Series Of Acute Myeloid Leukemia Research Articles

Case Series of Acute Myeloid Leukemia (AML) in Pregnancy – A Single Centre Experience over 5 years Review

Acute Myeloid Leukemia (AML) is a rare condition presenting in pregnancy and still poses a challenge in its management which involves maintaining a balance between maternal and fetal well-being. This study aims to create a local registry for hematological malignancy in pregnancy and to review outcomes of both maternal and fetal outcomes. Twelve pregnancies were identified from 11 patients. 3 pregnancies were terminated upon diagnosis and 1 miscarried. One maternal death prior to decision. Out of the 6 pregnancies continued, 5 received chemotherapy during pregnancy with all successfully achieving complete remission and were delivered at 33-37 weeks gestation. All babies delivered with good Apgar score with 1 reported neonatal death. One fetal anomaly reported in a patient exposed to Decitabine in early pregnancy. Chemotherapy can be safely administered during second or third trimester with delivery planned as close to term as possible. Decitabine should be avoided during pregnancy due to its potential teratogenicity.

Transcriptomic Features of Immune Exhaustion and Senescence Predict Outcomes and Define Checkpoint Blockade-Unresponsive Microenvironments in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Reinstating immunological control of AML is highly desirable to eradicate chemotherapy-resistant clones and provide long-term disease control. We recently identified bone marrow (BM) microenvironmental transcriptomic profiles that stratify patients with newly diagnosed AML into an immune-infiltrated and an immune-depleted subtype and that refine the accuracy of survival prediction beyond that afforded by current prognosticators (Vadakekolathu J et al., 2020). We have also shown that CD8 + T cells from patients with AML exhibit features of immune exhaustion and senescence (IES), including heightened expression of killer cell lectin-like receptor subfamily G member 1 (KLRG1) and B3GAT1 (encoding CD57) (Knaus H et al., 2018). Whether deranged T-cell functions affect the likelihood of responding to antitumor therapy, including immune checkpoint blockade (ICB), is an outstanding question in AML.In the current study, we analyzed 183 BM samples collected longitudinally at time of AML onset, response assessment and disease relapse from multiple cohorts of patients with AML treated with standard-of-care induction chemotherapy, and from 33 elderly AML patients with newly diagnosed or chemotherapy-refractory/relapsed AML treated with azacitidine, and the PD-1 checkpoint inhibitor pembrolizumab (NCT02845297). Primary patient specimens and associated clinical data were obtained via informed consent in accordance with the Declaration of Helsinki on research protocols approved by the Institutional Review Boards of the participating Institutions. RNA (150-200 ng) was extracted from BM aspirates and was processed on the nCounter FLEX analysis system (NanoString Technologies, Seattle, WA) using the PanCancer Immune profiling panel, as previously published (Vadakekolathu J et al., 2020). The correlation between transcriptomic features of IES, clinical characteristics, therapeutic response and patient outcome was validated using publicly available RNA-sequencing and NanoString data from 1,698 patients with AML, including samples from the TCGA-AML (n=147 cases), Beat-AML Master Trial (n=264 cases, of which 240 with survival data and 195 with chemotherapy response data) and Children's Oncology Group (COG)-TARGET AML series (n=145 cases).We initially showed that, compared with their non-senescent CD8 +CD57 -KLRG1 - counterpart, senescent CD8 +CD57 +KLRG1 + T cells are functionally impaired in terms of their ability to effect AML-blast killing mediated by an anti-CD33/CD3 bi-specific T-cell engager antibody construct (kindly provided by Amgen, USA; effector/target [E/T] ratio = 1:5). We then used gene set enrichment analysis (GSEA) to derive a transcriptomic signature of IES encompassing natural killer (NK)-cell and stem-like CD8 + T-cell markers, and showed that IES states correlate with lymphoid infiltration, adverse-risk molecular lesions (TP53 and RUNX1 mutations), experimental gene signatures of leukemia stemness (LSC17 score; Ng et al., 2016) and poor outcome in response to standard induction chemotherapy (Fig. 1A). In independent validation cohorts of children and adults with AML, the IES score was higher at baseline in patients with primary induction failure (following a standard 2 cycles of chemotherapy) compared with complete remission, increased in post-chemotherapy BM specimens, and predicted survival with greater accuracy than the ELN cytogenetic risk classifier (Fig. 1B). In the immunotherapy setting, high IES scores at baseline defined a checkpoint blockade-unresponsive AML tumor microenvironment and correlated with significantly shorter overall survival (9.1 versus 15.56 months in patients with high and low IES scores, respectively; HR = 3.32 (95% CI = 1.19-9.25); log-rank P = 0.021; Fig. 1C). Finally, the IES-related gene set also predicted for long-term outcomes and objective responses, based on RECIST criteria, to single-agent nivolumab or pembrolizumab, or combination anti-PD-1 + anti-CTLA-4, in 106 patients with melanoma (PRJEB23709 and GSE93157 series), a tumor type known to derive durable clinical benefit from ICB (Fig. 1D).Our findings encourage the pursuit of immune senescence reversal as a strategy to functionally reinvigorate T cells and could inform the delivery of ICB and other T cell-targeting immunotherapies to patients who are likely to benefit. [Display omitted] DisclosuresRadojcic: Syndax Pharmaceuticals: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Allakos: Membership on an entity's Board of Directors or advisory committees. Minden: Astellas: Consultancy. Tasian: Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Incyte Corporation: Research Funding.

