Acute megakaryoblastic leukaemia (AMKL) and transient myeloproliferative disorder (TMD) in Down syndrome: a multi-step model of myeloid leukaemogenesis

Children with Down syndrome (DS) have a marked increase in susceptibility to Acute Megakaryoblastic Leukaemia (DS-AMKL) and the closely linked neonatal preleukaemic syndrome, Transient Myeloproliferative Disorder (DS-TMD). The distinct stages of DS-TMD and DS-AMKL provide an excellent tractable model to study leukaemogenesis. This review focuses on recent studies describing clinical, haematological and biological features of DS-AMKL and DS-TMD. The findings from these studies suggest that mutations in the key haemopoietic regulator GATA1 (GATA binding protein 1) in DS-AMKL and DS-TMD may be useful in diagnosis and assessing minimal residual disease. These findings raise the possibility of population-based screening strategies for DS-TMD and the development of treatment to eliminate the preleukaemic TMD clone to prevent DS-AMKL. Advances in our understanding of perturbed haemopoiesis in DS, the role of GATA1 and of cooperating mutations are also discussed. These findings have implications for leukaemia biology more broadly given the frequency of acquired trisomy in other human leukaemias.     

Functional analysis of JAK3 mutations in transient myeloproliferative disorder and acute megakaryoblastic leukaemia accompanying Down syndrome

JAK3 mutations have been reported in transient myeloproliferative disorder (TMD) as well as in acute megakaryoblastic leukaemia of Down syndrome (DS-AMKL). However, functional consequences of the JAK3 mutations in TMD patients remain undetermined. To further understand how JAK3 mutations are involved in the development and/or progression of leukaemia in Down syndrome, additional TMD patients and the DS-AMKL cell line MGS were screened for JAK3 mutations, and we examined whether each JAK3 mutation is an activating mutation. JAK3 mutations were not detected in 10 TMD samples that had not previously been studied. Together with our previous report we detected JAK3 mutations in one in 11 TMD patients. Furthermore, this study showed for the first time that a TMD patient-derived JAK3 mutation (JAK3(I87T)), as well as two novel JAK3 mutations (JAK3(Q501H) and JAK3(R657Q)) identified in an MGS cell line, were activating mutations. Treatment of MGS cells and Ba/F3 cells expressing the JAK3 mutants with JAK3 inhibitors significantly decreased their growth and viability. These results suggest that the JAK3 activating mutation is an early event during leukaemogenesis in Down syndrome, and they provide proof-of-principle evidence that JAK3 inhibitors would have therapeutic effects on TMD and DS-AMKL patients carrying activating JAK3 mutations.     

Incidence and implication of additional chromosome aberrations in acute promyelocytic leukaemia with translocation t(15;17)(q22;q21): a report on 50 patients

Acute promyelocytic leukaemia (APL) is characterized by the translocation t(15;17)(q22;q21). Usually t(15;17) is the sole cytogenetic abnormality, but some patients show other chromosome aberrations in addition to t(15;17). The influence of additional chromosome aberrations on the clinical outcome of patients with t(15;17) is unclear. We have analysed 50 cases of APL carrying the translocation t(15;17). Additional chromosome aberrations were observed in 17/47 patients (36%) studied at initial diagnosis and in all three patients studied at relapse. In nine cases (18%) an additional chromosome 8 and in six cases (12%) an isochromosome of the long arm of the derivative chromosome 17 was observed. Various structural rearrangements in addition to t(15;17) were detected in nine patients (18%). Clinical follow-up data were available for 44 patients studied at diagnosis. A complete remission (CR) was achieved in 34 patients (77%). 10 patients (23%) died within 1 month after diagnosis due to infection or bleeding, eight (24%) relapsed within 10–18 months after initial diagnosis. 28 patients are alive 2–93 months after diagnosis (25 in first CR, two in second and one in third CR) (median follow-up 18.5 months). Bone marrow transplantation was performed in six patients (three in first CR, two in second CR, one in third CR), all are alive and in CR. An influence of secondary chromosome anomalies on prognosis was not observed. However, if a higher rate of long-term remission can be reached, specific secondary chromosome aberrations might turn out to be of prognostic value.     

t(4;11)(q21;p15) translocation involving NUP98 and RAP1GDS1 genes: characterization of a new subset of T acute lymphoblastic leukaemia

