Favorable outcome in children and adolescents with a high proportion of advanced phase disease using single/multiple autologous or matched/mismatched allogeneic stem cell transplantations.

We determined the indication, outcome, and risk factors of single and multiple hematopoietic stem cell transplantation(s) (HSCT) in children and adolescents mostly with advanced disease. Forty-one out of 483 patients (8.5 %; median age 9 years) diagnosed at the University of Leipzig with hematological and oncological diseases required HSCT from 1999 to 2011. Patients had overall survival (OS) of 63 ± 10 and 63 ± 16 %, event-free survival (EFS) of 57 ± 10 and 42 ± 16 %, relapse incidence (RI) of 39 ± 10 and 44 ± 18 % and nonrelapse mortality (NRM) of 4 ± 4 and 13 ± 9 % at 10 years after one or more allogeneic and autologous HSCT, respectively. One patient in CR1 and five with advanced disease received two HSCT. Four of the six patients maintained/achieved CR for a median of 13 months. Three died of progression and one of NRM. Two patients had a third HSCT and one survived in CR +231 days after HSCT. Risk factors for OS and EFS were disease stage at HSCT and EBMT risk score. Center (pediatric or JACIE accredited pediatric/adult) was not a determinant for survival. Pediatric single and multiple HSCT are important curative approaches for high-risk malignant diseases with low NRM. Efforts to reduce high RI remain the major aim.

Randomised Introduction of 2-CDA as Intensification during Consolidation for Children with High-risk AML--results from Study AML-BFM 2004.

BACKGROUND: The outcome in children and adolescents with high-risk (HR) acute myeloid leukemia (AML) is still unsatisfactory. Therefore, in study AML-BFM 2004 we aimed to improve outcome of HR-patients by adding moderately dosed 2-Chloro-2-Deoxyadenosine (2-CDA) to the respective consolidation treatment backbone without increasing toxicity. The aim was to improve prognosis especially in FAB M4/M5/MLL patients, who represent the largest subgroup of HR patients. PATIENTS AND METHODS: In total, 343 children and adolescents with HR-AML were randomized to receive or not 2-CDA (6 mg/m²/d, days 1, 3) in combination with cytarabine/idarubicine (AI=500 mg/m² cytarabine 5 days continuous infusion plus 7 mg/m²/d idarubicin, days 3 and 5). RESULTS: RESULTS for patients of the AI/2-CDA arm (n=168) vs. the AI-arm (n=175) were similar: 5-year overall survival 68±4 vs. 72±4%, plogrank=0.38, event-free survival 53±4 vs. 49±4%, plogrank=0.77; cumulative incidence of relapse at 5 years: 35±4 vs. 37±4%, p(Gray)=0.89. RESULTS in patients with MLL rearrangement or FAB M4/M5 were also similar in the treatment groups. In addition, toxicities did not differ between the two arms. CONCLUSION: We conclude that additional, moderate dose 2-CDA does not improve prognosis in HR-patients when given during consolidation treatment. Its effect might be too low in this multidrug regimen, where the strongest effects are achieved during induction, or the chosen dose of 2-CDA might have been too low.

Allogeneic stem cell transplantation for patients with advanced rhabdomyosarcoma: a retrospective assessment.

BACKGROUND: Allogeneic haematopoietic stem cell transplantation (allo-SCT) may provide donor cytotoxic T cell-/NK cell-mediated disease control in patients with rhabdomyosarcoma (RMS). However, little is known about the prevalence of graft-vs-RMS effects and only a few case experiences have been reported. METHODS: We evaluated allo-SCT outcomes of 30 European Group for Blood and Marrow Transplantation (EBMT)-registered patients with advanced RMS regarding toxicity, progression-free survival (PFS) and overall survival (OS) after allo-SCT. Twenty patients were conditioned with reduced intensity and ten with high-dose chemotherapy. Twenty-three patients were transplanted with HLA-matched and seven with HLA-mismatched grafts. Three patients additionally received donor lymphocyte infusions (DLIs). Median follow-up was 9 months. RESULTS: Three-year OS was 20% (s.e.±8%) with a median survival time of 12 months. Cumulative risk of progression was 67% (s.e.±10%) and 11% (s.e.±6%) for death of complications. Thirteen patients developed acute graft-vs-host disease (GvHD) and five developed chronic GvHD. Eighteen patients died of disease and four of complications. Eight patients survived in complete remission (CR) (median: 44 months). No patients with residual disease before allo-SCT were converted to CR. CONCLUSION: The use of allo-SCT in patients with advanced RMS is currently experimental. In a subset of patients, it may constitute a valuable approach for consolidating CR, but this needs to be validated in prospective trials.

