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Osteonecrosis occurs as an acute and long-term serious side effect in children, adolescents, and adults with acute lymphoblastic leukemia. It is associated with severe pain and reduced mobility, ultimately leading to joint destruction and significant long-term morbidity. The cumulative incidence ranges from 11 to 20% in adolescents and young adults. In symptomatic patients, multiple joints are frequently affected, which in turns poses a risk factor for the development of severe osteonecrosis. The genesis of leukemia-associated osteonecrosis is multifactorial. Risk factors include the use of corticosteroids and asparaginase. These exert their effects on the blood supply to the bone through hypercholesterolemia, hypertriglyceridemia, and hypertension. Bacteriemia, genetic susceptibility, and stem cell transplantation pose additional risk factors. The treatment of osteonecrosis is challenging and not evidence based. Preventive measurements have as yet mainly been tested in preclinical models.Osteonekrosen treten als schwerwiegende Komplikation bei Kindern, Jugendlichen und Erwachsenen mit akuter lymphoblastischer Leukämie akut unter Therapie aber auch als Spätfolge auf. Diese gehen mit starken Schmerzen, Einschränkungen der Mobilität, Gelenkdestruktion und erheblicher Langzeitmorbidität einher. Die kumulative Inzidenz bei Jugendlichen und jungen Erwachsenen beträgt 11–20 %. Bei symptomatischen Patienten sind häufig multiple Gelenke betroffen, die multiple Gelenkbeteiligung ist ein Risikofaktor für einen schweren Verlauf. Leukämieassoziierte Osteonekrosen haben eine multifaktorielle Genese. Risikofaktoren umfassen v. a. die Gabe von Kortikosteroiden und Asparaginase. Diese verursachen eine Hypertriglyzeridämie, Hypercholesterolämie und arterielle Hypertonie, die die Blutzufuhr zum Knochen beeinträchtigen können. Weitere Risikofaktoren sind Bakteriämien, genetische Faktoren und eine Stammzelltransplantation. Die Therapie ist herausfordernd und bisher nicht evidenzbasiert, präventive Ansätze wurden bisher vor allem in präklinischen Modellen getestet.

Acrylamid als potentiell krebserregende, mutagene und reproduktionsschädigende Prozess-kontaminante mit einer relativ geringeren Sicherheitsmarge der Exposition (Margin of Expo-sure (MOE)) stellt eine ernstzunehmende Bedrohung für die öffentliche Gesundheit dar. Daher gibt es Bemühungen, die Acrylamidkonzentration in Lebensmitteln so niedrig wie möglich zu halten. Erhitzte Kartoffelerzeugnisse wie Pommes frites stehen hierbei aufgrund der hohen Konzentration an freiem L-Asparagin besonders im Fokus. Die Zugabe einer Asparaginase (PreventASe L von DSM®) zur Verminderung von Ac-rylamid in tiefgekühlten (TK) Pommes frites wurde im industriellen Maßstab untersucht. Der Einsatz wurde an die komplexen industriellen Prozessparameter angepasst und ver-schiedene Enzymkonzentrationen (0; 0,1; 0,3; 0,5 und 1,0 Gew.-%) wurden getestet. Es erfolgte eine Verminderung der Acrylamidkonzentration von bis zu 59 %. Ein Einfluss auf die Farbe (Agtron-Bestimmung) wurde ausgeschlossen. Die sensorische Bestimmung eines sauren Geschmacks durch gebildete L-Asparaginsäure wurde durch zehn Panelisten als nicht signifikant (α= 0,05) eingestuft. Das Ergebnis beschreibt die erste erfolgreiche Be-handlung von TK-Pommes frites im industriellen Maßstab ohne Einfluss auf die sensori-schen Eigenschaften der Pommes frites. Die Wirkung von Phenolsäuren auf die Bildung von Acrylamid wird kontrovers diskutiert. Zur Bestimmung ihrer Wirksamkeit wurden Gemische aus den Phenolsäuren p-Cumar-, Kaffee- und Ferulasäure sowie Zimtsäure als Kontrolle mit Acrylamid erhitzt und auf eine mögliche Additionsreaktion untersucht. UPLC-HR-MS-qToF Messungen zeigten eine einfach addierte Bildung unter Wasserabspaltung bei allen vier Phenylacrylsäuren. Fragmen-tanalysen deuteten auf eine oxa-MICHAEL-Addition hin. Der Beweis erfolgte mittels Struk-turanalyse über NMR Spektrometrie. In den Modellsystemen wurden 3-Amino-2-oxo-phenylacrylate nachgewiesen. In Untersuchungen von realen Proben aus Kartoffeln und Süßkartoffeln konnte keine Adduktbildung nachgewiesen werden. Aufgrund ebenfalls geringer Acrylamidkonzentration wurde eine Konzentration unter der Nachweisgrenze diskutiert.

The treatment of acute lymphoblastic leukemia (ALL) in childhood and adolescence achieves nowadays cure rates of more than 80%. The detection of minimal residual disease (MRD) via molecular genetic methods provides - in comparison with conventional clinical and biological parameters - much more sensitive approaches to monitor individual treatment response. Here we will discuss the molecular background and technical developments in the framework of the BFM-study group.

