FOLFOXIRI Plus Cetuximab or Bevacizumab as First-Line Treatment of BRAFV600E-Mutant Metastatic Colorectal Cancer: The Randomized Phase II FIRE-4.5 (AIO KRK0116) Study.

PURPOSE: BRAFV600E mutation is associated with a poor outcome in metastatic colorectal cancer (mCRC). This clinical trial investigated the efficacy of triplet chemotherapy (fluorouracil, folinic acid, oxaliplatin, and irinotecan) combined with either cetuximab or bevacizumab in patients with previously untreated BRAFV600E-mutant mCRC. PATIENTS AND METHODS: In this controlled, randomized, open-label phase II trial, 109 patients were randomly assigned, 107 of whom were included into the full analysis set (FAS). Patients were randomly assigned in a 2:1 ratio to receive either FOLFOXIRI plus cetuximab in the experimental arm (n = 72) or FOLFOXIRI plus bevacizumab in the control arm (n = 35). The primary end point was objective response rate (ORR) according to RECIST 1.1., evaluated in patients treated according to protocol (ATP population). Progression-free survival (PFS), overall survival (OS), toxicity, and feasibility were analyzed as secondary end points. RESULTS: Eighteen patients discontinued study treatment before the first tumor assessment, thus resulting in the ATP population of 89 patients. In these patients, ORR was 51% (30/59) in the cetuximab-based experimental arm and 67% (20/30) in the bevacizumab-based control arm (odds ratio, 1.93; 80% CI, 1.06 to 3.52; P = .92 [one-sided]). In the full analysis set, median PFS was significantly inferior in the experimental arm (6.7 months v 10.7 months; hazard ratio [HR], 1.89; P = .006). Median OS analyzed at an event rate of 64.5% showed a trend toward shorter survival in cetuximab-treated patients (12.9 months v 17.1 months; HR, 1.4; P = .20). CONCLUSION: To our knowledge, FIRE-4.5 is the first prospective and randomized study investigating first-line treatment of BRAFV600E-mutant mCRC. FOLFOXIRI plus cetuximab does not induce a higher ORR when compared with FOLFOXIRI plus bevacizumab in first-line treatment of BRAFV600E-mutant mCRC. Bevacizumab-based chemotherapy remains the preferable first-line treatment of patients with BRAFV600E-mutant mCRC.

Surveillance of megakaryocytic function by measurement of CD61-exposing microparticles in allogeneic hematopoietic stem cell recipients.

Increasing evidence suggests that circulating microparticles (MP) exposing CD61 originate predominantly from megakaryocytes. Dramatic changes in megakaryocytic homeostasis are regularly observed following allogeneic hematopoietic stem cell transplantation (HSCT) and associated with transplantation-associated complications. We studied MP plasma levels prospectively in healthy subjects (n = 10) and allogeneic HSCT recipients (n = 19) twice weekly from the start of conditioning therapy up to day 30. A total of 224 measurement points were evaluated. MP were isolated, double-stained with annexin V and anti-CD61, and analyzed by flow cytometry. In uncomplicated HSCT, we found a correlation between platelet and CD61-exposing MP count, which resulted in a constant ratio of MP per platelet. The ratio was increased in patients with active hematological malignancies before transplantation and normalized during conditioning therapy. After take, the MP ratio increased, whereas infections and microangiopathic hemolytic anemia did not affect the ratio. In patients with GvHD, a decreased MP ratio was observed depending on the grade of GvHD, possibly indicating megakaryocytic damage. The MP ratio was able to discriminate between toxic, septic, and GvHD-induced hyperbilirubinemia. We first describe CD61+ MP levels during allogeneic HSCT and postulate that the MP ratio might be a useful biomarker for the surveillance of megakaryocytes during HSCT.

Endothelial cell-derived microparticles in allogeneic hematopoietic stem cell recipients.

