Littérature scientifique sur le sujet « Pediatric B-Cell Precursor »

AbstractImmunostimulatory DNA containing unmethylated cytosine-phosphate-guanosine (CpG) induces the development of T helper 1 (Th1) immune responses. The response of B cells to CpG stimulation involves increased proliferation, cytokine production, and costimulatory molecule expression. Similar effects have been observed following CpG stimulation of a variety of malignant B cells. Pediatric precursor B acute lymphoblastic leukemia (B-ALL) cells express low levels of costimulatory molecules and are generally poor stimulators of T-cell responses. In this study, we evaluated the impact of CpG stimulation on precursor B-ALL cell lines and pediatric patient-derived samples. The ability to respond to CpG oligodeoxynucleotides was determined by the level of Toll-like receptor 9 (TLR9) expression. In contrast to both nonleukemic B-cell precursors and mature B cells, the response of precursor B-ALL cells was characterized by increased CD40 expression but only small changes in CD86 levels and no induction of CD80 expression. CpG stimulation of ALL blasts produced increased levels of interleukin-6 (IL-6), IL-8, and IL-10 but no detectable IL-12p70 and led to a skewing of allogeneic T cells, with enhanced interferon γ (IFN-γ) production and reduced secretion of IL-5. These results demonstrate the functional relevance of CpG stimulation of precursor B-ALL cells and provide a rational basis for study of these agents for use in treatment of this disease.

B-cell lymphoblastic lymphoma (BCP-LBL) and B-cell acute lymphoblastic leukemia (BCP-ALL) are the malignant counterparts of immature B-cells. BCP-ALL is the most common hematological malignancy in childhood, while BCP-LBL accounts for only 1% of all hematological malignancies in children. Therefore, BCP-ALL has been well studied and treatment protocols have changed over the last decades, whereas treatment for BCP-LBL has stayed roughly the same. Clinical characteristics of 364 pediatric patients with precursor B-cell malignancies were studied, consisting of BCP-LBL (n = 210) and BCP-ALL (n = 154) patients. Our results indicate that based on the clinical presentation of disease, B-cell malignancies probably represent a spectrum ranging from complete isolated medullary disease to apparent complete extramedullary disease. Hepatosplenomegaly and peripheral blood involvement are the most important discriminators, as both seen in 80% and 95% of the BCP-ALL patients and in 2% of the BCP-LBL patients, respectively. In addition, we show that the overall survival rates in this cohort differ significantly between BCP-LBL and BCP-ALL patients aged 1–18 years (p = 0.0080), and that the outcome for infants (0–1 years) with BCP-LBL is significantly decreased compared to BCP-LBL patients of all other pediatric ages (p < 0.0001).

Abstract Until recently, 20% to 30% of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) could not be classified into any of the established molecular subtypes. Recent molecular studies of such cases have, however, further clarified their mutational spectrum and identified new oncogenic subtypes consisting of cases with DUX4 rearrangements, ETV6-RUNX1–like gene expression, MEF2D rearrangements, and ZNF384 rearrangements. In this review, we describe these new subtypes, which account for up to 50% of previously unclassified pediatric BCP-ALL cases.

Abstract PAX5, located on 9p13, belongs to the PAX gene family and encodes for a transcription factor essential for B lymphoid cell commitment. It functions both as a transcriptional activator and repressor of different target genes involved in lineages development. PAX5 has been recently reported to be target of aberrancies (including point mutation, deletions, and gene fusions), in approximately 30% of pediatric patients affected by BCP-ALL, the most frequent leukemia subset in children. Translocations are estimated to occur at an incidence of 2-3%, with a variety of partner genes, encoding for transcription factors (TEL, PML, FOXP1), kinases (JAK2), structural proteins (ELN, POM121) or molecules of unknown function (C20orf112, AUTS2). We performed a FISH-based study on an Italian cohort of BCP-ALL patients having 9p13 chromosomal rearrangement (as a hallmark of PAX5 rearrangement), and we identified novel PAX5 partner genes. Two pediatric patients were harboring a t(7;9)(q11.2;p13.2) with a PAX5/AUTS2 fusion transcript, thus confirming its recurrent alteration in pediatric B-ALL. Three novel partner genes of PAX5 were identified by FISH. SOX5 was found as a PAX5 partner in a pediatric patient harboring a dic(9;12)(p13;p13) chromosome. A further patient, showing a t(9;12)(p13;q34) translocation, revealed PAX5 as fused to a novel transcript isoform of CHFR, a gene widely expressed in a library of normal tissues. A third partner was identified in an adult B-ALL case, which showed a deletion within the short arm of chromosome 9, leading to the fusion of PAX5 to MLLT3. A fourth PAX-rearranged case, involving POM121C (different from the already described POM121) as fused to PAX5 in a t(7;9)(q11;p13) translocation, was identified by a RNAseq approach on BCP-ALL cases without known prognostic features. The breakpoint on chromosome 7q11 is similar to the one associated with PAX5/AUTS2. An accurate FISH analysis was performed on bone marrow cells of all cases to dissect the genomic breakpoints and the structure of the rearrangements. The fusion genes were cloned by 5’ and/or 3’ RACE PCR, confirmed by sequencing and verified by RT-PCR with specific primers on the source material. PAX5-translocated cases were further characterized by genome-wide Single Nucleotide Polymorphism array. Interestingly, Copy Number Variation analysis showed that a limited number of cooperative genetic lesions were present in addition to the translocation event, thus suggesting a primary role of the PAX rearrangement in leukemogenesis. We therefore hypothesize that PAX5 alterations may represent single genetic aberration events in a simple background, rather than being part of a complex scenario of cooperating genetic lesions involved in leukemogenesis. A common pathway for all PAX5 genomic lesions still need to be elucidated. Disclosures: No relevant conflicts of interest to declare.

