Littérature scientifique sur le sujet « MLL+ infant ALL »

10030 Background: Infant acute lymphoblastic leukemia (ALL) is the only subtype of childhood ALL whose outcome has not improved over the past two decades. The most important prognosticator is the presence of rearrangements in the Mixed Lineage Leukemia gene (MLL-r), however, many patients present with high-risk clinical features but without MLL-r. We recently identified two cases of infant ALL with high-risk clinical features resembling MLL-r, but were negative for MLL-r by conventional diagnostics. RNA sequencing revealed a partial tandem duplication in MLL (MLL-PTD). We thus aimed to determine if MLL-PTD, other MLL abnormalities, or other genetic or transcriptomic features were driving this subset of high-risk infant ALL without MLL-r. Methods: We obtained 19 banked patient samples from the Children’s Oncology Group (COG) infant ALL trial (AALL0631) from MLL wildtype patients as determined by FISH and cytogenetics. Utilizing deep RNA-sequencing, we manually inspected the MLL gene for MLL-PTD, while also performing automated fusion detection and gene expression profiling in search of defining features of these tumors. Results: 3 additional MLL-PTDs were identified, all in patients with infant T-cell ALL, whereas both index cases were in patients with infant B-cell ALL. Gene expression profiling analysis revealed that all five MLL-PTD infants clustered together. Eight infants (7 with B-cell ALL) were found to have Ph-like expression. Five of these 8 infants were also found to have an IKZF1/JAK2 expression profile; one of these five had a PAX5-JAK2 fusion detected. Two infants (including the one noted above) had novel PAX5 fusions, known drivers of B-cell leukemia. Additional detected fusions included TCF3-PBX1 and TCF4-ZNF384. Conclusions: MLL-PTDs were found in both B- and T-cell infant ALL. Though Ph-like ALL has been described in adolescents and young adults, we found a substantial frequency of Ph-like expression among MLL-WT infants. Further characterization of these infants is ongoing. If replicated in other infant cohorts, these two findings may help explain the poor prognosis of MLL-WT ALL when compared to children with standard risk ALL, and offer the possibility of targeted therapy for select infants.

Abstract MLL gene rearranged acute lymphoblastic leukemia (MLL) is an aggressive type of acute lymphoblastic leukemia (ALL) with a relatively poor response to treatment, predominantly occurring in infants (<1 years of age). Since conventional chemotherapy is failing in >50% of these patients, identification of novel therapeutic targets to direct therapy against is demanded. For this, understanding this disease and finding biological and molecular features characterizing MLL may be crucial. Here we report that infant MLL is characterized by silencing of the putative tumor suppressor fragile histidine triad (FHIT) gene. Gene expression profiling showed that FHIT expression is significantly reduced in MLL patients (n=20) as compared to patients diagnosed with conventional ALL (n=24) bearing germ line MLL genes. Using quantitative real-time PCR we confirmed FHIT mRNA expression to be low in MLL gene rearranged infant ALL (infant MLL) as compared to both infants and older children (non-infants) not carrying chromosomal abnormalities involving the MLL gene. Infant MLL patients (n=35) significantly (p<0.001) expressed ~15-fold less FHIT mRNA compared to infant ALL patients (n=8) and 11-fold less than non-infant ALL patients (n=22). Methylation specific PCR (MSP) analysis demonstrated that the FHIT promoter region is hypermethylated in all of the 36 infant MLL cases tested, resulting in strongly reduced FHIT mRNA as well as FHIT protein expression. In contrast, the incidence of FHIT promoter methylation in both infant and non-infant ALL patients carrying germ line MLL genes was markedly lower, with a frequency of 63% (5/8) and 56% (24/43) respectively. Suppression of FHIT expression could be reversed by exposing leukemia cells bearing MLL gene rearrangements to the demethylating drug 5′-aza-2′-deoxycytidine. These findings imply that MLL rearranged ALL is characterized by FHIT promoter hypermethylation. Currently experiments are being conducted to determine the biological significance of FHIT silencing in this type of cancer.