The bacterial enzyme asparaginase is an important antineoplastic drug used for the treatment of a variety of lymphoproliferative disorders and lymphomas, in particular acute lymphoblastic leukemia. Its capacity to modulate the plasmatic coagulation is a known clinical complication. As a side effect the asparaginase might pathologically activate or inactivate the intrinsic coagulation. The aim of the present research work is to analyze the action of different asparaginase drugs on the plasmatic thrombin generation in 118 individual normal plasma samples using the new ultra specific, ultra-sensitive and precise thrombin generation test recalcified coagulation activity assay (RECA). Three different asparaginases were utilized for the laboratory analytical experiments; two native Escherichia coli (E. coli) asparaginases and an E. coli asparaginase coupled to polyethylene glycol (PEG-asparaginase). The two native E. coli asparaginases only differed in their time of production (the first lot produced would be referred to as the old lot, whereas the second lot produced would be called the new lot). The RECA diagnosed the coagulation modulating (especially the prothrombotic) power of the present asparaginases. Therefore, the approximate (approx.) 200 % stimulatory concentration (SC200) or the approx. 50 % inhibitory concentration (IC50) was determined. The SC200 is the plasmatic asparaginase concentration that results in 200 % of the original thrombin generation. The IC50 is the asparaginase concentration that results in only 50 % of the original thrombin generation. The supplemented plasmas became procoagulant or anticoagulant, or stayed resistant to hemostasis modulation by asparaginase. The old lot asparaginase was procoagulant in every plasma sample with approx. SC200-values of 5.8 ± 5.4 unit per milliliter (U/ml) (mean value ± 1 standard deviation) [MV ± 1 SD]. By contrast, only 51 % of the plasma samples that were supplemented with the new lot asparaginase became procoagulant, 24.5 % of the supplemented plasmas had a combination of SC200 and IC50 (depending on coagulation reaction time), 21 % had just an IC50 (MV = 6 U/ml) and 3,5 % of the plasmas were resistant towards modulation of coagulation. This means that the new lot has less thrombotic potential compared to the old one. In contrast the analyzed IC50-values point out that it could have an anticoagulant effect on the plasmatic blood coagulation of the individual patient, which could lead to bleeding complications. The PEG-asparaginase has been described as a modified molecule of asparaginase with less side effects than unmodified native asparaginase. The comparative plasma samples supplemented with both asparaginase preparations demonstrated no difference between the native E. coli asparaginase and the PEG-asparaginase with regard to the blood coagulation. The approx. SC200-values of both asparaginases correlated with a correlation coefficient greater than 0.9 . This high correlation and the similar mean values indicated, that respective plasmatic thrombin generation the rather expensive modified PEG-asparaginase behaved similar as the native asparaginase. Thus the hemostatic system of every patient responds individually to certain asparaginase preparations. Using the RECA, the patient’s individual coagulation triggering response to asparaginase can be previewed and the drug dosage could be adjusted: it is suggested to diagnose the patient´s individual coagulation response to bacterial asparaginase prior to and during the administration of the drug. Patients with an increased risk for asparaginase-induced thrombosis should be treated with prophylactic to therapeutic concentrations of low molecular weight heparin (LMWH)., Das bakterielle Enzym Asparaginase ist ein sehr effektives Zytostatikum in der Therapie lymphoproliferativer Erkrankungen. Die Eigenschaft des Enzyms, die plasmatische Blutgerinnung zu verändern, ist eine bekannte klinische Komplikation. Nebenwirkungen der Asparaginase können sowohl Blutungen als auch disseminierte intravaskuläre Thrombosen sein. Das Ziel der vorliegenden laboranalytischen Arbeit war es, die Auswirkungen unterschiedlicher Asparaginasen auf die plasmatische intrinsische Thrombin-Generierung in 118 individuellen normalen Plasmaproben mittels eines neuen hochsensitiven, hochspezifischen Thrombin-Generierungstests, dem „recalcified coagulation activity assay“ (RECA) zu bestimmen. Für die Versuche wurden drei verschiedene Asparaginasen verwendet; zwei native E. coli Asparaginasen, die sich nur in ihrem Herstellungs- datum unterschieden (alte Charge/neue Charge) und eine modifizierte Form der Asparaginase, die Polyethylenglykol (PEG)-Asparaginase, eine E. coli Asparaginase gekoppelt an Polyethylenglykol. Mit Hilfe des RECA wurden die Auswirkungen (besonders die prothrombotische) der jeweiligen Asparaginasepräparation auf die plasmatische Gerinnung ermittelt. Dafür wurde für jedes normale Plasma die approximative 200 % stimulatory concentration (approx. SC200) oder die approximative 50 % inhibitory concentration (approx. IC50) bestimmt. Die approx. SC200 ist die Asparaginasekonzentration, bei der 200 % der Thrombin-Generierung von unsupplementiertem Plasma erreicht werden. Die approx. IC50 ist die Asparaginase-Konzentration, bei der nur noch 50 % der Thrombin-Generierung von unsupplementiertem Plasma erreicht werden. Die supplementierten Plasmen verhielten sich prokoagulatorisch, antikoagulatorisch oder Gerinnungs-resistent hinsichtlich der Hämostase-modulierenden Eigenschaft der Asparaginase. Bei der alten Charge Asparaginase zeigten alle Proben uneingeschränkt ein prothrombotisches Verhalten mit approx. SC200-Werten von 5,8 ± 5,4 U/ml (Mittelwert ± 1 Standardabweichung; Bereich: 1 - 25 U/ml). Im Gegensatz dazu demonstrierten bei der neuen Charge nur 51 % der untersuchten Plasmen ein prothrombotisches Verhalten, 24,5 % besaßen eine Kombination aus IC50 und SC200 (abhängig von der Gerinnungs-Reaktions-Zeit), 21 % der Proben verfügten über eine alleinige IC50 (Mittelwert = 6 U/ml) und 3,5 % der Plasmen erwiesen sich als gerinnungs- modulationsresistent. Dies bedeutet, dass die neue Charge Asparaginase im Vergleich zur alten weniger thrombogen wirkt. Hingegen deuten die ermittelten IC50-Werte jedoch an, dass die neue Charge empfindliche Plasmen stark antikoagulieren könnte. Die PEG-Asparaginase als modifizierte Form der nativen E. coli Asparaginase wird als weniger nebenwirkungsreich beschrieben. Hinsichtlich der Gerinnung ergab sich jedoch kein signifikanter Unterschied zwischen der nativen E. coli Asparaginase und der PEG-Asparaginase. Beide modulierten gleichermaßen die Gerinnung. Die approx. SC200-Werte von beiden Asparaginasen korrelierten mit einem Korrelationskoeffizienten größer 0,9. Diese hohe Korrelation und die ähnlichen Durchschnittswerte verdeutlichen, dass sich die (teurere) PEG-Asparaginase bezüglich der plasmatischen Thrombin-Generierung ähnlich wie die native E. coli Asparaginase verhielt. Die Ergebnisse zeigen, dass das Hämostase-System jedes Patienten individuell empfindlich auf die jeweilige Asparaginasepräparation reagiert. Mit Hilfe des RECA kann die individuelle prothrombotische Antwort des intrinsischen Gerinnungssystems jedes einzelnen Patienten auf Asparaginase vor Gabe bestimmt werden. Es sollte der differenzierte Gerinnungsstatus des individuellen Patienten und seine Veränderung durch Asparaginase in vitro vor und während der Therapie ermittelt werden. So kann insbesondere eine prokoagulatorische Tendenz des Zytostatikums auf das Patientenplasma diagnostiziert werden. Dies erlaubt vor Asparaginase-Gabe Risikopatienten zu erkennen und ggf. mit niedermolekularem Heparin prophylaktisch stärker entgegenzusteuern.

