[A Curatively Resected Case of Lateral Lymph Node Metastasis Five-Years after Initial Surgery for Rectal Cancer].

A 74-year-old male had undergone laparoscopic abdominoperineal resection for lower rectal cancer in July 2009. The pathological diagnosis was T2, N0, M0, pStage I (TNM 7th). Because of pathological venous invasion, adjuvant chemotherapy with Tegafur-uracil(UFT)plus Leucovorin for a year was performed. A CT examination revealed slowly growing peripheral right internal iliaclymph node. PET-CT demonstrated a 20mm right lateral lymph node(LLN)metastasis without other distant metastases. On diagnosis of solitary LLN metastasis of rectal cancer, the patient underwent surgical lymph node resection in September 2014. The pathological diagnosis was lymph node metastasis from rectal cancer. Subsequently, the patient received mFOLFOX6 adjuvant chemotherapy for 6 months. The patient remains alive without any recurrence 31 months after the second surgical treatment. lt is important to consider that LLN metastasis of Stage I rectal cancer might still occur a long time after the curative operation.

A case of an intramesenteric GIST accurately diagnosed and completely resected by PET-CT and laparoscopy after gastrectomy for gastric cancer.

We performed the accurate diagnosis and complete surgical resection of a gastrointestinal stromal tumor at the mesentery of the small bowel. Computed tomography (CT) in a 62-year-old man at 2 years after gastrectomy for gastric cancer showed a mesenteric tumor, with no other tumors noted. Positron emission tomography-computed tomography (PET-CT) showed a maximum standardized uptake value (SUV max) of 2.9 at the tumor. The presence of a single and low SUV max tumor allowed us to perform laparoscopic surgery. Partial resection of the tumor with an adequate margin was performed. The pathological findings showed c-kit positivity and a low Ki-67 proliferation index (<5%). In the present case, PET-CT and laparoscopic assessments were useful for accurately evaluating the surgical resectability of the mesenteric tumor after distal gastrectomy for gastric cancer. The low SUV max and laparoscopic findings led to complete surgical resection of a mesenteric tumor.

Restoration of SMAD4 by gene therapy reverses the invasive phenotype in pancreatic adenocarcinoma cells.

SMAD4 is a critical cofactor in signal transduction pathways activated in response to transforming growth factor-beta (TGF-beta)-related ligands, regulating cell growth and differentiation. The roles played by SMAD4 inactivation in tumours highlighted it as a tumour-suppressor gene. However, restoration of the TGF-beta antiproliferative pathway following SMAD4 gene transfer in null-tumour cell lines is controversial. Herein, we report the inhibitory effects of SMAD4 on pancreatic tumour invasion and angiogenesis. Adenoviral transfer of this gene in a panel of SMAD4 homozygous-deleted human pancreatic tumour cell lines restored SMAD4 protein expression and function. Although it did not affect proliferation significantly in vitro, SMAD4 inhibited in vivo tumour growth in immunodeficient mice. In this xenograft setting, differential suppression of tumour growth in vivo was mediated, at least in part, through downregulation of vascular endothelial growth factor and expression of gelatinases. We documented the reduced invasion and angiogenesis histologically and by intravital microscopy, and gained mechanistic insight at the messenger and protein level. Finally, we found a negative reciprocal regulation between SMAD4 and ETS-1. ETS-1 is considered a marker for tumour invasion. Upon SMAD4 deletion, we detected high expression levels of ETS-1 in pancreatic tumour cells, suggesting the shift of the pancreatic tumour toward an invasive phenotype.

DSCP1, a novel TP53-inducible gene, is upregulated by strong genotoxic stresses and its overexpression inhibits tumor cell growth in vitro.

TP53-inducible genes play crucial roles from many biological aspects including cell cycle control, DNA repair, and apoptosis. Herein we report the identification and characterization of a novel TP53-inducible gene, DSCP1 (damage stimulated cytoplasmic protein 1), localized at 17q11. The gene was expressed ubiquitously in normal adult tissues; its protein product was localized mainly in the cytoplasm with anchoring on unknown subcellular structures. Exogenous expression of TP53 induced expression of DSCP1, but more interestingly, DSCP1 was induced by strong genotoxic stresses not only in TP53-maintaining cells but also in TP53-dysfunctioning cells, although the induction was much more efficient in the former than in the latter. In cultured cancer cells, the basal expression level appeared to depend on the functional status of TP53. Moreover, exogenous overexpression of DSCP1 retarded cancer cell growth in vitro. These results indicate that DSCP1 is a stress-inducible gene in both a TP53 dependent and independent manner and that its protein product can inhibit cancer cell growth.

Contribution of gene expression to metabolic fluxes in hypermetabolic livers induced through burn injury and cecal ligation and puncture in rats.

