Significance of MUC1 and ß-Catenin Localization in Mucinous Carcinoma of the Breast.

BACKGROUND/AIM: There are two main subtypes of mucinous carcinoma (MC) based on the quantification of the mucinous component: the pure variant (pMC) and the mixed variant (mMC). pMC has been subdivided into pure A with a hypocellular variant, and pure B with a hypercellular variant. PATIENTS AND METHODS: We retrospectively analyzed the clinicopathological features of 99 patients with MC who were treated at our institution from January 2002 to December 2014. We evaluated the expression profiles of markers, including mucin (MUC) family members, in the patients groups representing different MC subtypes by performing immunohistochemistry to identify factors involved in the differentiation and progression of MCs. RESULTS: Among the 99 patients, 76 (76.8%) had pure mucinous carcinomas (pMC) and the other 23 (23.2%) had mixed mucinous carcinomas (mMC). Of the pMCs, 54 were pure A and 22 were pure B. The prognosis was worse for pure B than pure A and worse for mMC than pMC. Although there was no significant difference in clinicopathological factors between the pure A and pure B groups, immunohistochemical staining revealed differences in the localization of mucin MUC1 and ß-catenin. A comparison of the pMC and mMC cases revealed more lymphovascular invasion in mMC and differences in the localization of ß-catenin between the two groups. CONCLUSION: The patients' prognoses were significantly poorer depending on the histologic subtype (in the order pure A, pure B, and mixed). MUC1 localization and ß-catenin were revealed as independent predictors contributing to the poorer prognosis.

Association Between High Expression of Phosphorylated-STMN1 and Mesenchymal Marker Expression and Cancer Stemness in Breast Cancer.

BACKGROUND/AIM: In patients with breast cancer, the expression of stathmin1 (STMN1) has been significantly related to a poor prognosis, cancer aggressiveness, and expression of cancer stem cell markers. The STMN1 protein is closely regulated by phosphorylation in four sites. However, few studies have investigated the relationship between the expression of phosphorylated STMN1 (pSTMN1) and clinicopathological findings, including tumor-aggressive biomarkers, in patients with breast cancer. MATERIALS AND METHODS: The expression levels of four pSTMN1 (Ser16, Ser25, Ser38, and Ser63) were immunohistochemically analyzed in 213 breast cancer cases. The clinicopathological factors evaluated included epithelial-mesenchymal transition (EMT) markers and cancer stem cell markers. RESULTS: The cytoplasmic expression of pSTMN1 (Ser16, Ser25, Ser38, and Ser63) in normal breast tissues was low. The positive expression ratios of Ser25 (54.5%) and Ser38 (39.0%) were high compared to those of Ser16 (25.8%) and Ser63 (23.9%). The overexpression of pSTMN1 (Ser38) was associated with tumor-aggressive characteristics, such as triple-negative breast cancer (TNBC) phenotypes, high mesenchymal marker, and expression of cancer stem cell markers. CONCLUSION: STMN1 phosphorylation might be associated with clinicopathological factors, breast cancer subtypes, and expression of mesenchymal markers and breast cancer stem cell markers through the regulation of STMN1 function. Ser38 phosphorylation of STMN1 may be a novel biomarker for high-grade TNBC associated with mesenchymal marker expression and cancer stemness.

Conophylline Inhibits Hepatocellular Carcinoma by Inhibiting Activated Cancer-associated Fibroblasts Through Suppression of G Protein-coupled Receptor 68.

