Cardiac Rhabdomyoma in Four Göttingen Minipigs.

Göttingen minipigs are increasingly used as an alternative large animal model in nonclinical toxicology studies, and proliferative lesions in this species are rare. Here, we report four cases of cardiac rhabdomyoma in Göttingen minipigs, an incidental and benign mass in the heart. Three cases lacked gross observations and had a microscopic nodule in either the left ventricle or interventricular septum. The last case had a large, firm, raised nodule on a left ventricular papillary muscle noted at necropsy, with additional microscopic intramural masses in the left ventricular wall. In all cases, microscopic evaluation revealed well-circumscribed, expansile nodules composed of bundles of large, highly vacuolated, ovoid to polygonal cells with variable cytoplasmic processes radiating from a centrally located nucleus. Cells displayed patchy accumulation of intracytoplasmic, PAS-positive material and haphazardly arranged cytoplasmic cross-striations. There was no evidence of cardiac insufficiency or other data to suggest the masses were clinically meaningful. Cardiac rhabdomyomas have been reported in meat-hybrid swine, with a breed predisposition in red wattle. This lesion is well established in guinea pigs, but documentation in other laboratory species used in toxicologic studies is limited to two beagle dogs. To our knowledge, this is the first report of spontaneous cardiac rhabdomyoma in Göttingen minipigs.

Using Deep Learning Artificial Intelligence Algorithms to Verify N-Nitroso-N-Methylurea and Urethane Positive Control Proliferative Changes in Tg-RasH2 Mouse Carcinogenicity Studies.

In Tg-rasH2 carcinogenicity mouse models, a positive control group is treated with a carcinogen such as urethane or N-nitroso-N-methylurea to test study validity based on the presence of the expected proliferative lesions in the transgenic mice. We hypothesized that artificial intelligence-based deep learning (DL) could provide decision support for the toxicologic pathologist by screening for the proliferative changes, verifying the expected pattern for the positive control groups. Whole slide images (WSIs) of the lungs, thymus, and stomach from positive control groups were used for supervised training of a convolutional neural network (CNN). A single pathologist annotated WSIs of normal and abnormal tissue regions for training the CNN-based supervised classifier using INHAND criteria. The algorithm was evaluated using a subset of tissue regions that were not used for training and then additional tissues were evaluated blindly by 2 independent pathologists. A binary output (proliferative classes present or not) from the pathologists was compared to that of the CNN classifier. The CNN model grouped proliferative lesion positive and negative animals at high concordance with the pathologists. This process simulated a workflow for review of these studies, whereby a DL algorithm could provide decision support for the pathologists in a nonclinical study.

Nervous System Sampling for General Toxicity and Neurotoxicity Studies in the Laboratory Minipig With Emphasis on the Göttingen Minipig.

The use of minipigs as an alternative nonclinical species has increased in the last 20 years. The Society of Toxicologic Pathology (STP) has produced generic "best practice" recommendations for nervous system sampling in nonrodents during general toxicity studies (Toxicol Pathol 41[7]: 1028-1048, 2013), but their adaptation to the minipig has not been attempted. Here, we describe 2 trimming schemes suitable for evaluating the unique neuroanatomic features of the minipig brain in nonclinical toxicity studies. The first scheme is intended for general toxicity studies (Tier 1) to screen agents with unknown or no anticipated neurotoxic potential; this approach using 7 coronal hemisections accords with the published STP "best practice" recommendations. The second trimming scheme for neurotoxicity studies (Tier 2) uses 14 coronal hemisections and 2 full coronal sections to investigate toxicants where the nervous system is a suspected or known target organ. Collection of spinal cord, ganglia (somatic and autonomic), and nerves from minipigs during nonclinical studies should follow published STP "best practice" recommendations for sampling the central (CNS, Toxicol Pathol 41[7]: 1028-1048, 2013) and peripheral (PNS, Toxicol Pathol 46[4]: 372-402, 2018) nervous systems.

