Dissertations / Theses on the topic 'Lymphoblastic'

Acute lymphoblastic leukemia (ALL) is a neoplasia characterized by an abnormal, clonal and self-maintaining proliferation of lymphoid cells. In this three year of phd I tried to add some information to understand the complex molecular mechanisms underlying this disease. I performed my studies in the laboratory of "Oncoematologia Pediatrica, Dipartimento di Pediatria dell'Università  degli studi di Padova" and for three months in the laboratory of Prof. A. A. Ferrando, Columbia University, Irving Cancer Center, New York. The recombination, insertion and deletion of immunoglobulin (Ig) and T cell receptors (TCR) gene segments results in an individual gene sequence unique for each lymphocytes, named N-region. This genes junctional-region can be considered a fingerprint-marker specific of each lymphocytes and, consequentially, of each lymphoid neoplasia. It can be used to study the biological characteristics of leukemia or to analyze the minimal residual disease (MRD). The initial period of my Phd has been dedicated to the study of the t(4;11) positive ALL, in 32 children above the age of 1 year through the survival, immunophenotype and Ig/TCR pattern analysis. Immunophenotype data in t(4;11)-positive ALL from children 1 year or older demonstrate a more mature pattern of IGH rearrangements as compared to infant cases as like as the more mature age goes with a more mature lymphocyte (Chapter 1.1). Fundamental for the Ig/TCR analysis is to have enough exordia DNA to execute the panel of screening PCR. The screening of diagnosis DNA is hampered by lack of fresh material and consequent limited amount of genomic DNA available. We evaluate the possibility to perform the Ig/TCR screening starting from unstained glass slide smear of cells from bone marrow aspirate or from extracted small amount of DNA after a whole genome amplification with ?29 polymerase and random primers. We execute 476 PCR before and after the whole genome amplification. Results of PCR analysis, confirmed sequencing the PCR products, have a concordance of 98%. No false-positive results were obtained by PCR analysis after whole genome amplification (Chapter 1.2). The main topic of my Phd studies have been the prognostic value of MRD analysis in relapsed pediatric ALL patients. The treatment of ALL in children has improved over the last decades, nevertheless, disease recurrence remains the leading cause of treatment failure in the 20% of patients. In the AIEOP LAL REC2003 protocol, relapsed patient are stratified in class of risk: low-medium (S1-S2) and high risk (S3-S4) based on immunophenotype, time and site of relapse. We evaluate the prognostic value of MRD during the therapeutic treatment in high risk relapsed patients. We studied 60 relapsed patients classified in S3-S4, treated with AIEOP REC2003 protocol. The Ig/TCR clonal profile have been studied with screening PCR and homo-heteroduplex analysis in each patient; the MRD have been exanimate with RQ-PCR in different time-point during therapy: (TP1) after induction, (TP2) after re-induction, (TP3) and after consolidation after relapse. At three year, the EFS is 73, 45 and 19% respectively for patients with TP1 negative, positive not quantifiable (MRD < 10-4) or positive (MRD ? 10-4), (p < 0.05). The statistical significance of MRD predictive prognostic value have been confirmed by multivariate analysis. In high-risk relapsed patients we demonstrated that MRD evaluation was able to identify patients who cold benefit from the chemotherapy treatment and subset of patients who seem not to benefit from further consolidations, including allogenic HSCT (Chapter 2.1). Medullary (BM) relapse is currently defined still according to morphological criteria, when a blast count ? 25% is detected in a bone marrow sample after the achievement of complete remission. We evaluate the clinical significance of MRD monitoring in ALL pediatric patients as an indicator of impending hematological disease recurrence and determinate the critical levels of MRD which can predict relapse. The cumulative incidence of relapse in case of detection of positive and quantifiable MRD finding resulted 85.7%. The value is statistically significant in prediction when compared with the presence of a MRD negative or positive below the quantitative range. The prior identification of MRD as an signal of subsequent morphological hematological relapse could help to decide for a preventive chemotherapeutic treatment (Chapter 2.1). To understand the way to develop new therapeutic treatment for patient with high-risk characteristics, we embrace the hypothesis that a rare cancer stem cell population would be the origin of many malignant neoplasy, responsible of the growth, diffusion and resistance to treatment of tumor, providing a key target for novel curative therapies. In ALL patients this sub-population has not been clearly identify and characterized, anyway recent studies move the attention to the subpopulations characterized by surface antigens CD34/CD38/CD19. We analyzed the Ig/TCR clonal profile of CD34+CD38-CD19+ and CD34+CD38-CD19- subpopulations after sorting, comparing with the Ig/TCR pattern of patients' unsorted blasts. In 9/10 patients the subpopulations of progenitors, isolated and analyzed, share with total blast cells at least one specific clonal rearrangement (finger-print marker). This is a direct demonstration, through a genetic marker and not though a functional assay, of the origin fo blast cell from the subpopulation CD34+CD38-, in the 90% of cases from the sub-fraction CD34+CD38-CD19- (Chapter 3.1). We also studied the correlation between the frequency of CD34+CD38- at the diagnosis of ALL and the level of MRD after treatment. We analyzed 133 ALL patients finding and statistical significant association between the percentage of CD34+CD38- <1% and a low level of MRD at day 33 of chemotherapy (Chapter 3.1). During the last period of the phd I approached the study of WT1, a transcription factor important for normal cellular development and cell survival. The molecular mechanisms involved in disease progression and recurrence of T-ALL are poorly understood. Starting from the observation that in the 10% of T-ALL patients WT1 is mutated, the attention move toward it with genetic and proteomic studies to comprehend its involvement in the development of leukemia. My contribute concerned the study of WT1 gene promoter methylation status (Chapter 4.1).<br>La leucemia linfoblastica acuta (LLA) è una neoplasia caratterizzata da una proliferazione abnorme, clonale ed auto-mantenuta di cellule linfoidi. In questi tre anni di dottorato ho cercato di aggiungere qualche nozione per la comprensione dei complessi meccanismi molecolari che sottendono a questa malattia. L'attività  di ricerca si è svolta presso il Laboratorio di Oncoematologia Pediatrica, Dipartimento di Pediatria dell'Università  degli Studi di Padova e per tre mesi presso il Laboratorio del Prof. A. A. Ferrando, Columbia University Irving Cancer Research Center, New York. Ogni linfocita è contraddistinto a livello dei siti di ricombinazione, inserzione e delezione dei segmenti genici delle regioni variabili delle immunoglobuline (Ig) e del recettore delle cellule T (TCR), da una regione giunzionale detta N-region. Le regioni giunzionali possono essere considerate un fingerprint-marker specifico di ogni linfocita e di ogni neoplasia linfoide ed essere utilizzate per studiare le caratteristiche biologiche della leucemia o per l'analisi della malattia residua minima (MRD). I primi mesi di dottorato sono stati dedicati allo studio della LLA positiva per t(4;11) in 32 pazienti pediatrici di età  superiore a 1 anno attraverso l'analisi dell'immunofenotipo, riarrangiamenti Ig/TCR e prognosi. Dall'analisi è stato identificato un pattern di riarrangiamenti diverso tra i pazienti pediatrici e gli infant dovuto alla diversa maturità  della cellula nelle due classi di pazienti come se la maggiore età  dei pazienti andasse a pari passo con lo stadio maturativo del blasto leucemico (Capitolo 1.1). Fondamentale per l'analisi dei riarrangiamenti Ig/TCR è la disponibilità di materiale per l'estrazione del DNA e l'esecuzione del pannello di PCR di screening. Se si ha una quantità limitata di DNA non è quindi sempre possibile ricavare il pattern di clonalità . Abbiamo eseguito uno studio per valutare la possibilità di eseguire le PCR di screening della regione Ig/TCR su DNA amplificato con una tecnica di "whole genome amplification" basata sull'utilizzo della DNA polimerasi ?29 e di random primer. Il DNA utilizzato era estratto da cellule in sospensione di asiprato midollare, anche in quantità limitata, o da cellule su vetrino non colorato. Abbiamo eseguito 476 PCR prima e dopo "whole genome amplification". Il confronto dei risultati, dopo sequenziamento dei prodotti di PCR, ha mostrato una concordanza dei risultati del 98%. L'amplificazione dell'intero genoma non ha inficiato i risultati di PCR nella regione Ig/TCR (Capitolo 1.2). Il principale campo di interesse in questi anni di dottorato è stata lo studio del valore prognostico della MRM nei pazienti pediatrici LLA ricaduti. La quasi totalità dei pazienti pediatrici con leucemia linfoblastica acuta raggiunge la remissione completa continua ma esiste ancora un 20% di questi che va incontro ad una recidiva di malattia. Il protocollo AIEOP LAL REC 2003 stratifica i pazienti ricaduti in classi di rischio: basso-medio (S1-S2) e alto (S3-S4) a seconda dell'immunofenotipo, del tempo e della sede di ricaduta. Abbiamo valutato il valore prognostico della malattia residua minima durante la terapia nella classe di pazienti ad alto rischio. Sono stati studiati 60 pazienti ricaduti classificati come S3-S4, arruolati nel protocollo AIEOP LAL REC 2003. Per ogni paziente E' stato analizzato il profilo di clonalità  con PCR di screening e l'analisi heteroduplex dei riarrangiamenti Ig/TCR; è stata analizzata la malattia residua minima (MRM) attraverso RQ-PCR in diverse fasi della terapia: dopo l'induzione (TP1), dopo la re-induzione (TP2) e dopo il consolidamento (TP3) post-ricaduta. L'EFS a tre anni è del 73, 45 e 19% rispettivamente per i pazienti con MRD al TP1 negativa, positiva non quantificabile (MRD < 10-4) o positivo (MRD ? 10-4), (P < 0.05). Il valore prognostico predittivo statisticamente significativo dell'MRD è stato confermato dall'analisi multivariata. Abbiamo dimostrato che la quantificazione delle malattia residua minima permette di differenziare i pazienti precocemente e in modo efficace comprendendo quali pazienti rispondono alla terapia convenzionale e possano ricevere il trapianto di cellule staminali allogeniche e quali invece necessitino di terapie innovative (Capitolo 2.1). La ricaduta midollare è attualmente definita secondo criteri morfologici quando la conta dei blasti è ?25% dopo che il paziente ha raggiunto la remissione completa. Abbiamo valutato il potere della presenza di MRM come indicatore di successiva ricorrenza ematologica di ALL determinando se vi siano livelli critici di MRM predittivi di ricaduta. Abbiamo trovato che rilevare un prelievo positivo quantificabile durante il follow-up del paziente è associato a una cumulative relapse incidence dell'85.7%. Questo dato ha valore statisticamente significativo se confrontato con il valore predittivo di un prelievo negativo o positivo non quantificabile per MRM. Identificare anticipatamente la ricaduta potrebbe aiutare nel progettare un trattamento terapeutico preventivo di ricaduta morfologica (Capitolo 2.1). Nella prospettiva di comprendere su quali vie si potrebbero spostare le future strategie terapeutiche per pazienti che presentino caratteristiche di alto rischio, abbiamo abbracciato l'ipotesi attuale che l'origine di molte neoplasie maligne risieda in una ristretta popolazione di cellule staminali tumorali responsabile della crescita, diffusione tumorale e resistenza alla terapia. Nella LLA questa popolazione non è stata ancora chiaramente identificata e caratterizzata anche se studi recenti spingono l'attenzione sulle subpopolazioni caratterizzate dagli antigeni di superficie CD34/CD38/CD19. Abbiamo analizzato in 10 pazienti il profilo di clonalità  Ig/TCR delle popolazioni CD34+CD38-CD19+ e CD34+CD38-CD19- dopo sorting, confrontandolo con quello identificato nella popolazione totale dei blasti leucemici del paziente. In 9/10 pazienti le subpopolazioni di progenitori, isolate e analizzate, condividevano almeno un riarrangiamento genico clonale (fingerprint- marker) con i blasti leucemici. Questa è una dimostrazione diretta, attraverso un marcatore genetico, e non funzionale, dell'origine del blasti leucemici dalla popolazione CD34+CD38-, nel 90% dei casi nella sottopolazione CD34+CD38-CD19- (Capitolo 3.1). Abbiamo inoltre studiato se la frequenza del compartimento CD34+CD38- all'esordio LLA correlasse con il livello di MRM dopo chemioterapia. Abbiamo analizzato 133 pazienti LLA rilevando che una percentuale <1% di CD34+CD38- correla in modo statisticamente significativo con un basso livello di MRM rilevato dopo 33 giorni di terapia (Capitolo 3.1). Durante gli ultimi mesi di dottorato mi sono avvicinata allo studio di WT1, un fattore di regolazione dello sviluppo. I meccanismi molecolari implicati nello sviluppo e nella ricaduta della LLA ad immunofenotipo T sono scarsamente compresi. Partendo dall'osservazione che nel 10% dei pazienti con LLA-T il gene WT1 è mutato, abbiamo approcciato lo studio di questo fattore dal punto di vista genico e proteico, per comprendere il suo coinvolgimento nello sviluppo della leucemia. La parte del lavoro a cui ho contribuito ha riguardato l'analisi dello stato di metilazione del promotore del gene WT1 (Capitolo 4.1).