In dieser Untersuchung wurden Methoden zum Nachweis von drei nicht-synonymen SNPs im Asparaginsynthetase-Gen etabliert und deren Vorkommen untersucht. Mit Hilfe von Restriktionsenzymen wurden die SNP rs1049674 und rs3179866 des Gens der AS untersucht. Der SNP rs2070159 wurde mittels Allelspezifischer-Real-Time-PCR analysiert. Das Untersuchungsmaterial entstammte 36 pädiatrisch hämato-onkologischen Patienten, die unter ASPase-Therapie teilweise Nebenwirkungen erlitten, acht leukämischen Zelllinien und 31 Blutspendern. Die SNPs rs2070159 und rs3179866 ließen sich in keiner der Proben nachweisen. Der SNP rs1049674 war im hämato-onkologischen Kollektiv zu 44 %, im Blutspenderkollektiv zu 48 %, im Nebenwirkungskollektiv zu 60 % und im nebenwirkungsfreien Kollektiv zu 20 % vorhanden. Eine Korrelation von ASPase-Resistenz, AS-Expressionslevel und SNPs konnte nicht gefunden werden. Die Häufung des SNP rs1049674 im Nebenwirkungskollektiv sollte anhand einer größeren Fallzahl überprüft werden.

The enzyme L-asparaginase is a crucial component in the treatment of acute lymphoblastic leukaemia (ALL). As all asparaginases in clinical use are derived from microorganisms (with E.coli being the most frequent one), immunological reactions are the most important adverse events associated with asparaginase treatment. They range from clinically severe allergic shocks to silent enzyme inactivation without any clinical symptoms with the latter being particularly problematic in treatment schemes. For improving therapy success it is essential to identify patients prone to theses severe problems in advance. To date there is no such test being specific and sensitive enough to fulfil the purpose. Based on the assumption that the built antibodies are specific for epitopes, a lot of research has been done to classify the localisation within the enzyme more precisely. Concordant with previous results, the method used in this experiment (SPOTs Kit, enables synthesis of peptides on a cellulose membrane) showed the dominant sequence SVFDTLA (residues 252-258) when testing with a commercially available rabbit anti-E.coli-asparaginase antiserum but not with the serum of two healthy subjects. Considering the spatial position within the molecule and accessibility parameters, this sequence is highly likely to play a decisive role for immunological reactions as it blocks the enzymatic loop and thus the activity of the whole enzyme. The results achieved confirm the direction of research and help on the way of figuring out an epitope-specific assay. For statistically significant assertions further research has to follow., L-Asparaginase ist ein bakterielles Enzym, das erfolgreich in der Behandlung akuter lymphatischer Leukämien sowie einiger maligner Lymphome eingesetzt wird. Das Enzym bewirkt über eine hydrolytische L-Asparagin-Spaltung mit konsekutiver Verminderung der AS-Konzentration im Serum, dass Tumorzellen, welche essentiell auf das Vorhandensein der AS Asparagin angewiesen sind, keine weitere Möglichkeit zur Zellteilung mehr haben. Neben diesem im Rahmen der Chemotherapie erwünschten Effektes kommt es jedoch auch zu ungewollten Nebenwirkungen und Überempfindlichkeiten. Besonders letztere, welche von schweren allergischen Schocks bis hin zu stummer Enzyminaktivierung ohne klinische Zeichen reichen können, stellen medizinisch ein großes Problem dar. Gelänge es, die potentiell für solche Reaktionen prädisponierten Patienten frühzeitig zu erkennen, könnte so eine deutliche Verbesserung des Therapieerfolgs erzielt werden. Die bisher verwendeten immunologischen Nachweistests werden diesem Anspruch jedoch aufgrund zu geringer Sensitivität und Spezifität nicht gerecht. Auf der Annahme basierend, dass es sich bei den gebildeten AK um epitopenspezifische handelt, begannen eine Reihe von Forschungsarbeiten zur genaueren Lokalisation selbiger innerhalb des Enzyms. Als Grundlage diente hierbei unter anderem die mithilfe eines Restriktionsverdaus gewonnene Erkenntnis, dass eine Antikörper-Population bei vorzeitiger Enzyminaktivierung vornehmlich gegen das C-terminale Fragment (237-326) gebildet wird, und die Tatsache, dass auch das Phage Display in etwa zwei Drittel der Fälle eine Identifikation der Sequenz 252-258 zeigte. Das in der vorliegenden Arbeit verwendete SPOTs-Kit (kombinatorische Peptidsynthese auf Zellulose-Membran) zeigte ebenfalls erneut diese dominante Sequenz (SVFDTLA) bei Verwendung von käuflich erworbenem Anti-Asparaginase-Serum, nicht jedoch bei zwei Proben von gesunden Probanden. Unter Berücksichtigung der räumlichen Lage jener Sequenz im Molekül scheint diese tatsächlich eine entscheidende Rolle bei der immunologischen Reaktion der stummen Inaktivierung zu spielen, da die Bindung eines AK an eines der entsprechenden Epitope mit hoher Wahrscheinlichkeit die Bewegung der Enzymschleife und somit die Aktivität blockiert. Die gewonnenen Ergebnisse bestärken die bisher eingeschlagene Forschungsrichtung und liefern weitere Anhaltspunkte in Richtung Entwicklung eines Epitopen-spezifischen Assays. Um statistisch signifikante Aussagen treffen zu können, müsste jedoch an entsprechenden Stichproben weiter untersucht werden.