BACKGROUND: Alterations of microparticles derived from different cell types are described in a number of diseases associated with inflammation and hemostatic disorders. METHODS: In this prospective study, we firstly analyzed endothelial cell derived microparticles (EMP) in 19 hematopoietic stem cell recipients. Cultured human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor-alpha (TNF-alpha) served as positive controls. EMP were analyzed by fluorescent activated cell sorting (FACS), detecting the particels via expression of CD62 (E-selectin) and anionic phospholipids binding to annexin V. RESULTS: EMP were not significantly influenced by conditioning regimens with non-myeloablative chemotherapy and 4 Gy total body irradiation (TBI) or by myeloablative regimens containing 12 Gy TBI. During acute graft versus host disease (aGVHD), significantly higher levels of EMP were detected than in patients without aGVHD (18.5/microl s=10.1 vs. 14.6/microl SD = 11.5; P = 0.004) while infectious complications did not alter EMP levels significantly. Immunosuppressive therapy with corticosteroids tendentially elevated EMP levels. HUVEC treated with TNF-alpha 1 ng/ml, 10 ng/ml and 100 ng/ml released significantly more EMP than unstimulated cultures (30.0/microl ss = 13.6 vs. 126.8/microl SD = 66.9, P = 0.032 / vs. 683.3/microl SD = 349.9; P = 0.03 / vs. 489.3 s = 184.4; P = 0.013). CONCLUSIONS: Elevation of EMP during aGVHD might express severe endothelial cell injury within this complication after hematopoietic stem cell transplantation and might serve as a diagnostic test for early differentiation of aGVHD from other transplanted related complications.

Extensive local and systemic therapy in extraneural metastasized glioblastoma multiforme.

Cases of extracranial metastases of glioblastoma multiforme to sites such as bones, spleen, lung, liver and kidneys have been reported but available information about treatment of organ and bone metastases is extremely scarce. In this report a case of glioblastoma multiforme (GBM) of the temporal lobe with subsequent liver and bone metastases is described and the success of different chemotherapy regimens is discussed. Liver and bone metastases were effectively treated with temozolomide and later with carboplatin and docetaxel. Two years after first diagnosis symptomatic local recurrence occurred. Therefore a stereotactic fractionated radiotherapy was performed. As a result of relapse of liver metastases the patient received chemotherapy with adriamycin, cyclophosphamide and etoposide. Visceral metastases were stable, but nevertheless the patient died from local progression 3 years after first diagnosis. In conclusion, liver metastases of GBM can be effectively treated by chemotherapy. This case report suggests suitable substances which can be chosen according to clinical circumstances.

Diagnosis of hepatic veno-occlusive disease by plasminogen activator inhibitor-1 plasma antigen levels: a prospective analysis in 350 allogeneic hematopoietic stem cell recipients.

BACKGROUND: Veno-occlusive disease (VOD) is one of the most serious complications following allogeneic hematopoietic stem cell transplantation (HSCT) and is associated with a high mortality. We conducted a large trial in order to investigate the value of plasminogen activator inhibitor-1 (PAI-1) plasma antigen levels in VOD patients as PAI-1 has been described as a possible diagnostic marker of VOD. METHODS: In all, 350 stem cell recipients were included in our study. PAI-1 levels were analyzed prior to conditioning therapy and then weekly until eight weeks after HSCT. Transplantation-related complications (TRC) including VOD, microangiopathic hemolytic anemia (MAHA), and graft-versus-host disease (GVHD) were recorded weekly throughout the study. RESULTS: Maximum PAI-1 antigen levels were increased in all patients with VOD (n=15; mean 248 ng/ml; 95% CI 183-314 ng/ml). Maximum PAI-1 levels above 120 ng/ml showed a sensitivity of 100% and a specificity of 30.6% for VOD after HSCT. CONCLUSION: Our study underlines that maximum PAI-1 plasma antigen levels not exceeding 120 ng/ml have a strong negative predictive value in the diagnosis of VOD and thus represent a helpful non-invasive tool for exclusion of VOD after HSCT.

Peripheral dendritic cell chimerism in allogeneic hematopoietic stem cell recipients.

