- Email: [email protected]
Prevalence of unexpected pulmonary embolism at CT scan for cancer staging in patients with lung cancer
Abstracts / Thrombosis Research 129, Supplement 1 (2012) S155–S194
were generated that included age, race, gender, cancer VTE-risk group, prior VTE >90 days, prior medical hospitalization, and prior surgery >30 days before the index hospitalization. Results: Among 267,846 cancer cases hospitalized for a non-surgical condition, 1,839 (0.69%) met criteria for hospital-acquired VTE and 1,972 (0.79%) met criteria for post-discharge VTE. In a multivariable model that included age, race, sex, prior VTE and prior surgery, high VTE-risk cancers (HR=1.8, CI: 1.7-2.2) but not intermediate risk cancers (HR=1.1, CI: 0.9-1.4) were predicative of in-hospital acquired VTE. Major and extreme severity-ofillness were both predicative of hospital-acquired VTE (both had HR=1.6, CI: 1.2-2.2). For post-hospital VTE, significant predictors included a 5-14 day hospital stay (HR=1.2, CI: 1.1-1.3), a 15-30 day hospital stay (HR=1.3, CI: 1.1-1.5) and presence of a high VTE-risk cancer (HR=2.0; CI: 1.7-2.4), but not severity-of-illness (p>0.45). Conclusions: Presence of a high VTE-risk cancer was the strongest predictor of both in-hospital and post-hospital VTE in medical cancer patients. Major or extreme severity-of-illness predicted in-hospital acquired VTE but not post-discharge VTE. However, longer duration of hospitalization was a risk factor for post-discharge VTE. These findings suggest post-discharge prophylaxis should be strongly considered in patients who have both a high VTE-risk cancer and a hospital stay > 5 days.
PO-13 Breast cancer and comorbidity and risk of venous thromboembolism: a Danish nationwide population based cohort study A.G. Ording 1 , E. Horváth-Puhó 1 , M. Vyberg 2 , H.T. Sørensen 1 , T.L. Lash 1,3 1 Department of Clinical Epidemiology, Aarhus University Hospital, Aarhus, Denmark; 2 Institute of Pathology, Aalborg Hospital, Aalborg, Denmark; 3 Department of Epidemiology & Prevention, Wake Forest University School of Medicine, USA Introduction: Breast cancer patients are at increased risk of venous thromboembolism (VTE) because of their breast cancer, but also from other causes. Many breast cancer patients are older women with comorbidities, but the impact of comorbidities on the risk of VTE among breast cancer patients is unclear. Aim: We compared the interaction between breast cancer and comorbid diseases, measured by the Charlson comorbidity index (CCI), on the rate of VTE beyond that expected by the breast cancer and comorbidity acting alone. Materials and methods: We designed a registry-based matched-cohort study using the Danish Cancer Registry to identify all female incident breast cancer patients between 35 and 85 years of age in Denmark and diagnosed between 1995 and 2009. Five comparison cohort members from the general population were selected without replacement from the Central Person Registry for each breast cancer patient, matched to the breast cancer patient on age, and region of residence on the date of the cancer patient’s diagnosis. The cohorts were followed for five years to compare the VTE risk between CCI score categories with crude rates, Cox models adjusted for hypertension, alcoholism disease, atrial fibrillation, obesity, former or present use of NSAIDs, aspirin, and antipsychotics. The interaction contrast, which measures the excess rate of VTE that cannot be explained by the independent effects of breast cancer or the CCI score alone, was also calculated. Results: Out of 54,397 women with breast cancer and 271,985 matched comparison cohort members, 972 breast cancer patients and 2,382 matched women developed VTE within five years. The 5-year cumulative VTE rate was 2.3% in the breast cancer cohort and 1.1% in the matched cohort. Compared to breast cancer patients with a CCI score of 0, the HR for breast cancer patients with a CCI score of 1 increased to 1.77 (1.59, 1.97), to 1.96 (1.72, 2.22) for a CCI of 2 or 3, and to 2.03 (1.53, 2.70) for CCI score of ≥4. The five-year interaction contrast for breast cancer patients with a Charlson score of ≥4 was 6.2 per 1,000 person-years out of a total VTE rate of 14.8 per 1000 person-years. Interaction between breast cancer and high comorbidity therefore accounted for 42% of the total rate. Conclusion: In this large prospective study, breast cancer and comorbidity both increased the risk of VTE. A CCI score of ≥4 interacts with breast cancer to increase the risk of VTE beyond what can be expected by the breast cancer and comorbidity acting alone.
