Iatrogenic Ureteral Injury and Prophylactic Stent Use in Veterans Undergoing Colorectal Surgery.

INTRODUCTION: Iatrogenic ureteral injury (IUI) is an uncommon complication in colorectal surgery. Prophylactic ureteral stenting (PUS) gained acceptance to aid in intraoperative identification of the ureter. Despite its use, the benefit of pus to avoid IUI remains debatable. We sought to analyze the rates of IUI after colorectal surgery in veterans and to compare the outcomes after PUS using a large matched cohort. METHODS: The veterans affairs surgical quality improvement program database was queried for patients who underwent colorectal surgery from 2008-2015. To analyze the outcomes of PUS, we created two matched groups using propensity-score matching accounting for demographical and clinical cofactors to assess variable outcomes. Cross-tabulation was used to calculate rates of IUI and univariate and multivariate analyses were performed to evaluate risk factors associated with IUI. RESULTS: 27,448 patients were identified and 458 underwent PUS placement (1.6%). The majority of procedures were performed electively and with an open approach. Mean age was 65 y, 96.3% were male, and colorectal cancer was the most common indication. 45 patients (0.2%) were diagnosed with IUI. IUI incidence was higher in female patients, after left-sided colorectal resection, and in those undergoing open procedures. After matching, PUS use was associated with longer length of stay and operative time and increased creatinine levels from baseline. CONCLUSION: We demonstrated that the use of PUS is independently associated with increased operative time and change in creatinine levels. Although no IUI occurred in the PUS group, this finding was not statistically significant. The risk and/or benefit ratio of PUS should be considered for each individual case, with its selective use based on the presence of risk factors for IUI, such as female patients and left-sided resections.

IL-10 Signaling in the Tumor Microenvironment of Ovarian Cancer.

Unlike other malignancies, ovarian cancer (OC) creates a complex tumor microenvironment with distinctive peritoneal ascites consisting of a mixture of several immunosuppressive cells which impair the ability of the patient's immune system to fight the disease. The poor survival rates observed in advanced stage OC patients and the lack of effective conventional therapeutic options have been attributed in large part to the immature dendritic cells (DCs), IL-10 secreting regulatory T cells, tumor-associated macrophages, myeloid-derived suppressor cells, and cancer stem cells that secrete inhibitory cytokines. This review highlights the critical role played by the intraperitoneal presence of IL-10 in the generation of an immunosuppressive tumor microenvironment. Further, the effect of antibody neutralization of IL-10 on the efficacy of DC and chimeric antigen receptor T-cell vaccines will be discussed. Moreover, we will review the influence of IL-10 in the promotion of cancer stemness in concert with the NF-κB signaling pathway with regard to OC progression. Finally, understanding the role of IL-10 and its crosstalk with various cells in the ascitic fluid may contribute to the development of novel immunotherapeutic approaches with the potential to kill drug-resistant OC cells while minimizing toxic side effects.

Outcomes of cholecystectomy in US veterans with cirrhosis: Predicting outcomes using nomogram.

BACKGROUND: This study examines the outcomes of open and laparoscopic cholecystectomy (OC/LC) in veterans with cirrhosis and develops a nomogram to predict outcomes. METHODS: We analyzed the Veterans Affairs Surgical Quality Improvement Program to identify all patients with cirrhosis and ascites who underwent cholecystectomy from 2008 to 2015. Univariate and multivariate regression were used to identify predictors of morbidity and mortality. A predictive nomogram was constructed and internally validated. RESULTS: A total of 349 patients were identified. Overall, complications occurred in 18.7% of patients, and mortality was 3.8%. LC was performed in 58.9%, and 19.2% were preformed emergently. Overall, Model for End-Stage Liver Disease score was an independent factor of morbidity and mortality, while laparoscopic approach had a protective effect on morbidity. CONCLUSIONS: Although cholecystectomy is a high-risk operation in cirrhotic veterans, LC may have favorable outcomes than OC in selected patients. An easy-to-use nomogram to predict morbidity and mortality for cirrhotic patients undergoing cholecystectomy is proposed.

Postoperative outcomes of ventral hernia repair in veterans.