Severe injury activates many stress-related and inflammatory pathways that can lead to a systemic hypermetabolic state. Prior studies using perfused hypermetabolic rat livers have identified intrinsic metabolic flux changes that were not dependent upon the continual presence of elevated stress hormones and substrate loads. We investigated the hypothesis that such changes may be due to persistent alterations in gene expression. A systemic hypermetabolic response was induced in rats by applying a moderate burn injury followed 2 days later by cecum ligation and puncture (CLP) to produce sepsis. Control animals received a sham-burn followed by CLP, or a sham-burn followed by sham-CLP. Two days after CLP, livers were analyzed for gene expression changes using DNA microarrays and for metabolism alterations by ex vivo perfusion coupled with Metabolic Flux Analysis. Burn injury prior to CLP increased fluxes while decreases in gene expression levels were observed. Conversely, CLP alone significantly increased metabolic gene expression, but decreased many of the corresponding metabolic fluxes. Burn injury combined with CLP led to the most dramatic changes, where concurrent changes in fluxes and gene expression levels occurred in about 1/3 of the reactions. The data are consistent with the notion that in this model, burn injury prior to CLP increased fluxes through post-translational mechanisms with little contribution of gene expression, while CLP treatment up-regulated the metabolic machinery by transcriptional mechanisms. Overall, these data show that mRNA changes measured at a single time point by DNA microarray analysis do not reliably predict metabolic flux changes in perfused livers.

Effects of dehydroepiandrosterone administration on rat hepatic metabolism following thermal injury.

BACKGROUND: Severe burns cause dramatic alterations in liver and whole-body metabolism. Recently, there has been interest in using dehydroepiandrosterone (DHEA) as a treatment for trauma patients, and enhanced survival and immune function have been reported using DHEA in animal trauma models. The specific effects of DHEA on hepatic metabolism following burn injury have not been explored. MATERIALS AND METHODS: Male rats received either (1) a burn covering approximately 20% of the total body surface area or a sham burn or (2) burn injury followed by two intraperitoneal injections of DHEA or vehicle. After 4 days, the livers were isolated and perfused in vitro, and 28 metabolite fluxes were measured. Metabolic flux analysis was used to obtain the intracellular metabolic flux distribution and provide an overview of the metabolic state of the livers in each experimental group. RESULTS: Burn injury decreased the uptake of lactate and the production of beta-hydroxybutyrate and increased the deamination of glutamine to glutamate and asparagine to aspartate. DHEA, compared to vehicle treatment, decreased pentose phosphate pathway (PPP) fluxes and the uptake of several amino acids in burned rats. Furthermore, DHEA treatment restored liver metabolism in burned rats to a state that was very similar to that of the sham control group. CONCLUSIONS: DHEA administration appears to normalize hepatocellular metabolism in burned rats but also decreases the PPP flux, which may impair the liver's ability to recycle endogenous antioxidants. DHEA treatment combined with exogenous antioxidants should receive further consideration in the management of burn and trauma patients.

Evolution of intrahepatic carbon, nitrogen, and energy metabolism in a D-galactosamine-induced rat liver failure model.

A clearer picture of the hepatic metabolic pathways affected by fulminant hepatic failure (FHF) would help develop nutritional support and nonsurgical therapies for FHF. We characterized the evolution of hepatic metabolism in a rat model of FHF using an isolated perfused liver system together with a mass-balance model of intermediary metabolism. Principal component analysis (PCA) was used to identify potential new sensitive markers for FHF. To induce FHF, rats were given two D-galactosamine injections under fasting conditions. Controls were fasted only. Livers were harvested 1, 4, 8, and 12 h later and perfused with Eagle minimal essential medium supplemented with amino acids and bovine serum albumin, and equilibrated with 95% O2/5% CO2. At the 1 h time point, lactate release increased concomitant with a decrease in gluconeogenesis, TCA cycle and mitochondrial electron transport fluxes. At 4 h, amino acid metabolism and urea cycle fluxes were significantly depressed. By 8 h, gluconeogenesis had switched to glycolysis. By 12 h, amino acid metabolism was broadly inhibited, and there was a net release of many amino acids. Mass-balance analysis shows that the main source of ATP production in the FHF liver gradually changed from mitochondrial oxidative phosphorylation to glycolysis. PCA suggests that a linear combination of glucose, lactate, and glutamine concentrations in arterial plasma is a sensitive marker for FHF. We conclude that D-galactosamine causes early mitochondrial dysfunction while glycolytic ATP synthesis remains functional. Markers that are indirectly linked to these pathways may be used to evaluate the progression of FHF.

Quantitative effects of thermal injury and insulin on the metabolism of the skeletal muscle using the perfused rat hindquarter preparation.

Injury from a severe burn or trauma can propel the body into a hypermetabolic state that can lead to the significant erosion of lean muscle mass. Investigations describing this process have been somewhat limited due to the lack of adequate experimental models. Here we report the use of a perfused rat hindquarter preparation to study the consequences of a moderate burn injury (approximately 20% total body surface area), with or without the addition of exogenous insulin (12.5 mU/mL), on the fluxes of major metabolites across the isolated skeletal muscle. The metabolic flux data was further analyzed using metabolic flux analysis (MFA), which allows for the estimation of the impact of these conditions on the intracellular muscle metabolism. Results indicate that this model is able to capture the increased rate of proteolysis, glutamine formation, and the negative nitrogen balance associated with the burn-induced hypermetabolic state. The inclusion of exogenous insulin resulted in significant changes in several fluxes, including an increase in the metabolism of glucose and the flux through the pentose phosphate pathway, as well as a reduction in the metabolism of glutamine, alanine, and leucine. However, insulin administration did not affect the nitrogen balance or the rate of proteolysis in the muscle, as has been suggested using other techniques. The use of the perfused hindquarter model coupled with MFA is a physiologically relevant and experimentally flexible platform for the exploration of skeletal muscle metabolism under catabolic conditions, and it will be useful in quantifying the specific metabolic consequences of other therapeutic advances.