Thesis (M.S.)--University of California, San Diego, 2009.<br>Title from first page of PDF file (viewed July 22, 2009). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 63-69).

In chapter 1, bone structure, bone growth and development, osteoporosis in children and skeletal morbidities in children with acute lymphoblastic leukaemia (ALL) are discussed. After that, the mechanostat and the effect of whole body vibration (WBV) on bone health are considered. Finally, I examine diagnostic approaches to assess the musculoskeletal system. In chapter 2, the incidence and risk factors for skeletal morbidity in ALL children are determined. The medical records of all (n,186, male,110) children presenting with ALL between 1997 and 2007 and treated on UKALL97, UKALL97/01 or UKALL2003 were studied. Skeletal morbidity included musculoskeletal pain (MSP), fractures and osteonecrosis (ON). MSP was classified as any event of limb pain, muscle pain, joint symptoms or back pain that required radiological examination. Fractures and ON were confirmed by X-rays and MRI respectively. We found that skeletal morbidity, presenting as MSP, fractures or ON were reported in 88(47%) children of whom 56(63%) were boys. Of 88 children, 49(55%), 27(30%) and 18(20%) had MSP, fracture(s) or ON, respectively. Six (7%) had both fractures and ON. The median(10th,90thcentiles) age at diagnosis of ALL children without skeletal morbidity was 3.9years(1.4,12), which was lower than in those with skeletal morbidity at 8.2years(2.2,14.3) (p<0.00001,95%CI:1.7,4.4). Children with ALL diagnosed over 8years of age were at increased risk of developing fracture(s) (p=0.01,odds ratio(OR)=2.9,95%CI:1.3,6.5), whereas the risk of ON was higher in those who were diagnosed after 9years of age (p<0.0001,OR=15,95%CI:4.1,54.4). There was no gender-difference in the incidence of skeletal morbidity. Children who received dexamethasone had a higher incidence of skeletal morbidity than those who were treated with prednisolone (p=0.027,OR=2.6,95%CI:1.1,5.9). We concluded that the occurrence of skeletal morbidity in ALL children may be influenced by age and the type of glucocorticoids (GCs). These findings may facilitate the development of effective bone protective intervention. In chapter 3, the aim is to investigate the influence of physical activity, age and mineral homeostasis over the first 12months of chemotherapy on subsequent skeletal morbidity. We reviewed 56 children who presented with ALL between 2003 and 2007 and treated only on iv UKALL2003. The number of in-patient days over the first 12months of chemotherapy was collected and used as a surrogate marker of inactivity and lack of well-being. Data for serum calcium (Ca), phosphate (Pho), magnesium (Mg) and albumin were also collected over this period. Skeletal morbidity was defined as any episode MSP or fractures. We found that the median duration of in-patient days over the first 12months of treatment in children with no skeletal morbidity was 58days(40,100), whereas the median number of in-patient days during the first 12months in those children with any skeletal morbidity, MSP only or fractures only was 83days(54,131), 81days(52,119) and 91days(59,158), respectively (p=0.003). Children with skeletal morbidity and fractures particularly had lower levels of serum Ca, Mg and Pho compared with those without skeletal morbidity over the first 12 months of chemotherapy. There was a higher risk of skeletal morbidity in those who were diagnosed after the age of 8years (p=0.001,OR=16,CI:3,80). Multiple regression analysis showed that the incidence of skeletal morbidity only had a significant independent association with age at diagnosis (p=0.001) and the number of inpatient days (p=0.03) over the first 12months (r=23). All children who were diagnosed after the age of 8years with an inpatient stay of greater than 75 days in the first 12 months of the chemotherapy (n,14) had some form of skeletal morbidity (OR=64). The conclusion was that the incidence of skeletal morbidity in children receiving chemotherapy for UKALL2003 is associated with a higher likelihood of being older and having longer periods of inpatient stay. The close link between age and changes in bone mineral status may be one explanation for the increased bone morbidity in ALL children In chapter 4, the effects of two WBV regimens on endocrine status, muscle function and markers of bone turnover are compared. We recruited 10adult men with a median age of 33years(29,49), who were randomly assigned to stand on the Galileo platform (GP) (frequency (f)=18-22Hz, peak to peak displacement (D)=4mm, peak acceleration (apeak) =2.6-3.8g) or Juvent1000 (f=32-37Hz, 0.085mm,0.3g) platform (JP) three times/week for a period of eight weeks. The measurements were performed at five time points (T0, T1, T2, T3, T4) and performed in a four week period of run-in (No WBV), eight weeks of WBV and a four-week period of washout (No WBV). The measurements included anthropometries, body composition measured by Tanita, muscle function measured by Leonardo mechanography and biochemical markers of endocrine status and bone turnover. The immediate term effect of WBV at 22Hz was associated with an increase in serum growth hormone (GH), increasing v from 0.07μg/l(0.04,0.69) to 0.52μg/l(0.06,2.4) (p=0.06),0.63μg/l(0.1,1.18)(p=0.03) ,0.21μg/l (0.07,0.65) (p=0.2) at 5minutes, 20minutes and 60minutes after WBV, respectively in the GP group. The immediate term effect of GP at 18Hz was associated with a reduction in serum cortisol from 316nmol/l (247,442) at 60minutes pre-WBV to 173nmol/l(123,245)(p=0.01), 165nmol/l(139,276)(p=0.02) and 198nmol/l(106,294)(p=0.04) at 5minutes, 20minutes and 60minutes post-WBV, respectively. At 22 Hz, GP was associated with a reduction in serum cortisol from 269nmol/l(115,323) at 60minutes before WBV to 214nmol/l(139,394)(p=0.5), 200nmol/l(125,337)(p=0.08) and 181nmol/l(104,306)(p=0.04) at 5minutes, 20minutes and 60minutes post-WBV, respectively. Median serum cortisol decreased after eight weeks of WBV from 333nmol/l(242,445) to 270nmol/l(115,323)(p=0.04). Median serum of the carboxy-terminal telopeptide (CTX, bore resorption marker) reduced significantly after eight weeks of WBV from 0.42ng/ml(0.29,0.90) to 0.29ng/ml(0.18,0.44)(p=0.03). None of these changes were observed in the JP group. Therefore, WBV at a certain magnitude can stimulate GH secretion, reduce circulating cortisol and reduce bone resorption. These effects are independent of clear changes in muscle function and depend on the type of WBV that is administered. In chapter 5, the effect of WBV using GP on the bone health of children receiving chemotherapy for ALL was assessed. We recruited 16children with ALL with a median age of 7.8years(5-13.8; 9males), who were randomized either to receive side-alternating WBV (f=16-20Hz,D=2mm, apeak =1-1.6g)(n,9) or to stand on a still platform as a control group (n,7) for 9minutes, once/week for four months. Measurements were performed at baseline, two-month and four-month assessing bone health (DXA and p.QCT), body composition and muscle function by imaging and biochemical assessment. DXA BMC data were corrected for bone area and presented as BMC z-score. We found that the median compliance rate measured as a ratio of actual completed minutes and expected minutes of WBV was 55%(17,100). The median percentage change of total body BMC z score in the WBV group from baseline to four months dropped by 10%(-25,10)(p=0.1), whereas it was 87%(-203,4)(p=0.07) in the control group. The median lumbar spine BMC z-score (L2-L4) in the WBV group was -0.4(-1.3,0.3) and -0.3(-1.4,1.5) at baseline and four months, whereas the respective data in the control group were 0.04(-0.6,2.4) and -0.1(-1.1,1), respectively. The median percentage change in LS-BMC z-score declined from baseline to four-month by19%(-349,365)(p=0.1) vi and 75%(-1016,178)(p=0.1) in the WBV and control groups, respectively. We concluded that WBV is tolerated by children receiving chemotherapy.

