Clinical Features of Patients with ASXL1 and ASXL2 Mutations in Pediatric Acute Myeloid Leukemia

Abstract Background; Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Currently, a number of gene mutations have been implicated in the pathogenesis of both adult and pediatric AML, including mutations of CEBPA,NPM1, DNMT3A, IDH1/2, TET2 and EZH2 in addition to RAS, KIT and FLT3, because the recent development of massively parallel sequencing technologies. We have performed whole-exome sequencing of paired tumor-normal DNA from 19 patients, and identified 80 somatic mutations or 4.2 mutations per sample. Many of the recurrent mutations identified in this study involved previously reported targets in adult AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1, BCORL1, EZH2, and major cohesin components including SMC3 and RAD21. In addition to these mutations, we also identified disease-associated candidate genes of ASXL2, PAX5 and others. Recently, recurrent somatic mutations in ASXL1 have been reported to occur in patients with adult AML, and to be associated with adverse outcome. Another study suggested that ASXL1 or ASXL2 mutations were associated with a high incidence of relapse. To reveal the significance of these mutations, we performed mutational analysis of ASXL1 and ASXL2 in 184 pediatric AML patients. Methods; Between 2006 and 2010, 485 de novo pediatric AML patients aged <18 years old participated in the Japanese AML-05 study conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). Among them, 369 samples were available. We analyzed the first half of these samples which were registered in the order (184 samples). To estimate the frequency and prognostic impact of the ASXL1 and ASXL2 mutations in pediatric AML, we performed targeted sequencing of ASXL1 (exon 12) and ASXL2 (exon 12) genes using next-generation sequencer in 184 de novo AML patients including 51 patients with t(8;21). We validated the mutations in ASXL1 and ASXL2 by Sanger sequencing. Furthermore, we investigated the correlation among these mutations, other cytogenetic alterations and clinical characteristics. Results and Discussion; ASXL1 mutations were identified in 4 of 184 de novo pediatric AML patients (2.2 %) and all 4 ASXL1 mutation positive patients harbored t(8;21). Two of them relapsed, and one died after relapsed. On the other hand, ASXL2 mutations were identified in 10 of 184 de novo pediatric AML patients (5.4%) and 6 of them harbored t(8;21). Five of these 10 patients relapsed, and 2 of them died after relapsed. Especially, all of 4 patients without t(8;21) relapsed (2 in M5a and 2 with CBFA2T3-GLIS2 in M7), and 2 died after relapsed. Although only one ASXL2 patients with t(8;21) relapsed (1/6 or 17%), no ASXL2 positive patients with t(8;21) died. ASXL2 mutations were more observed in AML patients with t(8;21) (11.8%, 6/51), but not in 13 patients with inv(16). Overall survival of the patients with or without ASXL2 mutations were 80% and 66.7% (p=0.54), respectively. ASXL1 and ASXL2 mutations were mutually exclusive in this study. Conclusion; Ten of 184 patients (5.4%) had mutations of ASXL2 in pediatric AML, and the outcome of ASXL2 mutant patients with t(8;21) was favorable. Among the 51 pediatric AML patients with t(8;21), ASXL2 mutations were detected in 6 (11.8%) patients. All of them have been survived, suggesting that ASXL2 mutations in patients with t(8;21) may be associated with favorable prognosis in pediatric AML in contrast to adult AML. On the other hand, 4 (2.2%) of 184 patients had ASXL1 mutations, and all of them were t(8;21). In these 4 patients, 2 of them relapsed, and one died after relapsed. Although the number of patients is too small, ASXL1 mutations were not considered to be associated with favorable outcome. Both ASXL1 and ASXL2 mutations were detected at high frequency among pediatric AML patients with t(8;21) and mutual exclusive. As we consider that further study will be needed to clarify the significance of these mutations, we are now analyzing mutations in other exons of ASXL2, and would like to report these data in the annual meeting. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Whole-Exome Resequencing Identifies Somatic Mutations Of BCOR and BCORL1 Transcriptional Corepressor Genes and Major Cohesin Complex Component Genes In Pediatric Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.834.834 ◽

2013 ◽

Vol 122 (21) ◽

pp. 834-834

Author(s):

Norio Shiba ◽

Kenichi Yoshida ◽

Yasunobu Nagata ◽

Ayana Kon ◽

Yusuke Okuno ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Gene Mutations ◽