Proposal to Eliminate Urethane-Treated Positive Control Dose Groups in 26-Week Tg.rasH2 Carcinogenicity Studies.

Short-term (26 weeks) Tg.rasH2 mouse carcinogenicity studies have been conducted as an alternative model to the conventional 2-year mouse carcinogenicity studies, using urethane as a positive control material. In these studies, urethane was used at a dose of 1,000 mg/kg/dose, administered intraperitoneally on days 1, 3, and 5. Urethane consistently produces lung adenomas and carcinomas and hemangiosarcomas of the spleen, proving validity of the assay. We conducted 3 pilot studies at 3 different sites of Charles River Laboratories using a lower dose of urethane (500 mg/kg/dose), administered on days 1, 3, and 5, followed by a 12-week observation period. Our results demonstrate that a lower dose can be used successfully with fewer number of animals per sex to prove the validity of the assay. However, based on our cumulative experience with this model, we propose to eliminate positive control dose groups in future Tg.rasH2 carcinogenicity studies.

Myocardial fibrosis, inflammation, and altered cardiac gene expression profiles in rats exposed to a predator-based model of posttraumatic stress disorder.

People who are exposed to life-threatening trauma are at risk of developing posttraumatic stress disorder (PTSD). In addition to psychological manifestations, PTSD is associated with an increased risk of myocardial infarction, arrhythmias, hypertension, and other cardiovascular problems. We previously reported that rats exposed to a predator-based model of PTSD develop myocardial hypersensitivity to ischemic injury. This study characterized cardiac changes in histology and gene expression in rats exposed this model. Male rats were subjected to two cat exposures (separated by a period of 10 d) and daily cage-mate changes for 31 d. Control rats were not exposed to the cat or cage-mate changes. Ventricular tissue was analyzed by RNA sequencing, western blotting, histology, and immunohistochemistry. Multifocal lesions characterized by necrosis, mononuclear cell infiltration, and collagen deposition were observed in hearts from all stressed rats but none of the control rats. Gene expression analysis identified clusters of upregulated genes associated with endothelial to mesenchymal transition, endothelial migration, mesenchyme differentiation, and extracellular matrix remodeling in hearts from stressed rats. Consistent with endothelial to mesenchymal transition, rats from stressed hearts exhibited increased expression of α-smooth muscle actin (a myofibroblast marker) and a decrease in the number of CD31 positive endothelial cells. These data provide evidence that predator-based stress induces myocardial lesions and reprograming of cardiac gene expression. These changes may underlie the myocardial hypersensitivity to ischemia observed in these animals. This rat model may provide a useful tool for investigating the cardiac impact of PTSD and other forms of chronic psychological stress.Lay summaryChronic predator stress induces the formation of myocardial lesions characterized by necrosis, collagen deposition, and mononuclear cell infiltration. This is accompanied by changes in gene expression and histology that are indicative of cardiac remodeling. These changes may underlie the increased risk of arrhythmias, myocardial infarction, and other cardiac pathologies in people who have PTSD or other forms of chronic stress.

Scientific and Regulatory Policy Committee Points to Consider: Histopathologic Evaluation in Safety Assessment Studies for PEGylated Pharmaceutical Products.

Colorless, intracytoplasmic vacuoles occur in multiple tissues in animals following repeated administration of polyethylene glycol (PEG)-conjugated molecules. The extent of vacuolation depends on physical characteristics and molecular backbone of the PEG and the dose, product, drug target/pharmacology, and duration of exposure. The collective experience gathered from multiple nonclinical toxicology studies of PEGylated biopharmaceuticals indicates that in general, PEG-related vacuolation is not associated with demonstrable cell and tissue damage or dysfunction and is reversible with sufficient duration of drug-free periods. Existing data are insufficient to predict whether nonclinical animal species differ in their sensitivity to develop PEG-associated vacuoles; however, recent data suggest that there may be species differences. Recent comprehensive reviews have addressed the basic challenges in developing PEGylated pharmaceutical products, including general reference to and description of PEG-associated tissue findings. These manuscripts have identified gaps in our current understanding of PEG-associated vacuolation, including the lack of a widely accepted standardized histological terminology and criteria to record and grade the severity of vacuolation as well as insufficient knowledge regarding the nature of the contents of these vacuoles. The goal of this article is to help address some of the gaps identified above by providing points to consider, including a pictorial review of PEG-associated microscopic findings, when evaluating and reporting the extent, severity, and significance (adversity or lack of adversity) of PEG-associated cytoplasmic vacuolation in safety assessment studies. [Box: see text].