Treatment of hepatocellular carcinoma (HCC) is currently challenging. Cancer-associated fibroblasts (CAFs) promote the malignancy of HCC cells via production of cytokines. Conophylline (CnP), a vinca alkaloid obtained from Ervatamia microphylla leaves, has been reported to suppress activation of hepatic stellate cells and liver fibrosis in rats. We examined the efficacy of CnP in suppressing tumor growth in HCC. Specifically, we investigated whether CnP could inhibit CAFs, which were derived from HCC tissues in vitro and in vivo Same as previous reports, CAFs promoted proliferative and invasive ability of HCC cells. CnP suppressed α-smooth muscle actin expression of CAFs, and inhibited their cancer-promoting effects. CnP significantly suppressed CAFs producting cytokines such as IL6, IL8, C-C motif chemokine ligand 2, angiogenin, and osteopontin (OPN). Combined therapy with sorafenib and CnP against HCC cells and CAFs in vivo showed to inhibit tumor growth the most compared with controls and single treatment with CnP or sorafenib. Transcriptome analysis revealed that GPR68 in CAFs was strongly suppressed by CnP. The cancer-promoting effects of cytokines were eliminated by knockdown of GPR68 in CAFs. CnP inhibited the HCC-promoting effects of CAFs by suppressing several HCC-promoting cytokines secreted by CAFs expressing GPR68. Combination therapy with CnP and existing anticancer agents may be a promising strategy for treating refractory HCC associated with activated CAFs.

Inflammatory and immune checkpoint markers are associated with the severity of aortic stenosis.

Objective: Aortic stenosis (AS) is a disease characterized by narrowing of the aortic valve (AV) orifice. In relation to this disease, the purpose of this study was to elucidate the relationships among factors such as expression of programmed cell death-1 ligand (PD-L1, which is the ligand of PD-1 protein; together, they play a central role in the inhibition of T lymphocyte function), clinicopathologic characteristics, infiltrating immune cells, and disease severity. Methods: We performed immunohistochemical analysis on the surgically-resected AVs of 53 patients with AS. We used the resultant data to identify relationships among PD-L1 expression, disease severity, and the infiltration of immune cells including cluster of differentiation (CD8)-positive T lymphocytes, cluster of differentiation 163 (CD163)-positive macrophages, and forkhead box protein 3 (FOXP3)-positive regulatory T lymphocytes (Tregs). Results: PD-L1 expression in resected AVs was significantly associated with being nonsmoker, valve calcification, and the infiltration of CD8-positive T cells and CD163-positive macrophages. Disease severity and valve calcification were significantly associated with low infiltration of FOXP3-positive Tregs and high infiltration of CD8-positive T cells and CD163-positive macrophages. Moreover, calcified AVs with high PD-L1 expression showed active inflammation without FOXP3-positive Tregs but with high levels of CD8-positive T lymphocytes and CD163-positive macrophages. Conclusions: Immune cell infiltration in the AVs and expression of the immune checkpoint protein PD-L1 were associated with the calcification of AS and disease severity.

Role of PD-L1 Expression during the Progression of Submucosal Gastric Cancer.

INTRODUCTION: Programmed death-ligand 1 (PD-L1) expression is a prognostic marker for gastric cancer that correlates with tumor diameter and depth of penetration. But the role of PD-L1 and mechanism(s) employed in the initial phase of invasion in early gastric cancer is yet to be understood. OBJECTIVE: This study aims to elucidate the role of PD-L1 during the progression of gastric cancer, specifically invading the submucosa beyond the lamina muscularis mucosa. METHODS: Using 107 patients with pathological submucosal gastric cancer, we determined the expression of PD-L1 based on the staining of the cell membrane or cytoplasm of tumor cells in the central and invasive front of the tumor. Samples were categorized into 3 groups based on the intensity of PD-L1 expression. CD8+ lymphocytes expressing PD-1 and CD163+ macrophages were used to determine the number of cell nuclei at the invasive front, similar to PD-L1. CMTM6 levels were determined and used to stratify samples into 3 groups. RESULTS: PD-L1 expression was higher in the invasive front (26.2%) than in the central portion of the tumors (7.4%; p < 0.001). Moreover, lymphatic and vascular invasion were more frequently observed in samples with high levels of PD-L1 (lymphatic invasion: 60.7 vs. 35.4%, p = 0.0026, and vascular invasion: 39.3 vs. 16.5%, p = 0.0018). There was no correlation between PD-L1 expression and the levels of PD-1, CD8, CD163, and CMTM6. CONCLUSIONS: PD-L1-expressing cancer cells at the invasive front of gastric cancer influence the initial stages of tumor invasion and lymphovascular permeation in early-stage gastric cancers. Immune checkpoint signaling may be the driving force in the invasive front during the invasion of the submucosa beyond the lamina muscularis mucosa.

