The distribution of MLL breakpoints correlates with outcome in infant acute leukaemia

Acute leukaemia in early childhood ‐ and mainly infant leukaemia (IL) – is characterized by acquired genetic alterations, most commonly by the presence of distinct MLL rearrangements (MLL‐r). The aim of this study was to investigate possible correlations between clinical features and molecular analyses of a series of 545 childhood leukaemia (≤24 months of age) cases: 385 acute lymphoblastic leukaemia (ALL) and 160 acute myeloid leukaemia (AML). The location of the genomic breakpoints was determined in a subset of 30 MLL‐r cases. The overall survival of the investigated cohort was 60·5%, as determined by the Kaplan‐Meier method. Worse outcomes were associated with age at diagnosis ≤6 months (P < 0·001), high white blood cell count (P = 0·001), and MLL‐r (P = 0·002) in ALL, while children with AML displayed a poorer outcome (P = 0·009) regardless of their age strata. Moreover, we present first evidence that MLL‐r patients with poor outcome preferentially displayed chromosomal breakpoints within MLL intron 11. Based on the literature, most MLL‐r IL display a breakpoint localization towards intron 11, which in turn may explain their worse clinical course. In summary, the MLL breakpoint localization is of clinical importance and should be considered as a novel outcome predictor for MLL‐r patients.     

Azurophilic inclusions in lymphocytes and plasma cells: a case of Sanfilippo disease

Correct action upon re‐emergence of minimal residual disease in acute myeloid leukaemia (AML) patients has not yet been established. The applicability of demethylating agents and use of allogeneic stem cell transplantation will be dependent on pre‐relapse AML growth rates. We here delineate molecular growth kinetics of AML harbouring MLL partial tandem duplication (MLL‐PTD; 37 cases) compared to those harbouring MLL translocations (43 cases). The kinetics of MLL‐PTD relapses was both significantly slower than those of MLL translocation positive ones (median doubling time: MLL‐PTD: 24 d, MLL‐translocations: 12 d, P = 0·015, Wilcoxon rank sum test), and displayed greater variation depending on additional mutations. Thus, MLL‐PTD+ cases with additional RUNX1 mutations or FLT3‐internal tandem duplication relapsed significantly faster than cases without one of those two mutations (Wilcoxon rank sum test, P = 0·042). As rapid relapses occurred in all MLL subgroups, frequent sampling are necessary to obtain acceptable relapse detection rates and times from molecular relapse to haematological relapse (blood sampling every second month: MLL‐PTD: 75%/50 d; MLL translocations: 85%/25 d). In conclusion, in this cohort relapse kinetics is heavily dependent on AML subtype as well as additional genetic aberrations, with possibly great consequences for the rational choice of pre‐emptive therapies.     

急性白血病11q23/MLL基因易位重排及其临床意义

目的：研究11q23/MLL基因易位重排在急性白血病（AL）中的发生率、产生融合基因的常见类型及其临床意义.方法：用荧光原位杂交技术，MLL双色断裂分离重排探针检测50例AL患者（49例初治，1例难治）的11q23/MLL基因，用流式细胞仪检测免疫表型，对于11q23/MLL基因易位重排阳性的患者，用巢式RTPCR方法检测11q23/MLL基因易位重排形成的6种常见融合基因类型.结果：6例AL有11q23/MLL基因易位重排，发生率为12%，2例为AML-M5，4例为ALL且均为B-ALL.2例11q23/MLL基因易位重排阳性的AML M5患者融合基因均为MLL/AF9，其中1例为初治，发病时左侧小腿有白血病细胞浸润，本例患者化疗1疗程获CR;1例为难治性AL患者，于第3个疗程化疗后才达CR.4例11q23/MLL基因易位重排阳性的B-ALL患者中有2例于诊断后3周内死于全身衰竭和感染，化疗未获CR，其中1例患者的融合基因为MLL/ENL，1例未扩出融合基因产物;1例于诊断后第2天因DIC脑出血死亡，未进行化疗，其融合基因为MLL/AF9;1例发病时胸椎有白血病细胞浸润，1疗程化疗后获CR，其融合基因产物未扩出.结论：荧光原位杂交技术是检测AL11q23/MLL基因易位重排快速、灵敏的方法，巢式RT-PCR是检测11q23/MLL基因易位重排所产生的融合基因类型简便可行的方法;有11q23/MLL基因易位重排的AL患者临床症状凶险，预后差。     