Pigs in Toxicology: Breed Differences in Metabolism and Background Findings.

Both a rodent and a nonrodent species are required for evaluation in nonclinical safety studies conducted to support human clinical trials. Historically, dogs and nonhuman primates have been the nonrodent species of choice. Swine, especially the miniature swine or minipigs, are increasingly being used in preclinical safety as an alternate nonrodent species. The pig is an appropriate option for these toxicology studies based on metabolic pathways utilized in xenobiotic biotransformation. Both similarities and differences exist in phase I and phase II biotransformation pathways between humans and pigs. There are numerous breeds of pigs, yet only a few of these breeds are characterized with regard to both xenobiotic-metabolizing enzymes and background pathology findings. Some specific differences in these enzymes based on breed and sex are known. Although swine have been used extensively in biomedical research, there is also a paucity of information in the current literature detailing the incidence of background lesions and differences between commonly used breeds. Here, the xenobiotic-metabolizing enzymes are compared between humans and pigs, and minipig background pathology changes are reviewed with emphasis on breed differences.

Background Pathological Changes in Minipigs: A Comparison of the Incidence and Nature among Different Breeds and Populations of Minipigs.

Swine, especially the miniature swine or minipigs, are increasingly being used in preclinical safety assessment of small molecules, biopharmaceutical agents, and medical devices as an alternate nonrodent species. Although swine have been used extensively in biomedical research, there is a paucity of information in the current literature detailing the incidence of background lesions and differences in incidence between commonly used breeds. This article is a collaborative effort between multiple organizations to define and document lesions found in the common breeds of minipigs used for toxicological risk assessment in North America (NA) and the European Union (EU). We retrospectively assessed 10 years of historical control data from several institutions located in NA and EU, covering the period of 2004-2015. Here we report the background lesions with consideration of breed and geographical location. To our knowledge, this is the first report documenting spontaneous background lesions in commonly used breeds of swine in both NA and EU. This report serves as a resource to pathologists and will aid in interpretation of findings and differentiation of background from test article-related changes.

T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death.

T315, an integrin-linked kinase (ILK) inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt) and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy.

Multimodel assessment of BRCA1 mutations in Taiwanese (ethnic Chinese) women with early-onset, bilateral or familial breast cancer.

Although evidence suggests an importance of genetic factors in the development of breast cancer in Taiwanese (ethnic Chinese) women, including a high incidence of early-onset and secondary contralateral breast cancer, a major breast cancer predisposition gene, BRCA1, has not been well studied in this population. In fact, the carcinogenic impacts of many genetic variants of BRCA1 are unknown and classified as variants of uncertain significance (VUS). It is therefore important to establish a method to characterize the BRCA1 VUSs and understand their role in Taiwanese breast cancer patients. Accordingly, we developed a multimodel assessment strategy consisting of a prescreening portion and a validated functional assay to study breast cancer patients with early-onset, bilateral or familial breast cancer. We found germ-line BRCA1 mutations in 11.1% of our cohort and identified one novel missense mutation, c.5191C>A. Two genetic variants were initially classified as VUSs (c.1155C>T and c.5191C>A). c.1155C>T is not predicted to be deleterious in the prescreening portion of our assessment strategy. c.5191C>A, on the other hand, causes p.T1691K, which is predicted to have high deleterious probability because of significant structural alteration, a high deleterious score in the predictive programs and, clinically, triple negative characteristics in breast tumors. This mutant is confirmed by transcription activation and yeast growth-inhibition assays. In conclusion, we show as high a prevalence of germ-line BRCA1 mutation in high-risk Taiwanese patients as in Caucasians and demonstrate a useful strategy for studying BRCA1 VUSs.