Identification of optimal classification functions for biological sample and state discrimination from metabolic profiling data.

MOTIVATIONS: Classification of biological samples for diagnostic purposes is a difficult task because of the many decisions involved on the number, type and functional manipulations of the input variables. This study presents a generally applicable strategy for systematic formulation of optimal diagnostic indexes. To this end, we develop a novel set of computational tools by integrating regression optimization, stepwise variable selection and cross-validation algorithms. RESULTS: The proposed discrimination methodology was applied to plasma and tissue (liver) metabolic profiling data describing the time progression of liver dysfunction in a rat model of acute hepatic failure generated by d-galactosamine (GalN) injection. From the plasma data, our methodology identified seven (out of a total of 23) metabolites, and the corresponding transform functions, as the best inputs to the optimal diagnostic index. This index showed better time resolution and increased noise robustness compared with an existing metabolic index, Fischer's BCAA/AAA molar ratio, as well as indexes generated using other commonly used discriminant analysis tools. Comparison of plasma and liver indexes found two consensus metabolites, lactate and glucose, which implicate glycolysis and/or gluconeogenesis in mediating the metabolic effects of GalN.

Gene therapy for pancreatic cancer based on genetic characterization of the disease.

In order to develop an effective therapeutic intervention for patients with pancreatic cancer, we analyzed the association of loss of heterozygosity (LOH) with the clinicopathological features of the disease. Based on these results, we are developing a new gene therapy that targets the genetic character of pancreatic cancer, using mutant adenoviruses that are selectively replication-competent in tumor cells. LOH of 30% or more was observed on chromosome arms 17p (47%), 9p (45%), 18q (43%), 12q (34%), and 6q (30%). LOH of 12q, 17p, and 18q showed significant association with poor prognosis. These data strongly suggest that mutations of the putative tumor suppressor genes, TP53 and SMAD4, play significant roles in the disease progression. Based on this rationale, we are developing a new gene therapy that targets tumors without normal TP53 function. The E1B-55kDa-deleted adenovirus can selectively replicate in TP53-deficient human tumor cells, but not in cells with functional TP53. We evaluated the therapeutic effect of this E1B-55kDa-deleted mutant adenovirus on pancreatic cancer without normal TP53 function. The growth of a human pancreatic tumor in a severe combined immunodeficiency (SCID) mouse model was markedly inhibited by consecutive injections of the E1B-55kDa-deleted adenovirus. Furthermore, the replication-competent adenovirus is not only a strong weapon itself but it is also a useful carrier of genes that possess antitumor activities, as a virus vector specific to tumors without normal TP53 function.

The role of chromosome 18 abnormalities in the progression of pancreatic adenocarcinoma.

To date, the events that mediate tumor progression in pancreatic cancer are still poorly understood. Cytogenetic, allelotype, and somatic cell hybrid studies in human pancreatic adenocarcinoma have suggested that chromosome 18 may carry tumor suppressor genes (TSGs), including SMAD4. We previously identified that LOH of 18q at the SMAD4 locus, along with LOHs on 17p and 12q, positively associated with poor prognoses of pancreatic cancer patients. However, restoration of the SMAD4 gene did not suppress in vitro proliferation of pancreatic cancer cells that harbored homozygous deletion of this gene. An intraductal papillary mucinous neoplasm (IPMN ) is thought to be one of the premalignant lesions of the pancreas that progresses to carcinoma. Although there were frequent LOH (7/14, 50%) at the SMAD4 locus in IPMN samples, SMAD4 protein was observed immunohistochemically in tumor cells, and no mutations of the SMAD4 gene were observed, suggesting that it is the existence of a TSG in 18q, other than SMAD4, that suppresses cell growth. To functionally assess the activity of chromosome 18 in pancreatic cancer, we transferred a normal copy of the chromosome into pancreatic ductal carcinoma cells with and without completely inactivated SMAD4. In this study, in vitro growth of the hybrid cells was significantly suppressed compared with the parental cells, regardless of the initial SMAD4 status. To estimate the metastatic ability of the hybrids, we used a lung colonization model. At the end of the experiment, there was significant suppression of the number of surface metastases developing in mice injected with hybrids in comparison with those injected with parental cells. To identify and characterize genes that are involved in the progression of pancreatic cancer, we used micro-array expression analysis employing a 20k oligo-array system. It was revealed that there was increased expression of 4 genes relating to apoptosis in the 18 chromosome hybrids cells compared with the parental cells. We are now analyzing the function of these genes.