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Intestinal metaplasia (IM) is a risk factor for gastric cancer following infection with Helicobacter pylori. To explore the susceptibility of pure gastric IM to cancer development, we investigated genetic alterations in single IM gastric glands. We isolated 50 single IM or non-IM glands from the inflamed gastric mucosa of 11 patients with intramucosal gastric carcinoma (IGC) and 4 patients without IGC; 19 single glands in the noninflamed gastric mucosa of 11 individuals from our cohort and previous dataset were also included as controls. Whole-exome sequencing of single glands revealed significantly higher accumulation of somatic mutations in various genes within IM glands compared with non-IM glands. Clonal ordering analysis showed that IM glands expanded to form clusters with shared mutations. In addition, targeted-capture deep sequencing and copy number (CN) analyses were performed in 96 clustered IM or non-IM gastric glands from 26 patients with IGC. CN analyses were also performed on 41 IGC samples and The Cancer Genome Atlas-Stomach Adenocarcinoma datasets. These analyses revealed that polyclonally expanded IM commonly acquired CN aberrations (CNA), including amplification of chromosomes 8, 20, and 2. A large portion of clustered IM glands typically consisted of common CNAs rather than other cancer-related mutations. Moreover, the CNA patterns of clustered IM glands were similar to those of IGC, indicative of precancerous conditions. Taken together, these findings suggest that, in the gastric mucosa inflamed with H. pylori infection, IM glands expand via acquisition of CNAs comparable with those of IGC, contributing to field cancerization.Significance:This study contributes to our understanding of gastric intestinal metaplasia as a risk factor for gastric adenocarcinoma via their multifocal expansion and acquisition of CNAs and somatic mutations.

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Supplementary Table 1 List of antibodies, reagents and kits used in the study Supplementary Table 2 List of primers used in the study Supplementary Table 3 Comprehensive literature search of angiogenic regulators in HCC Supplementary Table 4 Correlation of 178 genes related to angiogenesis in HCC with SULF2 expression in human HCC microarray gene analysis

Supplementary Data from Expansion of Gastric Intestinal Metaplasia with Copy Number Aberrations Contributes to Field Cancerization

Existing antiangiogenic approaches to treat metastatic hepatocellular carcinoma (HCC) are weakly effectual, prompting further study of tumor angiogenesis in this disease setting. Here, we report a novel role for sulfatase 2 (SULF2) in driving HCC angiogenesis. Sulf2-deficient mice (Sulf2 KO) exhibited resistance to diethylnitrosamine-induced HCC and did not develop metastases like wild-type mice (Sulf2 WT). The smaller and less numerous tumors formed in Sulf2 KO mice exhibited a markedly lower microvascular density. In human HCC cells, SULF2 overexpression increased endothelial proliferation, adhesion, chemotaxis, and tube formation in a paracrine fashion. Mechanistic analyses identified the extracellular matrix protein periostin (POSTN), a ligand of αvβ3/5 integrins, as an effector protein in SULF2-induced angiogenesis. POSTN silencing in HCC cells attenuated SULF2-induced angiogenesis and tumor growth in vivo. The TGFβ1/SMAD pathway was identified as a critical signaling axis between SULF2 and upregulation of POSTN transcription. In clinical HCC specimens, elevated levels of SULF2 correlated with increased microvascular density, POSTN levels, and relatively poorer patient survival. Together, our findings define an important axis controlling angiogenesis in HCC and a mechanistic foundation for rational drug development. Cancer Res; 77(3); 632–45. ©2016 AACR.

Supplementary Table from Expansion of Gastric Intestinal Metaplasia with Copy Number Aberrations Contributes to Field Cancerization

Supplementary Figure 1 Schematic representation of experimental design for HCC carcinogenesis in mice. Supplementary Figure 2 Loss of Sulf2 impairs tumor growth and incidence in DEN-model of HCC carcinogenesis. Supplementary Figure 3 Conditioned media from SULF2-transfected HCC cells promotes endothelial cells viability and adhesion. Supplementary Figure 4 In silico analysis of HCC-angiogenic factors. Supplementary Figure 5 Expression of POSTN in Sulf2 knockout or wild type animals.

Intestinal metaplasia (IM) is a risk factor for gastric cancer following infection with Helicobacter pylori. To explore the susceptibility of pure gastric IM to cancer development, we investigated genetic alterations in single IM gastric glands. We isolated 50 single IM or non-IM glands from the inflamed gastric mucosa of 11 patients with intramucosal gastric carcinoma (IGC) and 4 patients without IGC; 19 single glands in the noninflamed gastric mucosa of 11 individuals from our cohort and previous dataset were also included as controls. Whole-exome sequencing of single glands revealed significantly higher accumulation of somatic mutations in various genes within IM glands compared with non-IM glands. Clonal ordering analysis showed that IM glands expanded to form clusters with shared mutations. In addition, targeted-capture deep sequencing and copy number (CN) analyses were performed in 96 clustered IM or non-IM gastric glands from 26 patients with IGC. CN analyses were also performed on 41 IGC samples and The Cancer Genome Atlas-Stomach Adenocarcinoma datasets. These analyses revealed that polyclonally expanded IM commonly acquired CN aberrations (CNA), including amplification of chromosomes 8, 20, and 2. A large portion of clustered IM glands typically consisted of common CNAs rather than other cancer-related mutations. Moreover, the CNA patterns of clustered IM glands were similar to those of IGC, indicative of precancerous conditions. Taken together, these findings suggest that, in the gastric mucosa inflamed with H. pylori infection, IM glands expand via acquisition of CNAs comparable with those of IGC, contributing to field cancerization. Significance: This study contributes to our understanding of gastric intestinal metaplasia as a risk factor for gastric adenocarcinoma via their multifocal expansion and acquisition of CNAs and somatic mutations.

