Biobanking of fresh-frozen endoscopic biopsy specimens from esophageal adenocarcinoma.

The process of preparing endoscopic esophageal adenocarcinoma samples for next-generation DNA/RNA sequencing is poorly described. Therefore, we assessed the feasibility and pitfalls of preparing esophageal adenocarcinoma endoscopic biopsies toward DNA/RNA samples suitable for next-generation sequencing. In this prospective study, four tumor biopsy samples were collected from consecutive esophageal cancer patients during esophagogastroduodenoscopy and fresh-frozen in liquid nitrogen. DNA and RNA were isolated from samples with a tumor percentage of at least 50%. For next-generation sequencing, double-stranded DNA (dsDNA) is required and high-quality RNA preferred. The quantity dsDNA and RNA quantity and quality were assessed with the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Biopsy samples of 69 consecutive patients with esophageal adenocarcinoma were included. In five patients (7%), the tumor percentage was less than 50% in all four biopsies. Using a protocol allowing simultaneous DNA and RNA isolation, the median dsDNA yield was 2.4 µg (range 0.1-12.0 µg) and the median RNA yield was 0.5 µg (range 0.01-2.05 µg). The median RNA integrity number of samples that were fresh-frozen within 30 minutes after sampling was 6.7 (range 4.2-8.9) compared with 2.5 (1.8-4.5) for samples that were fresh-frozen after 2 hours. The results from this study show that obtaining dsDNA and RNA for next-generation sequencing from endoscopic esophageal adenocarcinoma samples is feasible. Tumor percentage and dsDNA/RNA yield and quality emphasize the need for sampling multiple biopsies and minimizing the delay before fresh-freezing.

Influence of heparin and a low molecular weight heparinoid on specific endogenous and exogenous fibrinolytic factors during rest and exercise.

The effects of heparin (5,000 IU i.v.) and the low molecular weight heparinoid Org 10172 (Orgaran) (3,250 anti-Xa units i.v.) on components of the fibrinolytic system were studied in two double-blind, randomised, placebo-controlled, cross-over trials using healthy subjects. In study A (n = 6) the effects were studied during rest and standardized exercise and in study B (n = 6) during a low dose infusion of recombinant tissue-type plasminogen activator (rt-PA; 80 micrograms over 16 min). At rest, heparin and Org 10172 did not influence the plasma concentrations of endogenous t-PA antigen and activity, urokinase-type PA (u-PA) antigen, plasmin activatable pro-urokinase (scu-PA), active urokinase (tcu-PA) and plasminogen activator inhibitor-1 (PAI-1) antigen. Recombinant t-PA antigen and activity during rt-PA infusion were also not affected. During exercise, neither heparin nor Org 10172 influenced the area under the curve (AUC) of t-PA and u-PA antigen and t-PA activity when compared with placebo. Unexpectedly, after heparin the AUC of t-PA activity was 49% larger (range +19 to +245%) than after Org 10172 (p < 0.05). The last difference was considered spurious, scu-PA, tcu-PA and PAI-1 antigen levels at 2 min after termination of exercise were unaffected by both compounds (p > 0.05). Sulphated polysaccharides do not increase fibrinolytic activity of the plasma by changing the concentrations of the components of the fibrinolytic system.

Influence of chlorthalidone on the pharmacokinetics and pharmacodynamics of Org 10172 (Lomoparan), a low molecular weight heparinoid, in healthy volunteers.

The influence of chlorthalidone (100 mg PO) on the pharmacokinetics and pharmacodynamics of Org 10172 (IV bolus injection of 3250 anti-Xa units), a low molecular weight heparinoid, was studied in six healthy male volunteers using an open randomized two-way crossover design. Chlorthalidone produced a slight decrease in clearance of anti-Xa activity from 7.1 +/- 1.0 to 6.6 +/- 0.8 mL/min and a decrease of the volume of distribution from 0.20 +/- 0.05 to 0.16 +/- 0.04 L/kg, whereas the volume of distribution of antithrombin activity increased from 0.14 +/- 0.05 to 0.26 +/- 0.10 L/kg (all differences P less than .05). During the entire study period no adverse events occurred. In summary, chlorthalidone showed separate effects on different fractions of Org 10172. The clinical implication of the slight change observed in plasma anti-Xa activity is likely to be limited, whereas the 80% increase in distribution volume of plasma antithrombin activity can not be defined as yet in terms of clinical relevance.