Childhood acute lymphoblastic leukaemia (ALL) does not possess a propagating cell hierarchy, at least as defined by B-cell precursor immunophenotype. Indeed, many, or even all, leukaemic blasts may have the potential to propagate the disease. This unusual characteristic mirrors the substantial capacity for clonal expansion demonstrated by fully differentiated normal lymphoid cells. This Fellowship aimed to investigate the genetic programmes underlying the propagation of acute lymphoblastic leukaemia. An initial candidate approach confirmed the expression of PIWIL2, a gene critical to the maintenance of germline stem cells, in both cell line and primary ALL. Knockdown of PIWIL2 resulted in reduced cellular proliferation and significant prolongation of doubling time in two ALL cell lines, SEM (MLL/AF4) and 697 (E2A/PBX1). Unexpectedly, PIWIL2 was also found to be expressed in peripheral lymphoid cells from healthy donors, but not terminally differentiated cells of myeloid origin, suggesting that PIWIL2 may have a previously unidentified function in both normal and malignant lymphoid cells. A second project has developed an in vitro genome-wide RNAi screen to identify candidate genes involved in the clonal propagation of ALL. This project has assessed a serial re-plating assay using feeder cell co-culture to provide a surrogate niche environment. Initial results have demonstrated the feasibility of such an approach. The benefit of using a co-culture re-plating assay, as compared to a standard suspension culture approach, remains under investigation. ii Finally, this Fellowship developed a protocol for the lentiviral transduction of patient-derived leukaemic blasts and cloned and validated a novel lentiviral vector capable of in vitro analysis, in vivo disease monitoring and RNAi. With these, it will now be possible to validate candidate leukaemic propagation genes in vivo, using primary leukaemic material. The results of these studies will provide candidates for the development of novel therapeutic agents for children with ALL.

The t(12;21)(p13;q22) translocation is present in up to 25% of children with pre-B cell Acute Lymphoblastic Leukaemia (ALL). This translocation involves two transcription factors, TEL (ETV6) and AML (RUNX1), both of which have crucial roles in regulating haematopoiesis. Clinically, TEL-AML1 positive patients have good prognoses. However, late relapses, additional genetic lesions affecting prognosis, and long-term side-effects of chemotherapy remain a cause for concern. In light of recent studies showing genetic and functional heterogeneities in cells responsible for cancer clone maintenance and propagation, targeting common deregulated pathways may be critical for the success of novel therapies. Using Affymetrix GeneChip global gene expression analysis our laboratory previously identified three genes: Tbx2, E2f5 and Lif-R, specifically expressed in TEL-AML1 transduced mouse foetal liver haematopoietic progenitor cells (HPC) cells compared with control cells. Over-expression of these genes was confirmed by real-time qPCR and the specificity of target gene expression was evaluated in human TEL-AML1 positive and negative leukaemia cells. Pathway analysis of TEL-AML1 transcriptional target genes also demonstrated deregulated expression of genes associated with STAT3 signalling, known to be one of the most important pathways required for proliferation and maintenance of multipotency in cancer stem cells. In this study we demonstrate the importance of STAT3 activity in a mouse model of TEL-AML1 overexpression, in human TEL-AML1 positive leukaemia cells and primary human leukaemic samples. Our data indicate a central role for TEL-AML1 in maintaining activated STAT3. This is mediated by transcriptional induction of the Guanine nucleotide exchange factor, ARHGEF4, leading to RAC1 activation and consequent stimulation of STAT3. The latter is necessary for survival, proliferation and self-renewal of TEL-AML1 positive leukaemia through transcriptional induction of MYC expression. In conclusion, we show a novel signalling pathway important for maintenance of t(12;21) leukaemia that constitutes a promising novel therapeutic target for the treatment of this disease.

Dexamethasone (dex) is a key treatment for childhood acute lymphoblastic leukaemia (ALL), but is associated with significant variability in terms of toxicity and efficacy. In this project, the following variables were assessed to better understand how dex personalisation may be achieved: pharmacokinetics, intracellular dex accumulation, glucocorticoid receptor (GR) posttranslational modifications and B-cell maturation state. For pharmacokinetic studies, samples were collected from 154 patients randomised to short (10mg/m2 x 14 days) or standard (6mg/m2 x 28 days) dex induction therapy, as part of the UKALL 2011 trial, and analysed using a validated LC/MS method. Wide pharmacokinetic variability was observed, with AUC0-12h and Cmax significantly higher on the short compared to standard arm. However there was substantial overlap between the two arms, with a number of patients on the standard arm exhibiting higher exposures than those on short therapy. The UKALL 2011 trial found no statistical difference in terms of steroid-related toxicity or MRD response between short and standard dosing. These data suggest that the considerable dex pharmacokinetic variation identified may be a more important factor than variation in dosing regimen. For cellular pharmacology experiments, cell lines, primagraft and primary patient samples were studied. Dex sensitivity was assessed using Alamar Blue assays and GI50 values ranged from 2-1000nM. Western blotting indicated wildtype GR in all samples. Dex accumulation was assessed by LC/MS and flow cytometric analysis of dex-FITC. While patient samples exhibited large variability, dex accumulation was not significantly different between sensitive and resistant cells. Differential dex sensitivity was not accounted for by differences in GR posttranslational modifications, assessed using capillary isoelectric focusing. However, assessment of B-cell maturation using mass cytometry revealed a relationship with dex resistance. Importantly, >50% of patient cell samples had dex GI50 values greater than plasma concentrations observed on either arm of the UKALL 2011 trial. A combined approach incorporating pharmacokinetic assessments and cellular response in ALL cells may allow a more comprehensive understanding of dex pharmacology to optimise its clinical utility.

Acute leukemias, such as acute lymphoblastic leukemia (ALL), are aggressive cancers characterized by the rapid proliferation of malignant hematopoietic cells. Throughout the last 60 years, childhood ALL’s long-term survival rates increased from less than 10% to more than 90%. Despite these improvements, certain subtypes remain hard to manage (e.g. mixed lineage leukemia, MLL-r), and even new therapies frequently fail. Therefore, identifying leukemia-cell restricted antigens in these ALL subtypes remains crucial in the quest to develop novel diagnostic tools and specific treatments. Traditionally, ALL research has mostly been focusing on genomics and transcriptomics efforts, mainly due to the limited amounts of patient material, and technical difficulties throughout sample processing. The potential encompassed in the use of other -omics technologies has remained unexplored in the context of ALL. For the work presented in this thesis, I have focused on undertaking the first comprehensive characterisation of glycocalyx alterations that occur in ALL and particularly in MLL-r in primary, patient derived cancer cells. This work supports the concept that the glycocalyx of ALL and MLL-r cells undergoes dramatic alterations that clearly differentiate these cells from healthy precursor B- (pre-B) cells. These findings might open doors towards novel potential diagnostic and therapeutic targets. The studies encompassed in chapters III, IV and V were performed in collaboration with Prof. Eleonora Heisterkamp’s team at the Beckman Research Institute (City of Hope, CA, USA). I have performed the first multi-omics analyses of primary patient MLL-r cells by integrating data from the transcriptome, glycome, and proteome of these cells. These results revealed that MLL-r cells exhibit distinct glycosylation features that differentiate them from healthy pre-B cells, which I was able to correlate with alterations at the transcript level of relevant glycosyltransferases. In depth proteome analyses revealed an overall good correlation between proteomics and transcriptomics findings, but also uncovered numerous examples where significant changes were just found in one but not the other approach. Nevertheless, this integrated approach used for the systematic evaluation of MLL-r allowed to obtain significant data for putative novel diagnostic/therapeutic protein markers and revealed important features of the disease that remained elusive until now. I was also able to apply the developed integrated multi-omics workflow to investigate the protective role of the surrounding microenvironment and its impact in environmentmediated drug resistance (EMDR) of pre-B ALL cells, which remains a major obstacle for the efficacy of chemotherapeutics in patients. To date the relevance of glycoconjugates for the development of EMDR has been largely unexplored. I explored a long-term co-culture system using human pre-B ALL cells and mitotically inactivated supporting murine stromal cells (OP9 cells), where pre-B ALL cells were put under a selective pressure to survive in the presence of vincristine, a widely used chemotherapeutic drug. I have performed a multi-omics analyses to understand the effect vincristine-resistance has on the cells' glycocalyx. These results demonstrated both glycome-wide and glycoprotein site-specific alterations, which could potentially be employed to identify emerging drug-resistance at an earlier stage or possibly serve as treatment targets in pre-B ALL. Throughout these studies, I have observed a significant modulation of the sialylation profile on pre-B ALL cells in patients and during EMDR development. Unsurprisingly, changes in sialylation have frequently been linked with development and progression of many cancer types but remain largely unexplored in the context of pre-B ALL. I investigated the impact of the major sialyltransferase, ST6Gal1, on the glycome of pre-B ALL cells. ST6Gal1 is the transferase known to be largely responsible for attaching sialic acids in an a2-6 linkage onto N-glycans. Surprisingly, these results demonstrated that a ST6GAL1 knockout did not ablate the production of a2-6 sialylated N-glycans, unless these N-glycans carried a core fucose residue. Demonstrating for the first time how core-fucosylation regulates a2-6 sialylation also allowed me to unravel the existence of ST6Gal1 independent, alternative a2-6 sialylation pathways that are specific for non-fucosylated N-glycans. I demonstrated that ST6GalNAc3-6 are capable to produce a2-6 sialylated N-glycans in the absence of core-fucose and that ST6Gal1 is required to introduce a2-6 sialylation on corefucosylated N-glycans. These results challenge long standing dogmas in glycobiology while delivering a novel understanding of hitherto unknown mechanism that regulate protein glycosylation, which will have a significant impact on our understanding of glycosylation changes in health and disease.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>Institute for Glycomics<br>Griffith Health<br>Full Text

Thesis (Ph. D.)--West Virginia University, 2010.<br>Title from document title page. Document formatted into pages; contains x, 102 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Traditionally, response to treatment in hematological malignancies is evaluated by light microscopy of bone marrow (BM) smears, but due to more effective therapies more sensitive methods are needed. Today, detection of minimal residual disease (MRD) using immunological and molecular techniques can be 100 times more sensitive than morphology. The main aim of this thesis was to compare and evaluate three currently available MRD methods in childhood acute lymphoblastic leukemia (ALL): (i) real-time quantitative PCR (RQ-PCR) of rearranged antigen receptor genes, (ii) multicolor flow cytometry (FCM) of leukemia-associated immunophenotypes and (iii) real-time quantitative PCR of fusion gene transcripts (RT-PCR). In paper I, we assessed the applicability of RQ-PCR in a population-based cohort of childhood ALL diagnosed in Sweden between 2002-2006. Clonal IG/TCR rearrangements were identified in the 96% of the 279 ALL cases. Using RQ-PCR, the quantitative range of 10-3 was reached in 93% of B-cell precursor (BCP) ALL and 86% of T-cell ALL (T-ALL) by at least one target gene. In paper II, we compared MRD detection using both RQ-PCR and FCM in the context of NOPHO ALL-2000 protocol. By applying the stratification threshold of ≥0.1% MRD late during induction therapy (day 29), we could demonstrate that both methods can predict the risk of BM relapse but not extramedullary relapse. However, the threshold of ≥0.2% MRD appears to be more optimal using RQ-PCR in BCP ALL, whilst in T-ALL, the results indicate that RQ-PCR is preferable for MRD assessment. The stability of RNA in vitro is a critical factor when using sensitive molecular techniques such as MRD detection. In paper III, we evaluated the influence on MRD detection when blood is collected in tubes with RNA stabilization reagents (PAX gene Vacutatiner®) compared to collection in EDTA-tubes (non-stabilized). We analyzed 68 matched samples from chronic myeloid leukemia patients and the results indicated that non-stabilized blood processed within 30 hours is preferable for MRD detection. In paper IV, follow-up samples from eight children with Philadelphia positive (Ph+) ALL were evaluated with the three available MRD methods. MRD measured by the fusion gene transcripts (BCR-ABL1) appeared to be the most sensitive method, however, precise quantification can be difficult and the other methods are thus complementary. In conclusion, all three applied MRD methods are useful and correlate to each other, although not necessary exchangeable in individual patients. We also conclude that MRD assessment by RQ-PCR, based on rearranged IG/TCR genes and multicolor FCM are predictive for identification of high risk childhood ALL patients.

