Hideaki Kinugasa

Detection of K-ras gene mutation by liquid biopsy in patients with pancreatic cancer

Cell‐free circulating tumor DNA (ctDNA) in serum has been considered to be a useful candidate for noninvasive cancer diagnosis. The current study was designed to estimate the clinical usefulness of genetic analysis for ctDNA by digital polymerase chain reaction in patients with pancreatic cancer.

Mitochondrial SOD2 regulates epithelial–mesenchymal transition and cell populations defined by differential CD44 expression

Epithelial–mesenchymal transition (EMT) promotes cancer cell invasion, metastasis and treatment failure. EMT may be activated in cancer cells by reactive oxygen species (ROS). EMT may promote conversion of a subset of cancer cells from a CD44low-CD24high (CD44L) epithelial phenotype to a CD44high-CD24−/low (CD44H) mesenchymal phenotype, the latter associated with increased malignant properties of cancer cells. ROS are required for cells undergoing EMT, although excessive ROS may induce cell death or senescence; however, little is known as to how cellular antioxidant capabilities may be regulated during EMT. Mitochondrial superoxide dismutase 2 (SOD2) is frequently overexpressed in oral and esophageal cancers. Here, we investigate mechanisms of SOD2 transcriptional regulation in EMT, as well as the functional role of this antioxidant in EMT. Using well-characterized genetically engineered oral and esophageal human epithelial cell lines coupled with RNA interference and flow cytometric approaches, we find that transforming growth factor (TGF)-β stimulates EMT, resulting in conversion of CD44L to CD44H cells, the latter of which display SOD2 upregulation. SOD2 induction in transformed keratinocytes was concurrent with suppression of TGF-β-mediated induction of both ROS and senescence. SOD2 gene expression appeared to be transcriptionally regulated by NF-κB and ZEB2, but not ZEB1. Moreover, SOD2-mediated antioxidant activity may restrict conversion of CD44L cells to CD44H cells at the early stages of EMT. These data provide novel mechanistic insights into the dynamic expression of SOD2 during EMT. In addition, we delineate a functional role for SOD2 in EMT via the influence of this antioxidant upon distinct CD44L and CD44H subsets of cancer cells that have been implicated in oral and esophageal tumor biology.

Droplet digital PCR measurement of HER2 in patients with gastric cancer

Background:Although there are some new criteria for human epidermal growth factor receptor 2 (HER2) expression with immunohistochemistry/fluorescence in situ hybridisation (IHC/FISH) in gastric cancer, the method is still ambiguous and is somewhat dependent on the subjective qualities of the evaluator.Methods:We used droplet digital polymerase chain reaction (ddPCR) to evaluate HER2 amplification in formalin-fixed and paraffin-embedded (FFPE) samples and cell-free serum circulating tumour DNA (ctDNA) in 25 patients with gastric cancer.Results:The concordance rate of HER2 amplification examined in FFPE samples with ddPCR and IHC/FISH was 92% (23 out of 25). The concordance rate of FFPE with ctDNA was not high (62.5%); however, patients who were HER2-positive by ctDNA had significantly shorter survival compared with HER2-negative patients.Conclusions:Our results demonstrated that this ddPCR method was as effective as IHC/FISH and therefore might become a standard method for analysing not only FFPE but also ctDNA.

EGFR inhibitors prevent induction of cancer stem-like cells in esophageal squamous cell carcinoma by suppressing epithelial-mesenchymal transition

There exists a highly tumorigenic subset of esophageal squamous cell carcinoma (ESCC) cells defined by high expression of CD44. A novel therapy targeting these cancer stem-like cells (CSCs) is needed to improve prognosis of ESCC. CSCs of ESCC have a mesenchymal phenotype and epithelial-mesenchymal transition (EMT) is critical to enrich and maintain CSCs. EGFR, frequently overexpressed in ESCC, has pivotal roles in EMT induced by TGF-β in invasive fronts. Thus, EMT in invasive fronts of ESCC might be important for CSCs and EGFR could be a target of a novel therapy eliminating CSCs. However, effects of EGFR inhibitors on CSCs in ESCC have not been fully examined. EGFR inhibitors, erlotinib and cetuximab, significantly suppressed enrichment of CSCs via TGF-β1-mediated EMT. Importantly, EGFR inhibitors sharply suppressed ZEB1 that is essential for EMT in ESCC. Further, EGFR inhibitors activated Notch1 and Notch3, leading to squamous cell differentiation. EGFR inhibition may suppress expression of ZEB1 and induce differentiation, thereby blocking EMT-mediated enrichment of CSCs. In organotypic 3D culture, a form of human tissue engineering, tumor cells in invasive nests showed high expression of CD44. Erlotinib significantly blocked invasion into the matrix and CD44 high expressing CSCs were markedly suppressed by erlotinib in organotypic 3D culture. In conclusion, EMT is a critical process for generation of CSCs and the invasive front of ESCC, where EMT occurs, might form a CSC niche in ESCC. EGFR inhibitors could suppress EMT in invasive fronts and be one therapeutic option targeting against generation of CSCs in ESCC.