Partial in vivo neutralisation of plasma anticoagulant effects of Lomoparan (Org 10172) by protamine chloride.

In a cross-over study increasing doses of protamine hydrochloride (20-100 mg) or placebo were administered to six groups of four healthy male volunteers each, following a single intravenous dose of 3200 anti-Xa units of Org 10172. No neutralising effects were observed on the Org 10172 induced changes in the bleeding time, prothrombin time and thrombin time. A small and statistically not significant temporary decrease in anti-Xa activity was observed after doses of 80 and 100 mg protamine chloride. The anti-thrombin activity was dose-dependently and partly irreversibly neutralised by protamine chloride to a maximum of approximately 60%. This neutralisation correlated with the observed shorter prolongation of the thrombin time. The thrombin-generation inhibition activity was for approximately 35% neutralised by protamine chloride doses of 60-100 mg.

Heparin and its biocompatibility.

Heparin has been used in clinical practice since 1936 as anticoagulant for: the treatment of thromboembolic disorders, the prevention of deep vein thrombosis and pulmonary embolism and the maintenance of blood fluidity in extracorporal circuits. Its use in these indications has been complicated by an increased risk of hemorrhage such as major bleeding during the treatment of pulmonary embolism and wound hematoma after surgery. Bleeding problems associated with the use of heparin in extracorporal circuits are the following: hemorrhages after cardiopulmonary bypass, serious hemorrhagic complications in patients treated with hemodialysis during acute renal failure and in patients on chronic intermittent hemodialysis and increased occult blood loss from the gastrointestinal tract and from other sites. The precise contribution of the use of heparin to the enhanced bleeding in these conditions has not yet been established. The effects on platelets, coagulation factors and/or fibrinolytic activity by the exposure of blood to foreign surfaces together with uremia present in hemodialysis patients may also contribute to abnormalities in clinical hemostasis. Recently heparin fractions and a heparinoid of low molecular weight (LMW) have been developed because of their potential to diminish the hazard of hemorrhage while retaining their antithrombotic properties. Preliminary reports from pilot studies have confirmed the increased efficacy in preventing deep vein thrombosis (DVT) of some of the new LMW heparin(oid)s; however, improved safety with regard to bleeding still needs to be shown. The use of LMW heparins and of a new LMW heparinoid in acute and chronic hemodialysis has also been shown to be effective.(ABSTRACT TRUNCATED AT 250 WORDS)

A new low molecular weight heparinoid Org 10172 as anticoagulant in hemodialysis.

A low molecular weight heparinoid (Org 10172) was compared with unfractionated heparin in 36 patients on chronic hemodialysis in an open randomized cross-over study with three anti-coagulant treatment regimens for a single hemodialysis session. The anti-coagulant regimens were: a) standard heparin (3250-4750 I.U. heparin at start of hemodialysis followed by continuous infusion of 2000-2700 I.U. per hour); b) Org 10172 administered as a single intravenous bolus of 2400 anti-Xa units at start of dialysis; c) Org 10172 administered as a single bolus of 3200 anti-Xa units at start of dialysis. Plasma anti-Xa activity during hemodialysis was highest in regimen; d) and significantly lower when heparin was used. Mean beta-thromboglobulin concentrations changed to the same extent in the three groups. Plasma platelet factor 4 concentrations were higher after the use of heparin. The extracorporeal circuit was maintained patent in all groups; the volume of blood retained in the dialyzers did not differ markedly. Org 10172 proved safe and its anticoagulant effect was sufficient at the dose levels studied.

Prognostication and prediction using gene expression profiling in oesophageal cancer.

AIM: To evaluate current literature on gene expression profiling in oesophageal cancer. METHODS: We performed a review of the literature (2000-2010) on prognostication and prediction using gene expression analysis in oesophageal cancer. RESULTS: Seventeen papers comprising 638 patients were included. Gene expression profiles studied in relation to survival, lymph node metastasis and response to neoadjuvant therapy. Most studies included a limited number of patients. Several prognostic and predictive gene signatures were identified with different accuracies. In only one study, the gene signature was validated in a large, independent patient cohort. CONCLUSION: Gene expression profiling has potential clinical applications in oesophageal cancer. Especially a signature which is predictive for response to neoadjuvant treatment could be of great clinical value. To date, most published studies suffer from an underpowered training cohort or lack adequate validation. Clinicians should put effort in the collection of high quality tissue samples and should participate in biobank initiatives, considering the increasing availability and possibilities of sequencing technology.