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PO-14 Preoperative D-dimer levels predict long-term disease-free survival in patients with curatively intended resection for stage III colorectal cancer: a prospective cohort study K.G. Sunesen, M.T. Stender, O. Thorlacius-Ussing Dept. of Gastroenterological Surgery, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark Purpose: Plasma D-dimer levels predict overall survival and disease progression in patients with metastatic colorectal cancer (CRC). However, controversy exits regarding the prognostic value of preoperative D-dimer levels in patients with resectable CRC. We examined this issue in a prospective cohort study. Methods: We measured preoperative D-dimer levels in a consecutive series of patients with curative-intent resection for non-metastatic CRC. Samples for plasma D-dimer were immediately analyzed using the Auto Dimer® assay (Biopool® International, Umeå, Sweden). Patients were followed from date of surgery for CRC and until date of recurrence, death, or 5 years postoperative, whichever came first. Kaplan-Meier curves of disease-free survival were computed according to preoperative D-dimer levels (≤0.1, >0.1 to <0.3, and ≥0.3 mg/l). We used Cox-regression analysis to compute hazard ratios (HRs) of recurrence or death according to preoperative D-dimer levels with adjustment for sex, age, American Society of Anesthesiologists (ASA) physical status classification, and preoperative carcinoembryonic antigen (CEA) levels. All analyses were stratified by pathological CRC stage. Results: A total of 166 patients were included. Preoperative D-dimer levels were ≤0.1 mg/l in 47 (28%), >0.1 to <0.3 mg/l in 58 (35%), and ≥0.3 mg/l in 61 (37%) patients. Among 43 patients with stage III CRC, 5-year disease-free survival was 85% (CI: 51-96%), 38% (CI: 14-63%), and 24% (CI: 7-45%) in patients with preoperative D-dimer levels of ≤0.1 mg/l, >0.1 to <0.3 mg/l, and ≥0.3 mg/l, respectively. Among 123 patients with stage I-II CRC, corresponding figures were 74% (CI: 55-85%), 76% (CI: 60-86%), and 64% (CI: 48-76%). In the group of patients with stage III CRC, adjusted Cox-regression analysis revealed a substantial increased risk of recurrence or death among patients with preoperative D-dimer levels of >0.1 to <0.3 mg/l (HR: 6.3; CI: 1.3-31.1) and ≥0.3 mg/ l (HR: 13.2; CI: 2.2-78.6) when compared to patients with preoperative D-dimer level ≤0.1 mg/l. In the group of patients with stage I-II CRC, no difference in risk of recurrence or death was found according to preoperative D-dimer levels. Conclusion: Preoperative D-dimer level >0.1 mg/l was associated with a more than 6-fold increased risk of recurrence or death after curative-intent resection for stage III CRC. In contrast, patients with curative-intent resection for stage I-II CRC had low risk of recurrence or death regardless of preoperative D-dimer level.
