Wolf-Dieter Ludwig

Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOP-BFM ALL 2000 study

The Associazione Italiana di Ematologia Oncologia Pediatrica and the Berlin-Frankfurt-Münster Acute Lymphoblastic Leukemia (AIEOP-BFM ALL 2000) study has for the first time introduced standardized quantitative assessment of minimal residual disease (MRD) based on immunoglobulin and T-cell receptor gene rearrangements as polymerase chain reaction targets (PCR-MRD), at 2 time points (TPs), to stratify patients in a large prospective study. Patients with precursor B (pB) ALL (n = 3184) were considered MRD standard risk (MRD-SR) if MRD was already negative at day 33 (analyzed by 2 markers, with a sensitivity of at least 10(-4)); MRD high risk (MRD-HR) if 10(-3) or more at day 78 and MRD intermediate risk (MRD-IR): others. MRD-SR patients were 42% (1348): 5-year event-free survival (EFS, standard error) is 92.3% (0.9). Fifty-two percent (1647) were MRD-IR: EFS 77.6% (1.3). Six percent of patients (189) were MRD-HR: EFS 50.1% (4.1; P < .001). PCR-MRD discriminated prognosis even on top of white blood cell count, age, early response to prednisone, and genotype. MRD response detected by sensitive quantitative PCR at 2 predefined TPs is highly predictive for relapse in childhood pB-ALL. The study is registered at http://clinicaltrials.gov: NCT00430118 for BFM and NCT00613457 for AIEOP.

Risk-Adjusted Therapy of Acute Lymphoblastic Leukemia Can Decrease Treatment Burden and Improve Survival: Treatment Results of 2169 Unselected Pediatric and Adolescent Patients Enrolled in the Trial ALL-BFM 95.

The trial ALL-BFM 95 for treatment of childhood acute lymphoblastic leukemia was designed to reduce acute and long-term toxicity in selected patient groups with favorable prognosis and to improve outcome in poor-risk groups by treatment intensification. These aims were pursued through a stratification strategy using white blood cell count, age, immunophenotype, treatment response, and unfavorable genetic aberrations providing an excellent discrimination of risk groups. Estimated 6-year event-free survival (6y-pEFS) for all 2169 patients was 79.6% (+/- 0.9%). The large standard-risk (SR) group (35% of patients) achieved an excellent 6y-EFS of 89.5% (+/- 1.1%) despite significant reduction of anthracyclines. In the medium-risk (MR) group (53% of patients), 6y-pEFS was 79.7% (+/- 1.2%); no improvement was accomplished by the randomized use of additional intermediate-dose cytarabine after consolidation. Omission of preventive cranial irradiation in non-T-ALL MR patients was possible without significant reduction of EFS, although the incidence of central nervous system relapses increased. In the high-risk (HR) group (12% of patients), intensification of consolidation/reinduction treatment led to considerable improvement over the previous ALL-BFM trials yielding a 6y-pEFS of 49.2% (+/- 3.2%). Compared without previous trial ALL-BFM 90, consistently favorable results in non-HR patients were achieved with significant treatment reduction in the majority of these patients.

Diffusion‐weighted magnetic resonance imaging allows monitoring of anticancer treatment effects in patients with soft‐tissue sarcomas

To determine if diffusion‐weighted imaging (DWI) can be used as a surrogate marker of tumor response to anticancer therapy in patients with soft‐tissue sarcomas.

Standardization of flow cytometric minimal residual disease evaluation in acute lymphoblastic leukemia: Multicentric assessment is feasible†‡

Single‐laboratory experience showed that flow cytometric (FCM) assessment of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) is feasible in most patients and gives independent prognostic information. It is, however, not known whether FCM analysis can reliably be standardized for multicentric application.

Time point-dependent concordance of flow cytometry and real-time quantitative polymerase chain reaction for minimal residual disease detection in childhood acute lymphoblastic leukemia.

