SP vs. NSP: 32.7% vs 14.6%). Analysis of ALDH1-positive ACHN cells revealed that they have a higher sphere forming ability, self-renewal ability, tumorigenicity and express higher mRNA levels of CSC-LC property-related genes (e.g., ABC transporter genes, self-replication genes, anti-apoptosis genes, and so forth) than ALDH1-negative cells. Drug treatment or exposure to hypoxic condition induced a 2- to 3-fold increase in number of ALDH1-positive cells. In conclusion, the results suggest that the ALDH1-positive cell population rather than SP cells show CSC-LC properties in a RCC cell line, ACHN.

N-myc downstream regulated gene1/Cap43 overexpression suppresses tumor growth by hepatic cancer cells through cell cycle arrest at the G0/G1 phase

N-myc downstream regulated gene-1 (NDRG1)/Cap43 regulates tumor growth and metastasis in various carcinomas. In this study we examined whether and how NDRG1/Cap43 modulates tumor growth by human hepatocellular carcinoma (HCC) cells. NDRG1/Cap43 cDNA was used to transfect HCC cell lines (KIM-1), and stable transfectants overexpressing NDRG1/Cap43 (KIM-1/Cap43) were obtained. Cell cycle analysis showed that KIM-1/Cap43 cells were arrested in the G0/G1 phase. Western blot analysis demonstrated an increase in p21 in KIM-1/Cap43 cells in culture under full confluency as compared with KIM-1/Mock. When KIM-1 cells, which are very low in NDRG1/Cap43 expression, were treated with mimosine, a G0/G1 cell cycle blocker, expression of NDRG1/Cap43 was induced in a dose dependent manner, together with p21 induction and CDK4 reduction. In vivo, KIM-1/Cap43 cells showed markedly decreased tumor growth rates compared with those of KIM-1/Mock. Immunohistochemical staining demonstrated markedly higher p21 labeling index in the KIM-1/Cap43 tumor than KIM-1/Mock tumor, and lower CDK4 and Ki-67 labeling index in the KIM-1/Cap43 than KIM-1/Mock. In order to confirm suppressive effects of NDRG1/Cap43, we further established a stable transfectant expressing NDRG1/Cap43 (HAK-1B/Cap43) using another HCC cell line, HAK-1B. Western blot analysis demonstrated an increase in p21 and a decrease in CDK4 in HAK-1B/Cap43 cells in culture under full confluency as compared with HAK-1B/Mock. HAK-1B/Cap43 also showed decreased tumor growth rates as compared with its control counterpart in vivo. NDRG1/Cap43 overexpression thus induced cell cycle arrest at the G0/G1 phase accompanied by increased p21 and decreased CDK4 expression in HCC cells. NDRG1/Cap43 might play a key role in the cell cycle control of G0/G1 in HCC cells.

RECQL1 DNA Repair Helicase: A Potential Therapeutic Target and a Proliferative Marker against Ovarian Cancer

Objective This study analyzed the clinicopathological correlation between ovarian cancer (OC) and RECQL1 DNA helicase to assess its therapeutic potential. Methods Surgically resected OC from 118 retrospective cases, for which paraffin blocks and all clinical data were complete, were used in this study. RECQL1 and Ki-67 immunostaining were performed on sections to correlate RECQL1 staining with subtype and patient survival. Ten OC and two normal cell lines were then examined for RECQL1 expression and were treated with siRNA against RECQL1 to assess its effect on cell proliferation. Results Of the 118 cases of adenocarcinoma (50, serous; 26, endometrioid; 21, clear cell; 15, mucinous; 6, other histology), 104 (90%) showed varying levels of RECQL1 expression in the nuclei of OC cells. The Cox hazards model confirmed that diffuse and strong staining of RECQL1 was correlated with histological type. However, RECQL1 expression did not correlate with overall patient survival or FIGO stage. In vitro, RECQL1 expression was exceptionally high in rapidly growing OC cell lines, as compared with normal cells. Using a time-course analysis of RECQL1-siRNA transfection, we observed a significant inhibition in cell proliferation. Conclusions RECQL1 DNA helicase is a marker of highly proliferative cells. RECQL1-siRNA may offer a new therapeutic strategy against various subtypes of OC, including platinum-resistant cancers, or in recurrent cancers that gain platinum resistance.