Contrast-enhanced computed tomography (CECT) is generally used to evaluate the response to treatment of hepatocellular carcinoma (HCC); however, CECT is unsuitable for the early prediction of therapeutic effects and frequent monitoring. We aimed to investigate the usefulness of our simplified method for the quantification of tumor vascularity using contrast-enhanced ultrasound (CEUS) with perfluorobutane microbubbles [Sonazoid(®) (GE Healthcare, Oslo, Norway)] to predict the therapeutic effect of lenvatinib. Among the 13 patients studied, nine who had more than a 20% reduction in tumor vascularity within 2 weeks of starting treatment experienced complete response or partial response at 8–12 weeks as assessed by CECT. In contrast, three patients without reductions and one patient with only a slight decrease in tumor vascularity had a poor response to lenvatinib. Quantitative assessment of tumor vascularity by our simplified CEUS-based method could be a useful predictor of therapeutic responses to lenvatinib in patients with HCC.

Encoding the sodium iodide symporter (NIS) by an adenovirus (Ad) is a promising strategy to facilitate non-invasive imaging and radiotherapy of pancreatic cancer. However, insufficient levels of NIS expression in tumor cells have limited its clinical translation. To optimize Ad-based radiotherapy and imaging, we investigated the effect of Ad death protein (ADP) deletion on NIS expression. We cloned two sets of oncolytic NIS-expressing Ads that differed only in the presence or absence of ADP. We found that ADP expression negatively affected NIS membrane localization and inhibited radiotracer uptake. ADP deletion significantly improved NIS-based imaging in pancreatic cancer models including patient-derived xenografts, where effective imaging was possible for up to 6 weeks after a single virus injection. This study demonstrates that improved oncolysis may hinder the therapeutic effect of oncolytic viruses designed to express NIS. In vivo studies in combination with 131I showed potential for effective radiotherapy. This also highlights the need for further investigation into optimal timing of 131I administration and suggests that repeated doses of 131I should be considered to improve efficacy in clinical trials. We conclude that ADP deletion is essential for effective NIS-based theranostics in cancer., Graphical abstract, Sodium iodide symporter (NIS)-expressing oncolytic viruses enable tumor-specific imaging and radiotherapy for infected tumors. We designed a novel NIS-expressing adenoviral vector with deletion of the adenovirus death protein (ADP) gene. ADP deletion improved NIS expression and subsequent imaging capabilities highlighting the role of vector design in effective transgene expression.

BACKGROUND Many patients with hepatocellular carcinoma present with impaired hepatic function, which often requires interruption or withdrawal of lenvatinib due to associated adverse events. We aimed to identify pre-treatment predictors of tolerability and clinical outcome of lenvatinib therapy. METHODS Eighty patients who received lenvatinib at our institution between 2018 and 2020 were included in this study. We assessed essential factors associated with prolonged progression-free survival (PFS), using Cox proportional hazards model. We also investigated the correlation between the factor identified as contributing most to PFS and the relative dose intensity (RDI), response rate, and duration of treatment with lenvatinib. RESULTS Pre-treatment level of Mac-2-binding protein glycosylation isomer (M2BPGi) showed significant association with PFS (hazard ratio = 0.52, P = .0358). Low M2BPGi levels (

Gestational trophoblastic disease (GTD) is a heterogeneous group of lesions arising from placental tissue. Epithelioid trophoblastic tumor (ETT), derived from chorionic-type trophoblast, is the rarest form of GTD with only approximately 130 cases described in the literature. Due to its morphologic mimicry of epithelioid smooth muscle tumors and carcinoma, ETT can be misdiagnosed. To date, molecular characterization of ETTs is lacking. Furthermore, ETT is difficult to treat when disease spreads beyond the uterus. Here using RNA-Seq analysis in a cohort of ETTs and other gestational trophoblastic lesions we describe the discovery of LPCAT1-TERT fusion transcripts that occur in ETTs and coincide with underlying genomic deletions. Through cell-growth assays we demonstrate that LPCAT1-TERT fusion proteins can positively modulate cell proliferation and therefore may represent future treatment targets. Furthermore, we demonstrate that TERT upregulation appears to be a characteristic of ETTs, even in the absence of LPCAT1-TERT fusions, and that it appears linked to copy number gains of chromosome 5. No evidence of TERT upregulation was identified in other trophoblastic lesions tested, including placental site trophoblastic tumors and placental site nodules, which are thought to be the benign chorionic-type trophoblast counterpart to ETT. These findings indicate that LPCAT1-TERT fusions and copy-number driven TERT activation may represent novel markers for ETT, with the potential to improve the diagnosis, treatment, and outcome for women with this rare form of GTD.

