Yoshiaki Shinden

Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer.

Background:Functional microRNAs (miRNAs) in exosomes have been recognised as potential stable biomarkers in cancers. The aim of this study is to identify specific miRNAs in exosome as serum biomarkers for the early detection of recurrence in human colorectal cancer (CRC).Methods:Serum samples were sequentially obtained from six patients with and without recurrent CRC. The miRNAs were purified from exosomes, and miRNA microarray analysis was performed. The miRNA expression profiles and copy number aberrations were explored using microarray and array CGH analyses in 124 CRC tissues. Then, we validated exosomal miRNAs in 2 serum sample sets (90 and 209 CRC patients) by quantitative real-time RT–PCR.Results:Exosomal miR-17-92a cluster expression level in serum was correlated with the recurrence of CRC. Exosomal miR-19a expression levels in serum were significantly increased in patients with CRC as compared with healthy individuals with gene amplification. The CRC patients with high exosomal miR-19a expression showed poorer prognoses than the low expression group (P<0.001).Conclusions:Abundant expression of exosomal miR-19a in serum was identified as a prognostic biomarker for recurrence in CRC patients.

Identification of a bona fide microRNA biomarker in serum exosomes that predicts hepatocellular carcinoma recurrence after liver transplantation

BackgroundPredictive biomarkers for the recurrence of hepatocellular carcinoma (HCC) have great benefit in the selection of treatment options, including liver transplantation (LT), for HCC. The purpose of this study was to identify specific microRNAs (miRs) in exosomes from the serum of patients with recurrent HCC and to validate these molecules as novel biomarkers for HCC recurrence.MethodsWe employed microarray-based expression profiling of miRs derived from exosomes in the serum of HCC patients to identify a biomarker that distinguishes between patients with and without HCC recurrence after LT. This was followed by the validation in a separate cohort of 59 HCC patients who underwent living related LT. The functions and potential gene targets of the recurrence-specific miRs were analysed using a database, clinical samples and HCC cell lines.ResultsWe found that miR-718 showed significantly different expression in the serum exosomes of HCC cases with recurrence after LT compared with those without recurrence. Decreased expression of miR-718 was associated with HCC tumour aggressiveness in the validated cohort series. We identified HOXB8 as a potential target gene of miR-718, and its upregulation was associated with poor prognosis.ConclusionCirculating miRs in serum exosomes have potential as novel biomarkers for predicting HCC recurrence.

Integrated Multiregional Analysis Proposing a New Model of Colorectal Cancer Evolution.

Understanding intratumor heterogeneity is clinically important because it could cause therapeutic failure by fostering evolutionary adaptation. To this end, we profiled the genome and epigenome in multiple regions within each of nine colorectal tumors. Extensive intertumor heterogeneity is observed, from which we inferred the evolutionary history of the tumors. First, clonally shared alterations appeared, in which C>T transitions at CpG site and CpG island hypermethylation were relatively enriched. Correlation between mutation counts and patients’ ages suggests that the early-acquired alterations resulted from aging. In the late phase, a parental clone was branched into numerous subclones. Known driver alterations were observed frequently in the early-acquired alterations, but rarely in the late-acquired alterations. Consistently, our computational simulation of the branching evolution suggests that extensive intratumor heterogeneity could be generated by neutral evolution. Collectively, we propose a new model of colorectal cancer evolution, which is useful for understanding and confronting this heterogeneous disease.

Rapid intraoperative visualization of breast lesions with γ-glutamyl hydroxymethyl rhodamine green

We previously developed γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) as a tool to detect viable cancer cells, based on the fact that the enzyme γ-glutamyltranspeptidase (GGT) is overexpressed on membranes of various cancer cells, but is not expressed in normal tissue. Cleavage of the probe by GGT generates green fluorescence. Here, we examined the feasibility of clinical application of gGlu-HMRG during breast-conserving surgery. We found that fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors, even those smaller than 1 mm in size, from normal mammary gland tissues, with 92% sensitivity and 94% specificity, within only 5 min after application. We believe this rapid, low-cost method represents a breakthrough in intraoperative margin assessment during breast-conserving surgery.

