Evaluation of Intraoperative Autotransfusion Filtration for Hepatectomy and Pancreatectomy

Background Hepatectomy and pancreatectomy are often associated with significant intraoperative blood loss leading to postoperative anemia, which has been demonstrated to lead to increased perioperative morbidity, a prolonged hospital stay, and decreased overall survival. Cancer has remained an absolute contraindication to autotransfusion because of the unproven concern about reinfusion of malignant cells. Thus, the aim of this study was to test for the presence of malignant cells in autotransfused filtered blood in patients undergoing major pancreatic and liver resection. Methods A prospective study of 20 consecutive patients evaluated the presence of malignant cells from autotransfusion filtered blood after resection by flow cytometric and immunohistochemical methods. Results Ten patients underwent major hepatectomy for metastatic colorectal cancer, with a median blood loss of 500 mL (range, 200–700 mL). Three patients received a total of six units of packed red blood cells. Ten patients underwent pancreaticoduodenectomy for adenocarcinoma with a median blood loss of 400 mL (range, 200–1300 mL). Five patients received a total of nine units of packed red blood cells. Flow cytometry did not demonstrate the presence of any cytokeratin-positive carcinoma cells in filtered blood. Conclusions Intraoperative autotransfusion for major hepatectomy in metastatic colorectal cancer and pancreatectomy for adenocarcinoma is safe and should begin to be evaluated in a phase II study for efficacy.     

Intraoperative frozen section of sentinel nodes: a formal decision analysis

In the absence of data from randomized trials, a formal decision analysis was undertaken to quantify the relative value of the patients' quality of life with regard to performing intraoperative frozen section (FS) versus permanent section (PS) analysis of the sentinel lymph node (SLN) for breast cancer. Assumptions for this analysis were based on data from 203 nodal basins where the prevalence of nodal metastasis was 26.1 per cent; the FS sensitivity was 68 per cent and FS false-positive rate was 0.7 per cent. DATA 4.0 was used to model the decision analysis. The two branches of the tree represent the two surgical options of either FS analysis intraoperatively, accompanied by immediate axillary dissection for positive nodes versus PS analysis followed by reoperative axillary dissection. The expected utility (EU) with the FS strategy is 0.9736; the EU for PS is 0.9732, suggesting no definite preference for either procedure. Overall, the toss-up is very robust. The decision to choose FS versus PS should be based on individual patient risk and preference because the decision analysis indicated that these two options are equivalent with respect to patient quality of life.     

Discovery and evaluation of potent P1 aryl heterocycle-based thrombin inhibitors

In an effort to discover potent, clinically useful thrombin inhibitors, a rapid analogue synthetic approach was used to explore the P(1) region. Various benzylamines were coupled to a pyridine/pyrazinone P(2)-P(3) template. One compound with an o-thiadiazole benzylic substitution was found to have a thrombin K(i) of 0.84 nM. A study of ortho-substituted five-membered-ring heterocycles was undertaken and subsequently demonstrated that the o-triazole and tetrazole rings were optimal. Combination of these potent P(1) aryl heterocycles with a variety of P(2)-P(3) groups produced a compound with an extraordinary thrombin inhibitory activity of 1.4 pM. It is hoped that this potency enhancement in P(1) will allow for more diversification in the P(2)-P(3) region to ultimately address additional pharmacological concerns.     

E1A-induced apoptosis does not prevent replication of adenoviruses with deletion of E1b in majority of infected cancer cells

Apoptotic pathways are initiated as a cellular defense mechanism to eliminate adenovirus-infected cells. We have investigated how E1A-induced apoptosis interferes with viral replication in cancer cells. We found that E1B19K alone can efficiently suppress E1A-induced apoptosis in cancer cells. Viruses deleted for both E1B19K and E1B55K resulted in cellular DNA degradation. However, less than 20% of human lung cancer cells infected with a virus deleted for both E1B19K and E1B55 K had evidence of chromatin condensation and multiple-micronuclei formation (apoptotic hallmarks); these cells could not produce infectious viral particles. The majority of cancer cells infected with viruses deleted for the entire E1b gene did not undergo extended apoptosis and produced abundant viral progeny. Thus, only a fraction of cancer cells underwent apoptosis and did not allow E1b-deleted viruses to replicate, while the majority of cancer cells were resistant to E1A-induced apoptosis and could support virus-selective replication. The results of this study imply that, in addition to inhibiting E1A-induced apoptosis, E1B proteins may contribute other important roles in the viral life cycle. Our results also suggest that combining virus-induced apoptosis and selective viral replication into one vector will be a novel approach to destroy cancer cells.     

Colorectal cancer cell adhesion attenuates Ad-E2F-1 mediated apoptosis

BACKGROUND: The complex interplay of cell adhesion molecules, intracellular signaling, and tumor growth behavior have important implications for the failure of most conventional cancer therapies. Cell adhesion to the basement membrane has been shown to promote tumor cell survival. We hypothesize that the presence of matrix substrate contributes to chemoresistance through signaling via cell adhesion molecule. MATERIALS AND METHODS: RKO colorectal cancer cells express integrin beta1 to adhere to substrate. We measured apoptosis of the cells after infection with adenovirus vector containing the transgene E2F-1 (Ad-E2F-1), a potent tumor suppressor gene, and Ad-LacZ (as control), both under the control of the cytomegalovirus promoter. Cells were plated on Matrigel, an extracellular substrate similar to basement membrane and compared to tissue culture plastic. Apoptosis was assessed by flow cytometry-based TUNEL assay and cell proliferation was assessed by WST-1 assay. E2F-1 expression was confirmed by Western blot analysis. A function-blocking anti-beta1 integrin antibody was used to assess the contribution of beta1 on cell survival. RESULTS: At 120 h postinfection of RKO cells with 50 multiplicity of infection, cells plated on plastic underwent marked apoptosis in response to Ad-E2F-1 compared with Ad-LacZ control-treated cells (53% vs 1% apoptosis, respectively). However, when cells were plated on Matrigel, the same dose of E2F-1 was ineffective at inducing apoptosis (3% vs 1% apoptosis, comparing Ad-E2F-1 with Ad-LacZ control). The cell proliferation assay showed >3-fold cell survival in E2F-1-infected cells on Matrigel vs plastic (P < 0.004). By Western blot analysis, attenuation of apoptosis may be a result of reduction in transduction efficiency on Matrigel and function-blocking anti-beta1 integrin antibody does not abolish the decrease in apoptosis afforded by Matrigel. CONCLUSIONS: These data suggest that escape from adenoviral E2F-1-mediated apoptosis, at least in part, is related to reduction of intracellular E2F-1 expression. Interactions involving cellular adhesion via beta1 integrin to matrix proteins does not seem to contribute toward gene therapy resistance. Further studies will investigate other specific receptor-ligand interactions after gene and/or chemotherapy.     

Synthesis and evaluation of imidazole acetic acid inhibitors of activated thrombin-activatable fibrinolysis inhibitor as novel antithrombotics

Thrombin-activatable fibrinolysis inhibitor (TAFI) is an important regulator of fibrinolysis, and inhibitors of this enzyme have potential use in antithrombotic and thrombolytic therapy. Appropriately substituted imidazole acetic acids such as 10j were found to be potent inhibitors of activated TAFI and selective versus the related carboxypeptidases CPA, CPN, and CPM but not CPB. Further, 10j accelerated clot lysis in vitro and was shown to be efficacious in a primate model of thrombosis.