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Second central nervous system (CNS) relapses represent about 7.3% of subsequent recurrences of childhood acute lymphoblastic leukemia (ALL). In most children these subsequent CNS relapses occur during the first 18 months after diagnosis of the first relapse (mean 1.42 +/- 0.73 years). We present a patient who suffered a second ALL relapse in the CNS more than seven years after diagnosis of his first relapse. The leukemic clone was completely stable over more than ten years as shown by minimal residual disease techniques. Possible reasons for the recurrence of the leukemic clone after this very long period of dormancy (e.g. role of the disease site, immune system dysfunction) are discussed.

Purpose: We recently reported that children with acute leukemias who show increasing mixed chimerism (MC) after allogeneic stem-cell transplantation have a significantly enhanced risk of relapse. Here we present the results of a prospective multicenter study to investigate (1) whether relapse of acute lymphoblastic leukemia (ALL) can be determined in advance by serial analysis of chimerism, and (2) if outcome can be influenced by withdrawal of immunosuppression and/or by low-dose donor lymphocyte infusion when increasing MC is detected., Patients and Methods: Serial and quantitative analysis of chimerism was performed using a fluorescent-based short-tandem-repeat-polymerase chain reaction in 163 children with ALL., Results: One hundred one patients revealed complete chimerism (CC) or low-level MC (CC/low-level MC); increasing MC was found in 46 patients; and decreasing MC, in 16 patients. Relapse was significantly more frequent in patients with increasing MC (26 of 46) than in patients with CC/low-level MC (eight of 101) or in patients with decreasing MC (0 of 16; P <.0001). The probability of 3-year event-free survival (EFS) was 54% for all patients, 66% for patients with CC/low-level MC (n = 101), 66% for patients with decreasing MC (n = 16), and 23% for patients with increasing MC (n = 46; P <.0001). Of the 46 patients with increasing MC, 31 received immunotherapy. This group had a significantly higher 3-year EFS estimate (37%) than the 15 patients who did not receive immunotherapy (0%; P <.001)., Conclusion: Serial analysis of chimerism reliably identifies patients at highest risk to relapse. The 3-year EFS of patients with increasing MC without immunotherapy was 0%, by which overt relapse could be prevented in a considerable group of patients.

Purpose: Childhood Philadelphia chromosome positive (Ph(+)) acute lymphoblastic leukemia (ALL) has a poor prognosis. Because leukemia cell burden is reduced but not eradicated by polychemotherapy, improved treatment strategies should enhance those immune mechanisms responsible for the maintenance of complete remission. The aim of this study was to evaluate the protection of mice challenged with the syngeneic Ph(+) ALL cell line BM185 using genetically modified leukemia cell vaccines and immunomodulating oligonucleotides., Experimental Design: Because retroviral vectors are ineffective at transducing nondividing primary cells from human hematopoietic malignancies, we first evaluated nonviral techniques (electroporation and ballistic transfer) using minimalistic immunogenically defined gene expression vectors to generate B7.1 or granulocyte macrophage colony-stimulating factor (GM-CSF)-expressing BM185 cells. Subsequently, protective vaccination experiments with these cells were performed in a leukemia challenge mouse model., Results: Electroporation yielded a high transfection rate (82.6% for B7.1) with moderate GM-CSF secretion/1 x 10(6) cells (228 pg), whereas ballistic transfer led to a lower transfection rate (30.9%) with high GM-CSF secretion (614 pg). Secondly, we immunized mice with B7.1/interleukin 2- or B7.1/GM-CSF-expressing BM185 cell vaccines. We observed a better protection of mice that received the B7.1/GM-CSF vaccine compared with these receiving the B7.1/interleukin 2 vaccine. Protection was additionally enhanced by application of a double stem-loop immunomodulating oligonucleotide containing CpG motifs., Conclusion: Our data indicate that immunization with B7.1/GM-CSF-expressing cell vaccines generated by electroporation and application of double stem-loop immunomodulating oligonucleotide protected mice against a murine Ph(+) ALL challenge. Ultimately, this approach may also lead to clinical benefit in patients with Ph(+) ALL.