Autologous haematopoietic stem cell transplantation for systemic lupus erythematosus: data from the European Group for Blood and Marrow Transplantation registry.

OBJECTIVES: Patients with systemic lupus erythematosus (SLE) refractory to conventional immunosuppression suffer substantial morbidity and mortality due to active disease and treatment toxicity. Immunoablation followed by autologous stem cell transplantation (ASCT) is a novel therapeutic strategy that potentially offers new hope to these patients. METHODS: This retrospective survey reviews the efficacy and safety of ASCT in 28 SLE patients from eight centres reported to the European Group for Blood and Marrow Transplantation (EBMT) registry between 2001 and 2008. RESULTS: Median disease duration before ASCT was 52 (nine to 396) months, 25/28 SLE patients (89%) were female, age 29 (16-48) years. At the time of ASCT, eight (one to 11) American College of Rheumatology (ACR) diagnostic criteria for SLE were present and 17 (60%) patients had nephritis. Peripheral blood stem cells were mobilized with cyclophosphamide and granulocyte-colony stimulating factor in 93% of patients, and ex vivo CD34 stem cell selection was performed in 36%. Conditioning regimens were employed with either low (n = 10) or intermediate (18) intensities. With a median follow-up of 38 (one to 110) months after ASCT, the five-year overall survival was 81 ± 8%, disease-free survival was 29 ± 9%, relapse incidence (RI) was 56 ± 11% and non-relapse mortality was 15 ± 7%. Graft manipulation by CD34+ selection was associated with a lower RI (p = 0.001) on univariate analysis. There were five deaths within two years after ASCT: three caused by infection, one by secondary autoimmune disease and one by progressive SLE. CONCLUSIONS: Our data further support the concept of immunoablation and ASCT to re-induce long-term clinical and serologic remissions in refractory SLE patients even in the absence of maintenance therapy. This study also suggests a beneficial effect of ex vivo graft manipulation on prevention of relapses post-transplantation in SLE.

Key treatment questions in childhood acute lymphoblastic leukemia: results in 5 consecutive trials performed by the ALL-BFM study group from 1981 to 2000.

Between 1981 and 2000, 6 609 children (<18 years of age) were treated in 5 consecutive trials of the Berlin-Frankfurt-Münster (BFM) study group for childhood acute lymphoblastic leukemia (ALL). Patients were treated in up to 82 centers in Germany, Austria, and Switzerland. Probability of 10-year event-free survival (survival) improved from 65% (77%) in study ALL-BFM 81-78% (85%) in ALL-BFM 95. In parallel to relapse reduction, major efforts focused on reducing acute and late toxicity through advanced risk adaptation of treatment. The major findings derived from these ALL-BFM trials were as follows: 1) preventive cranial radiotherapy could be safely reduced to 12 Gy in T-ALL and high-risk ALL patients and eliminated in non-high-risk non-T-ALL patients, if it was replaced by high-dose and intrathecal methotrexate; 2) omission of delayed reintensification severely impaired outcome of low-risk patients; 3) 6 months less maintenance therapy caused an increase in systemic relapses; 4) slow response to an initial 7-day prednisone window was identified as adverse prognostic factor; 5) condensed induction therapy resulted in a significant improvement of outcome; 6) the daunorubicin dose in induction could be safely reduced in low-risk patients; 7) intensification of consolidation/reintensification treatment led to considerable improvement of outcome in high-risk patients.

Multiple viral infections after haploidentical hematopoietic stem cell transplantation in a child with acute lymphoblastic leukemia.