Abstract Natural killer (NK) cells represent a promising cell type in antitumor immunotherapy for efficacy and safety, particularly in the treatment of hematologic malignancies. NK cells have been shown to exert antileukemia activity in the context of haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Products have been developed to boost the activation of NK cells only when cross-linked by tumor cells, avoiding any off-target effect. Here, we tested the in vitro effect of different NK-cell engagers (NKCE), which trigger either NKp46 or NKp30 together with CD16A, and target either CD19 or CD20 to induce killing of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Target cells were NALM-16 and MHH-CALL-4 cell lines and four primary leukemias, while effector cells were resting NK cells derived from healthy donors and pediatric patients with leukemia after αβT/B-depleted haplo-HSCT. The NK cell–resistant MHH-CALL-4 was efficiently killed using all NKCEs. Boosting of NK activity against MHH-CALL-4 was also evident by degranulation and IFNγ production. Because of the lack of CD20 and high expression of CD19 on primary BCP-ALL, we focused on NKCEs targeting CD19. NKp46- and NKp30-based NKCEs displayed similar potency at inducing NK-cell activity, even when challenged with primary BCP-ALL blasts. Their efficacy was shown also using NK cells derived from transplanted patients. NKCE-induced activation against BCP-ALL can override HLA-specific inhibitory interactions, although the strongest response was observed by the alloreactive NK-cell subset. These data support the therapeutic use of NKp46/CD16A/CD19-NKCE to fight refractory/relapsed leukemia in pretransplantation or posttransplantation settings.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. Survival probability of pediatric ALL had been 10-20%, but the most recent clinical trials with multiagent chemotherapy have achieved overall survival probability of better than 80%. This is achieved because of better supportive care, treatment stratification based on relapse risk, and the biological features of leukemic cells. Diagnosis of ALL was based principally on morphological identification of leukemic blasts in bone marrow, and immunophenotype assessment by flow cytometry is necessary, and most pediatric ALL cases are clinically classified as B-cell precursor, T-cell ALL, or mature B-cell types. Key words: Acute Lymphoblastic Leukemia, ALL, Unilateral Facial palsy, pediatric ALL.

Abstract Terminal deoxynucleotidyl transferase (TdT)-positive cells in human bone marrow (BM) are a phenotypically inhomogeneous population of precursor cells. In their majority, these TdT+ cells are unambiguously committed to the B lineage, as evidenced by CD19 expression. However, TdT+ precursors that lack CD19 also exist and these may encompass a differentiation potential for the B as well as for other lineages. Because recent data suggested that CD19 expression is not the earliest differentiation event in B-cell ontogeny, we sought to reevaluate TdT+ lymphoid precursors in pediatric BM to define the phenotypic denominator of B-lineage affiliation upstream of CD19. Using four-color flow cytometry, we focused on the assessment of the CD79a antigen, which is highly B-cell specific and which may also be expressed very early in B-cell ontogeny. We found that a majority of TdT+ cells coexpressed CD19 and CD79a in addition to CD10 and CD34, whereas, in all investigated samples, some TdT+ precursors lacked CD19 but expressed CD79a, which suggestively indicates also their B-lineage affiliation. In contrast to the CD19+precursors, which were usually CD10hi and CD79b+, these CD19−CD79a+putative B-cell precursors preferentially expressed CD10 at low levels and were CD79b+ in only 41%. About 17% of these TdT+CD19−CD79a+ precursors also coexpressed CD33 and CD7, but not myeloperoxidase, CD14, or cytoplasmic CD3, which is discussed in the light of cellular activation rather than lineage promiscuity. Our data confirm that the earliest differentiation stages of B cells can be dissected upon expression of the lineage antigens CD79a and CD19 and imply that CD79a is earlier expressed than CD19. This raises the chance to follow the sequential events heralding B-cell commitment in the most immature precursors by correlating phenotypic and genetic differentiation markers. © 1998 by The American Society of Hematology.