PO-14 bis Prevalence of unexpected pulmonary embolism at CT scan for cancer staging in patients with lung cancer M. Verso 1 , G. Agnelli 1 , S. Mosca 2 , M. Fischer 2 , V. Marconi 2 , R. Chiari 3 , L. Crinò 3 1 Internal and Cardiovascular Medicine-Stroke Unit, Department of Internal Medicine, 2 Division of Radiology, 3 Division of Medical Oncology, University of Perugia, Italy Background: Venous thromboembolism (VTE) is a common complication in cancer patients. Symptomatic VTE is reported in about 7% of the patients with lung cancer. The incidence of unexpected pulmonary embolism (PE) is reported to be found in up to 4% of cancer patients undergoing to CT scan for cancer staging. Among cancer patients, a relative high rate of unexpected PE in patients with lung cancer was reported. Aim of the study: The aim of this study was to evaluate the prevalence of unexpected PE in a cohort of patients with lung cancer undergoing thoracic contrast-enhanced multi-detector (MD) CT scans for routine cancer staging. The PE was defined as unexpected if its diagnosis was made on routine CT imaging in absence of a clinical suspicion. Materials and methods: We performed a retrospective, cohort study. Consecutive adult patients with lung cancer (both in- or out-patient setting) referred to the Medical Oncologic Division of the University of Perugia, who underwent thoracic, contrast-enhanced, MDCT scan for routine cancer staging (both at cancer diagnosis or for cancer disease re-staging after
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Abstracts / Thrombosis Research 129, Supplement 1 (2012) S155–S194
chemotherapy) were included in the study. An independent adjudication committee, composed by two radiologists, unaware of the previous CT adjudication, reviewed the thoracic multi-detector CT scans. Results: A cohort of 158 patients with lung cancer were included in the study. Patients with unexpected PE on CT scan were 31 (19.6%), of which 17 were male (54.8%). Localization of lung cancer was central in 14 cases (45.2%) and peripheral in the remaining 17 patients. Unexpected PE was bilateral in 9 patents (29%); in the remaining cases the distribution of PE was on the left side in 11 patients (35%) and on right side in 11 patients. In the 87.1% of the PE cases a partial thrombotic obstruction of the arteries was documented. Twenty five out of 31 patients with positive CT scan for unexpected PE were managed as outpatients. In 4 patients unexpected PE was documented at the time of lung cancer diagnosis, while in 14 patients (45.2%) PE was associated with evidence on CT scan of cancer progression. in only 1 case (3.1%) PE was reported in a patient with remission of cancer disease. In about 50% of cases PE involved main and/or lobar arteries. Conclusions: In patients with lung cancer who underwent thoracic, contrast-enhanced, MDCT scans for routine cancer staging a rate of 19.6% of unexpected PE was observed. In 45% of cases PE was associated with CT evidences of progression of cancer disease. In 50% of unexpected PE main and/or lobar arteries were involved.
Poster Session 2: Tissue factor
PO-15 T cells, tissue factor and malignancy M.M. D’Elios, M. Benagiano, A. Amedei, C. Della Bella, E. Niccolai, D. Prisco Patologia Medica, University of Florence, Italy Introduction: Thrombosis significantly contribute to the morbidity and mortality of patients with malignancy, however the impact of cellular immunity on activation of coagulation cascade, oncogenesis and metastasis still needs to be clarified. Aim: We investigated the role of T-cell subsets in the induction of tissue factor (TF) production by human monocytes. Materials and methods: All experiments were done in triplicate in three consecutive experiments. T-cell stimulation enabled both unfractionated T cells and their CD4+ or CD8+ subsets to promote TF production. We derived T-cell clones from seven patients with gastric adenocarcinoma and we studied the ability to induce TF production of ten T helper (Th) 1, ten Th2 and ten Th17 clones. T-cell blasts of antigen-specific clones (800,000/ml) were cocultured for 16 h with autologous monocytes (400,000/ml) in the presence of medium, mitogen or the respective specific antigen. At the end of culture period, TF was quantitated by a specific enzyme-linked immunosorbent assay kit (American Diagnostica Inc, Greenwich, CT, USA). Results: After mitogen or antigen activation, all T-cell clones with T helper (Th) 1 and Th17 cytokine profile, but not Th2 clones, induced TF production. T-cell blasts from Th1 and Th17 activated clones were fixed with paraformaldehyde and added to monocytes in the presence of medium alone or their supernatants. Addition of either fixed Th1 or Th17 cells or their supernatants induced low TF production (mean 1.3 ng/mL + 0.13 SD), whereas addition of both cells and supernatants resulted in much higher TF synthesis (mean 2.9 ng/mL + 0.5 SD). No TF synthesis was induced by activated and fixed Th2 cells and/or their supernatants, whereas combined addition of fixed Th2 cells and Th1 or Th17 supernatants or IFN-gamma or IL-17 induced noticeable TF production. The addition of anti-IFN-gamma antibody, anti-IL-17 or Th2 supernatants to monocytes stimulated with activated and fixed Th1 cells and Th17 plus their respective supernatant resulted in a dose-dependent inhibition of TF synthesis, which was partially restored by neutralization of interleukin-4 (IL-4) or IL-10. Addition of recombinant IL-4, IL-13, or IL-10, but not IL-5, inhibited the Th1/Th17-induced TF production by monocytes. Summary: Data indicate that both CD8+ and CD4+ T cells, Th1/Th17, but not Th2, T cells can help TF production. Both cell-to-cell contact with activated T cells, Th1-type and Th17 cytokines, in particular IFN-gamma and IL-17, are crucial for optimal TF synthesis, whereas Th2-derived cytokines (IL-4, IL-13, and IL-10) are inhibitory.