Pediatric acute lymphoblastic leukemia (ALL) is the most common malignancy in children, which results from the malignant transformation of progenitor cells in the bone marrow into leukemic cells. The precise mechanisms for this transformation are not well defined, however recent studies suggest that aberrant regulation of gene expression or DNA methylation may play an important role. Hence, the aim of this thesis was to use novel methods to investigate genome-wide gene expression and DNA methylation patterns in a large collection of primary ALL cells from pediatric patients. With these studies, we aimed to increase the understanding of factors that regulate gene expression and DNA methylation in ALL. In the first study of the thesis we found that data obtained from genome-wide digital gene expression analysis enabled excellent cytogenetic subtype-specific classification of ALL cells and revealed new features of gene expression within the disease, such as prevalent antisense transcription and alternative polyadenylation. In the second study we used technology developed for large-scale single nucleotide polymorphism (SNP) genotyping for quantitative analysis of allele-specific gene expression (ASE), revealing widespread ASE in ALL cells. Analysis of DNA methylation in promoter regions of the genes displaying ASE using DNA-microarrays revealed frequent regulation of gene expression by DNA methylation. In the third study, using the same DNA methylation array, we identified differences in the DNA methylation patterns in ALL cells at diagnosis compared to healthy mononuclear cells from the bone marrow of the same children at remission. In the fourth study we measured the DNA methylation of &gt;450,000 CpG sites across the genome in a large collection of ALL samples and non-leukemic control cells. We found that ALL cells displayed highly divergent DNA methylation patterns depending on their cytogenetic subtype and widespread regions of differential methylation were enriched for repressive histone marks. DNA methylation levels at distinct regions in the genome were substantially increased at relapse compared to matched cells from diagnosis. Collectively, the results presented in this thesis provide new insights into the patterns of gene expression and epigenetic changes in ALL and further increase our understanding of the development and progression of the disease, which will hopefully lead to better treatment options in the future.

For many cancers research led to controversial results regarding frequency and identity of cancer stem cells, cells that self-renew and are able to reconstitute the full phenotype of the original malignancy. In some malignancies such as acute myeloid leukaemia the hierarchical stem cell model that suggests that only rare and immunophenotypically immature blasts exhibit stem cell characteristics, resembling the normal physiological haematopoietic hierarchy, has been well established. In contrast to that, the stochastic model states that all or at least a substantial proportion of malignant cells has stem cell potential whereby this is supported by extrinsic stimuli. Initially, several studies suggested that acute lymphoblastic leukaemia (ALL) also is organised in a hierarchy. However, using more immunocompromised mouse strains and refined transplantation techniques for in vivo xenotransplantation models, previous findings regarding frequency and restriction of stem cells to very early B-precursor cell stages have been challenged in more recent studies. In order to address the questions of identity and frequency of stem cells in ALL, a robust orthotopic mouse model with the most immunocompromised mouse strain currently available (NOD/scid IL2Rγnull; NSG) was established. Primary diagnostic patient ALLs or blasts harvested from engrafted mouse bone marrow were sorted for B-cell lineage differentiation markers CD10, CD19, CD20 or CD34 to purify candidate stem cell populations of different maturity. All transplanted cell populations, from the most immature CD34+CD19low stage to the already more differentiated stage of CD19+CD20high cells were able to reconstitute the original ALL in mouse bone marrow and followed a typical dissemination pattern with infiltration of the spleen. Furthermore, this more mature CD19+CD20high subpopulation proved self-renewal ability in serial transplantation experiments. To investigate, whether stem cells in ALL are a rare entity or more abundant, unsorted bulk leukaemia blasts were transplanted in limiting dilutions from 1 x 104 to 10 cells per mouse. Cell numbers required for engraftment varied between leukaemias but the leukaemia engrafted in mice when only 10 to 1,000 cells were transplanted. The limiting dilution experiments were repeated with sorted blast populations according to the surface antigens CD10, CD20 and CD34. Stem cell frequencies in all sorted populations were comparable and as few as 10 to 100 cells were sufficient to reconstitute the leukaemia in mice. Stem cells do not seem to be restricted to immature blast populations as in the hierarchical model but a broad spectrum of different blast immunophenotypes display stem cell capabilities. Furthermore, the frequency of stemness among unsorted bulk ALL cells as well as subpopulations of different maturity according to the blast immunophenotype is high and similar. The results from this thesis provide strong evidence for the stochastic cancer stem cell model in B precursor ALL.

Background: This thesis sits within the arena of weight status during and after childhood acute lymphoblastic leukaemia (ALL), with a particular focus on the prevalence of unhealthy weight status amongst (ALL), Saudi and UK populations. Each chapter in the thesis explores different aspects of unhealthy weight status in ALL which had been highlighted as gaps in the literature at a conference in Puebla, Mexico, at the end of 2006. A summary of each study is given below. Study 1: Background: This study estimated prevalence of unhealthy weight status and metabolic syndrome (MS) amongst Saudi survivors of standard risk ALL. Methods: We recruited 56 survivors, mean age 13.4 years (SD 4.1), a mean of 9.1 years (SD 4.1) post-diagnosis. The BMI for age was used to define weight status relative to national (Saudi) and international (Cole et al., International Obesity Task Force (IOTF), World Health Organisation (WHO), and Centre for Disease Control and Prevention (CDC)) reference data. We measured body composition by dual energy X-ray absorptiometry (DXA), waist circumference, blood pressure, lipid profile (HDL-C, Triglycerides), fasting glucose and insulin. Results: According to international definitions based on BMI for age, around half of the sample had unhealthy weight status. All of the approaches based on BMI for age underestimated over-fatness, present in 27/51 (53%) of the sample according to DXA. Prevalence of MS was 7.1% (3/42 of those over 9-years old) and 5.4% (3/56) by applying the International Diabetes Federation (IDF) definition and National Cholesterol Education Program Third Adult Treatment panel Guidelines (NCEP III), respectively. However, MS by the NCEP III definition was present in 19% of the overweight and obese survivors and 7.1% of the sample had at least two of the components of MS. Conclusions: Unhealthy body weight and over-fatness may be common amongst adolescent Saudi survivors of standard risk ALL, though overweight and obesity may be no more common than in the general Saudi adolescent population. Defining weight status using BMI underestimates over-fatness in this population, as in other populations. Study 2: Background: Underweight, overweight, and obesity at diagnosis may all worsen prognosis in childhood ALL, but no studies have estimated prevalence of unhealthy weight status at diagnosis in large representative samples using contemporary definitions of weight status based on BMI for age. Methods: Retrospective study which aimed to estimate prevalence of underweight, overweight, and obesity at diagnosis for patients with childhood ALL on three successive UK treatment trials: UKALL X (1985-1990, n 1033), UKALL XI (1990- 1997, n 2031), UKALL 97/97-99 (1997-2002, n 898) .The BMI for age was used to define weight status with both UK 1990 BMI for age reference data and the IOTF definitions. Results: Prevalence of underweight was 6% in the most recent trial for which data were available. Prevalence of overweight and obesity was 35% in the most recent trial when expressed using IOTF definitions; 41% when expressed relative to UK 1990 reference data. Conclusions: Even with highly conservative estimates >40% of all UK patients with ALL were underweight, overweight, or obese at diagnosis in the most recent trial for which UK data are available (UKALL 97/99, 1997-2002). Study 3: Background: This study tested the hypothesis that overweight/obesity at diagnosis of childhood ALL was related to risk of relapse. Methods and results: In a national cohort of 1033 patients from the UK there was no evidence that weight status at diagnosis was related significantly to risk of relapse: log ranks test (p value= 0.90) with overweight and obesity as the exposure (n 917); individual (p value= 0.42) and stepwise (p value= 0.96) proportional hazards models, with BMI z score as the exposure (n 1033). Conclusion: The study does not support the hypothesis that overweight/obesity at diagnosis impairs prognosis in childhood ALL in the UK. Study 4: Background: In the sample of Saudi patients recruited to study 1 we compared DXA whole body and lumbar spine bone mineral density (BMD) using manufacturers software with a body size correction which derived bone mineral content (BMC) for bone area and Apparent bone mineral density of lumbar spine (BMADLS). Methods and results: The survivors of ALL were from Saudi Arabia (n 51, mean age 13.5 years). With no corrections, 29 patients (57%) had lumbar spine BMD z score < -1.0 and 21 (41%) had whole body BMD z score < -2. After correction, by using BMC for bone area method only 6 (12%) had lumbar spine BMC z score <-1.0 and 4 (8%) had whole body BMC z score <-2. By using BMADLS method, 18 (35%) had BMC <-1.0 and 6 (11%) had BMC Z score <-2. Conclusions: Correction for body size seems essential to accurate interpretation of DXA bone health data in adolescent survivors of ALL. The three correction methods provided different conclusions, but bone health remains a concern after treatment for ALL.

The single most frequent chromosomal translocation associated with childhood Acute Lymphoblastic Leukaemia is the t(12;21) rearrangement, that creates a fusion gene between TEL (ETV6) and AML1 (RUNX1). Although TELAML1+ patients have a very good prognosis, relapses occur in up to 20% of cases and many patients face long-term side effects of chemotherapy. Our laboratory has previously shown that TEL-AML1 regulates Signal Transducer and Activator of Transcription 3 (STAT3) activation, which is critical for survival of the leukaemic cells. In this study, inhibition of STAT3 in TEL-AML1+ cells results in decreased SMAD7 gene expression. SMAD7 is an antagonist of TGF-β signalling, functioning through a negative feedback mechanism, but is also known to function in other biological pathways. In order to investigate the role of SMAD7 in TEL-AML1+ leukaemia, lentiviral mediated SMAD7 knockdown was performed in human TELAML1+ cell lines. SMAD7 silencing inhibited proliferation of TEL-AML1+ cell lines, eventually leading to growth arrest and apoptosis. Furthermore, our data showed that this effect is not mediated through TGF-β signalling, indicating that SMAD7 was functioning through an alternative pathway. We also observed growth arrest following SMAD7 knockdown in other ALL and AML subtypes. Furthermore, silencing of SMAD7 in TEL-AML1+ ALL cells transplanted into immunodeficient mice impaired disease progression in vivo, resulting in prolonged disease latency. To investigate the essential pathways regulated by SMAD7 in these leukaemic cells, we performed RNA-sequencing analysis on TEL-AML1+ cells following SMAD7 knockdown. Global gene expression analysis revealed SMAD7 to be a regulator of cholesterol biosynthesis, a pathway critical for leukaemia cell survival. Our 4 experiments establish a novel transcriptional pathway operating specifically in t(21;21) ALL, but regulating downstream pathways essential for ALL in general. This study highlights new therapeutic opportunities for ALL.