Terminal deoxynucleotidyl transferase (TdT)-positive cells in human bone marrow (BM) are a phenotypically inhomogeneous population of precursor cells. In their majority, these TdT+ cells are unambiguously committed to the B lineage, as evidenced by CD19 expression. However, TdT+ precursors that lack CD19 also exist and these may encompass a differentiation potential for the B as well as for other lineages. Because recent data suggested that CD19 expression is not the earliest differentiation event in B-cell ontogeny, we sought to reevaluate TdT+ lymphoid precursors in pediatric BM to define the phenotypic denominator of B-lineage affiliation upstream of CD19. Using four-color flow cytometry, we focused on the assessment of the CD79a antigen, which is highly B-cell specific and which may also be expressed very early in B-cell ontogeny. We found that a majority of TdT+ cells coexpressed CD19 and CD79a in addition to CD10 and CD34, whereas, in all investigated samples, some TdT+ precursors lacked CD19 but expressed CD79a, which suggestively indicates also their B-lineage affiliation. In contrast to the CD19+precursors, which were usually CD10hi and CD79b+, these CD19−CD79a+putative B-cell precursors preferentially expressed CD10 at low levels and were CD79b+ in only 41%. About 17% of these TdT+CD19−CD79a+ precursors also coexpressed CD33 and CD7, but not myeloperoxidase, CD14, or cytoplasmic CD3, which is discussed in the light of cellular activation rather than lineage promiscuity. Our data confirm that the earliest differentiation stages of B cells can be dissected upon expression of the lineage antigens CD79a and CD19 and imply that CD79a is earlier expressed than CD19. This raises the chance to follow the sequential events heralding B-cell commitment in the most immature precursors by correlating phenotypic and genetic differentiation markers. © 1998 by The American Society of Hematology.

Abstract Recent studies suggest that the majority of malignant cells found in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) arise from a rare population of leukemic progenitors. Little information is available on the presence of clonal rearrangements in cells at the stage of early precursor. To address this issue we analyzed clonality profile of early leukemic precursors sorted by flow-cytometry. Leukemic cells were obtained from bone marrow samples collected at diagnosis from 6 patients with childhood BCP-ALL. Furthermore, bone marrow cells were collected from 3 healthy children who were harvested for bone marrow donation. Three subpopulations of leukemic cells were investigated: total unsorted blasts, the sorted CD34+/CD38−/CD19+, and the sorted CD34+/CD38−/CD19− cells. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements were screened by polymerase chain reaction (PCR) in the sorted populations and in the bulk leukemic cells in order to identify molecular markers of clonal evolution. Sequence analysis was then performed on the N-region. Overall, a total of 38 different Ig/TCR gene rearrangements were identified in the 3 cell populations under study (total blasts, CD34+/CD38−/CD19+, and CD34+/CD38−/CD19−). Of them, 13 (34%) were found in the three populations; 12 (31%) were found in two of the three populations: 7 in total blasts and CD19+ subset, 3 in total blasts and CD19−, 2 in CD19− and CD19+; finally, 13 were found only in one subpopulation: 4 in total blast cell, 5 in CD19+, 4 in CD19−. In all the six patients studied, BCP-ALL progenitors CD34+/CD38−/CD19− and CD19+ and the bulk tumor blasts shared at least one Ig/TCR gene clonal rearrangement with the same N-region. In 5 out of 6 patients at least one rearrangement detected in the BCP-ALL progenitors was undetectable in total blasts. Conversely, in 3 patients the clonal rearrangement observed in the bulk leukemic cells was not identified in any of the two sorted ALL precursor populations. Clonal rearrangement was never detected in the samples from healthy bone marrow donors. Our findings confirm that clonal rearrangement may be detected at the stage of early B-lineage precursor CD34+/CD38−/CD19−, suggesting that leukemic transformation may occur at this stage or even before in BCP-ALL. We plan to extend this observation by repopulating studies in NOD/SCID mice.

