scholarly journals DNMT3Awt NPM1-Mutation Defines a Subgroup of MDS with Special Favorable Outcomes Towards Decitabine Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1724-1724
Author(s):  
Lingyun Wu ◽  
Xiao Li ◽  
Feng Xu ◽  
He Qi ◽  
Zheng Zhang ◽  
...  
Keyword(s):  
Median Survival ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Statistical Significance ◽  
Complete Response ◽  
Subsequent Treatment ◽  
Induction Treatment ◽  
Wild Type ◽  
Npm1 Mutation ◽  
Randomized Controlled Studies

Hypomethylating agents (HMA) (including decitabine and azacitidine) are considered standard of care for higher risk myelodysplastic syndrome (MDS). Clinical data showed only about 30% cases achieved complete response (CR) by decitabine. Mutations in the nucleophosmin-1 (NPM1) gene is one of the most common somatic mutations identified in de novo acute myeloid leukemia (AML) and have also been found in 1% to 5% of MDS patients although with different mutation locus (L287fs) when compared to that in AML (W288fs). Till now, the response and survival of NPM1-mutated MDS patients treated with HMA remains unknown. Here, we retrospectively analyzed higher risk MDSs who accepted decitabine therapy in our center. From December 2009 to July 2018, a total of 194 patients received decitabine induction treatment by 20mg/m2 intravenously for 5 consecutive days every 4-6 weeks. The median therapy course was four. Among them, twelve patients (6.2%) harbored NPM1/L287fs mutation. The median decitabine therapy cycle for the 12 NPM1 mutated patients was also four. To our interest, patients harboring NPM1 mutations achieved a relatively higher CR rate (6 of 12 cases, 50%) when compared to that of patients without NPM1 mutations (59 of 182 cases, 32.4%) , although without statistical significance (p = 0.304). Moreover, when the most common co-mutated genes DNMT3A (6 of 12 cases, 50%) (Figure 1a) was ruled out, patients harboring NPM1 mutation (DNMT3A wild type) achieved a CR rate of 83.3% (five of six), which is significantly higher than that of patients without NPM1 mutation (p = 0.018) (Figure 1b). Of note, when paired sequencing were analyzed, six patients who achieved CR by decitabine showed loss of mutated NPM1; One patients who achieved hematological improvement (HI) showed decreased variant allele frequency (VAF) of NPM1 mutation; Whereas two patients with no response (NR) showed unchanged NPM1 mutation (Figure 1d). Notably, a prolonged relapse-free survival (PFS) was observed in CR patients with NPM1 mutation and DNMT3A wild type (NPM1mut DNMT3Awt) even without any subsequent therapies after receiving 4-5 cycles of decitabine (Figure 1c). The median RFS of CR patients with NPM1mut DNMT3Awt was 66 months, which is significantly longer than that in patients without NPM1mutation (13.5M, p = 0.006) (Figure 1e). A remarkably prolonged median survival was also shown in patients harboring NPM1mut DNMT3Awt (median survival of 80M), which is significantly longer than that of patients without NPM1 mutation (18M) (p = 0.012) (Figure 1f). Interestingly, except with DNMT3A and PTPRD co-mutations, the response and survival of patients harboring NPM1 mutations treated with decitabine were favorable even when co-mutated with IDH2, NRAS, FLT3. In conclusion, NPM1 mutation with DNMT3A wild type defines a specific subgroup of MDS with a good response and prolonged survival by decitabine therapy, even when they were with some prognosis-poor co-mutations and without subsequent treatment. Enlarged sample of randomized controlled studies are needed to confirm our preliminary findings. Figure 1 Disclosures No relevant conflicts of interest to declare.