Surgical treatment results of intestinal and diffuse type gastric cancer. Implications for a differentiated therapeutic approach?

AIM: To study the outcome of patients who were surgically treated for primary gastric cancer with specific attention to differences in treatment results for intestinal and diffuse type tumours. METHODS: All patients who underwent a potentially curative gastric resection between 1995 and 2011 in our institute were included. Patient, tumour and treatment characteristics were obtained retrospectively. Binary logistic and Cox regression models were used for multivariate analysis. RESULTS: A consecutive series of 132 patients was included. Median follow-up was 53 months. There were no significant differences between patients with intestinal (N = 62) versus diffuse type (N = 70) gastric cancer with regard to the proportion of patients who underwent (neo)adjuvant treatment. Postoperative mortality was 2%. Pathological T- and N-stage were significantly more advanced for patients with diffuse type tumours. There was a significant difference in the percentage of microscopically irradical resections (2% versus 24%, p < 0.001) and median overall survival (129 versus 17 months, p < 0.001) between patients with intestinal type tumours and those with diffuse type tumours. On multivariate analysis, diffuse type histology was the only factor significantly associated with an R1 resection. In a multivariate Cox regression model, diffuse type histology was a significant adverse prognostic factor for overall survival. CONCLUSIONS: Striking differences were found between patients with diffuse type tumours and those with intestinal type tumours. These differences call for a differentiated approach in the potentially curative treatment of these two tumour types.

Mucosal uptake, mucosal transfer and retention of a therapeutic dose of iron.

The absorption of a pharmacological dose of iron was assessed by determination of mucosal uptake, mucosal transfer and retention of 33 mg (Fe(II) as ferrous sulphate and ferrous ascorbate. 20 subjects were studed in a cross-over trial, 11 with normal iron stores and 9 with iron deficiency according to the serum ferritin concentrations. The activity of 59Fe and 51Cr (administered as a non-absorbable indicator) was measured by whole-body counting. There was no difference in absorption between the two iron compounds in normal subjects. Absorption of ferrous ascorbate averaged 52% higher than ferrous sulphate in subjects with iron deficiency. The difference was the result of higher mucosal uptake, probably because oxidation of Fe(II) in the alkaline milieu of the intestine, which leads to formation of non-absorbable Fe(III) complexes, was prevented. The mucosal transfer fraction of both compounds was identical.

A cross-over comparison of the anti-clotting effects of three low molecular weight heparins and glycosaminoglycuronan.

1. The anti-clotting effects after intravenous administration of three low molecular weight (LMW) heparins, Fragmin (KABI 2165), Fraxiparine (CY 216), Clexane (PK 10169) and the LMW mixture of glycosaminoglycuronans Orgaran (Org 10172) were compared in a randomized cross-over study in 12 healthy male volunteers. 2. The time courses of the anti-Xa activity of Fragmin, Fraxiparine and Clexane (five subjects) were best fitted by a monoexponential function and had comparable half-lives of 1.9 h, 2.3 h and 2.8 h, respectively. The time courses of the anti-Xa activity of Orgaran and Clexane (four subjects) were described by a biexponential function with terminal half-lives of 56.8h and 27.7 h, respectively. They were longer than those of Fraxiparine and Fragmin. Orgaran injection was associated with a significantly smaller 'clearance' (0.8 +/- 0.2 l h-1) of the plasma anti-Xa activity compared with Fragmin (2.0 +/- 0.5), Fraxiparine (1.7 +/- 0.5) and Clexane (1.6 +/- 0.5). 3. In comparison with the three LMW heparins, the terminal half-life of plasma anti-IIa activity after Orgaran was longer and the 'clearance' of Orgaran was lower than that after Clexane. The area under the curve of the plasma anti-IIa activity after administration of Orgaran was negligible compared with that obtained after injection of the LMW heparins. 4. Orgaran caused the smallest and Fragmin the greatest prolongation of the activated partial thromboplastin time (Orgaran 5.8 +/- 1.2 s vs Fragmin 18.5 +/- 5.2 s) and the thrombin clotting time (Orgaran 2.9 +/- 1.7 s vs Fragmin 47.8 +/- 0.9 s).(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of Org 10172, a low molecular weight heparinoid, on antipyrine metabolism and the effect of enzyme induction on the response to Org 10172.