Decreased Expression of Fructose-1,6-bisphosphatase Associates with Glucose Metabolism and Tumor Progression in Hepatocellular Carcinoma

Fructose-1,6-bisphosphatase (FBP1), the rate-limiting enzyme in gluconeogenesis, is reduced in expression in certain cancers where it has been hypothesized to act as a tumor suppressor, including in hepatocellular carcinoma (HCC). Here, we report functional evidence supporting this hypothesis, providing a preclinical rationale to develop FBP1 as a therapeutic target for HCC treatment. Three independent cohorts totaling 594 cases of HCC were analyzed to address clinical significance. Lower FBP1 expression associated with advanced tumor stage, poor overall survival, and higher tumor recurrence rates. In HCC cell lines, where endogenous FBP1 expression is low, engineering its ectopic overexpression inhibited tumor growth and intracellular glucose uptake by reducing aerobic glycolysis. In patient specimens, promoter methylation and copy-number loss of FBP1 were independently associated with decreased FBP1 expression. Similarly, FBP1 downregulation in HCC cell lines was also associated with copy-number loss. HCC specimens exhibiting low expression of FBP1 had a highly malignant phenotype, including large tumor size, poor differentiation, impaired gluconeogenesis, and enhanced aerobic glycolysis. The effects of FBP1 expression on prognosis and glucose metabolism were confirmed by gene set enrichment analysis. Overall, our findings established that FBP1 downregulation in HCC contributed to tumor progression and poor prognosis by altering glucose metabolism, and they rationalize further study of FBP1 as a prognostic biomarker and therapeutic target in HCC patients. Cancer Res; 76(11); 3265-76. ©2016 AACR.

Circulating tumour cell-derived plastin3 is a novel marker for predicting long-term prognosis in patients with breast cancer

Background:Identification of promising biomarkers that predict the prognosis of patients with breast cancer is needed. In this study, we hypothesised that the expression of the epithelial–mesenchymal transition-related biomarker plastin3 (PLS3) in peripheral blood could be a prognostic factor in breast cancer.Methods:We examined PLS3 expression in breast cancer cell lines with epithelial and mesenchymal traits and in circulating tumour cells (CTCs) obtained from the peripheral blood of breast cancer patients. We investigated PLS3 expression in the peripheral blood of 594 patients with breast cancer to evaluate the clinical significance of PLS3 expression.Results:Robust PLS3 expression was observed in different breast cancer cell lines (Hs578t, MCF-7, MDA-MB-468, and MDA-MB-231) as well as in a bone marrow derived cancer cell line (BC-M1). In both the training (n=298) and validation (n=296) sets, PLS3 expression was observed in CTCs of patients with breast cancer. PLS3-positive patients showed significantly poorer overall and disease-free survival than PLS3-negative patients (P=0.0001 and 0.003, respectively). Subset analysis revealed that this prognostic biomarker was relevant in patients with stage I–III cancer, particularly in patients with luminal-type and triple-negative-type tumours.Conclusions:These data demonstrated that PLS3 was expressed in CTCs undergoing the epithelial–mesenchymal transition in patients with breast cancer. Furthermore, PLS3 may be an excellent biomarker for identifying groups at risk of recurrence or with a poor prognosis.

A highly accurate inclusive cancer screening test using Caenorhabditis elegans scent detection

Early detection and treatment are of vital importance to the successful eradication of various cancers, and development of economical and non-invasive novel cancer screening systems is critical. Previous reports using canine scent detection demonstrated the existence of cancer-specific odours. However, it is difficult to introduce canine scent recognition into clinical practice because of the need to maintain accuracy. In this study, we developed a Nematode Scent Detection Test (NSDT) using Caenorhabditis elegans to provide a novel highly accurate cancer detection system that is economical, painless, rapid and convenient. We demonstrated wild-type C. elegans displayed attractive chemotaxis towards human cancer cell secretions, cancer tissues and urine from cancer patients but avoided control urine; in parallel, the response of the olfactory neurons of C. elegans to the urine from cancer patients was significantly stronger than to control urine. In contrast, G protein α mutants and olfactory neurons-ablated animals were not attracted to cancer patient urine, suggesting that C. elegans senses odours in urine. We tested 242 samples to measure the performance of the NSDT, and found the sensitivity was 95.8%; this is markedly higher than that of other existing tumour markers. Furthermore, the specificity was 95.0%. Importantly, this test was able to diagnose various cancer types tested at the early stage (stage 0 or 1). To conclude, C. elegans scent-based analyses might provide a new strategy to detect and study disease-associated scents.

