Dissertations / Theses on the topic 'Leukemia'

Acute Myeloid leukemia (AML) is an aggressive haematological malignancy that mainly affects the elderly. Relapse is common and is thought to be due to the presence of chemotherapy resistant leukemic stem cells (LSC). Within the CD34+ disease (>5% of the blast cells expressing CD34) , two subtypes have been identified; an LMPP/GMPlike expanded type and a MPP/CMP-like expanded type, the former is the most common, accounting for around 80% of CD34+ AML. Both the GMP-like and LMPPlike expanded populations show LSC activity. To improve our understanding of the disease and gain better insight in to how to develop treatments, the molecular basis of the disease needs to be investigated. I investigated miRNAs in the GMP/LMPP-like expanded AML. miRNAs are small non-coding RNAs involved in the regulation of mRNA. In recent years miRNAs have been shown to be implicated in many different diseases. To investigate the role miRNAs play in AML, miRNA expression was profiled in leukemic and normal bone marrow. Bioinformatic analysis was then used to examine the different miRNA expression profiles between normal and leukemic marrow. Our study showed that miRNAs are dysregulated in AML. miRNAs from the miR-17-92 and its paralogous cluster miR-106b-92 were amongst the miRNAs to be found down regulated in AML As had been seen previously at an mRNA level, on an miRNA level the LSC populations more closely resembled more mature progenitor populations than HSC and MPP populations, however the LSC populations did display an aberrant stem cell-like miRNA signature.

Les dérivés de la cytosine sont d’importantes modifications épigénétiques dont le rôle dans l’évolution de la leucémie lymphoïde chronique (LLC) n’est pas totalement exploré. Dans ce contexte, notre première étude vise à examiner le niveau global de la 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) et 5-hydroxymethyluridine (5-hmU) dans des lymphocytes B purifiés de patients LLC (n=56) et d’individus sains (n=17). Les principaux acteurs de la régulation épigénétique (DNMT1/3A/3B, MBD2/4, TET1/2/3, SAT1) ont été évalués par PCR quantitative en temps réel. L’analyse a permis de mettre en exergue trois groupes de patients. En premier lieu, un groupe de patients stables (délai médian de progression [PFS] et délai au premier traitement [TFT] &gt;120 mois), avec un profil épigénétique similaire au groupe contrôle. Deuxièmement, un groupe intermédiaire (PFS=84; TFT=120 mois) qui présente une augmentation de la déméthylation de l’ADN expliquée par l'induction SAT1 / TET2 pendant la progression de la maladie. Troisièmement, un groupe de patients avec une forme active de la maladie (PFS=52; TFT=112 mois) qui présentent une hyperlymphocytose, une réduction du temps de doublement des lymphocytes et des modifications épigénétiques majeures. Au sein de ce groupe, une réduction est observée pour la 5-mCyt, 5-hmCyt, 5-CaCyt et serait associée à une diminution des DNMTs, TETs et MBDs au cours de la progression de la maladie. Les profils épigénétiques mis en évidence sont indépendants du statut mutationnel IGHV mais sont associés avec les anomalies cytogénétiques. Nous nous sommes également intéressés à cette association et nous avons montré dans la deuxième étude que les modifications des dérivées de la cytosine peuvent affiner le pouvoir pronostic des anomalies cytogénétiques. En conclusion, nos résultats suggèrent que les variations de la méthylation ainsi que des intermédiaires de la déméthylation de l’ADN sont impliqués dans la progression de la LLC<br>Cytosine derivatives are important epigenetic modifications whose role in the pathogenesis and evolution of chronic lymphocytic leukemia (CLL) is not fully explored. In this context, our first study aims to examine the global DNA level of 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) and 5-hydroxymethyluridine (5-hmU) in purified B lymphocytes of CLL patients (n = 56) and healthy individuals (n = 17). The main actors in epigenetic regulation (DNMT1 / 3A / 3B, MBD2 / 4, TET1 / 2/3, SAT1) were evaluated by quantitative real time PCR. The analysis highlighted three groups of patients. First, a group of patients with stable disease (median time to progression [PFS] and time to first treatment [TFT]&gt; 120 months), with an epigenetic profile similar to the control group. Secondly, an intermediate group (PFS = 84, TFT = 120 months) which shows an increase in DNA demethylation explained by SAT1 / TET2 induction during disease progression. Third, a group of patients with an active form of the disease (PFS = 52, TFT = 112 months) who have hyperlymphocytosis, a short lymphocyte doubling time, and major epigenetic changes. Within this group, a reduction is observed for 5-mCyt, 5-hmCyt, 5-CaCyt which is associated with a decrease in DNMTs, TETs and MBDs during disease progression. The identified epigenetic profiles are independent of IGHV mutational status but are associated with cytogenetic abnormalities. We were also interested in this association and we showed in the second study that modifications of cytosine derivatives levels can refine the prognostic power of cytogenetic abnormalities.In conclusion, our results suggest that methylation variations as well as DNA demethylation intermediates are involved in the progression of CLL

In Leukemias, recent developments have demonstrated that the Hedgehog pathway plays a key-role in the peculiar ability of self renewal of leukemia stem cells. The aim of this research activity was to investigate, through a first in man, Phase I, open label, clinical trial, the role and the impact, mainly in terms of safety profile, adverse events and pharmacokinetics, of a Sonic Hedgehog inhibitor compound on a population of heavely pretreated patients affected by AML, CML, MF, or MDS, resistant or refractory to standard chemotherapy. Thirty-five patients have been enrolled. The drug was administered orally, in 28 days cycles, without rest periods. The compound showed a good safety profile. The half life was of 17-35 hours, justifying the daily administration. Significant signs of activity, in terms of reduction of bone marrow blast cell amount were seen in most of the patients enrolled. Interestingly, correlative biological studies demonstrated that, comparing the gene expression profyiling signature of separated CD34+ cells before and after one cycle of treatment, the most variably expressed genes were involved in the Hh pathway. Moreover, we observed that many genes involved in MDR (multidrug resistance)were significantly down regulated after treatment. These data might lead to future clinical trials based on combinatory approaches, including, for instance, Hh inhibitors and conventional chemotherapy.

In Leukemias, recent developments have demonstrated that the Hedgehog pathway plays a key-role in the peculiar ability of self renewal of leukemia stem cells. The aim of this research activity was to investigate, through a first in man, Phase I, open label, clinical trial, the role and the impact, mainly in terms of safety profile, adverse events and pharmacokinetics, of a Sonic Hedgehog inhibitor compound on a population of heavely pretreated patients affected by AML, CML, MF, or MDS, resistant or refractory to standard chemotherapy. Thirty-five patients have been enrolled. The drug was administered orally, in 28 days cycles, without rest periods. The compound showed a good safety profile. The half life was of 17-35 hours, justifying the daily administration. Significant signs of activity, in terms of reduction of bone marrow blast cell amount were seen in most of the patients enrolled. Interestingly, correlative biological studies demonstrated that, comparing the gene expression profyiling signature of separated CD34+ cells before and after one cycle of treatment, the most variably expressed genes were involved in the Hh pathway. Moreover, we observed that many genes involved in MDR (multidrug resistance)were significantly down regulated after treatment. These data might lead to future clinical trials based on combinatory approaches, including, for instance, Hh inhibitors and conventional chemotherapy.