Abstract Introduction Acute lymphoblastic leukaemia (ALL) in infants has poor overall survival despite being characterized by very few genetic aberrations per case. The most common genetic change, present in over 75% of cases, is the rearrangement of the mixed lineage leukaemia (MLL/KMT2A) gene (MLL-R) that also occurs in AML and mixed phenotype acute leukaemia (MPAL). Because infant ALL is rare and distinctive, most Australian and New Zealand patients are enrolled on specific clinical trials. In earlier infant ALL trials, MRD was not used for risk stratification firstly because of the difficulty of finding sensitive markers for specific immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements and the secondly because, in infant ALL, Ig/TCR markers are often present in sub-clones that can be lost at relapse due to clonal selection. MRD testing is performed in the current Interfant 06 trial preferentially using markers based on the genomic breakpoint sequences of MLL gene rearrangements. The treatment of infant ALL remains very challenging with relatively poor survival rates attributable to both toxicity and relapse. The objectives of this study were therefore to analyse MRD data for Interfant 06 patients enrolled at ANZCHOG centres and to perform a pilot experiment evaluating gene expression for key genes in infant ALL samples on a microfluidics platform, as a basis for identifying potential targeted therapies. Methods MRD was measured in bone marrow DNA from Interfant 06 patients enrolled since 2006, using sensitive Real-time Quantitative PCR (PCR-MRD) patient-specific assays to detect either MLL gene rearrangements (in 17) and/or conventional immunoglobulin and T-cell receptor (Ig/TCR) markers (21). Gene expression levels for 90 genes important in childhood cancers were measured by Taqman-based microfluidic assays in duplicate using cDNA from 2 micrograms of total RNA from 10 infant ALL samples (5 MLL, 5 non-MLL) at diagnosis (6) or relapse (4). Results Patient-specific PCR-MRD tests were developed for 27 out of 28 Australian and New Zealand infant ALL patients. 17/18 MLL-R ALL patients had MLL-R assays and 21/28 patients had Ig/TCR MRD tests, with only 1 (non-MLL) patient having no MRD markers. There was a wide range of MRD responses to induction therapy (Figure 1). Bone marrow MRD at the end of induction was high (>1x10-3) in 44% of MLL-R ALL infants compared to 25% of non-MLL ALL infants and 15% of older children enrolled on ANZCHOG ALL8. In 8/13 MLL-R patients who had both types of marker, MRD levels were higher when measured by their MLL-R marker than by their Ig/TCR marker. In a set of 90 genes selected for expression analysis, higher levels were found for 17 genes in 2 or more of the 10 infant ALL samples evaluated. These more highly expressed genes included potential or known drug targets BCL2, ERBB2, ERBB4, ILRA2, CSF1R and PARP1. Conclusions The quantitation of MRD based on MLL rearrangements in ALL is effective and can also be used to monitor response to therapy in infant ALL as well as MLL-R cases of AML and MPAL. The combined application of MLL-R and Ig/TCR markers allowed 97% of infant ALL patients to be MRD monitored with a sensitive marker. In most patients with both type of MRD marker, higher levels of MRD were detected in end of induction samples using the MLL versus Ig/TCR tests. One interpretation is that Ig/TCR genes are rearranged after the MLL rearrangement in ALL sub-clones that are both more mature and more chemo-sensitive. This finding also confirms the current consensus that disease-related MLL-R markers provide better risk assessment than Ig/TCR markers. Our Fluidigm analysis has shown that quantitative measurement of multiple gene expressions is feasible on small RNA samples and can be used to rapidly screen for specific expression of genes coding for drug targets in ALL patients. Support: NHMRC Australia APP1057746, Sporting Chance Cancer Foundation. Figure 1. Comparison of MRD response to induction therapy in MLL-R infant ALL compared with non-MLL infant ALL (Interfant 06) and older children (ANZCHOG ALL8). Figure 1. Comparison of MRD response to induction therapy in MLL-R infant ALL compared with non-MLL infant ALL (Interfant 06) and older children (ANZCHOG ALL8). Disclosures No relevant conflicts of interest to declare.