L-Asparaginase ist ein bakterielles Enzym, das seit über 30 Jahren im Rahmen einer Polychemotherapie zur Behandlung akuter lympha-tischer Leukämien und einiger maligner Lymphome eingesetzt wird. Asparaginase katalysiert die Hydrolyse von L-Asparagin und bewirkt auf diese Weise eine Depletion des Asparaginspiegels im Plasma. Dadurch wird die Konzentration der für die Tumorzellen essentiellen Aminosäure so gering gehalten, so dass eine weitere Replikation der malignen Zellen nicht mehr möglich ist. Erhebliche Komplikationen im Rahmen der Behandlung mit Aspara-ginase stellen zum einen die unterschiedlich ausgeprägten Überem-pfindlichkeitsreaktionen und zum anderen die stumme Inaktivierung des Enzyms dar. Durch eine Vorhersage einer möglichen allergischen Reaktion bzw. durch die Möglichkeit, eine vorzeitige Inaktivierung des Enzyms zu er-kennen, könnte eine deutliche Verbesserung des Therapieerfolges er-zielt werden. Der bisher in der Klinik eingesetzte immunologische Test zum Nach-weis von gebildeten Antikörpern, die möglicherweise für eine Überem-pfindlichkeitsreaktion bzw. für die Inaktivierung des Enzyms verant-wortlich sind, ist auf Grund zu geringer Sensitivität und Spezifität pro-gnostisch nicht verwertbar. Von der Hypothese ausgehend, dass es sich bei der Antikörper-bildung um epitop-spezifische Antikörper handelt, wurden zunächst, ohne Kenntnisse über die genaue Lokalisation der Epitope, durch Re-striktionsverdau fünf Asparaginasefragmente erzeugt. Der Einsatz die-ser Fragmente mit bekannter Aminosäuresequenz als Antigen führte zu der Erkenntnis, dass bei Patienten, bei denen eine vorzeitige In-aktivierung des Enzyms im Serum festgestellt worden war, eine bestimmte Antikörperpopulation, nämlich gegen das C-terminale Frag-ment (237-326), gebildet wird. Eine eindeutige Korrelation zwischen Antikörpertitern gegen eines der anderen Fragmente und einer schnellen Inaktivierung , konnte nicht festgestellt werden. Diese Ergebnisse liefern eine vielversprechende Grundlage für die Weiterentwicklung des Assays durch genaues Epitop-Mapping, so dass die für immunologische Tests eingesetzten Fragmente, in Zukunft noch selektiver gestaltet werden können. Um statistisch signifikante Aussagen machen zu können, sind noch weitere Tests an explizit für diese Fragestellung gesammelten Serum-proben notwendig., L-asparaginase is a bacterial enzyme that has been used for over thirty years for polychemotherapy of acute lymphatic leukaemia and some malignant lymphomas. Asparaginase catalyzes the hydrolysis of L-asparaginase, which leads to depletion of asparagine in blood plasma. Since this amino acid is essential for tumor growth, the low concentration of asparagine consequently suppresses the replication of malignant cells. Major complications during therapy with asparaginase are hypersensitivity reactions and a so-called silent inactivation of the enzyme. Prediction of a potential hypersensitivity reaction or a silent inactivation would lead to a significant improvement in therapy with asparaginase. The immunological test that has been used to detect serum antibodies that might be responsible for hypersensitivity reactions or silent inactivation shows poor sensitivity and specificity. This test is therefore not helpful for predicting complications of aspariginase therapy. Based on the hypothesis that the antibodies are epitope specific, asparaginase-DNA was digested without knowledge of the precise location of the epitopes to obtain five asparaginase fragments with a known amino acid sequence. The use of these fragments as antigens allowed identification of an early antibody population against the c-terminal fragment (237-326) in patients with silent inactivation. A correlation between antibody titer against one of the other fragments and silent inactivation could not be found. These results establish a promising basis for further development of this assay. By performing distinct epitope mapping, the fragments used for immunological tests will be more selective in the future. Further tests in patients receiving aspariginase therapy will be necessary to statistically verify the predictive value of this new approach.

L-Asparaginase ist Bestandteil der Therapie der ALL und des NHL. Es katalysiert die Hydrolyse von L-Asparagin . Normale Körperzellen besitzen Asparagin Synthetase (ASYN), die die Synthese des L-Asparagins ermöglicht. Eine erhöhte Expression führt zur Resistenz gegenüber L-Asparaginase. Für eine Subgruppe der ALL (TEL/AML 1 pos.) konnte im MTT-Test eine höhere Sensitivität gegenüber L-Asparaginase nachgewiesen werden. Die Bestimmung der ASYN-mRNAKonzentration (RT-PCR) in Subgruppen mit/ohne Translokation konnte bestätigen, dass die TEL/AML1 positive Gruppe eine verminderte Expression besitzt. Desweiteren wurde ein Zusammenhang zwischen ASYN-Expression und Nebenwirkungen der L-Asparaginase überprüft. Ein matched-pair Vergleich von Patienten mit/ohne Nebenwirkungen ergab jedoch eine ausgeprägte Variabilität der ASYN-Expression, so dass eine prognostische Aussage bezüglich der Verträglichkeit der L-Asparaginase nicht möglich ist.

The detection of minimal residual disease (MRD) is a major prognostic factor for treatment in acute lymphoblastic leukemia (ALL) of childhood. Several groups showed the predictive value of MRD after 5 weeks of chemotherapy (at the end of induction therapy). Patients with more than 1 leukemic cells in 100 cells (or = 10(-2)) at this time-point have a significantly higher relapse rate. The MRD measurement has been shown to be an independent prognostic factor at several time points in the BFM study (ALL-BFM 90) as well as in the EORTC study. The aim of our investigations was the detection of MRD at the end of induction therapy within the COALL studies which is different from the above studies. In the COALL studies, therapy starts with a 1 week DNR prephase (24 h infusion on day one) and i.th. MTX. Induction therapy consisted of 3 drugs over a period of 4 weeks (Prednisolone, Vincristine and Daunorubicin), asparaginase is given later in consolidation. At the end of induction therapy, bone marrow was obtained for cytomorphologic and molecular analysis.We investigated bone marrow samples from 76 patients. All patients were in morphologic remission at the end. of induction therapy. For MRD analysis, DNA was isolated from bone marrow mononuclear cells. Clonal T-cell-receptor (TCR) or immunoglobulin gene (IgH) rearrangements were identified by PCR. Monoclonal products were either sequenced directly (TCR) or after excision from high resolution agarose gels. Subsequently patient-specific oligonucleotides for allele-specific PCR were generated. PCR analysis was performed with 1 microgram DNA for each reaction within a semiquantitative matter. This method reached sensitivities down to 10(-5).Eighty-four percent of the analysed samples were MRD positive at the end of induction therapy. 20 out of 76 patient samples (26%) were highly positive (or = 10(-2)), 28 patients had levels of about 10(-3) (37%), 16 had levels around 10(-4) (21%) and 12 patients had no detectable residual cells (16%). All analysed 15 T-ALL patients had detectable residual disease at this timepoint. Until now, 5/20 patients with very high MRD level at the end of induction therapy suffered a relapse.Patients with very high MRD level at the end of induction therapy showed an elevated risk of relapse, but the predictive value is much poorer than for example in the BFM 90 MRD-study. We suggest, that a high MRD level at this timepoint results from a different induction therapy compared to the BFM 90 study. In the COALL studies asparaginase is given only after induction therapy to decrease the risk of thrombosis. We would like to conclude that this differences were compensated later during therapy as the event free survival of both studies is similar. In conclusion, an optimal information from MRD studies is strongly associated with the given therapy. Therefore we initiated an additional MRD time-point after the first chemotherapy block in consolidation.