The CBFbeta-SMMHC fusion protein is expressed in acute myeloid leukemia (AML) samples with the chromosome inversion inv(16)(p13;q22). This fusion protein binds the transcription factor RUNX with higher affinity than its physiological partner CBFbeta and disrupts the core binding factor (CBF) activity in hematopoietic stem and progenitor cells. Studies in the Castilla laboratory have shown that CBFbeta-SMMHC expression blocks differentiation of hematopoietic progenitors, creating a pre-leukemic progenitor that progresses to AML in cooperation with other mutations. However, the combined function of cumulative cooperating mutations in the pre-leukemic progenitor cells that enhance their expansion to induce leukemia is not known. The standard treatment for inv(16) AML is based on the use of non-selective cytotoxic chemotherapy, resulting in a good initial response, but with limited long-term survival. Therefore, there is a need for developing targeted therapies with improved efficacy in leukemic cells and minimal toxicity for normal cells. Here, we used conditional Nras+/LSL-G12D; Cbfb+/56M; Mx1Cre knock-in mice to show that allelic expression of oncogenic N-RasG12D expanded the multi-potential progenitor (MPP) compartment by 8 fold. Allelic expression of Cbfbeta-SMMHC increased the MPPs and short-term hematopoietic stem cells (ST-HSCs) by 2 to 4 fold both alone and in combination with N-RasG12D expression. In addition, allelic expression of oncogenic N-RasG12D and Cbfbeta-SMMHC increases survival of pre-leukemic stem and progenitor cells. Differential analysis of bone marrow cells determined that Cbfb+/MYH11 and Nras+/G12D; vii Cbfb+/MYH11 cells included increased number of blasts, myeloblasts and promyelocytes and a reduction in immature granulocytes, suggesting that expression of N-RasG12D cannot bypass Cbfbeta-SMMHC driven differentiation block. N-RasG12D and Cbfbeta-SMMHC synergized in leukemia, in which Nras+/G12D; Cbfb+/MYH11 mice have a shorter median latency than Cbfb+/MYH11 mice. In addition, the synergy in leukemogenesis was cell autonomous. Notably, leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC showed higher (over 100 fold) leukemia-initiating cell activity in vivo than leukemic cells expressing Cbfbeta-SMMHC (L-IC activity of 1/4,000 and 1/528,334, respectively). Short term culture and biochemical assays revealed that pre-leukemic and leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC have reduced levels of pro-apoptotic protein Bim compared to control. The Nras+/G12D; CbfbMYH11 pre-leukemic and leukemic cells were sensitive to pharmacologic inhibition of MEK/ERK signaling pathway with increasing apoptosis and Bim protein levels but not sensitive to PI3K inhibitors. In addition, knock-down of Bcl2l11 (Bim) expression in Cbfbeta-SMMHC pre-leukemic progenitors decreased their apoptosis levels. In collaboration with Dr. John Bushweller’s and other research laboratories, we recently developed a CBFbeta-SMMHC inhibitor named AI-10-49, which specifically binds to CBFbeta-SMMHC, prevents its binding to RUNX proteins and restores CBF function. Biochemical analysis in human leukemic cells showed that AI-10-49 has significant specificity in reducing the viability of leukemic cells expressing CBFbeta-SMMHC (IC50= 0.83μM), and negligible toxicity in normal cells. Likewise, mouse Nras+/G12D; viii Cbfb+/MYH11 leukemic cells were sensitive to AI-10-49 (IC50= 0.93μM). By using the NrasLSL-G12D; Cbfb56M mouse model, we also show that AI-10-49 significantly prolongs the survival of mice bearing the leukemic cells. Preliminary mechanistic analysis of AI-10-49 activity has shown that AI-10-49 increased BCL2L11 transcript levels in a dose and time dependent manner in murine and human leukemic cells, suggesting that the viability through BIM-mediated apoptosis may be targeted by both oncogenic signals. My thesis study demonstrates that Cbfbeta-SMMHC and N-RasG12D promote the survival of pre-leukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define NrasLSL-G12D; Cbfb56M mice as a valuable genetic model for the study of inv(16) AML targeted therapies. For instance, the novel CBFbeta-SMMHC inhibitor AI-10-49 shows a significant efficacy in this mouse model. This small molecule will serve as a promising first generation drug for targeted therapy of inv(16) leukemia and also a very useful tool to understand mechanisms of leukemogenesis driving by CBFbeta-SMMHC.