The intrachromosomal amplification of chromosome 21 (iAMP21) was identified as a novel and prognositically important acquired chromosomal abnormality in childhood acute lymphoblastic leukaemia (ALL). It is defined by multiple copies of the RUNX1 gene, as seen by fluorescence in situ hybridisation (FISH), localised to a single abnormal duplicated chromosome 21 [dup(21)]. The morphological form of this chromosome is highly variable between patients and currently the only reliable method of detection is FISH with probes to RUNX1. Studies of 48 iAMP21 patients using detailed FISH techniques and array-based comparative genomic hybridisation highlighted an extensive region of chromosome 21 involvement. A minimum common region of amplification, between 33.19 and 39.80Mb, including RUNX1 was identified, together with a minimum common region of deletion, between 46.54 and 46.92Mb, in 100% and 77% of patients, respectively. This study established that there were unique patterns of imbalance, with evidence of deletions, inversions and amplification, displayed on the dup(21), between individual patients. This provided evidence of an abnormality that may have arisen from a breakage-fusion-bridge mechanism, possibly initiated by loss of a telomere. Results indicated that iAMP21 represents a distinct genetic subgroup of childhood ALL and is not secondary to a cryptic abnormality of chromosome 21. Two possible variant cases were identified both involving chromosome 15. The abnormality can be distinguished from other numerical abnormalities of chromosome 21 by exploiting the unique pattern of gain, amplification and deletion seen in these patients. This allowed for the development of diagnostic tests based on copy number using either FISH or multiplex ligation dependent probe amplification (MLPA), both of which successfully identified iAMP21 patients.

Acute Lymphoblastic Leukemia (ALL) is the most common childhood cancer. Disease relapse following treatment still occurs in a significant minority of children and the majority of adult patients. The inability to further intensify current treatments due to dose limiting toxicities of chemotherapeutic agents demands the development of new agents. One exciting new treatment, is the mTOR inhibitor everolimus. Preclinical studies using everolimus, while promising, revealed that resistance can emerge following prolonged treatment in vivo. This study uses ALL xenografts that have developed resistance to everolimus by long-term exposure ¬in vivo. This unique resource, combined with proteomic and transcriptome sequencing technology, allows a global approach to analyse the complex biological mechanisms behind the development of resistance to everolimus in ALL. The expression of RNA and protein, the cell cycle distribution of everolimus resistant xenografts as well as the Kaplan Meier survival curves was vastly different between the two ALL xenografts analysed in this study. This indicates that resistance to everolimus is likely to have developed through different mechanisms. The cell cycle distribution of everolimus resistant ALL xenografts also differed depending on the tissues from which they were isolated. Leukemia cells may home to different tissue specific microenvironments that express specific factors that support ALL growth and survival to varying degrees. Furthermore, while individual genes were dissimilar between the two xenografts, there was a common regulation in pathways involved in cellular adhesion and the cytoskeleton. Proteomic sequencing identified 3 proteins possibly involved in everolimus resistance; PDLIM1, Vimentin and Stathmin-1. These proteins are involved with the cytoskeleton and may have a role in the adhesion, migration and cell cycle, yet their exact role in the development of resistance to everolimus is yet to be confirmed. We were unable to correlate the possible mechanisms of resistance identified in the murine model to ALL patients after acute everolimus exposure. We identified a decrease in the expression of the oncogenic micro-RNA, miR-21, though, this was likely due to the immunosuppressive effects of everolimus and did not correlate to patient outcome.

In the past two decades, childhood acute lymphoblastic leukemia (ALL) cure rate has reached over 80% due to treatment advances, but some resistant ALL subtypes, such as those that carry the Philadelphia (Ph+) chromosome, still don’t respond to therapy. The presence of BCR-ABL in ALL children is correlated to a very poor prognosis, nevertheless, several long-scale studies have shown that Ph+ ALL is heterogeneous in terms of clinical parameters and patients respond differently to the therapy, what suggests the presence of additional mechanisms of leukemogenesis. A set of international studies with large series of patients have shown that an earlier remission after induction with glucocorticoids and intrathecal methotrexate (IT MTX) is correlated to a better outcome. In the present study we looked for secondary genetic lesions in a group of 78 consecutive Ph+ ALL children diagnosed in Italy between 2000 and 2010, specifically studying deletions in the IKZF1 gene and the miRNA profile according to the first therapy response. The IKZF1 gene was studied by PCR, direct sequencing and SNP array and a high incidence of deletions (70%) was detected in our children, specially the d4-7(62%). The miRNA signature was analysed by miRNA array comparing two groups of patients differentiated according to MRD/prednisone response. Particularly miR-221, 222 and miR-125b-2 are highly overexpressed in the poor responder group of patients. We showed in vivo that miR-125b-2 acts as oncogene and increases leukemia aggressiveness. Further studies will be necessary to understand how IKZF1, miR-221,222 and 125b act in cooperation with BCR-ABL to induce leukemia. In the future, novel target therapies can emerge as pro-apoptotic approaches to be added to the anti-tyrosine kinase drugs in order to improve response and survivor in Ph+ ALL.

HOXll, the prototypical member of the HOXll family (HOX11, HOXllLl and HOXllL2) was originally discovered as a transcriptional regulator aberrantly expressed in tumours with an immature T-cell phenotype (T-ALL) as a result of specific chromosomal translocations involving T-cell receptor loci. Subsequently, it was revealed that HOXll is required for normal spleen development since newborn Hoxll-/- mice exhibit asplenia. In both its normal and abnormal roles, HOXll has been postulated to function by binding regulatory elements within specific target genes to control gene transcription. However, very few genomic targets of HOX11 have been identified and little is known about its mode of action. In this study, we sought to further understand the role of HOX11 in controlling differentiation and cell growth by 1) determining the identity of genomic sequences that are directly bound by HOXll and 2) determining the identity of proteins which exist within HOXll-containing nuclear complexes. To identify direct HOXll target sequences, a whole genome PCR-based screening method was employed using immobilised recombinant HOXll that had first been expressed as a biologically active GST fusion protein. Using this approach, restriction enzyme-cleaved human genomic DNA was selected for high-affinity HOXll binding sites. Unexpectedly, almost all clones isolated contained sequences derived from satellite 2 DNA that, together with related satellite 3 DNA, is found on most chromosomes at transcriptionally inactive pericentromeric heterochromatin. The specific binding of HOXl1 to satellite 2 DNA was verified by bandshift assays using both recombinant HOXll protein and nuclear extract derived from the T-ALL cell line, ALL-SIL. DNA-protein complexes containing HOX11 were identified by their ablation upon addition of HOXl1 antibody. To confirm that HOXll associates with pericentromeric heterochromatin in vivo, HOXll was characterised in terms of its nuclear localisation during interphase in unsynchronised leukaemic T-cells (ALL-SIL) harbouring a translocation involving the HOXll locus. Using indirect immunofluorescence and confocal microscopy, HOXll antibody produced a punctate pattern of staining in the nucleus with discrete areas of dense staining superimposed on a diffuse distribution of HOXll protein. By dual staining, the bright HOXll foci correlated with centromeres since they overlapped with signals detected by an antibody specific for the centromeric protein CENP-B. Further evidence for a direct interaction of HOXll with satellite 2 DNA was provided by chromatin immunoprecipitation assay. In the presence of HOXll antibody, DNA fragments containing satellite 2 sequences were irnmunoprecipitated from sheared, cross-linked ALL-SIL chromatin but not from chromatin isolated from the HOXll-negative T-cell line PER-1 17. Finally, using a combination of immunoprecipitation with HOXll antibody, gel electrophoresis and mass peptide fingerprinting, a set of nuclear proteins were identified as potential HOXll interactors which are known to either localise to centromeric regions or act as regulators of gene expression. Together, these results implicate HOXl 1 in a functional interaction with centromeric heterochromatin, which may be a key feature of this oncoprotein in terms of both its T-cell transformation and transcriptional regulatory functions.

HOXll, the prototypical member of the HOXll family (HOX11, HOXllLl and HOXllL2) was originally discovered as a transcriptional regulator aberrantly expressed in tumours with an immature T-cell phenotype (T-ALL) as a result of specific chromosomal translocations involving T-cell receptor loci. Subsequently, it was revealed that HOXll is required for normal spleen development since newborn Hoxll-/- mice exhibit asplenia. In both its normal and abnormal roles, HOXll has been postulated to function by binding regulatory elements within specific target genes to control gene transcription. However, very few genomic targets of HOX11 have been identified and little is known about its mode of action. In this study, we sought to further understand the role of HOX11 in controlling differentiation and cell growth by 1) determining the identity of genomic sequences that are directly bound by HOXll and 2) determining the identity of proteins which exist within HOXll-containing nuclear complexes. To identify direct HOXll target sequences, a whole genome PCR-based screening method was employed using immobilised recombinant HOXll that had first been expressed as a biologically active GST fusion protein. Using this approach, restriction enzyme-cleaved human genomic DNA was selected for high-affinity HOXll binding sites. Unexpectedly, almost all clones isolated contained sequences derived from satellite 2 DNA that, together with related satellite 3 DNA, is found on most chromosomes at transcriptionally inactive pericentromeric heterochromatin. The specific binding of HOXl1 to satellite 2 DNA was verified by bandshift assays using both recombinant HOXll protein and nuclear extract derived from the T-ALL cell line, ALL-SIL. DNA-protein complexes containing HOX11 were identified by their ablation upon addition of HOXl1 antibody. To confirm that HOXll associates with pericentromeric heterochromatin in vivo, HOXll was characterised in terms of its nuclear localisation during interphase in unsynchronised leukaemic T-cells (ALL-SIL) harbouring a translocation involving the HOXll locus. Using indirect immunofluorescence and confocal microscopy, HOXll antibody produced a punctate pattern of staining in the nucleus with discrete areas of dense staining superimposed on a diffuse distribution of HOXll protein. By dual staining, the bright HOXll foci correlated with centromeres since they overlapped with signals detected by an antibody specific for the centromeric protein CENP-B. Further evidence for a direct interaction of HOXll with satellite 2 DNA was provided by chromatin immunoprecipitation assay. In the presence of HOXll antibody, DNA fragments containing satellite 2 sequences were irnmunoprecipitated from sheared, cross-linked ALL-SIL chromatin but not from chromatin isolated from the HOXll-negative T-cell line PER-1 17. Finally, using a combination of immunoprecipitation with HOXll antibody, gel electrophoresis and mass peptide fingerprinting, a set of nuclear proteins were identified as potential HOXll interactors which are known to either localise to centromeric regions or act as regulators of gene expression. Together, these results implicate HOXl 1 in a functional interaction with centromeric heterochromatin, which may be a key feature of this oncoprotein in terms of both its T-cell transformation and transcriptional regulatory functions.