Background Flow cytometric analysis of leukemia-associated immunophenotypes and polymerase chain reaction-based amplification of antigen-receptor genes rearrangements are reliable methods for monitoring minimal residual disease. The aim of this study was to compare the performances of these two methodologies in the detection of minimal residual disease in childhood acute lymphoblastic leukemia. Design and Methods Polymerase chain reaction and flow cytometry were simultaneously applied for prospective minimal residual disease measurements at days 15, 33 and 78 of induction therapy on 3565 samples from 1547 children with acute lymphoblastic leukemia enrolled into the AIEOP-BFM ALL 2000 trial. Results The overall concordance was 80%, but different results were observed according to the time point. Most discordances were found at day 33 (concordance rate 70%) in samples that had significantly lower minimal residual disease. However, the discordance was not due to different starting materials (total versus mononucleated cells), but rather to cell input number. At day 33, cases with minimal residual disease below or above the 0.01% cut-off by both methods showed a very good outcome (5-year event-free survival, 91.6%) or a poor one (5-year event-free survival, 50.9%), respectively, whereas discordant cases showed similar event-free survival rates (around 80%). Conclusions Within the current BFM-based protocols, flow cytometry and polymerase chain reaction cannot simply substitute each other at single time points, and the concordance rates between their results depend largely on the time at which they are used. Our findings suggest a potential complementary role of the two technologies in optimizing risk stratification in future clinical trials.

Monitoring treatment response of childhood precursor B-cell acute lymphoblastic leukemia in the AIEOP-BFM-ALL 2000 protocol with multiparameter flow cytometry: predictive impact of early blast reduction on the remission status after induction.

Treatment response is a strong outcome predictor for childhood acute lymphoblastic leukemia (ALL). Here, we evaluated the predictive impact of flow cytometric blast quantification assays (absolute blast count, BC, and blast reduction rate, BRR) in peripheral blood (pB) and/or bone marrow (BM) at early time points of induction therapy (days 0, 8 and 15) on the remission status in the AIEOP-BFM-ALL 2000 protocol. At the single parameter level (905 patients), the strongest predictive parameter for the remission status as a dichotomous minimal residual disease (MRD) parameter (positive/negative) has been provided by the BC at day 15 in BM (cutoff: 17 blasts/μl; 50 vs 15%; odds ratio: 5.6; 95% confidence interval: 4.1–7.6, P<0.001), followed by the BRR at day 15 in BM and by the BC at day 8 in pB (odds ratios: 3.8 and 2.6, respectively). In the multiple regression analysis (440 patients), BC in pB (d0 and d8) and in BM (d15) as well as BRR at day 8 in pB provided significantly contributing variables with an overall correct prediction rate of 74.8%. These data show that the quantitative assessment of early response parameters, especially absolute BCs at day 15 in BM, has a predictive impact on the remission status after induction therapy.

Loss of heterozygosity on chromosome 6q14-q24 is associated with poor outcome in children and adolescents with T-cell lymphoblastic lymphoma.

Deletions of chromosome 6q have been reported in several hematological malignancies, but data are not conclusive regarding their biological and prognostic impact. Therefore, we focused on pediatric patients diagnosed with T-cell lymphoblastic lymphoma (T-LBL) treated uniformly according to the NHL-BFM95 protocol. We used loss-of-heterozygosity (LOH) analysis of 25 microsatellite markers located on chromosome 6q14–q24. Fragment-length analysis was performed on ABI-PRISM3100 Genetic-Analyzer. Eligibility criterion was ⩾3 informative markers. Between April 1995 and March 2003, 185 T-LBL patients were treated according to the NHL-BFM95 protocol. Five-year event-free (EFS) and disease-free survival (DFS) were 79±3 and 87±3% (median follow-up 4.7 [1.2–10.1] years). Sixty-one patients were evaluable for LOH analysis, including 18 out of 23 patients with relapse. EFS and DFS were 67±6 and 69±6% for these 61 patients. Testing of 853 markers in the 61 patients identified the presence of LOH in 19 patients (31%): 13 of the 18 relapse patients and five of the 41 in complete remission (odds ratio 18.7, 95% confidence interval 4.7–75.3). One LOH-positive patient died from treatment-related toxicity. We conclude that LOH on chromosome 6q14–q24 may have conferred a high risk of relapse on our group of children with T-LBL treated according to the NHL-BFM95 protocol.