BACKGROUND: Relapse and graft-versus-host disease (GVHD) represent major causes of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Although leukocyte and T-cell chimerism analyses are performed routinely suggesting a predictive value on the patients outcome, little is known about chimerism of dendritic cells (DC) representing strong initiators of immune responses. METHODS: In this prospective study, peripheral DC1 (CD11c+) and DC2 (CD123+) chimerism was determined in hematopoetic stem cell recipients. DCs were isolated from peripheral blood by fluorescence activated cell sorting. Chimerism analyses were performed by fluorescent in situ hybridization or by polymerase chain reaction-based typing of short tandem repeats. RESULTS: At time of engraftment, DC chimerism analyses showed complete chimerism in 76.3% (DC1)/79.5% (DC2), mixed chimerism (MC) in 21.0% (DC1)/17.9% (DC2) and no chimerism in 2.7% (DC1)/2.6% (DC2) of the patients. Peripheral DC chimerism had no significant effect on relapse-free or overall survival. Although acute GVHD was observed more often in patients with MC for DC1/DC2 and chronic GVHD occurred more often in patients with MC for DC2, there was no statistically significant correlation. CONCLUSIONS: Although DCs as antigen presenting cells are supposed to have an impact on the induction of GVHD, there was no significant correlation between incidence of GVHD and DC chimerism after HSCT.

Transforming growth factor beta-1 released from platelets contributes to hypercoagulability in veno-occlusive disease following hematopoetic stem cell transplantation.

BACKGROUND: Hepatic veno-occlusive disease (VOD) is one of the most disastrous complications after allogeneic hematopoetic stem cell transplantation (HSCT). Thrombocytopenia with refractoriness to platelet transfusions suggests an increased platelet consumption in these patients. Interactions between platelets and endothelial cells might contribute to the hypercoagulable state at the sinusoidal endothelium as a central mechanism in the pathogenesis of VOD. STUDY DESIGN: The influence of activated platelets on cultured human endothelial cells was investigated in vitro. We focused on the release of plasminogen activator inhibitor-1 (PAI-1) from endothelial cells which has earlier been found to be significantly elevated in plasma of VOD patients. Endothelial cells isolated from human umbilical cords (HUVEC) were incubated with activated platelets. The release of PAI-1 in the presence or absence of specific antibodies was determined by ELISA technique. Tissue factor (TF) expression on endothelial cells was observed by flowcytometric analysis. RESULTS: HUVEC incubated with activated platelets were found to release significantly more PAI-1 compared to untreated cultures. The endothelial PAI-1-secretion after incubation of HUVEC with activated platelets was completely inhibited by an IgG monoclonal antibody against human transforming growth factor beta-1 (TGF beta-1). In contrast, PAI-1 production was not suppressed after inhibition of HUVEC-platelet-interaction by an IgG monoclonal antibody against CD154 (CD40L) expressed on the surface of activated platelets. An increased release of PAI-1 and an increased expression of tissue factor (TF) on the endothelial cell surface were observed after stimulation with TGF beta-1. CONCLUSION: TGF beta-1 released from activated platelets contributes to the hemostatic imbalance at the sinusoidal endothelium in patients with hepatic VOD by increase of endothelial cell PAI-1 production and TF expression. As a potent profibrotic cytokine, TGF beta-1 might further be involved in phlebosclerosis and sinusoidal fibrosis occurring in VOD.

Hemostatic complications in bone marrow transplantation: a retrospective analysis of 447 patients.

BACKGROUND: Hemostatic complications are not uncommon after bone marrow transplantation (BMT). However, little is known about the frequency, localization, determinants, and outcome of hemostatic events in autologous and allogeneic BMT. METHODS: Four hundred forty-seven patients (364 allogeneic, 83 autologous transplants) were evaluated retrospectively for the presence of hemostatic complications (bleeding, thrombosis, hepatic veno-occlusive disease [VOD], microangiopathic hemolytic anemia) from the start of conditioning therapy until June 2000. RESULTS: A total of 83.2% of the patients presented with at least one hemostatic complication during the investigational period. Most bleeding episodes occurred within the first 4 weeks after transplantation and were relatively mild. However, 27.1% of the patients hemorrhaged severely, generally doubling the overall mortality of the BMT recipients. Fatal gastrointestinal or intracerebral hemorrhages contributed to 1.1% of the events. Bleeding was strongly associated with prolonged thrombocytopenia and graft-versus-host disease (GVHD). Hemorrhagic cystitis may additionally have been triggered by the preceding conditioning regimens containing cyclophosphamide. Thromboembolic events occurred most frequently in allogeneic transplant recipients, for whom the incidence was 14.6%. Chronic GVHD and treatment with steroids were the major determining factors. The incidence of hepatic VOD in 4.7% of the allogeneic transplant recipients was associated with a high fatality rate. Busulfan conditioning increased the VOD risk 2.6-fold. Moderate or severe microangiopathic hemolytic anemia was associated with GVHD and occurred in 14.6% of the allogeneic transplant recipients, leading to an increased overall mortality. CONCLUSION: Hemostatic disturbances, commonly found in the course of transplantation, are associated with a high transplantation risk and closely related to thrombocytopenia and immunologic complications.