Mac-2-binding protein glycan isomer enhances the aggressiveness of hepatocellular carcinoma by activating mTOR signaling.

BACKGROUND: Wisteria floribunda agglutinin (WFA)+ Mac-2-binding protein (M2BPGi) is a novel serum marker for liver fibrosis. Although an elevated serum level of M2BPGi can predict development of hepatocellular carcinoma (HCC), the effect of M2BPGi on HCC remains unclear. There are no reports about the association of M2BPGi with HCC aggressiveness. We aimed to clarify the significance of M2BPGi in HCC. METHODS: The protein expression of M2BPGi and galectin-3, a ligand of M2BP, and the mRNA expression of M2BP were evaluated in surgically resected human HCC samples. M2BPGi-regulating signals in HCC cells were investigated using transcriptome analysis. The effects of M2BPGi on HCC properties and galectin-3/mTOR signaling were evaluated. RESULTS: M2BPGi and galectin-3 proteins co-localised in HCC cells, while M2BP mRNA was detected in cirrhotic liver stromal cells. mTOR signaling was upregulated in M2BPGi-treated HCC cells. Moreover, M2BPGi treatment induced tumour-promoting effects on HCC in vitro by activated mTOR signaling. In addition, M2BPGi bound to galectin-3 to induce membranous galectin-3 expression in HCC cells. In vivo, M2BPGi enhanced the growth of xenografted HCC. CONCLUSIONS: M2BPGi is produced in stromal cells of the cirrhotic liver. Furthermore, M2BPGi enhances the progression of HCC through the galectin-3/mTOR pathway.

Diagnostic value of 18F-FDG-PET to predict the tumour immune status defined by tumoural PD-L1 and CD8+tumour-infiltrating lymphocytes in oral squamous cell carcinoma.

BACKGROUND: Lately, immune checkpoint proteins, such as programmed death 1 (PD-1) and its ligand-1 (PD-L1), have garnered attention as a new target in oral squamous cell carcinoma (OSCC). Reportedly, fluoro-D-glucose (FDG)-uptake alteration by anti-PD-1 antibody treatment depicts the response in patients with lung cancer. This study aims to elucidate the correlations between tumour immune status, clinicopathological factors, 18F-FDG-uptake and cold tumour phenotypes as low PD-L1 expression/low CD8+tumour-infiltrating lymphocytes (TILs) in OSCC. METHODS: We performed immunohistochemical analysis of PD-L1, hypoxia-inducible factor 1 A (HIF-1A), glucose transporter type 1 (GLUT1), CD8, E-cadherin and Ki-67 on 59 operable OSCC samples. We assessed the correlations between these factors and preoperative 18F-FDG-uptake, clinicopathological characteristics and prognosis. RESULTS: Low expression of PD-L1 in OSCC correlated with cancer aggressiveness, poor prognosis, high 18F-FDG-uptake with HIF-1A/GLUT1 and low E-cadherin expression and low CD8. Cold tumour phenotypes as low PD-L1 tumour cells and low stromal CD8 correlated with the poor prognosis, high 18F-FDG-uptake and E-cadherin suppression. Furthermore, the high level of preoperative 18F-FDG-uptake in OSCC was an independent predictor of the cold tumour immune status. CONCLUSIONS: 18F-FDG-uptake is an independent predictor of cold tumour in OSCC. 18F-FDG-PET imaging could be a promising diagnostic tool to estimate tumour immune status.