Transcriptional Corepressor ◽

Whole Exome ◽

Recurrent Mutations ◽

Pediatric Aml ◽

Acute Myeloid

Abstract Background Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Currently, targeted sequencing efforts have identified several mutations that carry diagnostic and prognostic information such as RAS, KIT, and FLT3 in both adult and pediatric AML, and NPM1 and TET2 in adult AML. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across the entire genomes or protein-coding sequences in human cancers at a single-nucleotide level, which could be enabled the discovery of recurrent mutations in IDH1/2, and DNMT3A in adult AML. However, these mutations are extremely rare in pediatric AML. Methods To reveal a complete registry of gene mutations and other genetic lesions, whole-exome resequencing of paired tumor-normal DNA from 19 cases were analyzed with a mean coverage of approximately x100, and 82 % of the target sequences were analyzed at more than x20 depth on average. We selected various cases in age, FAB classification and karyotypes, including 5 cases with core-binding-factor AML, 6 cases with MLL-rearrangement and 2 acute megakaryoblastic leukemia cases. Results and Discussion A total of 80 somatic mutations or 4.2 mutations per sample were identified. As the mean number of somatic mutations reported in adult AML was about ten, somatic mutations in pediatric AML might be fewer than in adult AML. Many of the recurrent mutations identified in this study involved previously reported targets in AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1 and EZH2. On the other hand, several genes were newly identified in the current study, including BRAF, BCORL1, DAZAP1, CUL2, ASXL2, MLL2, MLL3, SMC3 and RAD21. Among these, what immediately drew our attention were SMC3 and RAD21, because they belong to the major cohesin components. Cohesin is a multimeric protein complex conserved across species and composed of four core subunits, i.e., SMC1, SMC3, RAD21, and STAG proteins, forming a ring-like structure. Cohesin is engaged in cohesion of sister chromatids during cell division, post-replicative DNA repair, and regulation of global gene expression through long-range cis-interactions. Furthermore, we also drew our attention to BCORL1, because it is a transcriptional corepressor, and can bind to class II histone deacetyllases (HDAC4, HDAC5, HDAC7), to interact with the CTBP1 corepressor, and to affect the repression of E-cadherin. BCOR is also a transcriptional corepressor and play a key role in the regulation of early embryonic development, mesenchymal stem cell function and hematopoiesis. To confirm and extend the initial findings in the whole-exome sequencing, we studied mutations of the above 8 genes, in pediatric AML (N = 190) using a high-throughput mutation screen of pooled DNA followed by confirmation/ identification of candidate mutations. In total, 32 mutations were identified in 31 of the 190 specimens of pediatric AML [BCOR (N = 7), BCORL1 (N = 7), RAD21 (N = 7), SMC3 (N = 5), SMC1A (N = 1), and STAG2 (N = 3)]. The mutually exclusive pattern of the mutations in these BCOR, BCORL1 and cohesin components genes was confirmed in this large case series, suggesting a common impact of these mutations on the pathogenesis of pediatric AML. The 4-year overall survival of these cases with major cohesin components gene mutations was relatively favorable (12/16 or 75.0%), but the outcome of cases with BCOR or BCORL1 cases was unfavorable (8/14 or 57.1%). Conclusion Whole exome resequencing unmasked a complexity of gene mutations in pediatric AML genomes. Our results indicated that a subset of pediatric AML represents a discrete entity that could be discriminated from the adult counterpart, in terms of the spectrum of gene mutations. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Genomic characterization of relapsed acute myeloid leukemia reveals novel putative therapeutic targets

Blood Advances ◽

10.1182/bloodadvances.2020003709 ◽

2021 ◽

Vol 5 (3) ◽

pp. 900-912

Author(s):

Svea Stratmann ◽

Sara A. Yones ◽

Markus Mayrhofer ◽

Nina Norgren ◽

Aron Skaftason ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Pediatric Patients ◽

De Novo ◽

Low Frequency ◽

Whole Genome ◽

Recurrent Mutations ◽

De Novo Aml ◽

Pediatric Aml ◽

Acute Myeloid

Abstract Relapse is the leading cause of death of adult and pediatric patients with acute myeloid leukemia (AML). Numerous studies have helped to elucidate the complex mutational landscape at diagnosis of AML, leading to improved risk stratification and new therapeutic options. However, multi–whole-genome studies of adult and pediatric AML at relapse are necessary for further advances. To this end, we performed whole-genome and whole-exome sequencing analyses of longitudinal diagnosis, relapse, and/or primary resistant specimens from 48 adult and 25 pediatric patients with AML. We identified mutations recurrently gained at relapse in ARID1A and CSF1R, both of which represent potentially actionable therapeutic alternatives. Further, we report specific differences in the mutational spectrum between adult vs pediatric relapsed AML, with MGA and H3F3A p.Lys28Met mutations recurrently found at relapse in adults, whereas internal tandem duplications in UBTF were identified solely in children. Finally, our study revealed recurrent mutations in IKZF1, KANSL1, and NIPBL at relapse. All of the mentioned genes have either never been reported at diagnosis in de novo AML or have been reported at low frequency, suggesting important roles for these alterations predominantly in disease progression and/or resistance to therapy. Our findings shed further light on the complexity of relapsed AML and identified previously unappreciated alterations that may lead to improved outcomes through personalized medicine.