The ATRA Question In AML: Lack Of Benefit Overall Or In Any Molecular Subgroup In The NCRI AML16 Trial

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 493-493 ◽  
Author(s):  
Alan K. Burnett ◽  
Robert K. Hills ◽  
Lone S. Friis ◽  
Lars Kjeldsen ◽  
Donald Milligan ◽  
...  
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Early Mortality ◽  
Significant Heterogeneity ◽  
Wild Type ◽  
Molecular Subgroup ◽  
Npm1 Mutation ◽  
Poor Risk ◽  
Good Standard

Abstract Background There is conflicting data on the effect of the addition of ATRA to chemotherapy in AML. Two large randomised trials showed no benefit (Estey et al Blood 1999; 93, 2478 (n=215); Burnett et al. Blood 2010, 115: 9482 (n=1075)), or benefit which was limited to patients with an NPM1 mutation (n=14) when given in combination with ICE (Idarubicin/Ara-C/Etoposide) (Schlenk et al, Leukemia 2004, 18; 1798 (n=242)) but not with DA alone. In an effort to prospectively clarify if this is predictive treatment for NPM1+ patients and whether the effect was etoposide dependent, we randomised 616 patients to DA vs ADE and ATRA vs no ATRA in a 2x2 factorial design. Methods Between August 2010 and May 2012, 616 patients were randomised. The median age was 67(53-82) years: 75% had de novo, 16% had secondary, and 8% had high risk MDS (marrow blasts 10-19%): 4%, 75% and 21% had favourable, intermediate or poor risk cytogenetics: ITD and NPM1 data was available on 422 and 404 patients, with mutation rates of 19% and 24%. A total of 56 patients (14% of those with data) were molecularly good risk (NPM1 mutant, ITD wild type). By Wheatley risk score, 24%, 40% and 36% had good, standard or poor risk disease. The demographic, cytogenetic, molecular and allocated treatments were balanced between the arms. Follow-up is complete to 1st January 2013 (median follow-up 18.7 months) Patients were given Daunorubicin 50mg/m2 days 1-3 + Ara-C 100mg/m2 bid days 1-10 (course 1) or days 1-8 (course 2). Those allocated ATRA were treated at 45mg/m2/day for 60 days; Etoposide in the ADE arm was given at 100mg/m2/day on days 1-5 of courses 1 and 2 of chemotherapy. Results The overall response rate (ORR) was 69% (CR 53%+ CRi 16%) and survival at 2 years was 35%. The ORR was not different between DA: 68% (CR 53%, CRi 15%) and ADE: 70% (CR 53%, CRi 17%), odds ratio (OR) 0.92 (0.65-1.30) p=0.6, although remission rates were non-significantly lower in patients given ATRA (ORR 66% (CR 53%, CRi 13%) vs 73% (CR 54%, CRi 19%), OR 1.39 (0.98-1.95) p=0.06) with significantly higher 30-day (16% vs 8%, p=0.005) and 60-day mortality (20% vs 12%, p=0.005). There were no differences in early mortality between ADE and DA arms. At 2 years, neither survival nor RFS differed between the arms: (ADE vs DA OS: 33% vs 36%, HR 1.07 (0.86-1.32) p=0.6; RFS: 23% vs 36% HR 1.15 (0.88-1.49) p=0.3; ATRA vs Not OS: 35% vs 35% HR 1.13 (0.91-1.40) p=0.3; RFS: 31% vs 30% HR 0.93 (0.71-1.20) p=0.6). Overall there was no interaction between the two treatments (OS, test for heterogeneity p=0.12). In an analysis stratified by Etoposide and by NPM1/ITD risk group there was no significant heterogeneity of the effect of ATRA (p=0.1, Figure). Importantly, when looked at by the underlying chemotherapy, no beneficial effect of ATRA in NPM1 mutant/ITD WT patients appeared for patients receiving ADE (p=1.0 for heterogeneity). Conclusions Neither the addition of Etoposide nor ATRA improves outcomes in this group of patients, with ATRA being associated with significantly greater early mortality. Importantly, an analysis by NPM1/FLT3 genotype fails to reproduce the large benefits seen by Schlenk et al in ATRA treated patients with an NPM1 mutant/ITD wild type genotype, either overall or for patients treated with ADE, thus failing to substantiate a benefit for ATRA in either context. Based upon the results of AML16, neither Etoposide nor ATRA improve outcomes for older patients given intensive DA chemotherapy. Acknowledgments This study received research support from Cancer Research UK, and the Cardiff Experimental Cardiff Medicine Centre. Disclosures: No relevant conflicts of interest to declare.