Nintedanib inhibits intrahepatic cholangiocarcinoma aggressiveness via suppression of cytokines extracted from activated cancer-associated fibroblasts.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a malignancy that is challenging to treat. Fibroblasts in ICC tissues have been identified as cancer-associated fibroblasts (CAFs) that promote the malignant behaviour of ICC cells. An antifibrotic drug nintedanib has been reported to suppress activated hepatic stellate cells in liver fibrosis. METHODS: We investigated whether nintedanib could suppress the cancer-promoting effect of CAFs derived from ICC tissues in vitro and in vivo. RESULTS: CAFs promoted the proliferation and invasion of ICC cells. Nintedanib suppressed activated CAFs expressing α-smooth muscle actin (α-SMA) and inhibited the ICC-promoting effects of CAFs. Nintedanib greatly reduced the levels of cancer-promoting cytokines, such as interleukin (IL)-6 (IL-6) and IL-8, secreted by CAFs. An in vivo study demonstrated that nintedanib reduced xenografted ICC growth and activated CAFs expressing α-SMA, and that combination therapy with nintedanib and gemcitabine against CAFs and ICC cells showed the strongest inhibition of tumour growth compared with the control and single-treatment groups. CONCLUSIONS: Nintedanib inhibited the cancer-promoting effect of CAFs via the suppression of CAF activation and secretion of cancer-promoting cytokines. Our findings suggest that therapeutic strategies combining conventional cytotoxic agents with nintedanib targeting CAFs are promising for overcoming refractory ICC with activated CAFs.

FDG-PET reflects tumor viability on SUV in colorectal cancer liver metastasis.

BACKGROUND: Liver resection is the most effective procedure for colorectal cancer liver metastasis (CRLM); however, early recurrence is an important problem that affects the postoperative prognoses of patients with CRLM. We previously suggested a therapeutic algorithm for CRLM using fluorodeoxyglucose-positron emission tomography (FDG-PET) and revealed the applicability of FDG-PET in predicting the prognosis after liver resection of CRLM. In this study, we assessed the correlation between FDG-PET and biological viability such as proliferation or metabolic activity. METHODS: We retrospectively evaluated 61 patients who underwent hepatectomy for CRLM. We assessed hypoxia inducible factor-1α (HIF-1α), pyruvate kinase isozyme M2 (PKM2), glucose transporter 1 (GLUT1), and Ki-67 expression via immunohistochemistry and evaluated the correlation between standardized uptake value (SUV) and these factors. RESULTS: High HIF-1α, PKM2, and GLUT1 expression were positively correlated with high SUV expression (P = 0.0444, 0.0296, and 0.0245, respectively). Ki-67 and SUV were also positively correlated (P = 0.00164). HIF-1α expression and PKM2 expression were significantly correlated (P = 0.0430), and PKM2 expression and GLUT1 expression were extremely significantly correlated (P < 0.0001). CONCLUSION: SUV reflected tumor proliferation or metabolic factors in CRLM. FDG-PET could be a useful modality for assessing tumor viability and may provide useful information regarding the appropriate treatment strategy for CRLM.

High Stromal TGFBI in Lung Cancer and Intratumoral CD8-Positive T Cells were Associated with Poor Prognosis and Therapeutic Resistance to Immune Checkpoint Inhibitors.

BACKGROUND: We investigated whether the expression of transforming growth factor-beta-induced protein (TGFBI) and intratumoral immune cells including CD8- and Forkhead box protein P3 (Foxp3)-positive T cells in clinical lung cancer patients could predict the therapeutic response to nivolumab. METHODS: Thirty-three patients who were treated with nivolumab were enrolled in this study. Immunohistochemical analyses of TGFBI, PD-L1, CD8, Foxp3, and vimentin expression were conducted. Serum concentrations of TGFBI and transforming growth factor-beta1 (TGF-ß1) were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Cancer TGFBI was not associated with prognosis and therapeutic response to nivolumab, but cancer stromal TGFBI and intratumoral CD8-positive T cells were associated with them. Therefore, we evaluated cancer stromal TGFBI and intratumoral CD8-positive T cells. The high-TGFBI-expression group had poorer clinical responses than did the low-TGFBI-expression group (p < 0.0001). The number of times nivolumab was administered in the high-CD8-expression group was significantly higher than that in the low-CD8-expression group (p = 0.0046). The high-CD8-expression group had better clinical responses than did the low-CD8-expression group (p = 0.0013). Interestingly, all patients in the high-TGFBI/low-CD8-expression group had progressive disease (PD). In contrast, all patients in the low-TGFBI/high-CD8-expression group had PR + SD (partial response + stable disease) by the Response Evaluation Criteria in Solid Tumors (RECIST 1.1). CONCLUSIONS: The dual evaluation of stromal TGFBI and intratumoral CD8-positive T cells could be a useful predictive marker for nivolumab.

