Phase I Study of Oncolytic Vaccinia Virus GL-ONC1 in Patients with Peritoneal Carcinomatosis.

Purpose: Peritoneal carcinomatosis is common in advanced tumor stages or disease recurrence arising from gastrointestinal cancers, gynecologic malignancies, or primary peritoneal carcinoma. Because current therapies are mostly ineffective, new therapeutic approaches are needed. Here, we report on a phase I study designed to assess safety, MTD, and antitumor activity of intraperitoneal administration of oncolytic vaccinia virus GL-ONC1 in advanced stage peritoneal carcinomatosis patients.Patients and Methods: GL-ONC1 was administered intraperitoneally every 4 weeks for up to four cycles at three different dose levels (107-109 pfu) following a standard 3+3 dose escalation design. GL-ONC1 was infused via an indwelling catheter that enabled repetitive analyses of peritoneal fluid biopsies. The primary study objective was safety of GL-ONC1 according to Common Terminology Criteria for Adverse Events, version 4.0 (CTCAEv4.0).Results: Patients with advanced-stage peritoneal carcinomatosis (n = 7) or advanced peritoneal mesothelioma (n = 2) received 24 doses of GL-ONC1. Adverse events were limited to grades 1-3, including transient flu-like symptoms and increased abdominal pain, resulting from treatment-induced peritonitis. No DLT was reported, and the MTD was not reached. Furthermore, no signs of viral shedding were observed. Importantly, in 8 of 9 study patients, effective intraperitoneal infections, in-patient replication of GL-ONC1, and subsequent oncolysis were demonstrated in cycle 1. All patients developed neutralizing activities against GL-ONC1.Conclusions: GL-ONC1 was well tolerated when administered into the peritoneal cavity of patients with advanced stage peritoneal carcinomatosis. Efficient tumor cell infection, in-patient virus replication, and oncolysis were limited to treatment cycle 1 (ClinicalTrials.gov number, NCT01443260). Clin Cancer Res; 24(18); 4388-98. ©2018 AACR.

Gastric Ulcer Complications after the Introduction of Proton Pump Inhibitors into Clinical Routine: 20-Year Experience.

BACKGROUND: The aim of this study was to analyze the admissions and the management of peptic ulcer disease (PUD) in a tertiary care surgical center. METHODS: We evaluated the medical records of all patients admitted to the University Hospital of Tübingen, Germany, for treatment of PUD during 1989-2008. Patients were included into the study if the diagnosis was verified endoscopically or surgically. Annual number of admissions, length of hospitalization, mortality rate, age, rate of non-steroidal anti-inflammatory drugs (NSAIDs) and proton pump inhibitor (PPI) medication, rate of Helicobacter pylori infection, and complications of PUD and surgery performed were recorded. Data were analyzed by descriptive analyses, Pearson's chi-square test, and regression analysis. RESULTS: This study included 614 admissions. The number of annual admissions (31 ± 12), the length of hospitalization (9 ± 3 days), and the mortality rate (5 ± 4% per year) remained constant, whereas the age increased (1989: 52 ± 14 years vs. 2008: 67 ± 16 years). The rates of patients with H. pylori infection (47 ± 28% per year), NSAIDs treatment (29 ± 15% per year), and PPI treatment (31 ± 27% per year) remained constant. The most frequent PUD complication was hemorrhage (42 ± 16% per year), followed by perforation (9 ± 8% per year). During 1999-2008, more hemorrhages (125 vs. 121; p < 0.05) and perforations (40 vs. 21; p < 0.05) were registered than during 1989-1998. The rate of emergency surgery increased from 70% during 1989-1998 to 87% during 1999-2008. In contrast, elective surgery decreased from 21% during 1989-1998 to 7% during 1999-2008. Ulcer excision and oversewing was the most frequent surgical procedure performed (59%), with decreasing rates of acid-reducing surgery. CONCLUSION: Despite recent advances in PUD management, ulcer hemorrhage and perforation remain a significant health burden and a surgical disease.

Personalized peptide vaccine-induced immune response associated with long-term survival of a metastatic cholangiocarcinoma patient.

