E. F. Van Leeuwen

Rearrangement status of the malignant cell determines type of secondary IgH rearrangement (V-replacement or V to Dj joining) in childhood B precursor acute lymphoblastic leukemia

Immunoglobulin heavy chain (IgH) oligoclonality in childhood B precursor acute lymphoblastic leukemia (ALL) as determined by Southern analysis is found in 30–50% of patients and has been shown to be the result of ongoing IgH rearrangement (mostly VH-replacement and VH to D–JH joining) after malignant transformation. It is unknown however, what determines the type of secondary rearrangement. Also the biological basis of the variable degree of oligoclonality observed in childhood ALL is poorly understood. We analyzed in detail the IgH rearrangement status of the leukemic cells for a random panel of 18 childhood B precursor ALL patients by polymerase chain reaction (PCR)/sequencing analysis and by Southern analysis. By Southern analysis 10/18 (55.6%) patients were considered oligoclonal and 8/18 (44.4%) monoclonal. In contrast, by PCR minor clonal rearrangements were detected in 14/18 (77.8%) patients. VH-replacement was found in 7/14 patients, VH to D–JH joining in 6/14 patients and an unusual type of secondary rearrangement, VH–D to JH joining, in one patient. Only a single type of secondary rearrangement was detected in each patient. The type of secondary rearrangement (VH-replacement or VH to D–JH joining) depended on the rearrangement status (VDJ/VDJ or VDJ/DJ, respectively) of the dominant leukemic clone as determined by Southern analysis. We found that in addition to a more ‘advanced’ IgH rearrangement status patients with VH-replacements also have a more ‘advanced’ TCRδ rearrangement status, which possibly reflects exposure of both the IgH locus and the TCRδ locus to recombinase activity in a preleukemic clone. Finally, we investigated a putative relationship between oligoclonality by Southern analysis and S-phase fraction of the leukemic cell population. We found a significantly lower percentage cells in S-phase for oligoclonal patients as compared to monoclonal patients. Our data add to the understanding of ongoing rearrangement of antigen receptor loci in childhood ALL and have implications for the monitoring of minimal residual disease by PCR.

What is the best predictor of the severity of ABO-haemolytic disease of the newborn?

In 80 newborn infants ABO-incompatible with their mothers, the lysis-inducing effect of the maternal IgG anti-A or anti-B antibodies in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay and the antigen density of A or B antigens on the red cells of the children were measured. On the basis of the results, the children were divided into two groups--24 children in whom increased haemolysis was to be expected, and 56 children in whom it was not. Signs of haemolysis and serological features of ABO haemolytic disease of the newborn (ABO-HDN) were compared in these two groups and a control group of 120 ABO-compatible infants. The effect of the maternal antibodies in the ADCC assay, the titres of maternal IgG anti-A or anti-B antibodies, the results of the direct antiglobulin test on the red cells in the cord blood, and the titre of IgG anti-A or anti-B antibodies in the serum of the infants were compared for their ability to predict the severity of ABO-HDN. This was also done for the combination of the ADCC assay results plus the A or B antigen density and the direct antiglobulin test plus the titre of maternal IgG anti-A or anti-B antibodies. The ADCC assay with maternal serum was the most sensitive assay to predict ABO-HDN, and the combination of the ADCC assay with A or B antigen density determination the most specific test.

Maternal antibodies against fetal blood group antigens A or B: lytic activity of IgG subclasses in monocyte-driven cytotoxicity and correlation with ABO haemolytic disease of the newborn.

IgG antibodies against blood group antigens A or B (anti‐A/B) are able to sensitize erythrocytes for destruction in an antibody‐dependent cell‐mediated (ADCC) assay with monocytes as effector cells. The activity of maternal IgG anti‐A/B in this test was compared with clinical signs of haemolytic disease of the newborn (HDN). When the ADCC was negative (less than 10% of the sensitized cells lysed), signs of increased red‐cell destruction in the children were never observed. In three cases with a strongly positive ADCC (>45% lysis), the children were severely affected and needed more than one exchange transfusion. In the cases with >10% but less than 45% lysis in the ADCC, there was no clear correlation between the result of the ADCC and the degree of lysis in the newborn infants. In these cases, the degree of lysis of the red cells of the infant was shown to be strongly influenced by the number of A/B antigens per red cell. There was a direct correlation between the degree of lysis in the ADCC and the titre of IgG3 anti‐A/B in the sera. There was comparable activity of maternal IgG anti‐A/B in the ADCC test in the 32nd week of pregnancy and at the moment of delivery.

Degradation of radioiodinated B cell monoclonal antibodies : inhibition via a FCγ-receptor-II-mediated mechanism and by drugs

Abstract Our aim is to treat patients with B cell malignancies with radioimmunotherapy using monoclonal antibodies (mAb) such as CD19, CD20 and CD22. In this study we investigated the rate of internalization and catabolism of these mAb. After 24 h at 37°C, 20% – 25% of initially cell-bound 125I-CD19 mAb and 125I-CD22 mAb was degraded in B cells, whereas almost no degradation occurred after binding of 125I-CD20 mAb. For B cells expressing Fcγ receptor II (FcγRII), isotype-dependent degradation was noted as the CD19 IgG1 mAb showed an enhanced degradation rate compared to the switch variant IgG2a. The effect of various pharmaceutical agents that delay the internalization or subsequent degradation of mAb was evaluated. The degradation was inhibited most effectively by a combination of etoposide and vinblastine, resulting in accumulation of radioactivity in the target cell. Also the simultaneous application of CD20 or CD22 with 125I-CD19 mAb or of CD20 with 125I-CD22 mAb proved to be a potent inhibitor of the rapid degradation of these mAb, by inhibiting internalization via an FcγRII-mediated mechanism. Both methods of reducing the degradation of radioiodinated mAb are expected to prolong irradiation of malignant B cells and consequently result in an enhanced therapeutic effect in vivo.

Maternal autoimmune thrombocytopenia and the newborn.

That a humoral factor was the causative agent in idiopathic thrombocytopenic purpura was suggested nearly 30 years ago after the observation that mothers with the disease often give birth to children who develop transient thrombocytopenia.1 Maternal platelet autoantibodies were presumed to pass into the fetal circulation. With present techniques2 3 platelet autoantibodies are detectable in most patients with idiopathic thrombocytopenic purpura. We have therefore tried to confirm the passage of autoantibodies through the placental barrier and determine their immunochemical characteristics. We used specific antiglobulin reagents labelled with fluorescein isothiocyanate.