Assessment of CEBPA Mutations Might Contribute to a Better Prognostic Assignment in Patients with Intermediate-Risk Cytogenetics Acute Myeloid Leukemia (AML).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2611-2611
Author(s):  
Marta Pratcorona ◽  
Montserrat Torrebadell ◽  
Neus Villamor ◽  
Maria Rozman ◽  
Mireia Camós ◽  
...  
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Complete Response ◽  
Prognostic Impact ◽  
Single Mutation ◽  
Intermediate Risk ◽  
Wild Type ◽  
Male Predominance ◽  
Cebpa Mutations ◽  
Wbc Count

Abstract Abstract 2611 Poster Board II-587 The prognostic heterogeneity of patients with intermediate-risk cytogenetics AML (AML-IR) is mostly clarified by determination of mutations of NPM1 gene (NPMmut) and internal-tandem duplication of FLT3 gene (FLT3-ITD). Nonetheless, other genetic lesions described in this population might contribute to a better prognostic categorization. In this context, we analyzed the presence of CEBPA mutations and associated features in patients with AML-IR lacking both NPMmut and FLT3-ITD. Overall, 136 patients (51% female; median age: 53, range: 17=74) diagnosed with de novo AML-IR (MRC definition) in our institution between 1994 and 2008 who received standard AML chemotherapy were included in the analysis. CEBPA mutations (CEBPAmut) were investigated by whole gene sequencing using 4 primer pairs according to previously reported methods (Fröhling et al, 2004). Sixty-five patients (48%) harbored NPM1 mutations, 30 of them having concomitant FLT3-ITD. Among NPM1 wild-type patients (NPMwt), FLT3-ITD, CEBPA and MLL mutations were detected in 18, 11, and 5 patients, respectively. Regarding cases with CEBPAmut, biallelic mutations were found in 8 patients, including 6 cases with combined mutations of N-terminal and bZIP domains and two homozygous mutations, whereas a single mutation in bZIP domain was found in the three remaining patients. As compared to patients with wild-type CEBPA (CEBPAwt), those with CEBPAmut were younger (29 vs. 53, p=0.027), and showed a trend to male predominance (73 vs. 46%, p=0.09) and lower WBC count at presentation (16 vs. 33.5 × 109/L, p=.095). Of note, CD7 antigen was aberrantly expressed in virtually all CEBPAmut cases (10/11), compared to only 21% of CEBPAwt patients (p<0.001). Moreover, an abnormal karyotype was observed in 4 patients with CEBPAmut. In the overall series, complete response rate (CR), survival (OS), and relapse incidence (RI) were 83%, 39±4% (5-yr), and 50±5% (5-yr), respectively. Independent favorable factors for survival were younger age (<median; RR: 1.9, 95% CI: 1.2-3, p=0.004), low WBC count at diagnosis (< median; RR: 1.7, 95% CI: 1.1–2.6, p=0.025) and NPMmut/FLT3-ITDneg status (RR: 2.4, 95% CI: 1.3–4.2, p=0.003). Remarkably, patients with CEBPAmut showed a favorable outcome, with a trend for a more prolonged survival, compared to patients with a high-risk NPM/FLT3 status (5-year OS in pts <60 years: 74±16% vs. 33±6%, p=0.087). Based on these results, patients were grouped in a favorable (i.e., either NPMmut /FLT3-ITDneg or CEBPAmut, FAV) or unfavorable molecular category (i.e., those with FLT3-ITD, double NPMwt and CEBPAwt configuration, or MLL abnormalities, UNFAV); these two groups had independent prognostic impact on OS (RR: 2.3, 95% CI: 1.3–4, p=0.001; see figure), RI (RR: 2.2, 95% CI: 1.2–4.3, p=0.016), and leukemia-free survival (RR: 2.2, 95% CI: 1.3–3.7, p=0.002). Importantly, the outcome of patients in the FAV group did not differ according to post-remission treatment (autologous vs. allogeneic stem-cell transplantation, SCT), whereas relapse risk was significantly higher in patients with unfavorable markers who received autologous SCT (60±13% vs. 18±12%, p=0.03). In summary, the assessment of CEBPA mutations, especially in patients lacking NPMmut and FLT3-ITD and expressing CD7 antigen, may refine the molecular prediction of prognosis and guide therapeutic strategy in patients with intermediate-risk AML. Disclosures: No relevant conflicts of interest to declare.