BACKGROUND & AIMS: We report a novel experimental immunotherapeutic approach in a patient with metastatic intrahepatic cholangiocarcinoma. In the 5year course of the disease, the initial tumor mass, two local recurrences and a lung metastasis were surgically removed. Lacking alternative treatment options, aiming at the induction of anti-tumor T cells responses, we initiated a personalized multi-peptide vaccination, based on in-depth analysis of tumor antigens (immunopeptidome) and sequencing. METHODS: Tumors were characterized by immunohistochemistry, next-generation sequencing and mass spectrometry of HLA ligands. RESULTS: Although several tumor-specific neo-epitopes were predicted in silico, none could be validated by mass spectrometry. Instead, a personalized multi-peptide vaccine containing non-mutated tumor-associated epitopes was designed and applied. Immunomonitoring showed vaccine-induced T cell responses to three out of seven peptides administered. The pulmonary metastasis resected after start of vaccination showed strong immune cell infiltration and perforin positivity, in contrast to the previous lesions. The patient remains clinically healthy, without any radiologically detectable tumors since March 2013 and the vaccination is continued. CONCLUSIONS: This remarkable clinical course encourages formal clinical studies on adjuvant personalized peptide vaccination in cholangiocarcinoma. LAY SUMMARY: Metastatic cholangiocarcinomas, cancers that originate from the liver bile ducts, have very limited treatment options and a fatal prognosis. We describe a novel therapeutic approach in such a patient using a personalized multi-peptide vaccine. This vaccine, developed based on the characterization of the patient's tumor, evoked detectable anti-tumor immune responses, associating with long-term tumor-free survival.

Metabolic alteration--Overcoming therapy resistance in gastric cancer via PGK-1 inhibition in a combined therapy with standard chemotherapeutics.

BACKGROUND AND OBJECTIVES: It can be assumed that PGK1 is involved in metastatic spread of gastric carcinomas. Furthermore PGK1 has a proven influence on the characteristics of tumor stem cells. The presence of malignant stem cells, regarding treatment resistance and recurrence, is of considerable importance. We hypothesized that inhibition of PGK1 makes these cells more sensitive to chemotherapeutic agents and therefore mediates an overcome of the existing therapy resistance. METHODS: All investigations were performed with human gastric adenocarcinoma cell lines. Small hairpin RNA knockdown of PGK1 via adenovirus-shPGK1 was used for PGK1-inhibition. Chemotherapeutic agents were 5-FU and mitomycin. FACS, qRT-PCR, and xCELLigence were performed. RESULTS: Using the medium-sole-control indicating the highest cell viability and Triton indicating the lowest, mitomycin and 5-FU alone showed a significant decrease in cell viability. The treatment with AdvshPGK1 alone already showed a better decrease. The simultaneous application of chemotherapeutics and adenovirus showed the strongest effect and is comparable to the effect of Triton. CONCLUSIONS: We showed a significant decrease in cell viability after the simultaneous application of chemotherapeutics and adenovirus. These results suggest that PGK1-inhibition is able to increase the vulnerability of gastric cancer cells and tumor stem cells to overcome the chemotherapeutic therapy resistance.

New telemetry device for the measurement of gastrointestinal motility in rats and comparison with standard equipment.

To perform stress-free recording of gastrointestinal motility in rats with strain gauge transducers, telemetry equipment had to be developed. We developed, programmed, and tested a new telemetry device that records gastrointestinal motility in freely moving rats using strain gauge transducers. The device can collect and transmit data in freely moving rats. Data are received and stored for later analysis with a regular PC. Linear calibration curves were obtained for the strain gauge transducers used. We compared data obtained with the new telemetry device with data gathered with standard equipment and could not find any statistically significant difference. Wired gastric and colonic contraction frequencies were 4.6 ± 0.3 per minute and 1.5 ± 0.3 per minute, whereas telemetric contraction frequencies were 4.4 ± 0.1 per minute and 1.25 ± 0.1 per minute. The new telemetry device is a very useful tool for the measurement of gastrointestinal motility in rats.

Laparoscopic surgery for benign and malign diseases of the digestive system: indications, limitations, and evidence.

