Academic literature on the topic 'Lymphoblastic'

Abstract Lymphoblasts in bone marrow samples, obtained from 43 children with acute lymphoblastic leukemia at diagnosis, were incubated with 1.0 mumols/L [3H] methotrexate for 24 hours in vitro. Nonexchangeable methotrexate and methotrexate polyglutamates were separated and quantitated. Event-free survival at 5 years was 38% +/- 9% for all 43 patients (27 failures), and 44% +/- 10% for the 35 with non-T, non-B- cell acute lymphoblastic leukemia (20 failures). Of these 35 children, those whose lymphoblasts accumulated more than 100 pmol methotrexate and 500 pmol methotrexate polyglutamates per billion cells experienced better 5-year event-free survival than those whose lymphoblasts did not (65% +/- 12% v 22% +/- 9%, P = .010). This difference characterized “good-risk” patients who were female (P = .014), less than age 7 at diagnosis (P = .005), or had low initial white blood cell counts (less than 20 X 10(9)/L, P = .018). Findings were similar for the 43 children with acute lymphoblastic leukemia and for the “good-risk” children in this total group. Thus, the ability of lymphoblasts to accumulate methotrexate and form methotrexate polyglutamates may be important to the curative properties of current therapy of acute lymphoblastic leukemia in children, particularly for “good-risk” patients. In such patients, inherent rather than acquired drug resistance may be the initial event leading to treatment failure.

Lymphoblasts in bone marrow samples, obtained from 43 children with acute lymphoblastic leukemia at diagnosis, were incubated with 1.0 mumols/L [3H] methotrexate for 24 hours in vitro. Nonexchangeable methotrexate and methotrexate polyglutamates were separated and quantitated. Event-free survival at 5 years was 38% +/- 9% for all 43 patients (27 failures), and 44% +/- 10% for the 35 with non-T, non-B- cell acute lymphoblastic leukemia (20 failures). Of these 35 children, those whose lymphoblasts accumulated more than 100 pmol methotrexate and 500 pmol methotrexate polyglutamates per billion cells experienced better 5-year event-free survival than those whose lymphoblasts did not (65% +/- 12% v 22% +/- 9%, P = .010). This difference characterized “good-risk” patients who were female (P = .014), less than age 7 at diagnosis (P = .005), or had low initial white blood cell counts (less than 20 X 10(9)/L, P = .018). Findings were similar for the 43 children with acute lymphoblastic leukemia and for the “good-risk” children in this total group. Thus, the ability of lymphoblasts to accumulate methotrexate and form methotrexate polyglutamates may be important to the curative properties of current therapy of acute lymphoblastic leukemia in children, particularly for “good-risk” patients. In such patients, inherent rather than acquired drug resistance may be the initial event leading to treatment failure.

Abstract Cancrum oris is an infectious disease, which involves the orofacial tissues and adjacent neighboring structures in its fulminant course. Cancrum oris can occur rarely in chemotherapy-induced neutropenic patients with acute lymphoblastic leukemia. This rare case report reveals that cancrum oris can be a sign of acute lymphoblastic leukemia relapse. It is characterized by gangrenous lesions on skin with fine-needle aspiration cytology smear from the lesion showing infiltration of lymphoblasts. Cancrum oris can occur rarely in chemotherapy-induced neutropenic patients with acute lymphoblastic leukemia, but the presentation as an early sign of extramedullary disease relapse has not been documented in literature. Here we report a case of cancrum oris in a 27-year-old woman who presented with redness and swelling of the upper lip and infranasal area. She had a known case of acute lymphoblastic leukemia diagnosed 6 months back and was on regular multiagent chemotherapy for the same. Clinical examination revealed gangrenous lesions on infranasal, intranasal, and area involving the upper lip. Complete blood count, bone marrow aspiration, and cytology did not show any lymphoblasts. Blood culture and swab culture from the gangrenous area were sterile. Patient did not get better even after 7 days of broad-spectrum antibiotic treatment. With a high degree of clinical suspicion of isolated extramedullary relapse, fine-needle aspiration cytology from marginal and gangrenous area was taken and revealed infiltration of soft tissues by lymphoblasts. After receiving four cycles of multiple chemotherapy drugs, the skin lesions resolved. Treatment of cancrum oris involves the correction of the underlying immune status, antibiotics, and surgical reconstruction. The rare possibility of relapse of acute lymphoblastic leukemia presenting as cancrum oris should be kept in mind in patients with acute lymphoblastic leukemia on chemotherapy with skin manifestations.