Fluorodeoxyglucose uptake is associated with low tumor-infiltrating lymphocyte levels in patients with small cell lung cancer.

OBJECTIVES: Positron emission tomography (PET) using 2-deoxy-2-[18F] fluoro-D-glucose (18F-FDG) is a clinically useful modality for cancer evaluation. The mechanism of 18F-FDG uptake within cancer cells involves the glucose transporter 1 (GLUT1) and hypoxia-inducible factor-1 α (HIF-1α). Although recent research has shown its clinical efficacy in small-cell lung cancer (SCLC), no suitable biomarker has been identified. We conducted a clinicopathological study to examine the relationship between tumor immunity and 18F-FDG uptake in patients with SCLC. MATERIALS AND METHODS: Tumor sections were stained by immunohistochemistry for GLUT1, HIF-1α, PD-L1, CD4, CD8, and Foxp3. The relationship between clinicopathological features and 18F-FDG uptake was analyzed. Student's t-test, χ2 test, non-parametric Spearman's rank test, and Kaplan-Meier method were used to evaluate associations between the variables. RESULTS: A total of 98 patients 78 men and 20 women who underwent 18F-FDG PET, were enrolled in this study. PD-L1 was expressed in 36.7% (36/98) of all patients; this was significantly associated with GLUT1 expression (p = 0.04). The accumulation of 18F-FDG was significantly higher in patients with low CD8 and CD4 TILs than in those with high TILs (p = 0.03 and p = 0.01, respectively). The uptake of 18F-FDG was not significantly associated with the expression of either Foxp3 or PD-L1. Multivariate analysis demonstrated that advanced stage, poor ECOG-PS, and high SUVmax were independent predictors of poor OS. Among patients with limited-stage disease, multivariate analysis confirmed high PD-L1 expression and a high SUVmax to be independent predictors of poor OS. However, only ECOG-PS was found to be an independent predictor of poor OS among patients with extensive-stage tumors. CONCLUSION: High SUVmax on 18F-FDG-PET is correlated with low expression of CD8(+) and CD4(+) TILs, but is an independent prognostic factor for OS, particularly in those with limited disease. Further studies are warranted to validate our findings.

Carboxypeptidase A4 accumulation is associated with an aggressive phenotype and poor prognosis in triple-negative breast cancer.

Using whole transcriptome analysis and a lentiviral short hairpin RNA screening library, carboxypeptidase A4 (CPA4) was identified as a novel marker in breast cancer and a therapeutic target in triple­negative breast cancer (TNBC) in the present study. Immunohistochemistry was used to evaluate the presence of CPA4, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, Ki67, epidermal growth factor receptor, cytokeratin 5/6, aldehyde dehydrogenase 1, cluster of differentiation (CD)44, CD24, claudins, E­cadherin, vimentin and androgen receptor in 221 cases of breast cancer, including 68 TNBC cases. The effects of CPA4 on the viability and migration ability of TNBC cells were analyzed using RNA interference methods. Increased CPA4 expression, specifically in the cytoplasm of cancer tissue cells, was detected. Furthermore, high CPA4 expression in TNBC cases was associated with low expression of E­cadherin and with the expression of cancer stem cell markers (high CD44/low CD24). Patients with TNBC and high levels of CPA4 expression had a significantly poorer prognosis compared with those with low CPA4 expression. Notably, viability and migration were reduced, but E­cadherin expression was upregulated in CPA4­suppressed TNBC cells. The present data suggested that CPA4 may be a novel inducer for epithelial­mesenchymal transition, which is characterized by the downregulation of E­cadherin and mesenchymal phenotypes. To conclude, CPA4 may be a marker for poor prognosis and a promising therapeutic target in TNBC with aggressive phenotypes.

