Małgorzata Dawidowska

T-cell acute lymphoblastic leukaemia: recent molecular biology findings

For many years, T‐cell acute lymphoblastic leukaemia (T‐ALL) has been considered and treated as a single malignancy, but divergent outcomes in T‐ALL patients receiving uniform treatment protocols encouraged intensive research on the molecular biology of this disease. Recent findings in the field demonstrate that T‐ALL is much more heterogeneous than originally believed and extremely diverse outcomes of patients require refinement of T‐ALL classification, leading to subtype‐specific adjustment of treatment. Many different biological features of T‐ALL blast cells have recently been found to contribute to disease development and patient outcome and their analysis could potentially be introduced into improved diagnostics and classification of the disease. This review focuses on five key issues of T‐ALL biology: chromosome aberrations, gene expression profiles, gene mutations, DNA methylation patterns, and immunoglobulin/T cell receptor (Ig/TCR) gene rearrangements. Additionally, molecular monitoring of minimal residual disease, by far the most reliable independent prognostic factor in T‐ALL, has been highlighted in the context of Ig/TCR gene rearrangements. Translation of this biological information into better prognostic classification and more effective treatment should lead to improvement of outcome in T‐ALL patients.

Methods of minimal residual disease (MRD) detection in childhood haematological malignancies

The appropriate management of haematological disorders must rely on a precise and long-term monitoring of the patient’s response to chemotherapy and radiotherapy. Clinical data are not sufficient and that is why in the last decade it became the most important to improve the knowledge of haematological diseases on the basis of molecular techniques and molecular markers. The presence of residual malignant cells among normal cells is termed minimal residual disease (MRD). Nowadays a great progress has been made in the treatment of malignant diseases and in the development of reliable molecular techniques, which are characterised by high sensitivity (10−3–10−6) and ability to distinguish between normal and malignant cells at diagnosis and during follow-up. Especially, MRD data based on quantitative analysis (RQ-PCR, RT-RQ-PCR) appear to be crucial for appropriate evaluation of treatment response in many haematological malignancies. Implementation of standardized approaches for MRD assessment into routine molecular diagnostics available in all oncohaematological centres should be regarded nowadays a crucial point in further MRD study development.

Implementation of the standard strategy for identification of Ig/TCR targets for minimal residual disease diagnostics in B-cell precursor ALL pediatric patients: Polish experience

Introduction:Minimal residual disease (MRD), detected based on immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements as markers of residual leukemic cells, is currently the most reliable prognostic factor in acute lymphoblastic leukemia (ALL). A feasibility study is presented of the standard strategy for the identification of Ig/TCR targets for MRD diagnostics in Polish ALL patients by identifying Ig/TCR gene rearrangement pattern using standard primer sets and protocols.Materials and Methods:The PCR-heteroduplex approach based on BIOMED-1 and BIOMED-2 protocols (recommended as the European standard) was used to detect IGH, IGK-Kde, TCRD, TCRG, and TCRB rearrangements in 58 Polish B-cell precursor ALL patients. Sequencing and homology analysis between the obtained and germline Ig/TCR sequences enabled identification of the rearrangements. The U-Gauss test was used for statistical analysis of the Ig/TCR rearrangement pattern in Polish patients compared with relevant data on other nationalities.Results:The following pattern was identified: IGH: 83% (VH-JH: 74%, DH-JH: 9%), IGK-Kde: 41%, TCRD: 78% (incomplete TCRD: 55%, Vδ2-Dδ3: 45%, Dδ2-Dδ3: 21%, Vδ2-Jα: 35%), TCRG: 50%, and TCRB: 13%. Considerable convergence of the Ig/TCR pattern in Polish patients and those of other nationalities (mainly West Europeans) was demonstrated. Statistically relevant differences were only found between the incidence of DH-JH in Polish (9%) and Dutch patients (24%; p<0.05) and Polish and Italian patients (19%; p<0.05), VH-JH in Polish (74%) and Chilean patients (100%; p<0.05), and TCRG in Polish (50%) and Brazilian patients (69%; p<0.05).Conclusions:The convergence of Ig/TCR patterns in Polish and European patients indicates that the strategy for Ig/TCR target identification based on standard primers and protocols might be directly used for the construction of Polish standards and recommendations for MRD diagnostics.