BACKGROUND: Ventral hernia repair is a common procedure with reported 15% to 37% morbidity and 0.3% to 1.4% mortality rates. This study examines the 30-day morbidity and mortality of open and laparoscopic ventral hernia repair in veterans, along with the impact of body mass index on these outcomes. METHODS: The Veterans Affairs Surgical Quality Improvement Program was queried for all ventral hernia repairs during the period 2008 to 2015. In this retrospective analysis, we compared outcomes of open ventral hernia repair versus laparoscopic ventral hernia repair and among different body mass index classes. RESULTS: A total of 19,883 patients were identified (92.6% male, mean age 59.7, 53.1% obese, and 71.6% with American Society of Anesthesiologists class ≥III). There were 95 (0.5%) mortalities, and complications occurred in 1,289 (6.5%) patients. Open ventral hernia repair was performed in 60.2%; 14.5% were recurrent, and 3.3% were performed as an emergency operation. When compared with open ventral hernia repair, the laparoscopic ventral hernia repair group had higher mean body mass index, less patients with American Society of Anesthesiologists class ≥III, fewer emergency operations, longer operative time, less complications, decreased mortality, and shorter duration of stay. Body mass index 35.00 to 49.99 was predictive of overall complications in the open ventral hernia repair group. CONCLUSION: Ventral hernia repair can be performed in the veteran population with outcomes comparable to those in the private sector. Morbid obesity has a negative impact on ventral hernia repair outcomes that is most prominent following open surgery. Laparoscopic ventral hernia repair may offer superior outcomes when compared to open ventral hernia repair and may be considered.

Recent trends in cholecystectomy in US veterans.

INTRODUCTION: We hypothesize that the recent trend in performing cholecystectomy in US Veterans shows wide adoption of the laparoscopic technique and improvement in the outcome following both laparoscopic (LC) and open cholecystectomy (OC). This study utilizes the Veterans Affairs Surgical Quality Improvement Program database to examine the status and outcome of cholecystectomy. METHODS: A retrospective review of veterans who underwent cholecystectomy between 2008 and 2015 was performed. Data analysis included patient demographics, operations, and postoperative outcomes. Cochran-Armitage trend analysis was used to assess significant changes in outcome over the study period. p ≤ 0.05 was considered significant. RESULTS: A total of 40,722 patients (average age of 61 years) were included in the study (males 85.6%). LC was performed in the majority of patients (86.4%). Patients in the OC group (13.6%) were more likely to have advanced age (≥ 65 years) (47.6% vs 32.0%, p < 0.001) and higher ASA class (III-V) (81.9% vs 65.4%, p < 0.001) than those in the LC group. Compared with LC, OC had higher mortality rates at 30 days (1.3% vs 0.3%; OR = 1.6, p = 0.03), 3 months (2.6% vs 0.7%; OR = 1.7, p < 0.001), 6 months (3.9% vs 1.1%; OR = 1.5, p < 0.001) and 1 year (5.7% vs 2.0%; OR = 1.5, p < 0.001); higher rates of morbidity, including pneumonia (OR = 1.9, p < 0.001), deep venous thrombosis (OR = 2.4, p = 0.02), reoperation (OR = 1.8, p < 0.001), and superficial (OR = 4.9, p < 0.001) and deep (OR = 1.5, p = 0.01) surgical site infections; and a longer length of stay (6.5 days vs 2.6 days, p < 0.001). Trend analysis showed a significant decrease in both mortality (p = 0.02) and morbidity (p < 0.001) for LC over the study period, but no improvement in mortality (p = 0.35) and a only a minimal improvement in morbidity (p = 0.04) for OC. CONCLUSION: In the recent era, LC has been widely performed in the VA with significant improvement in outcome. Efforts are needed to adopt alternative approaches to planned OC and to improve postoperative outcomes.

Engraftment of mesothelin chimeric antigen receptor using a hybrid Sleeping Beauty/minicircle vector into NK-92MI cells for treatment of pancreatic cancer.