The CBFbeta-SMMHC fusion protein is expressed in acute myeloid leukemia (AML) samples with the chromosome inversion inv(16)(p13;q22). This fusion protein binds the transcription factor RUNX with higher affinity than its physiological partner CBFbeta and disrupts the core binding factor (CBF) activity in hematopoietic stem and progenitor cells. Studies in the Castilla laboratory have shown that CBFbeta-SMMHC expression blocks differentiation of hematopoietic progenitors, creating a pre-leukemic progenitor that progresses to AML in cooperation with other mutations. However, the combined function of cumulative cooperating mutations in the pre-leukemic progenitor cells that enhance their expansion to induce leukemia is not known. The standard treatment for inv(16) AML is based on the use of non-selective cytotoxic chemotherapy, resulting in a good initial response, but with limited long-term survival. Therefore, there is a need for developing targeted therapies with improved efficacy in leukemic cells and minimal toxicity for normal cells. Here, we used conditional Nras+/LSL-G12D; Cbfb+/56M; Mx1Cre knock-in mice to show that allelic expression of oncogenic N-RasG12D expanded the multi-potential progenitor (MPP) compartment by 8 fold. Allelic expression of Cbfbeta-SMMHC increased the MPPs and short-term hematopoietic stem cells (ST-HSCs) by 2 to 4 fold both alone and in combination with N-RasG12D expression. In addition, allelic expression of oncogenic N-RasG12D and Cbfbeta-SMMHC increases survival of pre-leukemic stem and progenitor cells. Differential analysis of bone marrow cells determined that Cbfb+/MYH11 and Nras+/G12D; vii Cbfb+/MYH11 cells included increased number of blasts, myeloblasts and promyelocytes and a reduction in immature granulocytes, suggesting that expression of N-RasG12D cannot bypass Cbfbeta-SMMHC driven differentiation block. N-RasG12D and Cbfbeta-SMMHC synergized in leukemia, in which Nras+/G12D; Cbfb+/MYH11 mice have a shorter median latency than Cbfb+/MYH11 mice. In addition, the synergy in leukemogenesis was cell autonomous. Notably, leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC showed higher (over 100 fold) leukemia-initiating cell activity in vivo than leukemic cells expressing Cbfbeta-SMMHC (L-IC activity of 1/4,000 and 1/528,334, respectively). Short term culture and biochemical assays revealed that pre-leukemic and leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC have reduced levels of pro-apoptotic protein Bim compared to control. The Nras+/G12D; CbfbMYH11 pre-leukemic and leukemic cells were sensitive to pharmacologic inhibition of MEK/ERK signaling pathway with increasing apoptosis and Bim protein levels but not sensitive to PI3K inhibitors. In addition, knock-down of Bcl2l11 (Bim) expression in Cbfbeta-SMMHC pre-leukemic progenitors decreased their apoptosis levels. In collaboration with Dr. John Bushweller’s and other research laboratories, we recently developed a CBFbeta-SMMHC inhibitor named AI-10-49, which specifically binds to CBFbeta-SMMHC, prevents its binding to RUNX proteins and restores CBF function. Biochemical analysis in human leukemic cells showed that AI-10-49 has significant specificity in reducing the viability of leukemic cells expressing CBFbeta-SMMHC (IC50= 0.83μM), and negligible toxicity in normal cells. Likewise, mouse Nras+/G12D; viii Cbfb+/MYH11 leukemic cells were sensitive to AI-10-49 (IC50= 0.93μM). By using the NrasLSL-G12D; Cbfb56M mouse model, we also show that AI-10-49 significantly prolongs the survival of mice bearing the leukemic cells. Preliminary mechanistic analysis of AI-10-49 activity has shown that AI-10-49 increased BCL2L11 transcript levels in a dose and time dependent manner in murine and human leukemic cells, suggesting that the viability through BIM-mediated apoptosis may be targeted by both oncogenic signals. My thesis study demonstrates that Cbfbeta-SMMHC and N-RasG12D promote the survival of pre-leukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define NrasLSL-G12D; Cbfb56M mice as a valuable genetic model for the study of inv(16) AML targeted therapies. For instance, the novel CBFbeta-SMMHC inhibitor AI-10-49 shows a significant efficacy in this mouse model. This small molecule will serve as a promising first generation drug for targeted therapy of inv(16) leukemia and also a very useful tool to understand mechanisms of leukemogenesis driving by CBFbeta-SMMHC.

The human Philadelphia chromosome (Ph) arises from a translocation between chromosomes 9 and 22 [t(9;22)(q34;q11)]. The resulting chimeric BCR-ABLoncogene encodes a constitutively activated, oncogenic tyrosine kinase that induces chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). The BCR-ABL tyrosine kinase inhibitor (TKI), imatinib mesylate, induces a complete hematologic and cytogenetic response in the majority of CML patients, but is unable to completely eradicate BCR-ABL–expressing leukemic cells, suggesting that leukemia stem cells are not eliminated. Over time, patients frequently become drug resistant and develop progressive disease despite continued treatment. Two major reasons cause the imatinib resistance. The first one is the BCR-ABL kinase domain mutations which inhibit the interaction of BCR-ABL kinase domain with imatinib; the second one is the residual leukemia stem cells (LSCs) in the patients who are administrated with imatinib. To overcome these two major obstacles in CML treatment, new strategies need further investigation. As detailed in Chapter II, we evaluated the therapeutic effect of Hsp90 inhibition by using a novel water-soluble Hsp90 inhibitor, IPI-504, in our BCR-ABL retroviral transplantation mouse model. We found that BCR-ABL mutants relied more on the HSP90 function than WT BCR-ABL in CML. More interestingly, inhibition of HSP90 in CML leukemia stem cells with IPI-504 significantly decreases the survival and proliferation of CML leukemia stem cells in vitro and in vivo. Consistent with these findings, IPI-504 treatment achieved significant prolonged survival of CML and B-ALL mice. IPI-504 represents a novel therapeutic approach whereby inhibition of Hsp90 in CML patients and Ph+ ALL may significantly advance efforts to develop a cure for these diseases. The rationale underlying the use of IPI-504 for kinase inhibitor–resistant CML has implications for other cancers that display oncogene addiction to kinases that are Hsp90 client proteins. Although we proved that inhibition of Hsp90 could restrain LSCs in vitro and in vivo, it is still unclear how to define specific targets in LSCs and eradicate LSCs. In Chapter III, we took advantage of our CML mouse model and compared the global gene expression signature between normal HSCs and LSCs to identify the downregulation of Pten in CML LSCs. CML develops faster when Pten is deleted in Ptenfl/fl mice. On the other hand, Pten overexpression significantly delays the CML development and impairs leukemia stem cell function. mTOR is a major downstream of Pten-Akt pathway and it is always activated or overepxressed when Pten is mutated or deleted in human cancers. In our study, we found that inhibition of mTOR by rapamycin inhibited proliferation and induced apoptosis of LSCs. Notably, our study also confirmed a recent clinical report that Pten has been downregulated in human CML patient LSCs. In summary, our results proved the tumor suppressor role of Pten in CML mouse model. Although the mechanisms of Pten in leukemia stem cells still need further study, Pten and its downstream, such as Akt and mTOR, should be more attractive in LSCs study.