Download Full-text

The Clinical Features and Prognostic Impact of PRDM16 gene Expression in Adult Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.5228.5228 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5228-5228

Author(s):

Genki Yamato ◽

Hiroki Yamaguchi ◽

Hiroshi Handa ◽

Norio Shiba ◽

Satoshi Wakita ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Risk Group ◽

Gene Mutations ◽

Genetic Alterations ◽

Prognostic Impact ◽

Pediatric Aml ◽

Prdm16 Gene ◽

Acute Myeloid

Abstract Background Acute myeloid leukemia (AML) is a complex disease caused by various genetic alterations. Some prognosis-associated cytogenetic aberrations or gene mutations such as FLT3-internal tandem duplication (ITD), t(8;21)(q22;q22)/RUNX1-RUNX1T1, and inv(16)(p13q22)/CBFB-MYH11 have been found and used to stratify the risk. Numerous gene mutations have been implicated in the pathogenesis of AML, including mutations of DNMT3A, IDH1/2, TET2 and EZH2 in addition to RAS, KIT, NPM1, CEBPA and FLT3in the recent development of massively parallel sequencing technologies. However, even after incorporating these molecular markers, the prognosis is unclear in a subset of AML patients. Recently, NUP98-NSD1 fusion gene was identified as a poor prognostic factor for AML. We have reported that all pediatric AML patients with NUP98-NSD1 fusion showed high expression of the PR domain containing 16 (PRDM16; also known as MEL1) gene, which is a zinc finger transcription factor located near the breakpoint at 1p36. PRDM16 is highly homologous to MDS1/EVI1, which is an alternatively spliced transcript of EVI1. Furthermore, PRDM16 is essential for hematopoietic stem cell maintenance and remarkable as a candidate gene to induce leukemogenesis. Recent reports revealed that high PRDM16 expression was a significant marker to predict poor prognosis in pediatric AML. However, the significance of PRDM16 expression is unclear in adult AML patients. Methods A total of 151 adult AML patients (136 patients with de novo AML and 15 patients with relapsed AML) were analyzed. They were referred to our institution between 2004 and 2015 and our collaborating center between 1996 and 2013. The median length of follow-up for censored patients was 30.6 months. Quantitative RT-PCR analysis was performed using the 7900HT Fast Real Time PCR System with TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assay. In addition to PRDM16, ABL1 was also evaluated as a control gene. We investigated the correlations between PRDM16 gene expression and other genetic alterations, such as FLT3-ITD, NPM1, and DNMT3A, and clarified the prognostic impact of PRDM16 expression in adult AML patients. Mutation analyses were performed by direct sequence analysis, Mutation Biased PCR, and the next-generation sequencer Ion PGM. Results PRDM16 overexpression was identified in 29% (44/151) of adult AML patients. High PRDM16 expression correlated with higher white blood cell counts in peripheral blood and higher blast ratio in bone marrow at diagnosis; higher coincidence of mutation in NPM1 (P = 0.003) and DNMT3A (P = 0.009); and lower coincidence of t(8;21) (P = 0.010), low-risk group (P = 0.008), and mutation in BCOR (P = 0.049). Conversely, there were no significant differences in age at diagnosis and sex distribution. Patients with high PRDM16 expression tended to be low frequency in M2 (P = 0.081) subtype, and the remaining subtype had no significant differences between high and low PRDM16 expression. Remarkably, PRDM16 overexpression patients were frequently observed in non-complete remission (55.8% vs. 26.3%, P = 0.001). Patients with high PRDM16 expression tended to have a cumulative incidence of FLT3-ITD (37% vs. 21%, P = 0.089) and MLL-PTD (15% vs. 5%, P = 0.121). We analyzed the prognosis of 139 patients who were traceable. The overall survival (OS) and median survival time (MST) of patients with high PRDM16 expression were significantly worse than those of patients with low expression (5-year OS, 17% vs. 32%; MST, 287 days vs. 673 days; P = 0.004). This trend was also significant among patients aged <65 years (5-year OS, 25% vs. 48%; MST, 361 days vs. 1565 days, P = 0.013). Moreover, high PRDM16 expression was a significant prognostic factor for FLT3-ITD negative patients aged < 65 years in the intermediate cytogenetic risk group (5-year OS, 29% vs. 58%; MST, 215 days vs. undefined; P = 0.032). Conclusions We investigated the correlations among PRDM16 expression, clinical features, and other genetic alterations to reveal clinical and prognostic significance. High PRDM16 expression was independently associated with non-CR and adverse outcomes in adult AML patients, as well as pediatric AML patients. Our finding indicated that the same pathogenesis may exist in both adult and pediatric AML patients with respect to PRDM16 expression, and measuring PRDM16 expression was a powerful tool to predict the prognosis of adult AML patients. Disclosures Inokuchi: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria; Pfizer: Honoraria.

Download Full-text

Cytogenetic Diagnostics and Outcome in a Series of Thirty Three Greek Pediatric Acute Myeloid Leukemia Patients

Blood ◽

10.1182/blood.v112.11.4889.4889 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4889-4889

Author(s):

Kalliopi N Manola ◽

Agapi Parcharidou ◽

Vassilios Papadakis ◽

Maria Kalntremtziou ◽

Chryssa Stavropoulou ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Bone Marrow Cells ◽