Acute lymphoblastic leukaemia (ALL) is a heterogeneous disease of different immuno-phenotypically defined subtypes. Although successful conventional therapies are available, for a proportion of patients (approximately 30%) these are ultimately unsuccessful. ALL relapse is a result of the failure of the immune system to recognise these malignant cells and down regulation of crucial molecules required for cognate CD4+ T-cell recognition has been postulated as a means of immune escape. This study has concentrated on the augmentation of the immunogenicity of B-cell ALL to facilitate the generation of an anti-leukaemic immune response. The initial gene therapy approach to enhancing ALL immunogenicity relied upon the successful transfection of primary ALL cells with a range of constructs carrying immunomodulatory genes, and the development of a protocol to routinely establish ALL cell lines from primary cultures, thus enabling the use of 'low-yield' transfection systems to introduce recombinant DNA into ALL cells. This study was however unable to develop methodology to develop methodology to either transfect the ALL cells or to establish such cell lines in culture. This study does however demonstrate the effectiveness of physiological augmentation of ALL immunogenicity, showing that the CD154+ cell line, 40L.1, induces the upregulation of the expression of a number of crucial molecules involved in antigen recognition. The activation of ALL cells resulting from coculture with the 40L.1 cell line enhances allogeneic and autologous MLR responses to CD40-activated ALL cells and also induces CTL effectors which are capable of lysing wild-type autologous ALL cells. It is therefore conceivable that CD40-mediated activation of ALL could generate an active anti-tumour response in patients, following conventional therapy, reducing the incidence of relapse.

Tubulin-binding agents (TBAs) are employed widely in cancer treatment, including childhood acute lymphoblastic leukaemia (ALL). Despite their success, resistance to TBAs can be a major clinical problem. Mechanisms mediating resistance to TBAs that target the colchicine-binding site on β-tubulin, along with novel therapeutic strategies were investigated. 2-Methoxyestradiol (2ME2) is a multi-targeted TBA active in various cancer types, yet its efficacy and mechanisms(s) of action in haematological malignancies are not well evaluated. To improve understanding of mechanisms(s) of action and drug resistance, leukaemia cells, CCRF-CEM, were selected for resistance to 2ME2. These resistant cells displayed reduced drug-induced mitotic arrest and increased tubulin polymer levels. Moreover, the resistant cells also acquired β-tubulin mutations that affected microtubule stability and induced conformational change(s) to the 2ME2-binding site on β-tubulin. Two of the mutations resided in the colchicine-binding site, yet did not affect colchicine sensitivity, suggesting that 2ME2 and colchicine differ in their binding to β-tubulin. These 2ME2-resistant leukaemia cells have provided novel insights into microtubule stability and drug-target interactions to the tubulin molecule. Differential proteomics analysis identified alterations in the expression of cytoskeletal proteins in the 2ME2-resistant leukaemia cells, including a significant increase in class II β-tubulin. Furthermore, γ-enolase and galectin-1 were up-regulated and class IVb β-tubulin was down-regulated in 2ME2-resistant cells. Gene specific knockdown of class II β-tubulin expression in neuroblastoma cells using small interfering RNA increased sensitivity to 2ME2 and other colchicine-binding agents, suggesting that class II β-tubulin plays a functional role in drug resistance and sensitivity. In a clinically relevant NOD/SCID ALL mouse xenograft model, orally active 2ME2 and its analogue, ENMD-1198, increased survival rates in human ALL xenografts compared to control mice. ENMD-1198 showed better activity than 2ME2 against the ALL xenografts. Combining two microtubule-destabilising agents, ENMD-1198 and vincristine, demonstrated synergistic effect by causing microtubule disruption, angiogenesis and cell proliferation inhibition, and apoptosis induction in leukaemia cells. Importantly, ENMD-1198 and vincristine combination was more potent than single agent treatment in a vincristine-resistant ALL xenograft. In conclusion, this thesis identified novel mechanisms of resistance to 2ME2 and developed a new therapeutic strategy that could improve the treatment of drug refractory childhood ALL.

[Truncated abstract] The treatment of childhood acute lymphoblastic leukaemia (ALL) is one of the great success stories of paediatric oncology, transforming a universally fatal disease into one where 75 to 90% of children are now cured. Although in the past survival for children with T-cell ALL (T-ALL) lagged behind that of children with pre-B ALL, the use of contemporary intensified treatment strategies has significantly diminished this difference, with many investigators reporting similar cure rates for both groups of patients. Despite these marked improvements, numerous challenges still face physicians treating children with T-ALL. Firstly, there have been no additional major improvements in outcome over the last decade, despite additional treatment intensification. Secondly, effective regimens remain elusive for treating children with relapsed T-ALL or patients with resistant disease. Finally, there is a need to identify patients currently potentially overtreated and thus unnecessarily subjected to acute and long term toxicities without benefit. A major challenge therefore, is the identification of novel reliable prognostic markers, in order to identify patients at high risk of relapse and conversely those least likely to relapse, to guide therapy appropriately. Children predicted with a high risk of relapse would be candidates for intensification of therapy and/or novel experimental agents. Conversely, patients predicted to be at low risk of relapse could be offered clinical trials using reduced intensity therapy, thereby minimising toxicity. '...' Crucially, the 3-gene predictor was validated in a completely independent cohort of T-ALL patients, also treated on CCG style therapy. Our 3-gene predictor appears to identify a high risk group of patients which require alternative therapeutic strategies in order to attain a cure. This study has also identified a potential novel agent for the treatment of T-ALL, which may be used as an anthracycline potentiator or anthracycline-sparing agent. We hypothesised that genes associated with a relapse signature provide promising targets for novel therapies. We tested the hypothesis that CFLAR, an inhibitor of the extrinsic apoptotic pathway and a member of the 3-gene predictor may be involved in the development of resistance to chemotherapy. To test our hypothesis we used a novel agent, 2-cyano-3, 12-dioxooleana-1,9 (11)-dien-28-oic acid (CDDO), previously shown to inhibit CFLAR protein, in two cell lines established in our laboratory from paediatric patients diagnosed with T-ALL. We found that CDDO displayed single agent activity at sub-micromolar concentrations in both cell lines tested. Importantly, minimally lethal doses of CDDO resulted in significant enhancement of doxorubicin mediated cytotoxicity in one of the cell lines assessed. The findings presented as part of this thesis have revealed the value of gene expression analysis of childhood T-ALL for identifying novel prognostic markers. This study has shown that expression profiles may provide better prognostic information than currently available clinical variables. Additionally, genes that constitute a relapse signature may provide rational targets for novel therapies, as demonstrated in this study, which assessed a potential novel agent for the treatment of T-ALL.

The TEL-AMLl fusion is the result of t(12;21) (p13;q22), the most frequent chromosomal translocation in childhood B-cell ALL. Recent studies have found a positive correlation with TEL-AMLl and EpoR expression. Three possible mechanisms of up-regulation were investigated in this study: Gene expression analysis, DNA methylation and microRNAs. Microarray analysis compared TEL-AMLl positive and negative gene expression profiles to identify differentially expressed genes. Genes with well known roles in haematopoiesis were also examined. GATA-2 showed a similar expression profile to EpoR, with a higher expression found in TEL-AMLl positive cells. Over-expression of GATA-2 increased EpoR expression further in TEL-AMLl positive cells providing evidence of a link between GATA-2 and EpoR expression in the presence of the fusion protein. DNA methylation analysis of EpoR and GATA-2 promoter regions showed a much higher level of methylation in TEL-AMLl negative cells. After treatment with a demethylating agent EpoR levels did not increase indicating EpoR expression was not epigenetically controlled. However, demethylation of GATA-2 increased expression in both TEL-AMLl positive and negative samples indicating a direct role for DNA methylation with GATA-2 expression. MicroRNAs are short non-coding RNA molecules with the ability to post-transcriptionally regulate genes by binding to the 3•UTR. Taqman microRNA arrays allowed simultaneous analysis of 667 commonly found microRNAs in both TEL-AMLl positive and negative cells. Arrays were analysed and compared with target prediction results to identify microRNAs that could target EpoR and GATA-2. Over-expression of miR-362 showed down-regulation of EpoR protein levels. Over-expression of miR-650 showed down-regulation of both GATA-2 and EpoR. Together these results support a theory of an "EpoR friendly" environment in TEL-AMLl positive cells to aid and promote EpoR expression. This includes an increase in GATA-2 and a decrease in DNA methylation and microRNA expression. These factors are reversed in TEL-AMLl negative cells to hinder EpoR expression.

The morphological analysis of blood smear slides by haematologists or haematopathologists is one of the diagnostic procedures available to evaluate the presence of acute leukaemia. This operation is a complex and costly process, and often lacks standardized accuracy owing to a variety of factors, including insufficient expertise and operator fatigue. This research proposes an intelligent decision support system for automatic detection of acute lymphoblastic leukaemia (ALL) using microscopic blood smear images to overcome the above barrier. The work has four main key stages. (1) Firstly, a modified marker-controlled watershed algorithm integrated with the morphological operations is proposed for the segmentation of the membrane of the lymphocyte and lymphoblast cell images. The aim of this stage is to isolate a lymphocyte/lymphoblast cell membrane from touching and overlapping of red blood cells, platelets and artefacts of the microscopic peripheral blood smear sub-images. (2) Secondly, a novel clustering algorithm with stimulating discriminant measure (SDM) of both within- and between-cluster scatter variances is proposed to produce robust segmentation of the nucleus and cytoplasm of lymphocytic cell membranes. The SDM measures are used in conjunction with Genetic Algorithm for the clustering of nucleus, cytoplasm, and background regions. (3) Thirdly, a total of eighty features consisting of shape, texture, and colour information from the nucleus and cytoplasm of the identified lymphocyte/lymphoblast images are extracted. (4) Finally, the proposed feature optimisation algorithm, namely a variant of Bare-Bones Particle Swarm Optimisation (BBPSO), is presented to identify the most significant discriminative characteristics of the nucleus and cytoplasm segmented by the SDM-based clustering algorithm. The proposed BBPSO variant algorithm incorporates Cuckoo Search, Dragonfly Algorithm, BBPSO, and local and global random walk operations of uniform combination, and Lévy flights to diversify the search and mitigate the premature convergence problem of the conventional BBPSO. In addition, it also employs subswarm concepts, self-adaptive parameters, and convergence degree monitoring mechanisms to enable fast convergence. The optimal feature subsets identified by the proposed algorithm are subsequently used for ALL detection and classification. The proposed system achieves the highest classification accuracy of 96.04% and significantly outperforms related meta-heuristic search methods and related research for ALL detection.