Conclusion: The results obtained so are of potential interest for the development of novel future preventive or therapeutic strategies of thrombosis in patients with malignancy.
PO-16 Cryogenic- and room-temperature electron microscopy imaging of MDA231 breast cancer cells and microparticles using immunogold labeling L. Issman 1 , A. Aharon 2,3 , B. Brenner 2,3 , Y. Talmon 4 1 The Norman Seiden Multidisciplinary Graduate Program in Nanoscience & Nanotechnology, Technion; 2 Bruce Rappaport Faculty of Medicine, Technion; 3 Thrombosis and Hemostasis Unit, Dept. of Hematology, Rambam Health Care Campus; 4 The Department of Chemical Engineering, Technion, Haifa, Israel Cancer cell exposure to chemotherapy, irradiation, cytokines, or oxidative injury result in increased microparticles (MPs) secretion. MPs that present on their surface tissue factor (TF) play a central role in coagulation initiation and thrombus formation in-vivo. However, high-resolution features of MPs from cancer cells have not been elucidated in their native state by electron microscopy (EM). Our aim in this research is to characterize by high-resolution electron microscopy the cells of the MDA231 breast cancer cell line and the MPs shed from their surface by their morphology and TF localization. We also wish to better understand the shedding process by analyzing the high-resolution data. The use of EM allows acquisition of data in the macro, micro, and nano scale. Moreover, the correct implementation of cryogenic temperature electron microscopy (cryo-EM) can give high-resolution images of biological samples at their almost native state, as fast enough cooling vitrifies the specimen, thus preserving its nanostructure. The work at cryogenic conditions also prevents the drying of the specimen due to the high vacuum in the EM, making sample staining and drying unnecessary. In addition, the sample is physically fixed to prevent movement on the supra-molecular level, which may cause image blurring. Non-coated two-dimensionally cultured MDA231 breast cancer cells were imaged by high resolution scanning electron microscopy (HRSEM). In addition, we show results with anti-TF immunogold labeling of these cells which are in agreement with flow cytometry analysis we made. MPs were isolated from stimulated MDA231 cells by a 24-hour starvation period, and characterized in detail by cryogenic transmitting electron microscopy (cryo-TEM). These specimens were anti-TF immunogold labeled as well, thus enabling us to achieve better understanding of these particles. We have developed a methodology that leads to better understanding of MPs, and the cancerous cells, from which they originate, by cryogenic- and roomtemperature EM with the support of immunogold labeling. We show that by carefully choosing the imaging method, multiple levels of information can be acquired.
PO-17 Alternatively spliced tissue factor triggers breast tumor growth B. Kocatürk 1 , C. Tieken 1 , P.H. Reitsma 1 , V.Y. Bogdanov 2 , H.H. Versteeg 1 1 The Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands; 2 Department of Internal Medicine University of Cincinnati College of Medicine, Cincinnati, OH, USA Full Length Tissue Factor (flTF) influences breast cancer progression by cleavage of PAR2 and flTF on breast cancer cells, and constitutively associates with integrins which further facilitates PAR activation. A soluble isoform of TF(asTF), that arises from alternative splicing, does not facilitate PAR2 activation but leads angiogenesis by ligating integrins on endothelial cells. The lack of reagents specifically detect/block either isoform hampers interpretation of their roles in cancer growth. Thus, the role of asTF in cancer may be seriously underestimated. Since both asTF and flTF levels are increased in cancer, we aimed to determine their effect on breast tumor growth. Due to their different nature, we also investigated different mechanisms lying behind asTF and flTF dependant tumor growth. Breast cancer cell line MCF-7 was equipped with a FRT site allowing Flp recombinase-dependent integration of any gene of interest (empty vector, flTF and asTF) at the same genomic position. Localization of flTF and asTF was determined using confocal microscopy. Secretion of asTF from the cells was detected by acetone precipitation on culture media. In vitro cell proliferation was assessed using MTT assay or cell counting and expres-
