The KMT2A recombinome of acute leukemias in 2023.

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with de novo as well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2A gene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2A gene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5'-KMT2A, two patients had a 5'-KMT2A deletion, and one ETV6::RUNX1 patient had an KMT2A insertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2A recombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.

Complex MLL rearrangements in t(4;11) leukemia patients with absent AF4.MLL fusion allele.

The human mixed lineage leukemia (MLL) gene is frequently involved in genetic rearrangements with more than 55 different translocation partner genes, all associated with acute leukemia. Reciprocal chromosomal translocations generate two MLL fusion alleles, where 5'- and 3'-portions of MLL are fused to gene segments of given fusion partners. In case of t(4;11) patients, about 80% of all patients exhibit both reciprocal fusion alleles, MLL.AF4 and AF4.MLL, respectively. By contrast, 20% of all t(4;11) patients seem to encode only the MLL.AF4 fusion allele. Here, we analyzed these 'MLL.AF4(+)/AF4.MLL(-)' patients at the genomic DNA level to unravel their genetic situation. Cryptic translocations and three-way translocations were found in this group of t(4;11) patients. Reciprocal MLL fusions with novel translocation partner genes, for example NF-KB1 and RABGAP1L, were identified and actively transcribed in leukemic cells. In other patients, the reciprocal 3'-MLL gene segment was fused out-of-frame to PBX1, ELF2, DSCAML1 and FXYD6. The latter rearrangements caused haploinsufficiency of genes that are normally expressed in hematopoietic cells. Finally, patients were identified that encode only solitary 3'-MLL gene segments on the reciprocal allele. Based on these data, we propose that all t(4;11) patients exhibit reciprocal MLL alleles, but due to the individual recombination events, provide different pathological disease mechanisms.

Genetic polymorphism of NAD(P)H:quinone oxidoreductase is associated with an increased risk of infant acute lymphoblastic leukemia without MLL gene rearrangements.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a detoxification enzyme that protects cells against oxidative stress and toxic quinones. A polymorphism (C609T) in the gene produces in the heterozygous individuals (C/T) a reduction and in those homozygous for the variant allele (T/T) the abolishment of NQO1 protein activity. To assess whether NQO1 inactivating polymorphism (CT/TT) was a possible risk factor for infant acute lymphoblastic leukemia (iALL), we investigated the distribution of NQO1 genotype in 50 iALL patients, 32 with MLL gene rearrangements (MLL+) and 18 without (MLL-). As controls, 106 cases of pediatric ALL (pALL), and 147 healthy subjects were also studied. Compared to normal controls, the frequency of the low/null activity NQO1 genotypes was significantly higher in the iALL MLL- (72 vs 38%, P=0.006; odds ratio (OR) 4.22, 95% confidence interval (CI) 1.43-12.49), while no differences were observed in iALL MLL+ (44 vs 38%, P=0.553; OR 1.26, 95% CI 0.58-2.74). Similar results were observed when pALL were used as control. Our results indicate that only the iALL patients without MLL rearrangements had a significantly higher frequency of NQO1 genotypes associated with low/null activity enzyme, suggesting a possible role for NQO1 gene as an MLL-independent risk factor, in the leukemogenic process of this subtype of iALL.

Levels of L-asparagine in CSF after intramuscular administration of asparaginase from Erwinia in children with acute lymphoblastic leukemia.

PURPOSE: As part of a study on the pharmacokinetics associated with the administration of asparaginase (ASNase) from Erwinia to the CNS, we determined the levels of asparagine in the CSF of children with acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Twenty children received eight standard doses of intramuscular ASNase (10,000 IU/m2) every 3 days as part of induction therapy. In the postremission phase of therapy, the children were randomized to receive either 20 courses of high-dose intramuscular ASNase (25,000 IU/m2) weekly (n = 8) or four courses of standard-dose intramuscular ASNase (10,000 IU/m2) every 3 days (n = 12). RESULTS: All patients had detectable levels of L-asparagine in the CSF at the time of diagnosis. The levels of L-asparagine in CSF were undetectable in 15 of 20 (75%) and in seven of 19 (36.8%) children 3 and 5 days, respectively, after administration of standard-dose ASNase. After administration of high-dose ASNase, the levels of L-asparagine in the CSF were undetectable in five (62.5%) and two (25%) of eight children after 3 and 5 days, respectively. CONCLUSION: In this study 60% to 70% and 25% to 35% of children had complete depletion of L-asparagine from the CSF after 3 and 5 days, respectively, after administration of both schedules of ASNase from Erwinia. In the remaining patients, administration of ASNase may have resulted in a suboptimal antileukemic effect at the CNS level.