Impact of thrombophilic gene mutations and graft-versus-host disease on thromboembolic complications after allogeneic hematopoietic stem-cell transplantation.

BACKGROUND: Hemostatic events in patients undergoing allogeneic hematopoietic stem-cell transplantation (HSCT) increase the morbidity and mortality in this cohort. Little is known about the impact of graft-versus-host disease (GvHD) or of thrombophilic gene mutations/polymorphisms on these complications. STUDY DESIGN: Eighty-nine allogeneic stem-cell recipients and their donors were evaluated prospectively for the presence of the factor V G1691A mutation, the prothrombin G20210A mutation, the 5,10-methylenetetrahydrofolate-reductase (MTHFR) C677T mutation, the glycoprotein IIIa PI(a1/a2) polymorphism, the fibrinogen-beta-chain 455G/A polymorphism, the plasminogen activator inhibitor-1 -675 4G/5G polymorphism, and the angiotensin-converting enzyme intron 16 I/D polymorphism. These mutations/polymorphisms and GvHD parameters were correlated to hemostatic and toxic complications after transplantation. The data were compared with those of 128 healthy controls. RESULTS: The PAI-1 4G/4G polymorphism increases the risk for catheter thrombosis after HSCT 5.7-fold (32.2% vs. 71.4%, P<0.05). In patients with hepatic veno-occlusive disease, the frequency of the PAI-1 4G allele is also increased (83.3% vs. 55.1%, NS). Thrombophilic mutations/polymorphisms in donors do not influence complications in the corresponding recipients. The MTHFR TT genotype does not modify severity and duration of mucositis and aplasia in patients receiving methotrexate prophylaxis. Patients with chronic GvHD have a higher risk of thromboembolism (12.9% vs. 1.7%, P<0.05). CONCLUSION: Thrombophilic gene mutations have only a moderate influence on hemostatic complications in patients undergoing HSCT. This may be because of the overwhelming immunologic impact of GvHD on hemostasis in the allogeneic transplantation setting.

Microparticles for diagnosis of graft-versus-host disease after allogeneic stem transplantation.

BACKGROUND: The differentiation between acute graft-versus-host disease (aGvHD) and infection is still a clinical challenge in patients after allogeneic hematopoietic stem-cell transplantation (HSCT). Definitive diagnosis is based on histologic findings, but a simple blood test for differentiation is missing. METHODS: In a prospective study, we measured the plasma levels of erythrocyte-derived microparticles (EryMP) in 19 recipients during HSCT. Microparticles were isolated by differential centrifugation, double stained for glycophorin A (CD235) and annexin V, and analyzed by flow cytometry. RESULTS: Eight patients developed aGvHD (42%), 15 patients developed infectious complications (79%), and two patients developed microangiopathic hemolytic anemia (11%). The levels of EryMP, as measured before conditioning therapy (535 × 10(6)/L in median), were not affected by total body irradiation, high-dose chemotherapy, or in vivo T-cell depletion. EryMP levels were unaffected in uncomplicated patients during aplasia (522 × 10(6)/L in median; P=0.394) or after engraftment (480 × 10(6)/L in median; P = 0.594) and in patients with infectious complications or sepsis (586 × 10(6)/L in median; P = 0.606). In contrast, in patients who developed aGvHD after HSCT, a 1.7-fold increase in the plasma levels of EryMP was observed (880 ×1 0(6)/L in median; P<0.001 compared with the time before therapy and P = 0.015 compared with patients with infections or sepsis). CONCLUSION: Increased plasma levels of EryMP are present in patients who develop aGvHD but not in patients who develop infection or sepsis after HSCT. Therefore, EryMP are a potential, novel, blood marker that may be helpful in the diagnosis of this common complication after HSCT.