Allelic Imbalance at an 8q24 Oncogenic SNP is Involved in Activating MYC in Human Colorectal Cancer

AbstractBackground The rs6983267 at 8q24.21 has been established as a significant cancer-related single nucleotide polymorphism (SNP). The risk allele showed similarity to the binding site of transcription factor TCF4/LEF1 that activates transcription of MYC. However, little is known about the role of this SNP in increasing MYC activity in colorectal cancers (CRCs).MethodsThe genotypes of rs6983267 in peripheral blood and primary cancers, MYC activity and copy number (CN) alteration were examined in 107 CRCs. Next, we plotted the number of cancers cell lines exhibiting specific G/T genotypes in 746 cancer cell lines of the Sanger Institute database. Then we validated the relationship between the 8q24 SNP status and clinicopathologic parameters in 68 CRCs with loss of heterozygosity (LOH).ResultsThe MYC module activity was activated by either transcription in the risk allele (G) or by amplification in the non-risk allele (T). Then, we confirmed that the CN amplification dominantly occurred in the non-risk allele, whereas CN neutral LOH, which indicated uniparental disomy (UPD) was more frequently observed for the risk allele. Finally, we confirmed that risk allele dominant cases, either by amplification or by UPD, indicated a more malignant clinical phenotype than non-risk allele dominant cases.ConclusionsThe development of CRC requires MYC activation through retention of the risk allele, or amplification of the non-risk allele at the oncogenic SNP in the site of primary tumor.

An Integrative Analysis to Identify Driver Genes in Esophageal Squamous Cell Carcinoma.

Background Few driver genes have been well established in esophageal squamous cell carcinoma (ESCC). Identification of the genomic aberrations that contribute to changes in gene expression profiles can be used to predict driver genes. Methods We searched for driver genes in ESCC by integrative analysis of gene expression microarray profiles and copy number data. To narrow down candidate genes, we performed survival analysis on expression data and tested the genetic vulnerability of each genes using public RNAi screening data. We confirmed the results by performing RNAi experiments and evaluating the clinical relevance of candidate genes in an independent ESCC cohort. Results We found 10 significantly recurrent copy number alterations accompanying gene expression changes, including loci 11q13.2, 7p11.2, 3q26.33, and 17q12, which harbored CCND1, EGFR, SOX2, and ERBB2, respectively. Analysis of survival data and RNAi screening data suggested that GRB7, located on 17q12, was a driver gene in ESCC. In ESCC cell lines harboring 17q12 amplification, knockdown of GRB7 reduced the proliferation, migration, and invasion capacities of cells. Moreover, siRNA targeting GRB7 had a synergistic inhibitory effect when combined with trastuzumab, an anti-ERBB2 antibody. Survival analysis of the independent cohort also showed that high GRB7 expression was associated with poor prognosis in ESCC. Conclusion Our integrative analysis provided important insights into ESCC pathogenesis. We identified GRB7 as a novel ESCC driver gene and potential new therapeutic target.

Computed tomography Hounsfield units can predict breast cancer metastasis to axillary lymph nodes

BackgroundAxillary lymph node (ALN) status is an important prognostic factor for breast cancer. We retrospectively used contrast-enhanced computed tomography (CE-CT) to evaluate the presence of ALN, metastasis based on size, shape, and contrasting effects.MethodsOf 131 consecutive patients who underwent CE-CT followed by surgery for breast cancer between 2005 and 2012 in our institution, 49 were histologically diagnosed with lymph node metastasis. Maximum Hounsfield units (HU) and mean HU were measured in non-contrasting CT (NC-CT) and CE-CT of ALNs.ResultsOf 12 examined measurements, we found significant differences between negative and metastatic ALNs in mean and maximum NC-CT HU, and mean and maximum CE-CT HU (P < 0.05). We used a receiver operating curve, to determine cut-off values of four items in which significant differences were observed. The highest accuracy rate was noted for the cut-off value of 54 as maximum NC-CT HU for which sensitivity, specificity, and accuracy rate were 79.6%, 80.5% and 80.2%, respectively.ConclusionsCT HU of a patient with breast cancer are absolute values that offer objective disease management data that are not influenced by the screener’s ability.