Quantitative detection of DNMT3A R882H mutation in acute myeloid leukemia

Background DNMT3A mutations represent one of the most frequent gene alterations detectable in acute myeloid leukemia (AML) with normal karyotype. Although various recurrent somatic mutations of DNMT3A have been described, the most common mutation is located at R882 in the methyltransferase domain of the gene. Because of their prognostic significance and high stability during disease evolution, DNMT3A mutations might represent highly informative biomarkers for prognosis and outcome of disease. Methods We describe an allele-specific PCR with a Blocking reagent for the quantitative detection of DNMT3A R882H mutation providing the possibility to analyze the quantitative amount of mutation during the course of disease. Next, we analyzed 62 follow- up samples from 6 AML patients after therapy and allogeneic stem cell transplantation (alloSCT). Results We developed an ASB-PCR assay for quantitative analysis of R882H DNMT3A mutation. After optimization of blocker concentration, a R882H-positive plasmid was constructed to enhance the accuracy of the sensitivity of quantitative detection. The assay displayed a high efficiency and sensitivity up to 10−3. The reproducibility of assay analyzed using follow-up samples showed the standard deviation less than 3.1 %. This assay displayed a complete concordance with sequencing and endonuclease restriction analysis. We have found persistence of DNMT3A R882H mutations in complete remission (CR) after standard cytoreduction therapy that could be indicating presence of DNMT3A mutation in early pre-leukemic stem cells that resist chemotherapy. The loss of correlation between NPM1 and DNMT3A in CR could be associated with evolution of pre-leukemic and leukemic clones. In patients with CR with complete donor chimerism after alloSCT, we have found no DNMT3A R882H. In relapsed patients, all samples showed an increasing of both NPM1 and DNMT3A mutated alleles. This suggests at least in part the presence of NPM1 and DNMT3A mutations in the same cell clone. Conclusion We developed a rapid and reliable method for quantitative detection of DNMT3A R882H mutations in AML patients. Quantitative detection of DNMT3A R882H mutations at different time points of AML disease enables screening of follow-up samples. This could provide additional information about the role of DNMT3A mutations in development and progression of AML

Inhibition of IGF1-R overcomes IGFBP7-induced chemotherapy resistance in T-ALL

Background T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease with the need for treatment optimization. Previously, high expression of Insulin-like growth factor binding protein 7 (IGFBP7), a member of the IGF system, was identified as negative prognostic factor in adult T-ALL patients. Since aberrant IGFBP7 expression was observed in a variety of neoplasia and was relevant for prognosis in T-ALL, we investigated the functional role of IGFBP7 in Jurkat and Molt-4 cells as in vitro models for T-ALL. Methods Jurkat and Molt-4 cells were stably transfected with an IGFBP7 over-expression vector or the empty vector as control. Proliferation of the cells was assessed by WST-1 assays and cell cycle status was measured by flow-cytometry after BrDU/7-AAD staining. The effect of IGFBP7 over-expression on sensitivity to cytostatic drugs was determined in AnnexinV/7-AAD assays. IGF1-R protein expression was measured by Western Blot and flow-cytometric analysis. IGF1-R associated gene expression profiles were generated from microarray gene expression data of 86 T-ALL patients from the Microarrays Innovations in Leukemia (MILE) multicenter study. Results IGFBP7-transfected Jurkat cells proliferated less, leading to a longer survival in a nutrient–limited environment. Both IGFBP7-transfected Jurkat and Molt-4 cells showed an arrest in the G0/G1 cell cycle phase. Furthermore, Jurkat IGFBP7-transfected cells were resistant to vincristine and asparaginase treatment. Surface expression and whole protein measurement of IGF1-R protein expression showed a reduced abundance of the receptor after IGFBP7 transfection in Jurkat cells. Interestingly, combination of the IGF1-R inhibitor NPV-AEW541 restored sensitivity to vincristine in IGFBP7-transfected cells. Additionally, IGF1-R associated GEP revealed an up-regulation of important drivers of T-ALL pathogenesis and regulators of chemo-resistance and apoptosis such as NOTCH1, BCL-2, PRKCI, and TP53. Conclusion This study revealed a proliferation inhibiting effect of IGFBP7 by G0/G1 arrest and a drug resistance-inducing effect of IGFBP7 against vincristine and asparaginase in T-ALL. These results provide a model for the previously observed association between high IGFBP7 expression and chemotherapy failure in T-ALL patients. Since the resistance against vincristine was abolished by IGF1-R inhibition, IGFBP7 could serve as biomarker for patients who may benefit from therapies including IGF1-R inhibitors in combination with chemotherapy