After allogeneic hematopoietic stem cell transplantation (HSCT), viral infections/reactivations are a frequent complication, sometimes with fatal outcome. Thus, early diagnosis is recommended by screening of whole blood or plasma preparations using highly sensitive molecular techniques that test for the most common viral pathogens, such as Epstein-Barr virus, cytomegalovirus, and adenoviruses (ADVs). Despite this approach, not every reactivation/infection can be adequately detected or excluded, even with highly sensitive polymerase chain reaction. Particularly after toxic treatment, uncommon infections or infections resistant to first-line treatment can occur, even in unusual locations. Herein, we present the case of a child with Philadelphia chromosome-positive acute lymphoblastic leukemia after allogeneic HSCT who suffered from 5 different viral reactivations/infections, including acyclovir-resistant herpes simplex virus type 1 esophagitis, human herpesvirus 6 encephalitis, rotavirus gastroenteritis, respiratory syncytial virus pneumonia, and ADV esophagitis, despite routinely performed blood examinations for viral pathogens remaining unrevealing at all times.

Human leukocyte antigen distribution in German Caucasians with advanced Ewing's sarcoma.

BACKGROUND: Risk stratification criteria for patients with Ewing's sarcoma family of tumors (ESFT) are still limited. We hypothesized divergent human leukocyte antigen (HLA) patterns in ESFT patients and compared HLA-A, -B and -DR phenotype frequencies of patients with advanced ESFT with those of healthy controls. PATIENTS: HLA types of all German Caucasian patients with advanced ESFT and available HLA-A, -B and -DR data registered in the European Group for Blood and Marrow Transplantation, Paediatric Registry for Stem Cell Transplantation and the MetaEICESS data bases (study group, n=30) were retrospectively compared with HLA types of healthy German stem cell donors (control group, n=8 862 for single HLA frequencies and n=8 839 for allele combinations). Study group patients had been immuno-typed due to eligibility for allogeneic stem cell transplantation for high risk of treatment failure, and thus constituted a selected subgroup of ESFT patients. RESULTS: After Bonferroni correction for multiple testing (PC), phenotype frequencies of HLA-A24 remained significantly higher in the study group compared to controls (PC<0.05). Furthermore, several HLA combinations were significantly more frequent in the study group compared to controls (all PC<0.05). CONCLUSION: We report an increased incidence of circumscribed HLA patterns in German Caucasians with advanced ESFT. The possible clinical significance of this observation has to be re-assessed in prospective trials comprising larger ESFT patient numbers of all risk groups.

No improvement of survival with reduced- versus high-intensity conditioning for allogeneic stem cell transplants in Ewing tumor patients.

BACKGROUND: Outcomes of Ewing tumor (ET) patients treated with allogeneic stem cell transplantation (allo-SCT) were compared regarding the use of reduced-intensity conditioning (RIC) and high-intensity conditioning (HIC) regimens as well as human leukocyte antigen (HLA)-matched and HLA-mismatched grafts. PATIENTS AND METHODS: We retrospectively analyzed data of 87 ET patients from the European Group for Blood and Marrow Transplantation, Pediatric Registry for Stem Cell Transplantations, Asia Pacific Blood and Marrow Transplantation and MetaEICESS registries treated with allo-SCT. Fifty patients received RIC (group A) and 37 patients received HIC (group B). Twenty-four patients received HLA-mismatched grafts and 63 received HLA-matched grafts. RESULTS: Median overall survival was 7.9 months [±1.24, 95% confidence interval (CI) 5.44-10.31] for group A and 4.4 months (±1.06, 95% CI 2.29-6.43) for group B patients (P = 1.3). Death of complications (DOC) occurred in 4 of 50 (0.08) and death of disease (DOD) in 33 of 50 (0.66) group A and in 16 of 37 (0.43) and 17 of 37 (0.46) group B patients, respectively. DOC incidence was decreased (P < 0.01) and DOD/relapse increased (P < 0.01) in group A compared with group B. HLA mismatch was not generally associated with graft-versus-Ewing tumor effect (GvETE). CONCLUSIONS: There was no improvement of survival with RIC compared with HIC due to increased DOD/relapse incidence after RIC despite less DOC incidence. This implicates general absence of a clinically relevant GvETE with current protocols.