Treatment of pediatric acute lymphoblastic leukemia (ALL) is increasingly successful, achieving cure rates of over 80%. Early identification of patients with high risk for relapse has led to improved outcome, however, two third of patients encountering relapse were initially stratified into low-intermediate risk groups. The identification of better upfront prognostic factors remains an important challenge in childhood ALL. In this thesis, gene expression profiling (GEP) was applied to different research approaches aiming to dissect subgroups and to find novel therapeutic targets in B cell precursor ALL (BCP ALL). Among BCP ALL, the patients lacking major genomic aberrations (B-others) represent the subgroup that is most in need of in depth investigations in order to indentify new prognostic factors and improve of risk stratification. To advance biological knowledge of B-others we performed an integrated study of gene and non coding RNAs expression and genetic aberrancies. Chapter 1 reports a study on profiling by gene expression arrays of 145 Italian B-others BCP ALL patients and in a representative subcohort of patients microRNAs (miRNAs) expression profiling and genome-wide DNA copy number variation analysis. In this study we found that 25% of Italian B-others patients fits in a group with unique signature and is associated to a favourable outcome. MicroRNAs expression profiling of this group revealed an unique miRNAs signature characterized by over expression of hsa-miR-125b, -125b-2*, -99a, -100, -125a-3p and has-miR-491-5p. Over expression of cluster miR-125b-2 in region 21q21.1 goes along with over expression of genes in the same chromosomal region. Genome-wide analysis excluded copy number alteration of the 21q21.1 region. The frequent involvement of human chromosome 21 (Hsa21) aberrations in ALL (e.g. hyperdiploidy (HD), t(12;21) or iAmp21) and the involvement of the 21q21.1 region suggest a direct and functional contribution of Hsa21 genes to the malignant transformation of hematopoietic cells. There for there is high interest in studying ALL in children with Down Syndrome (DS), where trisomy 21 is constitutional and where the incidence of ALL is approximately 20-fold higher than in the general population. In Chapter 2 is presented a study of genomic analysis of a large group of DS ALLs that characterizes molecular abnormalities specific of this ALL group. Gene expression analysis revealed that DS ALL is a highly heterogeneous disease not definable as a unique ALL subtype with an enrichment of DNA damage and BCL6 responsive genes suggesting B-cell lymphocytic genomic instability. Surprisingly, only a single Hsa21 gene, SON was included in the DS ALL signature and it was only slightly upregulated. Furthermore gene expression data suggested that DS ALL and HD ALL are very different leukemias, reflecting the fundamental differences between constitutional and acquired trisomy such as the developmental stage in which the trisomy occurs and the fact that a constitutional trisomy is present both in the leukemia cells and in their microenvironment. The study further revealed that 62% of the DS ALL samples were characterized by aberrant expression of the type I cytokine receptor CRLF2. Two kind of aberrations involving CRLF2 were identified: a cryptic translocation involving IGH@ and CRLF2 in the pseudoautosomal region (PAR1) of the sex chromosomes and a deletion within PAR1. This aberration resulted in the P2RY8-CRLF2 fusion and leads to overexpression of CRLF2. Furthermore a novel activating somatic mutation, F232C, in CRLF2 was identified. We demonstrated that CRLF2 and mutated JAK2 cooperate in conferring cytokine independent growth to pro-B cells suggesting that the DS ALL children with CRLF2 aberrant expression may benefit from therapy blocking the CRLF2-JAK2 pathway. Since CRLF2 aberrations were found also among non DS patients, we further analyzed the incidence and prognostic impact of this potential new marker in BCP ALL Italian patients enrolled into the AIEOP-BFM ALL2000 study. Chapter 3, presents the study of a representative cohort of 464 non DS BCP ALL patients that was analyzed for the expression levels of CRLF2 and for the occurrence of CRLF2 rearrangements. In this study we found that the P2RY8-CRLF2 rearrangements in association with 20 times over expression of CRLF2 identifies BCP ALL patients with a very poor prognosis and, among them, an important subset of patients currently stratified in the intermediate risk need to be considered for treatment adaptation. Investigating the pathways highlighted by GEP analysis and testing drug effects require a substantial availability of leukemia samples. Primary ALL samples are difficult to culture in vitro and currently available cell lines poorly reflect the heterogeneous nature of the disease. Mouse xenotransplantation models are therefore widely used for in vivo testing and to amplify the number of leukemia cells to be used for various analyses. In Chapter 4 study we assessed the capability of xenografted samples to recapitulate their respective primary leukemia, and we investigated whether the murine microenvironment selects for leukemia initiating cells leading to a bulk tumor markedly different from the diagnostic patient sample. We analysed the gene expression profiles of 7 primary paediatric ALL samples at diagnosis as well as of their respective xenograft leukaemia samples after serial primary, secondary and tertiary passages in the NOD/SCID/huALL transplant model. In this study we demonstrated that the NOD/SCID/huALL transplant model recapitulates the primary human leukaemia, mimics the features of the primary malignancy and retains these characteristics over serial passages without selection for a subclone of the initial leukaemia. Chapter 5 reports on a study that investigated engraftment properties of 50 pediatric ALL samples transplanted into NOD/SCID mice. Time to leukemia (TTL) was determined for each patient sample engrafted as weeks from transplant to overt leukemia. The study shows that short TTL was strongly associated with high risk for early relapse, identifying a new independent prognostic factor. The high risk phenotype is reflected by a gene signature that identified patients with early relapse in an independent patient cohort. Gene expression profiling revealed a set of genes associated with this aggressive phenotype providing a potential strategy to identify these high-risk patients. Most importantly, pathways involving mTOR regulating cell growth were identified, providing targets for alternative therapeutic strategies for these high risk patients. Concluding, ten years after its introduction in oncohematology, GEP constitutes to be a valuable research tool, efficacious in subtype discovery, biomarkers identification and discoveries of deregulated molecular pathways.