Prognostic Impact of the Amount of CD34+ CD38- CD123+ Cells at Diagnosis In Acute Myeloid Leukemia: a Study From the GOELAMS

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 951-951
Author(s):  
François Vergez ◽  
Alexa Green ◽  
Jérôme Tamburini ◽  
Nathalie Gallay ◽  
Murielle Roussel ◽  
...  
Keyword(s):  
Multivariate Analysis ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Disease Free Survival ◽  
Complete Response ◽  
Leukemic Cells ◽  
Prognostic Impact ◽  
Npm1 Mutation ◽  
Number Of Patients ◽  
Wbc Count

Abstract Abstract 951 AML cells with CD34+CD38-CD123+ phenotype represent a subset enriched in leukemic stem cells. Moreover, this subpopulation has been described to be resistant to genotoxic agents as compared with leukemic bulk. However, the clinical impact of the amount of CD34+CD38-CD123+ remains poorly described. In this study we evaluated the prognostic impact of the amount of CD34+CD38-CD123+ cells detected at diagnosis in a series of AML patients treated by intensive chemotherapy according to trials from the French GOELAMS group. Quantification of blast cells with the CD34+CD38-CD123+ phenotype was achieved by flow cytometry in 111 patients less than 66 years old with de novo AML treated by 3+7-like chemotherapy. The characteristics of the patients are shown in table 1. Age, WBC count, NPM1 mutation and FLT3-ITD had no impact on achievement of complete response (CR) whereas, CD34+CD38-CD123+ (>15%) and unfavourable karyotype were significantly correlated with lack of CR. By logistic regression, CD34+CD38-CD123+ (>15%) retains significance for CR achievement with an OR of 0.3 (0.11-0.84) (p=0.02). For the 91 complete responders, age, WBC count, karyotype, NPM1 mutation had no impact on disease-free survival (DFS).Interestingly, patients with <1%, 1–15%, >15% CD34+CD38-CD123+ had a median DFS of 57.6 (SE 6.6), 11.2 (SE 7.5) and 9.2 (SE 13.4) months, respectively (p<0.0001, figure 1). FTL3-ITD was also significantly associated with a shorter DFS. In multivariate analysis, CD34+CD38-CD123+<1% was significantly associated with a longer DFS (p=0.00025). Age, %CD34+CD38-CD123+, karyotype, NPM mutation and FLT3-ITD significantly influenced overall survival (OS) whereas WBC count had no impact. Median OS was particularly impressive for patients with CD34+CD38-CD123+<1%. Indeed, median OS was 78.2 (SE 10.7), and 15.3 (SE 5.8) months for CD34+CD38-CD123+<1% vs others, respectively (p<0.0001, figure 1). Multivariate analysis for OS retains two significant factors: adverse karyotype (95%CI, 1.19–4.02, p=0.012) and CD34+CD38-CD123+<1% (95%CI, 0.12–0.52, p=0.00018). Moreover, although the number of patients was low in favourable karyotype (CBF), CD34+CD38-CD123+<1% had a major impact on both DFS, median 57.6 vs 10.2 months for >1% (HR, 0.19, 0.06–0.59, p=0.0038) and OS, median not reached vs 18.5 months (p=0.0025). This study emphasizes the prognosis impact of the CD34+CD38-CD123+ cell burden in AML patients, which is predictive for shorter OS and DFS when representing more than 1% of the leukemic cells, regardless of the usual prognosis categories. We provide here a new prognosis marker that may be easily translated to the clinical practice in AML although it remains to be validated on a large prospective cohort of patients. Moreover, new therapies targeting this subpopulation could help to improve outcome in AML patients.TableCharacteristics of patients.All patients N=111CD34+CD38-CD123+< 1 % N=40CD34+CD38-CD123+ 1-15% N=20CD34+CD38-CD123+> 15% N=20Gender, M/F50/6219/2124/276/14Age, median48 (20–65)47 (20–65)51 (20–64)50 (21–65)WBC,median (G/L)33.2 (1–254)13.8 (1–237.7)41 (1.3–254)32.9 (2.3–193.2)Favourable caryotype231184Intermediate caryotype6924378Unfavourable caryotype19568NPM1 mutation28/957/3116/455/19FLT3-ITD33/1007/3421/475/19Complete Response91 (82%)36 (90%)42 (81.4%)13 (65%)Relapse53 (58.2%)13 (36.1%)30 (71.4%)10 (76.9%)Allogeneic-SCT33 (36.3%)11 (30.6%)15 (35.7%)7 (53.8%) Disclosures: No relevant conflicts of interest to declare.