Abstract Tyrosine kinases play an important role in normal cellular growth and differentiation. Deregulation of tyrosine kinase activity can result in cellular transformation leading to the development of human cancer. The Mer receptor tyrosine kinase, initially cloned from a human B lymphoblastoid cell line, is expressed in a spectrum of hematopoetic, epithelial, and mesenchymal cell lines. Interestingly, while the RNA transcript of Mer is detected in numerous T and B lymphoblastic cell lines, Mer RNA is not found in normal human thymocytes, lymphocytes or in PMA/PHA stimulated lymphocytes. We have developed an anti-human Mer monoclonal antibody to further study the ectopic Mer expression in lymphoblasts. The antibody detects several forms of the Mer protein including a fully glycosylated 205 kD Mer protein in monocytes and a less glycosylated 170–180 kD Mer protein in acute lymphoblastic leukemia (ALL) cell lines. We analyzed lymphoblasts from 16 T cell ALL patients diagnosed between July 1995 and July 2004 at The Children’s Hospital, Denver for Mer protein surface expression. Of the T cell ALL patient samples, we found that 9/16 (56%) were positive for Mer protein surface expression. Although we were not able to statistically evaluate the clinical outcomes relative to Mer expression due to the study sample number, there was a statistically significant association between positive expression of Mer and lack of surface expression of CD3 (p = 0.035). We found that 7/9 (78%) of the Mer positive lymphoblasts lacked CD3 surface expression, while only 1/6 (17%) of the Mer negative samples lacked CD3 surface expression (see figure). Lymphoblasts that lack surface expression of CD3 represent an immature phenotype and have been associated with a decreased event free survival as compared with surface positive CD3 lymphoblasts. Further investigation of the ectopic expression of Mer in lymphoblasts may reveal the use of this novel Mer glycosylated protein as a prognostic marker and possibly a future therapeutic target in the treatment of childhood leukemia. Figure Figure

Abstract The identification of small numbers of leukemic cells in the cerebrospinal fluid (CSF) presents a diagnostic problem in the treatment of children with acute lymphoblastic leukemia (ALL). We adapted a latex sphere rosetting technique to allow us to identify simultaneously cell surface markers and cell morphology in 199 CSF samples from 34 patients and 14 control subjects. In patients without leukemic meningitis, the majority of CSF lymphocytes (69%) were found to be mature T cells positive for OKT11. A much smaller number of cells (8%) were found to be B cells positive for la. In these children, only 3% of CSF lymphoid cells expressed the common acute lymphoblastic leukemia antigen (CALLA). Similar results were found in the control subjects. By contrast, 28 CSF samples from nine children with varying numbers of CSF lymphoblasts had much greater proportions of CALLA- and la-positive CSF cells (24% to 96%). Leukemic meningitis was present in one of these patients and later developed in four others. However, three patients with small numbers of lymphoblasts present but with low proportions of CALLA-positive CSF cells (less than 5%) subsequently had normal CSF examinations. We found the use of this rosetting technique valuable in providing information complementary to that obtained from cell morphology alone about the possible malignant nature of small numbers of lymphoblast-like CSF cells seen on cytocentrifuge preparations in children with ALL.

The identification of small numbers of leukemic cells in the cerebrospinal fluid (CSF) presents a diagnostic problem in the treatment of children with acute lymphoblastic leukemia (ALL). We adapted a latex sphere rosetting technique to allow us to identify simultaneously cell surface markers and cell morphology in 199 CSF samples from 34 patients and 14 control subjects. In patients without leukemic meningitis, the majority of CSF lymphocytes (69%) were found to be mature T cells positive for OKT11. A much smaller number of cells (8%) were found to be B cells positive for la. In these children, only 3% of CSF lymphoid cells expressed the common acute lymphoblastic leukemia antigen (CALLA). Similar results were found in the control subjects. By contrast, 28 CSF samples from nine children with varying numbers of CSF lymphoblasts had much greater proportions of CALLA- and la-positive CSF cells (24% to 96%). Leukemic meningitis was present in one of these patients and later developed in four others. However, three patients with small numbers of lymphoblasts present but with low proportions of CALLA-positive CSF cells (less than 5%) subsequently had normal CSF examinations. We found the use of this rosetting technique valuable in providing information complementary to that obtained from cell morphology alone about the possible malignant nature of small numbers of lymphoblast-like CSF cells seen on cytocentrifuge preparations in children with ALL.