No difference in survival after HLA mismatched versus HLA matched allogeneic stem cell transplantation in Ewing sarcoma patients with advanced disease.

Patients with advanced Ewing sarcoma (AES) carry a poor prognosis. Retrospectively, we analyzed 66 AES patients treated with allogeneic stem cell transplantation (allo-SCT) receiving HLA-mismatched (group A, n = 39) versus HLA-matched grafts (group B, n = 27). Median age at diagnosis was 13 years, and 15 years (range 3-49 years) at allo-SCT. The two groups did not differ statistically in distribution of gender, age, remission status/number of relapses at allo-SCT, or risk stratum. 9/39 (23%) group A versus 2/27 (7%) group B patients developed severe acute graft versus host disease (GvHD). Of patients alive at day 100, 7/34 (21%) group A versus 9/19 (47%) group B patients had developed chronic GvHD. In group A, 33/39 (85%) versus 20/27 (74%) group B patients died of disease and 1/39 (3%) versus 1/27 (4%) patients died of complications, respectively. Altogether 12/66 (18%) patients survived in CR. Median EFS 24 months after allo-SCT was 20% in both groups, median OS was 27% (group A) versus 17% (group B), respectively. There was no difference in EFS and OS in AES patients transplanted with HLA-mismatched versus HLA-matched graft in univariate and multivariate analyses. In this analysis, CR at allo-SCT is a condition for survival (p < 0.02).

Posaconazole salvage treatment in paediatric patients: a multicentre survey.

While a paediatric dosage has not been defined, posaconazole is occasionally being used in children. We conducted a multicentre retrospective survey and identified 15 patients (median age 10 years [range 3.6-17.5]) who received posaconazole salvage therapy for proven (9 patients) or probable (6 patients) invasive fungal infections. Posaconazole was administered for a median of 32 days (range 4-262) at a median dosage of 21 mg/kg (range 4.8-33.3). None of the patients discontinued therapy due to adverse events, which were mostly mild and observed in 11 patients. Complete or partial responses were observed in 4/7 patients with zygomycosis, 3/4 patients with invasive mould infection, 1/2 patients with invasive aspergillosis and 1/2 patients with chronic disseminated candidiasis. We conclude from the data that posaconazole displays favourable safety and tolerance and may be useful for management of individual paediatric patients with invasive infections.

[MRI screening before stem cell transplantation--necessary?]. / MRT-Untersuchung des Neurokraniums vor Stammzelltransplantation--notwendig oder überflüssig?

PURPOSE: In the context of stem cell transplantation (SCT), we often observe neurological complications as a consequence of immune system suppression, conditioning therapy or prophylaxis and treatment of graft-versus-host disease. Furthermore, cerebral lesions in existence prior to transplantation can be found. The aim of this study was to evaluate the benefit of cerebral magnetic resonance imaging (MRI) prior to stem cell transplantation. PATIENTS AND METHOD: Cerebral MR examinations of 116 children and adolescents were performed before SCT. Patients ranged in age from 1.1 to 21.4 years (mean 12.6 years). All MR images were obtained by a 1.5 T system. The predefined short protocol included an axial T 1-weighted SE sequence and a coronary T 2-weighted TSE sequence. We evaluated existing cerebral lesions, the diameter of the ventricular system, and the paranasal sinuses. In the case of pathological findings, the short examination protocol was expanded. RESULTS: In 5 of 116 children (4.3 %) we observed prior to SCT findings requiring immediate treatment although the patients did not show any clinical symptoms (1 x aspergilloma, 1 x hemorrhage of vascular anomaly). An increased risk of bleeding caused by cavernoma or another vascular anomaly without hemorrhage also had to be taken into account. 32 of 116 patients (37.1 %) showed atrophic lesions. In 42 children (36.2 %), we observed affections of the paranasal sinuses. CONCLUSION: The imaging findings requiring immediate treatment even though the children did not show any clinical signs, justify cerebral MR examinations prior to stem cell transplantation.

