Christoph Reissfelder

Glypican-1 identifies cancer exosomes and detects early pancreatic cancer

Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1+ circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1+ crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1+ crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1+ crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1+ crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy.

Antigen-specific Tregs control T cell responses against a limited repertoire of tumor antigens in patients with colorectal carcinoma

Spontaneous antitumor T cell responses in cancer patients are strongly controlled by Tregs, and increased numbers of tumor-infiltrating Tregs correlate with reduced survival. However, the tumor antigens recognized by Tregs in cancer patients and the impact of these cells on tumor-specific T cell responses have not been systematically characterized. Here we used a broad panel of long synthetic peptides of defined tumor antigens and normal tissue antigens to exploit a newly developed method to identify and compare ex vivo the antigen specificities of Tregs with those of effector/memory T cells in peripheral blood of colorectal cancer patients and healthy subjects. Tregs in tumor patients were highly specific for a distinct set of only a few tumor antigens, suggesting that Tregs exert T cell suppression in an antigen-selective manner. Tumor-specific effector T cells were detectable in the majority of colorectal cancer patients but not in healthy individuals. We detected differences in the repertoires of antigens recognized by Tregs and effector/memory T cells in the majority of colorectal cancer patients. In addition, only effector/memory T cell responses against antigens recognized by Tregs strongly increased after Treg depletion. The selection of antigens according to preexisting T cell responses may improve the efficacy of future immunotherapies for cancer and autoimmune disease.

Infrahepatic inferior vena cava clamping for reduction of central venous pressure and blood loss during hepatic resection: a randomized controlled trial.

Objective:To evaluate the effectiveness and safety of infrahepatic inferior vena cava (IVC) clamping for reduction of central venous pressure (CVP) and blood loss during hepatic resection. Background:Low CVP during parenchymal transection has been widely accepted to reduce intraoperative hemorrhage via the hepatic veins and is commonly achieved by anesthesiological interventions such as fluid restriction. We hypothesized that infrahepatic clamping of the IVC may lower the intraoperative blood loss more effectively and, moreover, prevent potential adverse effects of fluid restriction such as hemodynamic instability. Methods:Patients scheduled for elective hepatic resection were enrolled and allocated randomly to CVP reduction by infrahepatic IVC clamping or anesthesiological interventions including primarily fluid restriction with additional use of diuretics, nitro compounds, and opioids (control group). The primary efficacy endpoint was total intraoperative blood loss. Analyses were done following intention-to-treat principles. The protocol was submitted to the clinicaltrials.gov registry (NCT00732979). Results:From April 2007 to December 2009, a total of 152 patients were randomized and 128 were eligible for final analyses. Baseline data were similar between both study groups. Despite higher CVP values during resection (4.0 ± 3.2 vs. 2.6 ± 1.8 mm Hg; P = 0.003), infrahepatic IVC clamping significantly reduced total intraoperative blood loss [550 (350.0–1150) mL vs. 900 (500–1500) mL; P = 0.02] and blood loss during parenchymal transection [150 (85–500) mL vs. 400 (200–700) mL; P = 0.006] compared with the control group. Postoperative mortality [4 (6.1%) vs. 2 (3.2%); P = 0.42] and total morbidity rates [38 (58.5%) vs. 37 (58.7%); P = 0.97] were comparable between both study groups. Although intraoperative hemodynamic instability occurred less frequently in patients with infrahepatic IVC clamping [0 vs. 4 (6.3%); P = 0.04], the incidence of pulmonary embolism was increased in this study arm [4 (6.1%) vs. 0; P = 0.04]. Conclusions:Infrahepatic IVC clamping is associated with significantly less intraoperative blood loss and may reduce the incidence of intraoperative hemodynamic instability. The potential association with postoperative pulmonary embolism represents a significant concern.

Overexpression of ZEB2 at the Invasion Front of Colorectal Cancer Is an Independent Prognostic Marker and Regulates Tumor Invasion In Vitro

Purpose: Epithelial-to-mesenchymal transition (EMT) plays a pivotal role in tumor invasion and dissemination. EMT occurs predominantly at the tumor edge where it is induced by cytokines, the extracellular matrix environment, or hypoxia. In the tumor cell, it is further mediated by several transcription factors and microRNAs. The aim of this study was to explore the expression of EMT-associated genes at the invasive front in colorectal cancer and to evaluate their prognostic significance. Experimental Design: We evaluated the expression of 13 EMT-associated genes at the invasion front of 30 colorectal liver metastases by quantitative real-time PCR. Immunostaining against zinc finger E-box–binding homeobox 2 (ZEB2) was carried out on 175 primary colorectal cancer specimens and 30 colorectal liver metastases and correlated to clinical and histopathologic data. DLD-1 cells were transfected with siRNA and subjected to migration and invasion assays. Results: Gene expression analysis and immunohistochemistry showed an upregulation of ZEB2 at the invasion front in primary colorectal cancer and liver metastases. Overexpression of ZEB2 at the invasion front correlated significantly with tumor stage in primary colorectal cancer. Moreover, univariate and multivariate analysis revealed overexpression of ZEB2 at the invasion front as an independent prognostic marker for cancer-specific survival. Downregulation of ZEB2 by siRNA decreased the migration and invasion capacity of DLD-1 cells in vitro. Conclusions: Overexpression of ZEB2 at the invasion front correlates with tumor progression and predicts cancer-specific survival in primary colorectal cancer. Therefore, ZEB2 may be interesting as biomarker and potential target for treatment of colorectal cancer. Clin Cancer Res; 17(24); 7654–63. ©2011 AACR.