Assessment of MR Imaging as a Tool to Differentiate between the Major Histological Types of Uterine Sarcomas

OBJECTIVES We retrospectively compared and quantified magnetic resonance (MR) images to distinguish major histological types of uterine sarcomas and malignant and benign tumors. METHODS MR images were obtained from patients who underwent preoperative examinations. We compared 25 pathologically confirmed uterine sarcomas (8 leiomyosarcomas, 11 carcinosarcomas, 6 endometrial stromal sarcomas) with 25 uterine leiomyomas. MR findings included tumor size, location, contour, signal intensity (SI), and contrast enhancement. Analysis focused on the contrast ratio (CR) of SI in T2-weighted images for the areas of lowest, highest, and main SI of each tumor as well as the contrast-enhanced ratio (CER) for the main solid part of each tumor in contrast-enhanced T1-weighted images. We evaluated diffusion-weighted (DW) images and apparent diffusion coefficient (ADC) values in 18 tumors (4 sarcomas, 14 leiomyomas). RESULTS Uterine sarcomas and leiomyomas differed significantly in tumor location, contour, hemorrhaging, necrotic and cystic components, CR for the area of lowest SI (P < 0.05), CR for the area of main SI (P < 0.01), and CER (P < 0.05). Leiomyosarcomas were larger than carcinosarcomas or endometrial stromal sarcomas, and the CR for the area of lowest SI of leiomyosarcomas (P < 0.05) was significantly lower. The CER for endometrial stromal sarcomas (P < 0.05) showed the most homogeneous enhancement. Hemorrhagic or necrotic and cystic components were found more often in larger tumors, although there was no significant difference in their occurrence between sarcoma types. All uterine sarcomas showed high intensity on DW images. The ADC values were lower of uterine sarcomas than leiomyomas, although the difference was not statistically significant. CONCLUSION Quantitative assessment using the CR or CER was useful for distinguishing benign and malignant uterine tumors as well as major histological types of uterine sarcomas.

Pegylated interferon-α2a inhibits proliferation of human liver cancer cells in vitro and in vivo.

Purpose We investigated the effects of pegylated interferon-α2a (PEG-IFN-α2a) on the growth of human liver cancer cells. Methods The effect of PEG-IFN-α2a on the proliferation of 13 liver cancer cell lines was investigated in vitro. Cells were cultured with medium containing 0–4,194 ng/mL of PEG-IFN-α2a, and after 1, 2, 3, or 4 days of culture, morphologic observation and growth assay were performed. After hepatocellular carcinoma (HCC) cells (HAK-1B and KIM-1) were transplanted into nude mice, various doses of PEG-IFN-α2a were subcutaneously administered to the mice once a week for 2 weeks, and tumor volume, weight, and histology were examined. Results PEG-IFN-α2a inhibited the growth of 8 and 11 cell lines in a time- and dose-dependent manner, respectively, although the 50% growth inhibitory concentrations of 7 measurable cell lines on Day 4 were relatively high and ranged from 253 ng/mL to 4,431 ng/mL. Various levels of apoptosis induction were confirmed in 8 cell lines. PEG-IFN-α2a induced a dose-dependent decrease in tumor volume and weight, and a significant increase of apoptotic cells in the tumor. Subcutaneous administration of clinical dose for chronic hepatitis C (3 μg/kg, 0.06 μg/mouse) was effective and induced about 30-50% reduction in the tumor volume and weight as compared with the control. Conclusions Although in vitro anti-proliferative effects of PEG-IFN-α2a were relatively weak, PEG-IFN-α2a induced strong anti-tumor effects on HCC cells in vivo. The data suggest potential clinical application of PEG-IFN-α2a for the prevention and treatment of HCC.

Malignant melanoma arising in a plaque-type blue nevus

Blue nevus is a type of dermal melanocytosis and has a variety of clinicopathological characteristics. A few particular variants are generally accepted as discrete clinicopathological entities. Plaque‐type blue nevus (PTBN) is one of the variants of blue nevus. PTBN presents at birth or arises in early childhood, and shows a combination of features found in common blue nevus and cellular blue nevus. In this report, we describe a malignant melanoma arising in association with a PTBN in a 65‐year‐old male. The tumor appeared as a well circumscribed but partially infiltrative upper‐abdominal mass, and was heavily pigmented and consisted of a spindle or epithelioid highly cellular component with mitotic figures and tumor necroses. In the skin around the tumor, multiple pigmented lesions were scattered around the trunk, and these had been recognized since childhood. Histologically, skin lesions showed sparse and wide distribution of the dendritic melanocytes with dense melanin pigments and melanophages between the collagen bundles from the reticular dermis to the abdominal skeletal muscle. We also discuss the histological features and clinical course in our patient in context with previous related literature.

Growth inhibitory effect of an injectable hyaluronic acid-tyramine hydrogels incorporating human natural interferon-α and sorafenib on renal cell carcinoma cells.