Introduction : Chronic myelogeneous leukemia (CML) is a myeloproliferative neoplasm that can progress into various conditions. Transformation of CML into acute lymphoblastic leukemia (ALL) is a rare case. Case : A 22-year-old male with history of CML since 2014 and positive BCR-ABL p210 in 2017 came with complaint of weakness. Physical examination showed hepatosplenomegaly. CBC results Hb 7.1 g/dL, WBC 290,620/μL, platelet 434,000/μL. Blood smear evaluation (BSE) suggested CML blastic crisis dd AML-M5. Patient’s condition got worse. CBC result showed WBC 96,770/μL and platelet 7,000/μL in 2 weeks later. BSE was dominated by mononuclear cells with scanty blue cytoplasm, no granules, no auer rods, loose chromatine and indistinct nucleoli, suggesting lymphoblasts with a proportion of 60%. Bone marrow aspiration (BMA) and immunophenotyping was done to confirm BSE. The BMA result was dominated by lymphoblast, consistent with ALL. The immunophenotyping result was CD10+, CD34+(0,99%), CD79a+, HLA-DR+, and CD20+. Molecular examination showed positive RUNX1 and NRAS while negative FLT3, NPM1 and del(5q). Discussion : BCR-ABL gene can be found both in CML and ALL. CML transformation into ALL had been reported to be related with deletion of a transcription gene. Diagnosis of ALL can be established by BMA and immunophenotyping. CD34+ expression of lymphoblast in ALL can be varied, but in this case was minimal. Conclusion : Patient with history of CML showed an ALL picture based on BSE, BMA and immunophenotyping suggesting CML transformation into ALL although CD34+ expression was minimal.

Lymphoblastic lymphoma (LBL) is the second most common non-Hodgkin's lymphoma in childhood. According to modern concepts LBL and acute lymphoblastic leukemia (ALL) are considered as manifestations of the same disease given the similar morphological substrate of the tumor – T and B lymphoblasts. The standard for the treatment of LBL is currently ALL-like riskadapted treatment protocols that allow achieving overall and event-free survival rates of 80–90%. The division into risk groups is based on the stage of the disease and the response to induction therapy. However, the problem of relapse/refractory course of the disease remains a serious problem due to the lack of sufficiently effective therapeutic options. Currently, there is a sufficient amount of clinical data that reliably shows that a number of molecular biological factors can be used to create a new system of into risk groups stratification of patients with LBL. This review focuses on the analysis of various factors that may be responsible for the prognosis of LBL in children.

Abstract Anthracylines play a very important role in the treatment of acute lymphoblastic leukemia (ALL) and relapsed ALL in childhood, however resistance to anthracyclines leads to a poor prognosis. In the present study, we have synthesized two new pinostilbene analogues, i.e. trans-3,4′-dihydroxy-5-methoxystilbene and (E)-resveratrol 4′-O-ß-D-glucopyranoside, which are able to overcome anthracycline resistance in childhood acute lymphoblastic leukemia (ALL) ex vivo and induce apoptosis in established leukemia cell lines via mitochondrial pathway. Apoptosis induction has been investigated by flowcytometric measurement of DNA-fragmentation [LC 50: 10 μM for (1) and (2)], mitochondrial membrane potential reduction and phosphatedylserin-staining on cell membrane surface. Cell death by necrosis could be excluded by a lactatdehydrogenase-release assay. For the first time we analysed the antileukemic and chemopreventive potentials of the pinostilbene analogues (1) and (2) ex vivo in a significant number of primary lymphoblasts of patients suffering from childhood ALL. Patients: Primary lymphoblasts isolated from 22 children with de novo ALL (median: 6.8 years; range 0,6–16.9 years) and relapsed ALL (median:7.2 years) were tested for ex vivo drug response with the anthracyclines daunorubicin (10 μmol/l) and doxorubicin (10 μmol/l) and two new pinostilbene analogues (1) and (2) (10μmol/l), according to their LC50 values in established cell lines. We could demonstrate in these primary cells that the pinostilbenes (1) and (2) were even more effective as compared with the anthracyclines. Out of 22 patients 14 were female (14 de novo ALL) and 8 were male (6 de novo ALL, 2 relapsed ALL). Within these cell populations following immunologic subgroups were found: c-ALL, pre-B-ALL, pro-B-ALL, T-ALL and pre-T-ALL. Results: Daunorubicin induced apoptosis in 6 out of 22 lymphoblast populations (response rate 27,3 %). A similar response rate was observed after treatment with doxorubicin: only 5 of 22 lymphoblast populations responded (22,7%). Nevertheless, far higher response rates were observed for (1) with 11/15 (73,3 %; p<0.005) and for (2) with 15/17 (88,2%; p<0.0002, all p-values by t-test). Interestingly, treatment of daunorubicin-resistant lymphoblasts resulted in significant apoptosis induction in 6 out of 10 cell populations after treatment with compound (1) (response rate 60 %) and in 6/6 after treatment with compound (2) (response rate 100%). Furthermore, pinostilbene (2) showed significant synergistic activity with daunorubicin in 2 of 3 lymphoblast populations. We clearly demonstrated that the ex vivo treatment of lymphoblasts from children with de novo and relapsed ALL with the new pinostilbenes (1) and (2) induced significantly higher response rates than daunorubicin or doxorubicin treatment. In conclusion, the high ex vivo sensitivity of anthracycline resistant leukemia cells to pinostilbene treatment reveals the great proapoptotic and chemopreventive potential of this new class of antileukemic agents.

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).
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.
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.