Risk-adapted therapy in acute lymphoblastic leukemia (ALL) of childhood relies on traditional risk factors such as age, white blood count, immunological subtype, chromosomal aberrations and response to treatment. In spite of risk-adapted therapy, however, 20-30% of the patients suffer a relapse and may have profited from more intensive therapy. On the other hand a third of the patients is probably overtreated considering cure rates of 30% with less intensive therapies in the seventies. Additional prognostic criteria are therefore urgently needed. In a retrospective Dutch study in childhood ALL drug sensitivity testing with the MTT assay was identified as a new highly significant prognostic factor.In study COALL-92 in 140 patients in vitro drug sensitivity was performed on initial bone marrow or blood samples and as in the Dutch study a score was derived from the sensitivity of the 3 drugs prednisolone, vincristine and asparaginase (PVA score). For each drug a score of 1 (highest) to 3 (lowest sensitivity) is given. A score of 3 therefore indicates the best, a score of 9 the worst sensitivity.Probability of event-free survival (pEFS) according to the PVA score was 0.94 for score 3 + 4, 0.80 for score 5-7, 0.35 for Score 8 + 9 (0.47 for score 7-9). For analysis within the low risk (LR) group (age 1-10 years, WBC25/nl, common or pre-B-ALL, remission day 28, no translocation 9;22 or 4;11) and high risk (HR) group 252 patients of the Dutch study and the COALL study were combined. In the LR group pEFS was 1.00 for score 3 + 4, 0.76 for score 5-7, 0.38 for score 8 + 9 (0.54 for score 7-9); in the HR group pEFS was 0.91 for score 3 + 4, 0.69 for score 5-7, 0.45 for score 8 + 9 (0.56 for score 7-9). Multivariate analysis identified the PVA score as independent prognostic factor. NEW STUDY COALL-97: In the new study COALL-97 patients are first stratified according to HR and LR criteria and then are stratified again according to the PVA score. LR and HR patients with a score of 3 + 4 receive less intensive treatment in reinduction therapy to reduce the cumulative dose of anthracyclines and to mitigate the high rate of infectious complications during this part of the protocol. LR patients with a score of 7-9 are treated according to the HR protocol; HR patients with a score of 8 + 9 receive BMT if an HLA identical family donor is available. In study COALL-97 the results of the minimal residual disease study and day 15 bone marrow will be compared with the PVA score.In vitro drug sensitivity testing is an independent prognostic factor which allows adjustment of therapy to the individual risk of relapse in addition to the traditional risk factors. It can be assumed that in patients with a favourable resistance profile therapy can be reduced without loss of efficacy and that patients with an unfavourable resistance profile might profit from more intensive therapy.

Long-term follow-up of cranial CT scans of children with acute lymphoblastic leukemia and evaluation of the influence of chemo- and radiotherapy on the CCT changes.CCT scans of 68 children with non-B-ALL were analyzed retrospectively for signs of atrophy and changes in density. Patients were treated between 1981 and 1990 at the St. Anna Childrens Hospital Vienna according to the ALL-BFM protocols. Children were examined with CCT in defined periods from diagnosis until 3 years after cessation of treatment. As a control group served 69 patients with solid tumors who had not received corticoids or cranial irradiation.At the initial examination 56% of the ALL-patients showed CCT changes, 85% of these patients had already received corticoids. In the control group only 20% of the CCTs were found abnormal (p = 0.005). In both groups an age-dependence was found: 64% of the ALL-patients under five years of age and 22% of the patients above 5 years had initial CCT changes (p = 0.001). In the control group 39% of the patients under five years of age and 7% of the older patients showed CCT changes at the beginning of treatment (p = 0.003). The highest incidence of abnormal CCT scans (68%) was seen during intensive chemo- and radiotherapy. Until the end of therapy the incidence of abnormal CCTs decreased to 32%. After cessation of antileukemic therapy 35% of the patients whose CNS-prophylaxis included cranial irradiation, and 12% of the non-irradiated patients had abnormal CCT scans.Corticoids can cause reversible signs of cerebral atrophy. In the assessment of CCTs a physiological age-dependence of the volume of the CSF compartment has to be taken into consideration. The main reason for non-reversible CCT changes is the CNS- prophylaxis, above all the cranial irradiation, whereby younger children seem to be particular vulnerable.

The aim of the present study was to determine the adverse effects of antineoplastic chemotherapy and radiotherapy on tooth development, i.e., on dentition in general and on craniofacial growth. The patients studied consisted of 26 children with solid tumors treated with chemotherapy alone and 34 patients with acute lymphoblastic leukemia (ALL) who were treated with intensive and follow-on radiation therapy (18 and 24 Gy) to the neurocranium as a constituent of central nervous system prophylactics in treatment protocols for ALL. The treatment protocol employed in these children is known as the BFM (Berlin, Frankfurt, Münster) protocol. At the time of diagnosis the children ranged in age from 3 to 52 months. All treated patients had microdontia, enamel dysplasia, thinning and tapering of apical roots portions, and marked shortening of the roots. These defects were more pronounced in children treated for ALL, but it was not possible to establish statistically relevant evidence on the influence of adjuvant radiotherapy in this group. This indicates that there is a catch up in craniofacial growth 9 years after therapy and that the most serious long-term adverse effects are on dentition.

Nine children of the ALL-REZ BFM 87 and 90 trial received L-Asparginase (L-ASP) as a continuous infusion for 48-72 hs (i.e. 25 therapy cycles). Seven patients had had an allergic reaction towards an i.m. application (i.m., 29 therapy cycles). Two further patients got L-ASP initially as continuous infusion. The i.m. applications were carried out 19 times with Erwinia and 10 times with E. coli-Asparaginase, the continuous infusions 15 times with Erwinia and 10 times with E. coli-Asparaginase. In case of four patients continuous infusions of the same L-ASP type (E. coli or Erwinia) was well tolerated, after there had been an allergic reaction after i.m. application. Allergic reactions after i.m. application occurred during 10 courses as local painful erythema, during five courses as urticaria, during four courses as a general exanthema during one course as difficult breathing and during a further course as drop in blood pressure. After continuous infusion of L-ASP urticaria and difficult breathing occurred once and a transient exanthema two times. There was no anaphylactic reaction in any case. These data show that i.m. application of L-ASP causes no life-threatening side effects but allergic reactions (local pain and swelling) which clearly impaired general condition. Continuous infusion is a pharmacologically equivalent alternative with less impairment of the patients' general condition.