Abstract MLL-rearranged infant acute lymphoblastic leukemia (ALL) remains the most aggressive type of childhood leukemia, displaying a unique gene expression profile. Here we hypothesized that this characteristic gene expression signature may have been established by potentially reversible epigenetic modifications. To test this hypothesis, we used differential methylation hybridization to explore the DNA methylation patterns underlying MLL-rearranged ALL in infants. The obtained results were correlated with gene expression data to confirm gene silencing as a result of promoter hypermethylation. Distinct promoter CpG island methylation patterns separated different genetic subtypes of MLL-rearranged ALL in infants. MLL translocations t(4;11) and t(11;19) characterized extensively hypermethylated leukemias, whereas t(9;11)-positive infant ALL and infant ALL carrying wild-type MLL genes epigenetically resembled normal bone marrow. Furthermore, the degree of promoter hypermethylation among infant ALL patients carrying t(4;11) or t(11;19) appeared to influence relapse-free survival, with patients displaying accentuated methylation being at high relapse risk. Finally, we show that the demethylating agent zebularine reverses aberrant DNA methylation and effectively induces apoptosis in MLL-rearranged ALL cells. Collectively these data suggest that aberrant DNA methylation occurs in the majority of MLL-rearranged infant ALL cases and guides clinical outcome. Therefore, inhibition of aberrant DNA methylation may be an important novel therapeutic strategy for MLL-rearranged ALL in infants.

Abstract Acute lymphoblastic leukemia (ALL) in infants (< 1 year) is characterized by a poor prognosis and a high incidence of MLL translocations. Several studies demonstrated the unique gene expression profile associated with MLL-rearranged ALL, but generally small cohorts were analyzed as uniform patient groups regardless of the type of MLL translocation, whereas the analysis of translocation-negative infant ALL remained unacknowledged. Here we generated and analyzed primary infant ALL expression profiles (n = 73) typified by translocations t(4;11), t(11;19), and t(9;11), or the absence of MLL translocations. Our data show that MLL germline infant ALL specifies a gene expression pattern that is different from both MLL-rearranged infant ALL and pediatric precursor B-ALL. Moreover, we demonstrate that, apart from a fundamental signature shared by all MLL-rearranged infant ALL samples, each type of MLL translocation is associated with a translocation-specific gene expression signature. Finally, we show the existence of 2 distinct subgroups among t(4;11)–positive infant ALL cases characterized by the absence or presence of HOXA expression, and that patients lacking HOXA expression are at extreme high risk of disease relapse. These gene expression profiles should provide important novel insights in the complex biology of MLL-rearranged infant ALL and boost our progress in finding novel therapeutic solutions.

Abstract MLL rearranged Acute Lymphoblastic Leukemia (ALL) represents an unfavorable and difficult to treat type of leukemia that often is highly resistant to glucocorticoids like prednisone and dexamethasone. As the response to prednisone largely determines the clinical outcome of pediatric ALL patients, overcoming resistance to these drugs may be an important step towards improved prognosis. Here we compared gene expression profiles between prednisone-resistant and prednisone-sensitive pediatric ALL patients to obtain gene expression signatures associated with prednisone resistance for both childhood (&gt;1 year of age) and MLL rearranged infant (&lt;1 year of age) ALL. Merging both signatures in search for overlapping genes associated with prednisone resistance in both patient groups we, found that elevated expression of MCL-1 (an anti-apoptotic member of the BCL-2 protein family) appeared to be characteristic for both prednisone-resistant ALL samples. To validate this observation, we determined MCL-1 expression using quantitative RT-PCR in a cohort of MLL rearranged infant ALL samples (n=23), and confirm that high-level MCL-1 expression significantly confers glucocorticoid resistance both in vitro and in vivo. Finally, down-regulation of MCL-1 in prednisone resistant MLL rearranged ALL cells by RNA interference (RNAi) markedly sensitized these cells to prednisone. Therefore we conclude that MCL-1 plays an important role in glucocorticoid resistance and that MCL- 1 suppressing agents co-administered during glucocorticoid treatment may be beneficial especially for MLL rearranged infant ALL patients.

Abstract MLL-rearranged acute lymphoblastic leukemia (ALL) represents an unfavorable type of leukemia that often is highly resistant to glucocorticoids such as prednisone and dexamethasone. Because response to prednisone largely determines clinical outcome of pediatric patients with ALL, overcoming resistance to this drug may be an important step toward improving prognosis. Here, we show how gene expression profiling identifies high-level MCL-1 expression to be associated with prednisolone resistance in MLL-rearranged infant ALL, as well as in more favorable types of childhood ALL. To validate this observation, we determined MCL-1 expression with quantitative reverse transcription–polymerase chain reaction in a cohort of MLL-rearranged infant ALL and pediatric noninfant ALL samples and confirmed that high-level MCL-1 expression is associated with prednisolone resistance in vitro. In addition, MCL-1 expression appeared to be significantly higher in MLL-rearranged infant patients who showed a poor response to prednisone in vivo compared with prednisone good responders. Finally, down-regulation of MCL-1 in prednisolone-resistant MLL-rearranged leukemia cells by RNA interference, to some extent, led to prednisolone sensitization. Collectively, our findings suggest a potential role for MCL-1 in glucocorticoid resistance in MLL-rearranged infant ALL, but at the same time strongly imply that high-level MCL-1 expression is not the sole mechanism providing resistance to these drugs.