Acute Lymphoblastic Leukemia (ALL) is the most common cancer in children, with close to 200 cases per year in the Nordic countries. Despite recent advances in modern chemotherapies, 20% of the ALL patients experience a relapse. ALL has traditionally been stratified into subtypes with different risk classification and therapy using large genomic aberrations such as translocations and aneuploidies. In recent years technological advances have enabled the detection of smaller genetic variants, such as point mutations and small insertions/deletions. This thesis focuses on the detection of these smaller variants and their potential impact for ALL. The present work includes four studies. In the first study we investigated the effects of whole genome amplification and non-indexed pooling strategies to maximize the output of targeted sequencing. We found that whole genome amplified DNA is equivalent to genomic DNA when screening for point mutations in targeted sequencing data. We were able to accurately detect variants in non-indexed pools with up to ten samples. The second study describes further work on non-indexed pools where we pooled samples in an overlapping scheme and identified carriers of rare variants. The third study describes the whole genome and RNA sequencing of four patients with ALL and validated the results in a cohort of 168 additional ALL patients. In the whole genome sequenced patients we found somatic mutations in both known cancer driver-genes (KRAS and NOTCH1) and in putative driver-genes (KMT2D and KIF1B) after analysis of the additional ALL patients. We validated point mutations genome-wide and observed a large number of C&gt;A mutations in one patient, in contrast to C&gt;T mutations that are more common in cancer in general. The fourth study analyzed the same cohort as the third study and expanded the target to 872 genes linked to cancer, ALL or epigenetic regulation recorded in the literature. We found distinctive differences between BCP-ALL and T-ALL both in number and types of mutations. In addition we observed an association between mutations in the Notch signaling pathway and relapse. These results will have an impact on future studies of cancer, and add another piece to the genetic puzzle of ALL.

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Most ALL cases originate from immature B-cells (BCP-ALL) and are characterized by reoccurring structural genetic aberrations. These aberrations hold information of the pathogenesis of ALL and are used for risk stratification in treatment. Despite increased knowledge of genetic aberrations in pediatric T-cell ALL (T-ALL), no reliable molecular genetic markers exist for identifying patients with higher risk of relapse. The lack of molecular prognostic markers is also evident in patients with relapsed ALL. During the last decades, aberrant epigenetic mechanisms including DNA methylation have emerged as important components in cancer development. Telomere maintenance is another important factor in malignant transformation and is crucial for long-term cell survival. Like DNA methylation, telomere length maintenance has also been implicated to reflect outcomes for patients with leukemia. In this thesis, the prognostic relevance of DNA methylation and telomere length was investigated in pediatric ALL at diagnosis and relapse. The telomere length (TL) was significantly shorter in diagnostic ALL samples compared to normal bone marrow samples collected at cessation of therapy, reflecting the proliferation associated telomere length shortening. Prognostic relevance of TL was shown in low-risk BCP-ALL patients where longer telomeres at diagnosis were associated with higher risk of relapse. Genome-wide methylation characterization by arrays in diagnostic T-ALL samples identified two distinct methylation subgroups denoted CIMP+ (CpG Island Methylator Phenotype high) and CIMP- (low). CIMP- T-ALL patients had significantly worse outcome compared to CIMP+ cases. These results were confirmed in a Nordic cohort treated according to the current NOPHO-ALL2008 protocol.  By combining minimal residual disease (MRD) status at treatment day 29 and CIMP status at diagnosis we could further separate T-ALL patients into risk groups. Likewise, the CIMP profile could separate relapsed BCP-ALL patients into risk groups, where the CIMP- cases had a significantly worse outcome compared to CIMP+ cases.  From these data we conclude that DNA methylation subgrouping is a promising prognostic marker in T-ALL, as well as in relapsed BCP-ALL two groups where reliable prognostic markers are currently missing. By elucidating the biology behind the different CIMP profiles, the pathogenesis of ALL will be further understood and may contribute to new treatment strategies.

The evolution of ALL from presentation to relapse is often accompanied by the emergence of resistance to commonly used chemotherapeutics, including dexamethasone (Dex), a synthetic glucocorticoid that has been the backbone of ALL treatment since the 1950s. In order to investigate the evolution of drug resistance we focused on the L707 cells, a matched Dex-sensitive presentation and Dex-resistant relapse pair from a patient with t(17;19) B ALL. A major genetic difference between presentation and relapse is a 5q deletion spanning 6 genes, including NR3C1, the glucocorticoid receptor and the site of action for Dex. It was found previously that the L707 presentation engrafts and proliferates faster than the relapse cells in the NSG mouse model. The loss of NR3C1 was hypothesised as the cause for reduced fitness of the relapse. This thesis investigated this, and the mechanisms behind the Dex resistance in the L707 relapse. Using shRNA approaches to look at the function of the genes in the relapse 5q deletion identified NR3C1 as the major driver of resistance but did not provide evidence that loss resulted in reduced fitness. Further in depth analysis of the L707 cells using microarrays has shown that the differences between presentation and relapse extend past the genetic differences, including alterations in transcriptional programmes. To identify further novel drivers of resistance, a whole genome in vivo CRISPR screen was carried out, implicating several genes in leukaemic fitness and Dex resistance as well as being a proof of concept for the use of these screens in primary material. Finally, an incidental finding that 697 pre B leukaemic cell line was Dex resistant in vivo, but not in vitro, and examination of these cells using RNA sequencing resulted in the finding that alterations in transcription induced by the murine microenvironment may be responsible for this change.

Malgré les avancés thérapeutiques, le pronostic des leucémies aiguës lymphoblastiques (LAL) reste mauvais. Notre laboratoire a montré que la calcineurine (Cn) est requise pour le maintien à long terme des LAL-T. Sa délétion est associée à la dérégulation de l'expression de plusieurs gènes, dont TRNP1 et NFIB, qui sont nouveaux dans le contexte des LAL-T. Nous avons montré que la suppression de leur expression a un effet délétère sur les cellules leucémiques de LAL-T in vitro et in vivo. Cn est aussi activée dans les LAL-B BCR-ABL+. Des travaux publiés ont proposé que son inhibition par la cyclosporine A (CsA) sensibilise les cellules leucémiques aux inhibiteurs de BCR-ABL. Nous montrons ici que cette sensibilisation est indépendante de l'inhibition de Cn. En effet, son inactivation génique n'affecte ni le maintien, ni la propagation de ces LAL-B in vivo. In vitro, la CsA sensibilise ces cellules leucémiques aux inhibiteurs de BCR-ABL en présence ou non de Cn. Le pronostic des patients atteints de LAL-B est mauvais lorsqu'ils présentent la délétion du gène codant pour le facteur de transcription Ikaros (IKZF1). Dans un modèle murin de LAL-B combinant BCR-ABL avec la perte d'un allèle de IKZF1 dans le lignage B, now montrons une accélération de l'émergence de la maladie et une dérégulation de gènes préférentiellement exprimés dans les cellules souches/progéniteurs hématopoïétiques précoces. Ceci suggère que la perte d'un allèle de IKZF1 permet la reprogrammation des progéniteurs B, cellules d'origine de ces leucémies, vers un stade plus primitif et indifférencié, augmentant ainsi l'agressivité de la maladie<br>Although acute Iymphoblastic leukemia (ALL) cure rates improved in the last decades, outcome for primary resistant and relapsed patients remains poor. Our laboratory showed that calcineurin (Cn) is essential to T-ALL Leukemia Initiating Cells activity. Its deletion is associated with strong anti-leukemic effects and to the deregulation of several genes, including TRNP1 and NFIB, which have never been reported in T-ALL. Here, we demonstrate that silencing of these genes is deleterious to T-ALL cells both in vitro and in vivo, unravelling new actors involved in leukemogenesis. Cn is activated in BCR-ABL+ B-ALL (Ph+ B-ALL) as well and its inhibition by cyclosporin A (CsA) was reported to sensitize leukemic cells to BCR-ABL inhibitors in a mouse model of the disease. We demonstrate here that this sensitization is due to off-target effects of CsA, since Cn genetic deletion did not affect BCR-ABL-induced B-ALL maintenance and propagation in two mouse models of the disease. Moreover, CsA similarly sensitized Cn+ and Cn- leukemic cells to BCR-ABL inhibitors in vitro. The outcome of B-ALL is particularly poor when patients present deletions of IKZF1, the gene encoding the transcription factor Ikaros, which are observed in &gt;800/0 of Ph+ B-ALL cases. Using a mouse model of BCR-ABL-induced B-ALL in which one copy of Ikaros can be specifically deleted in pro-B cells, we observed acceleration of leukemia onset and a stem-cell/early progenitor-like transcriptomic signature. Thus, the loss of one copy of Ikaros allows the reprogramming of the pro/pre-B cell of origin towards a more primitive, undifferentiated state, likely explaining the increased aggressiveness of the disease

Acute lymphocytic leukemia (ALL) is one of the most common types of pediatric cancers. Improvements in treatment within the last 20 years have resulted in reduced mortality and a greater focus upon quality of life. Several researchers have documented neuropsychological impairments in children following treatment for ALL; however, there have not been any comparative studies documenting differences in neuropsychological functioning based upon treatment modality despite the documented effects of radiation therapy and combined radiation/chemotherapy upon the developing brain. In addition, past studies have focused on unitary measures, ignoring the hierarchical relationship between basic cognitive functions and more abstract skills. This study examined the neuropsychological functioning of 81 children who were treated for ALL at a metropolitan children's hospital. All children were tested a minimum of two years after the final treatment session and were administered the NEPSY. Results do not support any interactions or main effects with the exception of the age of the child at diagnosis. Children diagnosed prior to the age of 5 showed greater impairments on tasks measuring attention, memory, and visuospatial reasoning in comparison to peers diagnosed after age 6.