A Phase II Study of Intravenous Azacitidine Alone in Patients with Myelodysplastic Syndromes NCT00384956.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1451-1451
Author(s):  
Richard Walgren ◽  
Crystal Dao ◽  
Frederieke Kreisel ◽  
Peter Westervelt ◽  
Camille Abboud ◽  
...  
Keyword(s):  
Median Time ◽  
Phase Ii ◽  
De Novo ◽  
Phase Ii Study ◽  
Study Drug ◽  
Complete Response ◽  
Subsequent Treatment ◽  
Open Label ◽  
Erythroid Response

Abstract Rationale: 5-Azacytidine (Aza), a DNA hypomethylating agent, has now been shown in 2 clinical trials involving high-risk MDS patients to provide a survival benefit over supportive/conventional care regimens. While one phase II study used a continuous 7-day IV infusion, Aza was administered subcutaneously (SQ) in most pre-approval studies. However, injection site reactions are not uncommon with SQ dosing, especially in thrombocytopenic patients. Aza given as a short intravenous (IV) infusion is anticipated to be efficacious from pharmacokinetic profiling and is FDA approved, but prospective efficacy data for short IV infusion are lacking. Study aim and design: To determine the efficacy of IV Aza when given as a short infusion, we have undertaken an open-label, single-arm, single-center phase II study of Aza in patients with MDS, either de novo or secondary, defined by FAB classification. Previously treated subjects were ineligible if they had already received Aza or decitabine. Treatment consisted of Aza 75 mg/m2 given as a 20 minute IV infusion once daily on Days 1–5 of a 28-day cycle. Response was evaluated by IWG 2000 criteria. After two cycles at the 75 mg/m2 dose, patients failing to achieve a CR were eligible for an increased dose of 100 mg/m2. After 6 cycles of therapy, patients must have demonstrated at least a hematologic improvement to continue on study. Study endpoints include determination of the complete response (CR) and partial response (PR) rates, and secondary endpoints examined the rates of hematological improvement, time to progression, and cytogenetic response. Results: Accrual began 8/17/06 with a target of 21 subjects. As of 7/31/07, 15 subjects have accrued with a median follow-up of 77 days (range 4 to 246). Subjects consisted of 9 males and 6 females with a median age of 69.6 yr (range 53 to 82). The median time from diagnosis is 213 days (range 0 days to 4 yr). By FAB criteria, subjects consist of 4 RA, 9 RAEB, 1 RAEB-t, and 1 CMML, and subjects are categorized by IPSS risk as 1 Low, 4 Int-1, and 10 Int-2. Two patients had therapy related MDS. The data remain preliminary with subjects having completed a mean of 3 cycles (range 1 to 6). None of the 5 subjects who have completed at least 4 cycles of therapy have achieved a CR. However, 2 (40%) of these subjects achieved a PR. Additionally, 1 (20%) patient had a major erythroid response, while another had a minor erythroid response. Median time to response was 2 months. Ten subjects remain on study, 1 patient withdrew due to progressive disease (in first week of therapy), and 4 deaths have occurred on study (2 due to sepsis, 1 each due to pneumonia and acute MI). No deaths were attributed to study drug. Common adverse events include nausea, emesis, and hematologic toxicities. Grade 2–3 nausea and grade 2–3 emesis each occurred in 5 subjects. Observed grade 3 or 4 hematologic toxicities included: anemia (n=7), thrombocytopenia (n=4), leukopenia (n=3), neutropenia (n=7), and febrile neutropenia (n=1). Hematologic toxicities have resulted in transient treatment delays (&lt; 4 weeks) and dose reduction, but hematologic toxicities have not prevented subsequent treatment on study. Conclusions: Although follow-up is short for assessment of efficacy, this is the first prospective study to report on efficacy and toxicity of short infusional Aza in the treatment of MDS.