La cura della leucemia linfoblastica acuta (ALL) sta migliorando con successo, raggiungendo un tasso di guarigione che va oltre l’80%. L’identificazione precoce dei pazienti con alto rischio di ricaduta ha portato ad un miglioramento generale dell’outcome, tuttavia, due terzi dei pazienti che incorrono nell’evento di ricaduta vengono inizialmente stratificati in gruppi a basso rischio o rischio intermedio. L’identificazione di migliori fattori prognostici rimane un’importante sfida nelle ALL pediatriche. In questa tesi, lo studio del profilo di espressione genica è stato applicato a diversi approcci di ricerca, con lo scopo di individuare sottogruppi e trovare nuovi target terapeutici nelle ALL a cellule precursori B (BCP ALL) Tra le BCP ALL, i pazienti privi delle aberrazioni genomiche più riccorrenti (B-others) rappresentano il sottogruppo che più necessita di studi approfonditi, tesi ad identificare nuovi fattori prognostici e migliorare la loro stratificazione nelle classi di rischio. Per aumentare le conoscenze biologiche riferite al gruppo dei B-others, è stato eseguito uno studio integrato di espressione genica, espressione di non coding RNAs e analisi delle aberrazioni genetiche. Il Capitolo 1 riporta lo studio mediante microarrays di espressione genica di 145 pazienti Italiani affetti da BCP ALL e, in una sotto-coorte rappresentativa, lo studio dell’espressione dei microRNAs (miRNAs) e l’analisi di variazione di DNA copy number estesa all’intero genoma. Da questo studio è emerso che il 25% dei pazienti Italiani di tipo B-others rientrano in un gruppo con una signature specifica e sono associati ad un outcome favorevole. Lo studio del profilo di espressione dei miRNAs rivela in questo gruppo una specifica signature di miRNAs caratterizzata dalla sovra espressione di hsa-miR-125b, -125b-2*, -99a, -100, -125a-3p e has-miR-491-5p. La sovra espressione del cluster miR-125b-2 nella regione 21q21.1 è accompagnata dalla concomitante sovra espressione dei geni nella stessa regione cromosomica. Le analisi sul genoma hanno portato ad escludere la presenza di alterazioni di DNA copy number nella regione 21q21.1. Il frequente coinvolgimento di aberrazioni a carico del cromosoma 21 nelle ALL (come nel caso di iperdiploidia (HD), t(12;21) o iAmp21) e il coinvolgimento della regione 21q21.1, suggeriscono un diretto e funzionale contributo dei geni nel cromosoma 21 alla trasformazione maligna delle cellule ematopoietiche. A questo proposito c’è un grande interesse nello studio delle ALL nei bambini affetti dalla Sindrome di Down (DS), nei quali la trisomia 21 è costituzionale e per i quali l’incidenza di ALL è approssimativamente 20 volte maggiore che nel resto della popolazione. Nel Capitolo 2 viene presentato uno studio di analisi genomica di un grande gruppo di DS ALL che mira a caratterizzare le anomalie molecolari specifiche di questo gruppo di ALL. L’analisi di espressione genica ha rivelato che le DS ALL sono leucemie molto eterogenee, non definibili come un unico sottotipo di ALL, con un arricchimento di geni rispondenti al signalling di BCL6 e di risposta al danno al DNA, che suggerisce un’instabilità genomica dei linfociti B. Sorprendentemente, solamente un gene appartenente al cromosoma 21, SON, è compreso nella signature delle DS ALL e risulta solo debolmente up-regolato. Inoltre, i dati di espressione genica suggeriscono che le DS ALL e le HD ALL sono leucemie molto diverse, riflettendo le differenze fondamentali tra trisomia costituzionale e acquisita, quali lo stadio di sviluppo nel quale la leucemia insorge e il fatto che la trisomia costituzionale è presente sia nelle cellule leucemiche che nel microambiente. Lo studio ha inoltre rilevato che il 62% delle DS ALL sono caratterizzate da un’aberrante espressione del recettore per le citochine di tipo I CRLF2. Due tipi di aberrazioni che coinvolgono CRLF2 sono state identificate: una traslocazione criptica che coinvolge il locus IGH@ e CRLF2 nella regione pseudoautosomale PAR1 dei cromosomi sessuali e una delezione in PAR1. Queste aberrazioni danno luogo alla formazione del trascritto di fusione P2RY8-CRLF2 che determina la sovra espressione di