Background Chronic myeloid leukemia (CML) is a relatively rare hematological malignancy with a constant incidence of approximately 90 new cases each year in Sweden (0.9 cases/100 000 inhabitants). The etiology is largely unknown but high doses of ionizing radiation are a known but rare risk factor. The treatment options were for a long time limited to chemotherapies i.e. hydroxyurea and busulfan, interferon’s and allogeneic hematopoietic stem cell transplantation and the median survival were only about four years. Since the beginning of the 21st century a new way of treating CML has been introduced, the tyrosine kinase inhibitors (TKI), leading to a rapid decrease in leukemic cells and symptoms. Due to the TKIs, the overall 5-year survival is nowadays approximately 85 % and CML patients have time to develop other diseases, including other malignancies. The aims of this thesis was to investigate the present and future prevalence of CML and the prevalence of other malignancies prior and subsequent to the diagnosis of CML, malignancies among first-degree relatives of persons with CML. In addition, the incidence of autoimmune and chronic inflammatory diseases among patients with CML was also investigated.   Methods From the Swedish CML register, data over nearly all Swedish CML patients from 2002 and forward were obtained for paper II-IV. For paper I, the Swedish cancer register was used to identify all Swedish CML patients since 1970 and the Swedish cause of death register was used to identify an eventual date of death for these patients. With a constant incidence and the relative survival rates for CML patients between 2006 and 2012 as a model, the present and future prevalence was calculated. For paper II-IV, data from the Swedish cancer register was used to identify other malignancies than CML. For paper II, information about autoimmune and chronic inflammatory diseases was retrieved from the Swedish national patient register. For paper II and IV, five controls matched for year of birth, gender and county of residence were randomly selected from the Swedish register of the total population. To calculate odds ratio (OR), conditional logistic regression was used. To calculate the risk of a second malignancy for paper III, Standardized incidence ratio (SIR) was used. In paper IV, first-degree relatives (parents, siblings and offsprings) for both cases and controls were retrieved from the Swedish multi-Generation Register, where persons born later than 1932 and registered in Sweden at some time since 1961 are registered.   Results Prevalence and survival As shown in paper I, the 5-year overall survival for CML patients increased remarkably from 0.18 to 0.82 between 1970 and 2012. The prevalence increased from 3.9 to 11.9 per 100 000 inhabitants in Sweden between 1985 and 2012. By assuming no further improvements in relative survival as compared to the survival rates between 2006 and 2012, the prevalence by 2060 is expected to increase to 22.0 per 100 000 inhabitants. This corresponds to 2 587 CML patients as compared to 1 137 CML patients in 2012.   Malignancies, autoimmune and chronic inflammatory diseases prior to CML In study II, more than 45 000 person-years of follow-up were evaluated in 984 CML patients diagnosed between 2002 and 2012. With an OR of 1.47 (95 % CI 1.20–1.82) and 1.55 (95 % CI 1.21–1.98), respectively, the prevalence of prior malignancies and autoimmune diseases were significantly increased as compared to matched controls. On the other hand, no association between CML and chronic inflammatory diseases was shown.   Second malignancies In 868 CML patients, diagnosed between 2002 and 2011, 52 malignancies were observed in the Swedish cancer register, as shown in paper III. When compared to expected rates in the background population, a significantly increased risk of second malignancies with a SIR of 1.52 (95 % CI 1.13–1.99) was shown. When looking at specific cancer types, gastrointestinal as well as nose and throat cancer were significantly increased.   Familial aggregation of malignancies 984 CML patients were identified in paper IV. However, 184 had a birth date prior to 1932, subsequently only 800 patients were analyzed. Among them, 4 287 first-degree relatives were identified, compared to 20 930 first-degree relatives of the matched controls. 611 malignancies were retrieved; no significant increase of malignancies in first-degree relatives of CML patients was shown (OR 1.06; 95 % CI: 0.96–1.16).   Conclusion Since CML patients nowadays have a high survival rate, the calculations in this thesis shows that the prevalence of CML will almost double by 2060. CML patients have an increased risk of developing malignancies prior and subsequent to the diagnosis of CML, suggesting a hereditary or acquired predisposition to develop cancer. Since there is no familial aggregation of malignancies in CML patients, a hereditary predisposition to develop cancer is unlikely to be part of the pathogenesis of CML, leaving an acquired predisposition more likely.

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).

Human acute nonlymphoblastic or myelogenous leukemia (ANLL or AML) is a malignant disease of the bone marrow involving hemopoietic (blood-forming) cells of the myeloid lineage. ANLL is a complex neoplastic disease, whose fundamental nature is only partially understood despite intensive research. The disease is complicated by its apparent heterogeneity in terms of the degree of differentiation of hemopoietic stem cell involvement in different patients and the cellular expression of immunologically defined surface markers. The presence of a common antigen in myelogenous leukemia (CAMAL) has been previously identified. This thesis examines the expression of the CAMAL marker in or on bone marrow (BM) and peripheral blood (PB) cells using a monoclonal antibody-based indirect immunoperoxidase slide test. Increased numbers of CAMAL-positive cells were found in or on BM and PB of myeloid leukemia patients (with acute or chronic forms of the disease) compared with those found in normals or most lymphoid malignancies. Results presented herein have demonstrated that fluctuations in CAMAL BM values (% positive cells) correlated with survival time prior to relapse. In a blind study, ANLL patients Whose CAMAL BM values decreased post-chemotherapy had significantly (p < 0.025) longer first remission times (x = 19.2 months) than patients with increasing or static CAMAL BM values (x = 6.8 months). CAMAL BM values were often observed to increase during remission, prior to relapse, suggesting the presence of residual subclinical disease. Addition of excess purified leukemia-derived CAMAL to an in vitro myeloid progenitor cell assay caused profound inhibition of normal CFU-c growth but had no inhibitory effect on CFU-c growth from myeloid leukemia patients in active disease states. Depletion of CAHAL from normal plasma and conditioned media (sources of numerous hemopoietic growth regulatory factors) caused significant inhibition of normal, but not myeloid leukemic, CFU-c growth. These results indicated that myeloid leukemic cells possessed apparent differences in responsiveness to CAMAL-mediated hemopoietic regulation compared to normal cells. Lack of responsiveness to inhibition by leukemia-derived CAMAL may facilitate dominance of the malignant clone over normal cells.<br>Science, Faculty of<br>Microbiology and Immunology, Department of<br>Graduate

Addendum attached to back facing leaves. Includes bibliographical references (leaves 172-199). The findings establish leukemia inhibitory factor, and possibly oncostatin M, as new members of a small but growing class of cytokines produced in an intracellularly active form and also suggest that the production of alternate transcripts and intercellularly-retained proteins may be a common and important feature of cytokines of the IL-6 and other families.

Thesis (Master Diploma(Medical Technology) -- Cape Technikon, Cape Town, 1990<br>Eighty six patients with acute l.eukaemia were studied using morphol.ogical., cytochemical. and immunol.ogical. techniques. The acute l.eukaemias were subdivided using the French-American-British (FAB) cl.assification. The immunophenotyping studies were compared with the morphological classification to assess their contribution to the diagnosis. Acute non-lymphoblastic leukaemia (ANLL) was diagnosed on the basis of morphol.ogy and cytochemical. criteria. In addition this group of patients was studied with antibodies directed against myel.omonocytic antigens. However, no further cl.inical.l.y useful. information was obtained. Patients whose bl.asts did not stain with Sudan black or myel.operoxidase were considered to have acute lymphoblastic leukaemia (ALL). After assessment with monocl.onal. antibodies directed against epitopes expressed on cel.l.s from the l.ymphoid lineage, these patients were subgrouped into non-T-ALL, common-ALL, B-ALL, T-ALL and l.ymphoblastic lymphoma categories. This study confirmed the val.ue of monocl.onal. antibodies for accurately assigning l.ineage to the acute l.eukaemias and particularly in those situations where conventional morphol.ogical. criteria and cytochemical. markers are inconclusive.

