Maria Malec

Karolinska University Hospital, Tukholma, Stockholm, Sweden

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Publications (10)73.92 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Minimal residual disease (MRD) assessment is a powerful prognostic factor for determining the risk of relapse in childhood acute lymphoblastic leukaemia (ALL). In this Swedish multi-centre study of childhood ALL diagnosed between 2002 and 2006, the MRD levels were analysed in 726 follow-up samples in 228 children using real-time quantitative polymerase chain reaction (RQ-PCR) of rearranged immunoglobulin/T-cell receptor genes and multicolour flow cytometry (FCM). Using an MRD threshold of 0·1%, which was the sensitivity level reached in all analyses, the concordance between RQ-PCR and FCM MRD values at day 29 was 84%. In B-cell precursor ALL, an MRD level of ≥0·1% at day 29 predicted a higher risk of bone marrow relapse (BMR) with both methods, although FCM was a better discriminator. However, considering the higher median MRD values achieved with RQ-PCR, a higher MRD cut-off (≥0·2%) improved the predictive capacity of RQ-PCR. In T-ALL, RQ-PCR was notably superior to FCM in predicting risk of BMR. That notwithstanding, MRD levels of ≥0·1%, detected by either method at day 29, could not predict isolated extramedullary relapse. In conclusion, the concordance between RQ-PCR and FCM was high and hence both methods are valuable clinical tools for identifying childhood ALL cases with increased risk of BMR.
    British Journal of Haematology 01/2011; 152(6):743-53. · 4.94 Impact Factor
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    ABSTRACT: Minimal residual disease (MRD) detection during the early treatment phase has become an important stratification parameter in many childhood acute lymphoblastic leukaemia (ALL) treatment protocols. Here, we aimed to address the applicability of rearranged antigen-receptor genes as potential MRD markers using real-time quantitative polymerase chain reaction (RQ-PCR) in a Swedish population-based cohort. From 334 childhood ALL cases diagnosed during 2002-2006, we analysed 279 diagnostic samples (84%) by screening for rearranged immunoglobulin (IG) and T-cell receptor (TCR) genes. Allele-specific oligonucleotides were designed, and the sensitivity and quantitative level was determined for each target. Overall, clonal IG/TCR rearrangements were detected in 97% (236/244) of B-cell precursor ALL (BCP ALL) and 94% (33/35) of T-ALL. A sensitive RQ-PCR analysis (< or = 10(-4)) was obtained in 89% (216/244) of BCP ALL and in 74% (26/35) of T-ALL, whereas two sensitive targets were only available in 47% (115/244) of BCP ALL and 29% (10/35) of T-ALL cases. With the stratification threshold of > or = 10(-3), which is applied in the current Nordic treatment protocol (NOPHO-ALL 2008) for the identification of high-risk patients, 93% of BCP ALL and 86% of T-ALL reached this quantitative range by at least one target gene. Taken together, this national retrospective study demonstrates that an IG/TCR target for MRD monitoring can be identified in the majority of childhood ALL cases, whereas identification of a second sensitive target gene needs to be improved.
    European Journal Of Haematology 11/2009; 84(2):117-27. · 2.55 Impact Factor
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    ABSTRACT: Molecular markers for minimal residual disease in B-lineage acute lymphoblastic leukemia were identified by determining, at the time of diagnosis, the repertoire of rearrangements of the immunoglobulin heavy chain (IGH) gene using segment-specific variable (V), diversity (D), and junctional (J) primers in two different studies that involved a total study population of 75 children and 18 adults. This strategy, termed repertoire analysis, was compared with the conventional strategy of identifying markers using family-specific V, D, and J primers for a variety of antigen receptor genes. Repertoire analysis detected significantly more markers for the major leukemic clone than did the conventional strategy, and one or more IgH rearrangements that were suitable for monitoring the major clone were detected in 96% of children and 94% of adults. Repertoire analysis also detected significantly more IGH markers for minor clones. Some minor clones were quite large and a proportion of them would not be able to be detected by a minimal residual disease test directed to the marker for the major clone. IGH repertoire analysis at diagnosis has potential advantages for the identification of molecular markers for the quantification of minimal residual disease in acute lymphoblastic leukemia cases. An IGH marker enables very sensitive quantification of the major leukemic clone, and the detection of minor clones may enable early identification of additional patients who are prone to relapse.
    The Journal of molecular diagnostics: JMD 04/2009; 11(3):194-200. · 3.48 Impact Factor
