MiR-200a regulates epithelial to mesenchymal transition-related gene expression and determines prognosis in colorectal cancer patients.

BACKGROUND: MicroRNAs (miRNAs) regulate the biological properties of colorectal cancer (CRC) cells and might serve as potential prognostic factors and therapeutic targets. In this study, we therefore globally profiled miRNAs associated with E-cadherin expression in CRC cells in an attempt to identify miRNAs that are associated with aggressive clinical course in CRC patients. METHODS: Two CRC cell lines (Caco-2 and HRT-18) with different E-cadherin expression pattern were profiled for differences in abundance for more than 1000 human miRNAs using microarray technology. One of the most differentially expressed miRNAs, miR-200a was evaluated for its prognostic role in a cohort of 111 patients and independently validated in 217 patients of the Cancer Genome Atlas data set. To further characterise the biological role of miR-200a expression in CRC, in vitro miR-200a inhibition and overexpression were performed and the effects on cellular growth, apoptosis and epithelial-mesenchymal transition (EMT)-related gene expression were explored. RESULTS: In situ hybridisation specifically localised miR-200a in CRC cells. In both cohorts, a low miR-200a expression was associated with poor survival (P<0.05). Multivariate Cox regression analysis identified low levels of miR-200a expression as an independent prognostic factor with respect to cancer-specific survival (HR=2.04, CI=1.28-3.25, P<0.002). Gain and loss of function assays for miR-200a in vitro led to a significantly differential and converse expression of EMT-related genes (P<0.001.) A low expression of miR-200a was also observed in cancer stem cell-enriched spheroid growth conditions (P<0.05). CONCLUSIONS: In conclusion, our data suggest that low miR-200a expression is associated with poor prognosis in CRC patients. MiR-200a has a regulatory effect on EMT and is associated with cancer stem cell properties in CRC.

Validation of C-reactive protein levels as a prognostic indicator for survival in a large cohort of pancreatic cancer patients.

BACKGROUND: Recent evidence indicates that the host inflammatory response has an important role in the tumour progression. Elevated C-reactive protein (CRP) levels have been previously associated with poor prognosis in several cancer types including small-scale studies in pancreatic cancer (PC) patients. The purpose of the present study was to validate the prognostic impact of plasma CRP levels at date of diagnosis on cancer-specific survival (CSS) in a large cohort of PC patients. METHODS: Data from 474 consecutive patients with adenocarcinoma of the pancreas, treated between 2004 and 2012 at a single centre, were evaluated retrospectively. CSS was analysed using the Kaplan-Meier method. To evaluate the prognostic significance of plasma CRP levels, univariate and multivariate Cox analyses were applied. RESULTS: High plasma CRP levels at diagnosis were significantly associated with well-established prognostic factors, including high tumour stage and tumour grade and the administration of chemotherapy (P<0.05). In univariate analysis, we observed that a high plasma CRP level was a consistent factor for poor CSS in PC patients (hazard ratio (HR)=2.21; 95% confidence interval (CI)=1.68-2.92, P<0.001). In multivariate analysis, tumour stage, grade, administration of chemotherapy, a high neutrophil-lymphocyte ratio and the highest quartile of CRP levels (HR=1.60, 95% CI=1.16-2.21; P=0.005) were identified as independent prognostic factors in PC patients. CONCLUSION: In conclusion, we confirmed a significant association of elevated CRP levels with poor clinical outcome in PC patients. Our results indicate that the plasma CRP level might represent a useful marker for patient stratification in PC management.

Increased neutrophil-lymphocyte ratio is a poor prognostic factor in patients with primary operable and inoperable pancreatic cancer.

BACKGROUND: The neutrophil-lymphocyte ratio (NLR) has been proposed as an indicator of systemic inflammatory response. Previous findings from small-scale studies revealed conflicting results about its independent prognostic significance with regard to different clinical end points in pancreatic cancer (PC) patients. Therefore, the aim of our study was the external validation of the prognostic significance of NLR in a large cohort of PC patients. METHODS: Data from 371 consecutive PC patients, treated between 2004 and 2010 at a single centre, were evaluated retrospectively. The whole cohort was stratified into two groups according to the treatment modality. Group 1 comprised 261 patients with inoperable PC at diagnosis and group 2 comprised 110 patients with surgically resected PC. Cancer-specific survival (CSS) was assessed using the Kaplan-Meier method. To evaluate the independent prognostic significance of the NLR, the modified Glasgow prognostic score (mGPS) and the platelet-lymphocyte ratio univariate and multivariate Cox regression models were applied. RESULTS: Multivariate analysis identified increased NLR as an independent prognostic factor for inoperable PC patients (hazard ratio (HR)=2.53, confidence interval (CI)=1.64-3.91, P<0.001) and surgically resected PC patients (HR=1.61, CI=1.02-2.53, P=0.039). In inoperable PC patients, the mGPS was associated with poor CSS only in univariate analysis (HR=1.44, CI=1.04-1.98). CONCLUSION: Risk prediction for cancer-related end points using NLR does add independent prognostic information to other well-established prognostic factors in patients with PC, regardless of the undergoing therapeutic modality. Thus, the NLR should be considered for future individual risk assessment in patients with PC.

