First report on the safety and efficacy of an extended half-life glycoPEGylated recombinant FVIII for major surgery in severe haemophilia A.

BACKGROUND: N8-GP (turoctocog alfa pegol) is an extended half-life glycoPEGylated recombinant factor VIII (FVIII) product developed for the prevention and treatment of bleeds in haemophilia A patients. AIM: This is a planned interim analysis of pathfinder™3, an international, open-label, Phase 3 trial evaluating the efficacy and safety (including immunogenicity) of N8-GP administered before, during and after major surgery in severe haemophilia A patients aged ≥12 years. METHODS: Sixteen patients who underwent 18 major surgical procedures (including synovectomy, joint replacement and ankle arthrodesis) were included here. Postoperative assessments were conducted daily for days 1-6, and once for days 7-14. Primary endpoint was N8-GP haemostatic efficacy, assessed after completion of surgery using a four-point scale ('excellent', 'good', 'moderate', 'none'). RESULTS: Haemostasis was successful (rated 'excellent' or 'good') on completion of surgery in 17 (94.4%) procedures and rated as 'moderate' (5.6%) for one surgery in a patient with multiple comorbidities who needed an intraoperative N8-GP dose (20.7 IU kg-1 ). In the postoperative period, three bleeds occurred (one during days 1-6; two during days 7-14); all were successfully treated with N8-GP. Mean N8-GP consumption on day of surgery was 80.0 IU kg-1 ; patients received a mean of 1.7 doses (median: 2, range: 1-3). No safety concerns were identified. CONCLUSION: The data showed that N8-GP was effective and well tolerated for the prevention and treatment of bleeds during major surgery; such FVIII products with extended half-lives may modify current treatment schedules, enabling fewer infusions and earlier patient discharge.

Changes in the amino acid sequence of the recombinant human factor VIIa analog, vatreptacog alfa, are associated with clinical immunogenicity.

BACKGROUND: Vatreptacog alfa, a recombinant human factor VIIa (rFVIIa) analog developed to improve the treatment of bleeds in hemophilia patients with inhibitors, differs from native FVIIa by three amino acid substitutions. In a randomized, double-blind, crossover, confirmatory phase III trial (adept(™) 2), 8/72 (11%) hemophilia A or B patients with inhibitors treated for acute bleeds developed anti-drug antibodies (ADAs) to vatreptacog alfa. OBJECTIVES: To characterize the formation of anti-vatreptacog alfa ADAs in hemophilia patients with inhibitors. METHODS/PATIENTS: This was a post hoc analysis of adept(™) 2. Immunoglobulin isotype determination, specificity analysis of rFVIIa cross-reactive antibodies, epitope mapping of rFVIIa single mutant analogs and pharmacokinetic (PK) profiling were performed to characterize the ADAs. RESULTS: Immunoglobulin isotyping indicated that the ADAs were of the immunoglobulin G subtype. In epitope mapping, none of the rFVIIa single mutant analogs (V158D, E296V or M298Q) contained the complete antibody epitope, confirming that the antibodies were specific for vatreptacog alfa. In two patients, for whom PK profiling was performed both before and after the development of ADAs, vatreptacog alfa showed a prolonged elimination phase following ADA development. During the follow-up evaluation, the rFVIIa cross-reactivity disappeared after the last vatreptacog alfa exposure, despite continued exposure to rFVIIa as part of standard care. CONCLUSIONS: Results from the vatreptacog alfa phase III trial demonstrate that the specific changes made, albeit relatively small, to the FVIIa molecule alter its clinical immunogenicity.

Recombinant factor VIIa analog in the management of hemophilia with inhibitors: results from a multicenter, randomized, controlled trial of vatreptacog alfa.

