Interphase FISH on TEL/AML1 positive acute lymphoblastic leukemia relapses – analysis of clinical relevance of additional TEL and AML1 copy number changes

Objectives: TEL/AML1 (ETV6/RUNX1) fusion resulting from the translocation t(12;21)(p13;q22) constitutes the most common chimeric fusion gene in initial childhood B‐cell precursor (BCP) acute lymphoblastic leukemia (ALL) (19–27%) and has been associated with good prognosis. Three secondary aberrations in TEL/AML1 positive ALL have been suspected to negatively influence outcome: deletion of the second TEL allele (T), gain of the second AML1 allele (A) and duplication of the derivative chromosome 21 (der(21), TA). Many studies have explored such aberrations in initial disease, while only few reports have investigated them in relapses. Methods: In this study, bone marrow samples from 38 children with relapsed TEL/AML1 RT‐PCR positive and negative BCP‐ALL were analyzed for these mutations by interphase fluorescence in situ hybridization and results were compared with published data. Results: In children with TEL/AML1 positive ALL relapse, additional (a) TEL loss, (b) combined AML1 and der(21) gain, (c) combined TEL loss and AML1 gain as well as (d) the occurrence of a subpopulation with the signal pattern 1T/3A/1TA appear to be related to higher peripheral blast counts (PBCs) at relapse diagnosis (a and d) or a tendency towards the occurrence of a subsequent relapse (b and c) (P‐values <0.05). Conclusions: Our data together with published results on TEL/AML1 positive ALL suggest that frequencies of additional TEL and AML1 mutations are, with the exception of loss of untranslocated TEL, higher in first relapses than in initial disease. They also show that it is important to consider combined mutations in the analysis of this leukemia entity.     

Comprehensive analysis of cooperative gene mutations between class I and class II in de novo acute myeloid leukemia

Acute myeloid leukemia (AML) has been thought to be the consequence of two broad complementation classes of mutations: class I and class II. However, overlap‐mutations between them or within the same class and the position of TP53 mutation are not fully analyzed. We comprehensively analyzed the FLT3, cKIT, N‐RAS, C/EBPA, AML1, MLL, NPM1, and TP53 mutations in 144 newly diagnosed de novo AML. We found 103 of 165 identified mutations were overlapped with other mutations, and most overlap‐mutations consisted of class I and class II mutations. Although overlap‐mutations within the same class were found in seven patients, five of them additionally had the other class mutation. These results suggest that most overlap‐mutations within the same class might be the consequence of acquiring an additional mutation after the completion both of class I and class II mutations. However, mutated genes overlapped with the same class were limited in N‐RAS, TP53, MLL‐PTD, and NPM1, suggesting the possibility that these irregular overlap‐mutations might cooperatively participate in the development of AML. Notably, TP53 mutation was overlapped with both class I and class II mutations, and associated with morphologic multilineage dysplasia and complex karyotype. The genotype consisting of complex karyotype and TP53 mutation was an unfavorable prognostic factor in entire AML patients, indicating this genotype generates a disease entity in de novo AML. These results collectively suggest that TP53 mutation might be a functionally distinguishable class of mutation.     

Multiple clonal MLL fusions in a patient receiving CHOP-based chemotherapy

MLL rearrangements were analysed in the blood of a patient receiving chemotherapy for diffuse large B‐cell lymphoma using inverse polymerase chain reaction targeting exon 12, parallel sequencing and a custom algorithm design. Of thirteen MLL rearrangements detected, five were capable of generating MLL fusion genes, including MLL‐MLLT3, the most common fusion in acute myeloid leukaemia (AML). Other fusions, all previously clinically unobserved, included MLL‐NKD1, a fusion to the negative regulator of Wnt/β‐catenin signaling, a pathway linked to leukaemic cell proliferation. The majority of the fusions exhibited clonal persistence from before treatment until 6 months post‐chemotherapy, suggesting the fusions may confer a survival advantage to the mutant clone. MLL breakpoints were partly clustered at a specific location, indicating commonality in the process of their formation. Further, the same MLL breakpoint location exhibited a 50–100‐fold increase in C to T transitions, consistent with attack by activation‐induced cytidine deaminase (AICDA). As is also observed in AML and acute lymphoblastic leukaemia, in this single patient setting, MLL is capable of interacting with multiple fusion partners. This finding defines a discrete site of MLL susceptibility to fragmentation, linked to possible deregulation of AICDA function.     