Mechanisms of leukemogenesis by MLL fusion proteins

Infant acute leukaemia is characterised by specific genetic rearrangements and a rapid onset of disease shortly after birth. The vast majority of these cases bear rearranged MLL alleles. However, many facets of MLL-rearranged leukaemia are largely unknown. Basically, there exists a fundamental and evolutionary conserved relationship between the family of MLL/Trithorax proteins and the regulation of HOX gene clusters. Therefore, direct MLL fusion proteins are per se able to deregulate HOX genes, except when reciprocal MLL fusion proteins come into play. This reviews discusses (i) the current situation in MLL-rearranged leukaemia, (ii) the molecular and genetic tools to functionally investigate the many different MLL fusions, (iii) the latency of disease development, (iv) a novel cancer mechanism that has been recently uncovered when different MLL fusion protein complexes were characterized, (v) mutated signalling pathways in MLL-rearranged leukaemia and (vi) presents new ideas on how a given MLL fusion protein may modulate existing signalling pathways in leukaemic cells. The hypothesis is posed that the many different fusion partners of MLL are critically distinct entities for which specific inhibitors should be identified in the future.     

CBL mutations do not frequently occur in paediatric acute myeloid leukaemia

RAS‐pathway mutations, causing a proliferative advantage, occur in acute myeloid leukaemia (AML) and MLL‐rearranged leukaemia. Recently, mutations in the Casitas B lineage lymphoma (CBL) gene were reported to be involved in RAS‐pathway activation in various myeloid malignancies, but their role in paediatric AML is still unknown. We performed mutation analysis of 283 newly diagnosed and 33 relapsed paediatric AML cases. Only two mutant cases (0·7%) were identified in the newly diagnosed paediatric AML samples, of which one was MLL‐rearranged. Both mutant cases showed CBL mRNA expression in the range of the non‐mutated cases. Phosphorylated extracellular signal‐regulated kinase (pERK) was not correlated with CBL protein expression (n = 11). In conclusion, we report a very low CBL mutation frequency in paediatric AML, which, together with the lack of difference in protein and mRNA expression, illustrates the limited role of CBL in paediatric AML.     

MLL‐rearranged acute lymphoblastic leukaemia stem cell interactions with bone marrow stroma promote survival and therapeutic resistance that can be overcome with CXCR4 antagonism

Infants with MLL‐rearranged (MLL‐R) acute lymphoblastic leukaemia (ALL) have a dismal prognosis. While most patients achieve remission, approximately half of patients recur with a short latency to relapse. This suggests that chemotherapy‐resistant leukaemia stem cells (LSCs) survive and can recapitulate the leukaemia. We hypothesized that interactions between LSCs and the bone marrow microenvironment mediate survival and chemotherapy resistance in MLL‐R ALL. Using primary samples of infant MLL‐R ALL, we studied the influence of bone marrow stroma on apoptosis, proliferation, and cytotoxicity induced by the FLT3 inhibitor lestaurtinib. MLL‐R ALL were differentially protected by stroma from spontaneous apoptosis compared to non‐MLL‐R ALL. Co‐culture of bulk MLL‐R ALL in direct contact with stroma or with stroma‐produced soluble factors promoted proliferation and cell cycle entry. Stroma also protected bulk MLL‐R ALL cells and MLL‐R ALL LSCs from lestaurtinib‐mediated cytotoxicity. Previous studies have demonstrated that CXCR4 mediates bone marrow microenvironment signalling. Using a xenograft model of MLL‐R ALL, we demonstrated that CXCR4 inhibition with AMD3100 (plerixafor) led to markedly enhanced efficacy of lestaurtinib. Therefore, the bone marrow microenvironment is a mediator of chemotherapy resistance in MLL‐R ALL and targeting leukaemia‐stroma interactions with CXCR4 inhibitors may prove useful in this high‐risk subtype of paediatric ALL.     

Translocation between chromosome 5q35 and chromosome 11q13 – an unusual cytogenetic finding in a primary refractory acute myeloid leukemia

Cytogenetic abnormalities are observed in approximately two‐thirds of patients with acute myeloid leukemia (AML). Chromosome rearrangements are associated with specific subtypes of AML and associated prognosis. We report a patient with AML, M2, who was primarily refractory to standard induction chemotherapy with idarubicin and cytarabine. Flow cytometry of a bone marrow aspirate showed aberrant expression of B‐cell markers including CD19. Cytogenetic studies disclosed a translocation between 5q35 and 11q13. Fluorescence in situ hybridization analyses demonstrated that neither the NSD1 nor MLL genes were involved in this case. Further study is required to define conclusively the genes involved and their contribution to pathogenesis in this case.     

