Childhood acute leukaemias are a heterogeneous group of malignant diseases. Based on cell origin, clinical manifestations, and molecular/chromosomal changes, we distinguish two main subtypes: acute myeloid leukaemia and acute lymphoblastic leukaemia. Acute lymphoblastic leukaemia (ALL) is the most frequent form of childhood leukaemia. Acute myeloid leukaemia (AML) is predominantly found in adults, being rarer in childhood. In the Czech Republic, the ALL is in childhood diagnosed approximately five times more often compared to AML. Despite the intensive research, aetiology of leukaemia has not been entirely clarified. So far, we only have knowledge of certain risk factors (ionising radiation, some chemicals and viruses) but in the vast majority of cases the aetiopathogenesis has not yet been made clear. Some of the answers may be provided by studies dealing with the presence of (pre)-leukaemic cells in a material archived prior to the clinical onset of the disease. Such are for example the so-called Guthrie cards, the dried blood samples collected immediately after birth and used in screening of the newborns for metabolic disorders. The better availability of material collected before the diagnosis of a secondary leukaemia (originally meant for the follow-up of the primary malignancy) might help us in better...

Acute leukemias are the main type of malignancy affecting children. They are defined by their precursor cell lineage: myeloid lineage for acute myeloid leukemia (AML) and lymphoid lineage for acute lymphoblastic leukemia (ALL). In this thesis, we use systems biology approaches to characterize transcription factor (TF) programs that define novel AML subtypes. We combine this approach with machine learning methods to group patients sharing similar TF programs and risk-stratify them. We identify a 9-cluster solution with statistically significant survival differences ranging from 84% for the best group to 41% for the worst. Each of the clusters is composed of patients with various cytogenetic aberrations that would not necessarily have been classified together. We identify top aberrantly activated TFs and potential master regulators or drug targets in each cluster. We also propose a novel stratification for FLT3-ITD patients with no other cytogenetic abnormalities. These patients are currently all classified as high-risk; however, we find a low-risk subtype and identify a TF signature that is predictive of risk in this subtype. Finally, we develop a binary classifier that is able to stratify the patients into two risk groups. We find that the activity of a large cluster of HOXA TFs is highly correlated with poor prognosis. In the second part, we characterize some mechanisms of relapse in B-ALL at a single-cell resolution focusing again on the patterns of activation and deactivation of TF activity in the course of the disease in matched trios of samples (diagnosis, remission and relapse). After a discussion on some of the technical aspects of differentiating normal cells and leukemic cells at a single-cell RNA sequencing resolution, we perform computational pseudo-lineage reconstruction based on groups of TFs whose activities rise and fall together through pseudotime. We find that each patient has unique mechanisms at the earliest pseudotimes but they seem to converge at the later pseudotimes into signatures in which the B-cell identity (in the case of B-ALL) gradually fades away. We also identify small populations of cells isolated at diagnosis in the later pseudotimes which is consistent with the view that many of the persistent cells in ALL pre-exist the malignancy and are selected by the treatment. This novel systems biology approach for characterizing clinical outcome in patients and defining lineage reconstruction identifies biochemical mechanisms and signaling pathways that are responsible for the development and maintenance of the malignancy and identifies potential therapeutic targets. The results exposed in this thesis will lead to a better understanding of some of the inner workings of pediatric acute leukemias and may lead to the development of improved targeted therapies.