BACKGROUND: We have previously demonstrated in vitro cytotoxicity of mesothelin-chimeric antigen receptor autologous T cells against pancreatic cancer cells using lentiviral vectors, but these vectors pose safety concerns. Here, we incorporated Sleeping Beauty and minicircle design enhancements into interleukin-2-secreting natural NK-92MI cells to eliminate both bacterial and viral components and address inhibition by the tumor microenvironment. METHODS: Parental (conventional deoxyribonucleic acid)-mesothelin-chimeric antigen receptor and minicircle-mesothelin-chimeric antigen receptor vectors were electroporated into NK-92MI cells and engraftment was visualized by immunofluorescence analysis with protein-L staining. Interferon-γ and granzyme B secretion were measured by enzyme-linked immunosorbent assay from cocultures of parental-mesothelin-chimeric antigen receptors and minicircle-mesothelin-chimeric antigen receptors with human pancreatic cancer cells, and cytotoxicity of chimeric antigen receptor NK-92MI cells was tested against three pancreatic cancer cell lines. RESULTS: Cloning of mesothelin-chimeric antigen receptor Sleeping Beauty into a minicircle vector removed its bacterial backbone and reduced its size with improved electroporation efficiency. Chimeric antigen receptor engraftment, Interferon-γ and granzyme B secretion, and specific lysis against all three pancreatic cancer lines were significantly increased with minicircle-mesothelin-chimeric antigen receptor versus parental-mesothelin-chimeric antigen receptor NK-92MI cells. CONCLUSION: We provide proof of concept that allogeneic mesothelin-chimeric antigen receptor NK-92MI cells with hybrid Sleeping Beauty and minicircle technologies provide increased engraftment and cytotoxicity in vitro with potential safety benefits when translated to the clinical arena.

Inhibition of Interleukin-10 in the tumor microenvironment can restore mesothelin chimeric antigen receptor T cell activity in pancreatic cancer in vitro.

BACKGROUND: Pancreatic cancer cells are known to shield themselves from immunosurveillance by secreting immune inhibitory cytokines such as Interleukin-10. Using mesothelin, a differentiating antigen that is overexpressed in pancreatic cancer, we assessed the negative effect of the tumor microenvironment on chimeric antigen receptor T cell-based immunotherapy and its reversal via depletion of Interleukin-10. METHODS: T cells cultured in pancreatic cancer-cell-conditioned medium were transduced with lentiviruses encoding mesothelin-chimeric antigen receptor in the presence or absence of anti-Interleukin-10-blocking antibody. RESULTS: Coculture supernatants of conditioned medium displayed significant inhibition of interferon γ and granzyme B secretion, both of which are crucial for induction of target cell cytotoxicity. In contrast, this inhibition was restored toward baseline when conditioned medium was Interleukin-10- depleted (p < .05 for both interferon γ and granzyme B). In addition, we observed a significant decrease in mesothelin-chimeric antigen receptor T cell-induced cytotoxicity of BxPC-3 target cells in the presence of conditioned medium. Furthermore, we observed a partial blunting of this inhibition when Interleukin-10 was depleted from the conditioned medium. CONCLUSION: Substantial reversal of tumor-derived immunosuppression may be achieved by blocking Interleukin-10 in the local microenvironment, allowing for more effective cytotoxicity of mesothelin-engrafted chimeric antigen receptor T cells and enhancing the potential for clinical application.

Enhanced phosphorylation of p53 by microRNA-26a leading to growth inhibition of pancreatic cancer.

PURPOSE: MicroRNA (miR)-26a has been identified as a tumor suppressor in pancreatic cancer cells. Although wild-type p53 controls cell-cycle progression, its mutant form normally present in pancreatic cancer loses this capability. Phosphorylation is known to restore wild-type activity to mutant p53. We, therefore, examined whether miR-26a treatment can restore wild-type functions of mutant p53 via phosphorylation, resulting in inhibition of cell growth. METHODS: The human pancreatic cancer cell line BxPc-3 harboring mutant p53 was used for colony formation, cell-cycle, and Western blotting assays. Gene profile analysis was conducted after transfection with pre-miR-26a. RESULTS: miR-26a expression significantly decreased cell proliferation by 80% along with marked inhibition of colony formation and cell migration. Cell-cycle inhibition at the G0/G1 interface was observed along with enhanced drug retention and increased chemosensitivity to gemcitabine. Mutant p53 was phosphorylated rapidly at its Ser9 and Ser392 residues, but not at Ser15 or Ser20. Gene profile analysis of pre-miR-26a-transfected cells showed a significant increase in gene transcripts promoting apoptosis and p53 activation, with decreased levels of genes involved in cell-cycle progression. CONCLUSION: Delivery of miR-26a may represent a novel strategy for inhibiting pancreatic cancer growth, at least in part by enhancing phosphorylation of mutant p53 to restore its wild-type functions.