Low expression of the putative tumour suppressor spinophilin is associated with higher proliferative activity and poor prognosis in patients with hepatocellular carcinoma.

BACKGROUND: Spinophilin, a multifunctional intracellular scaffold protein, is reduced in certain types of cancer and is regarded as a novel putative tumour suppressor protein. However, the role of spinophilin in hepatocellular carcinoma (HCC) has never been explored before. METHODS: In this study, we determined for the first time the expression pattern of spinophilin in human HCC by immunohistochemistry and quantitative reverse transcriptase-PCR analysis. In addition, we performed immunohistochemical analysis of p53, p14(ARF) and the proliferation marker Ki-67. Kaplan-Meier curves and multivariate Cox proportional models were used to study the impact on clinical outcome. Small interfering RNA (siRNA) was used to silence spinophilin and to explore the effects of reduced spinophilin expression on cellular growth. RESULTS: In our study, complete loss of spinophilin immunoreactivity was found in 44 of 104 HCCs (42.3%) and reduced levels were found in an additional 37 (35.6%) cases. After adjusting for other prognostic factors, multivariate Cox regression analysis identified low expression of spinophilin as an independent prognostic factor with respect to disease-free (hazard ratio (HR)=1.8; 95% confidence interval (CI)=1.04-3.40; P=0.043) and cancer-specific survival (HR=2.0; CI=1.1-3.8; P=0.025). Reduced spinophilin expression significantly correlated with higher Ki-67 index in HCC (P=0.014). Reducing spinophilin levels by siRNA induced a higher cellular growth rate and increased cyclin D2 expression in tumour cells (P<0.05). CONCLUSION: This is the first study of the expression pattern and distribution of spinophilin in HCC. According to our data, the loss of spinophilin is associated with higher proliferation and might be useful as a prognostic marker in patients with HCC.

The PDZ protein erbin modulates beta-catenin-dependent transcription.

Erbin is a member of the leucine-rich repeat and PDZ domain family that can regulate proliferation, differentiation and cell adhesion. As a binding partner of the receptor tyrosine kinase ErbB2, erbin targets this receptor to the basolateral membrane of polarized epithelial cells. In addition, erbin is known to inhibit the Ras-mediated activation of the mitogen-activated protein kinase pathway. Recently we identified the proto-oncoprotein beta-catenin as a ligand of the PDZ domain of erbin. Here we demonstrate that erbin acts as a negative regulator of the beta-catenin/T-cell-factor-dependent gene expression. In contrast, a mutant of erbin with a deletion of the N-terminal leucine-rich repeat allows the PDZ domain of erbin to increase the beta-catenin/T-cell-factor-dependent transcription. This mutant localizes to the nucleus and mimics a putative splice variant found in keratinocytes. Thus, erbin has the potential to act as an inhibitor as well as an activator of the beta-catenin-regulated gene expression.

DAPK2 is a novel E2F1/KLF6 target gene involved in their proapoptotic function.