The kinetics of relapse in DEK‐NUP214‐positive acute myeloid leukemia patients

Preemptive treatment of relapse of acute myeloid leukemia (AML) holds the promise to improve the prognosis of this currently highly lethal condition. Proposed treatment modalities applicable in preemptive cytoreduction (e.g., demethylating agents or standard chemotherapy) differ substantially in interval from administration to antileukemic effect. The t(6;9) balanced translocation, producing the DEK‐NUP214 fusion protein, is seen in only 1% of patients with AML. We hypothesized that in these patients, who relapse with a very high frequency, a more detailed knowledge of leukemic relapse growth kinetics would improve the personalized decision‐making regarding re‐administration of chemotherapy. Based on standardized quantitative PCR data, we therefore delineated the relapse kinetics in a cohort of 27 relapsing DEK‐NUP214‐positive patients treated in four different European countries. The prerelapse leukemic burden increased with a median doubling time of 13 d (range: 5–51 d, median: 0.71 logs/month, range: 0.18–1.91 logs/month), with FLT3‐ITD‐positive patients relapsing significantly faster than FLT3‐ITD‐negative ones (median: 0.9 vs. 0.6 logs/month, Wilcoxon rank sum test, P = 0.041). Peripheral blood and bone marrow were equally useful for minimal residual disease (MRD) detection, and thus, we found that with sampling intervals of 2 months, 94% of relapses would be detected with a median time from MRD detection to hematological relapse of 64 d. In conclusion, this data provide algorithms for handling the rare patients with DEK‐NUP214‐positive AML allowing for planning of both MRD follow‐up and, upon molecular relapse, the timing of cytoreduction or possibly transplant procedures.     

Mobilized peripheral blood stem cells compared with bone marrow from HLA-identical siblings for reduced-intensity conditioning transplantation in acute myeloid leukemia in complete remission: a retrospective analysis from the Acute Leukemia Working Party of EBMT

Reduced‐intensity conditioning (RIC)‐alloSCT is increasingly used for acute myelogenous leukemia. Limited data are available for the comparison of peripheral blood stem cells with bone marrow for RIC‐alloSCT. We used the European Group for Blood and Marrow Transplantation (EBMT) ALWP data to compare the outcome of mobilized peripheral blood stem cells (PBSC) (n = 1430) vs. bone marrow (BM) (n = 107) for acute myelogenous leukemia (AML) patients with complete remission that underwent RIC‐alloSCT from compatible sibling donors. The leukemia features, the disease status, and the time from diagnosis were similar between the two groups. Engraftment was achieved in 99% and 93% in the PBSC and BM groups, respectively (P < 0.0001). The day of engraftment was significantly earlier for the PBSC vs. the BM group, 15 (1–59) and 19 (5–69), respectively (P < 0.001). Acute GVHD, severe GVHD (grade III–IV) and chronic GVHD did not differ between the groups. leukemia‐free survival (LFS), relapse, and non‐relapsed mortality (NRM) were 51 ± 2%, 32 ± 1%, and 17 ± 1% vs. 50 ± 6%, 38 ± 6%, and 12 ± 3% for the PBSC and BM groups, respectively. Our results indicate faster engraftment, but no difference in GVHD, LFS, relapse, and NRM when comparing PBSC to BM grafts from sibling donors following RIC conditioning. This is the first study comparing PBSC to BM grafts in the RIC setting, analyzing a homogeneous population of patients with AML in remission. Whether PBSC should be preferred for advanced phases of the disease, where the outcome is dominated by relapse incidences, needs further investigation.     

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.     

Identification of emerging FLT3 ITD‐positive clones during clinical remission and kinetics of disease relapse in acute myeloid leukaemia with mutated nucleophosmin

FLT3 internal tandem duplication (ITD) mutations are frequently detected at diagnosis in cytogenetically normal acute myeloid leukaemia (CN‐AML) and predict unfavourable outcome. FLT3 ITD is an unstable aberration and may be lost or acquired at relapse. Recent whole genome sequencing studies have suggested that FLT3 ITD⁺ve AML relapse may evolve from small subclones undetectable at diagnosis by routine polymerase chain reaction (PCR). We developed a patient‐specific real‐time quantitative‐PCR (RQ‐PCR) to implement FLT3 ITD detection in six AML patients whose blasts carried wild‐type FLT3 at diagnosis and who relapsed with FLT3 ITD by routine PCR. Patient‐specific forward primers were designed after cloning and sequencing the FLT3 ITD in each case. The assay allowed retrospective detection of FLT3 ITD in diagnostic samples of 4/6 cases and to establish the kinetics of clonal evolution preceding relapse. After conventional chemotherapy, all patients had early relapse despite having been classified as NPM1⁺ve/FLT3 ITD^?ve at presentation, with shorter remissions being observed in four patients re‐classified as FLT3 ITD⁺ve by the new assay. Notably, FLT3 ITD clone became detectable by conventional PCR in three patients tested during remission after initial treatment. Our data underscore the need of identifying low FLT3 ITD levels, which are probably associated with relapse in otherwise good prognosis CN‐AML.     