A review of the existing grading schemes and a proposal for a modified grading scheme for prostatic lesions in TRAMP mice.

The transgenic adenocarcinoma of the mouse prostate (TRAMP) model is well established and offers several advantages for the study of chemopreventive agents, including its well-defined course of disease progression and high incidence of poorly differentiated carcinomas within a relatively short length of time. However, there is no consensus on the grading of prostatic lesions in these mice. In particular, agreement is lacking on the criteria for differentiating prostatic intraepithelial neoplasia (PIN) from well-differentiated adenocarcinoma, specifically as it relates to evidence of invasion. This differentiation is critical for evaluating the effects of putative chemopreventive agents on progression to neoplasia. Moreover, only one of the published grading schemes assigns numerical grades to prostatic lesions, which facilitate statistical analysis. Here, we review five currently available grading schemes and propose a refined scheme that provides a useful definition of invasion for the differentiation of PIN from well-differentiated adenocarcinoma and includes a numerical scoring system that accounts for both the most severe and most common histopathological lesions in each of the lobes of the prostate and their distributions. We expect that researchers will find this refined grading scheme to be useful for chemoprevention studies in TRAMP mice.

Histone deacetylase inhibitors stimulate histone H3 lysine 4 methylation in part via transcriptional repression of histone H3 lysine 4 demethylases.

This study investigates the mechanism by which histone deacetylase (HDAC) inhibitors up-regulate histone H3 lysine 4 (H3K4) methylation. Exposure of LNCaP prostate cancer cells and the prostate tissue of transgenic adenocarcinoma of the mouse prostate mice to the pan- and class I HDAC inhibitors (S)-(+)-N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide (AR42), N-(2-aminophenyl)-4-[N-(pyridine-3-yl-methoxycarbonyl)-aminomethyl]-benzamide (MS-275), and vorinostat led to differential increases in H3K4 methylation. Chromatin immunoprecipitation shows that this accumulation of methylated H3K4 occurred in conjunction with decreases in the amount of the H3K4 demethylase RBP2 at the promoter of genes associated with tumor suppression and differentiation, including KLF4 and E-cadherin. This finding, together with the HDAC inhibitor-induced up-regulation of KLF4 and E-cadherin, suggests that HDAC inhibitors could activate the expression of these genes through changes in histone methylation status. Evidence indicates that this up-regulation of H3K4 methylation was attributable to the suppressive effect of these HDAC inhibitors on the expression of RBP2 and other JARID1 family histone demethylases, including PLU-1, SMCX, and LSD1, via the down-regulation of Sp1 expression. Moreover, shRNA-mediated silencing of the class I HDAC isozymes 1, 2, 3, and 8, but not that of the class II isozyme HDAC6, mimicked the drug effects on H3K4 methylation and H3K4 demethylases, which could be reversed by ectopic Sp1 expression. These data suggest a cross-talk mechanism between HDACs and H3K4 demethylases via Sp1-mediated transcriptional regulation, which underlies the complexity of the functional role of HDACs in the regulation of histone modifications.

Targeting of the Akt-nuclear factor-kappa B signaling network by [1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (OSU-A9), a novel indole-3-carbinol derivative, in a mouse model of hepatocellular carcinoma.