Raman spectroscopic modeling of early versus late T-lymphocyte activation via differential spectral detection of receptor expression.

The proven efficacy of renal transplantation has made it the definitive treatment for end-stage renal disease. Despite its wide acceptance, transplantation has been limited by organ shortages. In the face of this, preservation of allograft longevity is essential. The predominately T cell-driven process of acute rejection (AR) can lead to graft dysfunction and even graft loss. As a marker for AR screening, serum creatinine has a low sensitivity and specificity. This has warranted the development of more accurate screening/diagnostic tools such as Raman Spectroscopy (RS) which has been demonstrated in previous studies to accurately quantify T cell activation. In this study we further explore its application by modeling the dynamic process of cell surface receptor expression during T cell activation. 50 mitogen (Concanavalin A and pokeweed) activated T cells were stained with CD69, CD25, and CD71 monoclonal antibodies (mAbs) at 48 and 72 hour time points. In parallel, 50 activated T cells were analyzed using RS at these same time periods. At 4 8h there was high expression of the CD69 cell surface receptor detected via mAb staining with no appreciable binding of CD25/CD71 fluorescent tag. In conjunction, 48 hour RS-analyzed T cells demonstrated a significant peak difference at the 1585 cm(-1) position which represented a 63% (p=0.01) increase in peak magnitude when compared with the 72 hour samples. By contrast, the 72 hour data demonstrated an attenuation of the CD69 expression and increased CD25/CD71 expression. The corresponding RS analysis showed two significant peak differences at the 903 cm(-1) and 1449 cm(-1) positions that were not present at 48 h. These differences in Raman shifts resulted in a 40% (p=0.04) and a 59% (p=0.001) increase in peak magnitudes at these positions, respectively. This study serves to further validate RS as a screening modality capable of not only detecting T cells early in the activation process through the spectral signatures associated with CD69, but also quantifying the persistent expression of CD25 and CD71. This provides a foundation for the development of a system capable of the accurate assessment of acute and maintenance immunosuppression efficacy at the molecular level.

MAGE-A3 with cell-penetrating domain as an efficient therapeutic cancer vaccine.

IMPORTANCE: In conjunction with chemotherapy, immunotherapy with dendritic cells (DCs) may eliminate minimal disease burden by generating cytotoxic T lymphocytes. Enhanced cytosolic bioavailability of tumor-specific antigens improves access to human leukocyte antigen (HLA) class I molecules for more efficient cytotoxic T lymphocyte generation. Various cell-penetrating domains (CPDs) are known to ferry covalently linked heterologous antigens to the intracellular compartment by traversing the plasma membrane. OBJECTIVE: To determine whether generating melanoma antigen family A, 3 (MAGE-A3), a tumor-specific cancer-testis antigen, as a fusion protein with CPD will enhance the cytosolic bioavailability of MAGE-A3. DESIGN: MAGE-A3 was amplified by polymerase chain reaction using complementary DNA from renal tissue and cloned in frame with a CPD (YARKARRQARR) at the amino-terminal end and hexahistidine at the carboxy-terminal end to generate CPD-MAGE-A3 in a pQE-70 expression vector. Cultures were grown in Escherichia coli BL21 Star (DE3-pLysS) cells followed by nickel-nitrilotriacetic acid affinity purification of recombinant proteins. MAIN OUTCOMES AND MEASURES: Measurement of DC membrane penetration of CPD-MAGE-A3 vs MAGE-A3 and determination of the effect of CPD-MAGE-A3 pulsing on DC phenotypic expression of cell-surface antigens. RESULTS: Media composition and isopropyl-d-thiogalactosidase induction were optimized to achieve high levels of protein expression followed by purification. Western blot analysis with MAGE-A3 antibodies recognized both MAGE-A3 and CPD-MAGE-A3 proteins, while CPD antibodies recognized only CPD-MAGE-A3. Purified CPD-MAGE-A3 exhibited more efficient DC membrane penetration than did MAGE-A3 alone as confirmed by immunofluorescence analysis. High-level expression of several unique DC markers (CD80, CD83, CD86, and HLA-DR) by flow cytometry was consistent with a mature DC phenotype, indicating that pulsing with CPD-MAGE-A3 did not alter specific cell-surface antigens required for T-cell activation. CONCLUSIONS AND RELEVANCE: We have demonstrated for the first time, to our knowledge, that cloning and purification of MAGE-A3 with CPD enhances its cytosolic bioavailability in DCs without altering cell-surface antigens, potentially making it a more potent therapeutic cancer vaccine compared with existing MAGE-A3 protein and peptide vaccines.