Normal Karyotype ◽

Acute Leukemias ◽

Early Treatment Response ◽

Pediatric Aml ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) accounting for approximately 17% of all childhood acute leukemias, arises either de novo or from a backround of myelodysplasia or previous chemotherapy. Cytogenetics is considered one of the most valuable prognostic determinants in AML while current risk–group classification in the limited cases of pediatric AML, is mainly based on cytogenetics and early treatment response. We reviewed the clinical and cytogenetic characteristics and the outcomes of 33 cases of childhood AML between 1997 and 2007 in order to investigate the incidence of the main FAB subtypes, the incidence of primary AML compared to secondary AML (s-AML) and the correlation between specific chromosome abnormalities and outcome in greek pediatric AML patients. Chromosome studies were performed on unstimulated bone marrow cells, derived from 33 pediatric AML patients, who were <18 years of age at the time of diagnosis. Eighteen patients were male and 15 were female. According to FAB classification one patient was classified as M0 (3%), 13 patients as M2 (39.4%), 4 as M3 (12.12%), 4 as M5 (12.12%), 2 as M6 (6.1%) and 4 as M7 (12.12%). No patient was classified as M4 while 5 patients with s-AML (15.15%) could not be classified. The median follow-up of all patients was 57.95 months (0.03–132.47). Overal survival and event free survival were 66,7% and 75,8% respectively. Eight patients with s-AML and 25 patients with primary AML were identified. The median age of patients with s-AML at diagnosis was 9.15 years while the median age of patients with primary AML was 7.2 years. Six out of 8 patients with s-AML died at a median follow up of 11.03 months. Nineteen out of 25 patients with primary AML are alive in complete remission (CR). Cytogenetic analysis was performed at diagnosis in 32 patients and results were obtained in 30 of them. The karyotype was abnormal in 21 out of 30 patients (70%). Normal karyotype was found in 9 patients, t(8;21)(q22;q22) in 5, t(15;17)(q22;q21) in 3, t(9;11)(p22;q23) in 3, −7/del(7q) in 5, del(9q) in 3, and complex karyotype in 4 patients. Three out of 4 patients with M3 are alive in CR with a median follow-up of 98.6 months while one with s-AML-M3 died 13 days post diagnosis. Three out of five patients with M2 and t(8;21), including 1 patient with s-AML, died at a median follow-up of 4.35 months. Three out of 5 patients with −7/del(7q) had s-AML and died in less than 4 years, while the two others are alive for more than 5 years, in CR. Although all patients with M7 had complex karyotypes, they are alive after a median follow-up of 96.73 months, 3 of them in CR and 1 in relapse. These results indicate that in greek patients, the main FAB subtypes show a distribution similar to that reported in the literature with the exception of M4 which is absent in our study but with a reported incidence of 20%. Pediatric patients with s-AML are older and their outcome is poor and is related to a higher probability of poor cytogenetic features compared to primary AML patients. Interestingly all patients with M7 had a good clinical course although they exhibited complex karyotypes.

Download Full-text

Acute Myeloid Leukemia with Coexpression of Lymphoid-Associated Antigens: Clinicobiological Associations and Prognostic Implications,

Blood ◽

10.1182/blood.v118.21.3596.3596 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3596-3596

Author(s):

Georgia Voutiadou ◽

Konstantina Kotta ◽

Barbara Tachynopoulou ◽

Apostolia Papalexandri ◽

Chryssanthi Vadikolia ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Critical Role ◽

Flow Cytometric Analysis ◽

Prognostic Impact ◽

Prognostic Implications ◽

Cytogenetic Profile ◽

De Novo Aml ◽

Acute Myeloid

Abstract Abstract 3596 Immune phenotyping plays a critical role in the diagnosis and classification of acute leukemia. Several studies have reported a variable proportion of patients with acute myeloid leukemia (AML) expressing lymphoid-associated antigens (LAA). The exact frequency and true clinical significance of this phenomenon remains undefined due to inconsistencies between series, likely related to methodological aspects or potential case selection biases. We retrospectively evaluated the expression of LAA in blast cells from 278 consecutive and unselected patients with AML diagnosed in our Department between 2002 and 2010. The patient cohort included 168 males and 110 females with a median age of 61 years (range, 10–88); 146/278 cases were above the age of 60. Within this cohort, 190 cases (68%) had de novo AML, whereas the remaining 88 cases (32%) concerned secondary AML (sAML) to either MDS (n=80) or other non-hematologic malignancies (n=8). Patients were treated uniformly according to age with Aracytin/Idarubicin induction regimens (“3+7” or “2+5” for ages \q60 or ≥60, respectively). The immunophenotype was determined by flow cytometric analysis of (mainly) bone marrow aspirate and/or peripheral blood samples utilizing a primary CD45/side scatter (SSC) gating procedure with antibodies against CD7, CD13, CD19, CD33, CD4, CD10, CD34, CD117, CD64, HLA-DR, CD20, CD2, CD15, CD56, CD14, CD8, MPO, CD3, CD79a, CD22, TdT and lysozyme; a cut-off value for positivity of 20% was adopted. Overall, we identified 153/278 cases (55%) expressing at least one LAA. The most commonly expressed LAAs were CD4 (outside AML with monocytic differentiation), CD56, CD7, CD2, CD10 and CD79a (in 39%, 33%, 29%, 14%, 10% and 8% of LAA+ AML cases, respectively); interestingly, all CD79a-positive cases co-expressed at least one more LAA. A significant association was identified between LAA expression and cytogenetic profile: in particular, at least one LAA was detected in 37/50 cases (74%) with adverse cytogenetics (SWOG unfavorable and/or monosomal karyotype), compared to 24/41 (58%) cytogenetically favorable cases and 68/134 (51%) cytogenetically intermediate risk cases (p=0.01). No other statistically significant associations were found for LAA expression (positive vs. negative) in respect to age and complete remission (CR) rate. Furthermore, the frequency of LAA-positive cases was identical (55%) in both de novo AML (105/190 cases) and sAML (48/88 cases). Monoparametric statistical analysis was also performed individually for each of the six more frequent LAAs. Significant associations (p<0.05) were identified between: (i) CD7 expression and adverse cytogenetics; (ii) CD10 expression and adverse cytogenetics as well as failure to achieve CR, at both cohort level as well as patients \q60 years with de novo AML; and (iii) CD2 expression and shorter overall and disease-free survival (DFS and OS, respectively). Cox-multivariate analysis identified CD2 expression in addition to advanced age, sAML and adverse cytogenetic profile as negative prognostic indicators (p=0.05) for both DFS and OS. In conclusion, expression of LAAs is frequent in AML, among both de novo AML and sAML cases, and significantly associated with adverse cytogenetics. Although the negative prognostic impact of CD2 expression is noteworthy, however, the precise prognostic implications of the expression of individual LAAs are hard to define on single institution retrospective series and will require evaluation in large prospective and well-controlled studies. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

