Phase II study of cetuximab in combination with docetaxel in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck after platinum-containing therapy: a multicenter study of the Arbeitsgemeinschaft Internistische Onkologie.

BACKGROUND: Cetuximab and docetaxel have single-agent activity in squamous cell carcinoma of the head and neck (SCCHN). The efficacy of their combination was evaluated in platinum-pretreated patients with recurrent and/or metastatic SCCHN. PATIENTS AND METHODS: A total of 84 patients were treated with docetaxel 35 mg/m(2) weekly for a maximum of 6 cycles and concomitant cetuximab 250 mg/m(2) weekly until disease progression or unacceptable toxicity. The primary endpoint was the objective response rate and secondary endpoints included the response rate in relation to platinum sensitivity, progression-free survival (PFS), overall survival (OS) and toxicity. RESULTS: Nine (11%) patients achieved a partial response and 34 (40%) stable disease, resulting in a disease control rate of 51%. Response to treatment was 49% in previously platinum-sensitive and 50% in previously platinum-resistant disease. The median PFS was 3.1 months and the median OS 6.7 months. The most common grade 3 or 4 adverse events were mucositis (8%), pneumonia (8%), fatigue (8%) and skin reactions (14%). Sepsis occurred in 3 patients. CONCLUSION: Cetuximab plus docetaxel is an active treatment regimen with moderate toxicity in SCCHN patients. However, no superiority in comparison with monotherapy could be shown. Responsiveness and survival were independent of previous platinum sensitivity.

[Haemostatic testing prior to elective surgery? Yes!]. / Laboranalytischer Ausschluss einer hämorrhagischen Diathese vor elektiven Eingriffen? Ja!

Haemorrhagic disorders must be excluded prior to any operation or other invasive procedure that has the potential to involve serious bleeding. When assessing the individual risk of bleeding, screening tests of hemostasis must be combined with the patient's clinical history and symptoms, and any history of bleeding must be explored under direct medical supervision using a standardized questionnaire. However, this bleeding history is neither very specific, nor is it particularly sensitive. Screening tests that have been found to be useful include platelet count, activated partial thrombo plastin time (aPTT), prothrombin time (PT) and clottable fibrinogen. No reliable, sensitive and specific screening test is however available today to screen for platelet dysfunction or von Willebrand disease. A specialized coagulation laboratory should be involved when the bleeding history or laboratory screening indicate a potential haemorrhagic disorder.

Circulating microemboli in patients with myeloproliferative disorders.

Myeloproliferative disorders (MPD) are associated with an increased risk for thrombembolic events. In this study, we examined the prognostic value of transcranial Doppler (TCD) microemboli detection regarding clinical events and correlated TCD findings with results of blood cell counts and platelet flow cytometry to gain insight into the composition of circulating microemboli in these patients. In a cohort of 42 patients with MPD TCD microemboli detection was performed on a single occasion and correlated with thrombembolic events during a prospective follow up of 29.7 +/- 7.3 month. In all patients, a complete blood count and in 17 patients platelet flow cytometry were performed on the day of the TCD examination. Microembolic signals (MES) were recorded in 15 (35.7%) patients, however, without any correlation with the type of MPD, blood cell counts, or thrombembolic events [9 (21.4%)]. MES positive and negative patients did not differ regarding the levels of activated platelets, platelet microaggregates, or microparticles. We found a strong trend for higher rates of platelet-neutrophil conjugates in MES positive patients (P = 0.09). Detection of MES by TCD on a single occasion in MPD patients has only limited prognostic value. MES do not correlate with the type of MPD, nor blood cell counts. Flow cytometry suggests that MES in MPD may consist of platelet-neutrophil aggregates.

Upregulation of GP IIb/IIIa receptors during platelet activation: influence on efficacy of receptor blockade.

INTRODUCTION: GP IIb/IIIa inhibitor doses high enough to inhibit platelet macroaggregation may not completely prevent formation of microaggregates. Platelets contain an internal pool of GP IIb/IIIa receptors which externalizes with activation and supports microaggregation. This study assesses microaggregation in the presence of the two GP IIb/IIIa inhibitors abciximab and tirofiban. METHODS: Citrated whole blood was preincubated with abciximab (5 microg/ml), tirofiban (50 ng/ml), or saline as control and activated with TRAP (5 microM) or ADP (2 microM) at 37 degrees C under constant stirring. Microaggregate formation and receptor expression were determined with flow cytometry. RESULTS: Within few seconds after TRAP-activation the platelet count dropped from 266,000/microl to 20,000/microl, the number of microaggregates increased from 3700/microl to 10,100/microl and the mean number of GP IIb/IIIa receptors increased from 53,000 to 65,000/platelet. With TRAP+abciximab the platelet count dropped from 259,000 to 113,000/microl, microaggregates increased from 2500 to 9300/microl, GP IIb/IIIa receptors from 56,000 to 77,000/platelet. Platelet microaggregate formation was reversible. With TRAP and tirofiban platelet count dropped to only 190,000/microl, there was no increase in platelet microaggregates, receptors increased to 66,000/platelet. Platelet activation with ADP gave similar results. CONCLUSIONS: During the early phase of activation additional GP IIb/IIIa receptors externalize to the platelet surface. Abciximab does not block these new receptors sufficiently to prevent microaggregate formation. However, the number of unblocked receptors is not high enough to maintain a stable aggregate, microaggregation is reversible. With tirofiban there is no microaggregate formation, possibly because the inhibitor rapidly binds to newly externalized receptors.