Additional mutations in IDH1/2‐mutated patients with acute myeloid leukemia

Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) frequently emerge in acute myeloid leukemia (AML), but the clinical features and molecular characteristics of IDH mutational status and other coexisting mutations have not been investigated in a large extensively characterized AML series. The aim of this study was to gain insight into the mutational profile of IDH-mutated patients, such as the frequency and clinical characteristics of coexisting mutated genes. We investigated 485 newly diagnosed AML patients (range 18-81years). DNA was extracted from bone marrow samples at the time of diagnosis. All samples were investigated with a panel of 49 mutational genes using next-generation sequencing (NGS). FLT3-ITD, NPM1, and CEBPA mutations were detected by Sanger PCR sequencing. We found 84 patients (17.3%) with IDH1 or IDH2 mutations. There were 40 IDH1R132 , 15 IDH2R140Q , 17 IDH2R172K , and 12 uncommon mutations. No patient was found to have both IDH1 and IDH2 mutations. Patients with IDH2R140Q mutations were more frequently older and presented with significantly lower average platelet counts, while IDH2R172K -mutated patients had significantly lower white blood cell (WBC) counts. On the background of IDH mutations, the presence of a normal karyotype showed a balanced distribution. The four most frequently coexisting mutated genes were NPM1, DNMT3A, TET2, and FLT3-ITD. The majority of coexisting mutated genes were involved in regulating transcription and DNA methylation. IDH mutation status had no effect on the CR rate, regardless of other molecular abnormalities. Isocitrate dehydrogenases mutations are associated with a complex coexisting mutation cluster in AML. Future investigation is needed to reveal the association between IDH mutations and other genetic abnormalities, which may have an impact on the progression and prognosis of disease.

Differences in the Mutational Landscape of Myeloid Malignancies (acute myeloid leukemia, myelodysplastic syndrome and myeloproliferative neoplasm) and Their Clinical Impact

Introduction: Myeloid malignancies are clonal disorders of hematopoietic stem cells and include acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN). Common biological markers have been described in the molecular pathogenesis, including gene mutations in splicing factors, epigenetic modifiers, transcription factors, signal pathways and tumor suppressors. These mechanisms have been associated with MDS and MPN progression to AML. Objectives: The main objective of this study is to identify differences in the mutational landscape of myeloid malignancies and describe mutation frequencies of genes and functional pathways in each neoplasm, as well as determine their clinical impact. Methods: This study involved a retrospective analysis of 430 patients with AML (209), MDS (106) and Philadelphia negative MPN (86) diagnosed in the Hospital Universitario 12 de Octubre (Spain). They were analyzed by a next generation sequencing (NGS)- panel for myeloid malignancies. The panel include 32 genes: CALR, ASXL1, EZH2, PHF6, DNMT3A 2, TET2, IDH1, IDH2, KDM6A, KMT2A, SF1, SF3A1, SF3B1, SRSF2, U2AF1, ZRSR2, PRPF40B, EPOR, FLT3, JAK2, KIT, SH2B3, MPL, CBL, HRAS, NRAS, KRAS, ETV6, RUNX1, VHL, TP53, PTEN. In addition, there were included 29 patients diagnosed with benign pathology that were referred to rule out MPN or congenital polyglobulia. Results: In the analyzed cohort we obtained a larger number of mutations in the more aggressive malignancies, AML and MDS. Mutations in epigenetic modifiers and signal pathways were the most frequent detected (31% and 24% respectively). The epigenetic modifiers were notably affected in AML (78%) and MDS (60.4%), whereas signal pathways were mutated more frequently in MPN (70.9%). Transcription factors, tumor suppressors and splicing factors mutations were more detected in AML and MDS (40%, 32%, 44% and 22%, 13%, 32% respectively). The mutation landscape obtained by genes was: Signal pathways: FLT3, NRAS, KIT, KRAS y SH2B3 were specially detected in AML (25%, 11%, 6%, 5% and 4% respectively). JAK2, CALR and MPL in MPN (38%, 15% and 6% respectively). Transcription factors: RUNX1, ETV6, PHF6, CEBPA and WT1 mutations were regularly observed in AML (21%, 6%, 6%, 6% and 5% respectively), and GATA1 in SMD (3.8%). Tumor suppressors: TP53 was particularly affected in AML (21%) and MDS (11%). Epigenetic modifiers: TET2 was notably mutated in MDS (32%), whereas ASXL1, DNMT3A, IDH2, IDH1 and EZH2 were in AML (21%, 21%, 17% 16% and 8% respectively). Splicing factors: SF3B1 was more frequently detected in MDS (18%) than AML (7%), whereas ZRSR2 presented a similar frequency in both pathologies (around 8%). U2AF1 was most commonly mutated in MPN (9%). SRSF2 was specially mutated in AML (23%). SF3A1 was altered in around 1%, similar in all three malignancies. With regard to survival studies, the presence of mutations in splicing factors (primarily in U2AF1) and its absence in signal pathways conferred an adverse outcome for overall survival (OS) in MPN. In MDS, gene mutations in tumor suppressors (especially TP53), U2AF1 splicing factor and EZH2 epigenetic modifier were associated with poor outcome. In our series of AML, gene mutations in tumor suppressors and TP53 were related to unfavorable prognosis in OS. Conclusion: The largest number of mutations and affected genes observed in AML suggest that leukemic transformation of MDS and MPN is conditioned by acquisition of new mutations. We observed different frequencies of mutations between AML, MDS and MPN that could guide the diagnostic and identify new targets of treatment. Further, some mutations have demonstrated differential prognostic impact. An extension of this study and the design of an algorithm with mutation data to elucidate a more accurate molecular prognosis will be presented at the meeting. This work has been financed thanks to the grant PI16/01225, PI 19/01518 and PI19/00730 from the Instituto de Salud Carlos III (Ministerio de Economia, Industria y Competititvidad) and cofinanced by the European Development Fund. Figure 1. Mutations detected (%) in AML, MDS and MPN classified by function. Table 1. Median overall survival of patients with MPN, MDS and AML according to gene state (mutated or not). Figure Disclosures No relevant conflicts of interest to declare.