EZH2 Mutations Are Associated with Low Blast Percentage in Bone Marrow and −7/Del(7q) Karyotype in De Novo Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v120.21.2544.2544 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2544-2544

Author(s):

Xiuli Wang ◽

Haiping Dai ◽

Qian WANG ◽

Qinrong Wang ◽

Yang Xu ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Conflicts Of Interest ◽

Univariate Analysis ◽

Myelogenous Leukemia ◽

Prognostic Impact ◽

Coding Region ◽

Acute Myeloid

Abstract Abstract 2544 Somatic mutation of the EZH2 gene is seen in myelodisplastic syndrome, myelofibrosis, and chronic myelomonocytic leukemia patients. The prevalence and prognostic impact of somatic mutations of EZH2 in patients with acute myelogenous leukemia (AML) remains unknown. In this study, we sought to determine the incidence and clinical implications of somatic EZH2 mutations in 714 patients with de novo AML by PCR amplification of the entire coding region followed by direct bidirectional DNA sequencing. EZH2 mutations were identified in 13/714 (1.8%) of AML patients and occurred almost exclusively in males (11/13, P=0.033). In univariate analysis, the presence of EZH2 mutations was significantly associated with lower blast percentage (21–30%) in bone marrow (P=0.0001) and −7/del(7q) (P=0.025). There was no difference in the incidence of mutations in 13 genes, including ASXL1, CBL, c-KIT, DNMT3A, FLT3, IDH1, IDH2, MLL, NPM1, NRAS, RUNX1, TET2, and WT1, between patients with and without EZH2 mutations. Complete remission, event-free survival or overall survival was similar between AML patients with and without EZH2 mutation (p>0.05). These results demonstrated EZH2 mutation as a recurrent genetic abnormality associated with lower blast percentage in BM and −7/del(7q) in de novo acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Prognostic Impact of the Interaction between DNMT3A mutation and Internal Tandem Duplication of the FLT3 Gene (FLT3-ITD) in Patients with De Novo Mutated NPM1 (NPM1mut) acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-115337 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1492-1492

Author(s):

Guadalupe Oñate ◽

Ana Garrido ◽

Jordi Esteve ◽

Rosa Coll ◽

Montserrat Arnan Sangerman ◽

...

Keyword(s):