Acute lymphoblastic leukaemia (ALL) is the most common cancer in children, and is characterised by the proliferation of immature lymphoid cells in the bone marrow. The fusion gene TEL/AML1, generated by the chromosome translocation t(12;21), is the most common single genetic aberration in B-lineage ALL, and is formed from the transcription factors TEL and AML1 (coded for by the genes ETV6 and RUNX1, respectively). However, it is still not clear how the presence of TEL/AML1 causes the development of leukaemia. In addition, the importance of a common secondary mutation in TEL/AML1+ leukaemia, the loss of the untranslocated ETV6 allele, is still subject to debate. The role of TEL/AML1 in malignant haematopoiesis has been previously studied in a variety of models, include in vivo mouse models, in vitro TEL/AML1+ cell lines, and ex vivo patient samples. Each of these models has contributed great amounts to our knowledge about TEL/AML1+ leukaemia; however, each type of model has its disadvantages. Here, a new model is presented which aims to complement these other models, based upon human embryonic stem cells (hESCs) expressing a TEL/AML1 transgene. These transgenic hESCs were shown to be capable of multipotent haematopoietic development, including towards B lymphocytes. The consequence of the loss of TEL in TEL/AML1+ leukaemia is not fully known. Here I provide two insights into this frequent secondary mutation. Firstly, a gene expression microarray revealed the transcriptional role of TEL and illuminated how its loss might contribute to progression of TEL/AML1+ leukaemia. Secondly, the functional role of TEL in proliferation and apoptosis was further investigated, which provided further insight about the clonal evolution of TEL/AML1+ leukaemia. In addition, new discoveries were made of potential partners for heterodimeric transcription factor complexes and targets for TEL repression, which provide new avenues for future studies. Taken together, the data presented in this thesis provides the basis for renewed study into TEL/AML1+ leukaemia which could address remaining questions about the disease.

Kreft er den vanligste medisinske dødsårsaken hos barn mellom 1 og 15 år. Akutt lymfatisk leukemi (ALL) er den vanligste kreftformen hos barn og i Norge får ca. 30-40 barn ALL hvert år. Behandlingen strekker seg over 2,5 år og består av 6-12 ulike cellegifter i forskjellige kombinasjoner i tillegg til støttebehandling og overvåkning. Behandlingen medfører mange bivirkninger, bl.a. nedsatt immunforsvar som gir økt risiko for infeksjoner. Den totale overlevelsen for barn med ALL er i dag på ca. 80-85%. Av de som dør etter å ha fått diagnosen skyldes dette for ca. 75% kreftsykdommen i seg selv hvorav de fleste skjer etter tilbakefall av sykdommen (residiv-relaterte dødsfall). De resterende 25% av dødsfallene skyldes bivirkninger av behandlingen, noe som kalles behandlingsrelatert død (TRD, treatment-related death). I denne studien har vi undersøkt forekomst, risikofaktorer og dødsårsaker hos 88 TRD-tilfeller blant 2700 pasienter med ALL i de fem nordiske landene Sverige, Danmark, Finland, Island og Norge. Forekomsten av TRD var 3,2% og denne holdt seg uendret fra den første ALL protokollen (NOPHO ALL 1992) til den neste (NOPHO ALL 2000). Risikofaktorer var kjønn (pike), behandlings-risikogruppe (høy-risk), T-celle sykdom, Down syndrom og gjennomgått beinmargstransplantasjon. Av dødsårsaker skyldtes 75% infeksjoner, 10% blødninger, 10% spesifikk organsvikt og 5% direkte kreftcelle-påvirkning (tumor byrde). Vi har ingen god forklaring på hvorfor noen blir mer syke av behandlingen enn andre. Det er derfor grunn til å tro at genetiske faktorer spiller inn. I de siste 10 årene har man forstått vesentlig mer om den naturlige genetiske variasjon hos menneske. Vi ville undersøke om ulike mønstre av en vanlig forekommende variasjon i det menneskelige genom, nemlig enkeltnukleotidpolymorfisme (eng.: single nuclotide polymorphism, SNP) kunne spille en rolle for risiko for alvorlige infeksjoner under leukemibehandlingen. Vi undersøkte 34000 SNP’er relatert til ALL sykdommen hos barn og fant 24 SNP’er som var assosiert til risiko for infeksjon under behandlingen. Ved hjelp av CART-analyse (classification and regression tree) identifiserte vi 4 SNP’er som inngikk i en SNP-profil som var i stand til å forutsi risiko for infeksjoner de første 50 dagene av behandlingen med stor nøyaktighet. SNP’ene tilhører genene OR51F1, CBR1, POLDIP3 og CCL11 som bl.a. regulerer cellegiftomsetning, cellevekst og betennelsesreaksjoner. Hvis disse funnene blir bekreftet i tilsvarende studier vil denne kunnskapen kunne brukes til bedre å «skreddersy» behandlingen til enkeltpasienter og dermed redusere forekomsten av alvorlige bivirkninger. I moderne genanalyser inngår ofte mange genvarianter samtidig. Det benyttes som regel DNA fra blodprøver, noe som kan være vanskelig å få tak i fra pasienter som ikke lenger er i live. Vi ønsket derfor å undersøke om det var mulig å analysere biologiske markører på arkivmateriale fra leukemipasienter, dvs. beinmargsutstryk og -biopsier. DNA ble ekstrahert fra beinmargsprøvene og deretter kvantitert. Pga. små mengder DNA fra hver pasientprøve ble det gjort hel-genom amplifisering (whole genome amplification, WGA) som et forsøk på å øke DNA mengden i prøvene. Deretter ble prøvene analysert for to typer genmarkører, 10 såkalte korte tandem repetisjoner (eng.: short tandem repeats, STRs) og 34000 SNP’er. Resultatene ble sammenliknet med tilsvarende markørundersøkelser på blod og vi fant at 90% av STR-markørene lot seg gjenskape fra arkivmaterialet. For SNP-markørene fikk vi tilfredsstillende resultat for bare 7 av 34 prøver, men to av prøvene ga svært godt resultat. WGA fungerte ganske bra for noen av prøvene, men dårligere sammenliknet med prøver uten WGA. Konklusjonen ble at bruk av gamle beinmargsprøver til multiple genanalyser er mulig, men analysemetoden må forbedres.<br>Cancer remains the leading cause of disease-related mortality among children aged 1-14 years in developed countries. Acute lymphoblastic leukaemia (ALL) is the most common malignancy in childhood and accounts for about 25% of all childhood cancers. In Norway about 30-40 children are diagnosed with ALL each year. Treatment comprises of a combination of 6-12 different chemotherapy drugs administered over a period of 2.5 years with additional supportive care. Side effects remain a great challenge for both patients and physicians. One of the most common side effects is immunosuppression which increases the risk of infection. Today, the overall survival rate in childhood ALL is 80-85%. About 75% of deaths related to ALL are caused by the disease itself, most after relapse, but about 25% of deaths are caused by treatment and are classified as treatment-related deaths (TRDs). In this study, the incidence, risk factors and causes of death were investigated for 88 TRDs among 2,700 patients in the Nordic countries Sweden, Denmark, Finland, Iceland and Norway. The incidence of TRD was 3.2%, which was stable in the two protocols, the Nordic Society of Paediatric Haematology and Oncology (NOPHO) ALL 1992 and ALL 2000 protocols. The risk factors identified were female gender, treatment risk-group, T-cell disease, Down syndrome and stem cell transplantation. Seventy-five percent of the deaths were related to infection, 10% to bleeding or thrombosis, 10% to organ failure and 5% to the tumour burden. We have no good explanation as to why some patients experience more severe infections than others, even when receiving the same drugs, drug dosage and supportive care. It is reasonable to believe that genetic factors play a role. In accordance with the increasing understanding of host genetic variation the past 10 years, we investigated 34,000 single nucleotide polymorphisms (SNPs) relevant in childhood ALL in a Danish ALL cohort including 69 children and detected 24 SNPs which associated with infections during induction treatment. Through CART-analysis (classification and regression tree) a four-SNP risk profile were identified as highly predictive of risk of infections during the 50 days induction treatment. The four SNPs belong to the genes R51F1, CBR1, POLDIP3 and CCL11 which regulate drug metabolism, cell-growth and inflammation. If these findings are replicated in larger studies, such knowledge may be useful for developing personalized medicine with more tailored therapy and supportive care, which will hopefully reduce the severity of side effects and increase overall survival. Studies involving multiple biological markers, such as SNPs and short tandem repeats (STRs), often use high-quality DNA extracted from blood samples. However, blood samples are not always easy to obtain from study participants, especially if the patient has died. Archival bone marrow samples from patients with leukaemia are available and represent a potential DNA source. We were interested in exploring whether such archival material is usable for the analysis and identification of multiple markers. DNA from 21 bone marrow smears and 13 bone marrow biopsies were extracted and quantified. Because of small amounts of DNA from each sample whole genome amplification (WGA) was applied. Ten different STR-markers and 34,000 SNPs were analysed and compared with corresponding blood samples. For the STR markers 90% detection rates were obtained. Shorter markers (107bp-242bp) from samples stored 0-3 years gave better results compared with longer markers (219bp-317bp) stored 4-10 years. Multiple SNP analysis was more complicated and only seven of 34 archival samples gave acceptable results (SNP call-rates above 50%). However, in two samples, nearly 100% of SNP-markers were detected. Although increasing the total amount of DNA, WGA reduced the analysis quality. In conclusion, DNA from archival bone-marrow samples might be used in multiple marker analysis, but adjustments of the laboratory set-up are essential to optimize this method.

Obesity is a recognized problem for children treated for acute lymphoblastic leukemia (ALL) and is present in roughly one fourth of children by the end of therapy. Obesity may lead to immediate health threats, such as an increased risk for cancer relapse, or may cause future heath issues such as diabetes, metabolic syndrome, hypertension, additional cancers, depression or cardiovascular disease. The purpose of this study was to determine if weight gain during two individual cycles of therapy (Induction or Delayed Intensification Cycle 1) were predictive of obesity (defined as body mass index ≥ 95th percentile for age and gender) at the end of treatment. This study retrospectively examined height and weight data from 1,017 childhood leukemia patients treated on Children's Oncology Group (COG) protocol number 1961. This study included patients that had fully completed therapy on protocol 1961 and who were between the ages of 2-20 years. Percentiles and z-scores for age and gender specific body mass index (BMI) were calculated using the height and weight measurements obtained at the beginning of each cycle of chemotherapy. Univariate and multivariate logistic regression analyses were performed. BMI z-score at the beginning of therapy and difference in BMI z-score during Induction were significant predictors (p<0.0001) of BMI ≥ 95th percentile at the end of maintenance in both males and females. A one unit increase in the difference of BMI z-score during Induction resulted in a 3.03 odds ratio (OR) for obesity at the end of therapy for males (95% CI, 1.90 to 4.84) and a 4.15 OR for females (95% CI, 2.32 to 7.43). The change in BMI z-score during Delayed Intensification I was not found to be significant in relationship to obesity at the end of therapy. Weight gain during Induction consisted of ≥ 20% increase in weight for 3.9% of the study participants. Weight gain during Induction therapy of childhood ALL treatment may be useful in predicting patients at increased risk for obesity development during therapy. Early identification of these at risk patients can assist with interventions aimed at normalizing weight gain during therapy.