[Initiation of oral anticoagulant treatment: comparison between different dosage regimens of warfarin and phenprocoumon]. / Initialphase der oralen Antikoagulanzientherapie: Vergleich verschiedener Dosierungen von Warfarin und Phenprocoumon.

A high "loading dosage" is often given during initiation of oral anticoagulant treatment in order to reach sufficient anticoagulation within short time. Increased bleeding risk as well as a transient prothrombotic tendency are complications of this treatment schedule. The aim of our study was to find proper dosage regimens of phenprocoumon and warfarin allowing initiation of oral anticoagulant treatment in a short time. For 50% of the patients 7.5 mg warfarin daily resulted in stable INR values within 4 days. Patients receiving higher (10 mg) or lower (5 mg) daily dosages of warfarin or 6 or 9 mg phenprocoumon daily during the first days of therapy reached the therapeutic range significantly later. Furthermore, no significant differences of prothrombin fragment F 1+2 were observed, indicating that no enhanced thrombin formation occurred. Thus, initiation of oral anticoagulant treatment using 7.5 mg warfarin daily is a simple and safe dosage regimen.

Comparison of GP IIB/IIIA inhibitors and their activity as measured by aggregometry, flow cytometry, single platelet counting, and the rapid platelet function analyzer.

BACKGROUND: GP IIb/IIIa inhibitors have primarily been used short-term e.g., during PTCA. They failed to show clinical benefit during long-term therapy. One reason might be the absence of a method to monitor inhibitor activity. This study compared platelet aggregometry, the rapid platelet function analyzer (RPFA) test, single platelet counting, and flow cytometric determination of receptor occupancy to measure GP IIb/IIIa-receptor inhibitor activity. METHODS: Increasing doses of abciximab, tirofiban, and eptifibatide were added to whole blood in vitro. Whole blood was used for the RPFA, for single platelet counting and flow cytometry. Platelet rich plasma was prepared for aggregometry. RESULTS: The correlation between aggregometry and RPFA results was linear for abciximab and eptifibatide. Tirofiban was a stronger inhibitor with the RPFA (IC(50) 7.7nM) than with aggregometry (IC(50) 19.6nM). The single platelet counting technique showed that even supratherapeutic concentrations of all three inhibitors could not completely suppress microaggregation. Abciximab concentrations that were equipotent to tirofiban with aggregometry were less potent with regards to the inhibition of microaggregation. This difference was more pronounced with TRAP induced microaggregation than with ADP. The flow cytometric receptor occupancy test showed that occupancy was 95% with 5 microg/ml abciximab and almost 97% with 10 microg/ml. Tirofiban reached a maximum receptor occupancy of 56%, eptifibatide 64%. CONCLUSIONS: While aggregometry is time consuming the RPFA provides results fast and with little variability. There is still a discrepancy between aggregometry and RPFA results for tirofiban. The single platelet counting technique detects the inhibition of microaggregation the relevance of which for the clinical outcome is not known. The flow cytometric receptor occupancy assay is best suited for abciximab.

Perioperative management of a patient with Fechtner syndrome.

Fechtner syndrome is a rare type of familial thrombocytopenia associated with large platelets, leukocyte inclusions, and features of Alport's syndrome. The bleeding tendency is usually mild, but severe hemorrhages have been reported. This is the case of a patient with Fechtner syndrome who was scheduled to undergo tonsillectomy. The patient had a history of easy bruising in childhood and a markedly prolonged bleeding time. Administration of DDAVP led to normalization of the bleeding time, and the patient underwent surgery without complications. With this approach the use of platelet concentrates could be avoided.

[The Wiskott-Aldrich syndrome in adulthood]. / Wiskott-Aldrich-Syndrom im Erwachsenenalter.