Constitutive activation of Akt and nuclear factor-kappaB (NF-kappaB) represents major cellular abnormalities associated with the development and progression of hepatocellular carcinoma (HCC). Based on the structure of indole-3-carbinol, a chemopreventive phytochemical, we developed a novel derivative, [1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (OSU-A9), that exhibits higher potency in inducing apoptosis by targeting the Akt-NF-kappaB signaling network. This study was aimed at assessing the antitumor activity of OSU-A9 using both in vitro and in vivo models of HCC, a malignancy in which the Akt-NF-kappaB signaling network plays major roles in pathogenesis and therapeutic resistance. Our data show that OSU-A9 was 100 times more potent than indole-3-carbinol in suppressing the viability of Hep3B, Huh7, and PLC5 HCC cells with IC(50) values ranging from 2.8 to 3.2 microM. OSU-A9 interfered with the interplay between Akt- and NF-kappaB-mediated oncogenic signaling, leading to changes in the functional status of diverse signaling effectors involved in cell cycle progression, apoptosis, angiogenesis, and metastasis. The in vivo efficacy of OSU-A9 was assessed in nude mice bearing luciferase-expressing Hep3B xenograft tumors. Daily oral treatments with OSU-A9 at 25 or 50 mg/kg for 56 days suppressed tumor growth by 67 and 80%, respectively, which was correlated with changes in intratumoral biomarkers pertinent to Akt-NF-kappaB signaling, and without apparent toxicity or evidence of hepatic biotransformation enzyme induction. Together, these findings indicate that OSU-A9 is a potent, orally bioavailable inhibitor of the Akt-NF-kappaB signaling network with a broad spectrum of antitumor activity that includes targets regulating multiple aspects of HCC pathogenesis and progression.

OSU-A9, a potent indole-3-carbinol derivative, suppresses breast tumor growth by targeting the Akt-NF-kappaB pathway and stress response signaling.

The molecular heterogeneity of human tumors challenges the development of effective preventive and therapeutic strategies. To overcome this issue, a rational approach is the concomitant targeting of clinically relevant cellular abnormalities with combination therapy or a potent multi-targeted agent. OSU-A9 is a novel indole-3-carbinol derivative that retains the parent compound's ability to perturb multiple components of oncogenic signaling, but provides marked advantages in chemical stability and antitumor potency. Here, we show that OSU-A9 exhibits two orders of magnitude greater potency than indole-3-carbinol in inducing apoptosis in various breast cancer cell lines with distinct genetic abnormalities, including MCF-7, MDA-MB-231 and SKBR3, with the half maximal inhibitory concentration in the range of 1.2-1.8 microM vis-à-vis 200 microM for indole-3-carbinol. This differential potency was paralleled by OSU-A9's superior activity against multiple components of the Akt-nuclear factor-kappa B (NF-kappaB) and stress response signaling pathways. Notable among these were the increased estrogen receptor (ER)-beta/ERalpha expression ratio, reduced expression of HER2 and CXCR4 and the upregulation of aryl hydrocarbon receptor expression and its downstream target NF-E2 p45-regulated factor (Nrf2). Non-malignant MCF-10A cells were resistant to OSU-A9's antiproliferative effects. Daily oral administration of OSU-A9 at 25 and 50 mg/kg for 49 days significantly inhibited MCF-7 tumor growth by 59 and 70%, respectively, without overt signs of toxicity or evidence of induced hepatic biotransformation enzymes. In summary, OSU-A9 is a potent, orally bioavailable inhibitor of the Akt-NF-kappaB signaling network, targeting multiple aspects of breast tumor pathogenesis and progression. Thus, its translational potential for the treatment or prevention of breast cancer warrants further investigation.

A potent indole-3-carbinol derived antitumor agent with pleiotropic effects on multiple signaling pathways in prostate cancer cells.