Conophylline suppresses pancreatic cancer desmoplasia and cancer-promoting cytokines produced by cancer-associated fibroblasts.

Despite recent advances in cancer treatment, pancreatic cancer is a highly malignant tumor type with a dismal prognosis and it is characterized by dense desmoplasia in the cancer tissue. Cancer-associated fibroblasts (CAF) are responsible for this fibrotic stroma and promote cancer progression. We previously reported that a novel natural compound conophylline (CnP) extracted from the leaves of a tropical plant reduced liver and pancreatic fibrosis by suppression of stellate cells. However, there have been no studies to investigate the effects of CnP on CAF, which is the aim of this work. Here, we showed that CAF stimulated indicators of pancreatic cancer malignancy, such as proliferation, invasiveness, and chemoresistance. We also showed that CnP suppressed CAF activity and proliferation, and inhibited the stimulating effects of CAF on pancreatic cancer cells. Moreover, CnP strongly decreased the various cytokines involved in cancer progression, such as interleukin (IL)-6, IL-8, C-C motif chemokine ligand 2 (CCL2), and C-X-C motif chemokine ligand 12 (CXCL12), secreted by CAF. In vivo, CAF promoted tumor proliferation and desmoplastic formation in a mouse xenograft model, CnP reduced desmoplasia of tumors composed of pancreatic cancer cells + CAF, and combination therapy of CnP with gemcitabine remarkably inhibited tumor proliferation. Our findings suggest that CnP is a promising therapeutic strategy of combination therapy with anticancer drugs to overcome refractory pancreatic cancers.

Mutant TP53 modulates metastasis of triple negative breast cancer through adenosine A2b receptor signaling.

PURPOSE: The identification of genes with synthetic lethality in the context of mutant TP53 is a promising strategy for the treatment of basal-like triple negative breast cancer (TNBC). This study investigated regulators of mutant TP53 (R248Q) in basal-like TNBC and their impact on tumorigenesis. EXPERIMENTAL DESIGN: TNBC cells were analyzed by RNA-seq, and synthetic-lethal shRNA knock-down screening, to identify genes related to the expression of mutant TP53. A tissue microarray of 232 breast cancer samples, that included 66 TNBC cases, was used to assess clinicopathological correlates of tumor protein expression. Functional assays were performed in vitro and in vivo to assess the role of ADORA2B in TNBC. RESULTS: Transcriptome profiling identified ADORA2B as up-regulated in basal-like TNBC cell lines with R248Q-mutated TP53, with shRNA-screening suggesting the potential for a synthetic-lethal interaction between these genes. In clinical samples, ADORA2B was highly expressed in 39.4% (26/66) of TNBC patients. ADORA2B-expression was significantly correlated with ER (P < 0.01), PgR (P = 0.027), EGFR (P < 0.01), and tumor size (P = 0.037), and was an independent prognostic factor for outcome (P = 0.036). In line with this, ADORA2B-transduced TNBC cells showed increased tumorigenesis, and ADORA2B knockdown, along with mutant p53 knockdown, decreased metastasis both in vitro and in vivo. Notably, the cytotoxic cyclic peptide SA-I suppressed ADORA2B expression and tumorigenesis in TNBC cell lines. CONCLUSIONS: ADORA2B expression increases the oncogenic potential of basal-like TNBC and is an independent factor for poor outcome. These data suggest that ADORA2B could serve as a prognostic biomarker and a potential therapeutic target for basal-like TNBC.

High stromal transforming growth factor ß-induced expression is a novel marker of progression and poor prognosis in gastric cancer.