Abstract CT035: TP53 abnormalities correlate with immune infiltration and are associated with response to flotetuzumab, an investigational immunotherapy, in acute myeloid leukemia

Abstract Background: Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. TP53 mutations and 17p (TP53) deletions occur in 37-46% of AML cases with adverse risk cytogenetics and are associated with primary induction failure (PIF), high risk of relapse and dismal prognosis. Herein, we aimed to determine whether TP53 abnormalities identify a patient subgroup that may benefit from immunotherapy approaches. Patients and Methods: We used the following transcriptomic data sets and patient cohorts (C) for in silico and wet-lab analyses: 1) The Cancer Genome Atlas (TCGA; 162 adult AML patients, 13 with TP53 mutations); 2) Beat AML Master Trial (281 adult AML patients, 17 with TP53 mutations); 3) HOVON (618 adult AML patients, 14 with TP53 mutations); 4) 24 diagnostic bone marrow (BM) samples from patients with TP53-wild-type AML (Bologna series); 5) 36 diagnostic BM samples from patients with TP53-mutated AML (Studien Allianz Leukämie [SAL] series); 6) 30 BM samples from patients with relapsed/refractory (R/R) AML (10 cases with TP53 mutations and/or 17p deletion) that received immunotherapy with flotetuzumab, a CD123×CD3 bispecific DART molecule (NCT02152956). Microenvironmental immune gene expression profiles (wet-lab cohorts, C4 and C5) were analyzed using the Pan-Cancer IO 360 Panel (NanoString Technologies, Seattle, WA). Immune cell type-specific and biological activity signature scores were computed as recently published (JITC 2017; 5: 18). Results: Compared with TCGA-AML cases (C1) with favorable-risk and intermediate-risk molecular features, all patients with TP53 mutations showed high levels of immune infiltration, including genes associated with adaptive immune responses and an interferon (IFN)-γ-dominant tumor microenvironment (TME), and a higher tumor mutational burden (14 versus 10 mutations on average in patients with TP53-mutated and TP53-wild-type AML, respectively; p=0.02). Similarly, 16 out of 17 (94%) TP53-mutated Beat AML cases (C2) expressed high levels of IFN signaling molecules, CD8 and markers of cytotoxicity (GZMB). Compared with patients with TP53-wild-type AML (C4), primary BM samples from patients with TP53-mutated AML (C5) showed higher levels of CD8A, markers of cellular senescence (EOMES, KLRD1, HRAS), IFN-γ-inducible genes (IRF1) and negative immune checkpoints including LAG3, IDO1, PDL1 and VSIR (VISTA). Interestingly, 10 patients with R/R AML (C6) had TP53 abnormalities and 5 of 6 patients evaluated for immune gene profiles had an immune-infiltrated TME. In patients with TP53 abnormalities, the overall response rate (ORR) was 40% to flotetuzumab (2 patients with CR, 1 patient with CRh, and 1 patient with morphologic leukemia-free state). The overall decrease of BM blasts averaged 42%. Stable disease was observed in 3 patients. Median overall survival (OS) was 3.5 months (range 1.25-21.25), which favorably compares with survival estimates for TP53-mutated cases with PIF in large AML series, such as HOVON (C3; median OS=1.16 months). Conclusions: This study provides evidence for a correlation between IFN-γ-dominant immune subtypes of AML and TP53 abnormalities. The ORR seen in this patient subgroup encourages further study of this immunotherapeutic approach. Citation Format: Jayakumar Vadakekolathu, Catherine Lai, Stephen Reeder, Sarah E. Church, Tressa Hood, John Muth, Heidi Altmann, Marilena Ciciarello, Antonio Curti, Peter J. Valk, Bob Löwenberg, Martin Bornhäuser, John F. DiPersio, Jan K. Davidson-Moncada, Sergio Rutella. TP53 abnormalities correlate with immune infiltration and are associated with response to flotetuzumab, an investigational immunotherapy, in acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT035.

Germline alterations in a consecutive series of acute myeloid leukemia.

Germline alterations in a consecutive series of acute myeloid leukemia.

Expression Profiling of Ribosome Biogenesis Factors Reveals Nucleolin as a Novel Potential Marker to Predict Outcome in AML Patients.