CRLF2. Inoltre una nuova mutazione somatica attivante, F232C, in CRLF2 è stata identificata. E’ stato dimostrato che CRLF2 e JAK2 mutato cooperano nel conferire capacità di crescita indipendente da citochine a cellule pro-B suggerendo che i bambini affetti da DS e ALL con un’espressione aberrante di CRLF2 possono trarre beneficio da terapie mirate a bloccare il pathway di CRLF2-JAK. Dal momento che le aberrazioni a carico di CRLF2 sono state trovate anche tra i pazienti non affetti dalla Sindrome di Down, è stata analizzata l’incidenza e l’impatto prognostico di questo potenziale nuovo marcatore nei pazienti Italiani con BCP ALL arruolati nello studio AIEOP-BFM ALL2000. Il Capitolo 3 presenta lo studio di una coorte rappresentativa di 464 pazienti con BCP ALL non affetti da DS che è stata analizzata per l’espressione di CRLF2 e per la presenza di riarrangiamenti a carico di CRLF2. Da questo studio è emerso che il riarrangiamento P2RY8-CRLF2 in associazione con la sovra espressione di CRLF2 (di almeno 20 volte maggiore che nel resto della coorte), identifica pazienti con una prognosi molto sfavorevole e, tra essi, un inportante sottogruppo di pazienti attualmente stratificati nella classe di rischio intermedia e che necessitano di essere considerati per un adeguamento della terapia. Per investigare i pathways emersi dalle analisi di espressione genica e per testare l’effetto dei farmaci è necessaria una grande disponibilità di cellule leucemiche. Le cellule da leucemia primaria sono difficili da coltivare in vitro e le linee cellulari attualmente disponibili non riescono a riflettere la natura eterogenea della malattia. Per questo motivo i modelli di xenotrapianto in topo sono ampiamente usati sia per lo studio in vivo che per amplificare il numero di cellule leucemiche da usare nelle varie analisi. Nello studio riportato nel Capitolo 4 è stata verificata la capacità delle cellule leucemiche ottenute da xenotrapianto di ricapitolare la loro rispettiva leucemia primaria ed è stata valutata la possibilità di una selezione da parte del microambiente murino per particolari cellule “inizianti” la leucemia che portino ad una massa tumorale marcatamente diversa da quella dei pazienti alla diagnosi. E’stato analizzato il profilo di espressione genica di 7 ALL primarie pediatriche alla diagnosi e le rispettive cellule leucemiche ottenute da xenotrapianto dopo un primo, un secondo ed un terzo passaggio seriale nel modello di trapianto di leucemia umana in topo NOD/SCID/huALL. In questo studio è stato dimostrato che il modello di trapianto NOD/SCID/huALL ricapitola la leucemia primaria umana, mima le caratteristiche del tumore primario e ne trattiene le caratteristiche durante i passaggi seriali senza selezionare per un sottoclone della leucemia primaria iniziale. Il Capitolo 5 riporta uno studio che ha investigato le proprietà di attecchimento di 50 ALL pediatriche trapiantate in topi NOD/SCID. Il tempo di attecchimento (Time To Leukemia – TTL) è stato determinato per ogni campione attecchito in termini di settimane trascorse dal trapianto alla manifestazione della leucemia. Lo studio ha mostrato che un breve TTL è fortemente associato con un alto rischio di ricaduta precoce, costituendo di fatto un nuovo marcatore prognostico indipendente. Il fenotipo di alto rischio è riflesso in una signature in grado di identificare pazienti incorsi precocemente nell’evento di ricaduta in una coorte di pazienti indipendente. Lo studio di espressione genica rivela una serie di geni associati con questo fenotipo aggressivo, mettendo a diposizione una potenziale strategia per identificare i pazienti ad alto rischio. In modo ancora più importante, pathways che regolano la crescita cellulare e che coinvolgono mTOR sono stati identificati, indicando dei target per strategie terapeutiche alternative per i pazienti ad alto rischio di riaduta. Concludendo, dieci anni dopo la sua introduzione in oncoematologia, lo studio del profilo di espressione genica si conferma essere un valido strumento di ricerca, efficace nella scoperta di nuovi sottotipi, nell’individuazione di biomarcatori e nel portare alla luce pathways molecolari deregolati.