Ritonavir-Mediated Induction of Apoptosis in Pancreatic Cancer Occurs via the RB/E2F-1 and AKT Pathways.

Recent observations suggest a lower incidence of malignancies in patients infected with HIV during treatment with Highly Active Anti-Retroviral Therapy (HAART) utilizing protease inhibitors. We investigated the effects of ritonavir, a FDA approved HIV protease inhibitor, on proliferation of pancreatic ductal adeno-carcinoma (PDAC) cell lines. Human PDAC cell lines BxPC-3, MIA PaCa-2, and PANC-1 were propagated under standard conditions and treated with serial dilutions of ritonavir. Ritonavir inhibited cell growth in a dose-dependent manner as well as activated the intrinsic apoptotic pathway in human pancreatic ductal adenocarcinoma (PDAC) cell lines. We observed down-modulation of cell-cycle promoting and up-regulation of cell-cycle inhibitory genes; enhanced interaction of retinoblastoma protein (RB) with E2F-1 transcription factor; inhibition of phosphorylation of RB, resulting in sequestration of E2F-1 and subsequent down-regulation of S phase genes; decreased interaction of E2F-1 with its consensus binding sites; inhibition of cell motility and invasiveness; and inhibition of the AKT pathway. Our results demonstrate a potential use of ritonavir as part of combination chemotherapy for PDAC. Since ritonavir is FDA approved for HIV, drug repositioning for PDAC would limit the costs and reduce risks.

Efficient lysis of epithelial ovarian cancer cells by MAGE-A3-induced cytotoxic T lymphocytes using rAAV-6 capsid mutant vector.

MAGE-A3 is highly expressed in epithelial ovarian cancer (EOC), making it a promising candidate for immunotherapy. We investigated whether dendritic cells (DCs) transduced with a rAAV-6 capsid mutant vector Y445F could elicit effective MAGE-A3-specific anti-tumor cytotoxic T lymphocyte (CTL) responses in vitro. MAGE-A3 was cloned and rAAV-6-MAGE-A3 purified, followed by proviral genome detection using real-time PCR. Immunofluorescence detection of rAAV-6-Y445F-MAGE-A3-transduced DCs demonstrated 60% transduction efficiency. Fluorescent in situ hybridization analysis confirmed chromosomal integration of rAAV vectors. Flow cytometric analysis of transduced DCs showed unaltered expression of critical monocyte-derived surface molecules with retention of allo-stimulatory activity. Co-culture of autologous T lymphocytes with MAGE-A3-expressing DCs produced CTLs that secreted IFN-γ, and efficiently killed MAGE-A3+ EOC cells. This form of rAAV-based DC immunotherapy, either alone or more likely in combination with other immune-enhancing protocols, may prove useful in the clinical setting for management of EOC.

EZH2-shRNA-mediated upregulation of p21waf1/cip1 and its transcriptional enhancers with concomitant downmodulation of mutant p53 in pancreatic ductal adenocarcinoma.

PURPOSE: Enhancer of zeste homologue 2 (EZH2), a component of the chromatin modification protein complex, is upregulated in pancreatic ductal adenocarcinoma (PDAC), whereas loss of p53 and its downstream target, p21(waf1/cip1), is also observed frequently. We sought to investigate the role of the p53-p21(waf1/cip1) pathway in relation to EZH2-mediated inhibition of PDAC. METHODS: The PANC-1 cell line was utilized in chromatin immunoprecipitation, gene profiling, Western blot, cell invasion, cell proliferation, and tumor xenograft assays. RESULTS: Western blot analysis with antibodies that recognize both wild-type and mutant p53 did not show any alterations in band intensity; however, antibody that detects only mutant p53 showed a band of significantly lesser intensity with EZH2 knockdown. Western blot analysis further revealed a significant upregulation of p21(waf1/cip1). Gene expression profile analysis indicated significantly enhanced transcripts of transcriptional inducers of p21(waf1/cip1), with downregulation of mutant p53 transcript, corroborating the Western blot analysis. PANC-1 cells expressing EZH2-short hairpin RNA displayed markedly attenuated growth in SCID mice. CONCLUSION: Downregulation of mutant p53 with concomitant enhanced expression of p21(waf1/cip1) and its transcriptional trans-activators may contribute toward EZH2-mediated suppression of PDAC.