Overall Survival ◽

Acute Myeloid Leukemia ◽

Complete Remission ◽

Myeloid Leukemia ◽

De Novo ◽

Prognostic Impact ◽

Intermediate Risk ◽

Mutational Status ◽

De Novo Aml ◽

Acute Myeloid

Abstract Introduction The association of NPM1mut and FLT3-ITD in de novo acute myeloid leukemia (AML) with intermediate-risk cytogenetics has different prognostic impact depending on the FLT3 allelic burden. Previous studies published by our cooperative group showed that patients with de novo AML of intermediate-risk cytogenetics with NPM1mut and FLT3-ITD low ratio (<0.5, FLT3low) at diagnosis presented an overall survival and relapse rate similar to those with NPM1mut and FLT3wt. Therefore, in the CETLAM-2012 protocol, patients with FLT3low NPM1mut AML are not considered for allogenic hematopoietic stem cell transplant (allo-HSCT) in first complete remission (CR1). Recent studies suggest that the co-occurrence of DNMT3A mutation in FLT3-ITD NPM1mut AML patients confers a worse prognosis regardless of FLT3-ITD ratio. We analysed our data to determine whether these findings were confirmed in our cohort, specifically in the low FLT3-ITD ratio patients, since this could have therapeutic implications. Methods and patients A total of 163 patients with de novo AML, intermediate-risk cytogenetics and NPM1mut were analysed (median age 53 years (18-72); male:female 72:91 (0.79)). Eighty patients (49%) harboured an FLT3-ITD, with a high allelic ratio in 42 of 76 patients with available ITD/wt ratio (55%). They were included in the AML-2003 (n=49) and AML-2012 (n=114) CETLAM protocols. Proportion of patients undergoing alloHSCT in CR1 is detailed in table 1. Bone marrow samples from diagnosis were studied for DNMT3A mutations as previously described. The definition of complete remission (CR), overall survival (OS), leukemia-free survival (LFS) and risk of relapse (RR) followed recommended ELN criteria. The Kaplan-Meier method was used to estimate the distribution of LFS and OS, for RR cumulative incidence was used. Results Out of the 163 patients with AML of intermediate risk cytogenetics and NPM1mut, 78 presented DNMT3A mutations (48%). Of these, 62 (79%) presented mutations in codon R882 or corresponded to DNA insertions/deletions while 16 (21%) harboured missense mutations. Presence of DNMT3A mutation did not associate with FLT3-ITD (ITD/85 DNMT3Awt vs ITD/78 DNMT3Amut, p=0.394). In the entire cohort, 5-year OS, LFS and RR were 58±4.5%, 59±4.6% and 27±13.9%. FLT3-ITD ratio confirmed its prognostic impact when analysing FLT3wt (n=83) vs FLT3low (n=34) vs FLT3high (n=42) patients (5-year OS of 68±6% vs 62±8.7% vs 37±8.6%; p=0.002; and 5-year RR of 18±9.4% vs 27±16.1% vs 41±23.2%; p=0.023). On the contrary, DNMT3Amut did not exert any effect on overall outcome (5-yr OS DNMT3Awt vs DNMT3Amut 61±6.2% vs 55±6.2%; p=0.234) When DNTM3A mutational status was considered, the impact of FLT3-ITD on outcome was mitigated in wild-type DNMT3A population. Thus, we found that DNMT3Awt patients presented no statistical differences in OS according to FLT3 mutational status or ratio: FLT3wt (n=46) vs FLT3-ITD (n=39) was 67±8.5% vs 57±8.2%; p=0.122, whereas FLT3wt (n=46) vs FLT3low (n=18) vs. FLT3high (n=19) was 67±8.5% vs. 66±11.5% vs 46±11.8%; p=0.088 (image 1A).This was also seen in relation to LFS and RR according to FLT3 ratio: 5-yr LFS of FLT3wt vs FLT3low vs FLT3high was 72±7.9% vs 61±12.6% vs 51±13.4%; p=0.244 and 5-year RR of the same groups: 19±8.8% vs 26±12.5% vs 27±21.9%; p=0.724 (image 2A). In the DNMT3Amut group, patients with FLT3-ITD (n=41) presented shorter OS than those with FLT3wt (n=37) with an OS of 37±10.7% vs 69±7.8%; p=0.028. When FLT3 ratio was considered, FLT3wt (n=37) vs FLT3low (n=16) vs FLT3high (n=23) showed an OS of 69±7.8% vs. 58±13.2% vs 27±13.1%; p=0.038 (image 1B). Similar results were seen in LFS according to FLT3 ratio (FLT3wt (n=29) vs FLT3low (n=16) vs FLT3high (n=20) 71±8.6% vs 53±12.9% vs 18±13.8%; p=0.012). Finally, we observed significant differences in the 5-year RR when considering DNMT3Amut patients in relation to FLT3 ratio (FLT3wt vs FLT3low vs FLT3high 18±10.6% vs 27±20% vs 54±28.8%; p=0.021)(image 2B). Conclusions In this study, patients with NPM1mut and FLT3-ITDlow presented a similar outcome to patients with NPM1mut and FLT3wt regardless of DNMT3A mutational status. These results support the modification of alloHCST policy in CR1 in CETLAM-2012, which do not consider alloHSCT for patients with FLT3low. On the other hand, concurrence of DNMT3A mutation may have an added negative effect in patients with NPM1mut and FLT3-ITDhigh, which should be further confirmed in larger studies. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Genomic Landscape and Prognosis in Pediatric Acute Myeloid Leukemia: A Study on the French ELAM02 Trial

Blood ◽

10.1182/blood.v128.22.1676.1676 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1676-1676

Author(s):

Alice Marceau-Renaut ◽

Nicolas Duployez ◽

Christine Ragu ◽

Arnaud Petit ◽

Odile Fenneteau ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Driver Mutations ◽