The present thesis is focused on the prognostic value of genetic variations and alterations in the initiation and development of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) patients. Several prognostic markers based on genetic or chromosomal aberrations are today used in clinic in these heterogeneous diseases. Novel biomarkers have been identified through next generation sequencing techniques and some of them may be useful as prognostic markers in clinical diagnostic. In papers I-IV we have investigated some of this markers in CLL and AML tumor cells. In papers I and III we investigated the prognostic value of the MDM2 SNP309 in relation to the presence of TP53 mutations in tumor cells from CLL and AML patients. The SNP309 G-allele was associated with a shorter overall survival in TP53 wildtype CLL and non-normal karyotype AML patients. Mutations in the TP53 gene were found in 6.2% in CLL and 21.7% in AML and were always associated with adverse overall survival. This was most significant observed among the AML patients, where the three year survival was zero. In paper II we investigated mutations in NOTCH1 and NOTCH2 as prognostic biomarkers in CLL. Notch1 and Notch2 play critical roles in lineage differentiation of white blood cells. We found mutation only in NOTCH1 in a frequency of 6.7% and our analysis revealed a shorter overall survival for these. NOTCH1 mutations were almost mutually exclusive with TP53 mutations and represented together 12.9% in CLL patients, and they may both be strong prognostic biomarkers in CLL. In paper IV we studied mutations in the tricarboxylic acid cycle. Metabolic disturbances in cancer cells have been known for many years, but recently mechanistic explanations have been identified. Hot spot mutations in IDH1/2 genes, result in neomorphic enzyme activities that results in global hypermethylation of the cancer cell genome. We found mutations in 21% of the AML patients. Among the CN-AML patients there is a lack of prognostic markers and in this subgroup we found patients with IDH2 mutations to have a shorter overall survival (3 vs. 21 months (p=0.009) for mutated and wild-type patients, respectively). Additionally, we also studied a SNP in the IDH1 gene, and both the IDH2 mutations and the SNP showed to have a potential as a new prognostic markers in CN-AML. In summary, the results in papers I-IV have a potential to function as novel prognostic biomarkers in the clinic for therapeutic considerations and may also be targets for novel drugs for CLL and AML patients.

Chronic myelogenous leukemia (CML) is a disease arising from a reciprocal translocation between chromosome 9 and 22. Chromosome 22 encodes the Bcr gene, while chromosome 9 gives rise to the proto-oncogene, c-abl. In CML, these two genes form a fusion protein known as Bcr/Abl, which contains a constitutively activated protein tyrosine kinase. The focus of my research has been to address genomic instability as it relates to CML.<br>Initially, I addressed genomic instability by utilizing Inter Simple Sequence Repeat Polymerase Chain Reaction (Inter-SSR PCR), in combination with primers which consist of a set of eight CA repeats. Using Inter-SSR PCR, I compared P190Bcr/Abl pre-leukemic mice (about 100 days, before onset of leukemia) and P190Bcr/Abl leukemic mice to control mice (BL6/CBA), and found an increased number of insertions and deletions in the pre-leukemic and leukemic mice compared to control. Next, I decided to again address genomic instability by utilizing the P190Bcr/Abl transgenic mice (line 623) and the Big Blue assay system (Stratagene). My results demonstrated a two to three fold increase in point mutation rate observed in P190Bcr/Abl x Big Blue pre-leukemic mice as compared to control mice (C57/BL6). These mice were then injected with the c-Abl specific kinase inhibitor, STI571 for ten consecutive days, to see if these mutation frequencies could be reversed. I observed a decrease in mutation frequencies upon injection with the inhibitor, in both the kidney and the spleen. I again utilized the InterSSR PCR system to address genomic instability in a P210 Bcr/Abl transgenic mouse with a tetracycline repressible system. My results showed an increase in insertions and deletions when the tetracycline is withdrawn from the P210Bcr/Abl transgenic mice and therefore Bcr/Abl is expressed. I examined genomic instability one last time by utilizing cDNA expression arrays to compare the P190Bcr/Abl pre-leukemic mice to the control mice (BL6/CBA). Using this system I noted an increase in several genes in the pre-leukemic mice, including several genes involved two genes involved in DNA repair, XPC and RAD23B . These experiments demonstrate that Bcr/Abl is inducing a mutator phenotype, seen as an increase in altered bands and point mutations, and that this can be reversed with the inhibitor ST1571. The results from the P210 Bcr/Abl inducible mice confirm previous results seen in the P190 Bcr

Chronic lymphocytic leukemia (CLL) exhibits remarkable clinical heterogeneity likely reflecting the underlying biological heterogeneity. The genetic landscape of CLL has been recently enriched with mutations within a number of genes proposed as novel prognostic markers. Mounting evidence also supports the pivotal role of the clonotypic B-cell receptor immunoglobulin (BcR IG) in the natural history of CLL. Interestingly, almost 30% of all CLL patients can be assigned to different patient subsets, each defined by expression of a distinct stereotyped BcR IG. Whether stereotyped subsets exhibit distinct clinical behavior is still an issue of debate. The aim of this thesis was to evaluate the prognostic relevance of recurrent gene mutations and to assess the clinicobiological associations and clinical impact of BcR IG stereotypy in CLL. In a cohort of 3490 patients, NOTCH1, SF3B1 and TP53 mutations were enriched within clinically aggressive cases carrying unmutated IGHV genes (U-CLL), frequently co-occurring with trisomy 12, del(11q) and del(17p), respectively. Of note, SF3B1 mutations increased in parallel with increasing timespan between diagnosis and mutational screening. NOTCH1 mutations, SF3B1 mutations and TP53 abnormalities (TP53abs, deletions and/or mutations) correlated with shorter time-to-first-treatment among early stage cases, while in multivariate analysis, only SF3B1 mutations and TP53abs retained independent significance. In a series of 8593 CLL patients, stereotyped subsets showed marked differences in demographics, clinical presentation, cytogenetic aberrations and gene mutational spectrum. Patients within a specific subset generally followed similar clinical courses, whereas patients in different stereotyped subsets—even when displaying similar IG somatic hypermutation status— experienced significantly different clinical outcome. In particular, subset #2 (IGHV3-21/IGLV3-21), the largest overall, was found to exhibit (i) a remarkably high incidence of SF3B1 mutations (44%), alluding to subset-biased acquisition of genomic aberrations, in the context of particular antigenic stimulation; and, (ii) a dismal clinical outcome, distinct from the remaining IGHV3-21 CLL. Our findings strongly support the adverse clinical impact of SF3B1 mutations in CLL in addition to TP53abs. BcR IG stereotypy also emerges as prognostically relevant, further highlighting that an immunogenetic sub-classification of CLL based on BcR IG configuration could refine patient risk stratification.

The problem of childhood leukemia is being discussed in the papers of the researchers from all parts of the world. This pathology is almost 1/3 of all malignancies in children and ranks first among the diseases of blood. The most common type of acute leukemia in children is acute lymphoblastic leukemia accounting for 80% of all the cases. The incidence of leukemia ranges from 3.2 to 4.4 per 100 thousand children under the age of 15 years. Peak incidence occurs at age of 2-5 years. 50% of childhood cancer deaths occur because of leukemia. Nowadays an extensive research work is being carried on to find out the epidemiology of leukemia for resolving funding measures for further implementation of the provision of highly specialized health care for children with cancer and hematoonkological diseases. The purpose of the study is to analyze the epidemiology of child leukemia population of Ukraine and Sumy region in 2010-2016.

