Yasunobu Nagata

Comprehensive analysis of genetic alterations and their prognostic impacts in adult acute myeloid leukemia patients.

To clarify the cooperative roles of recurrently identified mutations and to establish a more precise risk classification system in acute myeloid leukemia (AML), we comprehensively analyzed mutations in 51 genes, as well as cytogenetics and 11 chimeric transcripts, in 197 adult patients with de novo AML who were registered in the Japan Adult Leukemia Study Group AML201 study. We identified a total of 505 mutations in 44 genes, while only five genes, FLT3, NPM1, CEBPA, DNMT3A and KIT, were mutated in more than 10% of the patients. Although several cooperative and exclusive mutation patterns were observed, the accumulated mutation number was higher in cytogenetically normal AML and lower in AML with RUNX1-RUNX1T1 and CBFB-MYH11, indicating a strong potential of these translocations for the initiation of AML. Furthermore, we evaluated the prognostic impacts of each sole mutation and the combinations of mutations and/or cytogenetics, and demonstrated that AML patients could be clearly stratified into five risk groups for overall survival by including the mutation status of DNMT3A, MLL-PTD and TP53 genes in the risk classification system of the European LeukemiaNet. These results indicate that the prognosis of AML could be stratified by the major mutation status in combination with cytogenetics.

IDH1/2 mutations sensitize acute myeloid leukemia to PARP inhibition and this is reversed by IDH1/2-mutant inhibitors

Purpose: Somatic mutations in IDH1/2 occur in approximately 20% of patients with myeloid neoplasms, including acute myeloid leukemia (AML). IDH1/2MUT enzymes produce D-2-hydroxyglutarate (D2HG), which associates with increased DNA damage and improved responses to chemo/radiotherapy and PARP inhibitors in solid tumor cells. Whether this also holds true for IDH1/2MUT AML is not known. Experimental Design: Well-characterized primary IDH1MUT, IDH2MUT, and IDH1/2WT AML cells were analyzed for DNA damage and responses to daunorubicin, ionizing radiation, and PARP inhibitors. Results: IDH1/2MUT caused increased DNA damage and sensitization to daunorubicin, irradiation, and the PARP inhibitors olaparib and talazoparib in AML cells. IDH1/2MUT inhibitors protected against these treatments. Combined treatment with a PARP inhibitor and daunorubicin had an additive effect on the killing of IDH1/2MUT AML cells. We provide evidence that the therapy sensitivity of IDH1/2MUT cells was caused by D2HG-mediated downregulation of expression of the DNA damage response gene ATM and not by altered redox responses due to metabolic alterations in IDH1/2MUT cells. Conclusions: IDH1/2MUT AML cells are sensitive to PARP inhibitors as monotherapy but especially when combined with a DNA-damaging agent, such as daunorubicin, whereas concomitant administration of IDH1/2MUT inhibitors during cytotoxic therapy decrease the efficacy of both agents in IDH1/2MUT AML. These results advocate in favor of clinical trials of PARP inhibitors either or not in combination with daunorubicin in IDH1/2MUT AML. Clin Cancer Res; 24(7); 1705–15. ©2018 AACR.

Molecular features of early onset adult myelodysplastic syndrome

Myelodysplastic syndromes are typically diseases of older adults. Patients in whom the onset is early may have distinct molecular and clinical features or reflect a demographic continuum. The identification of differences between “early onset” patients and those diagnosed at a traditional age has the potential to advance understanding of the pathogenesis of myelodysplasia and may lead to formation of distinct morphological subcategories. We studied a cohort of 634 patients with various subcategories of myelodysplastic syndrome and secondary acute myeloid leukemia, stratifying them based on age at presentation and clinical parameters. We then characterized molecular abnormalities detected by next-generation deep sequencing of 60 genes that are commonly mutated in myeloid malignancies. The number of mutations increased linearly with age and on average, patients >50 years of age had more mutations. TET2, SRSF2, and DNMT3A were more commonly mutated in patients >50 years old compared to patients ≤50 years old. In general, patients >50 years of age also had more mutations in spliceosomal, epigenetic modifier, and RAS gene families. Although there are age-related differences in molecular features among patients with myelodysplasia, most notably in the incidence of SRSF2 mutations, our results suggest that patients ≤50 years old belong to a disease continuum with a distinct pattern of early onset ancestral events.