Adoptive Cell Transfer of Allogeneic Epstein-Barr Virus-Specific T Lymphocytes for Treatment of Refractory EBV-Associated Posttransplant Smooth Muscle Tumors: A Case Report

Posttransplant smooth muscle tumors (PTSMTs) are rare Epstein-Barr virus (EBV)-associated neoplasms, mostly occurring after solid organ transplantation. Current therapeutic strategies include surgery and reduction of immunosuppressive medication. We describe for the first time a novel treatment approach for PTSMT by adoptive cell transfer (ACT) of EBV-specific T cells to a 20-year-old patient with a medical history of cardiac transplantation, posttransplant lymphoproliferative disease, and multilocular PTSMT. During ACT, mild cytokine release syndrome occurred, while no unexpected safety signals were recorded. We observed in vivo expansion of EBV-specific T cells and reduction of EBV viremia. Best response was stable disease after 4 months with reduction of EBV viremia and normalization of lactate dehydrogenase levels. ACT with EBV-specific T cells may be a safe and efficacious therapeutic option for PTSMT that warrants further exploration

FLT3 mutations in Early T-Cell Precursor ALL characterize a stem cell like leukemia and imply the clinical use of tyrosine kinase inhibitors

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup

An alternative CYB5A transcript is expressed in aneuploid ALL and enriched in relapse

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogenous malignancy with poor prognosis in relapsed adult patients. The genetic basis for relapse in aneuploid subtypes such as near haploid (NH) and high hyperdiploid (HeH) BCP-ALL is only poorly understood. Pathogenic genetic alterations remain to be identified. To this end, we investigated the dynamics of genetic alterations in a matched initial diagnosis-relapse (ID-REL) BCP-ALL cohort. Here, we firstly report the identification of the novel genetic alteration CYB5Aalt, an alternative transcript of CYB5A, in two independent cohorts. Methods: We identified CYB5alt in the RNAseq-analysis of a matched ID-REL BCP-ALL cohort with 50 patients and quantified its expression in various molecular BCP-ALL subtypes. Findings were validated in an independent cohort of 140 first diagnosis samples from adult BCP-ALL patients. Derived from patient material, the alternative open reading frame of CYB5Aalt was cloned (pCYB5Aalt) and pCYB5Aalt or the empty vector were stably overexpressed in NALM-6 cells. RNA sequencing was performed of pCYB5Aalt clones and empty vector controls followed by differential expression analysis, gene set enrichment analysis and complementing cell death and viability assays to determine functional implications of CYB5Aalt. Results: RNAseq data analysis revealed non-canonical exon usage of CYB5Aalt starting from a previously undescribed transcription start site. CYB5Aalt expression was increased in relapsed BCP-ALL and its occurrence was specific towards the shared gene expression cluster of NH and HeH BCP-ALL in independent cohorts. Overexpression of pCYB5Aalt in NALM-6 cells induced a distinct transcriptional program compared to empty vector controls with downregulation of pathways related to reported functions of CYB5A wildtype. Interestingly, CYB5A wildtype expression was decreased in CYB5Aalt samples in silico and in vitro. Additionally, pCYB5Aalt NALM-6 elicited a more resistant drug response. Conclusions: Across all age groups, CYB5Aalt was the most frequent secondary genetic event in relapsed NH and HeH BCP-ALL. In addition to its high subgroup specificity, CYB5Aalt is a novel candidate to be potentially implicated in therapy resistance in NH and HeH BCP-ALL. This is underlined by overexpressing CYB5Aalt providing first evidence for a functional role in BCL2-mediated apoptosis