Acute myeloid leukemia (AML) is a heterogeneous disease. Prognosis is mainly influenced by patient age at diagnosis and cytogenetic alterations, two of the main factors currently used in AML patient risk stratification. However, additional criteria are required to improve the current risk classification and better adapt patient care. In neoplastic cells, ribosome biogenesis is increased to sustain the high proliferation rate and ribosome composition is altered to modulate specific gene expression driving tumorigenesis. Here, we investigated the usage of ribosome biogenesis factors as clinical markers in adult patients with AML. We showed that nucleoli, the nucleus compartments where ribosome production takes place, are modified in AML by analyzing a panel of AML and healthy donor cells using immunofluorescence staining. Using four AML series, including the TCGA dataset, altogether representing a total of about 270 samples, we showed that not all factors involved in ribosome biogenesis have clinical values although ribosome biogenesis is increased in AML. Interestingly, we identified the regulator of ribosome production nucleolin (NCL) as over-expressed in AML blasts. Moreover, we found in two series that high NCL mRNA expression level was associated with a poor overall survival, particular in elderly patients. Multivariate analyses taking into account age and cytogenetic risk indicated that NCL expression in blast cells is an independent marker of reduced survival. Our study identifies NCL as a potential novel prognostic factor in AML. Altogether, our results suggest that the ribosome biogenesis pathway may be of interest as clinical markers in AML.

Value of EVI1 Gene Expression Level in Adult Acute Lymphoblastic Leukemia (ALL): A Study from the Group for Research on Adult ALL (GRAALL)

Background:EVI1 gene overexpression is found in approximately 10% of acute myeloid leukemia (AML) patients, with a higher frequency seen in AML carrying chromosome 3q26 abnormality or MLL gene rearrangement, and associated with a dismal prognosis. Deregulation of EVI1 expression has also been reported in ALL, but its prognostic impact is unclear. Here, we retrospectively analyzed EVI1 expression in a large cohort of adult ALL patients, its correlation with ALL subsets, and its impact on patient outcome.Patients and Methods:EVI1 gene expression was measured by RQ-PCR detecting all splicing variants, with PBGD as control gene. We used dilutions of EVI1+ SKOV3 (kindly provided by Peter Valk, Rotterdam, The Netherlands) and EVI1-neg HL-60 cell line cDNA to build EVI1 and PBGD standard curves. Results were expressed as EVI1/PBGD ratio x 100. Blast samples from 354 patients treated in the GRAALL-2003/2005 and GRAAPH-2005 trials (191 B-cell precursor [BCP]-ALL, including 138 Ph-neg and 53 Ph+; 163 T-ALL) and 62 controls were analyzed. Immunophenotype results were centrally reviewed. In controls, median EVI1 expression level was 0.33% (Q1-Q3, 0.20-0.69). For prognostic analysis, we used the 1st and 99th percentiles of the controls (0.05% and 1.65%) to define patients with low and high EVI1 expression, respectively. Clinical endpoints were cumulative incidence of failure (CIF, failure meaning primary refractoriness or relapse) and event-free survival (EFS).Results: As illustrated in Figure 1, we observed that, as in one AML series, EVI1 expression may be up or down regulated in adult ALL. When compared to controls, the proportions of low and high EVI1 patients were 21 and 23% in Ph-neg BCP-ALL, 9 and 42% in Ph+ ALL, and 21 and 18% in T-ALL, respectively. In BCP-ALL patients, median EVI1 expression was similar to controls (0.53%; Q1-Q3, 0.11-1.88; p=0.15), but higher in the Ph+ as compared to the Ph-neg subgroup (0.93% versus 0.36%; p<0.001). In T-ALL patients, median expression tended to be lower than in controls (0.22%; Q1-Q3, 0.06-0.85; p=0.058). In these three ALL subgroups, EVI1 expression did not correlate with age or WBC. Among Ph-neg BCP-ALL patients, a lower expression was found in MLL-AF4+ t(4;11) cases (median, 0.04%; p<0.001), while no differences were observed for cases with t(1;19), an14q32, low hypodiploidy/near triploidy, complex karyotype, or IKZF1 deletion. Among T-ALL patients, a lower expression was found in cases with complex karyotype (median, 0.05%; p=0.03), while no differences were observed for cases with TLX1 overexpression, NOTCH1/FBXW7 mutation, N/K-RAS mutation or PTEN alteration. Only one patient had 3q26 abnormality (T-ALL with high EVI1 expression). Low or high EVI1 expression had no prognostic impact in Ph-neg as well as Ph+ BCP-ALL patients. In T-ALL, the proportion of patients with low EVI1 expression was more frequent in early and mature than in cortical T-ALL (33% and 33% vs 11%; p=0.002 and 0.028, respectively) and associated with a higher CIF (HR, 2.03; p=0.017) and shorter EFS (HR, 1.59; p=0.072). Low EVI1 expression was also significantly associated with an early T-cell precursor (ETP) phenotype (37.5% vs 18%; p=0.028). After adjustment on cortical and ETP phenotypes, as well as on 4-gene (NOTCH1/FBXW7/RAS/PTEN) genetic profiles, low EVI1 expression and high-risk genetic profiles remained independently associated with higher CIF (p=0.015 and <0.001, respectively) and shorter EFS (p=0.045 and 0.002, respectively).Conclusion: Overall, these results confirm that EVI1 gene expression is frequently deregulated in adult ALL. In BCP-ALL, down-regulation is observed in t(4;11) and up-regulation in BCR-ABL cases. Further studies are needed to confirm that, in T-ALL, a lower expression is associated with the early, mature and ETP phenotypes and independently predictive of a worse patient outcome. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A 16-Gene Signature Associated with High Levels of Wilms Tumor-1 (WT1) Expression Is an Adverse Prognostic Factor in Acute Myeloid Leukemia