EZH2 blockade by RNA interference inhibits growth of ovarian cancer by facilitating re-expression of p21(waf1/cip1) and by inhibiting mutant p53.

The enhancer of zeste homolog 2 (EZH2) methyltransferase is a transcriptional repressor. EZH2 is abnormally elevated in epithelial ovarian cancer (EOC). We demonstrated that EZH2 knockdown inhibited cell growth, activated apoptosis, and enhanced chemosensitivity. Further, silencing of EZH2 resulted in re-expression of p21(waf1/cip1) and down-regulation of mutant p53. Finally, EZH2 knockdown contributed to attenuated EOC growth in SCID mice.

Restoration of E-cadherin expression in pancreatic ductal adenocarcinoma treated with microRNA-101.

PURPOSE: To investigate the possibility of inhibiting the progression of pancreatic ductal adenocarcinoma (PDAC) by facilitating the expression of E-cadherin through the enforced expression of microRNA-101 (miR-101). METHODS: In situ hybridization was conducted with archival tissue using a double digoxigenin-labeled probe. Chromatin immunoprecipitation (ChIP) assay was conducted with EZ-Magna ChIPTM A. Gene profile analysis, Western blot, and immunoprecipitation assays were performed using standard protocols. RESULTS: We found that decreased miR-101 expression observed in archival patient tissues was significantly associated with poor prognosis indicated by low-intensity staining in high-grade tumors. ChIP assays using anti-enhancer of zeste homolog 2 (EZH2) antibodies indicated not only the interaction of EZH2 to the CDH1 (E-cadherin) promoter, but also that this interaction was significantly diminished in cells transfected with pre-miR-101. We observed a global downregulation of trimethylated lysine 27 of H3 histone (H3K27me3) along with upregulation of the enzymes histone deacetylase -1 and -2 with the re-expression of miR-101. Further, we observed lesser levels of transcriptional factors that inhibit the CDH1 promoter with pre-miR-101 treatment. Western blot analysis confirmed the enhanced E-cadherin expression. PANC-1 cells transduced with pre-miR-101 displayed markedly attenuated growth in SCID mice. CONCLUSION: These results suggest the potential therapeutic use of miR-101-enforced expression for inhibition of PDAC.

MicroRNA-101 inhibits growth of epithelial ovarian cancer by relieving chromatin-mediated transcriptional repression of p21(waf¹/cip¹).

PURPOSE: MicroRNA-101 (miR-101) expression is negatively associated with tumor growth and proliferation in several solid epithelial cancers. Enhancer of zeste homolog 2 (EzH2) appears to be a functional target of miR-101. We explore the role of miR-101 and its interaction with EzH2 in epithelial ovarian carcinoma (EOC). METHODS: In situ hybridization (ISH) for miR-101 was performed on EOC patient tissues and normal controls. EOC cell lines were transfected with miR-101 and subjected to growth analysis and clonogenic assays. Cell motility was assessed by Boyden chamber and wound-healing assays. P21(waf1/cip1) and EzH2 interaction was assessed by Chromatin Immunoprecipitation (ChIP) assay in MDAH-2774 cells. SCID mice were assessed for tumor burden after injection with miR-101 or control vector-treated MDAH-2774 cells. RESULTS: ISH analysis revealed a decrease in miR-101 expression in EOC compared with normal tissue. MiR-101 re-expression in EOC cell lines resulted in increased apoptosis, decreased cellular proliferation, invasiveness, and reduced growth of tumor xenografts. CHIP assays revealed that re-expression of miR-101 inhibited the interaction of EzH2 with p21(waf1/cip1) promoter. CONCLUSIONS: MiR-101 re-expression appears to have antitumor effects, providing a better understanding of the role of miR-101 in EOC.