BACKGROUND: Vatreptacog alfa, a recombinant factor VIIa (rFVIIa) analog with three amino acid substitutions and 99% identity to native FVIIa, was developed to improve the treatment of hemophilic patients with inhibitors. OBJECTIVES: To confirm the safety and assess the efficacy of vatreptacog alfa in treating bleeding episodes in hemophilic patients with inhibitors. PATIENTS AND METHODS: In this international, multicenter, randomized, double-blind, active-controlled, crossover, confirmatory phase III trial (adept(™) 2) in patients with hemophilia A or B and inhibitors, bleeds were randomized 3 : 2 to treatment with vatreptacog alfa (one to three doses at 80 µg kg(-1) ) or rFVIIa (one to three doses at 90 µg kg(-1) ). Treatment failures after three doses of trial product (TP) were managed according to the local standard of care. RESULTS: In the 72 patients enrolled, 567 bleeds were treated with TP. Both vatreptacog alfa and rFVIIa gave 93% effective bleeding control at 12 h. Vatreptacog alfa was superior to rFVIIa in secondary efficacy outcomes, including the number of doses used to treat a bleed and sustained bleeding control 24-48 h after the first dose. Eight patients (11%) developed antibodies against vatreptacog alfa, including four with cross-reactivity against rFVIIa and one with an in vitro neutralizing effect to vatreptacog alfa. CONCLUSIONS: This large randomized controlled trial confirmed the well-established efficacy and safety profile of rFVIIa, and showed that vatreptacog alfa had similar or better efficacy than rFVIIa. However, because of the development of anti-drug antibodies, a positive benefit-risk profile is unlikely to be achieved with vatreptacog alfa.

Impact of environmental and hereditary risk factors on the clinical manifestation of thrombophilia in homozygous carriers of factor V:G1691A.

BACKGROUND: Limited data exist on the clinical manifestations of homozygous factor (F)V:G1691A mutation (FV Leiden) and the impact of environmental and genetic risk factors. OBJECTIVES: To assess the contribution of these factors on the thrombophilic phenotype. PATIENTS AND METHODS: In a retrospective multicenter cohort study 165 individuals with homozygous FV:G1691A mutation, of whom 129 had previous venous thromboembolism (VTE), were included. To study the role of environmental risk factors, patients were compared by the use of a standardized questionnaire to 165 sex- and age-matched individuals (reference group A); of these, two had previous VTE. To assess the role of genetic risk factors, factor (F)II:G20210A and MTHFR:C677T were determined in individuals homozygous for FV:G1691A and in 177 healthy individuals without previous VTE (reference group B). RESULTS: The first VTE occurred significantly earlier in women (median age 25 years) than men (35.5 years). In 81% of women and 29% of men an environmental risk factor was present before first VTE. Oral contraceptives increased the risk of thrombosis 4-fold [odds ratio (OR) 4.0, 95% confidence interval (CI) 1.7, 10.4] in women with homozygous FV:G1691A. Postoperative and post-traumatic VTE as first manifestation occurred in 13% and 15% of surgical/traumatic events in patients and in 0.7% and 1.8% in reference group A, respectively (OR 19.7, 95% CI 2.5, 154 and OR 9.2, 95% CI 1.1, 79.4). Heterozygous FII:G20210A was more prevalent in symptomatic patients (11.7%) compared with reference group B (2.8%, OR 4.6, 95% CI 1.6, 13.2). The prevalence of homozygous MTHFR:C677T genotype was similar in patients and reference group B. CONCLUSIONS: Our study supports the concept of thrombophilia as a multifactorial disorder. The knowledge of coexisting factors predisposing to VTE is useful for medical advice for primary and secondary prophylaxis in these patients.

Risk of recurrent venous thrombosis in children with combined prothrombotic risk factors.

After a first episode of spontaneous venous thromboembolism (VTE), the risk of recurrence persists for many years. However, comprehensive data about the risk of recurrence in pediatric patients have hitherto not been reported. Thus, this study evaluated the risk of recurrent VTE among children in relation to the presence of single or combined-inherited and/or acquired causes of thrombophilia. A total of 301 patients aged neonate to 18 years (median, 6 years) who were referred for an objectively confirmed first episode of spontaneous VTE were followed prospectively for a median time of 7 years (range, 6 months to 15 years) after withdrawal of anticoagulation. All patients were studied for established acquired and inherited causes of thromboembolism. With reference to all 301 patients, one single prothrombotic risk factor was found in 176 subjects (58.5%), whereas combined defects were found in 20.6% (n = 62). Recurrent VTE occurred in 64 patients (21.3%) within a median time of 3.5 years (range, 7 weeks to 15 years) after withdrawal of anticoagulation, with a significantly shorter cumulative thrombosis-free survival in children carrying combined defects (P <.0001; chi-square, 42.2). The factor V G1691A mutation was present in the majority of patients with recurrent VTE. Including genetic defects, gender, and acquired risk factors, multivariate analysis showed that only the presence of prothrombotic defects increases the risk of recurrent VTE (single defect: odds ratio [OR], 4.6; 95% confidence interval [CI], 2.3-9.0; P <.0001; combined defect: OR, 24.0; 95% CI: 5.3-108.7; P <.0001). As a consequence of the data presented here, it is suggested that screening for genetic risk factors be done among pediatric patients with VTE.