Wilms Tumor-1 (WT1) expression level has long been found to be implicated in acute myeloid leukemia (AML) prognosis, though this is not reflected in current AML risk stratification. We hypothesized that a gene expression profile (GEP) associated with WT1expression could be of prognostic value.We analyzed two publically available AML GEP series in order to identify a gene signature associated with high-WT1 expression (hi-WT1). The first, herein called Netherlands series, comprised of 524 younger adult patients who have been treated according to sequential Dutch-Belgian Hemato-Oncology Cooperative Group and the Swiss Group for Clinical Cancer Research (HOVON/SAKK) AML-04, 04A, 29, 32, 42, and 43 protocols (GSE14468). The second series, herein called Germany series, consisted of 562 younger and older AML patients who were treated in the German AMLCG 1999 trial (GSE37642). We identified the hi-WT1 gene sets by comparing GEP among the highest and lowest quartiles of WT1 expression in both AML studies. About 62% of the probe sets in the Netherlands hi-WT1 set were found to be common with the Germany hi-WT1 set; 97% differed in the same direction. Moreover, a high degree of correlation of the fold differences was found among the two hi-WT1 sets (r2 = 0.81, p < 10-18), collectively suggesting a biological relevance for hi-WT1gene sets.In order to assess the prognostic implication of the hi-WT1 set, we used K-Nearest Neighborhood algorithm to generate various lists of hi-WT1 probe sets predicting event-free survival (EFS) as the favorable, and all others (dead, no remission, progressive disease/relapse) as the unfavorable events in the Netherlands series. Stepwise screening of the lists of 10 to 100 probe sets by Cox Regression identified a 16-gene subset of hi-WT1 set with distinct GEP and as the optimal predictor of overall survival (OS) and EFS in the Netherlands series. It comprised of GPR56, FAM30A, NGFRAP1, WBP5, LTK, PTP4A3, CD109, ZC3H12C, PYGB, CHIC1, HAVCR2, TMEM110, HAL, HDAC4, BLVRA, and P2RY2. In this series, the hi-WT1 cluster of patients showed lower 5y-probability of OS (10% vs 44%) and EFS (6% vs 38%) as compared to the remaining clusters. Accordingly, the hi-WT1 cluster showed shorter median OS (8.3 [CI 6.7-9.9] vs 31.3 [CI 17.1-45.5] months, P = 6 x 10-18) and EFS (4.9 [CI 3.2-6.5] vs 14.5 [CI 9.4-19.5] months, P = 3 x 10-16). Although the hi-WT1 cluster was associated with some of the cytogenetic and molecular aberrations including FLT3-ITD, it remained significant for both OS (P = 3 x 10-5) and EFS (P = 3 x 10-6) after adjustment for known AML risk factors.In order to validate our findings, we performed a supervised clustering of the Germany AML series using the 16-gene signature. The hi-WT1 cluster predicted both adverse OS and relapse-free survival (RFS) (Fig. 1), which remained statistically significant after adjustment for known AML risk factors (Table 1). The median OS was 7.1 (CI 5.7-8.5) months for the hi-WT1 cluster as compared to 20.1 (CI 15.2- 25.0) months for other cases (P = 2 x 10-13), and the median RFS was 5.8 (CI 4.8-6.8) vs 20.3 (CI 10.6-30.0) months, respectively (P = 2 x 10-11). Moreover, the rate of complete remission was significantly lower in hi-WT1 cluster as compared to other clusters (42% vs 61%, P = 2 x 10-5). The positive (PPV) and negative predictive value (NPV) of the marker for prediction of adverse OS were 90% and 34%, respectively. These values were found to be 88% and 38%, respectively, for prediction of adverse RFS. MetaCore analysis identified the Antigen Presentation by MHC-II as the most implicated biological pathway in hi-WT1sets, with many genes downregulated in the pathway.In brief, we identified a 16-gene signature associated with WT1 expression and demonstrated its adverse and independent prognostic impact in adult AML patients. These promising results should be validated in further trials and provide new clues to the molecular mechanisms underlying WT1regulation. [Display omitted] Table 1Multivariate analysis of OS and RFS in the Germany AML series. VariableOSRFSP valHR95% CI for HRP valHR95% CI for HRLowerUpperLowerUpperWT1 signature.0011.4851.1861.860.0001.8341.3262.536Age.0001.2911.1971.391.0011.1981.0811.327ELN2.0002.3951.7433.291.0003.1472.1244.663ELN3.0002.3951.7323.312.0002.1531.4053.299ELN4.0003.3062.3764.599.0006.4014.02710.175 DisclosuresNo relevant conflicts of interest to declare.

Hidden Mastocytosis in Acute Myeloid Leukemia With t(8;21)(q22;q22)

To assess the frequency of systemic mastocytosis (SM) in a large series of acute myeloid leukemia (AML) with t(8;21)(q22;q22). We retrospectively characterized 40 bone marrow aspirate smears and biopsy specimens from patients with AML with t(8;21) for the presence of SM. Cases were assessed for mast cell morphology and immunohistochemistry, as well as KIT exon 8 and 17 mutational assessment by reverse transcription polymerase chain reaction. Four patients met criteria for SM, 1 met criteria for myelomastocytic leukemia, and 8 demonstrated the benign finding of mast cell hyperplasia. We recommend examining all cases of AML with t(8;21) for the presence of SM via morphology, immunophenotyping, and KIT mutational analysis studies.