Ex vivo drug response profiling for response and outcome prediction in hematologic malignancies: the prospective non-interventional SMARTrial

Ex vivo drug response profiling is a powerful tool to study genotype-drug response associations and is being explored as a tool set for precision medicine in cancer. Here we conducted a prospective non-interventional trial to investigate feasibility of ex vivo drug response profiling for treatment guidance in hematologic malignancies (SMARTrial, NCT03488641 ). The primary endpoint to provide drug response profiling reports within 7 d was met in 91% of all study participants (N = 80). Secondary endpoint analysis revealed that ex vivo resistance to chemotherapeutic drugs predicted chemotherapy treatment failure in vivo. We confirmed the predictive value of ex vivo response to chemotherapy in a validation cohort of 95 individuals with acute myeloid leukemia treated with daunorubicin and cytarabine. Ex vivo drug response profiles improved ELN-22 risk stratification in individuals with adverse risk. We conclude that ex vivo drug response profiling is clinically feasible and has the potential to predict chemotherapy response in individuals with hematologic malignancies beyond clinically established genetic markers

Prognostic impact of <i>CEBPA </i>mutational subgroups in adult AML

Despite recent refinements in the diagnostic and prognostic assessment of CEBPA mutations in AML, several questions remain open, i.e. implications of different types of basic region leucin zipper (bZIP) mutations, the role of co-mutations and the allelic state. Using pooled primary data analysis on 1010 CEBPA-mutant adult AML patients, a comparison was performed taking into account the type of mutation (bZIP: either typical in-frame insertion/deletion (InDel) mutations (bZIP InDel), frameshift InDel or nonsense mutations inducing translational stop (bZIP STOP) or single base-pair missense alterations (bZIP ms), and transcription activation domain (TAD) mutations) and the allelic state (single (smCEBPA) vs. double mutant (dmCEBPA)). Only bZIP InDel patients had significantly higher rates of complete remission and longer relapse free and overall survival (OS) compared with all other CEBPA-mutant subgroups. Moreover, co-mutations in bZIP InDel patients (e.g. GATA2, FLT3, WT1 as well as ELN2022 adverse risk aberrations) had no independent impact on OS, whereas in non-bZIP InDel patients, grouping according to ELN2022 recommendations added significant prognostic information. In conclusion, these results demonstrate bZIP InDel mutations to be the major independent determinant of outcome in CEBPA-mutant AML, thereby refining current classifications according to WHO (including all dmCEBPA and smCEBPA bZIP) as well as ELN2022 and ICC recommendations (including CEBPA bZIP ms). (Figure presented.)</p

Point Mutations in the FLT3-ITD Region Are Rare but Recurrent Alterations in Adult AML and Associated With Concomitant KMT2A-PTD

FLT3-ITD mutations are common druggable alterations in patients with acute myeloid leukemia (AML) and associated with poor prognosis. Beside typical ITD mutations, point mutations and deletions in the juxtamembrane domain (JMD) have been observed. However, due to the low frequency of these alterations, there is only limited information on molecular and clinical associations. To evaluate the prognostic impact of non-ITD mutations in the FLT3 JMD region, we analyzed a large cohort of 1,539 adult AML patients treated in different protocols of the Study Alliance Leukemia, using next-generation sequencing. Non-ITD point mutations and deletions within the FLT3 JMD were identified with a prevalence of ~1.23% (n = 19). Both FLT3-ITD and non-ITD mutations were associated with a higher rate of NPM1 (42%–61%; p < 0.001) and DNMT3A mutations (37%–43%; p < 0.001), as well as an increased percentage of peripheral blood (54%–65%) and bone marrow blast cells (74%; p < 0.001), compared to FLT3-wild-type patients. Most significantly, AML patients with FLT3 non-ITD mutations had a higher rate of concomitant KMT2A-PTD mutations (37.5%; p < 0.001) as compared to FLT3-ITD (7%) or FLT3-wild-type cases (4.5%). In a multivariable analysis, FLT3 non-ITD mutations were not an independent prognostic factor. However, patients with dual FLT3 non-ITD and KMT2A-PTD mutations showed a trend for inferior outcome, which points at a functional interaction in this subset of AML