Origins of myelodysplastic syndromes after aplastic anemia

To the editor: The course of aplastic anemia (AA) is often complicated by the development of clonal disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and secondary myelodysplastic syndromes (sMDS).[1][1][⇓][2][⇓][3][⇓][4]-[5][5] Identification of patients at risk for development of

Array CGH identifies copy number changes in 11% of 520 MDS patients with normal karyotype and uncovers prognostically relevant deletions.

Array CGH identifies copy number changes in 11% of 520 MDS patients with normal karyotype and uncovers prognostically relevant deletions

Distinct clinical and biological implications of various DNMT3A mutations in myeloid neoplasms

Distinct clinical and biological implications of various DNMT3A mutations in myeloid neoplasms

Distinct gene alterations with a high percentage of myeloperoxidase-positive leukemic blasts in de novo acute myeloid leukemia

The myeloperoxidase (MPO)-positivity of blasts in bone marrow smears is an important marker for not only the diagnosis, but also the prognosis of acute myeloid leukemia (AML). To investigate the relationship between genetic alterations and MPO-positivity, we performed targeted sequencing for 51 genes and 10 chimeric gene transcripts in 164 newly diagnosed de novo AML patients; 107 and 57 patients were classified as AML with >50% MPO-positive blasts (MPO-high group) and ≤50% MPO-positive blasts, (MPO-low group), respectively. The univariate analysis revealed that RUNX1-RUNX1T1 (P < 0.001), the KIT mutation (P < 0.001), and CEBPA double mutation (P = 0.001) were more likely to be found in the MPO-high group, while the DNMT3A mutation (P = 0.001), FLT3 tyrosine kinase domain mutation (P = 0.004), and TP53 mutation (P = 0.020) were more likely to be present in the MPO-low group. Mutations in genes related to DNA hypermethylation signatures (IDH1, IDH2, TET2, and WT1 genes) were more frequent in the MPO-high group (P = 0.001) when patients with fusion genes of core-binding factors were excluded from the analysis. Our results suggest that MPO-positivity of blasts was related with the distinct gene mutation patterns among de novo AML patients.

Prognostic analysis according to the 2017 ELN risk stratification by genetics in adult acute myeloid leukemia patients treated in the Japan Adult Leukemia Study Group (JALSG) AML201 study

Many genetic alterations that are associated with the prognosis of acute myeloid leukemia (AML) have been identified, and several risk stratification systems based on the genetic status have been recommended. The European LeukemiaNet (ELN) first proposed the risk stratification system for AML in 2010 (ELN-2010), and recently published the revised system (ELN-2017). We validated the long-term prognosis and clinical characteristics of each ELN-2017 risk category in Japanese adult AML patients who were treated in the Japan Adult Leukemia Study Group (JALSG) AML-201 study. We demonstrated that the 3-risk category system of the ELN-2017 successfully discriminated the overall survival and complete remission rates in our cohort in comparison with the 4-risk category of the ELN-2010. However, there were still genetic categories in which stratification of patients into favorable or intermediate risk categories was controversial; the low allelic ratio of FLT3-ITD was not necessarily associated with a better prognosis in patients with FLT3-ITD, and cytogenetic abnormalities may affect the prognosis in patients with favorable genetic lesions such as NPM1 and CEBPA mutations. As many molecular targeting agents, such as FLT3 inhibitors, have been developed, we must continue to modify the genetic risk stratification system to match the progression of therapeutic strategies.