Clinical and laboratory features of pediatric acute myeloid leukemia with inversion of chromosome 16

To evaluate the clinical and laboratory features of pediatric inv(16) acute myeloid leukemia (AML) retrospectively. Dual color fluorescence in situ hybridization (D-FISH) using a LSI CBFβ inv(16) break apart probe labeled by Spectrum red and Spectrum green was performed in 15 acute myeloid leukemia cases, including 13 cases with or without abnormal eosinophils but with positive core binding factor β (CBFβ)-MYH11 fusion transcript detected by RT-PCR, and 2 cases with trisomy 8 (+8). The results were compared with the morphology, immunophenotype, karyotype and RT-PCR. Morphologically, 12 cases were diagnosed as M(4)EO, 2 as M(4), and 1 as M(2a). Immunophenotypically, all 13 AML cases with inv(16) showed positive expression of CD(13) and CD(33), but without the expression of any lymphoid lineage antigens. Karyotyping analysis with G-banding detected inv(16) in 10 AML cases, including 9 M(4)EO cases and 1 M(2a), but only 5 positive cases were detected using R-banding technique. Among them, 2 cases had simultaneous +8 and trisomy22 (+22), one had +22 only in addition to inv(16). D-FISH revealed a CBFβ-MYH11 rearrangement in 13 cases of AML with positive RT-PCR results, and the mean positive rate of cell detection was 55.15% (range 37.0% - 86.0%). The complete remission rate (CR) and median survival period in this series of inv(16) AML were 81.5%and 11 months, respectively, of whom, 8 cases were still in CR. Relapse and karyotypic evolution were seen in case 5 with +8, +22 in addition to inv(16). AML with inv(16) is a special subtype. Most cases belong to M(4)EO. Its prognosis is good in general, but it seems to be an unfavorable feature for AML with inv(16) and +8, +22 simultaneously, especially with karyotypic evolution. For detection of inv(16), G-banding technique is evidently superior to R-banding technique. D-FISH combined with RT-PCR are more sensitive and reliable than chromosome banding analysis.

Acquired mutations in the genes encoding IDH1 and IDH2 both are recurrent aberrations in acute myeloid leukemia: prevalence and prognostic value

AbstractSomatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) were recently demonstrated in acute myeloid leukemia (AML), but their prevalence and prognostic impact remain to be explored in large extensively characterized AML series, and also in various other hematologic malignancies. Here, we demonstrate in 893 newly diagnosed cases of AML mutations in the IDH1 (6%) and IDH2 (11%) genes. Moreover, we identified IDH mutations in 2 JAK2 V617F myeloproliferative neoplasias (n = 96), a single case of acute lymphoblastic leukemia (n = 96), and none in chronic myeloid leukemias (n = 81). In AML, IDH1 and IDH2 mutations are more common among AML with normal karyotype and NPM1mutant genotypes. IDH1 mutation status is an unfavorable prognostic factor as regards survival in a composite genotypic subset lacking FLT3ITD and NPM1mutant. Thus, IDH1 and IDH2 mutations are common genetic aberrations in AML, and IDH1 mutations may carry prognostic value in distinct subtypes of AML.

Mutual exclusion of ASXL1 and NPM1 mutations in a series of acute myeloid leukemias

Mutual exclusion of ASXL1 and NPM1 mutations in a series of acute myeloid leukemias

K313dup is a recurrent CEBPA mutation in de novo acute myeloid leukemia (AML)

The CEBPA gene codes for a transcription factor that has a pivotal role in controlling proliferation and differentiation of myeloid progenitors. Acquired CEBPA mutations have been found in acute myeloid leukemias (AML) with a good prognosis, and most of these patients have a normal karyotype. In this paper, we report four cases that displayed the same K313dup in the CEBPA gene. All four had an AML-M1 with CD7 positivity and T-cell receptor gamma chain (TCR-gamma) rearrangement. This mutation could represent nearly 10% of all CEBPA mutations described to date. K313dup disappeared in samples from patients in complete remission. In transfected cells, the K313dup mutant had reduced protein stability with respect to the wild-type protein. K313dup seems to be selected in leukemic cells, and its frequency in other AML series could be determined using the screening method reported in this paper.

Acute Myeloid Leukemia (AML) with 9q Aberrations Occuring within a Non-Complex Karyotype Is Highly Associated with CEBPA and NPM1 Mutations - A Joint Analysis of the German-Austrian AML Study Group (AMLSG) and Cancer and Leukemia Group B (CALGB).