Abstract Abstract 2547 Acute lymphoblastic leukemia (ALL) in children less than 1 year old is the relatively rare disease with specific biological features and poor outcome. It is also characterized by high incidence of MLL gene rearrangements. Immunophenotype of infants' leukemia varies due to presence or absence of MLL-rearrangements. Aim of the study. description of immunophenotype in infant acute lymphoblastic leukemia. Methods. Totally 421 cases of pediatric acute leukemia (AL) were studied. 81 patients (39 boys and 42 girls) aged from 5 days to 11 months were included in the study group. Their data was compared to 332 cases of acute leukemia in older children. Tumor cells immunophenotyping was performed by 6–8-color flow cytometry. Detection of various types of MLL-gene rearrangements was done by simultaneous application of chromosomal banding analysis, fluorescence in-situ hybridization, reverse-transcriptase polymerase chain reaction (PCR) and long-distance inverse PCR. Results. There were 54 (66.7%) ALL cases in the study group. ALL was found less frequently in infants than in older children (66.7% and 88.5% respectively, p<0.0001) while percentage of acute myeloid leukemia cases was higher in infants (27.2% and 10.0% respectively, p=0.0001). EGIL immunophenotypes distribution also differed between infants and older children. BI-ALL was the most common immunological ALL type in infant ALL (55.6% vs 3.3% in older age group, p<0.0001), while BII-ALL was notably less frequent compared with other age groups (33.3% and 77.1% respectively, p<0.0001). T-lineage ALL was also less frequent in infants (3.7% vs 14.3% in older age group) although difference did not achieve statistical significance (p=0.0536). Totally infant ALL were mainly presented by B-cell precursor ALL (BCP-ALL) – 51 patients (94.4%). Various types of MLL-rearrangements were found in 40 (74.1%) patients (pts) out of 54 infants ALL cases. Among them 21 pts (52.5%) carried MLL-AF4 fusion gene, 8 pts (20.0%) – MLL-MLLT1, 5 pts (12.5%) – MLL-MLLT3, 3 pts (7.5%) – MLL-EPS15, 1 pt (2.5%) – MLL-MLLT10, 1 pt (2.5%) – MLL-AFF3 and 1 pt (2.5%) had MLL-rearrangement with unidentified partner gene. Significant immunophenotypic differences were observed in patients with and without MLL gene rearrangements. Number of cases in those tumor cells expressed CD10, CD20, CD45, CD133, CD15, NG2 significantly varied between MLL-positive and MLL-negative groups (p=0.0001, p<0.0001, p=0.0008, p=0.0018, p=0.0306 and p<0.0001 correspondingly). NG2-positivity represented the highest overall correct prediction (OCP) rate for presence of MLL-rearrangements (95.5%). Diagnostic accuracy of CD20-negativity and CD45-positivity was slightly lower (87.5% and 86.3% respectively) while OCP for CD10-negativity (78.4%), CD133-positivity (75.0%) and CD15-positivity (66.7%) was not sufficient enough. Nevertheless CD10-positive BCP-ALL with MLL-rearrangements differed from CD10(+) cases in MLL-germline group. CD10 homogeneous expression was noted in 10 out of 11 MLL-germline patients and in 1 of 10 MLL-rearranged cases (p=0.0011). Although there were found no significant differences in CD22-positive patients' number, CD22(+)-cells percentage was significantly lower in MLL-positive cases (median 89.9%, range 25.2–99.7% and median 99.9%, range 96.0–99.9% respectively, p=0.0026). Thus CD20-negativity, CD10-negativity/low expression, high CD45, CD15, CD65 and NG2 expression, decreased CD22-expression are immunophenotypic signatures of MLL-rearranged infant ALL, although NG2 has the highest diagnostic efficacy. Interestingly there were no markers able to distinguish MLL-AF4-positive cases from patients carrying other types of MLL-rearrangements. Even NG2 expression intensity did not differ between these groups (p=0.2720). Except BCP-ALL pts, two cases of T-lineage ALL and one mature B-ALL (without other Burkitt lymphoma features) were found. Conclusion. Thus immunophenotype of ALL in children less than 1 year old differs significantly from patients of older age groups. Infants' B-cell precursor ALL immunophenotype varies greatly due to the presence of MLL gene rearrangements. Complex diagnostic immunophenotyping of infants' ALL allows predicting presence of MLL rearrangements and NG2 is the most applicable single marker. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2417 Leukemia is the commonest malignancy in infants, the most frequently occurring form of which is infant ALL. When ALL occurs in infants the disease is clinically aggressive and associated with poor outcome. MLL gene rearrangements producing transforming fusion oncoproteins are found in 75% of infant ALL and they are adverse prognostic factors. Infant ALL has never occurred in families except in monozygous twins, where concordance in leukemia occurrence is nearly 100%. Identical but non-germline genomic breakpoint junction sequences have pointed to an in utero origin of MLL gene rearrangements in these twin cases, where it is believed that metastasis of cells with the rearrangement occurs from one twin to the other via the placental circulation. Here we describe two highly novel siblings both deceased from precursor B cell infant ALL (ages at diagnosis: proband 160 d, sibling 121 d). Remarkably, the second of these two decedents is survived by a now 3 year-old monozygous twin who is as yet unaffected. MLLrearrangements in the leukemia blasts of both affected siblings were characterized by conventional cytogenetics and/or FISH, M-FISH, high resolution Illumina 610K Bead Chip SNP array and Southern blot analysis. MLL genomic breakpoint junction sequences and fusion transcripts were defined using panhandle PCR approaches, PCR with gene-specific primers and reverse transcriptase PCR. The twins were confirmed to be monozygous using the genotype calls from SNP array analysis of the peripheral blood and bone marrow of the unaffected and affected twins, respectively. Quantitative real-time PCR analysis of leukemia DNA was used to determine the allele specific sequences of the NQO1 (NADPH quinone oxidorecutase 1) gene, an inactivating polymorphism in which previously was implicated as a risk factor for MLL-rearranged infant ALL. The complex karyotype in the leukemia cells of the proband was 46, XX, der(2) t(2;3) (q3?;?), der(3) ?t(3;4;11), del(4) (q21), der(11) ?del(11) (p11.2) t(3;11) (?;q23).ish der(3) (5'MLL+), der(11) (3'MLL+) [14]/46, XX[8], suggesting extensive damage to the genome. Consistent with a three-way t(3;4;11) translocation, two alternately spliced 5'-MLL-MLLT2(AF-4)-3' fusion transcripts were identified, indicating disruption of the chromosome band 4q21 partner gene MLLT2. Also consistent with the three-way translocation, reverse panhandle PCR detected a 5'-partner-MLL-3' genomic breakpoint junction fusing 3' MLL to the upstream region of a highly novel chromosome 3 gene encoding a nucleotidyltransferase fold protein C3ORF31 (Accession no. NM_138807; Kuchta, 2009). Unlike in the proband, the ALL of the affected twin exhibited a simple t(4;11)(q21;q23) translocation, the reciprocal genomic breakpoint junctions of which fusing MLL and MLLT2 also have been characterized. Similar to non-familial infant ALL, the genomic breakpoint junctions in both infants contained sequence features of nonhomologous end joining DNA repair. The different MLL gene rearrangements in the leukemia cells in the affected siblings indicate that the translocations were not hereditary. Even though the NQO1 inactivating polymorphism is one genetic risk factor for infant ALL, the NQO1 genotype was wild-type in both affected siblings. Further studies in this uniquely afflicted family with two siblings who succumbed to infant ALL and a monozygous twin of one of the decedents surviving beyond infancy unaffected, will provide a one-time opportunity to capture novel mutations predisposing to the development of, and cooperating with, MLL translocations in infant ALL. The MLLT2 involvement in both cases has even further implications for the knowledge to be gained because the MLL-MLLT2 rearrangement occurs in 50% of infant ALL and adversely impacts outcome. Disclosures: Felix: Children's Hospital of Philadelphia: Methods and Kits for Analysis of Chromosomal Rearrangements Associated with Leukemia - U.S. Patent # 6,368,791 issued April 9, 2002.