Background and Objectives: Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient-specific targets for polymerase chain reaction (PCR)-based detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). Nevertheless, instability of these targets during the course of the disease has important implications for PCR-based MRD monitoring and may lead to false negative results., Design and Methods: Several studies have shown that Ig and TCR targets are reasonably stable in ALL between diagnosis and first relapse, but up to now, there are no data on the stability of these targets between first and second relapse. We, therefore, performed a PCR-study on bone marrow samples from 49 children with precursor B-ALL at first and second relapse. Homo-heteroduplex PCR analyses were used for identification of clonal IGH, IGK-Kde, TCRG and TCRD gene rearrangements. Clonal targets were studied by sequencing and/or comparative homo-heteroduplex analysis., Results: In 52% (25/48) of the patients, all PCR targets identified at first relapse were preserved at second relapse; in 92% (44/48) of the patients at least one target and in 73% (35/48) at least two targets remained stable. Best stability was found for IGH and TCRG gene rearrangements., Interpretation and Conclusions: Based on these first data about clonal stability of Ig and TCR targets between first and second relapse of childhood precursor B-ALL, we developed a stepwise strategy for appropriate selection of stable PCR targets for MRD monitoring. This strategy was applicable in 84% of the relapsed patients and resulted in at least one stable MRD-PCR target per patient in 98% of these children.

The aim of our study was to characterise, for the first time, the chemo- and radiation sensitivity of seven pediatric acute lymphoblastic leukemias xenotransplanted into immunodeficient NOD/SCID mice and to correlate the findings with the expression of three drug resistance proteins, P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP1) and lung resistance protein (LRP). Mice were treated with single drugs used in clinical protocols: daunorubicin, doxorubicin, cyclophosphamide, vincristine, cytarabine, asparaginase and methotrexate. Two ALL samples, established from primarily diagnosed patients, responded to 5 or 6 of the tested cytostatics, respectively, while 3 out of 5 ALLs from relapse patients were only sensitive towards 2-4 drugs tested. Daunorubicin was more efficient than doxorubicin. The response of xenografted ALL toward vincristine and cyclophosphamide was inversely correlated with the expression of P-gp, LRP and MRP1 (R2 = 0.71, 0.70 and 0.64 for vincristine and 0.44, 0.70 and 0.60 for cyclophosphamide). A good correlation could be detected between the expression of P-gp and LRP (R2 = 0.88), P-gp and MRP1 (R2 = 0.75) and LRP and MRP1 (R2 = 0.90). The highest co-expression of the drug resistance proteins in the leukemia ALL-SCID 6 coincided with a high resistance to radiation and chemotherapy. Prediction of the individual drug resistance profile of a patient on the basis of results from the ALL-SCID xenograft studies was not possible because of the relatively long time necessary and because of the changes in the expression of P-gp, LRP and MRP1 during the murine generations. We conclude that in the drug resistance phenotype of ALL not only the above mentioned proteins but a variety of different molecules are involved.

Purpose: As previous data had shown that only two-thirds of patients had the predicted activity time courses when PEG-asparaginase 1000 U/m(2) was used in reinduction after native E. coli asparaginase in induction treatment of acute lymphoblastic leukaemia (ALL), drug monitoring was performed with the use of a higher dose., Methods: Because one-third of patients had insufficient serum asparaginase activity time courses after a single dose of 1000 U/m(2) PEG-asparaginase during reinduction treatment, a dose of 2500 U/m(2) PEG-asparaginase, which is the approved dosage in Germany, was used in 39 reinduction and 20 relapse patients to determine whether prolongation of the activity time course may be possible with this higher dose, and to look for significant differences between reinduction and relapse patients., Results: After 1, 2 and 3 weeks, the mean activities were 1113 +/- 699 U/l, 231 +/- 259 U/l, and 13 +/- 35 U/l in the reinduction patients, and 1078 +/- 649 U/l, 165 +/- 195 U/l and 19 +/- 28 U/l in the relapse patients, respectively. There were a considerable number of patients with a substantially shortened activity time course in both groups. In 10 of 39 reinduction patients and in 7 of 24 doses during relapse treatment, only activities <100 U/l were found after 1 week with a further fast decline. No statistically significant differences between the two patient groups could be shown at any time-point., Conclusions: Comparison of these data with activities after 1000 U/m(2) PEG-asparaginase showed no prolongation of the time with activity in the therapeutic range with the higher dose. Therefore, for a longer duration of therapeutic activity, administration of further doses is mandatory.