Diagnosis and severity criteria for sinusoidal obstruction syndrome/veno-occlusive disease in pediatric patients: a new classification from the European society for blood and marrow transplantation.

The advances in hematopoietic cell transplantation (HCT) over the last decade have led to a transplant-related mortality below 15%. Hepatic sinusoidal obstruction syndrome/veno-occlusive disease (SOS/VOD) is a life-threatening complication of HCT that belongs to a group of diseases increasingly identified as transplant-related, systemic endothelial diseases. In most cases, SOS/VOD resolves within weeks; however, severe SOS/VOD results in multi-organ dysfunction/failure with a mortality rate >80%. A timely diagnosis of SOS/VOD is of critical importance, given the availability of therapeutic options with favorable tolerability. Current diagnostic criteria are used for adults and children. However, over the last decade it has become clear that SOS/VOD is significantly different between the age groups in terms of incidence, genetic predisposition, clinical presentation, prevention, treatment and outcome. Improved understanding of SOS/VOD and the availability of effective treatment questions the use of the Baltimore and Seattle criteria for diagnosing SOS/VOD in children. The aim of this position paper is to propose new diagnostic and severity criteria for SOS/VOD in children on behalf of the European Society for Blood and Marrow Transplantation.

The MLL recombinome of acute leukemias in 2017.

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

Genotype-outcome correlations in pediatric AML: the impact of a monosomal karyotype in trial AML-BFM 2004.

We conducted a cytogenetic analysis of 642 children with de novo acute myeloid leukemia (AML) treated on the AML-Berlin-Frankfurt-Münster (BFM) 04 protocol to determine the prognostic value of specific chromosomal aberrations including monosomal (MK+), complex (CK+) and hypodiploid (HK+) karyotypes, individually and in combination. Multivariate regression analysis identified in particular MK+ (n=22) as a new independent risk factor for poor event-free survival (EFS 23±9% vs 53±2% for all other patients, P=0.0003), even after exclusion of four patients with monosomy 7 (EFS 28±11%, P=0.0081). CK+ patients without MK had a better prognosis (n=47, EFS 47±8%, P=0.46) than those with MK+ (n=12, EFS 25±13%, P=0.024). HK+ (n=37, EFS 44±8% for total cohort, P=0.3) influenced outcome only when t(8;21) patients were excluded (remaining n=16, EFS 9±8%, P<0.0001). An extremely poor outcome was observed for MK+/HK+ patients (n=10, EFS 10±10%, P<0.0001). Finally, isolated trisomy 8 was also associated with low EFS (n=16, EFS 25±11%, P=0.0091). In conclusion, monosomal karyotype is a strong and independent predictor for high-risk pediatric AML. In addition, isolated trisomy 8 and hypodiploidy without t(8;21) coincide with dismal outcome. These results have important implications for risk stratification and should be further validated in independent pediatric cohorts.

Germline activating TYK2 mutations in pediatric patients with two primary acute lymphoblastic leukemia occurrences.

The contribution of genetic predisposing factors to the development of pediatric acute lymphoblastic leukemia (ALL), the most frequently diagnosed cancer in childhood, has not been fully elucidated. Children presenting with multiple de novo leukemias are more likely to suffer from genetic predisposition. Here, we selected five of these patients and analyzed the mutational spectrum of normal and malignant tissues. In two patients, we identified germline mutations in TYK2, a member of the JAK tyrosine kinase family. These mutations were located in two adjacent codons of the pseudokinase domain (p.Pro760Leu and p.Gly761Val). In silico modeling revealed that both mutations affect the conformation of this autoregulatory domain. Consistent with this notion, both germline mutations promote TYK2 autophosphorylation and activate downstream STAT family members, which could be blocked with the JAK kinase inhibitor I. These data indicate that germline activating TYK2 mutations predispose to the development of ALL.

Elevated serum insulin-like growth factor binding protein-2 is associated with a high relapse risk after hematopoietic stem cell transplantation in childhood AML.