In the ongoing trial ALL-BFM 90 for the treatment of childhood non-B cell acute lymphoblastic leukemia (ALL) 1468 unselected patients (pts) were enrolled from 84 centers in Germany and Switzerland from 4/90 to 12/93. Based on the results of the previous trial ALL/NHL-BFM 86 this treatment program focused especially on therapy modifications for average (MRG) and high risk (HRG) pts, on the evaluation of therapy response for prognosis, and on the identification of high risk pts by molecular genetics. For average risk pts consolidation therapy was intensified by the addition of L-asparaginase (L-ASP) on a randomized basis. In HRG induction and consolidation therapy was modified by introduction of early intensification elements that had proved to be effective in relapsed pts. This patient group was randomized for the evaluation of the effects of G-CSF administered in the intervals between the intensification elements. Distribution of the 1376 eligible pts into the three treatment arms SRG (standard risk), MRG, and HRG was as expected (17 pts not yet assigned): 385 pts (28.0%), 834 pts (60.6%), and 140 pts (10.2%), respectively. Treatment consisted of the 8-drug induction (Protocol I), consolidation (Protocol M), reinduction (Protocol II), and maintenance therapy (total therapy duration 24 months). The drug doses and combinations were only slightly modified compared to the previous study ALL-BFM 86 with the exception of the randomized L-ASP containing arm MRG-2 (Protocol M-A) and group HRG. Preventive cranial irradiation was reduced to 12 Gy and applied to MRG and HRG pts only. As in study ALL-BFM 86, the initial response to a 7-day exposure to prednisone and to the first intrathecal injection of MTX at diagnosis was evaluated at day 8 of treatment with regard to blast count in peripheral blood (PB). In addition, pts were now investigated for the presence of blasts in the bone marrow (BM) at day 15 of treatment to compare the prognostic power of both response parameters. Identification of translocation t(9; 22) and/or BCR-ABL rearrangement characterized a small subgroup of pts that were not detected by poor initial therapy response. These pts were enrolled in HRG for more intensive treatment including allogeneic bone marrow transplantation (BMT). After a median observation time of 22 months, the overall probability for event-free survival (p-EFS) is 82 +/- 2%. 11 pts (0.8%) died before complete remission (CR) was achieved, 15 pts (1.1%) died while in CR for reasons other than relapse.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in the coagulation system due to steroids and asparaginase during treatment for acute lymphoblastic leukemia (ALL) are well known side effects and may cause bleeding or thrombosis. We report the case history of a 7-year old girl who developed thrombosis of the sinus sagittalis superior during ALL-treatment. Diagnosis was made by computed tomography and magnetic resonance imaging after the child became symptomatic with seizure. Until this event the girl had been treated already for two weeks with prednisone and E. coli-asparaginase (4 infusions). This medication caused distinct hypofibrinogenemia (Fibrinogen 53 mg/dl), prothrombin time expressed as percent of normal values of 58% was also pathological, activated partial thromboplastin time of 35 sec, antithrombin III 120% and thrombocyte count 178 G/l were in normal range. We were not successful in the attempt to adjust the imbalance in the coagulation system by transfusion of fresh frozen plasma (FFP)--seizure happened during FFP-infusion, fibrinogen blood level could be elevated only slightly. Our patient stayed consequently asymptomatic, the clinical recovery was confirmed radiologically.

During or immediately following L-asparaginase (L-asp) therapy especially intracranial thromboembolic or hemorrhagic complications due to hemostatic imbalance have been observed. We report about 6 children who had intracranial thrombosis or hemorrhage after 3 to 8 doses of L-asp. Initial symptoms of the cerebral events were similar--seizure, coma, hemiparesis and disorientation. All patients were examined by cerebral computerized tomography (CT) or/and magnetic resonance imaging (MRI). Three patients had a dural sinus thrombosis, one had an intracranial hemorrhage and two a hemorrhagic infarction with typical findings in the cerebral CT or MRI. Two other patients were interpreted as having peripheral thrombosis. They showed nontypical hypodense cortical and subcortical areas without any contrast medium enhancement in CT and hyperintense areas in T2-weighted MRI scan. All patients recovered from neurological symptoms, and showed obvious regression of CT and MRI findings which correlate with the good prognosis of these complications. Both CT and MRI are useful in diagnosis and follow-up of cerebrovascular complications of L-asp therapy. The CT and MRI findings of the reported cases seem to reflect different appearances of the same entity, i.e. thrombosis of venous vessels of different size with or without congestive edema and hemorrhagic complication.

Between 1988 and 1990, 55 patients with first relapses of acute lymphoblastic leukemia (ALL) were treated with a modified BFM-protocol (ALL REZ I/88). The patients were divided according to time and site of relapse: relapses with bone marrow involvement up to 6 months after stopping front line therapy (group A), relapses with bone marrow involvement beyond 6 month after therapy (group B) and isolated extramedullary relapses at any time (group C). During therapy the patients received alternating courses of polychemotherapy including infusions of intermediate dose methotrexate (1 g/m2 in 36 hours). The maintenance treatment consisted of daily oral thioguanine and biweekly intravenous (IV) MTX. The overall second remission rate was 89% (group A: 90%, group B: 86%, group C: 93%) and the probability of event free survival (EFS) at 4 years is 0.28 +/- 0.13 (group A: 0.22 +/- 0.12, group B: 0.24 +/- 0.18, group C: 0.57 +/- 0.15). We conclude, that with the treatment regimen applied, long lasting second remission can be achieved in about one third of patients even after intensive front line therapy. The most unfavourable prognoses were seen in patients with early bone marrow relapses (group A).

A 30-year-old man complained of high fever, resistant to antibiotics, and progressive loss of strength for five weeks. The peripheral blood showed pancytopenia (leucocytes 2200/microliters, platelets 45,000/microliters, haemoglobin 10.7 g/dl). There was also hepatosplenomegaly, abdominal lymph node enlargement, pleural and pericardial effusions and slight excess of lymphocytes in the cerebrospinal fluid. Histological examination of the bone marrow suggested a small-cell pleomorphic T-cell lymphoma, but Hodgkin's disease was also considered. Splenectomy was performed to confirm the diagnosis and treat the pancytopenia. The blood count rapidly returned to normal and the suggestion of Hodgkin's disease was excluded. Intensive chemotherapy (BMFT-ALL protocol) was followed by regression of nearly all the abnormalities, but marrow infiltration persisted. This treatment was discontinued after phase II of induction. After an intercurrent cytomegalovirus infection maintenance treatment with alpha-interferon (up to 5 million units daily) was started. The partial remission remained stable for 22 months. The lymphoma then relapsed but was held in check by further chemotherapy (Dexa-PAMB protocol). For the past 11 months the patient has remained in stable partial remission on treatment with alpha-interferon (3 million units every other day). Frequent infections (sinusitis, diarrhoea, abscess) require close supervision.