Death-associated protein kinase 2 (DAPK2) belongs to a family of proapoptotic Ca(2+)/calmodulin-regulated serine/threonine kinases. We recently identified DAPK2 as an enhancing factor during granulocytic differentiation. To identify transcriptional DAPK2 regulators, we cloned 2.7 kb of the 5'-flanking region of the DAPK2 gene. We found that E2F1 and Krüppel-like factor 6 (KLF6) strongly activate the DAPK2 promoter. We mapped the E2F1 and KLF6 responsive elements to a GC-rich region 5' of exon 1 containing several binding sites for KLF6 and Sp1 but not for E2F. Moreover, we showed that transcriptional activation of DAPK2 by E2F1 and KLF6 is dependent on Sp1 using Sp1/KLF6-deficient insect cells, mithramycin A treatment to block Sp1-binding or Sp1 knockdown cells. Chromatin immunoprecipitation revealed recruitment of Sp1 and to lesser extent that of E2F1 and KLF6 to the DAPK2 promoter. Activation of E2F1 in osteosarcoma cells led to an increase of endogenous DAPK2 paralleled by cell death. Inhibition of DAPK2 expression resulted in significantly reduced cell death upon E2F1 activation. Similarly, KLF6 expression in H1299 cells increased DAPK2 levels accompanied by cell death that is markedly decreased upon DAPK2 knockdown. Moreover, E2F1 and KLF6 show cooperation in activating the DAPK2 promoter. In summary, our findings establish DAPK2 as a novel Sp1-dependent target gene for E2F1 and KLF6 in cell death response.

PU.1 binding to the p53 family of tumor suppressors impairs their transcriptional activity.

The transcription factor PU.1 is essential for terminal myeloid differentiation, B- and T-cell development, erythropoiesis and hematopoietic stem cell maintenance. PU.1 functions as oncogene in Friend virus-induced erythroleukemia and as tumor suppressor in acute myeloid leukemias. Moreover, Friend virus-induced erythroleukemia requires maintenance of PU.1 expression and the disruption of p53 function greatly accelerates disease progression. It has been hypothesized that p53-mediated expression of the p21(Cip1) cell cycle inhibitor during differentiation of pre-erythroleukemia cells promotes selection against p53 function. In addition to the blockage of erythroblast differentiation provided by increased levels of PU.1, we propose that PU.1 alters p53 function. We demonstrate that PU.1 reduces the transcriptional activity of the p53 tumor suppressor family and thus inhibits activation of genes important for cell cycle regulation and apoptosis. Inhibition is mediated through binding of PU.1 to the DNA-binding and/or oligomerization domains of p53/p73 proteins. Lastly, knocking down endogenous PU.1 in p53 wild-type REH B-cell precursor leukemia cells leads to increased expression of the p53 target p21(Cip1).

Nerve regeneration in diabetic rats.

This study evaluated the capacity of diabetic rats to recover the ability to walk after nerve repair or nerve graft of the posterior tibial nerve at thigh level. Functional recovery of the posterior tibial nerve was evaluated by walking track analysis during regeneration in streptozotocin-induced diabetic rats. Surgical procedures were performed 8 weeks after induction of diabetes. The nerve repair was epineurial. The nerve graft was a 1.5 cm segment orthotopically replaced. There was no significant difference in functional recovery between normal and diabetic rats for both the nerve repair and nerve graft groups at 6, 12, and 24 weeks after nerve reconstruction. It is concluded that the presence of diabetes is not a contraindication for nerve reconstruction.

Finger polydactyly.

Polydactyly is one of the most common congenital differences. Duplications of the index finger, central rays, and small digit each have unique characteristics and associations. Complex anomalies such as the mirror hand and pentadactyly represent specialized forms of polydactyly. The goal of reconstructing a functional hand is met by appreciating the anatomic variations and systemic implications, then employing the challenging technical and intellectual concepts described.

Repair of midline vertical lower lip deficiency after mandible reconstruction.

BACKGROUND: When lower lip incisions are combined with anterior oromandibular resection and composite flap reconstruction, increased potential exists for abnormal healing of the lower lip. The aim of this study is to describe these deformities and operative techniques for their correction. METHODS: A retrospective review of all patients undergoing oromandibular reconstruction with osteocutaneous free tissue transfer at Indiana University Medical Center and Affiliated Hospitals during the 5-year period between January 1991 and 1996 was performed. Patients requiring secondary lower lip revision or repair were identified. Patient characteristics, lip deformities, and operative correction techniques were detailed. RESULTS: Sixty patients underwent 62 free tissue transfers for oromandibular reconstructions. Five patients (7.1%) required reoperation for lower lip deformities related to abnormal wound healing. All affected patients had lower lip split incisions for access to the oropharynx and composite anterior floor of mouth and mandibular resection and reconstruction. Each patient had successful repair of the lower lip deformity and correction of related functional deficits using lower lip tissue. CONCLUSION: Lower lip deformities occur in a subset of patients undergoing anterior mandibular reconstruction. Horizontally redundant lower lip tissue can be mobilized to repair the secondary aesthetic and functional deficits.

Cost-effective analysis of surgical palliation versus endoscopic stenting in the management of unresectable pancreatic cancer.

