M Dworzak

Drug-induced immunophenotypic modulation in childhood ALL: implications for minimal residual disease detection.

Assessment of minimal residual disease (MRD) by flow cytometry is considered to be based on the reproducibility of the leukemic immunophenotype detected at diagnosis. However, we previously noticed modulation of surface antigen expression in acute lymphoblastic leukemia (ALL) during the early treatment. Hence, we investigated this in 30 children with B-cell precursor ALL consecutively enrolled in the AIEOP-BFM ALL 2000 protocol. Quantitative expression of seven antigens useful in MRD monitoring was studied at diagnosis and compared to that measured at different time points of remission induction therapy. Downmodulation in the expression of CD10 and CD34 occurred at follow-up. By contrast, upmodulation of CD19, CD20, CD45RA, and CD11a was observed, while the expression of CD58 remained stable. Despite this, we could unambiguously discriminate leukemic cells from normal residual B cells. This holds true when bone marrow (BM) samples from similarly treated T-ALL patients, but not from healthy donors, were used as reference. Our results indicate that immunophenotypic modulation occurs in ALL during the early phases of BFM-type protocols. However, the accuracy of MRD detection by flow cytometry seems not negatively affected if adequate analysis protocols are employed. Investigators should take this phenomenon into account in order to avoid pitfalls in flow cytometric MRD studies.

Incidence and relevance of secondary chromosome abnormalities in childhood TEL / AML1 + acute lymphoblastic leukemia: an interphase FISH analysis

The aim of the present study was to determine the frequency and clinical relevance of the most common secondary karyotype abnormalities in TEL/AML1+ B-cell precursor acute lymphoblastic leukemia (ALL) as assessed with fluorescence in situ hybridization (FISH) analyses. Screening of 372 patients who were enrolled in two consecutive Austrian childhood ALL multicenter trials identified 94 (25%) TEL/AML1+ cases. TEL deletions, trisomy 21 and an additional der(21)t(12;21) were detected in 52 (55%), 13 (14%) and 14 (15%) TEL/AML1+ patients, respectively. The 12p aberrations (P=0.001) and near tetraploidy (P=0.045) were more common in TEL/AML1+ patients, whereas the incidence of diploidy, pseudodiploidy, hypodiploidy, low hyperdiploidy, near triploidy, del(6q), chromosome 9 and 11q23 abnormalities was similar among TEL/AML1+ and TEL/AML1− patients. None of the TEL/AML1+ patients had a high hyperdiploid karyotype. Univariate analysis indicated that among TEL/AML1+ patients those with a deletion of the nontranslocated TEL allele had a worse prognosis than those without this abnormality (P=0.034). We concluded that the type and incidence of the most common secondary aberrations in TEL/AML1+ ALL can be conveniently identified with little additional effort during interphase screening with appropriate TEL and AML1 FISH probes. We also provided preliminary evidence that the deletion of the nontranslocated TEL allele may adversely influence the clinical course of TEL/AML1+ ALL.

Structure of the human TNF receptor 1 (p60) gene (TNRF1) and localization to chromosome 12p13

Abstract Clones encoding the entire coding and 3′ untranslated region of the human type I tumor necrosis factor receptor (p60) gene (TNFR1) were isolated by hybridization using probes derived from TNFR-1 cDNA. The gene was characterized by restriction mapping, DNA blot analysis and sequence analysis. The coding region and the 3′ untranslated region are distributed over 10 exons. Each of the four repeats, comprising the extracellular ligand binding domain and characterizing a receptor superfamily, is interrupted by an intron. However, the intron-exon structure is not conserved in the nerve growth factor receptor gene, another member of this superfamily. By PCR analysis of human-mouse somatic cell hybrids and in situ hybridization using biotinylated genomic TNFR1 DNA, we localized the gene to human chromosomal band 12p13. This corresponds to the homologous murine gene localized at the distal region of mouse chromosome 6.

PAX5/ETV6 fusion defines cytogenetic entity dic(9;12)(p13;p13).

Recurrent chromosomal abnormalities present in malignant cells often define subentities with unique biological and clinical features. The molecular identification of genes involved in genetic alterations has led to the characterization of fusion genes with neoplastic properties. However, for many nonrandom translocations including the dic(9;12)(p11–13;p11–12), the molecular equivalent has not as yet been identified. The dicentric translocation dic(9;12) is a recurrent chromosome abnormality that accounts for close to 1% of childhood acute lymphoblastic leukemia (ALL). This specific alteration occurs almost exclusively in B-progenitor ALL, and unlike many other nonrandom translocations, is associated with an excellent prognosis. In this work, we provide strong evidence that the PAX5/ETV6 fusion transcript defines the clinical and biological entity that is associated with the presence of a dic(9;12) chromosome. As the PAX5 and ETV6 genes are localized at 9p13 and 12p13, respectively, the cytogenetic description of the dic(9;12)-PAX5/ETV6 rearrangement should be refined to dic(9;12)(p13;p13).