Abstract At present, long-term survival rates in childhood acute lymphoblastic leukemia (ALL) easily exceed 80%. However, the prognosis for infants (&lt;1 year) with ALL barely reaches 50%. Infant ALL is characterized by chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene that occur in about 80% of the cases. The most frequent MLL translocations in infant ALL include t(4;11), t(11;19) and t(9;11). In about 20% of the infant ALL cases no MLL rearrangements are observed. Recent gene-expression profiling characterized MLL-rearranged ALL as a unique type of leukemia, that is genetically clearly separable from other ALL subtypes. As epigenetic modifications affect gene-expression, we hypothesized that the specific gene-expression profiles associated with MLL-rearranged ALL may well be driven by epigenetic changes. The best-studied epigenetic event in hematological malignancies constitutes the transcriptional silencing of (tumor suppressor) genes by promoter CpG island hypermethylation. To explore the DNA methylation patterns underlying MLL-rearranged infant ALL, we applied Differential Methylation Hybridization (DMH) using both 9k (Huang, 2002) and 244k CpG island microarrays (Agilent) on primary infant ALL samples carrying t(4;11) (n=21), t(11;19) (n=17), t(9;11) (n=6) or wild-type MLL genes (n=13). The resulting DNA methylation patterns were compared with the patterns found in healthy pediatric bone marrow samples (n=8). In addition, relapse material from three infants with MLL-rearranged ALL was included and compared with the corresponding patient sample obtained at diagnosis. Both CpG island microarray platforms demonstrate that t(4;11) and t(11;19) characterize extensively hypermethylated leukemias, whereas t(9;11)-positive and translocation-negative infant ALL epigenetically resemble normal bone marrow. When the CpG array data (Agilent) were compared with available gene expression profiles (Affymetrix), we found that 95% of the genes from the top 100 of genes most significantly hypermethylated in t(4;11)- or t(11;19)-positive infant ALL were indeed down-regulated. Using the t(4;11)-positive cell line models SEMK2 and RS4;11, we demonstrate that the majority of these hypermethylated genes could be demethylated by the demethylating agent zebularine. Among t(4;11)- and t(11;19)-positive infant ALL samples, two subgroups could be identified displaying either more or less pronounced methylation patterns. Heavy methylation appeared to be associated with a significantly reduced relapse-free survival (p=0.03). Encouraged by these data, we analyzed relapse samples from t(4;11)- and t(11;19)-positive infant ALL patients, and found that these samples were even more extensively hypermethylated than the corresponding initial infant ALL samples. We here present, for the first time to our knowledge, a global view of the methylome in infant patients with MLL-rearranged ALL. We demonstrate that severe promoter CpG hypermethylation is present in t(4;11)- and t(11;19)-positive infant ALL. Of main therapeutic interest is our finding that the degree of DNA methylation among t(4;11)- and t(11;19)-positive infant ALL patients is related to relapse-free survival. Therefore, MLL-rearranged infant ALL patients with heavily methylated leukemias in particular should be considered candidates for therapies including inhibitors of DNA methylation in order to reverse the malignant phenotypes of these leukemias, and improve prognosis. Since MLL-rearranged infant ALL patients are even more hypermethylated at relapse, inhibition of aberrant DNA methylation might also be of vital importance at this stage of disease. Based on these data, we propose to initiate clinical trials using demethylating agents for patients with relapsed MLL- rearranged infant ALL. Meanwhile, we are investigating the in vitro cytotoxicity of various demethylating agents in our laboratory to pave the way for future clinical trials.