Two cases of T acute lymphoblastic leukaemia (T-ALL) with an identical t(4;11)(q21;p15) translocation were identified within a prospective study on the biological and clinical features of adult ALL patients enrolled into the therapeutic protocol ALL0496 of the GIMEMA Italian Group. In both cases, the molecular characterization showed an involvement of the NUP98 gene on 11p15 which rearranges with the RAP1GDS1 gene on 4q21. The morphological and immunological features of the leukaemic cells, as well as the clinical behaviour and response to induction therapy, were the same in both patients. Based on the available data, the t(4;11)(q21;p15) translocation involving the NUP98-RAP1GDS1 fusion gene emerges as a new highly specific genetic abnormality that characterizes a subset of T-ALL.     

A novel t(15;17) translocation in acute myeloid leukaemia not associated with PML/RARα rearrangement

We report a novel t(15;17)(q15;q11) translocation in acute myeloid leukaemia (AML M4) which was not associated with PML/RARα rearrangement or with acute promyelocytic leukaemia (APL) morphology. The leukaemia behaved in refractory fashion, with 70% blasts in the bone marrow after the first course of chemotherapy. In view of the refractory behaviour of the leukaemia, the patient was treated with high dose chemotherapy and autologous stem cell rescue. The patient is alive and well 22 months post‐autologous stem cell transplant. This case demonstrates that while cytogenetic analysis provides important diagnostic and prognostic information, the precise location of the chromosomal breakpoints is critical in the interpretation of cytogenetic results.     

Interphase FISH to detect PBX1/E2A fusion resulting from the der(19)t(1;19)(q23;p13.3) or t(1;19)(q23;p13.3) in paediatric patients with acute lymphoblastic leukaemia

Approximately 6% of paediatric patients with precursor B-cell acute lymphoblastic leukaemia (B-ALL) harbour a rearrangement involving the gene regions of PBX1 (1q23) and E2A (19p13.3) which is visualized cytogenetically either as a der(19)t(1;19)(q23;p13.3) or the less common balanced t(1;19)(q23;p13.3). Unfortunately, no commercial dual-colour, double fusion fluorescence in situ hybridization (D-FISH) strategies are available to detect this recurrent anomaly. Therefore, we have created a D-FISH assay to detect these translocations and monitor minimal residual disease. This probe set was created using four bacterial artificial chromosomes (BACs) corresponding to the PBX1 gene region at 1q23 and four BACs corresponding to the E2A gene region at 19p13.3. We analysed 30 negative bone marrow controls and 20 diagnostic and post-treatment specimens from 13 paediatric B-ALL patients with a cytogenetically defined 1;19 translocation. Once unblinded, the results demonstrated that our D-FISH method effectively identified all diagnostic samples as abnormal and identified disease in four post-treatment samples that were previously considered to be normal by conventional cytogenetic analysis. The development of this FISH strategy for the detection of der(19)t(1;19)(q23;p13.3) and t(1;19)(q23;p13.3) proved to be an effective technique, allowing both the detection of disease in diagnostic samples and in post-treatment samples.     

Cell lineage specific involvement in acute promyelocytic leukaemia (APL) using a combination of May-Grünwald-Giemsa staining and fluorescence in situ hybridization techniques for the detection of the translocation t(15;17)(q22;q12)

Acute promyelocytic leukaemia (APL) is strongly associated with the translocation t(15;17) which therefore provides a reliable marker to assess the potential involvement of different cell lineages. Six cases with morphologically, cytogenetically and molecularly proven APL were analysed at diagnosis or relapse by combining fluorescence in situ hybridization (FISH) with standard May-Grünwald-Giemsa (MGG) staining at the single cell level on bone marrow and blood smears. With the FICTION technique, combining immunophenotyping with FISH, haemopoietic precursor cells were identified using monoclonal antibodies against CD34, B- and T-lymphocytes could be identified with CD19 and CD3. In addition, HLA-DR-positive cells were studied for the presence of t(15;17).
Morphologically identified myeloblasts were relocated on the smear after FISH and were found to be PML/RARA positive in 91%, abnormal promyelocytes in 97%. In contrast, a positive signal was obtained in only 18% of PMN. Erythroblasts, lymphocytes and plasma cells did not show a PML/RARA rearrangement. Accordingly, all cells expressing CD3 or CD19 were PML/RARA negative. CD34 positive precursor cells identified by FICTION were PML/RARA positive in 97%. HLA-DR-positive cells contained a PML/RARA rearrangement in 24% of cells in one case and were negative in the two other cases investigated. These data indicate that APL appears to originate from a level of haemopoietic precursor cells but is restricted to the myeloid lineage. The low proportion of PML/RARA-positive PMN points to the impairment of blast cell differentiation beyond the promyelocyte stage but also emphasizes the existence of normal residual haemopoietic stem cells from which PML/RARA-negative PMN must be derived.     