NG2 expression in MLL rearranged acute myeloid leukaemia is restricted to monoblastic cases

Expression of NG2 has been reported in the majority of paediatric acute leukaemia (AL) cases with MLL rearrangement. We demonstrated 7. 1 positivity in 2/3 paediatric and 4/11 adult MLL rearranged acute myeloid leukaemia (AML) but in 0/28 adult AML without MLL rearrangement, thus extending the 100% specificity to adult cases. Positivity correlated with stage of maturation arrest since it was found in 0/6 immature AML but in 6/8 monoblastic cases. These data demonstrate that, if NG2 expression in AL is the (in)direct result of MLL rearrangement, such activation is restricted to a monoblastic population in AML. They also have practical implications for NG2 diagnostic screening strategies.     

Therapy-Related Acute Myeloid Leukemia 6 Years after Clonal Detection of inv(11)(q21q23) and MLL Gene Rearrangement

Results of recent studies with animal models suggest that expression of MLL fusion proteins promotes acute leukemogenesis. However, the most potent MLL fusion proteins are not sufficient for the development of acute myeloid leukemia (AML). The clinical data on the pathogenesis of this type of leukemia are limited. We analyzed the case of a patient with therapy-related AML with MLL rearrangement. The patient initially developed AML with t(8;21). Although the patient achieved complete remission with chemotherapy, an abnormal karyotype, inv(11)(q21q23), was detected. After 6-year persistence of a clone with the inversion 11 karyotype in the bone marrow, secondary AML developed. Results of fluorescence in situ hybridization analysis combined with magnet-activated cell sorting analysis showed that MLL rearrangement was detected in CD34+ and CD13+ fractions but not in a CD3+ fraction of the bone marrow. There were 2 important clinical findings. One was that MLL rearrangement was not sufficient for the development of leukemia. The other was that MLL rearrangement targets specific lineages.     

Apoptosis and leukaemia

Defects in the intrinsic ability of haematopoietic progenitor cells to undergo apoptosis may allow the cell to acquire further mutations, survive inappropriately and eventually become malignant. Additionally, this defect could account for the resistance to cell death, observed in leukaemic cells, following treatment with chemotherapy. This review discusses some of the molecules known to influence apoptosis in leukaemic cells, particularly the novel fusion proteins produced as a result of leukaemia-associated chromosomal translocations. The ultimate aim of understanding how apoptosis is altered in leukaemia cells is so that the process can be modulated to overcome resistance to chemotherapy and improve clinical outcomes. The relationship of leukaemia-related fusion proteins such as PML-RAR α , BCR-ABL, E2A-HLF, AML1-ETO and the various MLL fusions to the biochemical pathways involved in apoptosis are discussed as well as the consequences for therapeutic applications.     

HMGA2 as a potential molecular target in KMT2A‐AFF1‐positive infant acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) in infants is an intractable cancer in childhood. Although recent intensive chemotherapy progress has considerably improved ALL treatment outcome, disease cure is often accompanied by undesirable long‐term side effects, and efficient, less toxic molecular targeting therapies have been anticipated. In infant ALL cells with KMT2A (MLL) fusion, the microRNA let‐7b (MIRLET7B) is significantly downregulated by DNA hypermethylation of its promoter region. We show here that the expression of HMGA2, one of the oncogenes repressed by MIRLET7B, is reversely upregulated in infant ALL leukaemic cells, particularly in KMT2A‐AFF1 (MLL‐AF4) positive ALL. In addition to the suppression of MIRLET7B, KMT2A fusion proteins positively regulate the expression of HMGA2. HMGA2 is one of the negative regulators of CDKN2A gene, which encodes the cyclin‐dependent kinase inhibitor p16^INK4A. The HMGA2 inhibitor netropsin, when combined with demethylating agent 5‐azacytidine, upregulated and sustained the expression of CDKN2A, which resulted in growth suppression of KMT2A‐AFF1‐expressing cell lines. This effect was more apparent compared to treatment with 5‐azacytidine alone. These results indicate that the MIRLET7B‐HMGA2‐CDKN2A axis plays an important role in cell proliferation of leukaemic cells and could be a possible molecular target for the therapy of infant ALL with KMT2A‐AFF1.     

Oncogenesis in utero: fetal death due to acute myelogenous leukaemia with an MLL translocation

The incidence of translocations involving the 11q23 gene MLL is markedly increased in leukaemias that occur in infants < 1 year of age. Epidemiological and molecular data have demonstrated that at least some of these translocations occur in utero . In this report we describe a case of fetal death at 36 weeks of gestation. At autopsy the fetus was found to have widely disseminated acute myelogenous leukaemia (AML), FAB subtype M5. Molecular cytogenetic studies of nuclei recovered from paraffin-embedded tissue sections demonstrated that the leukaemic cells contained an MLL translocation. This is the first detailed report, to our knowledge, of fetal death due to acute leukaemia, and directly demonstrates oncogenesis in utero .