Pediatric precursor T lymphoblastic leukemia and lymphoblastic lymphoma: Differences in the common regions with loss of heterozygosity at chromosome 6q and their prognostic impact

Deletions on chromosome 6q are frequently reported in hematological malignancies. However, their biological or prognostic impact has not yet been clarified. This study analyzed loss of heterozygosity (LOH) at chromosome 6q and compared the LOH findings in pediatric precursor T lymphoblastic lymphoma (T-LBL) with the LOH findings in precursor-T lymphoblastic leukemia (T-ALL). For LOH analyses, a set of 25 microsatellite-markers on 6q14-q24 were examined. All patients were treated uniformly according to ALL-BFM-type treatment-strategy. A total of 1671 markers were successfully analyzed from 108 T-LBL patients. LOH was detected in 21 T-LBL patients. There was clear association between LOH at 6q and an increased risk of relapse. In comparison, 3109 markers were successfully analyzed from 127 T-ALL-patients. LOH was detected in 16 patients, but was not associated with increased relapse-rate. The localization of the common LOH regions identified for T-LBL and T-ALL samples did not overlap. Therefore patterns of LOH at 6q and the prognostic impact of LOH differ between T-ALL and T-LBL. These results hint at biologic differences between the two diseases.

Expression and prognostic significance of different mRNA 5′-end variants of the oncogene EVI1 in 266 patients with de novo AML: EVI1 and MDS1/EVI1 overexpression both predict short remission duration

Rearrangements of chromosome band 3q26.2 lead to overexpression of the EVI1 gene and are associated with a poor prognosis in myeloid malignancies. EVI1 is also overexpressed in some cases without 3q26 rearrangements. To uncover its prognostic significance in this patient group, however, it may be necessary to distinguish among several known 5′‐end variants of its mRNA. According to a recent report, overexpression of the transcript variant EVI1_1d was associated with shortened survival in acute myeloid leukemia (AML), but overexpression of MDS1/EVI1, whose protein product differs structurally and functionally from that of all other known EVI1 5′‐end variants, was not. The aim of the present study was to determine, for the first time, the expression and prognostic significance of all known EVI1 5′‐end variants in AML. Quantitative RT‐PCR was used to measure the expression of EVI1_1a, EVI1_1b, EVI1_1d, EVI1_3L, and MDS1/EVI1 in 266 samples from patients with de novo AML. To correlate expression of the EVI1 5′‐end variants with survival parameters, regression analyses were performed. 41/266 patients (15.4%) overexpressed at least one, but more often several or all, EVI1 transcript type(s). High expression of each of the EVI1 mRNA variants, including MDS1/EVI1, was significantly associated with shortened continuous complete remission in the total patient population as well as in the subgroups of patients with intermediate risk or normal cytogenetics. The present study therefore shows that high levels of each of the known EVI1 mRNA 5′‐end variants represents an adverse prognostic factor in de novo AML without 3q26 rearrangements. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.© 2008 Wiley‐Liss, Inc.

Distinction of acute lymphoblastic leukemia from acute myeloid leukemia through microarray-based DNA methylation analysis.

Patterns of DNA methylation are substantially altered in malignancies compared to normal tissue, with both genome-wide hypomethylation and regional increase of cytosine methylation at dinucleotides of cytosine and guanine, i.e., CpG dinucleotides. While genome-wide hypomethylation renders chromosomes instable, hypermethylation of CpGs in promoter regions is generally associated with transcriptional silencing, e.g., of tumor suppressor genes. To investigate whether disease-specific methylation profiles exist for different entities of acute leukemia, a microarray-based DNA methylation analysis simultaneously assessing 249 CpG dinucleotides originating from 57 genes was employed. Hereby, samples from precursor B-cell acute lymphoblastic leukemia (ALL) could be distinguished from cases of acute myeloid leukemia by virtue of N33, EGR4, CDC2, CCND2, or MOS hypermethylation in ALL.