Normal karyotype pediatric B-cell precursor ALL patients are heterogeneous with respect to chemotherapy response, relapse rates and prognosis and the reason is unknown. These patients are treated with a standard protocol, and stratified using MRD methodology that shows they have variable responses and predicted outcomes. This study aims to determine the reasons behind such heterogeneity. This study shows that through miRNA profiling, miR-221/222 are differentially expressed in normal karyotype patients and are up regulated in Poor Responder patients. Through proliferation, apoptosis, viability assays and cell cycle analysis, proliferation advantage was identified as the main cause of resistance to glucocorticoid treatment in miR-221/222 over expressing cell lines. SMAD1 and CREBBP were down regulated in miR-221/222 over expressing cell lines implicating the dysregulation of TGFβ and glucocorticoid-receptor pathways, while NLK was identified as a novel target of miR-221/222 through which resistance to chemotherapy is mediated.

La Leucemia Linfoblastica Acuta (LLA) è il tumore più frequente in età pediatrica. Nonostante l’alto successo terapeutico raggiunto, una percentuale di pazienti non risponde alle terapie convenzionali. Numerosi studi hanno mostrato come il microambiente midollare giochi un importante ruolo nella progressione tumorale. Il nostro scopo è stato quello di identificare i pahways cruciali coinvolti nel cross-talk tra le cellule leucemiche e il microambiente stromale e che possano essere dei potenziali target terapeutici. Abbiamo studiato AttivinaA, molecola della superfamiglia del TGF-β, all’interno della nicchia midollare leucemica. Questa molecola è nota nel contesto dei tumori solidi per la sua capacità di promuovere la progressione tumorale attraverso la regolazione della motilità e invasività cellulare. Abbiamo qui definito per la prima volta AttivinaA come un fattore associato alla leucemia: all’esordio di leucemia, questa molecola è altamente espressa nel midollo leucemico e risulta essere prodotta a più alti livelli dalle cellule stromali mesenchimali (MSC) in seguito a co-coltura con le cellule leucemiche. Anche nel contesto della B-LLA, abbiamo dimostrato che AttivinaA promuove la motilità ed invasività cellulare in presenza o meno del fattore chemiotattico CXCL12, fondamentale nella localizzazione delle cellule staminali ematopoietiche (HSC) e delle cellule leucemiche all’interno della nicchia midollare. In particolare, il meccanismo d’azione di AttivinaA dipende dall’incremento dei livelli di calcio intracellulare e della polimerizzazione dell’actina che sono importanti regolatori della riorganizzazione del citoscheletro e del movimento cellulare. Interessante, AttivinaA modula in modo specifico le proprietà biologiche delle cellule leucemiche in quanto svolge un effetto contrario sulle HSC, favorendo quindi le cellule leucemiche nella competizione per la nicchia. Come già riportato in letteratura, abbiamo trovato livelli ridotti di CXCL12 nel midollo leucemico e abbiamo osservato che AttivinaA è responsabile almeno in parte di questa riduzione a causa di un suo effetto diretto sulla secrezione della chemochina da parte delle MSC. Tuttavia, essendo AttivinaA capace di aumentare la migrazione cellulare anche verso concentrazioni subottimali di CXCL12, questi dati suggeriscono un possibile meccanismo tramite il quale le cellule leucemiche persistono all’interno della nicchia midollare distruggendo l’ematopoiesi sana. I nostri dati in vitro circa il ruolo pro-migratorio e pro-invasivo di AttivinaA sono stati confermati anche in vivo. I processi di regolazione dei flussi di calcio e della riorganizzazione del citoscheletro da parte di AttivinaA sono importanti per stimolare anche la vescicolazione da parte delle cellule. Abbiamo dimostrato che AttivinaA è in grado di aumentare il rilascio di vescicole extracellulari (VE) da parte delle cellule di B-LLA. Tali vescicole trasportano al loro interno l’oncogene t(1;19), tipico delle cellule dalle quali esse originano. Sia AttivinaA sia le VE da essa indotte conferiscono chemioresistenza alle cellule leucemiche, diminuendo in maniera significativa la loro sensibilità al farmaco Asparaginasi, che viene poi ripristinata bloccando la via di segnalazione di AttivinaA. Il meccanismo alla base della chemioprotezione esercitata dalle VE può essere spiegato dalla presenza al loro interno di microRNA differenzialmente espressi in seguito al trattamento con AttivinaA, tra cui il miRNA-491-5p precedentemente associato a chemioresistenza all’Asparaginasi nella leucemia pediatrica. Complessivamente, i nostri dati suggeriscono che AttivinaA è una molecola chiave della nicchia leucemica, che conferisce un vantaggio migratorio alle cellule leucemiche e le protegge dai farmaci convenzionali attraverso la produzione di VE. Il nostro lavoro potrebbe portare dunque allo sviluppo di nuovi farmaci in grado di agire sul cross-talk stroma-leucemia.