1. We have investigated the effect of repeated s.c. Org 10172 (a low molecular weight heparinoid; Lomoparan) treatment (1000 anti-Xa units twice daily for 5 days) on antipyrine (500 mg orally) metabolism, and the effect of enzyme induction by pentobarbitone (100 mg for 12 days) on the pharmacokinetics and pharmacodynamics of Org 10172 following an intravenous bolus injection of 3250 anti-Xa units. 2. Org 10172 treatment caused a small increase in the formation rates of all antipyrine metabolites (P less than 0.05), while the overall kinetics of antipyrine did not change significantly. 3. Oxidative enzyme induction by pentobarbitone, as demonstrated by an increased clearance of antipyrine, was associated with an increase in the area under the anti-thrombin activity vs time curve (P less than 0.05). No influence was seen on the kinetics of plasma anti-Xa and thrombin generation inhibiting (TGI) activity. 4. The pharmacodynamics of Org 10172, as determined by clotting tests, was not influenced by enzyme induction. 5. The clinical relevance of these observations is likely to be limited.

Interaction of ORG 10172, a low molecular weight heparinoid, and digoxin in healthy volunteers.

Potential pharmacokinetic and pharmacodynamic interactions between a new low molecular weight heparinoid Org 10172 (bolus injection of 3250 anti-Xa units) and digoxin (0.25 mg once daily for 8 days) were studied in 6 healthy male volunteers using an open, randomised three-way cross-over design. Digoxin produced a slight increase in clearance of anti-Xa activity from 4.3 to 4.8 ml.min-1, while plasma antithrombin and thrombin generation inhibiting (TG1) activity remained unchanged. Digoxin did not affect the actions of Org 10172 on the clotting tests. In the presence of Org 10172 there was a reduction in the AUC of digoxin during one dosing interval after the seventh digoxin tablet from 20 to 17 ng.ml-1.h, and a significant reduction in the average serum digoxin concentration. Since renal digoxin clearance was not significantly changed this probably might be due to a change in the non-renal clearance of digoxin. Atrio-ventricular node conduction, as measured by PR-time intervals, remained unchanged during all three treatments. In conclusion, although the pharmacokinetics of Org 10172 and digoxin were slightly changed by the combination, it is probably safe to administer Org 10172 and digoxin simultaneously. The clinical relevance of the slight decrease in plasma anti-Xa activity levels cannot yet be defined.

Pharmacokinetic considerations on Orgaran (Org 10172) therapy.

Pharmacokinetic investigations on Orgaran (Org 10172) have been conducted by monitoring the following biological effects: plasma anti-Xa, anti-IIa and IIa-generation-inhibiting (IIaGI) activities. In addition, a limited number of studies were conducted on the basis of concentrations of the No-affinity glycosaminoglyc(uron)an (NoA-GAG) fraction as determined by a competitive binding assay. In humans, widely different pharmacokinetic profiles for various biological effects were observed, with relatively short elimination half-lives for the anti-IIa and IIaGI activities of 4.3 +/- 3.5 and 6.7 +/- 3.2 h, respectively, but a relatively long elimination half-life of anti-Xa activity of 24.5 +/- 9.6 h. These differences in half-life mainly reflect differences in the rate of elimination of individual components of Orgaran. Rapid elimination of some of these components may explain why twice daily dosing is required for optimal thrombosis prophylaxis with Orgaran. In a comparative study in healthy male volunteers, the pharmacokinetics of the following low molecular weight heparin(oid)s were determined after intravenous administration: Orgaran (3,750 anti-Xa units), Fragmin (5,000 anti-Xa units), Fraxiparine (7,500 IC units) and Clexane (40 mg). Between these products, wide differences in pharmacokinetics were observed. Particularly, the half-lives of anti-Xa activity and IIaGI activity were much longer for Orgaran than for the other products. At the same time, a relatively low area under the curve of anti-IIa activity was observed. The absolute bioavailability of Orgaran following subcutaneous administration was determined on the basis of plasma anti-Xa and IIaGI activities and the NoA-GAG fraction concentrations. Absorption from subcutaneous tissues was found to be close to 100%, which is significantly higher than of heparin; a finding which indicates that the subcutaneous route is reliable for the administration of Orgaran. The elimination of Orgaran components occurs by renal and possibly non-renal routes. With respect to anti-Xa activity, about 50% of the total clearance can be accounted for by urinary excretion. Therefore, in severe renal failure, a reduction of the maintenance dose of Orgaran would seem to be indicated. Studies on the influence of enzyme induction as a result of treatment with pentobarbital suggest that the pharmacokinetics of Lomoparan are relatively insensitive to changes in hepatic function. In a number of studies, the influence of conditions such as age, body weight and drug interactions were studied. Generally, only minor changes in the pharmacokinetic parameters of Orgaran were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Gastrointestinal blood loss in haemodialysis patients during use of a low-molecular-weight heparinoid anticoagulant.