The laparoscopic technique was introduced in gastrointestinal surgery in the mid 1980s. Since then, the development of this technique has been extraordinary. Triggered by technical innovations (stapling devices or coagulation/dissecting devices), nowadays any type of gastrointestinal resection has been successfully performed laparoscopically and can be performed laparoscopically dependent on the patient's condition. This summary gives an overview over 30 years of laparoscopic surgery with focus on today's indications and evidence. Main indications remain the more common procedures, e.g., appendectomy, cholecystectomy, bariatric procedures or colorectal resections. For all these indications, the laparoscopic approach has become the gold standard with less perioperative morbidity. Regarding oncological outcome there have been several high-quality randomized controlled trials which demonstrated equivalency between laparoscopic and open colorectal resections. Less common procedures like esophagectomy, oncological gastrectomy, liver and pancreatic resections can be performed successfully as well by an experienced surgeon. However, the evidence for these special indications is poor and a general recommendation cannot be given. In conclusion, laparoscopic surgery has revolutionized the field of gastrointestinal surgery by reducing perioperative morbidity without disregarding surgical principles especially in oncological surgery.

CCK1-receptor stimulation protects against gut mediator-induced lung damage during endotoxemia.

BACKGROUND/AIMS: Cholecystokinin 1-receptor (CCK1-R) activation by long chain fatty acid (LCFA) absorption stimulates vago-vagal reflex pathways in the brain stem. The present study determines whether this reflex also activates the cholinergic anti-inflammatory pathway, a pathway known to modulate cytokine release during endotoxemia. METHODS: Mesenteric lymph was obtained from wild type (WT) and CCK1-R knockout (CCK1-R(-/-)) mice intraperitoneally challenged with Lipopolysaccharid (LPS) (endotoxemic lymph, EL) and intestinally infused with vehicle or LCFA-enriched solution. The lymph was analyzed for TNFα, IL-6 and IL-10 concentration and administered to healthy recipient mice via jugular infusion. Alveolar wall thickness, myeloperoxidase (MPO) and TUNEL positive cells were determined in lung tissue of recipient mice. RESULTS: LCFA infusion in WT mice reduced TNFα concentration in EL by 49% compared to vehicle infusion, but had no effect in CCK1-R(-/-) mice. EL significantly increased the alveolar wall thickness, the number of MPO-positive and TUNEL-positive cells compared to control lymph administration. LCFA infusion in WT, but not in CCK1R(-/-) mice, significantly reduced these pathological effects of EL. CONCLUSION: During endotoxemia enteral LCFA absorption reduces TNFα release into mesenteric lymph and attenuates histomorphologic parameters of lung dysfunction. Failure to elicit this effect in CCK1R(-/-) mice demonstrates that anti-inflammatory properties of LCFAs are mediated through CCK1-Rs.

Phosphoglycerate kinase 1 as a promoter of metastasis in colon cancer.

Oxidative stress due to intratumoral hypoxia in solid cancer has been shown to be associated with increased mortality. Phosphoglycerate kinase 1 (PGK1) is an enzyme of the glycolytic pathway, which is regulated by hypoxia-inducible factor-1α (HIF-1α) and has been described for its role in tumor progression and metastasis in several malignancies. We investigated whether the expression of PGK1 varies between metastatic and non-metastatic colon cancer. We compared PGK1 expression in colon cancer patients either with or without metastasis via polymerase chain reaction (PCR) and immunohistochemistry. Microarray analysis was performed to test altered gene expression after PGK1 silencing, using isolates from HCT116 cell lines. PCR results showed an increased expression of PGK1 in colon cancer tissue from metastatic patients in comparison to patients with no metastasis (fold change 2.6, p<0.001). Immunohistochemical staining of PGK1 showed stronger staining in metastatic tissue in comparison to non-metastatic cancer tissue according to a semi-quantitative evaluation. Microarray and subsequent pathway analysis provided 4 genes of interest (CYR61, FOS, JUN and EGR1) used for pathway proposal. The results indicate that increased expression of PGK1 in colon cancer tissue is associated with metastasis. Furthermore, we propose several genes induced by PGK1 that could account for cell migration, mainly EGR1 and CYR61 together with the transcription factors FOS and JUN.

Induction of tumor stem cell differentiation--novel strategy to overcome therapy resistance in gastric cancer.