Most hepatocellular carcinomas (HCCs) develop on the basis of chronic hepatitis, but the mechanism of epigenetic regulation in inflammatory hepatocarcinogenesis has yet to be elucidated. Among de novo DNA methyltransferases (DNMTs), DNMT3B has lately been reported to act specifically on actively transcribed genes, suggesting the possibility that it plays a role in the pathogenesis of cancer. We confirmed that DNMT3B isoforms lacking its catalytic domain were highly expressed in HCCs compared with non-tumorous liver tissue. To elucidate the role of DNMT3B in hepatocarcinogenesis, we generated a genetically engineered mouse model with hepatocyte-specific Dnmt3b deletion. The liver of the Dnmt3b-deficient mice exhibited an exacerbation of thioacetamide-induced hepatitis, progression of liver fibrosis and a higher incidence of HCC compared with the liver of the control mice. Whole-genome bisulfite sequencing verified a lower CG methylation level in the Dnmt3b-deficient liver, demonstrating differentially methylated regions throughout the genome. Transcriptome analysis revealed decreased expression of genes related to oxidative phosphorylation in the Dnmt3b-deficient liver. Moreover, primary hepatocytes isolated from the Dnmt3b-deficient mice showed reduced mitochondrial respiratory capacity, leading to the enhancement of oxidative stress in the liver tissue. Our findings suggest the protective role of DNMT3B against chronic inflammation and HCC development via maintaining mitochondrial homeostasis.

Hepatocellular carcinoma (HCC) developing after hepatitis C virus (HCV) eradication is a serious clinical concern. However, molecular basis for the hepatocarcinogenesis after sustained virologic response (SVR) remains unclear. In this study, we aimed to unveil the transcriptomic profile of post-SVR liver tissues and explore the molecules associated with post-SVR carcinogenesis. We analysed 90 RNA sequencing datasets, consisting of non-cancerous liver tissues including 20 post-SVR, 40 HCV-positive and 7 normal livers, along with Huh7 cell line specimens before and after HCV infection and eradication. Comparative analysis demonstrated that cell cycle- and mitochondrial function-associated pathways were altered only in HCV-positive non-cancerous liver tissues, whereas some cancer-related pathways were up-regulated in the non-cancerous liver tissues of both post-SVR and HCV-positive cases. The persistent up-regulation of carcinogenesis-associated gene clusters after viral clearance was reconfirmed through in vitro experiments, of which, CYR61, associated with liver fibrosis and carcinogenesis in several cancer types, was the top enriched gene and co-expressed with cell proliferation-associated gene modules. To evaluate whether this molecule could be a predictor of hepatocarcinogenesis after cure of HCV infection, we also examined 127 sera from independent HCV-positive cohorts treated with direct-acting antivirals (DAAs), including 60 post-SVR-HCC patients, and found that the elevated serum Cyr61 was significantly associated with early carcinogenesis after receiving DAA therapy. In conclusion, some oncogenic transcriptomic profiles are sustained in liver tissues after HCV eradication, which might be a molecular basis for the liver cancer development even after viral clearance. Among them, up-regulated CYR61 could be a possible biomarker for post-SVR-HCC.

BACKGROUND/PURPOSE Lenvatinib was recently approved as a novel agent for hepatocellular carcinoma. To maximize the therapeutic effect of anticancer drugs, it is essential to maintain treatment intensity by avoiding dose reduction or discontinuation. We aimed to identify essential factors contributing to achieve sufficient treatment intensity of lenvatinib. METHODS Seventy-one patients who received treatment with lenvatinib were included in this study. We used the delivered dose intensity/body surface area ratio (DBR) to measure treatment intensity of lenvatinib. RESULTS 2M-DBR (DBR for the first 60 days) of lenvatinib (≥206.7) was strongly correlated with objective response and was the significant factor contributing to prolonged progression-free survival (PFS). Patients with high 2M-DBR had significantly prolonged PFS compared with those who had low 2M-DBR (P

International audience; The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease; however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of many well-characterized GLI target genes (BCL2, MYCN, PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g. E2F1, BMP1, CDK2) strongly down-regulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are up-regulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Furthermore, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 co-ordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis.

Recent genetic analyses revealed genetic heterogeneity in hepatocellular carcinoma (HCC), although it remains unclear how genetic alterations contribute to the multistage progression of HCC, especially the early step from hypovascular liver nodules to hypervascular HCC. We conducted multiregional whole-genome sequencing on HCCs with a nodule-in-nodule appearance, consisting of inner hypervascular HCC surrounded by hypovascular HCC arising from a common origin, and identified point mutations, structural variations, and copy-number variations in each specimen. According to the genetic landscape of the inner and outer regions, together with the pathological and radiological findings, we examined the stepwise evolution of cancer cells from slow-growing HCC to rapid-growing HCC. We first demonstrated that most tumor cells consisting of hypovascular well-differentiated HCCs already harbored thousands of point mutations and even several structural variations, including chromosomal translocations and chromothripsis, as the trunk events. Telomerase reverse transcriptase (TERT)-associated aberrations, including promoter mutations, chromosomal translocation, and hepatitis B virus DNA integration, as well as abnormal methylation status, were commonly detected as the trunk aberrations, while various liver cancer-related genes, which differed in each case, had additionally accumulated in the inner dedifferentiated nodules. Further, differences in the trunk and branch mutational signatures suggested a multistep contribution to the mutagenesis in each case. In conclusion, genomic alterations associated with the TERT gene could be the key driver events to form the hypovascular HCC, and additional case-specific driver mutations accumulate during the progression phase, forming intra- and inter-tumoral heterogeneity, confirming the importance of genetic testing before targeting therapy. These data shed light on the process of multistep hepatocarcinogenesis and will be helpful toward investigating new therapeutic strategies for HCC. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Lenvatinib was recently approved as a novel first-line molecular targeted agent (MTA) for treating hepatocellular carcinoma (HCC). The importance of relative dose intensity (RDI) has been shown in the treatment of various types of cancers. However, RDI may not accurately reflect the treatment intensity of lenvatinib, as it is the first oral MTA where the dose is based on the patient&rsquo, s weight. We aimed to evaluate the utility of 2M-DBR (the delivered dose intensity/body surface area ratio at 60 days) by comparing the relationship between 2M-DBR, 2M-RDI (RDI at 60 days), and the therapeutic response. The therapeutic response to lenvatinib was evaluated in 45 patients who underwent computed tomography 8&ndash, 12 weeks after treatment initiation. We also investigated the clinical factors associated with high 2M-DBR. The area under the receiver operating characteristic of 2M-DBR that predicts the response to lenvatinib was higher than that of 2M-RDI (0.8004 vs. 0.7778). Patients with high 2M-DBR achieved significantly better objective responses and disease control rates than those with low 2M-DBR (p &lt, 0.0001 and 0.0008). Patients with high 2M-DBR experienced significantly longer progression-free survival (PFS) than those with low 2M-DBR (p = 0.0001), while there was no significant correlation between 2M-RDI levels and PFS (p = 0.2198). Patients who achieved higher levels of 2M-DBR had a significantly better modified ALBI grade (p = 0.0437), better CONUT score (p = 0.0222), and higher BTR (p = 0.0281). Multivariate analysis revealed that high 2M-DBR was the only significant factor associated with longer PFS. In conclusion, 2M-DBR could be an important factor that reflects treatment intensity and useful for predicting the response to lenvatinib against HCC, instead of 2M-RDI.