Indole-3-carbinol has emerged as a promising chemopreventive agent due to its in vivo efficacy in various animal models. However, indole-3-carbinol exhibits weak antiproliferative potency and is unstable in acidic milieu. Thus, this study was aimed at exploiting indole-3-carbinol to develop potent antitumor agents with improved chemical stability. This effort culminated in OSU-A9 {[1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol}, which is resistant to acid-catalyzed condensation, and exhibits 100-fold higher apoptosis-inducing activity than the parent compound. Relative to indole-3-carbinol, OSU-A9 displays a striking qualitative similarity in its effects on the phosphorylation or expression of multiple signaling targets, including Akt, mitogen-activated protein kinases, Bcl-2 family members, survivin, nuclear factor-kappaB, cyclin D1, p21, and p27. The ability of OSU-A9 to concurrently modulate this broad range of signaling targets underscores its in vitro and in vivo efficacy in prostate cancer cells. Nevertheless, despite this complex mode of mechanism, normal prostate epithelial cells were less susceptible to the antiproliferative effect of OSU-A9 than PC-3 and LNCaP prostate cancer cells. Treatment of athymic nude mice bearing established s.c. PC-3 xenograft tumors with OSU-A9 at 10 and 25 mg/kg i.p. for 42 days resulted in a 65% and 85%, respectively, suppression of tumor growth. Western blot analysis of representative biomarkers in tumor lysates revealed significant reductions in the intratumoral levels of phosphorylated (p-) Akt, Bcl-xL, and RelA, accompanied by robust increases in p-p38 levels. In conclusion, the ability of OSU-A9 to target multiple aspects of cancer cell survival with high potency suggests its clinical value in prostate cancer therapy.

OSU-A9 inhibits pancreatic cancer cell lines by modulating p38-JAK-STAT3 signaling.

Pancreatic cancer is an aggressive malignancy that is the fourth leading cause of death worldwide. Since there is a dire need for novel and effective therapies to improve the poor survival rates of advanced pancreatic cancer patients, we analyzed the antitumor effects of OSU-A9, an indole-3-carbinol derivative, on pancreatic cancer cell lines in vitro and in vivo. OSU-A9 exhibited a stronger antitumor effect than gemcitabine on two pancreatic cancer cell lines, including gemcitabine-resistant PANC-1 cells. OSU-A9 treatment induced apoptosis, the down-regulation of Akt phosphorylation, up-regulation of p38 phosphorylation and decreased phosphorylation of JAK and STAT3. Cell migration and invasiveness assays showed that OSU-A9 reduced cancer cell aggressiveness and inhibited BxPC-3 xenograft growth in nude mice. These results suggest that OSU-A9 modulates the p38-JAK-STAT3 signaling module, thereby inducing cytotoxicity in pancreatic cancer cells. Continued evaluation of OSU-A9 as a potential therapeutic agent for pancreatic cancer thus appears warrented.

Palliative Gastrectomy Prolongs Survival of Metastatic Gastric Cancer Patients with Normal Preoperative CEA or CA19-9 Values: A Retrospective Cohort Study.

Background. Palliative gastrectomy has been suggested to improve survival of patients with metastatic gastric cancer, but limitations in study design and availability of robust prognostic factors have cast doubt on the overall merit of this procedure. Methods. The characteristics and clinical outcomes of 173 patients diagnosed between 2008 and 2012 were analyzed to determine the value of palliative gastrectomy and to identify potential prognostic factors. Results. Median overall patient survival was 6.5 months. To attenuate potential selection bias, patients with adequate performance and survival time of ≥ 2 months since diagnosis were included for risk factor analysis (n = 137). The median overall survival was longer for patients who were younger than 60 years, had better performance status (8.7 versus 6.4 months, P = 0.015), received systemic chemotherapy, or had palliative gastrectomy in univariate analyses. Gastrectomy (P = 0.002) remained statistically significant in multivariate analyses. Subgroup analysis showed that patients aged < 60 years, CEA < 5 ng/mL or CA19-9 < 35 U/mL, obtained a survival advantage from palliative gastrectomy. In fact, palliative gastrectomy doubled overall survival for patients who had normal CEA and/or normal CA19-9. Conclusions. Palliative gastrectomy prolongs the survival of metastatic gastric cancer patients with normal CEA and/or CA19-9 level at the time of diagnosis.

The role of adjuvant chemotherapy for patients with stage II and stage III gastric adenocarcinoma after surgery plus D2 lymph node dissection: a real-world observation.