Therapy of acute leukemia may be improved by a more accurate assessment of the effects of treatment on tumor burden and by anticipating relapse with greater precision. The sensitivity limit of assessing residual disease by morphology is usually 5%. Several alternative approaches are available to study minimal residual disease, defined as the presence of leukemic cells not detectable by morphology. These include studies of chromosomal abnormalities by conventional karyotyping, flow cytometry, in situ hybridization and polymerase chain reaction (PCR), investigation of gene rearrangements by Southern blotting and PCR, and immunological methods. Some of these techniques enable the detection of 1 leukemic cells among 10 000 or more normal cells. In the following, the advantages and limitations of sensitive methods for detecting small numbers of leukemic cells are reviewed. The rationale for monitoring residual disease in acute leukemia and the initial results of studies correlating minimal residual disease and clinical outcome are discussed.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

[Truncated abstract] Childhood acute lymphoblastic leukaemia (ALL) accounts for the largest number of cases of childhood cancer (25-35%) and is the primary cause of cancer related morbidity. Today more than 76% of children with ALL are alive and disease free at 5 years. Approximately one in 900 individuals between the ages of 16 and 44 years is a survivor of childhood cancer. In contrast, those patients who relapse with childhood ALL currently have a 6-year event free survival of 20-30%. The short arm of chromosome 9p is mutated or deleted in many cancers including leukaemia. Aberrations of the INK4A/ARF and INK4B loci at the 9p21 band are linked to the development and progression of cancer. In murine cancer models there is evidence to suggest that mutations of Ink4a/Arf and p53 gene loci promote resistance to chemotherapeutic drugs known to trigger apoptosis. The initial aim of this project was to develop an accurate, reproducible method to detect deletions at the INK4A/ARF locus in patient bone marrow specimens. This technique was then applied to detect the incidence of deletions of this locus in childhood ALL specimens. The hypothesis developed was that deletion at the INK4A/ARF locus at diagnosis in childhood ALL is an independent prognostic marker and is involved in disease progression. A secondary aim of this study was to determine which deletions at the INK4A/ARF and INK4B loci are the most relevant in leukaemogenesis in childhood ALL. ... This study has shown that deletion of the INK4A/ARF locus is an independent prognostic indicator in childhood ALL. In addition, the frequency of deletion at the INK4A/ARF and INK4B loci is increased at relapse compared to diagnosis in childhood ALL. In the relapse study group, deletion of the p16INK4A gene at diagnosis was associated with a decreased median time to relapse compared to other genes analysed. Murine studies suggest that such deletions may result in an increased resistance to chemotherapy. If the findings from this study are confirmed in a larger cohort, it is expected that therapeutic interventions based on assessment of the p16INK4A gene in diagnostic childhood ALL specimens will be implemented to prevent relapse in standard risk patients and help to improve the outcome in high risk patients.