Consequences of mutant TET2 on clonality and subclonal hierarchy

Somatic mutations in TET2 are common in myelodysplastic syndromes (MDS), myeloproliferative, and overlap syndromes. TET2 mutant (TET2MT) clones are also found in asymptomatic elderly individuals, a condition referred to as clonal hematopoiesis of indeterminate potential (CHIP). In various entities of TET2MT neoplasia, we examined the phenotype in relation to the strata of TET2 hits within the clonal hierarchy. Using deep sequencing, 1781 mutations were found in 1205 of 4930 patients; 40% of mutant cases were biallelic. Hierarchical analysis revealed that of TET2MT cases >40% were ancestral, e.g., representing 8% of MDS. Higher (earlier) TET2 lesion rank within the clonal hierarchy (greater clonal burden) was associated with impaired survival. Moreover, MDS driven by ancestral TET2MT is likely derived from TET2MT CHIP with a penetrance of ~1%. Following ancestral TET2 mutations, individual disease course is determined by secondary hits. Using multidimensional analyses, we demonstrate how hits following the TET2 founder defect induces phenotypic shifts toward dysplasia, myeloproliferation, or progression to AML. In summary, TET2MT CHIP-derived MDS is a subclass of MDS that is distinct from de novo disease.

Clonal PIGA mosaicism and dynamics in paroxysmal nocturnal hemoglobinuria

Somatic PIGA gene mutations initiate pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH) [1, 2] and exemplify evolutional somatic adaptability of hematopoietic stem cells (HSCs). The resultant glycosylphosphatidylinositol (GPI) anchor-deficient phenotype appears to offer a growth and/or survival advantage over other unaffected HSCs [3, 4] in the context of immune-mediated bone marrow failure [5] explaining the unique association of PNH with aplastic anemia (AA). Recently, additional mutations in genes other than PIGA were identified in PNH clones, offering an explanation for intrinsic expansion drive of PNH clones irrespective of or after the initial immune escape occurred [6]. Multiple PIGA mutations have been discovered at very low frequencies in healthy individuals [7], as well as in isolated reports of occasional PNH patients [8, 9] and thus observation did not lead to the appreciation that the phenomenon of an oligoclonal disease origin may be ubiquitous and potentially essential for the pathogenesis of PNH. PIGA mutant cells fail to expand in engineered animal models [10], suggesting that additional permissive conditions and factors are required for PNH clone evolution. Using next generation sequencing (NGS) of the PIGA gene and cell sorting by flow cytometry, our goal was to determine if we could improve PIGA mutant clone detection and gain insight into PNH clonal dynamics through longitudinal monitoring. According to theoretical stipulations ~20% patients may harbor >1 PIGA mutant hematopoietic stem cell (HSC) [11]. The recently described acquisition of multiple lesions at HLA loci in AA represents an analogous scenario of immune evasion and somatic adaptability of HSCs [12, 13]. We set out to experimentally challenge this hypothesis, and determine if, in addition to the classic two-hit scenario characterized by a founder PIGA mutation followed by a subclonal driver hit, there are more complex, potentially mosaic, clonal dynamics; whereby multiple PIGA clones compete for dominance. We also asked whether stratifying AA/PNH and classic PNH patients would provide additional insight as to the process of clonal selection. Patients with PNH and AA were diagnosed according to current clinical guidelines and following informed consent enrolled into this study, as detailed in supplemental data. Using flow cytometry to identify PNH clones, 133 patients with PNH (N= 33), AA with small PNH clones >0.5% of WBCs (N= 33), and AA/PNH (N= 67) were studied (Table S1). Mean WBC PNH clone sizes were 77%, 0.22%, and 27%, respectively. DNA was extracted and subjected to multi-amplicon deep sequencing of PIGA using primers covering all exons of the PIGA gene (Table S2). Using deep NGS analysis, 223 mutations in the PIGA gene were detected (Fig. 1a). Frameshift (35%, N= 78) and missense mutations (34%, N= 77) were most common, followed by splice site (14%, N= 31), nonsense (13%, N= 30), and nonframeshift insertion/deletion (3%, N= 7) mutations. As detection rates correlate with PNH clone size in WBCs (P < 0.0001, chi-square, Figure S1), sorting PNH positive and negative cells by flow cytometry (Figure S2; PNH N= 12, AA/PNH N= 17; mean purity 97.3%, SD * Michael J. Clemente michaelwork4@gmail.com