BACKGROUND: The influence of adjuvant chemotherapy on the survival of gastric adenocarcinoma patients in a stage-specific manner is controversial. METHODS: To further explore this topic, we retrospectively analyzed the impact of adjuvant chemotherapy on the clinical outcomes of 77 stage II and 117 stage III patients diagnosed between January 2008 and December 2012. RESULTS: All 194 patients underwent radical operation plus D2 dissection, and were followed a median time of 23.3 (range 0.4-80.2) months. Median patient age was 67.7 (range 33.9-97.5) years. Adjuvant chemotherapy prolonged the relapse-free survival [22.9 (95 % confidence interval 9.4-36.4) vs. 14.2 (95 % CI 8.6-19.8) months, P = 0.009] and overall survival [32.3 (95 % CI 22.6-42.0) vs. 13.4 (95 % CI 9.5-17.2) months, P < 0.001] for patients with stage III, but not stage II, disease. Higher overall survival from adjuvant chemotherapy in stage II patients with node involvement did not reach the level of statistical significance (P = 0.102). To reduce the selection bias, 142 patients aged <75 years were included in a subgroup analysis in which the benefit of adjuvant chemotherapy on relapse-free survival and overall survival were demonstrated for patients with stage III disease. CONCLUSIONS: Adjuvant chemotherapy prolongs relapse-free and overall survival for patients with stage III gastric cancer in a real-world situation. Tailoring therapy based on different characteristics for patients with stage II gastric cancer may produce better outcomes.

BX795, a TBK1 inhibitor, exhibits antitumor activity in human oral squamous cell carcinoma through apoptosis induction and mitotic phase arrest.

TANK-binding kinase 1 (TBK1), a member of IκB Kinase (IKK)-related kinases, plays a role in regulating innate immunity, inflammation and oncogenic signaling. This study aims to investigate the role of BX795, an inhibitor of TBK1, in a panel of oral squamous cell carcinoma (OSCC) cell lines. The antitumor effects and mechanisms of BX795 were assessed by MTT assays, flow cytometry, Western blotting, and confocal microscopy. BX795 exhibited a dose-responsive antiproliferative effect on OSCC cells with relative sparing of normal human oral keratinocytes. The compound caused apoptosis as evidenced by PARP cleavage, the presence of pyknotic nuclei in the TUNEL assay, and fragmented DNA tails in the Comet assay. BX795 inhibits Akt and NF-κB signaling, arrests cells in the mitotic phase, and increases generation of autophagy in oral cancer cells. Interestingly, the antiproliferative activity of BX795 does not correlate with TBK1 protein expression level in OSCC cells. We propose that the TBK1-independet effect is related to mitotic phase arrest. Pleiotropic anticancer activity with relative sparing of normal oral keratinocytes underscores the potential value of BX795 and warrants its further study in oral squamous cell carcinoma therapy.

Hepatitis C infection is associated with hepatic toxicity but does not compromise the survival of patients with diffuse large B cell lymphoma treated with rituximab-based chemotherapy.

The influence of hepatitis C virus (HCV) infection on the outcome of patients with diffuse large B cell lymphoma (DLBCL) treated with rituximab-based chemotherapy is controversial. We retrospectively analyzed the characteristics and clinical outcomes of 168 patients with DLBCL diagnosed between January 2005 and December 2011. Twenty-nine patients who were HCV-positive before lymphoma treatment were compared with 139 patients who did not have HCV infection. The median follow-up duration was 3.0 (0.07-8.02) years. HCV infection resulted in more hepatic toxicity in both univariate (p=0.001) and multivariate (p=0.003) analyses. In addition, HCV-positive DLBCL patients were more likely to have treatment delay (20.1% vs. 0.7%, p<0.001). For patients who developed hepatic toxicity during immunochemotherapy, HCV-positive patients had significantly higher folds of aspartate aminotransferase elevation (p=0.042) and total bilirubin elevation (p=0.012) compared with those who were HCV negative. However, HCV did not influence the 5-year progression-free survival rate (p=0.412) or 5-year overall survival rate (p=0.410). In conclusion, HCV infection is associated with increased hepatic toxicity and delayed chemotherapy without compromised survival in DLBCL patients treated with rituximab-based chemotherapy.