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

Hyperleukocytosis is a complication of various leukemias and can result in life-threatening leukostasis. Critical white blood cell (WBC) counts are conventionally defined as higher than 100 × 109/l in acute myeloid leukemia and > 300 × 109/l in acute lymphatic leukemia and other leukemic disorders (e. g. chronic myeloid leukemia). Leukocytapheresis is a therapeutic tool to reduce leukocyte counts in patients with symptomatic or threatening leukostasis until induction chemotherapy works. In patients with temporary contraindications against cytotoxic drugs, e.g. during pregnancy, leukocytapheresis can be used as a bridging therapy until conventional chemotherapy can be started. Therapeutic leukocytapheresis should be performed in specialized centers by experienced, well-trained staff. Thorough monitoring of the patients is extremely relevant. During a single procedure, WBC count can be reduced by 10–70%. Treatment should be repeated daily and can be discontinued when the symptoms of leukostasis have been resolved and/or leukocyte counts have fallen below the critical thresholds. There are no prospective studies evaluating the clinical efficacy of therapeutic leukocytapheresis in patients with hyperleukocytosis. It can be concluded from retrospective studies that leukocytapheresis might have some beneficial effect in early morbidity and mortality of patients with newly diagnosed AML but has no influence on overall long-term survival. Induction chemotherapy is the most important treatment in these patients and must never be postponed. / Leukozytose ist eine Komplikation verschiedener Leukämien und kann zur lebensbedrohlichen Leukostase führen. Als kritische Leukozytenzahlen gelten im Allgemeinen Werte über 100 × 109/l bei akuten myeloischen Leukämien und über 300 × 109/l bei akuter lymphatischer Leukämie und anderen Leukämieformen (z. B. chronisch-myeloische Leukämie). Mittels therapeutischer Leuko zytapherese können pathologisch erhöhte Leukozytenwerte bei Patienten mit symptomatischer oder drohender Leukostase reduziert werden, bis die Wirkung der Induktions-Chemotherapie einsetzt. Bei Patienten mit vorübergehenden Kontraindikationen gegen Zytostatika, wie z.B. in der Schwangerschaft, dient die Leukozytapherese zur Überbrückung des Zeitraums, bis die konventionelle Chemotherapie begonnen werden kann. Leukozytapheresen sollten nur in spezialisierten Zentren von erfahrenem, geschultem Personal durchgeführt werden. Eine sorgfältige Überwachung der Patienten ist von besonderer Bedeutung. Während einer Behandlung kann die Leukozytenzahl um 10–70% reduziert werden. Die Behandlung sollte täglich wiederholt werden, bis die Leukostasesymptomatik abgeklungen bzw. die Leukozytenzahl unter die kritische Interventionsschwelle abgefallen ist. Es mangelt an prospektiven, randomisierten, kontrollierten Studien, die den klinischen Effekt der therapeutischen Leukozytapherese bei Patienten mit Leukostase evaluieren. Retrospektive Studien lassen auf eine therapeutische Wirksamkeit der Leukozytapherese hinsichtlich Frühmorbidität und –mortalität bei Patienten mit neu diagnostizierter AML schließen. Ein Einfluss dieser Therapie auf das Gesamtüberleben von AML-Patienten konnte nicht nachgewiesen werden. Die entscheidende Therapie für diese Patienten ist die Induktions-Chemotherapie, die deshalb auch keinesfalls verzögert werden sollte. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010. / Härtill 5 uppsatser.

[Truncated abstract] Acute lymphoblastic leukaemia (ALL) is the most common form of cancer that affects children and the leading cause of child cancer-related death. There have been dramatic improvements in the 5-year event free survival (EFS) for childhood ALL in recent years, with EFS reaching 75-90% for some forms of the disease. Despite this success, treatment for the disease is aggressive with numerous long and short-term side effects. Many cases of ALL are characterised by chromosomal defects including translocations, variations in chromosome number and the deletion of the tumour suppressor genes. Although these gross chromosomal changes have been extensively studied in childhood ALL, the cascade of altered gene expression that results from these changes has not. Further improvements in survival and the quality of life of survivors relies on a better understanding of the underlying biology of ALL. The primary aim of this study was to determine the gene expression profile of pre-B ALL specimens and normal, or non-malignant, control cells using microarrays in order to further examine the underlying biology of childhood ALL. ... Analysis of the ALL profile with two normal haematopoietic populations demonstrated that ALL specimens have a profile similar to that of CD34+ cells. Specifically, specimens of the MLL subtype had a profile that uniformly resembled that of CD34+ cells. Other subgroups contained specimens with profiles that ranged in similarity to that of CD34+ cells, however, the gene expression profile of all ALL specimens analysed more closely resembled the CD34+ cells than the more differentiated CD19+IgM- cells. This study identified exceptionally high expression of connective tissue growth factor (CTGF/CCN2) in ALL specimens compared to control cells. CTGF expression was v restricted to B-lineage ALL specimens, however, specimens containing the E2A-PBX1 translocation showed low or no expression. Protein studies by Western blot analysis demonstrated the presence of CTGF in ALL cell-conditioned media. The study presented here provides insight into the biology of ALL including the observation that ALL cells have an immature gene expression profile similar to that of CD34+ cells and the possible existence of an autocrine loop involving CTGF. The findings may also have clinical application in the future treatment of ALL, such as the use of metabolic inhibitors or the blocking of CTGF expression. This study provides an important insight into many aspects of ALL disease biology and may offer potential new therapeutic targets for the treatment of ALL.