Postoperative course and clinical significance of biochemical blood tests following hepatic resection

Hepatic resection continues to be associated with substantial morbidity. Although biochemical tests are important for the early diagnosis of complications, there is limited information on their postoperative changes in relation to outcome in patients with surgery‐related morbidity.

The Predictive Value of Postoperative Clinical Risk Scores for Outcome After Hepatic Resection: A Validation Analysis in 807 Patients

BackgroundAlthough early postoperative risk stratification might allow a more precise prediction of outcomes after hepatic resection, evaluation of different postoperative clinical risk indices has been lacking.MethodsA total of 1,219 patients underwent hepatic resection between January 2002 and 2010, and 807 patients were eligible for final analyses. The model for end stage liver disease (MELD) score, the “50–50 criteria,” and the International Study Group of Liver Surgery (ISGLS) definition of posthepatectomy liver failure (PHLF) were assessed as clinical risk scores on postoperative day 5. Risk factors for morbidity and mortality were analyzed using multivariate logistic regression analyses.ResultsThe overall morbidity and mortality rates were 43 and 6%, respectively. Sensitivity of the MELD score, the 50–50 criteria and the PHLF for prediction of morbidity and mortality were 55, 6, 23 and 71, 26, 65%. On multivariate analyses MELD score [odds ratio (OR) 2.06; 95% confidence interval (95% CI) 1.41–3.02] and PHLF (5.61; 2.73–11.55) were associated with morbidity, whereas this association did not reach statistical significance for the 50–50 criteria (3.64; 0.78–17.02). The 50–50 criteria (16.45; 3.50–77.25) and PHLF (13.80; 4.27–44.61) were identified as powerful, independent predictors of mortality. This association was less strong for the MELD score (2.86; 0.98–8.31).ConclusionPostoperative clinical risk scores are associated independently with outcome after hepatic resection. Owing to lack of sensitivity only the MELD score can be recommended for early prediction of overall morbidity, whereas the MELD score and the PHLF enabled adequate risk stratification regarding perioperative mortality.

Surgery for locally recurrent rectal cancer in the era of total mesorectal excision: is there still a chance for cure?

Objective:To evaluate the perioperative outcome and long-term survival of patients who underwent surgical resection for recurrent rectal cancer within a multimodal approach in the era of total mesorectal excision (TME). Background:Introduction of TME has reduced local recurrence and improved oncological outcome of patients with rectal cancer. Local recurrence after TME still occurs in 2% to 8% of patients and presents a challenge to surgical and medical oncologists. However, there has been very limited data on the perioperative and long-term outcome of patients who are operated for local recurrence in the era of TME. Methods:A total of 107 patients who were identified from a prospective rectal cancer database underwent surgical exploration for recurrent rectal cancer after previous TME between October 2001 and April 2009. Risk factors of perioperative morbidity were analyzed using a multivariate logistic regression model. Independent predictors of disease-specific survival were identified by a Cox proportional hazards regression model, as were those of local recurrence and disease recurrence at any site. Results:Surgical resection was performed in 92 patients and negative resection margins were achieved in 54 (58.7%) of these. Recurrent disease was located intraluminally and extraluminally in 35 (38.0%) patients and 57 (62.0%) patients, respectively. A total of 19 (20.6%) patients had metastatic extrapelvic disease at the time of surgery. Perioperative surgical morbidity and in-hospital mortality accounted for 42.4% and 3.3%, respectively. On multivariate analysis, partial sacrectomy was associated with surgical morbidity (P = 0.004). Three- and 5-year disease-specific survival rates were 61% and 47%. Three-year survival rate of patients with extrapelvic disease who underwent R0 resection was 42%. On multivariate analysis, surgical morbidity (P = 0.001), presence of extrapelvic disease (P = 0.006), and noncurative (R1; R2) resection (P < 0.0001) were identified as independent adverse predictors of disease-specific survival, whereas a transabdominal resection (as opposed to an abdominoperineal resection/pelvic exenteration) was associated with a more favorable prognosis (P = 0.04). Conclusions:Surgical resection of local recurrence from rectal cancer in the era TME can be carried out with acceptable morbidity and curative resection rates. Curative resection remains the major prognostic factor and may enable long-term survival even in patients with extrapelvic disease.