Involvement of platelets in extrahepatic metastasis of hepatocellular carcinoma

Recently, a relationship between platelets and cancer metastasis has been reported. The aim of this study is to elucidate the risk factors for extrahepatic metastasis (EHM), with emphasis on association with platelets in patients, with hepatocellular carcinoma (HCC).

Use of non-invasive serum glycan markers to distinguish non-alcoholic steatohepatitis from simple steatosis

Serum glycans have been reported to be promising diagnostic markers for many inflammatory diseases and cancers. The aims of this study were to investigate whole glycan expression in patients with non‐alcoholic fatty liver diseases and to evaluate the potential use of glycan profiles as new clinical biomarkers to distinguish non‐alcoholic steatohepatitis (NASH) from simple steatosis (SS).

Prognotic impact of serum follistatin in patients with hepatocellular carcinoma

Follistatin (FST) is a glycoprotein expressed in most organs, which interacts with activins or other members of the transforming growth factor beta family. Recently, several reports have shown that FST regulates a variety of processes during tumor progression. Here, serum FST in patients with liver diseases was measured, and its clinical utility as a biomarker was assessed.

Low frequency of drug-resistant virus did not affect the therapeutic efficacy in daclatasvir plus asunaprevir therapy in patients with chronic HCV genotype-1 infection.

BACKGROUND The efficacy of a direct-acting antiviral agent (DAA) is compromised by the development of drug resistance. The associations between resistance-associated virus (RAV) and therapeutic outcomes have not been well-understood. METHODS A total of 30 patients with HCV genotype-1b were enrolled and treated for 24 weeks with asunaprevir (ASV) and daclatasvir (DCV). Viral sequences in non-structural (NS) regions 3 and 5A in serum and liver tissue before treatment were examined with direct sequencing, next-generation sequencing (NGS) and the PCR-invader method to evaluate the importance of drug-resistance in the prediction of the outcomes of ASV plus DCV therapy. RESULTS Of 30 patients (22 treatment-naive patients, 2 interferon-intolerant patients and 6 non-responders), 25 patients (83.3%) achieved sustained virological response (SVR) 24 weeks after the treatment. Viral breakthrough occurred in three treatment-naive patients and one non-responder. One treatment-naive patient experienced viral relapse. Among 25 patients without RAV, 24 obtained SVR, whereas 5 patients had RAV with a 1.3 to 88% frequency, resulting in various therapeutic outcomes. As for HCV compartments, similar RAVs were detected in serum and liver tissue for a patient obtaining SVR despite HCV NS5A Y93H and another developed viral breakthrough although no RAV was detected. Direct sequencing could not detect RAVs in low frequency (1.3 to 12%) for three of four patients. CONCLUSIONS Low frequency of RAVs might not affect the outcomes of ASV plus DCV therapy. Deep sequencing and PCR-invader methods can detect clinically significant RAVs for ASV plus DCV therapy.

Alteration of N-glycan profiles in patients with chronic hepatitis and hepatocellular carcinoma

Most of the modification of N‐glycosylation reported in cancers including hepatocellular carcinoma (HCC) were based on the examinations of a small number of patients or particular proteins. The aim of this study is to reveal changes in whole serum N‐glycan profiles systematically during the process of hepatocarcinogenesis and to elucidate their clinical application.

Clinical utility of serum fucosylated hemopexin in Japanese patients with hepatocellular carcinoma

Aim:  Hepatocellular carcinoma (HCC) is a common clinical problem all over the world. Fucosylated hemopexin (Fuc‐Hpx) is a newly reported glycoprotein for the diagnosis of HCC, however, its clinical implications are unclear. The aim of this study was to elucidate the clinical utility of Fuc‐Hpx in Japanese patients with HCC.