A sensitive model for prediction of relapse in adult acute myeloid leukaemia with t(8;21) using white blood cell count, CD56 and MDR1 gene expression at diagnosis

Acute myeloid leukaemia (AML) carrying t(8;21) has an overall favourable prognosis. However, relapse occurs and the impact of multidrug resistance gene (MDR1) expression on recurring disease in this group of patients is not known. We determined quantifiable MDR1 expression in the bone marrow of 28 AML patients with t(8;21) by a validated real-time polymerase chain reaction assay. Using MDR1 expression, white blood cell count and CD56 expression at diagnosis we observed complete concordance of predicted and observed relapses. A calculated logit out of these three variables was a strong independent prognostic factor for overall (P = 0.007) and disease-free survival (P = 0.002).     

A study in three European IBD cohorts confirms that the ATG16L1 c.898A > G (p.Thr300Ala) variant is a susceptibility factor for Crohn’s disease

Background and aimsA recent study reported that a nonsynonymous SNP rs2241880 (c.898A > G, p.Thr300Ala) within ATG16L1 confers susceptibility to Crohn's disease (CD). We analyzed ATG16L1 c.898A > G in three independent European inflammatory bowel disease (IBD) cohorts from Germany, Hungary and the Netherlands.MethodsIn total, we included 910 European IBD patients and compared the ATG16L1 c.898A > G genotype frequency with 707 ethnically matched healthy controls. We included patients from 3 populations originating from Germany (CD n = 310; ulcerative colitis [UC] n = 179), Hungary (CD n = 147; UC n = 117), and the Netherlands (CD n = 157). Subtyping analysis was performed in respect to CARD15 alterations and clinical characteristics.ResultsWe found a highly significant association of c.898A > G to CD. The association was significant (p = 0.0005) for the total CD cohort but also for the individual populations from Germany (p = 0.02) and Netherlands (p = 0.02) whereas in the Hungarian CD patients a clear trend was observed (p = 0.19; OR 1.227, 95% CI 0.910; 1.654). No association was found between c.898A > G and UC. No statistical interactions were observed between ATG16L1 c.898A > G and CARD15 variants. Furthermore no association to a CD subphenotype was detected.ConclusionsWe confirm that ATG16L1 variant c898A > G confers a risk variant for CD but is not associated with a distinct CD phenotype.     

A Phase I/II study of the safety and efficacy of the addition of sirolimus to tacrolimus/methotrexate graft versus host disease prophylaxis after allogeneic haematopoietic cell transplantation in paediatric acute lymphoblastic leukaemia (ALL)

Sirolimus has been shown to have activity against human acute lymphoblastic leukaemia at serum levels used for immunosuppression. We hypothesized that the addition of sirolimus to a tacrolimus/methotrexate graft- versus -host disease (GVHD) prophylaxis regimen would decrease relapse after haematopoietic stem cell transplantation and initiated a phase I/II study to demonstrate safety, feasibility, and efficacy. The study cohort included 18 patients in high-risk (HR) first complete remission (CR1), 16 in HR CR2, 17 in intermediate risk (IR) CR2, and 12 in CR3+. The 2-year event-free survival (EFS) of the cohort was 66% (standard error 6·4). EFS of risk groups was 74%, 81%, 44% and 46% for CR1, IR CR2, HR CR2 and CR3+ patients respectively, and did not differ by stem cell source. Cumulative incidence of acute GVHD grade II–IV and III–IV was 38% and 21% respectively, while the cumulative incidence of chronic GVHD was 32%. Cumulative incidence of transplant-related mortality and relapse was 10% and 25% respectively. Significant toxicities included veno-occlusive disease [seven patients (11%)], transplant-associated microangiopathy (three patients), and idiopathic pneumonitis (one patient). In summary, sirolimus-based GVHD prophylaxis can be given safely in this population and early survival results are promising. A phase III trial to test whether sirolimus decreases relapse and improves outcome after transplantation for ALL is ongoing.