Insulin-like growth factor binding protein (IGFBP)-2 has mitogenic effects in normal and neoplastic cells. The purpose of this study is to examine the diagnostic and prognostic significance of elevated IGFBP-2 levels in children with AML after hematopoietic stem cell transplantation (HSCT) at relapse and continuous complete remission (CCR). In 27 children with AML (mean age 13.6+/-5.3 years; patients in remission n=15 with relapse n=12) serum parameters of IGFBP-2, IGFBP-3, IGF-I and IGF-II were analyzed up to 18 months after HSCT by RIA. AML-patients with evidence of relapse demonstrated a continuous increase of IGFBP-2 levels during the follow-up. At day 100 after HSCT, IGFBP-2 concentrations were significantly higher in patients with relapse than in children without relapse (7.4+/-4.0 standard deviation score (SDS) vs 3.9+/-1.7 SDS; P=0.01). Serum IGFBP-2 was identified as an independent factor for the prediction of relapse. Furthermore, the probability of relapse-free survival (RFS) in patients with IGFBP-2 >4.5 SDS at day 100 after HSCT was 31% compared to patients with IGFBP-2 <4.5 SDS was 72% (P=0.004). Patients with IGFBP-2 concentration up to 4.5 SDS more likely developed a relapse and had a poorer outcome. Identification of these patients allows a more individualized and aggressive adjuvant treatment and follow-up.

Minimal residual disease after intensive induction therapy in childhood acute lymphoblastic leukemia predicts outcome.

We investigated the level of minimal residual disease (MRD) in 26 children with B-lineage acute lymphoblastic leukemia (ALL) after intensive induction therapy. A quantitative semi-nested polymerase chain reaction (PCR) detecting the clone-specific rearranged immunoglobulin heavy chain genes was developed to improve sensitivity and specificity of amplification. In all patients, one leukemic cell could be detected in a background of 10(5) normal blood mononuclear cells. All patients investigated were in complete remission at the end of induction therapy as evaluated by morphologic criteria. Nineteen patients (73%) had no detectable residual leukemic cells using the sensitive semi-nested PCR. Seven patients (27%) were PCR positive. Three had a low level (<2 x 10(-5) leukemic cells per bone marrow cell), while four patients had a high level (>2 x 10(5)) of detectable residual leukemic cells. All patients with low or undetectable levels of residual leukemia remained in complete remission at a median of 63 months from diagnosis (range 40-80 months), while all four patients with a high level of residual leukemia subsequently relapsed at a median of 21 months from diagnosis (range 13-37 months). The patient groups with undetectable or low, and high level of MRD did not differ significantly in other clinical or genetic features with prognostic significance. We conclude that the level of MRD at the end of the intensive induction therapy period is predictive of outcome in childhood B lineage ALL. If confirmed by large prospective studies, the level of MRD might be useful in stratifying patients into high and low risk categories.

Tandem application of flow cytometry and polymerase chain reaction for comprehensive detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Children with acute lymphoblastic leukemia (ALL) with > or = 0.01% leukemic cells in the bone marrow after remission induction are at a greater risk of relapse. The most promising methods of detecting minimal residual disease (MRD) are flow cytometric identification of leukemia-associated immunophenotypes and polymerase chain reaction (PCR) amplification of antigen-receptor genes. However, neither assay can be applied to all patients. Moreover, both assays carry the risk of false-negative findings due to clonal evolution. The simultaneous use of both assays might resolve these problems, but the correlation between the methods is unknown. We studied serial dilutions of normal and leukemic cells by flow cytometry and PCR amplification of IgH genes and found the two methods highly sensitive (one leukemic cell among 10(4) or more normal cells), accurate (r2 was 0.999 for flow cytometry and 0.960 for PCR by regression analysis) and concordant (r2 = 0.962). We then examined 62 bone marrow samples collected from children with ALL in clinical remission. In 12 samples, both techniques detected MRD levels > or = 1 in 10(4). The percentages of leukemic cells measured by the two methods correlated well (r2 = 0.978). Of the remaining 50 samples, 48 had MRD levels < 1 in 10(4). In only two samples results were discordant: 2 in 10(4) and 5 in 10(4) leukemic cells by PCR but < 1 in 10(4) by flow cytometry. We conclude that immunologic and molecular techniques can be used in tandem for universal monitoring of MRD in childhood ALL.