Prevalence of a 23bp insertion in exon 3 of the endothelial cell protein C receptor gene in venous thrombophilia.

BACKGROUND: The endothelial cell protein C receptor (EPCR) enhances protein C activation by the thrombin-thrombomodulin complex. As evidence is accumulating that EPCR is an important component of the protein C anticoagulant pathway, polymorphisms in the EPCR gene might be candidate risk factors predisposing to venous thromboembolism (VTE). Recently, a 23bp insertion in exon 3 of the EPCR gene has been identified, which duplicates the preceding 23 bases and results in a STOP codon downstream from the insertion point. However, the clinical significance of this mutation in VTE remains to be clarified. METHODS AND RESULTS: In this study we evaluated the EPCR 23bp insertion in 889 patients with documented VTE and in 500 healthy controls. The prevalence of the EPCR insertion among patients was 0.1%, which was not significantly different compared to controls (0.6%, p = 0.1). CONCLUSIONS: Our findings showed that the EPCR 23bp insertion is very rare in both patients with VTE and the general population and failed to support an association between the EPCR 23bp insertion and an increased risk of VTE.

Increased lipoprotein (a) levels as an independent risk factor for venous thromboembolism.

Elevation of serum lipoprotein (a) (Lp[a]) is a known risk factor predisposing to cardiovascular and cerebrovascular disease. However, little is known about the role of increased Lp(a) in venous thromboembolism (VTE). This study evaluated the role of Lp(a) among a panel of established hereditary thrombogenic defects in patients with VTE. A total of 685 consecutive patients with at least one episode of VTE and 266 sex- and age-matched healthy controls were screened with regard to activated protein C resistance, protein C, protein S, and antithrombin deficiency, elevated serum levels of Lp(a), and the factor V G1691A, MTHFR C677T, and prothrombin G20210A mutations. Elevated Lp(a) levels above 30 mg/dL were found in 20% of all patients, as compared to 7% among healthy controls (P <.001, odds ratio [OR] 3.2, 95% confidence interval [CI], 1.9-5.3). The coexistence of FV G1691A and elevated Lp(a) was significantly more prevalent among patients with VTE than in the control group (7% versus 0.8%; P <.001, OR 9.8, 95% CI, 2.4-40.7). No other established prothrombotic risk factor was found to be significantly combined with increased Lp(a). These data suggest that Lp(a) concentrations greater than 30 mg/dL are a frequent and independent risk factor for VTE. Furthermore, elevated Lp(a) levels might contribute to the penetrance of thromboembolic disease in subjects being affected by other prothrombotic defects, such as FV G1691A mutation.

Pregnancy-associated risk for venous thromboembolism and pregnancy outcome in women homozygous for factor V Leiden.

INTRODUCTION: To evaluate the pregnancy-associated risk of venous thromboembolism and the risk of stillbirth and miscarriage a multicenter, retrospective and controlled study was conducted in women carrying the homozygous factor V Leiden mutation and in an agematched control group of women from the normal population. PATIENTS AND METHODS: In 64 homozygous (median age 44 years, range 21-75 years) and in 52 control women from five different centers data on venous thromboembolism and pregnancy outcome were obtained. RESULTS: The 64 homozygous women had in total 212 pregnancies, the 52 control women had 118 pregnancies. In homozygous women 65% of pregnancies ended with delivery of a viable infant, 15% with fetal loss (3.3% stillbirth, 12% miscarriage) and 20% by pregnancy termination. In the control women 75% of pregnancies ended with delivery of a viable infant, 12% with fetal loss (1.7% stillbirth, 10% miscarriage) and 13% by pregnancy termination. The differences were statistically not significant. Venous thromboembolism occurred significantly more often in the homozygous women, in 4.2% (9/212) during pregnancy and in 4.7% (10/212) after delivery or pregnancy termination. None of the control women had a thromboembolic episode. CONCLUSION: Our data indicate that women with homozygous factor V Leiden have a high probability for a favorable pregnancy outcome. The increased risk for venous thromboembolism during pregnancy and after delivery would favor heparin prophylaxis during and after pregnancy in women homozygous for factor V Leiden.