BACKGROUND AND OBJECTIVES: Transforming growth factor ß-induced (TGFBI) protein is a secreted extracellular matrix protein with conflicting roles in cancer, acting as a tumour suppressor and a promoter, which appears to be tissue specific. The role of TGFBI in gastric cancer (GC) remains unclear, which we aimed to investigate using the clinical samples as well as an in vitro coculture model of GC. METHODS: The clinical significance of TGFBI was assessed in 208 GC samples using immunohistochemistry. Molecular function of TGFBI in the GC cells was examined by small interfering RNA-mediated TGFBI downregulation in the gastric fibroblasts cocultured with the GC cells. RESULTS: TGFBI expression was localised mainly in the cancer stroma and not in the noncancerous gastric tissue or the GC cells. High TGFBI expression was significantly associated with poor prognosis and cancer progression. Downregulation of TGFBI in the cocultured gastric fibroblasts inhibited the invasion and migration abilities of the GC cells. CONCLUSIONS: High stromal TGFBI expression might be a useful predictive marker for poor prognosis in GC patients. Furthermore, TGFBI in the cancer stromal cells is a promising target for GC treatment.

High expression of forkhead box protein C2 is associated with aggressive phenotypes and poor prognosis in clinical hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the major causes of tumor death; thus, the identification of markers related to its diagnosis and prognosis is critical. Previous studies have revealed that epithelial-to-mesenchymal transition (EMT) is involved in tumor invasion and metastasis, and the forkhead box protein C2 (FOXC2) has been shown to promote tumor cell proliferation, invasion, and EMT. In the present study, we examined the clinicopathological significance of FOXC2 and EMT-related markers in clinical HCC specimens and identified factors related to the diagnosis and prognosis of HCC. METHODS: The expression of FOXC2 and EMT-related markers was evaluated by immunohistochemistry in 84 cases of hepatocellular carcinoma. RESULTS: A high expression of FOXC2 was observed in 26 of 84 cases, and expression was significantly correlated with background liver cirrhosis, poor tumor differentiation, high serum AFP, and elevated cell proliferation markers. In addition, this high expression was related to the induction of the Cadherin switch and vimentin expression and was an independent predictor for poor prognosis. CONCLUSION: The high expression of FOXC2 in HCC is correlated with tumor malignancy and poor prognosis, suggesting that FOXC2 may be an important prognostic factor for HCC.

High expression of carcinoembryonic antigen and telomerase reverse transcriptase in circulating tumor cells is associated with poor clinical response to the immune checkpoint inhibitor nivolumab.

The present study aimed to enrich circulating tumor cells (CTCs) from blood samples using a new size-sorting CTC chip. The present study also set out to identify a blood sensitivity marker for the immune checkpoint inhibitor nivolumab in patients with advanced, pre-treatment lung cancer. The CTC sorting efficacy of the chip was investigated and the large cell fraction of blood samples from 15 patients with pre-treatment lung cancer who were later administered nivolumab were purified. The expression levels of carcinoembryonic antigen (CEA), human Telomerase Reverse Transcriptase (hTERT), cytokeratin19 (CK19), and programmed death ligand-1 (PD-L1) were investigated to clarify the association between these CTC markers and the clinical response to nivolumab. The CTC chip effectively enriched cells from lung cancer cell line PC-9. The large cell fraction had a high expression of CEA and hTERT, with the former being significantly associated with the clinical response to nivolumab. The expression of CEA and hTERT in CTCs derived from the blood of a patient with lung cancer were also validated. The evaluation of CEA and possibly hTERT in CTCs collected by the CTC chip may represent a promising predictive blood marker for sensitivity to nivolumab. To the best of our knowledge this is the first report to describe the predictive CTC marker for nivolumab in pre-treatment patients.

Elucidation of the Anatomical Mechanism of Nodal Skip Metastasis in Superficial Thoracic Esophageal Squamous Cell Carcinoma.