Numerous reports have demonstrated a tumor inhibitory effect of polyunsaturated fatty acids (PUFAs). However, the molecular mechanisms modulating this phenomenon are in part poorly understood. Here, we provide evidence of a novel antitumoral mechanism of the PUFA arachidonic acid (AA). In vivo and in vitro experiments showed that AA treatment decreased tumor growth and metastasis and increased apoptosis. Molecular analysis of this effect showed significantly reduced expression of a subset of antiapoptotic proteins, including BCL2, BFL1/A1, and 4-1BB, in AA-treated cells.Wedemonstrated that down-regulation of the transcription factor gliomaassociated protein 1 (GLI1) in AA-treated cells is the underlying mechanism controlling BCL2, BFL1/A1, and 4-1BB expression. Using luciferase reporters, chromatin immunoprecipitation, and expression studies, we found that GLI1 binds to the promoter of these antiapoptotic molecules and regulates their expression and promoter activity. We provide evidence that AA-induced apoptosis and down-regulation of antiapoptotic genes can be inhibited by overexpressing GLI1 in AA-sensitive cells. Conversely, inhibition of GLI1 mimics AA treatments, leading to decreased tumor growth, cell viability, and expression of antiapoptotic molecules. Further characterization showed thatAArepresses GLI1 expression by stimulating nuclear translocation of NFATc1, which then binds the GLI1 promoter and represses its transcription. AA was shown to increase reactive oxygen species. Treatment with antioxidants impaired the AAinduced apoptosis and down-regulation of GLI1 and NFATc1 activation, indicating that NFATc1 activation and GLI1 repression require the generation of reactive oxygen species. Collectively, these results define a novel mechanism underlying AA antitumoral functions that may serve as a foundation for future PUFA-based therapeutic approaches. Fil: Comba, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Almada, Luciana Victoria. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados Unidos Fil: Tolosa, Ezequiel J.. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados Unidos Fil: Iguchi, Eriko. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados Unidos Fil: Marks, David L.. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados Unidos Fil: Vara Messler, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Silva, Renata Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Fernandez-Barrena, Maite G.. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados Unidos Fil: Enriquez-Hesles, Elisa. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados Unidos Fil: Vrabel, Anne L.. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados Unidos Fil: Botta, Bruno. Sapienza University; Italia Fil: Di Marcotulio, Lucia. Sapienza University; Italia Fil: Ellenrieder, Volker. University Medical Center Göttingen; Alemania Fil: Eynard, Aldo Renato. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Pasqualini, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Fernandez Zapico, Martin Ernesto. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados Unidos

AIM Recent advances in next-generation sequencing (NGS) technologies allow for evaluation of genetic alterations in various cancer-related genes in daily clinical practice. Archival formalin-fixed paraffin-embedded (FFPE) tumor tissue is often used for NGS-based clinical sequencing assays; however, the success rate of NGS assays using archival FFPE tumor tissue is reported to be lower than that using fresh tumor tissue. We aimed to evaluate the feasibility and safety of ultrasound (US)-guided liver tumor biopsy for NGS-based multiplex gene assays. METHODS We compared the success rate of NGS assays between archival FFPE tumor tissues and US-guided liver tumor biopsy tissues, and summarized the treatment progress of the patients. RESULTS Next-generation sequencing assays using US-guided liver biopsy samples were successful in all patients (22/22), whereas the success rate with archival FFPE tumor tissue was 84.8% (151/178, P

Purpose Genomic testing has increased the quantity of information available to oncologists. Unfortunately, many identified sequence alterations are variants of unknown significance (VUSs), which thus limit the clinician’s ability to use these findings to inform treatment. We applied a combination of in silico prediction and molecular modeling tools and laboratory techniques to rapidly define actionable VUSs. Materials and Methods Exome sequencing was conducted on 308 tumors from various origins. Most single nucleotide alterations within gene coding regions were VUSs. These VUSs were filtered to identify a subset of therapeutically targetable genes that were predicted with in silico tools to be altered in function by their variant sequence. A subset of receptor tyrosine kinase VUSs was characterized by laboratory comparison of each VUS versus its wild-type counterpart in terms of expression and signaling activity. Results The study identified 4,327 point mutations of which 3,833 were VUSs. Filtering for mutations in genes that were therapeutically targetable and predicted to affect protein function reduced these to 522 VUSs of interest, including a large number of kinases. Ten receptor tyrosine kinase VUSs were selected to explore in the laboratory. Of these, seven were found to be functionally altered. Three VUSs (FGFR2 F276C, FGFR4 R78H, and KDR G539R) showed increased basal or ligand-stimulated ERK phosphorylation compared with their wild-type counterparts, which suggests that they support transformation. Treatment of a patient who carried FGFR2 F276C with an FGFR inhibitor resulted in significant and sustained tumor response with clinical benefit. Conclusion The findings demonstrate the feasibility of rapid identification of the biologic relevance of somatic mutations, which thus advances clinicians’ ability to make informed treatment decisions.