Background: In a previous study we showed that AML with deletion 9q (9q-) occurring in the context of a non-complex karyotype is associated with CEBPA loss-of-function mutations; their prevalence was 41% (Frohling et al., Genes Chromosomes Cancer 2005; 42:427). We hypothesized that disruption of CEBPA function and loss of a critical segment of 9q cooperate in leukemogenesis. This is consistent with the model of leukemogenesis, in which mutations from different complementation groups cooperate, e.g., CEBPA mutations, representing the class II mutation impairing differentiation, occur simultaneously with 9q- associated with loss and/or mutation of an as yet unidentified gene that confers the proliferative advantage (class I mutation). 9q- is also associated with t(8;21)/RUNX1/RUNX1T1, another class II mutation. Importantly, in initial studies screening for NPM1 mutations, a few patients (pts) with 9q- were reported to harbor NPM1 mutations.Objective: To evaluate the incidence and clinical significance of mutations in the CEBPA and NPM1 genes in a large series of AML with 9q aberrations.Methods: Fifty-seven pts exhibiting a 9q aberration on chromosomal banding analysis were screened for CEBPA and NPM1 mutations. Pts were classified into three groups:1.those with 9q- occurring as a sole aberration or together with one additional abnormality other than t(8;21) (n=35);2.pts with 9q- occurring within a complex karyotype, defined as ≥3 abnormalities (n=10); and3.pts with 9q- secondary to t(8;21) (n=12).Results: The frequencies of CEBPA and NPM1 mutations in group 1 were 49% and 29%, respectively. Strikingly, either a CEBPA or NPM1 mutation was identified in 27 of the 35 (77%) pts within this subgroup. CEBPA and NPM1 mutations did not occur concurrently. In contrast, only one CEBPA and no NPM1 mutations were detected in group 2; no pt in group 3 had mutations in CEBPA or NPM1. Although the number of pts is still limited, within group 1, pts with CEBPA mutations and those with NPM1 mutations had higher CR rates, 86% and 80%, respectively, than pts with wild-type CEBPA and NPM1, whose CR rate was 57%. Overall survival of 9q- pts with CEBPA mutations was significantly better than that of the remaining pts comprising those with NPM1 mutations and pts with wild-type CEBPA and NPM1 (p=0.03).Conclusions: Abnormalities of 9q occurring in AML pts with a non-complex karyotype and without t(8;21) are highly associated with CEBPA or NPM1 gene mutations. CEBPA and NPM1 mutations are mutually exclusive, a finding that further supports the hypothesis that both CEBPA and NPM1 mutations act as class II mutations, which cooperate with a class I mutation affecting a thus far unknown gene on 9q. Currently, additional pts with 9q- are under investigation.

Distinct gene expression profiles of acute myeloid/T-lymphoid leukemia with silenced CEBPA and mutations in NOTCH1

AbstractGene expression profiling of acute myeloid leukemia (AML) allows the discovery of previously unrecognized molecular entities. Here, we identified a specific subgroup of AML, defined by an expression profile resembling that of AMLs with mutations in the myeloid transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα), while lacking such mutations. We found that in these leukemias, the CEBPA gene was silenced, which was associated with frequent promoter hypermethylation. The leukemias phenotypically showed aberrant expression of T-cell genes, of which CD7 was most consistent. We identified 2 mechanisms that may contribute to this phenotype. First, absence of Cebpa led to up-regulation of specific T-cell transcripts (ie, Cd7 and Lck) in hematopoietic stem cells isolated from conditional Cebpa knockout mice. Second, the enhanced expression of TRIB2, which we identify here as a direct target of the T-cell commitment factor NOTCH1, suggested aberrantly activated Notch signaling. Putatively activating NOTCH1 mutations were found in several specimens of the newly identified subgroup, while a large set of control AMLs was mutation negative. A gene expression prediction signature allowed the detection of similar cases of leukemia in independent series of AML.

Adverse prognostic impact of CD36 and CD2 expression in adult de novo acute myeloid leukemia patients

Background and objectives: A consecutive series of acute myeloid leukemias (AML) patients was analyzed in conditions which reduce the inter-assay variations (the same flow cytometer, the same observers and the same panel of monoclonal antibodies) in order to investigate the prognostic information provided by flow cytometry. Design and methods: Two hundred and sixty-six bone marrow (BM) samples from 326 patients enrolled in the LMA-99 protocol from the CETLAM group were studied by multiparametric flow cytometry. Immunophenotyping studies were performed on erythrocyte-lysed BM samples. Antigen expression of leukemic cells was analyzed using triple stainings with fluorochrome-conjugated combinations of monoclonal antibodies. Results: CD2 was positive in 21 cases (8%); an associated inv(16) was detected in eight CD2+ cases (38%). Two-year overall survival (OS) rate for CD2+/inv(16)+ patients was 75%, whereas it was 0% for CD2+/inv(16)− patients and 47% for CD2− patients ( p = 0.0001). CD36 was expressed in 37% of patients ( n = 98). Two-year leukemia-free survival (LFS) rate was 34% for CD36+ patients and 55% for CD36− patients ( p = 0.001). In the multivariate analysis, CD2+ (RR = 8.4; p = 0.0001) and adverse karyotype (RR = 10.2; p = 0.0001) were associated with a lower CR rate, CD36+ (RR = 1.5; p = 0.03), CD2+ (RR = 2; p = 0.04) and adverse karyotype (RR = 4; p = 0.0001) were associated with a lower OS and CD36+ (RR = 2; p = 0.002) and adverse karyotype (RR = 3.5; p = 0.005) predicted a lower LFS. Conclusions: CD2+ patients had a very poor OS when CD2/inv(16)+ cases were excluded. CD36 and CD2 expression at diagnosis can provide prognostically important information in adult de novo AML.

CEBPα Mutations in Childhood Acute Myeloid Leukemia.