were generated that included age, race, gender, cancer VTE-risk group, prior VTE >90 days, prior medical hospitalization, and prior surgery >30 days before the index hospitalization. Results: Among 267,846 cancer cases hospitalized for a non-surgical condition, 1,839 (0.69%) met criteria for hospital-acquired VTE and 1,972 (0.79%) met criteria for post-discharge VTE. In a multivariable model that included age, race, sex, prior VTE and prior surgery, high VTE-risk cancers (HR=1.8, CI: 1.7-2.2) but not intermediate risk cancers (HR=1.1, CI: 0.9-1.4) were predicative of in-hospital acquired VTE. Major and extreme severity-ofillness were both predicative of hospital-acquired VTE (both had HR=1.6, CI: 1.2-2.2). For post-hospital VTE, significant predictors included a 5-14 day hospital stay (HR=1.2, CI: 1.1-1.3), a 15-30 day hospital stay (HR=1.3, CI: 1.1-1.5) and presence of a high VTE-risk cancer (HR=2.0; CI: 1.7-2.4), but not severity-of-illness (p>0.45). Conclusions: Presence of a high VTE-risk cancer was the strongest predictor of both in-hospital and post-hospital VTE in medical cancer patients. Major or extreme severity-of-illness predicted in-hospital acquired VTE but not post-discharge VTE. However, longer duration of hospitalization was a risk factor for post-discharge VTE. These findings suggest post-discharge prophylaxis should be strongly considered in patients who have both a high VTE-risk cancer and a hospital stay > 5 days.
PO-13 Breast cancer and comorbidity and risk of venous thromboembolism: a Danish nationwide population based cohort study A.G. Ording 1 , E. Horváth-Puhó 1 , M. Vyberg 2 , H.T. Sørensen 1 , T.L. Lash 1,3 1 Department of Clinical Epidemiology, Aarhus University Hospital, Aarhus, Denmark; 2 Institute of Pathology, Aalborg Hospital, Aalborg, Denmark; 3 Department of Epidemiology & Prevention, Wake Forest University School of Medicine, USA Introduction: Breast cancer patients are at increased risk of venous thromboembolism (VTE) because of their breast cancer, but also from other causes. Many breast cancer patients are older women with comorbidities, but the impact of comorbidities on the risk of VTE among breast cancer patients is unclear. Aim: We compared the interaction between breast cancer and comorbid diseases, measured by the Charlson comorbidity index (CCI), on the rate of VTE beyond that expected by the breast cancer and comorbidity acting alone. Materials and methods: We designed a registry-based matched-cohort study using the Danish Cancer Registry to identify all female incident breast cancer patients between 35 and 85 years of age in Denmark and diagnosed between 1995 and 2009. Five comparison cohort members from the general population were selected without replacement from the Central Person Registry for each breast cancer patient, matched to the breast cancer patient on age, and region of residence on the date of the cancer patient’s diagnosis. The cohorts were followed for five years to compare the VTE risk between CCI score categories with crude rates, Cox models adjusted for hypertension, alcoholism disease, atrial fibrillation, obesity, former or present use of NSAIDs, aspirin, and antipsychotics. The interaction contrast, which measures the excess rate of VTE that cannot be explained by the independent effects of breast cancer or the CCI score alone, was also calculated. Results: Out of 54,397 women with breast cancer and 271,985 matched comparison cohort members, 972 breast cancer patients and 2,382 matched women developed VTE within five years. The 5-year cumulative VTE rate was 2.3% in the breast cancer cohort and 1.1% in the matched cohort. Compared to breast cancer patients with a CCI score of 0, the HR for breast cancer patients with a CCI score of 1 increased to 1.77 (1.59, 1.97), to 1.96 (1.72, 2.22) for a CCI of 2 or 3, and to 2.03 (1.53, 2.70) for CCI score of ≥4. The five-year interaction contrast for breast cancer patients with a Charlson score of ≥4 was 6.2 per 1,000 person-years out of a total VTE rate of 14.8 per 1000 person-years. Interaction between breast cancer and high comorbidity therefore accounted for 42% of the total rate. Conclusion: In this large prospective study, breast cancer and comorbidity both increased the risk of VTE. A CCI score of ≥4 interacts with breast cancer to increase the risk of VTE beyond what can be expected by the breast cancer and comorbidity acting alone.