Prothrombin G20210A gene mutation and further prothrombotic risk factors in childhood thrombophilia.

Risk factors for venous thrombosis in adults are the prothrombin G20210A and the factor V (FV) G1691A mutations and hereditary deficiencies of protein C, protein S and antithrombin. However, data are limited on the relevance of these risk factors for thrombosis in children and adolescents. We therefore investigated 261 patients aged 0 to 18 (median 5.7 years, 48% male) with venous thrombosis and controls (n=370) for the presence of prothrombotic risk factors including the prothrombin G20210A mutation. The following frequencies of hereditary risk factors (patients versus controls), odds ratios (OR) and 95% confidence intervals (CI), or results of Fisher's exact test, respectively, were found: prothrombin G20210A, 4.2% versus 1.1%, OR/CI 4.1/1.3 to 12.8; FV G1691A, 31.8% versus 4. 1%, OR/CI 11.0/6.2 to 19.7; protein C deficiency, 9.2% versus 0.8%, OR/CI 12.4/3.7 to 41.6, protein S deficiency, 5.7% versus 0.8%, OR/CI 7.5/2.1 to 26.0; antithrombin deficiency in 3.4% in the patients, but not in the controls, P=0.0003. The prothrombin mutation was combined with the heterozygous FV G1691A mutation (2. 3%) or protein C deficiency (0.3%) in the patients, but not in the controls (prothrombin and FV mutation, P=0.0048; prothrombin and protein C deficiency, not significant). The carrier frequencies and ORs of all hereditary risk factors showed a non-significant trend toward higher prevalences in patients suffering spontaneous thrombosis, compared with those with an additional underlying disease. In conclusion, the prothrombin G20210A and the FV G1691A mutation, deficiencies of protein C, protein S, and antithrombin are important risk factors for venous thrombosis during childhood and adolescence.

[Activated protein C resistance and venous thrombophilia: molecular genetic prevalence study in the German population]. / APC-Resistenz und venöse Thrombophilie: Molekulargenetische Prävalenzstudie in der deutschen Bevölkerung.

BACKGROUND AND OBJECTIVE: Resistance against activated protein C (APC), caused by factor V R506Q mutation (factor V Leiden mutation), is among the most important hereditary clotting defects that are associated with an increased risk of venous thrombosis. As there are hardly any data for Germany regarding APC resistance that have been validated by genetic analysis, this study was undertaken to determine the prevalence of factor V Leiden (fVL) mutations in a sizeable group of patients in Germany with venous thromboembolism (VTE) and a control group of healthy persons. PATIENTS AND METHODS: 1200 consecutive patients (689 females, 511 males) from various regions of Germany were examined who, at an age between 0.1 and 45 years, had developed primary deep vein thrombosis (DVT) and/or pulmonary embolism (PE), as confirmed by imaging tests. The control group consisted of 740 healthy persons (332 females and 408 males; median age 33 years) for whom there was no evidence in their personal or family history of TE. Analysis of the fV-1691 genotype was by Mnll-restriction analysis of genomic fV DNA fragments, amplified by polymerase chain reaction. RESULTS: The prevalence in the control group was 7.5% the for heterozygotic fV:Q506 mutant (25 females, 30 males). For the patients the prevalence of the fV R506Q mutation was 27.2% (32.1% heterozygotes [165 females, 112 males]; 4.1% homozygotes [33 females, 16 males], i.e. significantly higher than in the healthy controls (P < 0.0001). In 81.3% of the patients with fV:Q506 DVT in the leg-pelvic vein region was found as the first manifestation, thrombosis in an atypical site in 14.4% and isolated PE in 4.3%. The first manifestation had occurred spontaneously in 36% of patients with the fV:Q506 mutant (44 females, 75 males), in 53.1% of homozygotes and in 33.5% of heterozygotic carriers of the mutation. CONCLUSION: The fV Leiden mutation due to APC resistance is the most common cause of venous thrombosis and apparently one of the most common inherited diseases.