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.     

A phase I/II study of sunitinib and intensive chemotherapy in patients over 60 years of age with acute myeloid leukaemia and activating FLT3 mutations

Acute myeloid leukaemia (AML) with FLT3 mutation has a dismal prognosis in elderly patients. Treatment with a combination of FLT3 inhibitors and standard chemotherapy has not been extensively studied. Therefore, we instigated a phase I/II clinical trial of chemotherapy with cytosine arabinoside (Ara‐C)/daunorubicin induction (7+3) followed by three cycles of intermediate‐dose Ara‐C consolidation in 22 AML patients with activating FLT3 mutations. Sunitinib was added at predefined dose levels and as maintenance therapy for 2 years. At dose level 1, sunitinib 25 mg daily continuously from day 1 onwards resulted in two cases with dose‐limiting toxicity (DLT), prolonged haemotoxicity and hand‐foot syndrome. At dose level ?1, sunitinib 25 mg was restricted to days 1–7 of each chemotherapy cycle. One DLT was observed in six evaluable patients. Six additional patients were treated in an extension phase. Thirteen of 22 patients (59%; 8/14 with FLT3–internal tandem duplication and 5/8 with FLT3‐tyrosine kinase domain) achieved a complete remission/complete remission with incomplete blood count recovery. For the 17 patients included at the lower dose level, median overall, relapse‐free and event‐free survival were 1·6, 1·0 and 0·4 years, respectively. Four out of five analysed patients with relapse during maintenance therapy lost their initial FLT3 mutation, suggesting outgrowth of FLT3 wild‐type subclones.     

Persistence of DNMT3A R882 mutations during remission does not adversely affect outcomes of patients with acute myeloid leukaemia

Somatic mutation of the DNMT3A gene at the arginine R882 site is common in acute myeloid leukaemia (AML). The prognostic significance of DNMT3A R882 mutation clearance, using traditional diagnostic next generation sequencing (NGS) methods, during complete remission (CR) in AML patients is controversial. We examined the impact of clearing DNMT3A R882 mutations at diagnosis to the detectable threshold of ?3% during CR on outcome in 56 adult AML patients. Mutational remission, defined as clearance of pre‐treatment DNMT3A R882 and all other AML‐associated mutations to a variant allele frequency ?3%, occurred in 14 patients whereas persistent DNMT3A R882 mutations were observed in 42 patients. There were no significant differences in disease‐free or overall survival between patients with and without DNMT3A R882 mutation clearance. Patients with persistent DNMT3A R882 who cleared all other AML mutations and did not acquire new mutations (n = 30), trended towards longer disease‐free survival (1·6 vs. 0·6 years, P = 0·06) than patients with persistence of DNMT3A R882, in addition to other mutations or acquisition of new AML‐associated mutations, such as those in TET2, JAK2, ASXL1 and TP53 (n = 12). These data demonstrate that DNMT3A R882 mutations, as assessed by traditional NGS methods, persist in the majority of AML patients in CR.     

The applicability of the WHO classification in paediatric AML. A NOPHO‐AML study

The World Health Organization (WHO) classification of myeloid leukaemia was revised in 2008. It incorporates newly recognized entities and emphasizes the pivotal role of cytogenetic abnormalities. The aim of this study was to evaluate the usability of the WHO classification when applied to a large population‐based paediatric acute myeloid leukaemia (AML) cohort. We included children diagnosed with de novo AML, 0–18 years of age from the Nordic countries and Hong Kong from 1993 to 2012. Data were retrieved from the Nordic Society for Paediatric Haematology and Oncology AML database and patients classified according to the WHO 2008 classification. A successful karyotype was available in 97% of the cases. AML with recurrent genetic abnormalities were present in 262 (41%) and 94 (15%) were classified as AML with myelodysplasia‐related changes (AML‐MDS). WHO classifies patients with monosomy 7 and del(7q) into one group. We found that ?7 (n = 14) had significantly poorer outcome than del(7q) (n = 11); 5‐year event‐free survival 26% vs. 67%, (P = 0·02), and 5‐year overall survival 51% vs. 90%, (P = 0·04). The largest group was the highly heterogeneous AML not otherwise specified (NOS) (n = 280) (44%). In conclusion, the WHO classification allocated 15% to AML‐MDS, 44% to NOS and grouped together entities with clearly different outcome, therefore limiting the applicability of the current WHO classification in children with AML.     