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    ABSTRACT: Classical Hodgkin lymphoma (cHL) is characterized histologically by a minority of malignant Hodgkin Reed-Sternberg cells surrounded by abundant inflammatory cells, generally believed to be of major importance in the pathophysiology of the disease. Here, we present data that link inflammatory cell-derived arachidonic acid metabolites, the cysteinyl leukotrienes (CysLT), to the pathogenesis of cHL. Two HL cell lines, L1236 and KMH2, were shown to express functional CysLT(1) receptors, responding with a robust calcium signal upon leukotriene (LT) D(4) challenge. LTD(4) stimulated protein release of tumor necrosis factor-alpha, interleukin-6 and -8 by L1236 cells and interleukin-8 by KMH2 cells. Importantly, all these LTD(4)-induced effects were blocked by the CysLT(1) receptor-specific antagonist zafirlukast. Immunohistochemical studies of cHL biopsies and microarray analysis of microdissected cells revealed that the CysLT(1) receptor is expressed also by primary Hodgkin Reed-Sternberg cells. As these cells are surrounded by CysLT-producing eosinophils, macrophages and mast cells, our results suggest the CysLTs as mediators in the pathogenesis of cHL, contributing to the aberrant cytokine network of this lymphoma.
    International Journal of Cancer 09/2008; 123(10):2285-93. · 6.20 Impact Factor
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    ABSTRACT: Detection of minimal residual disease (MRD) in follow-up samples from patients with ALL is essential for evaluation of treatment response. We applied multicolor flow cytometry and real-time quantitative PCR (RQ-PCR) to compare MRD results in 71 follow-up samples from 22 children treated for ALL. When results obtained by flow cytometry and RQ-PCR were grouped into positive-negative categories, a significant level of agreement was found in 72% of samples (P<0.001). However, if a cutoff level of 0.01% was applied, the concordance was 89%. MRD could be quantified in 19 samples by both methods, showing a strong correlation (P<0.01). Nevertheless, MRD levels differed more than five-fold between both methods in 4/19 samples. In 20 (28%) samples, the two techniques showed discordant results. Most discordant results (17/20) were due to the limited sensitivity of flow cytometry analysis within the range 0.01-0.001%; remaining discordant results were due to the instable or subclonal IG/TCR gene rearrangements or a limited quantitative range of the applied RQ-PCR targets. Although concordant results could be obtained by flow cytometry and RQ-PCR analysis, MRD levels may differ. Therefore, MRD data obtained by these two techniques are not yet easily exchangeable.
    Leukemia 10/2004; 18(10):1630-6. · 10.16 Impact Factor
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    ABSTRACT: Classical Hodgkin's lymphoma (HL) is a malignant disorder characterized by a small number of tumor cells and inflammatory cells. Both the tumor cells and the inflammatory cells produce cytokines which are thought to contribute to the clinical parameters of HL. Quantification of these cytokines at a protein level is still somewhat imprecise. We, therefore, used a method to quantify cytokine mRNA expression in HL cell lines and lymph node biopsies. We used real-time quantitative polymerase chain reaction (RQ-PCR) to investigate mRNA expression for interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-8, IL-12p35, IL-12p40, IL-13, IL-15, interferon (IFN)-gamma and tumor necrosis factor (TNF-alpha) in lymph node tissue from 15 patients with classic Hodgkin's lymphoma (c-HL) and one with lymphocyte predominance (LP) HL. HL-derived cell lines L1236, L540, and L428 were also investigated. Reactive lymphatic tissue (n=6) and peripheral blood mononuclear cells (PBMC) from healthy donors (n=4) before and after stimulation were used as controls. In 5 c-HL samples the cytokine expression in T lymphocytes was also studied by flow cytometry. All c-HL samples (but not LP) expressed IL-13 mRNA. This cytokine was not found in non-stimulated PBMC or in reactive lymphatic tissue. Expression of IL-10, IL-1beta, IL-15 and IL-12p35 mRNA was higher in HL samples than in PBMC and reactive lymphatic tissue. Expression of IL-10, IL-1beta, TNF-alpha and IFN-gamma mRNA was significantly higher in the EBV+ HL samples (n=6) than in the EBV- cases. All HL cell lines showed high expression of IL-13, IL-12p35, TNF-alpha and IL-15 mRNA. IFNg mRNA levels were high in L428 and L540 cells, IL-10 in L1236 cells and L540 cells, IL-5 in L428 cells and IL-4 in L1236 cells. Cytokine mRNA levels can be measured by RQ-PCR using a limited amount of tissue. This method gives valuable information on biological variation between different HL samples and may contribute to unraveling the complex cytokine network contributing to the clinical and biological heterogeneity of this disease.
    Haematologica 07/2004; 89(6):679-85. · 5.94 Impact Factor
  • Leukemia 05/2004; 18(4):884-6. · 10.16 Impact Factor