BACKGROUND: Lymph node metastasis (LNM) is a standard mechanism of cancer progression in esophageal squamous cell carcinoma (ESCC). We aimed to clarify the anatomical mechanism of skip nodal metastasis to mediastinal zones by analyzing the relationship between LNM to sentinel zones and lymphatic vessel counts in the muscle layer adjacent to the outer esophagus. METHODS: We examined the surgical records of 287 patients with ESCC who underwent potentially curative surgery (three-field lymphadenectomy) and whole esophagi, including pharynges and stomachs from 10 cadavers, to determine the number of lymphatic vessels in the intra-outer longitudinal muscle layer adjacent to the outer esophagus of the cervical (Ce), upper thoracic, middle thoracic (Mt), lower thoracic (Lt), and abdominal esophagi (Ae). RESULTS: The frequency of LNM to the middle mediastinal and supraclavicular zones, including the Mt and Ce, respectively, was lower than to the upper and lower mediastinal and abdominal zone in patients with superficial and advanced thoracic ESCC. In cadavers, the lymphatic vessel counts of the intra-outer longitudinal muscle layer in the Mt and Ce were significantly lower than those of the Lt and Ae, suggesting that lymphatic flow toward the outside of the Mt and Ce was not more abundant than to other sites. CONCLUSION: Our anatomical data suggested that the absence of intra-muscle lymphatic vessels in the middle mediastinal and supraclavicular zones causes skip LNM in patients with thoracic ESCC. Thus, standard esophagectomy with lymph node dissection, including distant zones, may be appropriate for treating patients with superficial thoracic ESCC.

Re-evaluation of MIB-1 immunostaining for diagnosing hyalinizing trabecular tumour of the thyroid: semi-automated techniques with manual antigen retrieval are more accurate than fully automated techniques.

Hyalinizing trabecular tumour (HTT) immunohistochemically shows cell membranous immunoreactivity for MIB-1. This aberrant immunoreactivity is an important factor for the diagnosis of HTT. However, fully automated stainers frequently fail to confirm the immunoreactivity. The aim of this study is to investigate the cause of false negative cell membranous immunoreactivity for MIB-1 in HTT using fully automated stainers, to determine potential reasons for the problem, and to establish methods confirming cell membranous immunoreactivity for MIB-1 in HTT. Six participating institutions examined immunoreactivity for MIB-1 in 10 HTT cases using two approaches: fully automated and semi-automated methods. In the latter, antigen retrieval was carried out using manual methods adopted for routine assays at each institute. The autostainers used included the BOND-MAX, BOND-III, Benchmark XT, and Omnis systems. Using fully automated methods, institute E showed cell membranous MIB-1 positivity in all HTT cases. In contrast, at institute D, all HTT cases were negative. The positive rates of the remaining four institutes ranged from 10% to 20%. The incidence of positive cases using semi-automated methods was 100%, 90%, 90%, 30%, 80%, and 100% at institutes A, B, C, D, E, and F, respectively. We assert that antigen retrieval should be conducted manually for diagnosis of HTT; furthermore, definitively diagnosed HTT should be prepared as the external positive control.

Small cholangiolocellular carcinoma that was difficult to distinguish from cholangiocellular carcinoma: a case report.

BACKGROUND: Cholangiolocellular carcinoma (CoCC) is thought to be derived from hepatic progenitor cells. Because of its origin, CoCC has diverse clinicopathological and imaging findings. Here, we report a case of small CoCC that was difficult to diagnose preoperatively. CASE PRESENTATION: A 62-year-old woman was confirmed with a small liver nodule in the left lobe 2 years after a sustained virological response of hepatitis C virus. The size of the nodule was 11.9 × 6.1 mm, and 6 months later, the size increased to 12.5 × 7.8 mm. The doubling time of this tumor was 285 days. The tumor revealed peripheral early enhancement and delayed internal staining in dynamic computed tomography images and marked high intensity in diffusion-weighted magnetic resonance imaging scans. These imaging findings resembled those of cholangiocellular carcinoma (CCC). The tumor was removed by laparoscopic lateral sectionectomy. Pathological findings revealed that the tumor was composed of small cuboidal cells and showed irregular anastomosis small grand. Immunohistochemical findings showed that the tumor cells were negative for Hep-par 1 and positive for cytokeratin 19. Epithelial membrane antigen staining was positive for the membranous side of the lumen. According to these pathological findings, the tumor was diagnosed as CoCC. CONCLUSION: Although some characteristic imaging findings are reported for CoCC, they are not specific because of the variety in pathological findings. Especially, small CoCCs might have poor characteristic imaging findings and may be difficult to distinguish from CCC in the images. However, slow tumor growth might be one of the characteristics to suspect the possibility of a CoCC.