Chemotherapy followed by modified donor lymphocyte infusion as a treatment for relapsed acute leukemia after haploidentical hematopoietic stem cell transplantation without in vitro T‐cell depletion: superior outcomes compared with chemotherapy alone and an analysis of prognostic factors

We retrospectively compared the antileukemic effects of chemotherapy alone and chemotherapy followed by modified donor lymphocyte infusion (DLI) in 82 patients with relapsed acute leukemia after haploidentical hematopoietic stem cell transplantation (HSCT) without in vitro T‐cell depletion. We also investigated prognostic factors in patients receiving chemotherapy followed by modified DLI. Thirty‐two patients received chemotherapy alone, and the remaining 50 patients received chemotherapy followed by modified DLI. In patients receiving chemotherapy followed by modified DLI, complete remission rate was significantly higher (64.0% vs. 12.5%, P = 0.000), the incidence of relapse was significantly lower (50.0% vs. 100.0%, P = 0.000), and disease‐free survival was significantly improved (36.0% vs. 0.0%, P = 0.000) compared with patients receiving chemotherapy alone. Multivariate analysis demonstrated that patients with chronic graft‐versus‐host disease (GVHD) after intervention (P = 0.000) and patients receiving chemotherapy followed by modified DLI (P = 0.037) were associated with a lower relapse rate. Furthermore, in patients receiving chemotherapy followed by modified DLI, multivariate analysis demonstrated that chronic GVHD after modified DLI (P = 0.039) and duration of minimal residual disease (MRD) (?) ≥4 months after modified DLI (P = 0.001) were associated with a lower relapse rate. Our study is the first to suggest that chemotherapy followed by modified DLI is associated with stronger antileukemic effects and better survival in relapsed acute leukemia after haploidentical HSCT without in vitro T‐cell depletion. Furthermore, our study suggests that lack of chronic GVHD and duration of MRD (?) <4 months after modified DLI are associated with higher relapse rates in patients receiving chemotherapy followed by modified DLI.     

hMICL and CD123 in combination with a CD45/CD34/CD117 backbone – a universal marker combination for the detection of minimal residual disease in acute myeloid leukaemia

Real‐time quantitative polymerase chain reaction (qPCR) has been extensively validated for the detection of minimal residual disease (MRD) in acute myeloid leukaemia (AML). Meanwhile, multicolour flow cytometry (MFC) has received less attention because the so‐called leukaemia‐associated immunophenotypes (LAIPs) are generally of lower sensitivity and specificity, and prone to change during therapy. To improve MRD assessment by MFC, we here evaluate the combination of human Myeloid Inhibitory C‐type Lectin (hMICL, also termed C‐type lectin domain family 12, member A, CLEC12A) and CD 123 (also termed interleukin‐3 receptor alpha, IL3RA) in combination with CD34 and CD117 (KIT), as an MRD assay in pre‐clinical and clinical testing in 69 AML patients. Spiking experiments revealed that the assay could detect MRD down to 10^?4 in normal bone marrow with sensitivities equalling those of validated qPCR assays. Moreover, it provided at least one MFC MRD marker in 62/69 patients (90%). High levels of hMICL/CD123 LAIPs at the post‐induction time‐point were a strong prognostic marker for relapse in patients in haematological complete remission (P < 0·001). Finally, in post induction samples, hMICL/CD123 LAIPs were strongly correlated (r = 0·676, P = 0·0008) to applied qPCR targets. We conclude the hMICL/CD123‐based MFC assay is a promising MRD tool in AML.     

Serum B-cell maturation antigen is elevated in multiple myeloma and correlates with disease status and survival

Although TNFRSF17 (also designated as B‐cell maturation antigen (BCMA)) is expressed on tumour cells in B‐cell malignancies, it has not been found in serum. The present study found that BCMA concentrations were higher in the supernatants of cultured bone marrow mononuclear cells from multiple myeloma (MM) patients than in healthy subjects. Serum BCMA levels were measured in samples from MM patients (n = 209), monoclonal gammopathy of undetermined significance (MGUS) individuals (n = 23) and age‐matched controls (n = 40). BCMA was detected in the serum of untreated MM patients (n = 50) and levels were higher than in MGUS patients (P = 0·0157) and healthy subjects (P < 0·0001). Serum BCMA levels were higher among patients with progressive disease (n = 80) compared to those with responsive disease (n = 79; P = 0·0038). Among all MM patients, overall survival was shorter among patients whose serum BCMA levels were above the median (P = 0·001). We also demonstrated that sera from mice with human MM xenografts contained human BCMA, and levels correlated with the change in tumour volume in response to melphalan or cyclophosphamide with bortezomib. These results suggest that serum BCMA levels may be a new biomarker for monitoring disease status and overall survival of MM patients.