PURPOSE: Metastases are a frequent finding in gastric cancer and are associated with poor prognosis. A recently discovered link between metabolic changes, differentiation, and therapy resistance due to tumor stem cells could depict a novel approach in cancer research and therapy. Phosphoglycerate kinase 1 (PGK1) is a metabolic enzyme and is known to be involved in enabling gastric cancer cells to be invasive and to disseminate. In this study, we investigated if PGK1 is a promising candidate in inducing stem cell differentiation in gastric cancer. MATERIALS AND METHODS: MKN45 gastric cancer cells were used due to their known cancer stem cell population, which is defined by the surface marker CD44. MKN45 cells were separated between CD44+ and CD44- cells and, in equal parts, incubated with shRNA anti-PGK1 using fluorescence-activated cell sorting (FACS) analysis; they were then injected into nude mice to evaluate their tumor growth behavior in vivo. Further, the invasive potential of gastric cancer cells was evaluated in vitro using the xCelligence analyzing system. RESULTS: CD44+ gastric cancer cells treated with and without shRNA anti-PGK1 were capable to cause tumor growth in vivo, whereas tumor growth in CD44+ cells treated with shRNA anti-PGK1 was considerably smaller in comparison with that in CD44+ cells without treatment. CD44- cells did not show any noticeable tumor growth in vivo. By targeting PGK1, the invasive potential of gastric cancer cells was impressively reduced in vitro. In all our cells, which were targeted with shRNA anti-PGK1, we did not find any change that is in accordance with the phenotype of the cells using FACS analysis. CONCLUSIONS: Our findings suggest that targeting the key metabolic enzyme PGK1 in gastric cancer cells may open a new chapter in cancer treatment, which is well worth for further exploration in combination with recent chemotherapy, and might be a promising possibility to overcome therapy resistance in gastric cancer.

Lactate influences the gene expression profile of human mesenchymal stem cells (hMSC) in a dose dependant manner.

BACKGROUND/AIMS: Wounds, especially non-healing wounds are characterized by elevated tissue lactate concentrations. Lactate is known for being able to stimulate collagen synthesis and vessel growth. Lately it has been shown that lactate, in vivo, plays an important role in homing of stem cells. With this work we aimed to show the influence of lactate on the gene expressionprofile of human mesenchymal stem cells (hMSC). MATERIALS AND METHODS: hMSCs were obtained from bone marrow and characterized with fluorescence-activated cell sorting (FACS) analysis. Subsequently the hMSCs were treated with either 0, 5, 10 and 15 mM lactate (pH 7,4) for 24 hours. RNA Isolation from stimulated hMSCs and controls was performed. The Microarray analysis was performed using AffymetrixHuGene 1.0 ST Gene Chip. Selected targets were subsequently analysed using quantitative real time PCR (RTq-PCR). RESULTS: We were able to show that lactate in moderate concentrations of 5 respectively 10 mM leads to an anti-inflammatory, anti-apoptotic but growth and proliferation promoting gene expression after 24 h. In contrast, high lactate concentrations of 15 mM leads to the opposed effect, namely promoting inflammation and apoptosis. Hypoxia induced genes did not show any significant regulation. Contrary to expectation, we were not able to show any significant regulation of candidates associated with glycolysis. CONCLUSION: We were able to show that lactate alters gene expression but does not change the cell phenotype, which might be helpful for further investigations of new treatment strategies for chronic non-healing wounds as well as tumor-therapy and neuronal plasticity.

Impact of endotoxin exposure after exhausting exercise on the immune system in solid organ transplant recipients.

Subsequent to prolonged exhausting exercise a transient immunosuppression is often observed in athletes. This so-called "open window" results in a reduced resistance of the athletes to viral and bacterial infections after an exhaustive exercise bout. Concerning the effect of bacterial endotoxin contact after exhausting exercise in transplant recipients, who are innately immunosuppressed by their medication, no data exists at present. After performing 81 km cycling, including ascending more than 1800 m in altitude, peripheral blood from 10 male kidney transplant recipients and from 10 healthy controls matched for age and gender was obtained. Simulating contact of the athletes with a pathogen post-exercise, the blood samples were incubated with Lipopolysaccharides (LPS). Thereafter microarray analysis was performed. Microarray analysis revealed a markedly oppositional pattern of gene expression in transplant recipients compared with their controls after LPS incubation. Especially immune response genes were significantly over-represented in controls immediately after the exhaustive exercise bout with LPS stimulation, whereas numerous apoptotic genes were over-represented in transplant recipients. Merging our previous data with these recent findings it should be discussed if transplant recipients need to reduce their immunosuppressive medication before performing exhaustive exercise.