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PO-14 Preoperative D-dimer levels predict long-term disease-free survival in patients with curatively intended resection for stage III colorectal cancer: a prospective cohort study K.G. Sunesen, M.T. Stender, O. Thorlacius-Ussing Dept. of Gastroenterological Surgery, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark Purpose: Plasma D-dimer levels predict overall survival and disease progression in patients with metastatic colorectal cancer (CRC). However, controversy exits regarding the prognostic value of preoperative D-dimer levels in patients with resectable CRC. We examined this issue in a prospective cohort study. Methods: We measured preoperative D-dimer levels in a consecutive series of patients with curative-intent resection for non-metastatic CRC. Samples for plasma D-dimer were immediately analyzed using the Auto Dimer® assay (Biopool® International, Umeå, Sweden). Patients were followed from date of surgery for CRC and until date of recurrence, death, or 5 years postoperative, whichever came first. Kaplan-Meier curves of disease-free survival were computed according to preoperative D-dimer levels (≤0.1, >0.1 to <0.3, and ≥0.3 mg/l). We used Cox-regression analysis to compute hazard ratios (HRs) of recurrence or death according to preoperative D-dimer levels with adjustment for sex, age, American Society of Anesthesiologists (ASA) physical status classification, and preoperative carcinoembryonic antigen (CEA) levels. All analyses were stratified by pathological CRC stage. Results: A total of 166 patients were included. Preoperative D-dimer levels were ≤0.1 mg/l in 47 (28%), >0.1 to <0.3 mg/l in 58 (35%), and ≥0.3 mg/l in 61 (37%) patients. Among 43 patients with stage III CRC, 5-year disease-free survival was 85% (CI: 51-96%), 38% (CI: 14-63%), and 24% (CI: 7-45%) in patients with preoperative D-dimer levels of ≤0.1 mg/l, >0.1 to <0.3 mg/l, and ≥0.3 mg/l, respectively. Among 123 patients with stage I-II CRC, corresponding figures were 74% (CI: 55-85%), 76% (CI: 60-86%), and 64% (CI: 48-76%). In the group of patients with stage III CRC, adjusted Cox-regression analysis revealed a substantial increased risk of recurrence or death among patients with preoperative D-dimer levels of >0.1 to <0.3 mg/l (HR: 6.3; CI: 1.3-31.1) and ≥0.3 mg/ l (HR: 13.2; CI: 2.2-78.6) when compared to patients with preoperative D-dimer level ≤0.1 mg/l. In the group of patients with stage I-II CRC, no difference in risk of recurrence or death was found according to preoperative D-dimer levels. Conclusion: Preoperative D-dimer level >0.1 mg/l was associated with a more than 6-fold increased risk of recurrence or death after curative-intent resection for stage III CRC. In contrast, patients with curative-intent resection for stage I-II CRC had low risk of recurrence or death regardless of preoperative D-dimer level.