BACKGROUND: Ductal carcinoma of the pancreas is unresectable for cure in the majority of patients. We reviewed our results and cost effectiveness of surgical and endoscopic biliary bypass for unresectable pancreatic cancer to evaluate the comparable outcomes. METHODS: Between 1990 and 1992, 136 patients were managed operatively or endoscopically for pancreatic carcinoma. Excluding potentially curative resections and patients without follow-up, 34 patients endoscopically stented and 32 patients surgically bypassed were evaluated. RESULTS: Mean patient age was older (72.1 vs. 69.3 years) but average performance status was comparable (0.8 vs. 0.9 Eastern Cooperative Oncology Group grading) in the medical treatment group. The initial hospital stay was significantly longer for surgical patients (mean 14 vs. 7 days, p < 0.001), with higher average charges ($18,325 vs. $9,663). Twelve stented patients required rehospitalization (average charge of $4,029), and eight surgical patients were readmitted (average charge of $6,776). An average of 1.7 stent changes (average charge $1,190) were required. Mean survival was longer for the stented group (9.7 vs. 7.3 months, p = 0.13). CONCLUSIONS: Endoscopic stenting for unresectable pancreatic cancer provides equivalent duration of survival at reduced cost and shorter hospital stay, although subsequent stent changes are necessary. When curative resection is not possible, endoscopic biliary drainage should be considered a good first choice for palliative management.

Effects of tissue expansion on secondary ischemic tolerance in experimental free flaps.

The effects of tissue expansion on free flap tolerance and metabolic response to secondary ischemia were evaluated. A total of 178 male syngeneic Lewis rats were used: 28 in perfusion study and 75 donor and 75 recipient animals in flap survival study. Animals were organized in three experimental groups: control, sham operation, and expansion group. Sham group animals had the expander implanted but not insufflated. After 4 weeks of tissue expansion, 3 x 5-cm epigastric free flaps were transplanted to recipient animals. Twenty-four hours later, secondary ischemia was produced by 3-hour venous occlusion. Flap survival, perfusion, and enzyme activities were determined. Pre-expanded skin flaps had an increase in perfusion of approximately 700% as measured by fluorescein levels compared with control flaps (p < 0.001) and demonstrated a better success rate (76%) compared with those of the control (40%) (p < 0.05) and sham (28%) groups (p < 0.05). Glutathione peroxidase, glutathione reductase, and glucose 6-phosphate dehydrogenase of the antioxidant defense systems significantly increased in skin in both the sham and the expansion groups. In response to secondary ischemia, the control and sham groups exhibited a decrease in enzyme activities of the glutathione redox cycle, whereas the expansion group showed no significant changes from the elevated baseline activities. Tissue expansion improved flap tolerance to secondary ischemia by increasing flap circulation and probably by augmenting tissue metabolic response to oxidative stress.

Free radical damage in acute nerve compression.

Nerve compression causes injury by local ischemia and direct mechanical distortion. Peripheral nerves in diabetes mellitus are more prone to injury than those of nondiabetics. We sought to determine whether reperfusion-induced, oxygen-derived free radical injury occurs in peripheral nerves subjected to acute compression in normal and chronically diabetic rats. Female Sprague-Dawley rats weighing 250 to 275 g (N = 347) were divided into two groups: normal and streptozocin-induced diabetics. A total of 187 normal and 160 diabetic nerves were analyzed. After 8 weeks of untreated hyperglycemia, the sciatic nerves of normal and diabetes mellitus rats were subjected to one of three operations: a sham operation, 24-hour compression alone, and 24-hour compression followed by 1-hour reperfusion (CR). Nerve compression was established by banding the right sciatic nerve with a Silastic tubing, 1 cm long and 0.62 mm internal diameter, which was secured with 6-0 nylon suture. In the CR group, after 24 hours of compression, the tubings were released for 1 hour to permit reperfusion. Nerve tissue within the zone of compression underwent biopsy examination and was frozen for subsequent analysis. Blood flow to the nerve was quantified by injecting fluorescein (10 mg/kg intravenously) 10 minutes before harvest and measuring tissue levels fluorometrically. Compression with the Silastic tubing significantly reduced neural blood flow by 75%. Blood flow improved but failed to return to baseline levels after tubing release in diabetes mellitus nerves while perfusion returned to baseline in non-diabetes mellitus nerves. Nerve homogenate was assayed for malonyldialdehyde, an indicator of lipoperoxidation, as well as enzymes of cellular defense and glucose metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Preoperative highly selective catheter localization of occult small-intestinal hemorrhage with methylene blue dye.