HISTORY AND ADMISSION FINDINGS: A 24-year-old man with thrombocytopenia was referred for surgical resection of a bleeding polyp of the sigmoid. Examination showed a small haematoma and petechiae on both lower legs. The patient reported that several male family members also had a thrombocytopenic bleeding tendency. INVESTIGATIONS: Laboratory tests revealed thrombocytopenia (4000 platelets/ml, with small platelets: mean platelet volume [MPV] 5.6 ml). Serum immunoglobulins were normal. A mutation in the Wiskott-Aldrich (W-A) protein gene (intron 7 + 5 G-->A) was demonstrated both in the patient and his 26-year-old brother. DIAGNOSIS, TREATMENT AND COURSE: The diagnosis of W-A syndrome was made and, with perioperative administration of platelets, the polyp was resected without complication. CONCLUSION: Most patients with the W-A syndrome die by the time they are aged 10 years, unless appropriate treatment is given. This patient and his brother had a mutation of the W-A protein gene that unusually was in an intron rather than in an exon. Structurally normal W-A proteins were still being formed. This may explain the mild course and late onset of the disease.

Effect of glycoprotein IIb/IIIa inhibitors on CD62p expression, platelet aggregates, and microparticles in vitro.

Flow cytometry can detect platelet activation (CD62p), aggregate formation, microparticle formation, and glycoprotein IIb/IIIa (GP IIb/IIIa) receptor occupancy in one sample at the level of single particles. We studied the effect of GP IIb/IIIa inhibitors on platelet activation with flow cytometry in vitro. Citrated whole blood was incubated with increasing concentrations of three different GP IIb/IIIa inhibitors (c7E3, DMP728, XJ757), then thrombin or adenosine diphosphate (ADP) was added, and after 1 minute the sample was fixed. Samples with thrombin but without c7E3 had a decrease in platelet count, from a mean of 260,000 platelets/microl to 56,000 platelets/microL, and aggregates increased. Samples with concentrations of c7E3 that resulted in 80% or more receptor blockade had no decrease in platelet count, and no aggregates were formed, but the number of CD62p-positive single platelets increased from 1200 to 7400 platelets/microL. The two other inhibitors (DMP 725, XJ757) or ADP instead of thrombin gave similar results. Microparticle formation did not change with platelet activation in the presence of a GP IIb/IIIa inhibitor. With small inhibitor doses resulting in <80% receptor blockade, the number of aggregates did not change or was even higher than that in samples without inhibitor. GP IIb/IIIa inhibitors do prevent aggregate formation but they do not prevent activation of platelets. With GP IIb/IIIa inhibition, more activated single platelets remain in the blood. One may expect an increasing number of circulating, activated platelets with the use of GP IIb/IIIa inhibitors.

Pharmacology of warfarin and clinical implications.

The history of oral anticoagulants started in the 1920s in North Dakota and Alberta when a new type of hemorrhagic disease struck cattle in these areas. The group of Karl Paul Link finally succeeded in isolating the causative agent, dicumarol. It was not before the 1940s, that dicumarol or its derivatives were introduced to medicine. Acenocoumarol, phenprocoumon, and warfarin are the most commonly used oral anticoagulants. There is no known difference in the pharmacodynamic activity of these agents on the vitamin K metabolism. They are completely absorbed from the gastrointestinal tract and are firmly bound to plasma albumin and metabolized in the liver. The different elimination half-lives of the coumarins have several implications for patient management. Absorption, protein binding, and anticoagulant activity of oral anticoagulants are affected in many different ways. Also the different pharmacological properties of coumarins require different strategies in the clinical management of patients.

Quantitative assessment of platelets, platelet microparticles, and platelet aggregates with flow cytometry.

With fluorescent beads it has become possible to determine absolute numbers of cells in a given sample instead of relative percentages on a standard flow cytometer. This study assesses the ability to count platelets, microparticles, and aggregates with a flow cytometer. Whole blood was stimulated with 0.1 U thrombin per milliliter. Platelet and microparticle counts decreased, while the number of aggregates increased. Unactivated whole blood was diluted with buffer and showed a corresponding decrease in the concentration of platelets, microparticles, and aggregates. The platelet count on the flow cytometer was always in good correlation with counts on an automated blood analyzer. Only the cytometer, and not the automated analyzer, was able to detect and count microparticles and aggregates. In highly diluted samples of unactivated whole blood there was a spurious relative increase in CD62p-positive platelets because of a surplus of anti-CD62p antibodies and a relative increase in microparticles. Flow cytometry is a valuable method for counting platelets, aggregates, and microparticles in unstimulated and activated blood samples. If the platelet count changes and drops to less than 50% of the count for which the amount of antibody and the cytometer settings have initially been adjusted, care has to be taken to avoid misinterpretation.