Existing antiangiogenic approaches to treat metastatic hepatocellular carcinoma (HCC) are weakly effectual, prompting further study of tumor angiogenesis in this disease setting. Here, we report a novel role for sulfatase 2 (SULF2) in driving HCC angiogenesis. Sulf2-deficient mice (Sulf2 KO) exhibited resistance to diethylnitrosamine-induced HCC and did not develop metastases like wild-type mice (Sulf2 WT). The smaller and less numerous tumors formed in Sulf2 KO mice exhibited a markedly lower microvascular density. In human HCC cells, SULF2 overexpression increased endothelial proliferation, adhesion, chemotaxis, and tube formation in a paracrine fashion. Mechanistic analyses identified the extracellular matrix protein periostin (POSTN), a ligand of avb3/5 integrins, as an effector protein in SULF2-induced angiogenesis. POSTN silencing in HCC cells attenuated SULF2-induced angiogenesis and tumor growth in vivo. The TGFb1/SMAD pathway was identified as a critical signaling axis between SULF2 and upregulation of POSTN transcription. In clinical HCC specimens, elevated levels of SULF2 correlated with increased microvascular density, POSTN levels, and relatively poorer patient survival. Together, our findings define an important axis controlling angiogenesis in HCC and a mechanistic foundation for rational drug development. Fil: Chen, Gang. Mayo Clinic; Estados Unidos. Wenzhou Medical University; China Fil: Nakamura, Ikuo. Mayo Clinic; Estados Unidos Fil: Dhanasekaran, Renumathy. Mayo Clinic; Estados Unidos. University of Stanford; Estados Unidos Fil: Iguchi, Eriko. Schulze Center For Novel Therapeutics, Mayo Clinic; Estados Unidos Fil: Tolosa, Ezequiel Julian. Schulze Center For Novel Therapeutics, Mayo Clinic; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina Fil: Romecin, Paola A.. Schulze Center For Novel Therapeutics, Mayo Clinic; Estados Unidos Fil: Vera, Renzo Emanuel. Schulze Center For Novel Therapeutics, Mayo Clinic; Estados Unidos. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina Fil: Almada, Luciana Victoria. Schulze Center For Novel Therapeutics, Mayo Clinic; Estados Unidos Fil: Miamen, Alexander G.. Mayo Clinic; Estados Unidos Fil: Chaiteerakij, Roongruedee. Mayo Clinic; Estados Unidos Fil: Zhou, Mengtao. Wenzhou Medical University; China Fil: Asiedu, Michael K.. Mayo Clinic; Estados Unidos Fil: Moser, Catherine D.. Mayo Clinic; Estados Unidos Fil: Han, Shaoshan. Mayo Clinic; Estados Unidos Fil: Hu, Chunling. Mayo Clinic; Estados Unidos Fil: Banini, Bubu A.. Mayo Clinic; Estados Unidos Fil: Oseini, Abdul M.. Mayo Clinic; Estados Unidos Fil: Chen, Yichun. Mayo Clinic; Estados Unidos Fil: Fang, Yong. Mayo Clinic; Estados Unidos Fil: Yang, Dongye. Mayo Clinic; Estados Unidos Fil: Shaleh, Hassan M.. Mayo Clinic; Estados Unidos Fil: Wang, Shaoqing. Mayo Clinic; Estados Unidos Fil: Wu, Dehai. Mayo Clinic; Estados Unidos Fil: Song, Tao. Mayo Clinic; Estados Unidos Fil: Lee, Ju-Seog. Anderson Cancer Center; Estados Unidos Fil: Thorgeirsson, Snorri S.. National Cancer Institute; Estados Unidos Fil: Chevet, Eric. Université Rennes; Francia Fil: Shah, Vijay H.. Mayo Clinic; Estados Unidos Fil: Fernandez-Zapico, Martin E.. Schulze Center For Novel Therapeutics, Mayo Clinic; Estados Unidos Fil: Roberts, Lewis R.. Mayo Clinic; Estados Unidos

Pancreatic cancer is the third leading cause of cancer mortality in the U.S. with close to 40,000 deaths per year. Pancreatic ductal adenocarcinoma (PDAC) represents approximately 90 percent of all pancreatic cancer cases and is the most lethal form of the disease. Current therapies for PDAC are ineffective and most patients cannot be treated by surgical resection. Most research efforts have primarily focused on how genetic alterations cause, alter progression, contribute to diagnosis, and influence PDAC management. Over the past two decades, a model has been advanced of PDAC initiation and progression as a multi-step process driven by the acquisition of mutations leading to loss of tumor suppressors and activation of oncogenes. The recognition of the essential roles of these genetic alterations in the development of PDAC has revolutionized our knowledge of this disease. However, none of these findings have turned into effective treatment for this dismal malignancy. In recent years, studies in the areas of chromatin modifications, and non-coding RNAs have uncovered mechanisms for regulating gene expression which occur independently of genetic alterations. Chromatin-based mechanisms are interwoven with microRNA-driven regulation of protein translation to create an integrated epigenetic language, which is grossly dysregulated in PDAC. Thus in PDAC, key tumor suppressors that are well established to play a role in PDAC may be repressed, and oncogenes can be upregulated secondary to epigenetic alterations. Unlike mutations, epigenetic changes are potentially reversible. Given this feature of epigenetic mechanisms, it is conceivable that targeting epigenetic-based events promoting and maintaining PDAC could serve as foundation for the development of new therapeutic and diagnostic approaches for this disease.