BCL11B, FLT3, NOTCH1 and FBXW7 mutation status in T-cell acute lymphoblastic leukemia patients.

T-cell acute lymphoblastic leukemia is a heterogeneous malignancy originating from developing lymphocyte precursors likely due to mutations in genes regulating thymocyte differentiation. Here, we characterized mutation status of BCL11B and FLT3 genes, presumably involved in T-ALL, together with FBXW7 and NOTCH1 as known players in T-ALL in 65 pediatric T-cell acute lymphoblastic leukemia patients. We also aimed at the assessment of prognostic value of NOTCH1 and FBXW7 mutations in ALL-IC BFM 2002 protocol. FLT3 and BCL11B mutations were detected in 3% and 2% of patients, respectively. FBXW7 mutations were observed in 8% of patients, while NOTCH1 was mutated in 40%. No correlation was found between NOTCH1 and FBXW7 mutations and traditionally used clinical factors or molecular features. In total we have detected nine mutations, which have not been previously described by others. Eight of them were found in NOTCH1 and one in BCL11B gene. Observed frequencies of NOTCH1 and FBXW7 are in line with previous reports, thus confirming postulated participation of these two genes in T-ALL pathomechanism. Moreover, we report on mutation frequency of FLT3 and BCL11B, not extensively studied in T-ALL so far. Finally, we suggest a putative role of BLC11B as an oncogene in T-ALL pathogenesis.

Infant acute bilineal leukemia

Most cases of acute leukemia can be assigned to the myeloid, B or T lineage. There are rare cases of acute leukemia, which cannot be clearly classified, because either blasts express antigens of more than one lineage (acute biphenotypic leukemias) or distinct blast populations of two lineages co-exist (acute bilineal leukemias, aBLL). We present a 10-month-old infant with de novo aBLL, characterized by blasts of monocytic and B-cell precursor lineages. All leukemic cells harbored identical complex MLL gene rearrangement. Despite poor initial response, both to acute lymphoblastic leukemia (ALL) induction treatment and acute myeloid leukemia induction blocks, the child reached complete clinical remission with minimal residual disease negative status and was transplanted. Unfortunately, 16 months from HSCT the patient experienced BM relapse with all blasts characterized by pro-B-ALL immunophenotype. This case report illustrates that aBLL is a very aggressive type of acute leukemia that should be individually treated and monitored, particularly in children less than 1 year of age.

T-cell acute lymphoblastic leukemia from miRNA perspective: Basic concepts, experimental approaches, and potential biomarkers

T-cell acute lymphoblastic leukemia (T-ALL) is a rare, aggressive and heterogeneous malignancy originating from T-cell precursors. The mechanisms of T-ALL pathogenesis related to non-protein coding part of the genome are currently intensively studied. miRNAs are short, non-coding molecules acting as negative regulators of gene expression which shape phenotype of cells in a complex and context-specific manner. miRNAs may act as oncogenes or tumor suppressors; several miRNAs have been related to drug resistance and treatment response in various malignancies. Here we present the review of the state-of-the-art knowledge on the role of miRNAs in T-ALL pathogenesis, with detailed overview of the studies reporting on miRNAs with oncogenic and tumor suppressor potential. We discuss whether miRNAs might be considered candidate biomarkers of prognosis in T-ALL and leukemia subtype-specific markers. We also describe experimental approaches and a typical workflow applied in research on the involvement of miRNAs in oncogenesis.

Association of germline genetic variants in RFC, IL15 and VDR genes with minimal residual disease in pediatric B-cell precursor ALL