Transcription factor CCAAT/enhancer binding protein alpha(C/EBPα) is essential for granulocyte differentiation. CEBPα mutations have been described in 7–10% of adult patients with acute myeloid leukemia (AML) at initial diagnosis and conferred a favorable prognosis. CEBPα mutations in childhood AML has not been reported yet. In this study, we sought to assess the frequency of CEBPα mutration, its clinicohematologic correlation and to analyze the cooperating mutations, including FLT3 and N-ras mutations in childhood AML. CEBPα mutations were analyzed in 117 children (age one day to 17 years, median 5 years) with de novo AML. CEBPα mutation status was examined by performing DNA polymerase chain reaction (PCR) followed by direct sequencing for each PCR product. CEBPα mutations were detected in 7 (6.0%) of 117 patients. Of the 7 patients with CEBPα mutations, 4 had FAB subtype of M2, 2 M1 and 1 M4. All had intermediate cytogenetics and comprised 11.5% (7/61) of the intermediate cytogenetic risk group. Five of the 7 mutations occurred in both N-terminal part and bZIP domain, one had N-terminal frameshift mutation and the remaining one had an inframe insertion in the bZIP domain. Clonal analysis using expand long template PCR assay with a primer pair covering entire coding sequence was performed in all 5 patients carrying two different mutations. The results demonstrated that one had homozygous combined mutation and 4 had heterozygous biallelic mutations with the cloned alleles carrying single mutations at N-terminal or bZIP domain in the different alleles or harboring combined mutations in the same alleles. None of the remission marrow samples obtained from 5 patients who had CEBPα mutations at diagnosis carried the mutations. FLT3-ITD mutations were detected in 2 of the 7 patients with CEBPα mutations compared to 15 of the 109 patients without CEBPα mutations. None of the patients carrying CEBPα mutations had FLT3-TKD mutations. Two of the 7 CEBPα(+) patients also harbored N-ras mutations compared to 10 of the 110 patients without CEBPα mutations. Taken together, 4 of 7 CEBPα(+) patients had cooperating mutations including FLT3-ITD or N-ras as compared to 27 in 109 CEBPα(−) patients (P=0.081). There were no statistical differences in age, WBC count, LDH level, 5-year overall survival and event-free survival between patients with and without CEBPα mutations; similar results were obtained if we only analyzed patients with intermediate cytogenetics. The present data showed that the frequency of CEBPα mutations in our series of childhood AML was similar to that in adults. CEBPα mutations were frequently associated with FLT3-ITD or N-ras mutations supporting the two-hit hypothesis for the pathogenesis of AML.

A distinctive nuclear morphology in acute myeloid leukemia is strongly associated with loss of HLA-DR expression and FLT3 internal tandem duplication.

In a 5-year survey of nonpromyelocytic/nonmonocytic acute myeloid leukemias (AMLs) diagnosed in the University of Washington Hematopathology Laboratory, we identified 19 cases containing distinctive, cup-like nuclear indentation in 10% or more of the blasts ('AML-cuplike'). Fourteen of these cases (74%) demonstrated near-complete loss of HLA-DR expression, while the other five cases showed partial loss of HLA-DR. A total of 16 of the cases (84%) demonstrated internal tandem duplication (ITD) of the Flt3 gene. When compared to a selected set of AMLs lacking this nuclear morphology, AML-cuplike was significantly more likely to lack HLA-DR and CD34 expression, to express CD123 without CD133, to have a normal karyotype, and to harbor the Flt3 ITD. To characterize AML-cuplike in an unselected series of AMLs, we analyzed 42 consecutive nonpromyelocytic/nonmonocytic AMLs diagnosed in our laboratory during a 6-month period in 2002. Strikingly, in this unselected series, there was a statistically significant coincidence of invaginated nuclear morphology, loss of HLA-DR, and presence of the Flt3 ITD beyond that expected if these three features were unrelated, suggesting that AMLs with these three features may represent a distinct AML subset.

Polymerase Chain Reaction-Based Diagnosis of Del(5q) in Acute Myeloid Leukemia and Myelodysplastic Syndrome Identifies a Minimal Deletion Interval

Loss of all or part of the long arm of human chromosome 5 is a recurrent abnormality in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), especially after chemotherapy for a prior malignancy. It is one of the worst prognostic indicators in AML, associated with chemotherapy resistance and short survival. These deletions center at band 5q31, which has thus been proposed as the location of a tumor suppressor gene; this site is to be distinguished from that observed in 5q- syndrome, centering at 5q32. To define the molecular extent and the clinical prevalence of 5q31 deletions, we collected a series of AML and MDS cases of mixed karyotype, taking care to exclude MDS cases with 5q- syndrome. The samples were analyzed for loss of heterozygosity (LOH) using a panel polymerase chain reaction (PCR)-based microsatellite markers from 5q, comparing malignant cells with normal tissue derived from lymphoblastoid cell lines or buccal mucosa scrapings. Losses were detected in seven of 29 matched samples, including four of 17 with MDS, and three of 12 with AML; six of these seven also had a cytogenetically-visible del(5q) or -5. The one case without a cytogenetic deletion showed molecular loss of three contiguous markers, with retention of flanking markers interleukin-9 (IL-9) and D5S414, and thus contained a small deletion that is below cytogenetic resolution. PCR failed to detect 5q loss in two cases with large cytogenetic deletions, but both had been treated and contained low percentages of malignant cells in the samples. This study thus led to the identification of a case with a minimal deletion for the 5q31 tumor suppressor gene, specified by IL-9-D5S414, that is approximately 1 Mb (2 cM) in extent. Additionally, we demonstrate that PCR-based allelotyping is a reliable method for the detection of chromosomal deletion in myeloid malignancy, providing the specimens contain a high proportion of malignant cells. These studies will help to identify the tumor-suppressor gene at 5q31, and will help to develop molecular methods for diagnosis and monitoring of these disorders.