Effectiveness of triclosan-coated PDS Plus versus uncoated PDS II sutures for prevention of surgical site infection after abdominal wall closure: the randomised controlled PROUD trial

BACKGROUND Postoperative surgical site infections are one of the most frequent complications after open abdominal surgery, and triclosan-coated sutures were developed to reduce their occurrence. The aim of the PROUD trial was to obtain reliable data for the effectiveness of triclosan-coated PDS Plus sutures for abdominal wall closure, compared with non-coated PDS II sutures, in the prevention of surgical site infections. METHODS This multicentre, randomised controlled group-sequential superiority trial was done in 24 German hospitals. Adult patients (aged ≥18 years) who underwent elective midline abdominal laparotomy for any reason were eligible for inclusion. Exclusion criteria were impaired mental state, language problems, and participation in another intervention trial that interfered with the intervention or outcome of this trial. A central web-based randomisation tool was used to randomly assign eligible participants by permuted block randomisation with a 1:1 allocation ratio and block size 4 before mass closure to either triclosan-coated sutures (PDS Plus) or uncoated sutures (PDS II) for abdominal fascia closure. The primary endpoint was the occurrence of superficial or deep surgical site infection according to the Centers for Disease Control and Prevention criteria within 30 days after the operation. Patients, surgeons, and the outcome assessors were masked to group assignment. Interim and final analyses were by modified intention to treat. This trial is registered with the German Clinical Trials Register, number DRKS00000390. FINDINGS Between April 7, 2010, and Oct 19, 2012, 1224 patients were randomly assigned to intervention groups (607 to PDS Plus, and 617 to PDS II), of whom 1185 (587 PDS Plus and 598 PDS II) were analysed by intention to treat. The study groups were well balanced in terms of patient and procedure characteristics. The occurrence of surgical site infections did not differ between the PDS Plus group (87 [14·8%] of 587) and the PDS II group (96 [16·1%] of 598; OR 0·91, 95% CI 0·66-1·25; p=0·64). Serious adverse events also did not differ between the groups-146 of 583 (25·0%) patients treated with PDS Plus had at least one serious adverse event, compared with 138 of 602 (22·9%) patients treated with PDS II; p=0·39). INTERPRETATION Triclosan-coated PDS Plus did not reduce the occurrence of surgical site infection after elective midline laparotomy. Innovative, multifactorial strategies need to be developed and assessed in future trials to reduce surgical site infections. FUNDING Johnson & Johnson Medical Limited.

Tumor-specific cytotoxic T lymphocyte activity determines colorectal cancer patient prognosis

The composition of tumor-targeted T cell infiltrates is a major prognostic factor in colorectal cancer (CRC) outcome; however, the functional role of these populations in prolonging patient survival remains unclear. Here, we evaluated 190 patients with CRC for the presence of functionally active tumor-infiltrating lymphocytes (TILs), the tumor specificity of these TILs, and the correlation between patient TILs and long-term survival. Using intracytoplasmic cytokine staining in conjunction with HLA multimers loaded with tumor peptide and antigen-specific cytokine secretion assays, we determined that TNF-α expression delineates a population of tumor antigen-specific (TA-specific) cytotoxic T lymphocytes (CTLs) present within tumors from patients with CRC. Upregulation of TNF-α expression in TILs strongly correlated with an increase in the total amount of intratumoral TNF-α, which is indicative of tumor-specific CTL activity. Moreover, a retrospective multivariate analysis of 102 patients with CRC, which had multiple immune parameters evaluated, revealed that increased TNF-α concentration was an independent prognostic factor. Together, these results indicate that the prognostic impact of T cell infiltrates for CRC maybe largely based on subpopulations of active TA-specific T cells within the tumor, suggesting causal implication for these cells in patient survival. Additionally, these results support the use of intratumoral TNF-α, which is indicative of T cell function, as a prognostic parameter for CRC.

Invasion front-specific expression and prognostic significance of microrna in colorectal liver metastases

The tumor edge of colorectal cancer and its adjacent peritumoral tissue is characterized by an invasion front‐specific expression of genes that contribute to angiogenesis or epithelial‐to‐mesenchymal transition. Dysregulation of these genes has a strong impact on the invasion behavior of tumor cells. However, the invasion front‐specific expression of microRNA (miRNA) still remains unclear. Therefore, the aim of the present study was to investigate miRNA expression patterns at the invasion front of colorectal liver metastases. Laser microdissection of colorectal liver metastases was performed to obtain separate tissue compartments from the tumor center, tumor invasion front, liver invasion front and pure liver parenchyma. Microarray expression analysis revealed 23 miRNA downregulated in samples from the tumor invasion front with respect to the same miRNA in the liver, the liver invasion front or the tumor center. By comparing samples from the liver invasion front with samples from pure liver parenchyma, the tumor invasion front and the tumor center, 13 miRNA were downregulated. By quantitative RT‐PCR, we validated the liver invasion front‐specific downregulation of miR‐19b, miR‐194, let‐7b and miR‐1275 and the tumor invasion front‐specific downregulation of miR‐143, miR‐145, let‐7b and miR‐638. Univariate analysis demonstrated that enhanced expression of miR‐19b and miR‐194 at the liver invasion front, and decreased expression of let‐7 at the tumor invasion front, is an adverse prognostic marker of tumor recurrence and overall survival. In conclusion, the present study suggests that invasion front‐specific downregulation of miRNA in colorectal liver metastases plays a pivotal role in tumor progression. (Cancer Sci 2011; 102: 1799–1807)