Between September and August 1991 818 previously untreated children and adolescents up to 18 years of age with acute lymphoblastic leukemia were entered into two modified BFM-protocols. Patients with B-ALL were excluded. From 1981 to 1987 524 patients were entered into the randomized multicenter study ALL VII/81 (modified ALL-BFM 81 protocol). Patients were divided into three risk groups standard (SR), medium (MR), high risk (HR) using the BFM risk factor. In a connecting study from 1988 to 1991 294 patients were registered on the stratified and randomized multicentric trial ALL VIII/87 (modified ALL-BFM 86 study). The main modification in study ALL VII/81 concerned the duration of treatment. Patients were randomized into two groups. The first group received as a late reinduction protocol III and then therapy was stopped. The second group received 6-MP and MTX for another six months. The other whole treatment strategy of ALL-BFM 81 was adopted. In protocol ALL VIII/87 the only modification was the reduction of the MTX dosage from 5 g/m2 to 1 g/m2 with an infusion time of 24 hours (leucovorin rescue 15 mg/m2 after 48 and 54 hours). The probability of the event-free-survival (EFS) for study ALL VII/81 was 59%. CNS events were significantly more frequent in standard risk patients with intermediate dose MTX (4 x 0.5 g/m2) compared with the irradiation group (18 Gy). The EFS for SR patients amounts to 61%, for MR patients to 59% and for HR patients to 36%. There was no significant difference of EFS for the two groups with different duration of therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Treatment according to the ALL/NHL-BFM 90 protocol I (induction phase) caused multiple and severe coagulation changes in all 14 patients of our study. Glucocorticoids alone made Fibrinogen drop to 148 mg/dl, AT III and Protein C rise to 136% or even 179% respectively. After day 12, immediately following the start of therapy with Coli-Asparaginase (ASP), fibrinogen continued to drop to reach its lowest average value of 46 mg/dl on day 24. Anticoagulant factors like plasminogen (lowest average value: 36%), AT III (47%) and Protein C (93%) dropped abruptly. These alterations were reversed after discontinuation of Glucocorticoids and ASP. During consolidation (protocol II) similar alterations are observed as in protocol I when Glucocorticoids are applied alone. However, after Erwinia-ASP there is no fall in AT III, plasminogen, and Protein C as is observed in protocol I with Coli-ASP. 2. Severe hemorrhages or thromboses are uncommon as compared to the degree of coagulation changes which can be regularly observed. Complications occur more often in girls. Most of them are seen during the 2nd or 3rd week of simultaneous ASP-Glucocorticoid therapy. 3. To avoid twofold alteration of hemostasis it should be considered to apply Glucocorticoids and ASP separately and to replace Coli-ASP by Erwinia-ASP. The efficacy of prophylactic replacement of decreased coagulation factors has not yet been confirmed. Immunologic and infectious side effects have to be taken into consideration. 4. More definite recommendations can be given when each suspected bleeding and/or thrombosis is confirmed by imaging procedures, when it is documented and registered, and when coagulation studies are performed during the critical phase.

Dose intensity (DI) plays an important role in the treatment of neoplastic diseases. The individual DI within a protocol may vary considerably and thus may be an important prognostic factor. In 213/305 patients treated in the cooperative study COALL-85 for childhood acute lymphoblastic leukemia the following parameters of individual therapy intensity were analyzed: Total time for intensive treatment, cumulative doses of single drugs, mean relative dose (= relation between received and prescribed doses of all drugs), mean relative dose intensity ( = mean relative dose/time) as well as frequency and duration of leukocytopenia. Therapy for LR (low-risk) and HR (high-risk) patients were separately analyzed by both life-table method and multivariate regression analysis. Neither length of time, mean relative dose intensity nor the other parameters had any significant influence on prognosis within the HR protocol. The only significant prognostic factor was the remission status on day 28 (p less than 0.001 in multivariate analysis). In contrast patients treated with the LR protocol had significantly fewer relapses if treatment resulted in leukocytopenic episodes (probability for event free survival (EFS) = 0.76 in patients with one or two leukocytopenic episodes compared to 0.52 in patients with none). Patients with a mean relative dose greater than 0.9 showed a higher EFS of borderline significance than patients with mean relative dose less than = 0.9 (0.72 vs 0.49, p = 0.09). We would like to conclude, that treatment protocols with very intensive and prolonged combination chemotherapy have a certain margin of safety in DI without disadvantage for the patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Management of E.-coli L-asparaginase induced anaphylaxis mainly consists of application of i.v. epinephrine but also in the withdrawal of this drug. Despite of this firm recommendation we have shown in 7 patients with E. coli L-asparaginase induced anaphylaxis, that further application of the drug is possible in the presence of a continuous epinephrine infusion (0.01-0.02 mg/kg) started one hour before and finished one hour after the concomittant infusion of the E.-coli L-asparaginase. In none of the patients there was a second event of anaphylaxis even though most of the patients still had to continue on E.-coli L-asparaginase with an average of more than 6 infusion.

Prognostic factors to estimate the risk of relapse are crucial for risk-adapted therapy in acute lymphoblastic leukemia (ALL). In a cooperative multicenter treatment study for childhood ALL (COALL-03-85) the prognostic relevance of the bone marrow (BM) blast count at day 28 was evaluated. Treatment was adjusted to the initial risk factors; patients with high risk (white blood count (WBC) greater than or equal to 25/nl, age greater than or equal to 10 years, T- or NULL-ALL) received intensified therapy consisting of rotation of 6 non cross-resistant drug combinations with 12 different agents. After 4 weeks 289/305 (94.8%) children were in complete remission (CR); one child died of infection, and 15 (14 high-risk patients) still had more than 5% blasts in the BM. Twelve of these 15 patients were in remission after 2 to 4 weeks additional treatment. Poor responders often had a high initial WBC, age above 10 years of T- or NULL-ALL. In spite of continuation of intensive therapy all children with more than 10% blasts in the BM on day 28 suffered an early relapse except 2 who were transplanted in first remission. Event-free survival for the poor responders is 0.15 compared to 0.71 (p = 0.0001) for the good responders (median observation time 48 months). In multivariate analysis remission status on day 28 was the only significant prognostic factor in high-risk patients above one year of age; traditional risk factors as initial WBC, age above 10 years, hepatosplenomegaly, and immunological subtype were of no prognostic significance in this study. (ABSTRACT TRUNCATED AT 250 WORDS)