Mutational Profiling ◽

Genomic Landscape ◽

Diagnostic Samples ◽

Pediatric Aml ◽

Generation Sequencing ◽

Acute Myeloid

Abstract Background. Acute Myeloid Leukemia (AML) is a rare and genetically heterogeneous disease that constitutes 15 to 20% of childhood leukemia. Despite major treatment improvement over the past decades pediatric AML remains a challenging disease with poor outcome compared to acute lymphoid leukemia (ALL). About 50% of these patients relapse after standard intensive chemotherapy. Molecular analysis pointed out the prognostic impact of gene mutation such as FLT3-ITD, NPM1 or CEBPA; and new categories of regulators like epigenetic modifiers. More recently mutational profiling studies revealed distinct molecular subgroups with prognostic significant and stratification in adult AML. Nevertheless cytogenetic and mutational profiles are quite different between adult and pediatric AML. Extensive genomic studies have not been reported to date in pediatric AML. In this context it is of importance to identify additional genetic or molecular abnormalities to better understand leukemogenesis and also to predict outcome and serve as novel therapeutic targets. Methods. We performed a mutational analysis on diagnostic samples from patients enrolled in the French National Multicenter ELAM02 trial. 438 patients with de novo AML (except AML3) were enrolled between march 2005 and December 2011 (median age: 8,22yrs [0-18.61]; median WBC: 15.4G/l [0.4-575]; cytogenetic subgroups: CBF-AML[n=97], NK-AML [n=109], MLL-AML[n=95], MRC2 other[n=77], MRC3 [n=55], failure [n=5]). Diagnostic samples were prospectively collected and 386 of the 438 patients (88%) were studied by next-generation sequencing (Miseq, Illumina with haloplex librairy and ion Proton, thermofischer with ampliseq librairy) including 36 genes frequently reported in myeloid malignancy. Two different technologies of next generation sequencing (NGS) were used, allowing direct validation. FLT3-ITD was detected and quantified by Genescan analysis. Results. We identified 579 driver mutations involving 36 genes or regions in 386 patients (mean 1.5 per case), with at least 1 driver mutation in 291 patients (75%) and 2 or more driver mutations in 44% of samples. The number of mutation identified at diagnosis in cytogenetic subgroup is significantly lower in MLL-AML (0.44 mutation/patient; p<10-4). Mutations involving genes from the tyrosine kinase pathways (i.e RAS, FLT3, KIT, PTPN11, JAK2, MPL, CBL) were the most frequent and represent 56.3% of all aberrations. Among them N-RAS was detected in 26.4% of all cases, followed by FLT3-ITD, KIT and K-RAS in 14.8%, 12.4% and 12.2% respectively. We identified 64 driver mutations in the group of transcription factors (CEBPA, RUNX1, GATA, ETV6), 60 in the combined group of chromatin modifier (ASXL1, EZH2, BCOR) and DNA methylation (DNMT3A, IDH, TET2), 59 in the group of tumor suppressor genes (WT1, PHF6, TP53) 36 mutations in NPM1 gene, and few mutations in cohesion and spliceosome sub-groups. Identified mutations are indicated in the figure according cytogenetic subgroups. Among the 438 patients, 398 (91%) were in complete remission (CR) after two courses (induction and first consolidation), the 5-year overall survival (OS) is 71.5% [65-78] and the 5-year leukemia free survival (LFS) is 56.6% [49.7-63.5]. In univariate analysis, we found that FLT3-ITD, mutations in RUNX1, WT1 and PHF6 were associated with reduced LFS (p=0.0003 for FLT3-ITD, p=0.01 for RUNX1, p=0.02 for WT1 and p=0.025 for PHF6) and reduced OS (p=0.0003 for FLT3-ITD, p=0.0003 for RUNX1, p=0.015 for WT1 and p=0.04 for PHF6). Mutations in NPM1 is associated with an improved 5-yr LFS (p=0.014) and 5-yr OS (p=0.005). Multivariate analysis revealed that FLT3-ITD, RUNX1 and PHF6 were independently associated with an adverse outcome and NPM1 with an improved outcome. Conclusions. We performed an extensive mutational study in de novo pediatric AML enrolled in the ELAM02 trial. We described the genomic landscape of 386 patients and showed the frequency of different mutations according cytogenetics. Interestingly we found mutations in genes involved in constitutional or pre-leukemic disease such as PTPN11, RUNX1, MPL or ETV6. We found that FLT3-ITD, RUNX1 and PHF6 mutations predict poor outcome although NPM1 mutations predict a better outcome. Mutational profiling reveals useful information for risk stratification and therapeutic decisions. Figure Figure. Disclosures Baruchel: Amgen: Consultancy.

Download Full-text

Prognostic Impact of Anti-Apoptotic and Multidrug Resistance Phenotypes in Elderly Acute Myeloid Leukemia Patients.

Blood ◽

10.1182/blood.v106.11.2363.2363 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2363-2363

Author(s):

María-Belén Vidriales ◽

Lilia E. Suarez ◽

Jose García-Laraña ◽

Raimundo García-Boyero ◽

María-José Moreno ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Multidrug Resistance ◽