Effect of protracted high-dose L-asparaginase given as a second exposure in a Berlin-Frankfurt-Münster-based treatment: results of the randomized 9102 intermediate-risk childhood acute lymphoblastic leukemia study--a report from the Associazione Italiana Ematologia Oncologia Pediatrica.

PURPOSE: To assess in a randomized study the therapeutic effect of the addition of high-dose L-asparaginase (HD ASP) in the context of a Berlin-Frankfurt-Münster (BFM)-based chemotherapy regimen for intermediate risk (IR) childhood acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: From March 1991 to April 1995, a total of 705 patients, with 59% of the cohort of patients fewer than 15 years old, with newly diagnosed non-B ALL, enrolled onto the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) ALL-91 study, were assigned to the IR group. Patients in remission at the beginning of the reinduction phase were randomized either to the standard treatment (SD ASP arm) or the experimental treatment (HD ASP arm; weekly intramuscular administration of HD ASP 25,000 IU/m(2) repeated for a total of 20 weeks). Most of the patients (90%) were treated with Erwinia chrysanthemi L-asparaginase product. RESULTS: Among the 610 patients randomized to the SD ASP arm (n = 322) or to the HD ASP arm (n = 288), relapse occurred at a median time of 24 months after randomization in 76 (24%) and in 64 children (22%), respectively. Most of the relapses occurred in the marrow (100 isolated, 21 combined). There was no significant difference between the disease-free survival in the two treatment arms (P =.64), with estimated values at 7 years from randomization of 72.4% (SE 3.1) v 75.7% (SE 2.6) in the SD ASP and HD ASP arms, respectively. CONCLUSION: No advantage was observed for IR ALL children treated with BFM-based intensive chemotherapy who received protracted E chrysanthemi HD ASP during reinduction and the early continuation phase.

Clonal stability in children with acute lymphoblastic leukemia (ALL) who relapsed five or more years after diagnosis.

Although most relapses of childhood acute lymphoblastic leukemia (ALL) occur 24-36 months after first CR has been achieved, few patients relapse 5 or more years after CR achievement. The assessment of clonality has proved to be useful in determining whether even those very late events represent the reoccurrence of the original clone or alternatively a secondary leukemia. To gain further information on clonal stability in such late relapse, we performed detailed comparative Southern blotting and PCR analyses of TcRdelta and TcRgamma gene rearrangements in five ALL at presentation and subsequent relapse which occurred more than 5 years after diagnosis. At least one stable rearranged allele of the TcRdelta and TcRgamma loci was traced in all cases at presentation and clinical relapse despite a wide heterogeneity of the pattern of rearrangements. Our study extends to a larger series of patients previous findings which have sought to analyze the phenomenon of clonal evolution in children relapsed after more than 5 years of CCR. With respect to the potential pitfalls in monitoring minimal residual disease in childhood ALL for the presence of clonal evolution, our results highlight the combination of two target genes (such as TcRgamma and TcRdelta) as a tool to reduce false negative MRD results.

Clonal variegation and dynamic competition of leukemia-initiating cells in infant acute lymphoblastic leukemia with MLL rearrangement.

Distinct from other forms of acute lymphoblastic leukemia (ALL), infant ALL with mixed lineage leukemia (MLL) gene rearrangement, the most common leukemia occurring within the first year of life, might arise without the need for cooperating genetic lesions. Through Ig/TCR rearrangement analysis of MLL-AF4+ infant ALL at diagnosis and xenograft leukemias from mice transplanted with the same diagnostic samples, we established that MLL-AF4+ infant ALL is composed of a branching subclonal architecture already at diagnosis, frequently driven by an Ig/TCR-rearranged founder clone. Some MLL-AF4+ clones appear to be largely quiescent at diagnosis but can reactivate and dominate when serially transplanted into immunodeficient mice, whereas other dominant clones at diagnosis can become more quiescent, suggesting a dynamic competition between actively proliferating and quiescent subclones. Investigation of paired diagnostic and relapse samples suggested that relapses often occur from subclones already present but more quiescent at diagnosis. Copy-number alterations identified at relapse might contribute to the activation and expansion of previously quiescent subclones. Finally, each of the identified subclones is able to contribute to the diverse phenotypic pool of MLL-AF4+ leukemia-propagating cells. Unraveling of the subclonal architecture and dynamics in MLL+ infant ALL may provide possible explanations for the therapy resistance and frequent relapses observed in this group of poor prognosis ALL.