Liver damage induced by coumarin anticoagulants.

Except for bleeding complications, relevant adverse effects of coumarin anticoagulants are comparatively rare considering the widespread use of these substances. Here we present the case of a 56-year-old woman who developed recurrent episodes of severe hepatitis following repeated exposure to phenprocoumon (Marcumar; Roche, Grenzach-Wyhlen, Germany) and warfarin (Coumadin; DuPont Pharma, Bad Homburg, Germany) after replacement of the mitral valve with a mechanical prosthesis. The diagnosis of "coumarin-induced hepatitis" is compatible with the time relationship between start of the drug and the onset of hepatopathy (first episode 8 months, second episode 4 weeks, and third episode 7 days), the rapid improvement following discontinuation of the drug, recurrence of liver dysfunction after re-exposure to the drug, and liver histology. After anticoagulant therapy was changed to heparin and acenocoumarol (Sintrom; Ciba-Geigy, Basel, Switzerland), the patient's general state was markedly improved and her liver values became almost normal. This case will be discussed and compared with other reports of coumarin-induced hepatic lesions. Although liver damage induced by coumarin derivates is rare, it is important to be aware of the hepatotoxic potential of these drugs, which, in most cases, mimics the clinical presentation of viral hepatitis.

Study of the prothrombin gene 20201 GA variant in FV:Q506 carriers in relationship to the presence or absence of juvenile venous thromboembolism.

The G20210A transition of the prothrombin gene has been identified as a common but probably mild hereditary risk factor for venous thromboembolism (VTE). However, the prothrombin gene variant might contribute to the penetrance of thromboembolic disease in many patients with other prothrombotic defects, such as the FV:R506Q mutation. In this investigation, the A20210 allele was found in 9 of 450 healthy controls (2%). Among 89 asymptomatic FV:Q506 carriers, 3 subjects were doubly affected (3.4%). In contrast, of 263 unrelated carriers of the FV:Q506 mutant with a history of juvenile VTE, 30 also had the prothrombin gene G20210A variant (11.4%), including 25 of 220 patients who were heterozygous (11.4%) and 5 of 43 homozygous (11.6%) for FV:Q506. Thus, the A20210 allele of the prothrombin gene is significantly overrepresented in symptomatic FV:Q506 carriers compared with healthy controls (P<0.0001) and asymptomatic relatives carrying the FV mutant (P=0.02). Persons homozygous for the 20210A allele were not found. A statistically significant increase in the prevalence of more unusual sites of venous thrombosis at clinical onset was found in doubly affected patients (9 of 30; 30%) compared with patients without the prothrombin gene variant (26 of 233; 11. 1%) (P=0.004). First VTE occurred spontaneously in 53.3% of all doubly affected patients (16 of 30) and in 28.3% of all simply affected patients (66 of 233) (P=0.005). Among patients with VTE preceded by circumstantial risk factors, the A20210 allele was found in 7.7% (14 of 181). We conclude that the A20210 allele of the prothrombin gene is frequently coinherited in symptomatic FV:Q506 carriers and possibly influences age, site, and type of thrombotic onset manifestation in these patients.

Prospective evaluation of the thrombotic risk in children with acute lymphoblastic leukemia carrying the MTHFR TT 677 genotype, the prothrombin G20210A variant, and further prothrombotic risk factors.

