Henk Behrendt

Phenotypic and karyotypic properties of hyperdiploid acute lymphoblastic leukaemia of childhood

Summary. The DNA/cell content was measured by flow cytometry in samples obtained from 98 unselected children with acute lymphocytic leukaemia (ALL) at diagnosis. The frequency of anomalies in modal DNA content was compared to that encountered in acute childhood non‐lymphocytic leukaemia (ANLL) and disseminated non‐Hodgkins lymphoma (NHL). In ALL the most frequent (35%) aberration in DNA content was an increase by 20% relative to the modal value of normal white blood cells. This subcategory, referred to as hyperdiploid ALL (HD‐ALL), was characterized by a close association with the expression of the c‐ALL surface marker (20/20 patients) and characteristic numerical chromosome changes, including tri‐ or tetrasomy of chromosome 21. Moreover, patients with hyperdiploid ALL had a much lower peripheral leucocyte count (P= 0.001) than those with diploid disease and a varying proportion of their leukaemic cells existed in the peripheral blood as morphologically normal lymphocytes expressing the c‐ALL antigen. Within the standard risk category, patients with HD‐ALL had a longer disease‐free survival than those with diploid disease (P= 0.058). It is concluded that routine analysis by flow cytometry can conveniently and consistently detect ALL patients with hyperdiploid chromosome numbers. Hyperdiploid ALL constitutes a fairly large subtype of childhood ALL with specific biological and karyotypic properties, possibly associated with favourable prognosis.

Chromosome Studies on Acute Nonlymphocytic Leukaemia in Children

Summary: Cytogenetic studies have been carried out on 17 children with acute nonlymphocytic leukaemia (ANLL). Of the 16 patients analysed at diagnosis, 11 had acquired clonal chromosome abnormalities. Four out of seven cases with acute myeloid leukaemia (M2) had 8;21 translocations, two of which were variants. Comparisons with other data on ANLL confirmed the association between the 8;21 translocation and the younger age groups. There are indications that the Netherlands may be a high incidence area for this translocation. Differences in the type of chromosome anomalies between childhood and adult ANLL were evident suggesting that different aetiologic factors may be involved.

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.

Imbalance in the ribonucleotide pools of lymphoid cells from acute lymphoblastic leukemia patients

The intracellular purine and pyrimidine ribonucleotide concentrations were determined in the lymphoid cells from peripheral blood and/or bone-marrow of 29 patients with acute lymphoblastic leukemia (ALL), as well as in the mononuclear cells from peripheral blood of 12 patients with ALL in remission. The lymphoid cells of ALL patients showed an imbalance in the nucleotide pool compared with normal lymphocytes, whereas the nucleotide pool of mononuclear cells from patients with ALL in remission had normal values. The imbalance in the lymphoid cells from ALL patients involved decreased ratios of purine:pyrimidine, adenine:guanine and uracil:cytosine nucleotides, and an increased amount, together with a changed composition, of the UDP sugars. When compared with tonsil-derived B lymphocytes and thymocytes, ALL lymphoid cells have an increased amount (absolute and relative) and a change composition of the UDP sugars. Significant differences were found between the mean values for the immunologically defined subgroups of ALL and between the mean values for patients with a high or a low percentage of blast cells. However, individual patients cannot be classified according to their nucleotide pattern, because of the overlapping ranges. The results of this study may be useful for the design of new therapeutic regimens.

PCR-positivity in harvested bone marrow predicts relapse after transplantation with autologous purged bone marrow in children in second remission of precursor B-cell acute leukaemia.

Purging of autologous bone marrow (BM) grafts of children in second remission after a relapse of precursor B acute lymphoblastic leukaemia (ALL) in the BM has been carried out in our laboratory since 1987, initially by complement mediated cell lysis. This protocol was extended by performing an immunorosette depletion before lysis with complement. The aim of the present study was to assess by polymerase chain reaction the presence of residual leukaemic cells in the BM grafts before and after purging. The results were then correlated to clinical outcome. In 24/28 patients a PCR product was obtained by amplification of IgH and/or TcR junctional regions. BM before purging was available for analysis in 13 patients. We found that leukaemic cells could be detected in 8/13 (62%) of these grafts before purging . All these eight patients experienced a relapse, regardless of whether the purging procedure had been successful (defined as achievement of PCR‐negativity) or not. In contrast, none of the five patients with PCR‐negative grafts before purging relapsed (P = 0.0008). One patient died due to transplant‐related toxicity. Of the remaining 23 patients, nine patients received a PCR‐positive BM graft after purging. All these nine patients experienced a relapse as compared to 6/14 whose BM was PCR‐negative after purging (P = 0.0072). Two of eight PCR‐positive BM grafts could be purged to PCR‐negativity. Thus, improvements both in treatment of leukaemia and in purging efficacy are still needed.

Palmar flexion creases in childhood neoplasia.