In patients with occult lower gastrointestinal bleeding, locating of the source of the hemorrhage can be quite difficult. While multiple diagnostic tests may confirm a small bowel source of bleeding, precise localization allowing a directed, conservative surgical resection may be problematic. We describe three patients each of whom presented with hemorrhage from the small intestine. The preoperative use of highly selective angiographic catheter placement combined with intraoperative methylene blue dye injection allowed us to precisely locate the source of hemorrhage and to resect a conservative length of small intestine. This technique has been successful in the three patients herein, and we believe that it should be included in the options available to the surgeon.

Age and tolerance to secondary ischemia in rat epigastric flaps.

The use of microvascular procedures is increasing as the population continues to age. The purpose of this study was to observe the survival of skin flaps after ischemic injury. Skin flaps (n = 50) underwent either 3 hours of primary (1 degree) or secondary (2 degrees) venous occlusion in young (2-3 mo) and old (18-22 mo) rats. Skin flap survival was assessed on postoperative day 7. Survival rates for young and old after 3 hours of 1 degree ischemia was 100% and 90% (ns). Survival rats for young and old after 2 degrees ischemia were 67% and 47% (ns).

Spectrum and management of major complications of laparoscopic cholecystectomy.

Laparoscopic cholecystectomy has become the most prevalent method of treating uncomplicated, symptomatic cholelithiasis in the United States and elsewhere. As experience with this procedure grows, certain pitfalls are becoming apparent. Since October 1990, we have treated 22 patients for major injuries incurred during laparoscopic cholecystectomy, including 15 women and 7 men (range: 23 to 85 years). One patient had previous upper abdominal surgery; no other patient had any relative contraindication to laparoscopic surgery. The most frequent site of injury (19 patients) was the extrahepatic biliary tract. There was one fatal duodenal perforation. All but two patients whose injuries went unrecognized at laparoscopy were symptomatic during the immediate postoperative period. The biliary injuries included complete transection of the common hepatic or common bile duct in 10 patients, complete ductal occlusion in 3, a cystic duct stump leak in 2, and a partially retained gallbladder with a contained intraperitoneal bile leak in 2. The site and extent of biliary injuries were delineated with transhepatic or endoscopic retrograde cholangiography. Reconstruction or repair of the biliary tract was accomplished with Roux-en-Y hepaticojejunostomy or cholangiojejunostomy in 11 and 1 patients, respectively, completion cholecystectomy in 2, and temporary transhepatic stenting, primary choledochocholedochostomy, and primary choledochorrhaphy over a T-tube in 1 patient each. One patient with a cystic duct stump leak was managed successfully with endoscopic sphincterotomy, whereas another required operative ligation. Laparoscopic injuries during cholecystectomy can lead to serious morbidity and mortality, thus emphasizing the need for adequate training and credentialing for surgeons and for a heightened clinical awareness of the potential complications, their long-term sequelae, and how to avoid them.

Efficacy of intraoperative enteroscopy in diagnosis and prevention of recurrent, occult gastrointestinal bleeding.

The role and effectiveness of intraoperative enteroscopy in the evaluation of gastrointestinal (GI) bleeding of obscure origin is not clearly defined. Our aim was to determine if intraoperative enteroscopy is effective in identifying a source, which would lead to therapy and prevent recurrent gastrointestinal hemorrhage. Forty-four patients (median age: 64 years) underwent intraoperative enteroscopy. Median number of preoperative blood transfusions, duration of bleeding (months), and prior hospitalizations for GI hemorrhage were 19, 15, and 2, respectively. Many patients had risk factors associated with bleeding. All had undergone an extensive preoperative evaluation. Intraoperative enteroscopy was completely negative in 13 (30%). A site-specific source was seen in the small bowel in 31 patients (70%); 27 patients had lesions amenable to segmental resection with or without other means of definitive management. Only 6 of 31 patients (19%) had lesions that were actively bleeding. Twenty-three (52%) patients have had recurrent bleeding requiring transfusion (median follow-up: 21 months). Although intraoperative enteroscopy identified specific mucosal abnormalities in 70% of patients, the therapeutic efficacy in preventing recurrent hemorrhage was only 41%. Intraoperative enteroscopy is an effective tool in selected patients with occult GI bleeding and correctly identifies a treatable source and prevents recurrent bleeding in 41% of patients.