Experience after 100 patients treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

AIM: To investigate perioperative patient morbidity/mortality and outcome after cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). METHODS: Of 150 patients 100 were treated with cytoreductive surgery and HIPEC and retrospectively analyzed. Clinical and postoperative follow-up data were evaluated. Body mass index (BMI), age and peritoneal carcinomatosis index (PCI) were chosen as selection criteria with regard to tumor-free survival and perioperative morbidity for this multimodal therapy. RESULTS: CRS with HIPEC was successfully performed in 100 out of 150 patients. Fifty patients were excluded because of intraoperative contraindication. Median PCI was 17 (1-39). In 89% a radical resection (CC0/CC1) was achieved. One patient died postoperatively due to multiorgan failure. Neither PCI, age nor BMI was a risk factor for postoperative complications/outcome according to the DINDO classification. In 9% Re-CRS with HIPEC was performed during the follow-up period. CONCLUSION: Patient selection remains the most important issue. Neither PCI, age nor BMI alone should be an exclusion criterion for this multimodal therapy.

The gut is not only the target but a source of inflammatory mediators inhibiting gastrointestinal motility during sepsis.

BACKGROUND: Sepsis is a common problem in intensive care patients leading to multi-organ failure and gastrointestinal paralysis. AIM: The aim of the study was to investigate whether the gastrointestinal tract is not only the target but also the source of inflammatory mediators inhibiting gastrointestinal motility. METHODS: Mesenteric lymph was obtained from rats in which a sepsis was induced by lipopolysaccharide (LPS) intraperitoneally. Gastrointestinal motility was recorded following mesenteric lymph- or TNFα infusion into the jugular vein of separate healthy recipient rats using the strain gauge transducer technique. RESULTS: Infusion of sepsis lymph significantly impairs gastric and colonic motility, decreasing the motility-index in the stomach and colon by about 57% and 21% respectively in comparison to baseline motility. Among other inflammatory mediators, TNFα plays an important role in mediating the inhibitory effect of mesenteric lymph on gastrointestinal motility during sepsis. CONCLUSIONS: The gastrointestinal tract is the source and the target of inflammatory mediators released during sepsis causing paralytic ileus.

Cytoreductive surgery and HIPEC in peritoneal recurrent ovarian cancer: experience and lessons learned.

PURPOSE: Peritoneal recurrence of ovarian cancer is frequent after primary surgery and chemotherapy and has poor long-term survival. De novo cytoreductive surgery is crucial with the potential to improve prognosis, especially when combined with hyperthermic intraperitoneal chemotherapy (HIPEC). METHODS: The sampled data of 40 consecutive patients were retrospectively analyzed. Thirty-one patients were treated with cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy. RESULTS: No patient was lost in the perioperative period, and the combined procedure was performed with acceptable morbidity. Colon-preserving cytoreductive surgery was associated with reduced morbidity. CONCLUSIONS: Patients suffering from peritoneal recurrence of ovarian cancer should be considered for radical reoperation with HIPEC in a center with expertise in multimodal therapeutic options. Organ-preserving cytoreductive surgery allows complete cytoreduction with the goal of decreasing morbidity.

Simvastatin reduces tumor cell adhesion to human peritoneal mesothelial cells by decreased expression of VCAM-1 and ß1 integrin.