Cellular origin of platelet-derived microparticles in vivo.

INTRODUCTION: Microparticles (MP), presumably of platelet origin, are the most abundant microparticles in blood. To which extent such MP may also directly originate from megakaryocytes, however, is unknown. During hematopoietic stem cell transplantation, patients undergo total body irradiation which leads to an irreversible destruction of hematopoiesis. MATERIAL AND METHODS: We studied the levels of "platelet-derived" MP (PMP) in 13 patients before and after total body irradiation with 12 Gy (4 Gy for 3 days, dose rate 4.5 cGy/min). PMP were isolated and double-stained with annexin V and anti-CD61. In 6 patients, we additionally analyzed MP exposing P-selectin or CD63. RESULTS: PMP rapidly declined upon total body irradiation, which was 2.4-fold faster than platelet disappearance. In contrast, the kinetics of MP exposing P-selectin or CD63 was comparable to platelets. CONCLUSIONS: Since CD61-positive MP disappear faster than platelets or MP exposing P-selectin or CD63, our data indicate that MP exposing P-selectin or CD63 are likely to originate from platelets, whereas at least a major fraction of CD61-exposing MP is likely to originate from megakaryocytes in vivo.

Evaluation of C-reactive protein, interleukin-6, and procalcitonin levels in allogeneic hematopoietic stem cell recipients.

BACKGROUND: Prompt detection of transplant-related complications (TRC) as infections, acute graft-versus-host disease (aGVHD), microangiopathic hemolytic anemia, or veno-occlusive disease following allogeneic hematopoietic stem cell transplantation (HSCT) is essential. PATIENTS AND METHODS: We conducted a prospective trial on clinical significance of C-reactive protein (CRP), interleukin-6 (IL-6) and procalcitonin (PCT) serum levels in TRC. A total of 350 stem cell recipients were admitted. CRP, IL-6 and PCT were analyzed prior to conditioning and weekly until 8 wk after HSCT. TRC were recorded weekly throughout the study. RESULTS: CRP (4.4 mg/dL vs. 12.8 mg/dL; P < 0.001), IL-6 (93 ng/mL vs. 1.138 ng/mL; P < 0.001) and PCT (0.8 ng/dL vs. 5.7 ng/dL; P < 0.001) were increased in infectious complications. Only PCT differentiated between infection and other TRC. Exclusive aGVHD did not increase CRP (4.4 mg/dL vs. 5.7 mg/dL; n.s.), IL-6 (93 ng/mL vs. 153 ng/mL; n.s.) and PCT (0.8 ng/dL vs. 0.8 ng/dL; n.s.). CRP (6.1 mg/dL vs. 3.1 mg/dL; P < 0.001) and IL-6 (295 ng/mL vs. 122 ng/mL; P = 0.001) were decreased during steroid therapy, but not PCT (2.3 ng/dL vs. 2.0 ng/dL; n.s.). CONCLUSION: Our study confirmed CRP, IL-6 and PCT serum levels as helpful markers for TRC. PCT can differentiate infection from GVHD despite steroid therapy. Further trials are needed focusing on the identification of patients who benefit from early risk stratification.

Platelet function rather than plasmatic coagulation explains hypercoagulable state in cholestatic liver disease.

BACKGROUND/AIMS: As compared to other chronic liver diseases, cholestatic disorders are associated with a better outcome of variceal bleeding and less blood loss at transplantation, suggesting the presence of a hypercoagulable state. We have assessed plasmatic coagulation and platelet function in patients with cholestatic and non-cholestatic liver disease. METHODS: Thirty-seven patients with chronic cholestatic liver disease (primary biliary cirrhosis (PBC)/primary sclerosing cholangitis (PSC)), 53 patients with chronic hepatitis C (HCV) or alcoholic cirrhosis (C2), and 62 healthy controls were studied. RESULTS: Thrombelastography revealed a hypercoagulable state in non-cirrhotic patients with PBC/PSC, but not in those with HCV (ma-value: 6.54[6.25-6.92, 95%CI] vs. 5.39[5.11-5.58], P < 0.05) possibly due to higher fibrinogen levels in PBC/PSC patients (369[329-418]mg/dl vs. 263[250-275]mg/dl, P < 0.05). PFA-100 closure time was prolonged in HCV/C2 patients with advanced cirrhosis, but not in cirrhotic patients with PBC/PSC (Child B; epinephrine stimulation: 192[161-229]s vs. 132[105-158]s, P < 0.05). Flow cytometric studies of platelet receptors and granules revealed a higher surface expression of CD42b (112[105-119]% vs. 100[95-104]%, P < 0.05) and LIBS-1 (261[184-348]% vs. 121[92-145]%, P < 0.05) in patients with PBC/PSC than in those with HCV/C2. CONCLUSIONS: These results indicate that platelet function differs between patients with cholestatic and non-cholestatic liver disease and is stable or even hyperactive in patients with PBC and PSC.