PO-14 bis Prevalence of unexpected pulmonary embolism at CT scan for cancer staging in patients with lung cancer M. Verso 1 , G. Agnelli 1 , S. Mosca 2 , M. Fischer 2 , V. Marconi 2 , R. Chiari 3 , L. Crinò 3 1 Internal and Cardiovascular Medicine-Stroke Unit, Department of Internal Medicine, 2 Division of Radiology, 3 Division of Medical Oncology, University of Perugia, Italy Background: Venous thromboembolism (VTE) is a common complication in cancer patients. Symptomatic VTE is reported in about 7% of the patients with lung cancer. The incidence of unexpected pulmonary embolism (PE) is reported to be found in up to 4% of cancer patients undergoing to CT scan for cancer staging. Among cancer patients, a relative high rate of unexpected PE in patients with lung cancer was reported. Aim of the study: The aim of this study was to evaluate the prevalence of unexpected PE in a cohort of patients with lung cancer undergoing thoracic contrast-enhanced multi-detector (MD) CT scans for routine cancer staging. The PE was defined as unexpected if its diagnosis was made on routine CT imaging in absence of a clinical suspicion. Materials and methods: We performed a retrospective, cohort study. Consecutive adult patients with lung cancer (both in- or out-patient setting) referred to the Medical Oncologic Division of the University of Perugia, who underwent thoracic, contrast-enhanced, MDCT scan for routine cancer staging (both at cancer diagnosis or for cancer disease re-staging after
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Abstracts / Thrombosis Research 129, Supplement 1 (2012) S155–S194
chemotherapy) were included in the study. An independent adjudication committee, composed by two radiologists, unaware of the previous CT adjudication, reviewed the thoracic multi-detector CT scans. Results: A cohort of 158 patients with lung cancer were included in the study. Patients with unexpected PE on CT scan were 31 (19.6%), of which 17 were male (54.8%). Localization of lung cancer was central in 14 cases (45.2%) and peripheral in the remaining 17 patients. Unexpected PE was bilateral in 9 patents (29%); in the remaining cases the distribution of PE was on the left side in 11 patients (35%) and on right side in 11 patients. In the 87.1% of the PE cases a partial thrombotic obstruction of the arteries was documented. Twenty five out of 31 patients with positive CT scan for unexpected PE were managed as outpatients. In 4 patients unexpected PE was documented at the time of lung cancer diagnosis, while in 14 patients (45.2%) PE was associated with evidence on CT scan of cancer progression. in only 1 case (3.1%) PE was reported in a patient with remission of cancer disease. In about 50% of cases PE involved main and/or lobar arteries. Conclusions: In patients with lung cancer who underwent thoracic, contrast-enhanced, MDCT scans for routine cancer staging a rate of 19.6% of unexpected PE was observed. In 45% of cases PE was associated with CT evidences of progression of cancer disease. In 50% of unexpected PE main and/or lobar arteries were involved.
Poster Session 2: Tissue factor
PO-15 T cells, tissue factor and malignancy M.M. D’Elios, M. Benagiano, A. Amedei, C. Della Bella, E. Niccolai, D. Prisco Patologia Medica, University of Florence, Italy Introduction: Thrombosis significantly contribute to the morbidity and mortality of patients with malignancy, however the impact of cellular immunity on activation of coagulation cascade, oncogenesis and metastasis still needs to be clarified. Aim: We investigated the role of T-cell subsets in the induction of tissue factor (TF) production by human monocytes. Materials and methods: All experiments were done in triplicate in three consecutive experiments. T-cell stimulation enabled both unfractionated T cells and their CD4+ or CD8+ subsets to promote TF production. We derived T-cell clones from seven patients with gastric adenocarcinoma and we studied the ability to induce TF production of ten T helper (Th) 1, ten Th2 and ten Th17 clones. T-cell blasts of antigen-specific clones (800,000/ml) were cocultured for 16 h with autologous monocytes (400,000/ml) in the presence of medium, mitogen or the respective specific antigen. At the end of culture period, TF was quantitated by a specific enzyme-linked immunosorbent assay kit (American Diagnostica Inc, Greenwich, CT, USA). Results: After mitogen or antigen activation, all T-cell clones with T helper (Th) 1 and Th17 cytokine profile, but not Th2 clones, induced TF production. T-cell blasts from Th1 and Th17 activated clones were fixed with paraformaldehyde and added to monocytes in the presence of medium alone or their supernatants. Addition of either fixed Th1 or Th17 cells or their supernatants induced low TF production (mean 1.3 ng/mL + 0.13 SD), whereas addition of both cells and supernatants resulted in much higher TF synthesis (mean 2.9 ng/mL + 0.5 SD). No TF synthesis was induced by activated and fixed Th2 cells and/or their supernatants, whereas combined addition of fixed Th2 cells and Th1 or Th17 supernatants or IFN-gamma or IL-17 induced noticeable TF production. The addition of anti-IFN-gamma antibody, anti-IL-17 or Th2 supernatants to monocytes stimulated with activated and fixed Th1 cells and Th17 plus their respective supernatant resulted in a dose-dependent inhibition of TF synthesis, which was partially restored by neutralization of interleukin-4 (IL-4) or IL-10. Addition of recombinant IL-4, IL-13, or IL-10, but not IL-5, inhibited the Th1/Th17-induced TF production by monocytes. Summary: Data indicate that both CD8+ and CD4+ T cells, Th1/Th17, but not Th2, T cells can help TF production. Both cell-to-cell contact with activated T cells, Th1-type and Th17 cytokines, in particular IFN-gamma and IL-17, are crucial for optimal TF synthesis, whereas Th2-derived cytokines (IL-4, IL-13, and IL-10) are inhibitory.