In a randomised cross-over study we assessed total blood loss in 14 dialysis patients using 59Fe as a marker for measurement in a whole-body counting system. In one period the patients received standard heparin, in the other ORG 10172, a new low-molecular-weight-heparinoid. Our results show no significant difference between the two study periods with regard to blood loss and dialyser blood retention. In some patients a delayed bleeding ('oozing') from the puncture site was noticed as a side-effect of treatment with the low-molecular-weight-heparinoid. We conclude that this heparinoid is effective as an anticoagulant in regular dialysis treatment, but it seems to have no advantage over standard heparinisation with regard to occult bleeding. This may be related to the prolonged plasma anti-Xa activity (30.8 h) of this compound compared to standard heparin in dialysis patients.

Studies of interaction of a low-molecular-weight heparinoid (Org 10172) with cloxacillin and ticarcillin in healthy male volunteers.

Pharmacokinetic and pharmacodynamic interactions between Org 10172 (intravenous bolus injection of 3,250 anti-Xa units), which is a low-molecular-weight heparinoid, cloxacillin (500 mg orally four times daily for 3 days), and ticarcillin (4,000 mg intravenously four times daily for 2 days) were evaluated in two separate studies with healthy male volunteers (n = 18). Both cloxacillin and ticarcillin caused a significant increase in elimination half-life of anti-Xa activity, i.e., from 31 +/- 10 to 54 +/- 23 h and from 27 +/- 6 to 42 +/- 13 h, respectively (P less than 0.05). Ticarcillin decreased clearance (11%) and increased apparent volume of distribution (35%) (P less than 0.05), while for cloxacillin, these differences did not reach statistical significance. These changes in disposition of Org 10172 by the penicillins were not accompanied by important pharmacodynamic changes as evaluated by coagulation tests, platelet aggregation, and bleeding time. Cloxacillin appeared to influence blood coagulation (prolongation of the activated partial thromboplastin time and shortening of thrombin time; P less than 0.05) and facilitated thrombin-induced platelet aggregation, which coincided with a shorter bleeding time during the combined treatment in comparison with the time during treatment with Org 10172 alone (P less than 0.05). In conclusion, the disposition of Org 10172 was slightly changed by cloxacillin and ticarcillin, and, unexpectedly, cloxacillin appeared to have mild procoagulant effects.

Safety and pharmacokinetics of the low molecular weight heparinoid Org 10172 administered to healthy elderly volunteers.

1. In a cross-over study a new low molecular weight heparinoid Org 10172 was administered to 12 elderly male and female volunteers. It was well tolerated and no adverse effects occurred. 2. The absolute bioavailability of Org 10172 as measured by plasma anti-Xa activity, glycosaminoglycuronans with no affinity to antithrombin III (NoA-GAG) and thrombin generation inhibiting activity approached 100% in both sexes. 3. The half-life of elimination of its anti-Xa activity (19.2 +/- 6.1 h) was similar to that found previously in young volunteers. Org 10172 was further characterised by a rapid disappearance from the circulation of its anti-thrombin activity (t1/2 1.8 +/- 0.6 h) and of the NoA-GAG (t1/2 3.5 +/- 2.1 h). 4. Its thrombin generation inhibiting activity was of intermediate duration (t1/2 elimination 6.2 +/- 4.0 h).

Interaction of the combined medication with the new low-molecular-weight heparinoid Lomoparan (Org 10172) and acenocoumarol.

A high intravenous dose of the low-molecular-weight heparinoid Lomoparan (Org 10172) was administered to 6 healthy males in a steady state of anticoagulation (Thrombotest) by acenocoumarol. Prothrombin time, activated partial thromboplastin time and Stypven time were prolonged to a degree which was greater than that expected on the base of the summation of the effects by each drug alone. This effect was observed for a period of up to 1 h. The Thrombotest was affected for up to 5 h after the intravenous administration of Org 10172, therefore it is deemed unsuitable for monitoring the combined effects of these two anticoagulants during this period. Acenocoumarol did not affect the pharmacokinetic parameters of Org 10172 with the exception of a slight reduction of the clearance of plasma anti-Xa activity.