Peritoneal carcinomatosis describes cancer metastasis onto the surface of the peritoneum. It is frequently caused by ovarian and colorectal cancer. Once a tumor has penetrated the peritoneum, cancer cells disseminate into the abdominal cavity. Additionally, surgery can account for the spread of free tumor cells. Their subsequent adhesion to mesothelial cells (HMCs) initiates peritoneal carcinomatosis. Therefore, this study analyzed the effect of simvastatin on tumor cell adherence. HMCs were isolated from human greater omentum. Fluorescence-labeled tumor cells (SKOV-3, OvCar-29, OAW42, FraWü; ovarian/HT29; colorectal) were incubated on confluent mesothelial monolayers with 10 µM simvastatin for 48 h. Adhesion was quantified using a fluorescence reader. Expression of the adhesion molecules VCAM-1, ICAM-1 and ß1 integrin chain under the influence of simvastatin 0.1-100 µM for 24-72 h was analyzed using flow cytometry. Simvastatin significantly reduced the adhesion of all ovarian cancer cells and HT29 to HMCs (P≤0.001). Concomitantly simvastatin decreased the expression of VCAM-1 on HMCs. ICAM-1 and ß1 integrin chain expression on ovarian cancer cells was also clearly reduced. By contrast, the expression of the analyzed adhesion molecules on HT29 cells remained unchanged. Simvastatin clearly inhibits tumor cell adhesion to HMCs. In the case of ovarian cancer cell lines it appears to be mediated by decreased expression of both VCAM-1 on HMCs and the integrin α4ß1 on tumor cells. As an example of adhesion molecule down-regulating drugs, simvastatin may provide a novel therapeutic approach to the prevention of peritoneal carcinomatosis.

Phagocytosis of dying tumor cells by human peritoneal mesothelial cells.

Peritoneal carcinomatosis is an advanced form of metastatic disease characterized by cancer cell dissemination onto the peritoneum. It is commonly observed in ovarian and colorectal cancers and is associated with poor patient survival. Novel therapies consist of cytoreductive surgery in combination with intraperitoneal chemotherapy, aiming at tumor cell death induction. The resulting dying tumor cells are considered to be eliminated by professional as well as semi-professional phagocytes. In the present study, we have identified a hitherto unknown type of 'amateur' phagocyte in this environment: human peritoneal mesothelial cells (HMCs). We demonstrate that HMCs engulf corpses of dying ovarian and colorectal cancer cells, as well as other types of apoptotic cells. Flow cytometric, confocal and electron microscopical analyses revealed that HMCs ingest dying cell fragments in a dose- and time-dependent manner and the internalized material subsequently traffics into late phagolysosomes. Regarding the mechanisms of prey cell recognition, our results show that HMCs engulf apoptotic corpses in a serum-dependent and -independent fashion and quantitative real-time PCR (qRT-PCR) analyses revealed that diverse opsonin receptor systems orchestrating dying cell clearance are expressed in HMCs at high levels. Our data strongly suggest that HMCs contribute to dying cell removal in the peritoneum, and future studies will elucidate in what manner this influences tumor cell dissemination and the antitumor immune response.

Immunonutrition with long-chain fatty acids prevents activation of macrophages in the gut wall.

BACKGROUND: Immune cells and inflammatory mediators are released from the gastrointestinal tract into the mesenteric lymph during sepsis causing distant organ dysfunction. Recently, it was demonstrated that macrophages in the gut wall are controlled by the vagus nerve, the so-called cholinergic anti-inflammatory pathway. AIM: This study aims to investigate whether an enteral diet with lipid prevents the activation of leukocytes in the gut wall. METHODS: Mesenteric lymph was obtained from rats, receiving an enteral infusion of glucose or glucose + lipid before and after lipopolysaccharide (LPS) injection. Immune cells in mesenteric lymph were analyzed with fluorescence-activated cell sorting before and after LPS injection. Mesenteric lymph leukocytes from rats receiving enteral glucose with or without lipid were stimulated in vitro with LPS and tumor necrosis factor (TNF)α was measured in the supernatant. RESULTS: The release of macrophages from the gut during sepsis was not significantly different in animals enterally treated with glucose or lipid. However, the release of TNFα from mesenteric lymph leukocytes after in vitro LPS stimulation was more than 3-fold higher in the glucose group compared to the lipid-treated group. CONCLUSIONS: During sepsis, activated macrophages are released from the gut into mesenteric lymph. However, an enteral diet with lipid is able to suppress the inflammatory cytokine release from mesenteric lymph leukocytes.