Селективна доставка протиракових агентів до ракових клітин без шкоди для здорових клітин є основною метою нової терапії лейкемії у дітей на основі наночасток. Низка досліджень показують, що рецептори ліпопротеїдів (особливо рецептор до ліпопротеїдів високої щільності) дуже активні на поверхні злоякісних лейкозних клітин, тому можуть використовуватися в якості каналів для доставки протиракових агентів. Ліпосомальний сульфат вінкристину був першою наноформацією, що отримала схвалення FDA для лікування Ph+ гострої лімфобластної лейкемії у дорослих. Діти переносять тижневу дозу 2,25 мг/м2 ліпосомального сульфату вінкристину з доказом клінічної активності, але без нейротоксичности, що обмежує дозу. Ліпосомальний доксорубіцин і пегільований (покритий поліетиленгліколем) інкапсульований в ліпосоми доксорубіцин володіють вражаючим профілем безпеки, особливо щодо гострої серцевої токсичності, при лейкозі у дітей. Пегільована формула L-аспарагінази знижує імуногенність, збільшує період напіввиведення з кровотоку і може використовуватися у пацієнтів з гіперчутливістю до непегільованих продуктів.<br>Селективная доставка противораковых агентов к раковым клеткам без вреда для здоровых клеток является основной целью новой терапии лейкемии у детей на основе наночастиц. Ряд исследований показывают, что рецепторы липопротеидов (особенно рецептор к липопротеидам высокой плотности) очень активны на поверхности злокачественных лейкозных клеток, поэтому могут использоваться в качестве каналов для доставки противораковых агентов. Липосомальный сульфат винкристина был первой наноформацией, получившей одобрение FDA для лечения Ph+ острой лимфобластной лейкемии у взрослых. Дети переносят недельную дозу 2,25 мг/м2 липосомального сульфата винкристина с доказательством клинической активности, но без нейротоксичности, ограничивающей дозу. Липосомальный доксорубицин и пегилированный (покрытый полиэтиленгликолем) инкапсулированный в липосомы доксорубицин обладают впечатляющим профилем безопасности, особенно в отношении острой сердечной токсичности, при лейкозе у детей. Пегилированная формула L-аспарагиназы снижает иммуногенность, увеличивает период полувыведения из кровотока и может использоваться у пациентов с гиперчувствительностью к непегилированным продуктам.<br>Selective delivery of anti-cancer agents to cancer cells without harming the healthy cells is a major goal of novel nanoparticle-based pediatric leukemia therapy. Some studies are show that lipoprotein receptors (especially the HDL receptor) are highly active on the surface of malignant leukemic cells, that’s why may be used as conduits for the delivery of anti-cancer agents. Liposomal vincristine sulfate was the first nanoformulation to get approval by the FDA to treat Ph+ ALL in adults. Children tolerate 2.25 mg/m2/dose of weekly liposomal vincristine sulfate with evidence for clinical activity without dose‐limiting neurotoxicity. Liposomal doxorubicin and pegylated (polyethylene glycol coated) liposome-encapsulated doxorubicin has an impressive safety profile, particularly regarding acute cardiac toxicity, in childhood leukemia. Pegylated formula of L-asparaginase decreases immunogenicity, increases circulating half-life and can be used in patients with hypersensitive to un-pegylated products.

Abstract Childhood acute lymphoblastic leukemia (ALL) is the most common cancer in children. The overall survival rate has reached to 90%. However, ALL still presents a significant disease burden and is a major cause for deaths in children. Recently, both inherited germline variants related to ALL susceptibility and somatic genetic variants forming novel subgroups of ALL have been discovered. In this thesis two families with familial ALL were studied. Constitutional heterozygous microdeletion at chromosome 7p12.1p13, including IKZF1, was discovered in the first family with intellectual impairment, overgrowth, and susceptibility to childhood ALL. In the second family, constitutional chromosome translocation was revealed in two individuals with childhood ALL and, subsequently, in seven unaffected family members. The balanced reciprocal translocation t(12;14)(p13.2;q23.1) resulted in breakpoints on two genes; ETV6 on chromosome 12 and RTN1 on chromosome 14. Only a few familial and sporadic ALL cases with germline variants in either IKZF1 or ETV6 have been published, thus supporting the significant role of these constitutional variants in childhood ALL predisposition. Inherited bone marrow failure syndromes (IBMFS) may predispose to childhood leukemia, including ALL. Two unrelated patients were diagnosed with bone marrow failure without the symptoms of classical IBMFS. Neither patient had any signs of developmental delay or congenital anomalies. Exome sequencing revealed identical c.1457del(p.(Ile486fs)) mutation on the ERCC6L2 gene in both patients. A few patients with IBMFS and ERCC6L2 variants have been described in previous studies. Some of them also had congenital craniofacial anomalies and developmental delay that were not detected in the patients in this thesis. The ALL cohort study on genetic variation of mitochondrial DNA (mtDNA) included 36 children. Metabolic change where malignant cells uncouple energy production from oxidative phosphorylation (OXPHOS) is one of the established hallmarks of cancer. In the cohort in this study, 22% of patients harbored nonsynonymous variants on mtDNA in the protein-coding genes of OXPHOS enzyme complexes. The somatic non-neutral variants were found in patients with a poor prognosis cytogenetic marker. The results support the hypothesis that cancer cells harbor mtDNA variants that may affect the cell metabolism<br>Tiivistelmä Akuutti lymfoblastileukemia (ALL) on lasten yleisin syöpä. Vaikka nykyisin noin 90 prosenttia paranee, ALL aiheuttaa huomattavan paljon sairastavuutta ja on merkittävä lasten kuolinsyy. Vastikään on löydetty perinnöllisiä geneettisiä muutoksia, jotka altistavat lapsuusiän ALL:lle. Tutkimuksen kohteena oli kaksi perhettä, joissa vähintään kaksi lasta on sairastunut ALL:aan. Ensimmäisessä perheessä havaittiin lapsuusiän ALL:aan sairastuneilla kehityshäiriöisillä sisaruksilla äidiltä periytyvä heterotsygoottinen deleetio kromosomissa 7p12.1p13, jossa sijaitsee IKZF1-geeni. Toisessa perheessä perinnöllinen kahden kromosomin translokaatio todettiin kahdella lapsuusiän ALL:aan sairastuneella sekä seitsemällä perheenjäsenellä. Balansoitu translokaatio t(12;14)(p13.2;q23.1) aiheuttaa katkaisukohdan ETV6-geeniin kromosomissa 12 ja RTN1-geeniin kromosomissa 14. Tähän mennessä on julkaistu vain muutamia tutkimuksia potilaista, joilla on ollut perinnöllinen muutos joko IKZF1- tai ETV6-geenissä. Näillä geeneillä oletetaan olevan tärkeä merkitys perinnöllisessä alttiudessa sairastua lapsuusiän ALL:aan. Perinnölliset luuytimen toimintahäiriöt voivat altistaa leukemialle, kuten ALL:lle. Kahdella lapsella todettiin luuytimen toimintahäiriö, mutta ei muita oireita, jotka voisivat liittyä tyypillisiin perinnöllisiin luuytimen toimintahäiriöihin. Eksomisekvensoinnissa todettiin identtinen, homotsygoottinen mutaatio c.1457del(p.(Ile486fs)) ERCC6L2-geenissä. Kirjallisuuslähteiden mukaan vain muutamalla potilaalla on todettu ERCC6L2-geenin muutoksesta johtuva luuytimen toimintahäiriö. Osalla heistä on ollut synnynnäisiä kallon ja kasvojen anomalioita sekä kehityshäiriö, jollaisia tähän tutkimukseen osallistuneilla potilailla ei todettu. Potilaskohorttitutkimuksessa tutkittiin mitokondriaalisen DNA:n (mtDNA) muutoksia ALL:aan sairastuneilla lapsilla. Syöpäsolut eivät hyödynnä mitokondrion elektroninsiirtoketjua energian tuotantoon, ja tämä aineenvaihdunnan muutos on tunnustettu syövän ominaisuus. Tutkimuksessa havaittiin, että 22 prosentilla potilaista ilmeni diagnoosivaiheessa poikkeavia mtDNA:n muutoksia, jotka olivat elektroninsiirtoketjun entsyymien alayksiköitä koodaavissa geeneissä. Muutoksia todettiin useimmiten potilailla, joilla oli leukemiasoluissa huonon ennusteen geneettinen tekijä. Havaitut muutokset voivat mahdollisesti vaikuttaa leukemiasolun energia-aineenvaihduntaan

Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder characterized by the presence of the BCR-ABL1 oncogene, deriving from the t(9;22)-(q34;q11) balanced translocation. The deriving BCR-ABL1 fusion protein has tyrosine kinase activity, and it is capable of inducing the proliferation and expansion of myeloid precursors. Tyrosine kinase inhibitors, drugs capable of blocking the malignant proliferation of cells by directly targeting the BCR-ABL1 kinase activity, have dramatically changed patients’s outcome and improved survival rate. Dormant CML-Leukemia Stem Cells (LSCs) are intrinsically refractory to TKIs and for this reason, only few patients can discontinue TKI therapy. CML LSCs are transcriptionally silent and probably not detectable by qRT-PCR. Several studies tried to find a specific marker able to detect CML LSCs and distinguish them from normal hematopoietic stem cells (HSCs). CD26 (dipeptidylpeptidase IV) was very recently identified as the most specific surface marker for flow-cytometry quantification and isolation of CML LSCs mainly in the bone marrow while rare samples of peripheral blood have been tested so far. Thus, principal aim of this study was to evaluate the presence of circulating LSCs in peripheral blood samples of patients during TKI therapy, avoiding in this way the need of routine bone marrow aspirate. Secondary we investigated a correlation, if any, between LSCs value and degree of molecular response. The analysis was conducted on 202 CML subjects. We have tested by flow-cytometry analysis the expression on peripheral blood cells of stem cells markers such as CD45, CD34, CD38 and, in particular, we have exploited the co-expression of the PE-conjugated anti-CD26. In our experiments we confirmed that CD26 is a specific marker able to discriminate normal HSCs from CML LSCs also in peripheral blood and that it is not expressed on LSCs of other myeloid disorders. The results of our study also showed that circulating CD26+ LSCs are easily detected in CML patients while on TKIs treatment and when comparing bone marrow and peripheral blood actual amount of CD26+ LSCs in the same patient, values were superimposable. However, no correlation was found between CD26+ LSCs count and BCR-ABL/ABLIS ratio molecular analysis. This study assesses for the first time that flow-cytometry analysis of circulating CD45+/CD34+/CD38-/CD26+ LSCs is a powerful tool that could be of great help to identify the residual leukemic cells during follow-up of CML patients, just with routine peripheral blood testing. Further future investigations are required to establish possible roles of flow-cytometry LSCs detection during management of CML patients and to evaluate a potential relationship between undetectable CML LSCs and safe treatment discontinuation.

Les leucémies aiguës myéloïdes (LAM) sont des maladies hématologiques hétérogènes caractérisées par une prolifération clonale des blastes myéloïdes qui s’infiltrent dans la moelle osseuse (MO), le sang et d’autres organes. Identifiée comme le type le plus courant de leucémie aiguë chez l’adulte avec 80% des cas, la LAM est synonyme de rechute et de mauvais pronostic, avec 70% des patients étant confrontés à une mortalité dans l’année suivant le diagnostic. La présence des cellules souches leucémiques (CSL) a été associée à une résistance à la chimiothérapie et à une rechute dans la LAM. Le microenvironnement tumoral a été décrit pour son rôle clé dans la régulation des CSL par l’interaction des voies de signalisation. La voie des Bone Morphogenetic Proteins (BMP) est fortement impliquée dans la régulation des cellules souches hématopoïétiques (CSH), mais elle a également été reconnue pour réguler les CSL. Ici, nous avons identifié des concentrations élevées de BMP2 et BMP4 dans la MO des patients atteints de LAM au moment du diagnostic. De plus, nous avons identifié pour la première fois une nouvelle cascade de signalisation impliquant la liaison de BMP4 au récepteur BMPR1A, qui induit l’expression de ΔNp73 et NANOG. L’activation de cette signalisation favorise un phénotype proche des cellules souches dans les cellules leucémiques. Par conséquent, nous avons émis l’hypothèse que cette voie est responsable de la capacité de résistance des cellules leucémiques à la chimiothérapie. En outre, nous avons identifié BMPR1A/ΔNp73/NANOG comme marqueurs potentiels du pronostic dans la LAM, en raison de leurs surexpressions au moment du diagnostic associé à une rechute dans les trois ans. Lorsque nous avons analysé des échantillons de LAM lors d’une rechute, nous avons constaté des taux plus élevés de l’isoforme ΔNp73 par rapport à ceux de patients au moment du diagnostic. D’autre part, nous avons identifié une forte expression du récepteur BMPR1A, ΔNp73, NANOG, SOX2 et ID1 dans les cellules leucémiques primaires résistantes à court terme. Ces résultats sont en corrélation avec ce que nous avons observé dans les cellules résistantes de LAM, où BMPR1A, ΔNp73, NANOG et ID1 semblent être impliqués dans la capacité de résistance des cellules de LAM face à la chimiothérapie. La modulation et le ciblage des éléments de la voie BMP et des gènes associés identifiés au travers de notre étude représentent donc une approche prometteuse pour le développement de stratégies thérapeutiques innovantes et plus efficaces contre les LAM<br>Acute myeloid leukemias (AML) are heterogeneous hematological malignancies characterized by a clonal proliferation of myeloid blasts which infiltrate the bone marrow, blood and other organs. Identified as the most common type of acute leukemia in adults with 80% of cases, AML is associated with high relapse and poor prognosis where 70% of patients face mortality within one year after diagnosis. Leukemic stem cell (LSCs) presence has been related to resistance to chemotherapeutic agents and relapse in AML. The tumor microenvironment has been described for its key role regulating LSCs through the crosstalk of signaling pathways. Bone Morphogenetic Proteins (BMP) pathway is highly involved in hematopoietic stem cell (HSC) regulation, but has also been recognized to regulate LSCs. Here, we have identified high concentrations of BMP2 and BMP4 in bone marrow (BM) AML samples at diagnosis. Furthermore, we have identified for the first time a new signaling cascade, involving the binding of BMP4 to BMPR1A receptor, which induces the expression of ΔNp73 and NANOG. Activation of this signaling promotes a stem-like phenotype in leukemic cells. Therefore, we hypothesized that this signaling is responsible for the resistant capacity of leukemic cells to chemotherapy. In addition, we have reported BMPR1A/ΔNp73/NANOG as potential AML prognosis markers, due to their overexpression at diagnosis associated to an increased rate of relapse of AML patients within three years. When we analyzed AML samples at relapse, higher levels of ΔNp73 isoform were found compared to patients at diagnosis. Moreover, we have identified high expression of the BMPR1A receptor, ΔNp73, NANOG, SOX2 and ID1 in short-term resistant primary leukemic cells. These results correlate with what we observed in AML resistant cells, where BMPR1A, ΔNp73, NANOG and ID1 seem to be implicated in driving the resistant capacity of AML cells to drug therapy. Therefore, modulation and targeting of the BMP pathway elements and related genes identified with our study, represent a promising approach towards the development of new and more effective therapeutic strategies against AML