Aim To examine the effect of branched-chain amino acid (BCAA) therapy for patients with unresectable hepatocellular carcinoma (HCC) treated with sorafenib. Methods Seventy-eight subjects with unresectable HCC with a serum level of albumin of 3.5 g/dL or less treated with sorafenib were evaluated. They were classified into two groups: those receiving BCAA granules (n = 34; BCAA group) or a regular diet (n = 44; control group). We compared overall survival and administration period of sorafenib, and analyzed absolute changes in serum levels of albumin during sorafenib therapy in 41 patients who continued sorafenib therapy for 1 month or more with a follow up of more than 3 months. Results Median survival time (MST) in BCAA and control groups was 350 and 143 days (P = 0.007), respectively. Median administration period of sorafenib in the two groups was 59 and 41 days (P = 0.018). In the 41 patients described above, at 1 month, there was no significant change in the serum level of albumin between the two groups, but at 3 months, the difference in the absolute change in the serum level of albumin in the two groups reached significance (P = 0.023). In these subgroup analyses, the administration period of sorafenib as well as the MST in the BCAA group were significantly longer than those in the control group (P = 0.020 and = 0.004). Conclusion BCAA treatment during sorafenib therapy in HCC patients is useful for maintaining hepatic functional reserve, which may help to avoid early discontinuance of sorafenib therapy and improve survival.

We herein report the case of a 43-year-old man with distinct gastropathy and hypoproteinemia treated with H. pylori eradication therapy. Most reported cases of protein-losing gastropathy are divided into Menetrier's disease (MD) and diffuse varioliform gastritis (DVG). Our patient presented with leg edema due to marked hypoalbuminemia, which we ascribed to distinct gastropathy with novel endoscopic findings resembling cap polyposis in the colon, apparently different from both MD and DVG. H. pylori eradication therapy promptly induced the normalization of laboratory data and mucosal healing. Our case together with two previously published similar cases may contribute to establishing an association between cap-polyposis-like-gastropathy with hypoproteinemia and H. pylori.

The value of serum cholinesterase (ChE) level as a predictive marker in sorafenib therapy for advanced hepatocellular carcinoma (HCC) has not yet been investigated. The present retrospective study therefore analyzed the impact of the serum ChE level in 93 patients with advanced HCC treated with sorafenib. Patients were categorized into two groups: group A with pretreatment serum ChE ≥140 IU/l (n=46) and group B with pretreatment serum ChE

Little is known about acute interstitial pneumonia (AIP) induced by sorafenib therapy in patients with advanced hepatocellular carcinoma (HCC). Here, we present three patients with advanced HCC who developed AIP during sorafenib therapy, with fatal complications in two cases. Case 1 was a 76-year-old man who developed dyspnea. Chest CT showed interstitial pneumonia. Sorafenib was discontinued immediately, and prednisolone was started. His pneumonia resolved. A drug-induced lymphocyte stimulation test for sorafenib was positive. Case 2 was a 75-year-old man and case 3 was a 77-year-old man, both of whom developed high-grade fever and hypoxemia during sorafenib therapy, and were diagnosed with AIP. In spite of high-dose steroid therapy, their respiratory failure worsened and both patients died. In all three cases, serum KL-6 or surfactant protein D concentrations were elevated, and blood and sputum cultures did not grow pathogens. All three patients were smokers with restrictive lung disease on preoperative respiratory function testing, but did not have respiratory symptoms before sorafenib therapy. The clinical features of these three cases suggest that male gender, older age, smoking history, and lung disease are associated with acute sorafenib-induced AIP in patients with advanced HCC.

We report three cases of resected hepatocellular carcinomas with nodules showing different signal intensities in the hepatobiliary phase of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid-enhanced MRI (EOB-MRI). One case involved a nodule-in-nodule type hepatocellular carcinoma that showed high signal intensity for the outer tumor and low intensity for the inner tumor in the hepatobiliary phase of EOB-MRI. The inner tumor was more dedifferentiated than the outer. The other two cases involved similar nodules, which showed different signal intensities in the hepatobiliary phase of EOB-MRI. In all three cases, the expression of OATP8 showed good correlation with high signal intensity in the hepatobiliary phase of EOB-MRI, whereas MRP2, MRP3, or both were also highly expressed. However, in the two nodules showing low intensities, the expression of one excreting transporter was independently high even though that of OATP8 was not high. The expression of excreting transporters is usually characterized by passive correspondence to OATP8 expression levels; nevertheless, it sometimes shows expression independent of OATP8.