Detection of minimal residual disease: methods and relationship to outcome in T-lineage acute lymphoblastic leukemia.

The molecular basis of acute lymphoblastic leukemia (ALL) of both B-cell and T-cell lineages seems better understood using polymerase chain reaction (PCR) methods. The analysis of clone-specific junctional regions of rearranged genes for both Immunoglobulin (Ig H) and T-cell receptor (TcR) is the most sensitive tool for detection of minimal residual disease (MRD) in ALL. Because of the heterogeneity of all ALL patients examined in several studies, the detection of MRD at different times of treatment has not as yet been correlated with disease outcome. In contrast, T-ALL is a homogeneous disease characterized by expansion of a single clone showing a specific Rearranged junctional region of TcR delta and/or gamma genes. The use of a clone-specific probe allows detection of residual leukemia throughout treatment. However, 60 % of patients with T-ALL relapse during treatment or towards the end of therapy, with resurgence of the original leukemic clone. It is possible that the detection of MRD at a specific time-point after diagnosis, as well as at the beginning of maintenance, may help to identify a group of T-ALL patients at high risk of relapse. The correlation between detection of MRD and treatment phase may be used in the future to evaluate whether treatment regimens can be improved allowing for stratification, based on PCR-mediated detection of MRD.

[Acute lymphoblastic leukemia in children. Results of treatment in Sicily 1987-1992]. / Leucémie aiguë lymphoblastique de l'enfant. Résultats du traitement en Sicile entre 1987 et 1992.

OBJECTIVES: For several years, children in Sicily with acute lymphoblastic leukaemia have been treated locally at the University of Catania. We compared the results of locally treated children with the results obtained at other centres. METHODS: The diagnosis of acute lymphoblastic leukaemia (ALL) was made in 78 children in the haematology and oncology division of the University of Catania, Sicily, From January 1987 to December 1992. Patients diagnosed before December 1990 were treated with the protocol ALL 87 including prednisone, vincristine, daunorubicine, L-asparaginase and intrathecal methotrexate. Total duration of treatment was 25-26 months. For patients diagnosed after December 1990, the protocol 90-91 used the same drugs for induction and three intrathecal drugs (methotrexate, cytarabine and prednisone) as well as cyclophosphamide to prolong induction in intermediary risk patients. Only high risk patients received cerebral irradiation. Total duration of treatment was 2 years. Full follow-up data were available for 76 patients. RESULTS: Survival rate without relapse was 66% and the 5-year survival rate was 82.7%. These results were comparable with those published by other international groups. In addition, particular attention was given to psychological support to decrease the deleterious effects of both the disease and the treatment protocols. CONCLUSION: These results demonstrate that ALL children can be cared for locally in Sicily without risking poorer outcome. It would be preferable to treat these children as near as possible to their area of residence in order to diminish the psychological trauma resulting from long-term hospitalization far from their family.

Pulse pressure variation as a predictor of fluid responsiveness in mechanically ventilated patients with spontaneous breathing activity: a pragmatic observational study.

INTRODUCTION: Pulse pressure variation predicts fluid responsiveness in mechanically ventilated patients passively adapted to the ventilator. Its usefulness in actively breathing ventilated patients was examined only by few studies with potential methodological shortcomings. This study sought to describe the performance of pulse pressure variation as a predictor of fluid responsiveness in hypotensive critically ill patients who trigger the ventilator. METHODS: We studied forty two hypotensive, mechanically ventilated patients with documented spontaneous breathing activity in whom a fluid challenge was deemed necessary by the attending physician. All patients were ventilated with a Maquet Servo-i Ventilator in different ventilatory modes with a flow-regulated inspiratory trigger set on position 4. Pulse pressure variation, mean and systolic arterial pressure were observed before and after the fluid challenge, which consisted in the intravenous administration of a 250 ml bolus of 6% hetastarch. Fluid responsiveness was defined as a more than 15% increase in arterial pressure after volume expansion. RESULTS: The area under the receiver operator characteristic curve for pulse pressure variation was 0.87 (95% CI 0.74 -0.99; p<0.0001) and the grey zone limits were 10% and 15%. Pulse pressure variation was correlated with increase in systolic arterial pressure (r2=0.32; p<0.001) and mean arterial pressure (r2=0.10; p=0.037). CONCLUSIONS: Pulse pressure variation predicts fluid responsiveness in patients who actively interact with a Servo-i ventilator with a flow-regulated inspiratory trigger set on position 4.