Polycomb group (PcG) of proteins are a group of highly conserved epigenetic regulators involved in many biological functions such as embryonic development, stem cell self-renewal, cell proliferation, and cancer. PHD finger protein 19 (PHF19) is an associated factor of Polycomb Repressor Complex 2 (PRC2) that has been proposed to regulated its activity in embryonic stem cells. PHF19 has been shown to be up-regulated in different human cancers as well as cancer cell lines. In particular, myeloid leukemia cell lines show increased levels of PHF19, yet little is known about its function. Here, we have characterized the role of PHF19 in myeloid leukemia cell lines. We have demonstrated that PHF19 depletion decreases cell proliferation and induces erythroid differentiation. Mechanistically, we have demonstrated that PHF19 regulates the proliferation of chronic myeloid leukemia cell lines through its interaction with cell cycle regulator p21. Furthermore, we have observed that MTF2, a PHF19 homolog, occupies PHF19 target genes when PHF19 is depleted. Taken together, our results show that PHF19 is a key transcriptional regulator in myeloid leukemic cell lines and suggest that PHF19 inhibition could be a potential target to be explored for myeloid leukemia treatment.<br>El complejo de proteínas Polycomb (PcG), es un grupo de reguladores epigenéticos altamente conservados que participan en distintas funciones biológicas como el desarrollo embrionario, la auto renovación de las células madre, la proliferación y están involucradas también en cáncer. La proteína PHD finger 19 (PHF19), es un factor asociado al complejo represor Polycomb 2 (PRC2). PHF19 ha sido propuesta como reguladora de la actividad de PRC2 en células madre embrionarias. También se ha visto que esta sobreexpressada en diferentes canceres y líneas celulares cancerígenas. Nosotros hemos demostrado que la eliminación de PHF19 disminuye la proliferación de las líneas celulares mieloides cancerígenas. Hemos demostrado que la depleción de PHF19 en las células de leucemia crónica mieloide las induce a diferenciarse hacia eritrocitos. Mecánicamente, hemos demostrado que PHF19 regula la proliferación de esta línea celular mediante su interacción con el regulador de ciclo celular p21. Además, hemos observado que MTF2, un homólogo de PHF19, se deposita en aquellos genes donde previamente estaba PHF19. En conjunto, nuestros resultados muestran que PHF19 es un factor transcripcional clave en líneas celulares mieloides y sugieren que la inhibición de PHF19 podría ser una potencial diana para ser explorada para el tratamiento de la leucemia mieloide.

<p>Despite major advances in our understanding of the biology of childhood acute myeloid leukemia (AML) and the development of new cytotoxic drugs, the prognosis of long-term survival is still only 60-65 %.</p><p>In the present research, we studied the pharmacokinetics of drugs used in the induction therapy of childhood AML and performed in vitro drug sensitivity testing of leukemic cells from children with AML.</p><p>The aims of the studies were to correlate the results of the analysis to biological and clinical parameters and to identify subgroups of AML with specific drug sensitivity profiles in order to better understand why treatment fails in some patients and how therapy may be improved.</p><p>Blood samples were analysed to study the pharmacokinetics of doxorubicin (n=41), etoposide (n=45) and 6-thioguanine (n=50). Doxorubicin plasma concentration and total body clearance were correlated to the effect of induction therapy, and doxorubicin plasma concentration was an independent factor for complete remission, both in univariate and multivariate analysis including sex, age, and white blood cell count at diagnosis. For etoposide and 6-thioguanine no correlation was found between pharmacokinetics and clinical effect. Children with Down syndrome (DS) tended to reach higher blood concentrations of etoposide and thioguanine nucleotides, indicating that dose reduction may be reasonable to reach the same drug exposure as in children without DS.</p><p>Leukemic cells from 201 children with newly diagnosed AML, 15 of whom had DS, were successfully analysed for in vitro drug sensitivity by the fluorometric microculture cytotoxicity assay (FMCA). We found that samples from children with DS were highly sensitive to most drugs used in AML treatment. In non-DS children, the t(9;11) samples were significantly more sensitive to cytarabine (p=0.03) and doxorubicin (p=0.035) than other samples. The findings might explain the very favorable outcome reported in children with DS and t(9;11)-positive AML. A specific drug resistance profile was found for several other genetic subgroups as well. A detailed study of MLL-rearranged leukemia showed that cellular drug sensitivity is correlated both to partner genes and cell lineage, findings that support the strategy of contemporary protocols to include high-dose cytarabine in the treatment of patients with MLL-rearrangement, both in AML and acute lymphoblastic leukemia (ALL).</p><p>Our results indicate that drug resistance and pharmacokinetic studies may yield important information regarding drug response in different sub-groups of childhood AML, helping us to optimize future chemotherapy in childhood AML.</p>