Minimal residual disease (MRD) enables reliable assessment of risk in acute lymphoblastic leukemia (ALL). However, little is known on association between MRD status and germline genetic variation. We examined 159 Caucasian (Slavic) patients with pediatric ALL, treated according to ALL-IC-BFM 2002/2009 protocols, in search for association between 23 germline polymorphisms and MRD status at day 15, day 33 and week 12, with adjustment for MRD-associated clinical covariates. Three variants were significantly associated with MRD: rs1544410 in VDR (MRD-day15); rs1051266 in RFC (MRD-day33, MRD-week12), independently and in an additive effect with rs10519613 in IL15 (MRD-day33). The risk alleles for MRD-positivity were: A allele of VDR (OR = 2.37, 95%CI = 1.07–5.21, P = 0.03, MRD-day15); A of RFC (OR = 1.93, 95%CI = 1.05–3.52, P = 0.03, MRD-day33 and MRD-week12, P < 0.01); A of IL15 (OR = 2.30, 95%CI = 1.02–5.18, P = 0.04, MRD-day33). The risk for MRD-day33-positive status was higher in patients with risk alleles in both RFC and IL15 loci than in patients with risk alleles in one locus or no risk alleles: 2 vs. 1 (OR = 3.94, 95% CI = 1.28–12.11, P = 0.024), 2 vs. 0 (OR = 6.75, 95% CI = 1.61–28.39, P = 0.012). Germline variation in genes related to pharmacokinetics/pharmacodynamics of anti-leukemic drugs and to anti-tumor immunity of the host is associated with MRD status and might help improve risk assessment in ALL.

Molecular Aspects of Hematologic Malignancies

Acute lymphoblastic leukemia (ALL) is the most common but also the most successfully treated malignancy in children. Current cure rates of approximately 85 % have been reached through multi-agent therapeutic regimens and particularly through risk-stratification enabling therapy individualization. Nevertheless, relapse is still the main cause of treatment failure. Therefore, the main effort is now focused on improving the outcome of high risk ALL subtypes, i.e., Ph ? ALL, infant ALL, ALL with MLL gene rearrangements, hypodiploid ALL, some T-ALL subsets, recurrent and refractory leukemia. Recent research using advanced molecular techniques, in particular microarray-based genomic gene expression profiling (GEP) and high resolution single nucleotide polymorphism (SNP) microarray approaches, resulted in the identification of novel genetic factors with a potential impact on ALL classification and treatment. The main goal is now M. Dawidowska (&) M. D. Kraszewska Department of Molecular and Clinical Genetics, Institute of Human Genetics, Polish Academy of Sciences, Strzeszyńska 32, 60-479 Poznań, Poland e-mail: ma.dawidowska@wp.pl M. D. Kraszewska e-mail: m.kraszewska@gmail.com K. Derwich Department of Pediatric Oncology, Hematology and Transplantology, Poznań University of Medical Sciences, Szpitalna 27/33, 60-572 Poznań, Poland e-mail: kderwich@poczta.onet.pl T. Szczepański Department of Pediatric Hematology and Oncology, Medical University of Silesia, 3 Maja 13/15, 41-800 Zabrze, Poland e-mail: szczep57@poczta.onet.pl M. Witt et al. (eds.), Molecular Aspects of Hematologic Malignancies, Principles and Practice, DOI: 10.1007/978-3-642-29467-9_1, Springer-Verlag Berlin Heidelberg 2012 3 to translate these findings on ALL blast biology and those on pharmacogenetics of patient response to therapy into improved diagnostics, prognostic classification, and treatment of this malignancy.

Translocation t(8;14)(q24;q11) with concurrent PTEN alterations and deletions of STIL/TAL1 and CDKN2A/B in a pediatric case of acute T-lymphoblastic leukemia: A genetic profile associated with adverse prognosis

We report a pediatric case of acute T‐lymphoblastic leukemia (T‐ALL) with NOTCH1wt, FBXW7wt, STIL/TAL1, and PTEN (exons 2, 3, 4, 5) monoallelic deletions, biallelic CDKN2A/B deletion, and a minor t(8;14)(q24;q11)‐positive subclone. Undetectable by a flow cytometric minimal residual disease assay, the t(8;14)(q24;q11) subclone expanded as detected by fluorescence in situ hybridization from 5% at diagnosis to 26% before consolidation and 100% at relapse bearing a monoallelic deletion (exons 2, 3) with a new frameshift mutation of PTEN and the same set of remaining molecular alterations. This case documents an unfavorable prognostic potential of a co‐occurrence of this set of molecular genetic events and addresses risk stratification in T‐ALL.

Isolation of Mononuclear Cells from Human Blood and Bone Marrow by Density Gradient Centrifugation

Isolation of mononuclear cells (MNC) does not belong to the set of molecular procedures, however, it is the first step of isolation of nucleic acids from peripheral blood or bone marrow in many protocols used for molecular analyses in hemato-oncological diseases. In this chapter, the protocol for isolation of MNCs by density gradient centrifugation is presented. This basic protocol might be subjected to modifications, depending on the specific needs of particular applications.