To examine the effect of branched-chain amino acid (BCAA) therapy for patients with unresectable hepatocellular carcinoma (HCC) treated with sorafenib.Seventy-eight subjects with unresectable HCC with a serum level of albumin of 3.5 g/dL or less treated with sorafenib were evaluated. They were classified into two groups: those receiving BCAA granules (n = 34; BCAA group) or a regular diet (n = 44; control group). We compared overall survival and administration period of sorafenib, and analyzed absolute changes in serum levels of albumin during sorafenib therapy in 41 patients who continued sorafenib therapy for 1 month or more with a follow up of more than 3 months.Median survival time (MST) in BCAA and control groups was 350 and 143 days (P = 0.007), respectively. Median administration period of sorafenib in the two groups was 59 and 41 days (P = 0.018). In the 41 patients described above, at 1 month, there was no significant change in the serum level of albumin between the two groups, but at 3 months, the difference in the absolute change in the serum level of albumin in the two groups reached significance (P = 0.023). In these subgroup analyses, the administration period of sorafenib as well as the MST in the BCAA group were significantly longer than those in the control group (P = 0.020 and = 0.004).BCAA treatment during sorafenib therapy in HCC patients is useful for maintaining hepatic functional reserve, which may help to avoid early discontinuance of sorafenib therapy and improve survival.

We aimed to compare clinical outcomes and safety of radiofrequency ablation (RFA) in single hepatocellular carcinoma (HCC) patients aged75 years (elderly group) versus patients aged75 years (control group).There were 130 patients in the elderly group and 238 in the control group. Clinical outcomes including overall survival (OS), recurrence free survival (RFS) and local tumor progression (LTP), and safety were analysed for these two groups after initial RFA.The mean (+/- standard deviation [SD]) tumor diameter in the elderly and the control groups was 2.13 +/- 0.86 cm and 1.92 +/- 0.63 cm, respectively; the mean (+/- SD) observation period was 2.5 +/- 1.8 years and 3.2 +/- 2.0 years, respectively. The 1 and 3 year OS rates were 90.0 and 64.1%, respectively, in the elderly group and 97.6 and 83.7%, respectively, in the control group (P=0.001); the corresponding RFS rates were 66.9 and 21.3%, respectively, in the elderly group and 80.5 and 40.0%, respectively, in the control group (P=0.001). The 1 and 3 year LTP rates were 15.0 and 43.0%, respectively, in the elderly group and 8.3 and 26.3%, respectively, in the control group (P=0.002). In terms of duration of hospitalization (P=0.807) and serious adverse events related RFA (P=0.670), there was no significant difference between these two groups.The clinical outcomes in the elderly group were poorer than those in the control group, although RFA in the elderly patients was a safe procedure.

To compare the overall survival (OS) in patients without recurrent hepatocellular carcinoma (HCC) after initial radiofrequency ablation (RFA) and in those with recurrence, treated with repeat-RFA.We compared OS between patients without recurrence (group A; n=150) and those with recurrence treated with repeat-RFA (group B; n=130).One-, 3- and 5-year OS after initial RFA was 92.6%, 84.1% and 81.0%, respectively, in group A, and 99.0%, 84.1% and 61.8% in group B (p=0.296). There was no significant difference in OS between group A patients and patients with a single recurrent HCC (p=0.834). On multivariate analysis, serum albumin3.5 g/dl and first HCC recurrence, comprising of two or three nodules were significant predicting factors of poorer OS.Even if HCC recurs after initial RFA, survival comparable to that of patients without recurrence can be achieved in patients with a single recurrent tumor treated with repeat-RFA.

Goals: To elucidate whether long-term supplementation with branched-chain amino acid (BCAA) granules improves overall survival (OS) and recurrence-free survival (RFS) after radiofrequency thermal ablation (RFA) in patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC)≤3 cm in diameter with up to 3 nodules and a serum albumin level before RFA of ≤3.5 g/dL. Background: Whether BCAA treatment after curative RFA for patients with HCV-related HCC improves OS and RFS remains unclear. Study: We compared the OS rate and the RFS rate between the BCAA group (n=115) and the control group (n=141). We also examined factors contributing to OS and RFS. Results: The 1 and 3 years OS rates after RFA were 94.0% and 70.0%, respectively, in the BCAA group, and 94.0% and 49.8%, respectively, in the control group (P=0.001). The corresponding RFS rates 1 and 3 years after RFA were 61.8% and 28.0%, respectively, in the BCAA group, and 52.0% and 12.0%, respectively, in the control group (P=0.013). In the multivariate analysis, in terms of OS, BCAA treatment, and serum albumin level of ≥3.4 g/dL, and in terms of RFS, age 70 years or older, BCAA treatment, and a serum albumin level of ≥3.4 g/dL were significant independent factors, respectively. Conclusions: BCAA treatment may improve OS and RFS after RFA in patients with HCV-related HCC≤3 cm in diameter with up to 3 nodules and a serum albumin level before RFA of 3.5 g/dL.

In our previous study, we classified the radicality (R grading) of percutaneous radiofrequency ablation (RFA) therapy for single hepatocellular carcinoma (HCC) according to the extent of the ablated margin, and demonstrated that this grading system was useful for predicting local tumor progression (LTP) after RFA. The aim of this study was to measure the overall survival (OS), the recurrence free survival (RFS), and the distant recurrence (DR) rate for each R grade (A-D), and to examine the relationship between clinical outcome and R grading.This study involved 368 patients with solitary HCC who had undergone RFA. The mean tumor diameter was 2.0 ± 0.7 cm. We calculated the post-RFA cumulative OS, RFS, and DR rate for each R grade and analyzed the factors contributing to clinical outcomes.In the multivariate analysis, significant factors were as follows: tumor size2 cm, serum albumin3.5 g/dL, prothrombin time70 %, HCC recurrence within 1 year, and R grading (grade A) in OS; cause of liver disease (hepatitis B), gamma glutamyl transpeptidase (GGT)80 IU/L, platelet count10 × 10(4)/mm(3), and R grading (grade A or B) in RFS; GGT80 IU/L, platelet count10 × 10(4)/mm(3), and R grading (grade A or B) in DR. In patients with sufficient Lipiodol accumulation (n = 219), very similar results were obtained. However, in patients with grade A and B (n = 232), R grade was not a significant independent factor linked to OS, although grade A patients had lower LTP rate.Our proposed R grading system appears to be useful for predicting clinical outcomes after RFA.