Impact of thrombophilic gene mutations on thrombosis risk in patients with gastrointestinal carcinoma.

BACKGROUND: Patients with malignancies have an increased risk for thromboembolic events due to the release of tissue factor by the tumor, damage to the vessel wall, and immobilization. Moreover, tumors may improve their growth and metastatic spread by utilizing the coagulation system. To date, no information is available on the additional role of prothrombotic mutations in these patients. METHODS: The prevalence of the factor V Leiden mutation (FVL) and the prothrombin G20210A mutation and of homozygosity for the methylenetetrahydrofolate reductase (MTHFR) C677T substitution has been analyzed in a cohort of 175 patients with gastrointestinal adenocarcinoma by the polymerase chain reaction-restriction fragment length polymorphism technique. RESULTS: 6.9% of the patients were heterozygous for FVL, 5.7% were heterozygous for the prothrombin mutation, and 9.7% were homozygous for the MTHFR C677T mutation was detected in 9.7% of patients. Compared with the normal population, we found an increased prevalence of the prothrombin G20210A substitution (5.7% vs. 0.8%, P = 0.028). Thromboses were absent in 147 patients (Group A), whereas 28 of the patients suffered from thromboses during the period following tumor diagnosis (Group B). In Group A, 6.8% of the patients and 21.4% of the patients in Group B had a thrombosis before the diagnosis of cancer (P = 0.025, odds ratio [OR] 3.7). Heterozygous FVL was present in 4.8% of the patients in Group A and in 17.9% of the patients in Group B (P = 0.026, OR 4.4). In patients with thromboses before the detection of the tumor, the risk was elevated 6.3-fold (25.0% vs 5.0%, P = 0.015). Heterozygosity for the prothrombin mutation and homozygosity for the MTHFR C677T substitution did not increase the incidence of thromboses. CONCLUSIONS: We demonstrated a significant effect of FVL on thrombosis in patients with malignant disease. Most thromboses occurred during the first months after tumor diagnosis, implicating diagnostic and therapeutic procedures as important nongenetic risk factors for venous thromboembolism. Our data also indicate that the prothrombin G20210A mutation may be a possible cofactor in cancer pathogenesis.

Factor XIII activity levels in patients with allogeneic haematopoietic stem cell transplantation and acute graft-versus-host disease of the gut.

Severe graft-versus-host disease (GvHD) of the gut clinically resembles Crohn's disease and ulcerative colitis. As low plasma levels of factor XIII (FXIII) have been described in chronic inflammatory bowel disease (CIBD) and as beneficial effects of FXIII concentrates in CIBD have been reported, we studied the FXIII plasma activity levels in patients undergoing allogeneic stem cell transplantation (SCT). In 20 of 22 patients with an uncomplicated course of SCT, FXIII stayed within the normal range (median 102 iu/dl, range 74-122), but was significantly reduced with the lowest FXIII levels on d 0 and 7 (d 0: median 83 iu/dl, range 55-165, d 7: median 83, range 70-101). In 20 of 22 patients with histologically proven GvHD of the gut, FXIII levels far below the normal range were observed (median 50, range 21-87) with a strong correlation between FXIII activity levels and degree of GvHD (r = -0.908; P < 0.001). We conclude that FXIII is consumed in patients with GvHD of the gut. As FXIII plays a a crucial role in haemostasis and wound healing, a study on the potential benefit of FXIII substitution in patients with severe GvHD of the gut might be rewarding.