Relative and absolute changes of activated platelets, microparticles and platelet aggregates after activation in vitro.

Standard flow cytometers provide relative numbers of activated platelets, microparticles, and platelet aggregates. With fluorescent beads it is now possible to determine absolute numbers. Whole blood and platelet-rich plasma were incubated with agonists (ADP, collagen, thrombin). CD62p expression, microparticle and platelet aggregate formation were measured. Flow-Count Fluorospheres((R)) were added to calculate absolute concentrations. After activation there was an increase in the percentage of CD62p-positive platelets. However, the total number of platelets decreased and therefore the absolute number of CD62p-positive platelets did not increase but decreased. The number of CD62p-positive platelets decreased not as much as the number of CD62p-negative platelets, which explains why the relative percentage of CD62p-positive platelets increased. A similar increase in percent and decrease in absolute counts was found for microparticles. Platelet aggregates increased both in relative and absolute numbers. These results suggest that the detection of activated platelets by flow cytometry has to be complemented by the determination of the absolute concentrations to avoid misinterpretation.

Reactivity of platelets from healthy individuals and from patients with hematologic diseases following incubation with pegylated recombinant human megakaryocyte growth and development factor in vitro.

Platelets express the receptor for thrombopoietin. It is possible that thrombopoietin modulates platelet reactivity. We examined the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet activation in vitro using flow cytometry. We compared samples from healthy individuals and from patients with various hematologic diseases (AML, myeloma, postchemotherapy). Citrated whole blood was incubated with PEG-rHuMGDF (10 or 100 ng/ml), then a mild stimulus was added (0.1 U thrombin/ml). Blood from healthy individuals showed a significantly higher degree of platelet activation (CD62p expression), microparticle generation, and aggregate formation after incubation with PEG-rHuMGDF+thrombin versus thrombin alone (p < 0.05). However, this difference could not be shown for platelets from patients with thrombocytopenia or other hematologic diseases. The use of PEG-rHuMGDF should be safe and not cause an additional risk of thromboocclusive disease in these patients.

[Visceral leishmaniasis with an unusually long incubation time]. / Viszerale Leishmaniose mit ungewöhnlich langer Inkubationszeit.

HISTORY: A 25-year-old woman of Yugoslavian origin came to Germany two years before and did not leave Germany since this time. She developed a phlebothrombosis during pregnancy which was treated surgically and with subsequent heparinisation. The pregnancy had to be terminated by section because of abnormal liver functions and increased blood pressure. These values returned to normal within two months. Further tests again showed raised liver function tests (GOT 57 U/l, GPT 71 U/l) and antibodies against smooth muscle and actin. Autoimmune hepatitis was diagnosed and prednisolone given (100 mg daily). In the subsequent 4 months the patient progressively lost more weight and a pancytopenia developed. Suspected of having a systemic haematological syndrome she was admitted to hospital. FINDINGS: Physical examination was unremarkable except for hepato- and splenomegaly (spleen 15.6 cm in diameter by sonography). Laboratory tests showed hypergammaglobulinaemia (50 g/l, 53%), increased WBC count, as well as decreased haemoglobin concentration and platelet count (900 WBC/microliter, Hb 10.9 g/l, 146,000 platelets/microliter). Bone marrow puncture unexpectedly revealed a large number of Leishmania donovani. TREATMENT AND COURSE: Five-valent antimony was administered (sodium stibogluconate 20 mg/kg daily intravenously as bolus for 14 days). She has been free of symptoms since then (follow-up period of one year). CONCLUSION: Visceral leishmaniasis is a rare disease in Europe. Incubation periods of several years have been reported and the infection can be easily mistaken for other chronic liver disease, in this case for an autoimmune hepatitis. Leishmaniasis should be included in the differential diagnosis of unclear liver disease if there is a suggestive history (country of origin or journey into an endemic area).

Comparison of beta-thromboglobulin, flow cytometry, and platelet aggregometry to study platelet activation.

This study compares granule membrane protein (GMP)-140 expression measured by flow cytometry, release of beta-thromboglobulin (beta-TG), and platelet aggregometry as markers of platelet activation in vitro. Whole blood was activated with different concentrations of thrombin. There was a significant increase in beta-TG plasma levels after stimulation with 0.01 and 0.04 U thrombin/ml. There was also an increase in GMP-140 expression, but interindividual variability was high. Aggregometry of platelet-rich plasma did not detect platelet activation and formation of platelet aggregates with 0.05 and 0.1 U thrombin/ml, while flow cytometry showed an early and significant increase of GMP-140 expression with these doses. Beta-TG release is a more sensitive marker of platelet activation than GMP-140 while flow cytometry is easier to perform and less susceptible to artifacts.