Significant Influence ◽

Myeloid Leukemia ◽

De Novo ◽

Response Analysis ◽

Poor Performance Status ◽

Prognostic Impact ◽

Cd34 Expression ◽

Acute Myeloid

Abstract Elderly patients with acute myeloid leukemia (AML) have an unfavorable outcome, which has been related to both the poor performance status of many of these patients and the biological characteristics of the malignant clone, including the expression of multidrug resistance (MDR) phenotypes. We have quantitatively analyzed by flow cytometry the expression of apoptosis- (bcl-2, bax, APO2.7) and MDR- (P-gp, MRP, LRP) associated proteins in a group of 117 elderly (>65 years) uniformly treated de novo AML patients according to the Pethema LMA-98 protocol. Upon analyzing factors influencing the response to induction therapy, as expected, patients with good and intermediate cytogenetics (n=76) more frequently achieved morphological complete remission (mCR) (63%) than patients having poor cytogenetics (n=23) (mCR: 30%). In addition, CD34 expression also influenced response; thus, 81% (21/26) of CD34-negative patients achieved mCR vs only 46% (42/91) of CD34-positive cases (p=0,008). Neither age nor WBC counts showed a significant influence in response rate. As far as apoptosis and MRD proteins is concerned, interestingly, responding cases showed a lower expression of the bcl-2, MRP and LRP proteins (bcl-2 RFI (relative fluorescence intensity) 10±5.5 vs 13±6, p=0.02; MRP RFI 1.9±0.8 vs 2.7±1.8, p=0.008; LRP RFI 6.2±4.9 vs 8.7±7.5, p=0.01 in responding vs non-responding patients). By contrast, expression of APO 2.7, Bax, and MDR-1 did not influenced response. Analysis of relapse free survival showed that only the number of cycles of chemotherapy required to achieve mCR had prognostic influence (p=0.008), with no significant influence for age, WBC counts, CD34 expression, or cytogenetics. In turn, a high percentage of early apoptotic cells in bone marrow at diagnosis (p=0.01), as well as a low bcl-2/bax ratio (p=0.05), and low MRP expression (p=0.04) were associated with a prolonged RFS. Moreover, upon grouping AML patients according to the expression of MRP and the bcl-2/bax ratio (> and < of the mean of both parameters), only patients with both low MRP expression and low bcl-2/bax ratio (n=16) achieved a plateau phase in the RFS curve after 20 months of follow-up, while the remaining patients showed a continuous relapse trend. In summary, our results show that, in addition to high bcl-2 and bcl-2/bax ratio, a high expression of the LRP and MRP multidrug resistant proteins have an adverse prognostic influence in elderly AML patients. The combination of these parameters contribute to identify distinct groups of patients at a different risk of relapse.

Download Full-text

Acute Myeloid Leukemia (AML) with t(6;9)(p23;q34) Defines a Very Poor Risk Leukemia Subgroup with Distinguishing Clinicopathological Features: A United States (US) Cytogenetics Intergroup Study of 62 AML and MDS Cases.

Blood ◽

10.1182/blood.v104.11.567.567 ◽

2004 ◽

Vol 104 (11) ◽

pp. 567-567

Author(s):

Marilyn L. Slovak ◽

Clara D. Bloomfield ◽

Holly Gundacker ◽

Gordon Dewald ◽

Frederick R. Appelbaum ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Fusion Gene ◽

Young Patients ◽

Disease Free Survival ◽

Clinicopathological Features ◽

Published Data ◽

Pediatric Aml ◽

Acute Myeloid

Abstract The t(6;9)(p23;q34) translocation, which results in the formation of a chimeric fusion gene DEK/CAN on the der(6) chromosome, is a rare recurring cytogenetic aberration reported in patients (pts) with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Because the abnormality is an infrequent finding in AML with most reports describing 2 to 8 cases, the US Intergroup Cytogenetics Consortium investigated the frequency and clinical, pathologic and cytogenetic characteristics of t(6;9) leukemia among pts registered to 19 different treatment protocols. Among 6567 pts with evaluable karyotypes, 62 (0.9%) had t(6;9): 30 on pediatric trials (mean 12 yrs; 15/15 male/female) and 32 on adult trials (mean 38 yrs; 21/11 male/female), compared to the mean age of 8 yrs for pts on pediatric AML/MDS trials and 54 yrs for pts on adult AML/MDS trials. Three cases (5%) showed a complex (3- or 4-way) variant translocation and only 7 (11%) of the 62 pts showed secondary aberrations: 3 (10%) of 30 pediatric cases and 4 (13%) of 32 of the adult cases. The majority of t(6;9) cases were classified as FAB-M2 (34%), M4 (31%) or M1 (19%). Although the immunophenotyping (N=7) and morphology data (N=17) were limited, increased basophilia and Auer rods were observed and the blasts showed CD13, CD15, and CD33 expression, in agreement with a previously reported preliminary study (Am J Clin Pathol107:430–437,1997). Four pts (1 pediatric and 3 adults) had MDS. Among the remaining 58 pts, 25 (78%) adults had previously untreated AML (16 de novo, 2 secondary, and 7 unknown secondary/de novo status) while all 29 pediatric AML patients had de novo AML. For the 54 patients with previously untreated AML, complete remission rates were slightly higher, but not statistically significantly (p=.20) in children (69%), when compared to adults (52%). Disease-free survival (DFS) (combined median 8.8 mo, 95% CI, 5.1–13.7) and overall survival (OS) (combined median 11.9 mo, 95% CI, 10.0–14.3) were poor regardless of age, a finding in distinct contrast to the t(8:21) favorable risk group also commonly observed in M2/M4 AML. Kaplan-Meier estimates of 3-yr survival were 25% for pediatric cases and 9% for adults. Analysis of stem cell transplantation (SCT) was inconclusive due to the small number of transplanted patients (N=15), but suggested that allogeneic SCT might be associated with better OS than no SCT (hazard ratio [HR] 0.39 after SCT, 95% CI 0.14 – 1.11), while autologous SCT might not (HR 1.49, 95% CI 0.57–3.85). Based on this study of t(6;9), largest to date and previously published data, AML with t(6;9) leukemia is a distinct AML subgroup with distinguishing clinicopathological features including poor outcome in relatively young patients, not explained by other known poor prognostic factors that warrants novel therapeutic strategies. Similar to other recurring cytogenetic abnormality subtypes of de novo acute myeloid leukemia of the WHO classification, t(6;9) may warrant a specific leukemia disease subtype.

Download Full-text