The formation of specific IgE antibodies to L-Asp was studied in 27 children with ALL. Increased titers of specific IgE antibodies were significantly more frequent (p less than 0.001) prior to L-Asp infusions followed by allergic reactions than in those without reactions. After passive sensitization of basophile granulocytes of a healthy volunteer using patient's serum with the highest titers of specific IgE antibodies to L-Asp a significant release of histamine was observed after exposure of cells to L-Asp. In 1 of 8 patients with allergic reactions titers of specific IgE antibodies were elevated, while there was not detectable activation of the complement system. In conclusion, elevated titers of specific IgE antibodies to L-Asp are frequently detectable prior to L-Asp infusions which are associated with allergic reactions. In addition to activation of the classical complement pathway specific IgE antibodies and mediator release from mast cells may contribute to clinical symptoms. In a small number of patients this mechanism alone may be responsible for the allergic reaction.

Rabbits, treated with injections of 4000 IU of L-Asparaginase, develop the clinical and chemical signs of hypoparathyroidism. A simultaneous vaccination with vaccinia virus (strain "Elstree") markedly increase the tetanic symptoms ("conditioned deficiency"). L-Asparaginase may influence the cellular immunity by hypocalcemia. Two mechanisms are discussed: 1. the suppression of the phagocytosis, recognizable by the absence of signs for vaccinal allergy by deficiency of macrophages in the intradermal test with inactivated small-pox vaccine. 2. the inhibition of the PHA-induced lymphocyte transformation caused by deficiency of calcium ions.

In 25 children with lymphoblastic leukemia is shown that prophylactic treatment with allopurinol, 48 hours before initial cytostatic therapy, always prevents hyperuricemia. In 4 patients was renounced on this measurement and 2 of 4 patients died of the sequeles of hyperuricemia. In severe ill leukemic patients the parental use of allopurinol is recommended. 2 additional patients developed during remission therapy or after cessation of chemotherapy a moderate hyperuricemia which had to be treated, over years, with allopurinol.

L-asparaginase therapy is often limited by allergy or toxicity and probably in some cases by antibody mediated inactivation of the enzyme. These problems can be avoided by extracorporeal application of l-asparaginase. As the enzyme is a stable tetramer with high molecular weight it cannot pass through the dialysis membrane in contrast to the amino acid l-asparagin which is destroyed. The resulting l-asparagin depletion of the plasma is sufficient for therapeutic success. Effective extracorporeal l-asparaginase therapy is demonstrated in two patients with ALL who were resistant to other chemotherapy and could not be treated intravenously because of allergy and toxicity.

The pharmacological alteration of immunological reactivity is considered. The immunosuppressive drugs may be classified according to their chemical class and their antigen- or cycle-dependency. A relative specificity towards the lymphoid system is cyclophosphamide. New vistas are opened by the modulation of immunological reactivity by cyclic nucleotides. The possibilities afforded by the combined use of immunosuppressive drugs have not been fully exploited. A short therapeutic program including donor antigen, procarbazine and antilymphocyte serum induces immunological tolerance to allogeneic skin grafts in mice and bone marrow allografts in dogs. Immunosuppressive properties of microorganisms are exemplified.

Since 1979 in a cooperative study (COALL-80) 110 children with acute lymphoblastic leukemia (ALL) and 13 children with high grade malignant non Hodgkin's lymphoma (NHL) have been treated with a less aggressive modification of the Westberlin Study Program BFM 76/79. Asparaginase has been delayed from the initial 4 drugs regimen and interposed in between induction therapy and treatment of central nervous system (CNS). Induction therapy that way was well tolerated and realized mostly on an outpatient basis. The expected 2 1/2 years disease free survival rate for ALL is 86% and no worse than the results of the original BFM study. We were able to define by age (greater than 2, less than 7 years), initial blast count (less than 25000/mm3) and immunological typing (cALL Ag+) a great group of patients (52%) which has remained in continuous remission as a whole. For this group further reduction of therapy intensity is planned (omission of cyclophosphamide during CNS-phase and use of intermediate dose methotrexate (MTX-mHD) instead of CNS-irradiation).

In order to reduce the toxicity of the otherwise very effective childhood ALL treatment protocol BFM 76/79 asparaginase was omitted from the 4-drug induction regimen and placed as single drug before the intensification phase. In addition the effect of 3-monthly intermediate dose methotrexate (ID-MTX) pulses versus conventional vincristine (VCR) pulses during maintenance therapy was studied. Of 145 evaluable patients 124 (85.5%) survive in continuous and complete remission (CCR) at a median observation time of 21 (1,5-47) months. The expected rate of patients in CCR at 4 years is 75% respectively 80% after exclusion of 4 patients with fatal complications and one remission failure. These relapse free survival data re equal to the BFM 76/79 and 79/81 results. There was very few therapy related morbidity and no mortality from the COALL-80 induction therapy modification. The intensification phase, however, which was adopted from the BFM study without modification was difficult to manage and was not free of life threatening mostly infectious complications which were fatal in 4 cases. ID-MTX pulses did not prove superior to conventional VCR pulses. The relapse rate in patients with the c-ALL subtype was markedly lower than in any other subtype, remarkably also than in the undifferentiated type.

During l-Asparaginase (l-Asp) treatment the development of specific antibodies of IgG isotype is frequently observed. In most instances elevated IgG antibodies to l-Asp activate the complement system and induce allergic reactions following l-Asp infusion. However, in some cases no adverse reactions and no activation of complement are noticed, despite the presence of elevated anti-l-Asp levels. We studied the development of specific IgG antibodies to l-Asp in different subclasses in 12 children who had produced high levels of specific IgG. Results showed that all patients had elevated levels of IgG1. In 5 cases we were able to demonstrate the development of specific IgG3 antibodies and in 1 case of IgG4 antibody. Patients with high levels of IgG3 (above 100 AU) had the highest risk for subsequent anaphylaxis. Thus, subclass-specific determination of antibodies to l-Asparaginase might improve the estimation of the risk of anaphylaxis prior to 1-Asp infusions.