UNLABELLED Immunotherapy including interferon-alpha (IFN-α) is one of the treatment options for metastatic renal cell carcinoma (mRCC) patients. Despite clinical benefits for the selected patients, IFN-α therapy has some problems, such as poor tolerability and dose-limiting adverse effects. In addition, the frequent injections reduce a patients quality of life and compliance. Recently, an injectable and biodegradable hydrogel system to prolong drug release is reported. In this study, we investigated the anticancer effect of IFN-α (Sumiferon®)-incorporated hyaluronic acid-tyramine (HA-Tyr) hydrogels in human RCC-xenografted in nude mice. We also evaluated the synergistic efficacy of IFN-α-incorporated HA-Tyr hydrogels+sorafenib in this model. IFN-α-incorporated HA-Tyr hydrogels+sorafenib most effectively inhibited tumor growth on human RCC cells xenografted in nude mice. In addition, IFN-α-incorporated HA-Tyr hydrogels+sorafenib inhibited the proliferation of tumor in nude mice by inducing apoptosis and the suppression of angiogenesis. Our results suggest a possibility that HA-Tyr hydrogel drug delivery system prolongs the biological half-life of natural human IFN-α and enhances its anticancer effects on human RCC cells. STATEMENT OF SIGNIFICANCE The scope of this study is to provide an alternative approach to improve the anticancer efficacy in renal cell carcinoma (RCC) treatment by using hyaluronic acid-tyramine (HA-Tyr) hydrogel drug delivery system. We investigated the anticancer effect of natural interferon-α (IFN-α)-incorporated HA-Tyr hydrogels in RCC cells. We also evaluated the synergistic efficacy of natural human IFN-α-incorporated HA-Tyr hydrogels+sorafenib. We demonstrated that HA-Tyr hydrogel system is able to release natural human IFN-α in sustained manner and enhances its anticancer effects on human RCC cells. In addition, we suggested that IFN-α-incorporated HA-Tyr hydrogels+sorafenib exhibited most effectively anticancer effects. Hence, we believe that this approach could be applied to treatment with RCC in the future.

The expression of arginase-1, keratin (K) 8 and K18 in combined hepatocellular-cholangiocarcinoma, subtypes with stem-cell features, intermediate-cell type

Aims The WHO classification describes that combined hepatocellular-cholangiocarcinoma, subtypes with stem-cell features, intermediate-cell subtype (CHC-INT) is composed of tumour cells with features intermediate between hepatocytes and cholangiocytes. However, we previously reported that CHC-INT showed a high positive rate of biliary markers, but the expression of hepatocyte paraffin (HepPar)-1 was low. In this study, we examined the expression of other hepatocyte markers, such as arginase-1 (Arg-1), keratin (K) 8 and K18 in CHC-INT in order to examine the utility of pathological diagnosis in CHC-INT. Methods We performed immunohistochemistry (IHC) of Arg-1, K8 and K18 using 32 previously diagnosed as CHC-INT. Immunoreactivity was evaluated with grading from 0 to 4 according to the distribution area of positive cells. The obtained findings of Arg-1, K8 and K18 were compared with those of K7, K19 and HepPar-1. Results Out of the 32 cases, 22 (68.8%) cases were positive for Arg-1. Twenty-five (78.1%) were positive for K8. The IHC scores of Arg-1 and K8 were significantly higher than those of HepPar-1, but significantly lower than those of K7 and K19. The K18 expression was widely observed in all cases (100%). The IHC score of Arg-1 and K8 in CHC-INT was intermediate between hepatocellular carcinoma and cholangiocarcinoma. Conclusions Arg-1 and K8 were good markers to identify intermediate cells between hepatocytes and cholangiocytes. These can be useful markers for pathological diagnosis of CHC-INT, which usually has wide histological diversities, in combination with other hepatocytic and/or cholangiocytic markers.

Des-γ-carboxyprothrombin (DCP) and NX-DCP expressions and their relationship with clinicopathological features in hepatocellular carcinoma.

Aim Des-γ-carboxyprothrombin (DCP) has been used as a tumor marker for hepatocellular carcinoma (HCC). Recently the DCP/NX-DCP ratio, calculated by dividing DCP by NX-DCP, has been reported useful in detecting HCC. The purpose of this study is to clarify the significance of DCP and NX-DCP expression in HCC tissues. Methods HCC and non-HCC tissue samples were obtained from 157 patients and were immunohistochemically examined for DCP and NX-DCP expression using anti-DCP antibody and anti-NX-DCP antibody. DCP and NX-DCP expression scores were calculated by multiplying staining intensity grade by percentage of stained area. Serum DCP and NX-DCP levels were determined in 89 patients. We evaluated the relationship between tumor expression, serum level, and pathomorphological findings. Results Intrahepatic metastasis (im) was significantly more frequent in cases with high DCP expression than in cases with low DCP expression. High NX-DCP expression was associated with significantly lower histological grade, and less frequent im or portal vein invasion (vp) than low NX-DCP expression. Serum DCP was correlated with DCP expression, but serum NX-DCP was not correlated with NX-DCP expression. DCP-positive (≥40 mAU/L), NX-DCP-positive (≥90 mAU/L), and DCP/NX-DCP ratio-positive (≥1.5) cases were associated with significantly larger tumor size and more frequent vp than negative cases. DCP was rarely expressed, but NX-DCP was frequently expressed in non-cancerous liver tissues. Patients with NX-DCP expression-negative tumors showed a lower survival rate than those with NX-DCP expression-positive tumors (p = 0.04), whereas the survival in serum NX-DCP-positive cases was lower than that of serum negative cases (p = 0.02). Conclusions DCP and NX-DCP were produced in HCC tissues, but differed in expression level and biological properties. DCP expression, serum DCP or NX-DCP level, and DCP/NX-DCP ratio were closely related to malignant properties of HCC.