Multiparameter phenotype mapping of normal and post-chemotherapy B lymphopoiesis in pediatric bone marrow

We studied the differentiation profiles of B cell precursors (BCP) in normal and post-chemotherapy pediatric bone marrow (BM) using multiparameter flow cytometry. The goal of our study was to draw a comprehensive phenotypic map of the three major maturational BCP stages in BM. By correlating lineage-associated markers, CD45RA, and several adhesion molecules, the stage-specific patterns were found to differ in certain details from previously published concepts. Among the earliest BCP, a subset of CD34+CD10lo precursors was repeatedly observed in addition to the well characterized CD34+CD10hiCD19+ majority of cells. Only two-thirds of these CD34+CD10lo cells expressed CD19. However, uniformity of phenotypic features, absence of T lineage markers, and the regeneration kinetics after chemotherapy suggest the B lineage affiliation of the CD34+CD10lo precursors in general. In the more mature BCP, expression of CD10, CD20, cytoplasmic and surface μ chains (cμ and sμ) was observed to overlap more than previously recognized. We found that CD20 and cμ appear early during B cell ontogeny (already on CD34+ BCP), and that CD10 is lost late, following the onset of sμ expression. Differences between normal and post-chemotherapy BM specimens regarding the phenotypic appearance of BCP were exclusively due to differences in the subset composition, as post-chemotherapy samples showed a preponderance of immature stages. Our observations may build a framework for comparing leukemic cells with their normal counterparts to define possible leukemia-associated aberrations useful for residual disease studies.

Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients

Invasive fungal disease (IFD) is a life-threatening event in immunocompromised patients, and there is an urgent need for reliable screening methods facilitating rapid and broad detection of pathogenic fungi. We have established a two-reaction real-time PCR assay permitting highly sensitive detection of more than 80 fungal pathogens, covering a large spectrum of moulds, yeasts and Zygomycetes. To assess the clinical potential of the assay, more than 600 consecutive specimens from 125 pediatric patients carrying a high risk of IFD were analyzed. An excellent correlation between PCR positivity and the presence of proven, probable or possible fungal infection according to the European Organization for Research and Treatment of Cancer criteria was demonstrated, as revealed by the sensitivity of the assay of 96% (95% CI: 82–99%). The negative predictive value of the panfungal PCR assay presented was 98% (95% CI: 90–100%), while the specificity and the positive predictive value were 77% (95% CI: 66–85%) and 62% (95% CI: 47–75%), respectively. The results indicate that molecular screening of patients during febrile neutropenic episodes by the assay presented could help prevent unnecessary toxicity resulting from empirical antifungal treatment in individuals who may not be at risk of imminent fungal disease. Our observations raise the possibility that rapid species identification may be required to increase the positive predictive value for impending fungus-related disease.

Modulation of antigen expression in B-cell precursor acute lymphoblastic leukemia during induction therapy is partly transient: Evidence for a drug-induced regulatory phenomenon. Results of the AIEOP-BFM-ALL-FLOW-MRD-Study Group†

Background: Changes of antigen expression on residual blast cells of acute lymphoblastic leukemia (ALL) occur during induction treatment. Many markers used for phenotyping and minimal residual disease (MRD) monitoring are affected. Glucocorticoid (GC)‐induced expression modulation has been causally suspected, however, subclone selection may also cause the phenomenon.

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.

Flow Cytometric Detection of Minimal Residual Disease in Acute Lymphoblastic Leukemia

Abstract Assessment of minimal residual disease (MRD) during the first months of therapy gives information on the timely response to treatment, and proves to be a powerful and independent indicator of treatment outcome in patients with acute lymphoblastic leukemia (ALL). Immunological evaluation by flow cytometry (FCM) is one of the most attractive approaches to this. The present review summarizes the historical development of this approach over the last 20 years, and shows that current methodology is based on the existence of leukemia-associated patterns of derangement in antigen expression with respect to normal differentiation or location of occurrence. Recent clinical studies are summarized which proved that FCM is applicable to more than 90% of patients with ALL and gives prognostic information comparable to polymerase chain-reaction (PCR)-based technology. Ongoing efforts based on parallel application of both technologies are explained which are designed to clarify which approach bears the best cost-relevance ratio in order to be broadly used in the future for risk assessment and tailoring of treatment modalities. Concluding perspectives relate to further technical developments like usage of peripheral blood (PB) instead of bone marrow (BM), absolute quantification, or strategic placement of investigative time-points, which may allow to simplify the MRD approach and thus augment its economic efficiency.

Detection of Residual Disease in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia by Comparative Phenotype Mapping: Method and Significance

The present review summarizes our efforts in developing a novel immunologic approach (“Comparative Phenotype Mapping”) targeted at assessing minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients. The method relies on quantitatively aberrant, leukemia-associated antigen expression patterns which allow to discriminate leukemic from normal BCP using a limited panel of antibody combinations and multidimensional flow cytometry. In an analysis of 63 follow up bone marrow samples of patients with BCP-ALL we show that this approach enables to efficiently detect MRD. Further clinical observation revealed that the patients which were MRD-positive by flow cytometry (although in morphological remission) had a very high probability of early disease recurrence compared to the good chances of a relapse-free survival (RFS) in the MRD-negative cohort (RFS 0.0 vs. 0.76 at 3 years). Comparative Phenotype Mapping thus proves to be a reliable method for MRD detection in BCP-ALL. Concluding remarks relate to the optional applications of the method as well as to future perspectives. An ongoing large prospective study which we are now conducting on the basis of Comparative Phenotype Mapping will clarify the clinical significance of MRD detection in ALL patients by this method, and will determine its value compared to related as well as molecular-genetic techniques.