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    ABSTRACT: Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytic leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer (EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n=278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.
    Leukemia 01/2004; 17(12):2318-57. · 10.16 Impact Factor
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    ABSTRACT: The analysis of minimal residual disease (MRD) has assumed a growing role in the follow-up of patients with acute lymphoblastic leukemia (ALL). We have applied multiparameter flow cytometry (FC) with 'live-gate' analysis and allele-specific oligonucleotide (ASO)-PCR detecting leukemia-specific T cell receptor gamma and delta gene rearrangements for MRD follow-up in 30 ALL patients. The comparison of results obtained in 89 follow-up samples from 23 patients showed significantly consistent results in 70 samples (78%); (P < 0.001). Bone marrow samples taken during the first phase of treatment (during or immediately after induction) showed a lower level of consistency when compared to samples taken during later phases of treatment (69% vs 85% consistent results, respectively). Some of the discrepant results were due to low cellularity of the samples obtained for FC and some due to the presence of PCR inhibitors. Of 29 patients evaluated at the end of the induction treatment, 18 (62%) had detectable levels of MRD and six of these patients suffered relapse. In all these patients MRD levels by FC increased preceding relapse. Our results suggest that FC offers a MRD detection tool that can be easily applied in clinical practice and is as informative as molecular methods.
    Leukemia 05/2001; 15(5):716-27. · 10.16 Impact Factor
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    ABSTRACT: Prospective studies on the detection of minimal residual disease (MRD) in acute leukemia patients have shown that large-scale MRD studies are feasible and that clinically relevant MRD-based risk group classification can be achieved and can now be used for designing new treatment protocols. However, multicenter international treatment protocols with MRD-based stratification of treatment need careful standardization and quality control of the MRD techniques. This was the aim of the European BIOMED-1 Concerted Action 'Investigation of minimal residual disease in acute leukemia: international standardization and clinical evaluation' with participants of 14 laboratories in eight European countries (ES, NL, PT, IT, DE, FR, SE and AT). Standardization and quality control was performed for the three main types of MRD techniques, ie flow cytometric immunophenotyping, PCR analysis of antigen receptor genes, and RT-PCR analysis of well-defined chromosomal aberrations. This study focussed on the latter MRD technique. A total of nine well-defined chromosome aberrations with fusion gene transcripts were selected: t(1;19) with E2A-PBX1, t(4;11) with MLL-AF4, t(8;21) with AML1-ETO, t(9;22) with BCR-ABL p190 and BCR-ABL p210, t(12;21) with TEL-AML1, t(15;17) with PML-RARA, inv (16) with CBFB-MYH11, and microdeletion 1p32 with SIL-TAL1. PCR primers were designed according to predefined criteria for single PCR (external primers A <--> B) and nested PCR (internal primers C <--> D) as well as for 'shifted' PCR with a primer upstream (E5' primer) or downstream (E3' primer) of the external A <--> B primers. The 'shifted' E primers were designed for performing an independent PCR together with one of the internal primers for confirmation (or exclusion) of positive results. Various local RT and PCR protocols were compared and subsequently a common protocol was designed, tested and adapted, resulting in a standardized RT-PCR protocol. After initial testing (with adaptations whenever necessary) and approval by two or three laboratories, the primers were tested by all participating laboratories, using 17 cell lines and patient samples as positive controls. This testing included comparison with local protocols and primers as well as sensitivity testing via dilution experiments. The collaborative efforts resulted in standardized primer sets with a minimal target sensitivity of 10-2 for virtually all single PCR analyses, whereas the nested PCR analyses generally reached the minimal target sensitivity of 10-4. The standardized RT-PCR protocol and primer sets can now be used for molecular classification of acute leukemia at diagnosis and for MRD detection during follow-up to evaluate treatment effectiveness.
    Leukemia 12/1999; 13(12):1901-28. · 10.16 Impact Factor

Publication Stats

926 Citations
73.92 Total Impact Points

Institutions

  • 1999–2011
    • Karolinska University Hospital
      • Department of Hematology
      Tukholma, Stockholm, Sweden
  • 2009
    • University of New South Wales
      • Children’s Cancer Institute of Australia
      Kensington, New South Wales, Australia
  • 2004
    • Karolinska Institutet
      Solna, Stockholm, Sweden