The aim of the present study was to compare overall survival between stage IVA or stage IVB hepatocellular carcinoma (HCC) patients who received transcatheter arterial chemoembolization (TACE) and those who were treated with sorafenib. This retrospective comparative study included 55 patients with stage IVA or IVB HCC in whom TACE was performed as an initial treatment (the TACE group) and 56 patients with stage IVA or IVB HCC to whom sorafenib was administered (the sorafenib group). We compared the overall survival between these two groups. In the TACE group, there were 46 stage IVA HCC patients and 9 stage IVB HCC patients. In the sorafenib group, there were 26 stage IVA HCC patients and 30 stage IVB HCC patients. Median overall survival times were 6.6 months in the TACE group and 9.2 months in the sorafenib group. The 1- and 2-year overall survival rates were 34.4 and 14.2%, respectively, in the TACE group and 34.0 and 6.7%, respectively, in the sorafenib group. In terms of overall survival, there was no significant difference between the two groups (P=0.814). In subgroup analyses, according to HCC stage [stage IVA (P=0.266) or stage IVB (P=0.183)] and Child-Pugh classification [Child-Pugh A (P=0.915) or Child-Pugh B (P=0.676)], there were also no significant differences between the two groups. In conclusion, our study results suggest that TACE could serve as a first-line treatment for stage IV HCC patients as well as sorafenib therapy.

Background The clearance of hepatitis C virus infection by interferon therapy significantly reduces the incidence of hepatocellular carcinoma and death in elderly chronic hepatitis patients. However, there are few reports concerning the efficacy and safety of pegylated interferon-alpha2b plus ribavirin combination therapy in elderly patients. The aims of the present study were to examine the effect and safety of pegylated interferon-alpha2b plus ribavirin combination therapy in 427 patients with chronic hepatitis C infection. We compared the rates of sustained virological response--defined as the absence of detectable hepatitis C virus in serum 24 weeks after the treatment ended--and the treatment discontinuation rate between 319 younger patients aged < 65 years and 108 elderly patients aged ≥ 65 years. We also examined the factors contributing to a sustained virological response. Results There was no significant difference in the sustained virological response rate between younger patients and elderly patients according to their hepatitis C virus genotype (41.5% (100/241) and 40.7% (35/86) for genotype 1; P = 0.899, 89.7% (70/78) and 86.4% (19/22) for genotype 2; P = 0.703, respectively). There was also no significant difference in the treatment discontinuation rate between the two age groups (10.3% (33/319) and 13.9% (15/108), respectively; P = 0.378). There were no serious adverse events requiring hospitalization. The factors contributing significantly to a sustained virological response in elderly patients were gender, hepatitis C virus genotype, platelet count, and the presence of a rapid or early virological response (undetectable hepatitis C virus in serum at weeks 4 or 12 of treatment, respectively). However, upon multivariate analysis, the presence of an early virological response was the only significant factor (odds ratio: 0.115, 95% confidence interval: 0.040- 0.330, P < 0.001). Conclusions The efficacy and safety of pegylated interferon-alpha2b plus ribavirin combination therapy in elderly patients are not always inferior to those in younger patients. Obtaining an early virological response may be essential to achieve a sustained virological response in elderly patients with chronic hepatitis C infection.

Background After the result of ToGA study, trastuzumab was introduced in Japan, which is the first molecular targeting therapy in the era of gastric cancer. Aims To investigate HER2 overexpression in advanced gastric cancer (stage 4) by immunohistchemistry using endoscopic biopsy samples. To discuss the feasibility and problems of trastuzumab plus capecitabine/cisplatin (HXP) therapy. Methods We investigated HER2 overexpression in 35 patients of advanced gastric cancer from March 2011 to December 2011. Among these, 12 patients revealed HER2 positive by endoscopic biopsy, and 9 patients were introduced HXP. As a rule, we investigated the overexpression of HER2 protein in all cases of unresectable gastric cancer by immunohistchemistry. Basically, we carried out endoscopic biopsy from different several sites (if possible six sites) of the main tumor, considering the heterogeneity of HER2 overexpression. Results We introduced HXP for five males and four females. Median age was 67. Tumor location: body/antrum/cardia = 6/2/1. Histology: well/moderately/poorly = 1/5/3. In four cases, HXP was carried out as the first-line chemotherapy, whereas the others had been received previous chemotherapy such as S-1 plus cisplatin (SP) therapy. Effect evaluation: CR/PR/SD/PD/NE = 0/2/3/2/2. Remarkable adverse events due to trastuzumab were seen in only one case; fever and chills considered as infusion reaction. On the other hand, six cases showed renal dysfunction. No one showed serious adverse events regarded as grade 3/4. Discussion Trastuzumab was well tolerated without significant adverse events in our clinical experience. Trastuzumab seems to be safe for elder patients. Due to renal dysfunction, whenever necessary we reduced or interrupted cisplatin administration and continued trastuzumab. There is no evidence for the efficacy of trastuzumab beyond second-line therapy and there remains a question whether it is effective to continue trastuzumab beyond PD. Therefore, future clinical trials can be expected in this regard.