Conclusion: The results obtained so are of potential interest for the development of novel future preventive or therapeutic strategies of thrombosis in patients with malignancy.
PO-16 Cryogenic- and room-temperature electron microscopy imaging of MDA231 breast cancer cells and microparticles using immunogold labeling L. Issman 1 , A. Aharon 2,3 , B. Brenner 2,3 , Y. Talmon 4 1 The Norman Seiden Multidisciplinary Graduate Program in Nanoscience & Nanotechnology, Technion; 2 Bruce Rappaport Faculty of Medicine, Technion; 3 Thrombosis and Hemostasis Unit, Dept. of Hematology, Rambam Health Care Campus; 4 The Department of Chemical Engineering, Technion, Haifa, Israel Cancer cell exposure to chemotherapy, irradiation, cytokines, or oxidative injury result in increased microparticles (MPs) secretion. MPs that present on their surface tissue factor (TF) play a central role in coagulation initiation and thrombus formation in-vivo. However, high-resolution features of MPs from cancer cells have not been elucidated in their native state by electron microscopy (EM). Our aim in this research is to characterize by high-resolution electron microscopy the cells of the MDA231 breast cancer cell line and the MPs shed from their surface by their morphology and TF localization. We also wish to better understand the shedding process by analyzing the high-resolution data. The use of EM allows acquisition of data in the macro, micro, and nano scale. Moreover, the correct implementation of cryogenic temperature electron microscopy (cryo-EM) can give high-resolution images of biological samples at their almost native state, as fast enough cooling vitrifies the specimen, thus preserving its nanostructure. The work at cryogenic conditions also prevents the drying of the specimen due to the high vacuum in the EM, making sample staining and drying unnecessary. In addition, the sample is physically fixed to prevent movement on the supra-molecular level, which may cause image blurring. Non-coated two-dimensionally cultured MDA231 breast cancer cells were imaged by high resolution scanning electron microscopy (HRSEM). In addition, we show results with anti-TF immunogold labeling of these cells which are in agreement with flow cytometry analysis we made. MPs were isolated from stimulated MDA231 cells by a 24-hour starvation period, and characterized in detail by cryogenic transmitting electron microscopy (cryo-TEM). These specimens were anti-TF immunogold labeled as well, thus enabling us to achieve better understanding of these particles. We have developed a methodology that leads to better understanding of MPs, and the cancerous cells, from which they originate, by cryogenic- and roomtemperature EM with the support of immunogold labeling. We show that by carefully choosing the imaging method, multiple levels of information can be acquired.
PO-17 Alternatively spliced tissue factor triggers breast tumor growth B. Kocatürk 1 , C. Tieken 1 , P.H. Reitsma 1 , V.Y. Bogdanov 2 , H.H. Versteeg 1 1 The Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands; 2 Department of Internal Medicine University of Cincinnati College of Medicine, Cincinnati, OH, USA Full Length Tissue Factor (flTF) influences breast cancer progression by cleavage of PAR2 and flTF on breast cancer cells, and constitutively associates with integrins which further facilitates PAR activation. A soluble isoform of TF(asTF), that arises from alternative splicing, does not facilitate PAR2 activation but leads angiogenesis by ligating integrins on endothelial cells. The lack of reagents specifically detect/block either isoform hampers interpretation of their roles in cancer growth. Thus, the role of asTF in cancer may be seriously underestimated. Since both asTF and flTF levels are increased in cancer, we aimed to determine their effect on breast tumor growth. Due to their different nature, we also investigated different mechanisms lying behind asTF and flTF dependant tumor growth. Breast cancer cell line MCF-7 was equipped with a FRT site allowing Flp recombinase-dependent integration of any gene of interest (empty vector, flTF and asTF) at the same genomic position. Localization of flTF and asTF was determined using confocal microscopy. Secretion of asTF from the cells was detected by acetone precipitation on culture media. In vitro cell proliferation was assessed using MTT assay or cell counting and expres-