The reported incidence of thromboembolism in children with acute lymphoblastic leukemia (ALL) treated with L-asparaginase, vincristine, and prednisone varies from 2.4% to 11.5%. The present study was designed to prospectively evaluate the role of the TT677 methylenetetrahydrofolate reductase (MTHFR) genotype, the prothrombin G20210A mutation, the factor V G1691A mutation, deficiencies of protein C, protein S, antithrombin, and increased lipoprotein (a) concentrations in leukemic children treated according to the ALL-Berlin-Frankfurt-Muenster (BFM) 90/95 study protocols with respect to the onset of vascular events. Three hundred and one consecutive leukemic children were enrolled in this study. Fifty-five of these 301 subjects investigated had one established single prothrombotic risk factor: 20 children showed the TT677 MTHFR genotype; 5 showed the heterozygous prothrombin G20210A variant; 11 were carriers of the factor V G1691A mutation (heterozygous, n = 10; homozygous, n = 1); 4 showed familial protein C, 4 protein S, and 2 antithrombin type I deficiency; 9 patients were suffering from familially increased lipoprotein (a) [Lp(a)] concentrations (>30 mg/dL). In addition, combined prothrombotic defects were found in a further 10 patients: the FV mutation was combined with the prothrombin G20210A variant (n = 1), increased Lp(a) (n = 3), protein C deficiency (n = 1), and homozygosity for the C677T MTHFR gene mutation (n = 1). Lp(a) was combined with protein C deficiency (n = 2) and the MTHFR TT 677 genotype (n = 2). Two hundred eighty-nine of the 301 patients were available for thrombosis-free survival analysis. In 32 (11%) of these 289 patients venous thromboembolism occurred. The overall thrombosis-free survival in patients with at least one prothrombotic defect was significantly reduced compared with patients without a prothrombotic defect within the hemostatic system (P <.0001). In addition, a clear-cut positive correlation (P <.0001) was found between thrombosis and the use of central lines. However, because the prothrombotic defects diagnosed in the total childhood population studied were all found within the prevalences reported for healthy Caucasian individuals, the interaction between prothrombotic risk factors, ALL treatment, and further environmental factors is likely to cause thrombotic manifestations.

Multicentre evaluation of combined prothrombotic defects associated with thrombophilia in childhood. Childhood Thrombophilia Study Group.

UNLABELLED: To evaluate the role of multiple established and potential causes of childhood thrombophilia, 285 children with a history of thrombosis aged neonate to 18 years (first thrombotic onset) were investigated and compared with 185 healthy peers. APC-resistance (FV:Q506), protein C, protein S, antithrombin, heparin cofactor II (HCII), histidine-rich glycoprotein (HRGP), and prothrombin (F.II), factor XII (F.XII), plasminogen, homocysteine and lipoprotein (a) (Lp(a)) were investigated. In 59% of patients investigated one thrombotic defect was diagnosed, 19.6% showed two thrombotic risk factors, while in 21.4% of children investigated no risk factor could be identified. Single defects comprised established causes of inherited thrombophilia: FV:Q506 (homozygous n = 10, heterozygous n = 69), protein C (homozygous n = 1; heterozygous n = 31), heterozygous type I deficiency states of protein S (n = 7), antithrombin (n = 7) and homocystinuria (n = 6); potentially inherited clotting abnormalities which may be associated with thrombophilia: F.XII (n = 3), plasminogen (n = 2), HCII (n = 1), increased HRGP (n = 4); new candidate risk factors for thrombophilia: elevated plasma levels of Lp(a) (n = 26), F.II (n = 1). Heterozygous FV:Q506 was found in combination with heterozygous type I deficiency states of protein C (n = 2), protein S (n = 13), antithrombin (n = 8) and HCII (n = 1), increased Lp(a) (n = 13), and once each with elevated levels of F.II, moderate hyperhomocysteinemia, fibrinogen concentrations > 700 mg/dl and increased HRGP. In addition to the association with FV:Q506, heterozygous protein C type I deficiency was combined with deficiencies of protein S (n = 2), antithrombin (n = 1), and increased Lp(a) (n = 3). One patient showed protein C deficiency along with familially increased von Willebrand factor > 250%. Besides coexistence with FV:Q506 and protein C deficiency, protein S deficiency was combined with decreased F.XII and increased Lp(a) in one subject each. Furthermore, we found combinations of antithrombin deficiency/elevated Lp(a), hyperhomocysteinemia/Lp(a), deficiency of HCII/plasminogen, and plasminogen deficiency along with increased Lp(a) each in one. Increased prothrombin levels were associated with fibrinogen concentrations > 700 mg/dl and with HCII deficiency in one child each. Carrier frequencies of single and combined defects were significantly higher in patients compared with the controls. CONCLUSION: In conclusion, data of this multicentre evaluation indicate that paediatric thromboembolism should be viewed as a multifactorial disorder.

The course of fibrinolytic proteins in children with malignant bone tumours.