About 90% of Caucasian individuals in the general population may observe two transverse palmar flexion creases when their fingers are slightly bent. A small minority may find in one hand a single transverse crease or the usual two creases, of which one seems to cut across the palm to the ulnar margin. Those unusual creases are called simian‐ or Sydney‐creases, respectively. Normal healthy people hardly ever observe them in both hands. We observed those unusual creases, however, in more than 50% of children suffering from different types of malignant neoplasia, quite often in both hands. The difference between patients and controls of similar ages is highly significant. Among patients with childhood acute lymphocytic leukemia the variant creases were mostly of the Sydney type. They most frequently occurred in those patients in whom the disease had become manifest at an early age. Since fathers as well as mothers of the patients showed significantly higher frequencies of unusual creases, the phenomenon seems to be a familial one. A most singular effect is the striking incidence of those creases in younger siblings of the patients. With regard to crease variance our data are essentially similar for lymphoproliferative disorders (ALL and NHML) and embryonic malignant tumors. This might be the first indication of a common host factor in patients with ALL or embryonic tumors occurring in early childhood. We have postulated that this factor may be a regulatory one associated with cellular growth and differentiation in early fetal palmar pads as well as with cell‐mediated immune response to early pediatric tumors. The observation of palmar flexion creases may prove to be rewarding in future studies of cellular defense mechanisms in young patients with neoplasia. Cancer 43:749–759, 1979.

Numerical changes in the various peripheral white blood cells in children as a result of antineoplastic therapy.

The effects of various cytostatic drug regimens on the numbers of the individual white-cell types were studied retrospectively. 131 children with solid tumors were classified into six groups according to the type of tumor. All children within the same group received similar treatment. Differential counts were performed manually, as well as with the Hemalog D. The number of lymphocytes usually decreased rapidly after initial treatment and remained at the low level throughout the period of therapy. In the majority of the children the lymphocyte count became normal between 1 and 12 months after cessation of therapy. In contrast, granulocyte and monocyte counts were not affected in some children, whereas in other children the numbers of these cell types decreased. When there was a decrease in number, the number was soon restored and, in nearly all children, before the end of therapy.

Triple immunofluorescence staining for prediction of relapse in childhood precursor B acute lymphoblastic leukaemia.

In this study we describe a fast and sensitive method using three‐colour immunofluorescence for the detection of cells with phenotypes that are rare in normal bone marrow (BM) but occur frequently in children with precursor B acute lymphoblastic leukaemia. We show that, in the first year after initiation of therapy, in 17/18 patients (10 patients were analysed after first diagnosis and nine patients after first BM relapse; one patient was analysed on both occasions) the percentage of CD10+CD19+ cells and CD20−CD22+ cells in the CD34+ cell population indicated the likelihood of relapse. A suppression of cells expressing these phenotypes after initiation of therapy was followed by an outgrowth of normal precursor B cells after 12 months. Therefore this early test for impending relapse (which occurred 10–28 months after starting chemotherapy) was only applicable in the first year after beginning the treatment. However, despite this predictive value, comparison of fluorescence data with PCR results obtained from the same BM samples indicated that only a subpopulation of the CD34+CD10+CD19+ and CD34+CD20−CD22+ cells above the determined threshold value represented malignant cells. A large prospective study to confirm the predictive value of this three‐colour immunofluorescence assay is warranted.

Abnormal nucleotide pattern in the eosinophils of a patient with acute lymphoblastic leukemia associated with eosinophilia.

In this article we present a patient with acute lymphoblastic leukemia (ALL) associated with eosinophilia, in which the eosinophilia preceded a meningeal and bone-marrow relapse of ALL. We analysed the purine and pyrimidine nucleotide content of the eosinophils (92% pure) and compared the nucleotide pattern with that of eosinophils from healthy donors and from patients with eosinophilia not associated with leukemia. The ratios of purine:pyrimidine and of uracil:cytosine nucleotides were decreased compared with those in eosinophils from healthy donors and from patients with eosinophilia with other aetiologies. The total nucleotide concentration was increased, especially the concentration of UDP-sugars and pyrimidine nucleotides. The decrease in these ratios and the increase in concentration of the nucleotides and the UDP-sugars were also detected in leukemic cells of patients with ALL (de Korte et al., Leukemia Res. 10, 389-396 (1986) compared to normal lymphocytes. We suggest a malignant character of the eosinophils in our patient with ALL associated with eosinophilia, in contrast with the non-malignant state suggested previously for these cells.

Mitoxantrone is effective in treating childhood T-cell lymphoma/T-cell acute lymphoblastic leukemia

Background. T‐cell malignancies in childhood are highly malignant diseases usually treated with intensive multidrug chemotherapy. In these regimens, anthracyclines, which are considerably cardiotoxic, are used. Mitoxantrone is structurally related to the anthracyclines, but it is considered to be less cardiotoxic. Therefore, the effectiveness of mitoxantrone in treating childhood T‐cell malignancies was studied.