Long-term results of the Italian Association of Pediatric Hematology and Oncology (AIEOP) Studies 82, 87, 88, 91 and 95 for childhood acute lymphoblastic leukemia.

We analyzed the long-term outcome of 4865 patients treated in Studies 82, 87, 88, 91 and 95 for childhood acute lymphoblastic leukemia (ALL) of the Italian Association of Pediatric Hematology and Oncology (AIEOP). Treatment was characterized by progressive intensification of systemic therapy and reduction of cranial radiotherapy. A progressive improvement of results with reduction of isolated central nervous system relapse rate was obtained. Ten-year event-free survival increased from 53% in Study 82 to 72% in Study 95, whereas survival improved from 64 to 82%. Since 1991, all patients were treated according to Berlin-Frankfurt-Muenster (BFM) ALL treatment strategy. In Study 91, reduced treatment intensity (25%) yielded inferior results, but intensification of maintenance with high-dose (HD)-L-asparaginase (randomized) allowed to compensate for this disadvantage; in high-risk patients (HR, 15%), substitution of intensive polychemotherapy blocks for conventional BFM backbone failed to improve results. A marked improvement of results was obtained in HR patients when conventional BFM therapy was intensified with three polychemotherapy blocks and double delayed intensification (Study 95). The introduction of minimal residual disease monitoring and evaluation of common randomized questions by AIEOP and BFM groups in the protocol AIEOP-BFM-ALL 2000 are expected to further ameliorate treatment of children with ALL.

New insights to the MLL recombinome of acute leukemias.

Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.

Acute neurotoxicity in children with B-lineage acute lymphoblastic leukemia (B-ALL) treated with intermediate risk protocols.

BACKGROUND: This study describes the incidence of acute neurotoxicity (NT) in children with B-lineage acute lymphoblastic leukemia (ALL) treated with three intermediate risk protocols that differ in the intensity of central nervous system (CNS) "prophylaxis. " PROCEDURE: A total of 122 patients (64 boys; median age 5.3 years) with B-lineage ALL without CNS leukemia diagnosed between February 1987 and December 1997 were enrolled in the intermediate risk (IR) protocols: Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP)-ALL 87 (n = 33), 91 (n = 51), and 95 (n = 38). Presymptomatic CNS therapy consisted of intrathecal methotrexate (six doses) and cranial irradiation (18 Gy) in the IR AIEOP 87 study, and extended triple intrathecal therapy with methotrexate, cytarabine, and prednisone depending on age in the IR AIEOP-ALL 91 and 95 protocols (20 and 17 total doses, respectively). World Health Organization (WHO) grade 4 acute neurotoxicity criteria were employed. Patients with neurologic symptoms, in addition to physical examination, underwent EEG, computed tomography (CT) and/or magnetic resonance imaging (MRI), and lumbar puncture to exclude CNS leukemia and infection. RESULTS: Acute NT was not reported in AIEOP-ALL 87 treated patients, but we observed acute NT in 3 out of 51 (5.8%) AIEOP-ALL 91 patients, and in 7 out of 38 (18.4%) AIEOP-ALL 95 patients. CONCLUSIONS: There was an increased incidence of acute NT in our patients with ALL treated with current intermediate risk protocols. The intensification of treatment, however, bettered event free survival (EFS) to 58%, 72% and 85% in IR AIEOP 87, 91 and 95 studies, respectively.

Low-grade haemolysis and assessment of iron status during the steady state in G6PD-deficient subjects.

We evaluated the iron status of 50 Sicilian patients with G6PD deficiency under steady-state conditions and compared our results with those for 50 control patients. We studied haemolysis and iron indices to evaluate the iron balance. These patients could be considered to be at risk of iron overload as a result of increased bone marrow activity. Reticulocytosis and macrocytosis with reduced levels of haptoglobin were found in the G6PD-deficient subjects, both of which are evidence of a moderate haemolysis. Iron status within the normal range, without iron overload or iron deficiency, was found.