Ogni giorno, circa mille miliardi di nuove cellule vengono prodotte dal midollo osseo e messe in circolazione nel sangue periferico per far fronte alle necessità del nostro organismo. Tutte le cellule che compongono il nostro sangue, derivano da una stessa cellula-madre, chiamata cellula staminale ematopoietica, un precursore primordiale e immaturo che ha la capacità di espandersi e la potenzilità di differenzare a eritrociti, linfociti, leucociti, piastrine. Il processo di differenziamento che va dalla cellula staminale alle cellule mature circolanti è detto emopoiesi. La leucemia è un tipo di tumore circolante che può avere origine da una cellula qualunque del sangue, a un qualsiasi stadio differenziativo, la quale inizia a proliferare all’impazzata, sfuggendo ad ogni controllo, e prende il sopravvento sulle altre cellule sane. Il risultato è l’espansione aberrante di un singolo tipo cellulare, a discapito degli altri compartimenti, che causa una disfunzione del sistema emopoietico. La leucemia non può essere considerata un’ unica malattia, poichè ne esistono molti diversi tipi, a seconda del tipo cellulare interessato, e del tipo di mutazione genica che la caratteizza. Nel nostro laboratorio, presso il Centro di Ricerca M. Tettamanti dell’Opedale S. Gerardo di Monza, ci occupiamo delle leucemie del bambino. In particolare, nel mio periodo di dottorato, mi sono occupata di studiare una particolare forma di leucemia linfoblastica acuta, detta infant LLA, ad insorgenza nel primo anno di vita, che presenta il riarrangiamento del gene MLL. Questa leucemia rappresenta la forma prevalente in età neonatale, ed è purtoppo una malattia molto aggressiva con prognosi infausta che in Italia colpisce circa 10-12 nuovi nati all’anno. In particolare, i casi che presentano la traslocazione t(4;11) con espressione del prodotto di fusione MLL-AF4 rappresentano il sottogruppo genetico a incidenza maggiore in età neonatale. Per moltissimi aspetti, sia dal punto di vista clinico che biologico, la LLA infant rappresenta una malattia peculiare, diversa dalle altre forme di leucemia dell’età pediatrica e adulta. In questi quattro anni di dottorato mi sono occupata nello specifico di due progetti. Nel primo, in collaborazione con il Prof. Jacobsen in Svezia, ci siamo proposti di identificare, isolare e caratterizzare dal punto di vista biologico e funzionale la ‘cellula staminale leucemica’ della LLA infant con riarrangiamento del gene MLL. Nel secondo studio, pubblicato su ‘Leukemia’ abbiamo voluto valutare se il solo riarrangiamento del gene MLL fosse di per se sufficiente per dare origine alla malattia, oppure se alterazioni genetiche ulteriori, secondarie al riarrangiamento di MLL che costituisce il primo evento, fossero necessarie per la manifestazione clinica della leucemia. La composizione cellulare di ogni singolo tumore umano è altamente eterogenea, e non tutte le cellule che lo compongono sono capaci di sostenerne la crescita. Per spiegare queste osservazioni è stata postulata l’esistenza di un sottogruppo ristretto di cellule, dette staminali tumorali (CSC), o staminali leucemiche (LSC) nel caso si tratti di leucemia, che hanno la potenzialità di auto-rigenerarsi e di riprodurre l’intera massa tumorale. Secondo il modello della ‘cellula staminale tumorale’, in tutti i tumori solo un ristretto gruppo di cellule è capace di generare l’intera massa tumorale e sostenerne la crescita all’infinito. Poter bersagliare in modo specifico questa cellula significa eradicare definitivamente la malattia, che altrimenti inevitabilmente si rigenererebbe. Questa ipotesi è in contrasto con il precedente modello stocastico secondo cui ogni cellula che compone il tumore è potenzialmente capace di sostenerne la crescita: tuttavia la eterogenicità tumorale, e la sua bassa tumorigenicità sono dovute alla scarsa probalilità per una cellula di iniziare il ciclo cellulare e proliferare. Tuttavia recenti studi mettono in discussione l’ipotesi stessa dell’esistenza di una CSC, ed attualmente l’opinione scientifica è divisa riguardo al fatto se il modello gerarchico della CSC, piuttosto che il classico modello stocastico, siano più adatti per spiagere il meccanismo biologico alla base dell’insorgenza dei tumori umani. Infatti, benchè il concetto di CSC sia intuitivamente immediato, la sua effettiva identificazione e caratterizzazione si è rivelata inaspettatamente complicata, e ad oggi, non esiste una definizione universale di CSC. Il termine ‘leucemia’ comprende in realtà tantissimi tipi differenti di tumore, e tale eterogeneità si riflette sulla LSC. La specifica alterazione genetica, la cellula di origine (cioè la cellula