Acute Lymphoblastic Leukemia (ALL) is the most common type of cancer in children. About 80% of the cases arises from precursor B cells (BCP-ALL), which abnormally accumulate as a consequence of genetic alterations associated to differentiation inhibition and abnormal expansion. Despite the 85% survival rate, a total of 10-15% of patients retains leukemic stem cells and their progenitors in the bone marrow (BM), thereby relapsing following treatment cessation. The importance of BM microenvironment for cancer progression has been widely recognized in recent years. In this study, we aimed to identify the crucial pathways involved in the bi-directional leukemia-stroma cross-talk that could be an attractive target for future antileukemic therapy. We focused our attention on the characterization of ActivinA, a TGF-β family member, within the BM leukemic niche. Here, we identified ActivinA as a new crucial factor exploited by leukemic cells to create a self-reinforcing niche: indeed, this molecule was highly expressed in the BM plasma of leukemic patients. Furthermore, we reported that BCP-ALL cells, along with the highly pro-inflammatory environment of leukemic BM, induced a strong increase in the molecule secretion by Mesenchymal Stromal Cells (MSCs). In accordance with its protumoral role in solid tumors, ActivinA strongly induced both random and CXCL12-driven migration of cells also in the context of BCP-ALL. We observed that ActivinA selectively stimulated these leukemic cell biological properties with a calcium- and actin polymerization-mediated mechanism as this molecule showed an opposite effect on Hematopoietic stem cells (HSCs). According to the literature, we found reduced CXCL12 levels in the leukemic BM, but ActivinA enhanced cell migration also towards suboptimal CXCL12 concentrations, suggesting a possible mechanism by which leukemic cells could persist in the BM niche, displacing healthy hematopoiesis. Our in vitro data about the pro-migratory and pro-invasive role of ActivinA were confirmed also in vivo. By using a xenograft mouse model of human BCP-ALL, we demonstrated the ability of ActivinA to enhance both BM engraftment and metastatic potential intro extra-medullary sites of leukemic cells. Notably, the regulation of calcium influx and cytoskeleton organization by ActivinA is an important process to stimulate also cell vesiculation. Recent studies have shown that cancer extracellular vesicles (EVs) can mediate cell-cell communication and potentially contribute to tumor progression. Therefore, we investigated whether ActivinA was able to influence vesiculation by leukemic cells. We demonstrated that ActivinA increased the production of both exosomes and MVs by BCP-ALL cells. We found that EVs transport the t(1;19) fusion transcript, typical of cells from which they originate. We then studied the biological effects by which ActivinA-induced leukemia EVs can actively promote BCP-ALL disease, focusing our attention on resistance to therapy. Firstly, we demonstrated that ActivinA significantly decreased the sensitivity of leukemic cells to the anti-leukemic drug Asparaginase (ASNase) which was re-stored by blocking ActivinA signaling. Interestingly, also ActivinA-induced leukemia EVs conferred resistance to leukemic cells. To understand the mechanism underlying EV chemoprotection, we explored their miRNA cargo and identified differentially expressed miRNAs induced by ActivinA treatment. Of these, miR-491-5p has been previously reported to be associated with ASNase chemoresistance in childhood leukemia. The discovery of ActivinA signaling between BCP-ALL cells and MSCs adds significant insights into the mechanisms of communication in the leukemic niche. Moreover, ActivinA-induced leukemia EVs seem to play a crucial role in sustaining leukemic cells, by conferring them drug resistance. Our data provide a new concept to develop alternative therapeutic strategies that include targeting of the leukemic niche in BCP-ALL.