UNLABELLED: To evaluate the role of fibrinolytic and proteolytic proteins in children and adolescents suffering from Ewing sarcoma or osteosarcoma with respect to postoperative complications and late outcome, a prospective two-arm two-centre study was conducted. Plasminogen, plasminogen activator inhibitor (PAI)-1, tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) were investigated in the pre-surgical period and in the postoperative follow-up period in children suffering from Ewing sarcoma (ES; n = 36) or osteosarcoma (OS; n = 39). In addition, the factor V mutation (FV) Q506, protein C, protein S, antithrombin and lipoprotein (a) were determined. All children received LMWH (EnoxaparinR) 1 mg/kg s.c. once daily over a period of 6 weeks to 3 months. Besides a short-lasting increase of PAI-1 in patients with OS on day 1 and in children with Es on day 14, a small and significant but clinically irrelevant difference was found on days 7-10 for plasminogen, t-PA and u-PA. No thromboembolic complications occurred in patients treated with LMWH and having a prothrombotic genetic risk factor. Within one year of surgery 7 out of 36 patients with ES and 5 out of 39 children with OS showed a relapse of their disease. Prior to the first local tumour therapy, 5 out of 7 children with ES and relapse had elevated u-PA concentrations compared with 2 out of 5 children in the OS group. No such differences were found for PAI-1- or t-PA antigen. CONCLUSION: The role of u-PA as a possible follow-up marker for a poorer outcome in children with ES should be evaluated in a prospective multicentre study.

Cerebral venous sinus thrombosis in infancy and childhood: role of genetic and acquired risk factors of thrombophilia.

UNLABELLED: Over a 3 year period the R506Q mutation in the factor V (FV) FV:Q506 gene, FV, factor XII (FXII), prothrombin, protein C, protein S, antithrombin, heparin cofactor II, anticardiolipin antibodies and lipoprotein (a) (Lp(a)) were measured in 32 infants and children with sinus thrombosis. Heterozygous FV:Q506 (n=5), homozygous FV:Q506 (n=2), homozygous FXII deficiency (n=1), protein C deficiency type I (n=5), protein C deficiency type II (n=1), antithrombin deficiency type I (n=1) increased Lp (a) (n=5), activated protein C-resistance without mutation in the FV gene (n=2), and increased anticardiolipin IgG antibodies (n=2) were diagnosed in the children investigated. In a further two patients we found combinations of increased Lp(a) with moderate hyperhomocystinaemia and heterozygous plasminogen deficiency with heterozygous FXII deficiency. In addition, increased anticardiolipin IgG antibodies were found in combination with heterozygous FV:Q506 (n=1) and protein C type I deficiency (n=2) respectively. Out of 32 patients with venous sinus thrombosis, 3 showed additional peripheral venous vascular occlusion. Contributing factors were present in 31 out of 32 patients investigated. Family members of 10 affected children had suffered from venous thrombo-embolism prior to the study. CONCLUSION: Our data suggest that additional contributing factors may promote manifestation of cerebral venous sinus thrombosis in infants and children with an inherited prothrombotic state. Further prospective studies are required to evaluate their potential role as "triggering" agents.

Protease levels in breast, ovary, and other gynecological tumor tissues: prognostic importance in breast cancer.

Proteolytic destruction of basement membrane and tumor surrounding is a prerequisite of invasion and metastasis. In 587 frozen samples of malignant and nonmalignant tissue of breast, uterus, vulva, and ovary, levels of urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1) were examined with enzyme-linked immunosorbent assay (ELISA) and cathepsin D (cath D) with radioimmunoassay. UPA, PAI-1 and cath D were raised in malignant tissue with significantly higher levels in breast cancer (uPA, PAI-1) and ovarian cancer (cath D). TPA levels were lower in malignant tissue. In 393 primary breast cancer samples, uPA, PAI-1, and cath D were not related to other prognostic factors, whereas tPA levels were significantly raised in prognostic more favorable carcinomas. Over a follow-up period up to 46 months (median 30 months) the log-rank test showed in the whole group of breast cancer patients a significantly higher rate of relapse (p < 0.05) and death (p < 0.001) with tPA levels < 2.5 ng/mg. PAI-1 levels > 3 ng/mg were associated with shorter overall (p < 0.02; p = 0.01), disease-free (p < 0.008; p < 0.01), and metastasis-free (p < 0.04; p = 0.005) survival in all patients and in the node-negative subgroup, respectively. Higher uPA and cath D levels were not associated with rate of relapse or death over this follow-up period. The prognostic value of tumor-associated proteases could be of interest also in ovarian and cervical cancer.