inizialmente colpita dal primo evento trasformante da cui il tumore ha avuto luogo), le diverse metodologie in vivo applicate, l’influenza del microambiente midollare e l’alta variabilità fenotipica tra diversi pazienti affetti dalla medesima forma di leucemia, hanno portato inevitabilmente alla generazione di risultati apparentementente contrastanti tra i vari gruppi di ricerca che indipendentemente si sono riproposti di identificare la LSC. Il modello sperimentale d’elezione per identificare la LSC è il trapianto in vivo di blasti leucemici in topi umanizzati immunocompromessi, capaci di accettare cellule umane senza rigettarle e permetterne l’attecchimento. Nel periodo all’estero trascorso nel laboratorio del Porf. Jacobsen a Lund, mi sono occupata di mettere a punto il modello di trapianto in vivo di cellule leucemiche da pazienti affetti da infant LLA in topi unamizzati. Previa irradiazione subletale del topo ricevente, che ha il duplice scopo di eliminare le cellule circolanti murine mature e creare spazio per le cellule umane trapiantate, i blasti leucemici vengono iniettati nel topo per via sistemica attraverso la vena della coda.. Se l’attecchimento è avvenuto, già dopo poche settimane, i topi mostreranno alti livelli di ricostituzione nel midollo e nei compartimenti extramidollari (come milza, fegato e sangue periferico), con conseguente manifestazione dei sintomi clinici della malattia (quali splenomegalia, pelo arruffato, letargia, anoressia, perdita di peso e decesso). L’attecchimento viene valutato eseguendo periodiche biopsie midollari (dopo 6, 9, 12 o più settimane dal trapianto) dalla sede femorale, in anestesia generale, e successivamente la presenza di cellule leucemiche umane è rilevata nell’aspirato midollare tramite analisi successive. Nel primo lavoro inviato a ‘Cancer Cell’ abbiamo osservato che nella LLA infant MLL-AF4 positiva la LSC risiede nella frazione CD19+, costituita da linfociti B a diversi stadi maturativi. Infatti, la frazione CD19- è composta sia da cellule staminali normali residue (non leucemiche) che danno luogo a una ricostituzione normale in vivo, che da cellule leucemiche (MLL riarrangiate), tuttavia prive di potenziale leucemogenico, poiché incapaci di ripopolare il midollo di un topo irradiato e dare origine alla leucemia. Inoltre abbiamo dimostrato che all’interno della frazione CD19+, esistono molteplici sottopolazioni LSC con diverso potenziale leucemogenico e diverse caratteristiche biologiche e funzionali in vivo (es. penetranza, latenza di insorgenza della malattia e fenotipo della leucemia risultante). In conclusione, i nostri risultati dimostrano che, a differenza del modello convenzionale, esistono molteplici sottopopolazioni LSC con caratteristiche biologiche e funzionali distinte, che possono essere distinte tra loro e dalla restante popolazione leucemica (non tumorigenica) sulla base dell’espressione di determinati antigeni di superficie. Nel secondo studio pubblicato su ‘Leukemia’, ci siamo chiesti se la sola presenza della traslocazione del gene MLL fosse di per se sufficiente per la manifestazione clinica della malattia, o se mutazioni accessorie cooperanti fossero necessarie. Per fare ciò abbiamo analizzato a livello genomico un gruppo consistente di pazienti affetti da LLA infant MLL-AF4 positiva, tramite tecniche sofisticate di screening su tutto il genoma ad alta risoluzione, e abbiamo osservato che, contrariamente ai pazienti affetti da altri tipi di LLA pediatrica ad insorgenza dopo l’anno, nella LLA infant MLL-AF4 positiva non si rilevano mutazioni aggiuntive. Questi risultati dimostrano che il solo riarrangiamento di MLL è necessario e sufficiente per la manifestazione, in tempi brevi, della malattia conclamata, a differenza di altre forme di leucemia del bambino e dell’adulto, in cui mutazioni genetiche aggiuntive sono indispensabili. Inoltre tali evidenze ribadiscono ulteriormente che la LLA infant con riarangiamento di MLL è una malattia nica dal punto di vista biologco, diversa dagli altri tipi di leucemia, e che che il meccanismo di leucemogenesi può essere distinto. Considerando l’alta mortalità, l’alto rischio di ricaduta, la prognosi infausta della LLA infant MLL positiva e l’urgente necessità di avere a disposizione nuove ed efficaci terapie antitumorali, questi studi sono di rilevante importanza, in quanto non solo aiutano a comprendere meglio i meccanismi di insorgenza della LLA infant e fare luce sulla biologia e l’aggressività di questa malattia, ma rappresentano anche il punto di partenza per identificare nuove strategie terapeutiche al fine bersagliare la LSC ed eradicare la malattia in modo definitivo.