Count Recovery in AML Patients Achieving a Complete Response.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2062-2062 ◽  
Author(s):  
Steven Lawrence Rosinski ◽  
Farhad Ravandi ◽  
Stefan Faderl ◽  
Guillermo Garcia-Manero ◽  
Sherry Pierce ◽  
...  
Keyword(s):  
Recovery Time ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Complete Response ◽  
Platelet Recovery ◽  
Neutrophil Recovery ◽  
Secondary Aml ◽  
Recovery Times ◽  
Md Anderson ◽  
Count Recovery

Abstract Abstract 2062 Poster Board II-39 AML is typically defined as “de novo” or “secondary”, the latter referring to patients diagnosed only after persistent blood count abnormalities (AHD) or after prior “chemotherapy” (PCT) for other illnesses. Patients with secondary AML may have a different bone marrow microenvironment leading to prolonged neutrophil and platelet recovery times following induction chemotherapy. Accordingly, we compared time from start of chemotherapy to neutrophil recovery (>1,000/μl) and platelet recovery (>100,000/μl) in 424 patients who achieved a complete response (CR) following treatment with ara-C-containing induction therapy at MD Anderson Hospital from 1995 to 2008. We divided the 424 patients as follows: (1) no AHD, no PCT (236 patients); (2) AHD, no PCT (131 patients); (3) PCT, no AHD (28 patients); and (4) AHD and PCT (29 patients). Because time to recovery may also be influenced by cytogenetics and age we subdivided patients in each of the four groups according to age (< vs. ≥ 60) and cytogenetics (normal vs. complex or -5/-7). Despite very differing CR rates, time to neutrophil recovery in patients achieving CR while statistically longer in PCT patients (p=0.05) was from a medical standpoint essentially uninfluenced by AHD and PCT status (table, median delays 2 days with PCT). Platelet recovery was affected by such status (p<0.001) being delayed by a median of 6-8 days in patients who had received PCT. Age had no effect on time to neutrophil (p=0.42) or platelet (p=0.23) recovery, while complex or -5/-7 cytogenetics had a statistically significant (p=0.002 neutrophils, 0.009 platelets) but medically insignificant (median delays of 2 days) effect on recovery time. There was no interaction between age or cytogenetics and AHD or PCT status. A similar analysis in patients who do not achieve CR would be of interest, but might be confounded by the difficulty in distinguishing between the effects of chemotherapy and those of residual AML on count recovery. Our data suggest that older patients, patients with complex or -5/-7 cytogenetics, and patients with secondary AML should not be excluded from clinical trials because of concern about prolonged time to count recovery. Group CRs CR Rate Median Days to > 1000 Neut Median Days to > 100,000 Plt No AHD no PCT 236 68% 27 28 AHD no PCT 131 50% 27 30 PCT no AHD 28 44% 29 34 AHD + PCT 29 35% 30 36 Age < 60 255 64% 28 29 Age ≥ 60 169 48% 26 30 Normal cyto 281 69% 27 29 Complex cyto or -5/-7 143 41% 29 31 Disclosures: No relevant conflicts of interest to declare.

Disruption of the ASXL1 Gene Is Prevalent In PMF: Analysis of Molecular Genetics and Clinical Phenotypes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3074-3074
Author(s):  
Brady L Stein ◽  
Donna M Williams ◽  
Michael A McDevitt ◽  
Christine L. O'Keefe ◽  
Ophelia Rogers ◽  
...  
Keyword(s):  
Molecular Genetics ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Single Nucleotide Polymorphism Array ◽  
Myeloproliferative Neoplasms ◽  
Direct Sequencing ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Jak2 V617f ◽  
Wild Type

Abstract Abstract 3074 Background: The myeloproliferative neoplasms, PV, ET and PMF, share phenotypic features and molecular lesions, yet PMF distinguishes itself by its unfavorable natural history and rate of leukemic evolution. These distinctions may occur as a result of cooperating genomic lesions specific to PMF compared to PV or ET. We performed single nucleotide polymorphism array (SNP-A)-based karyotyping in 210 MPN patients and identified 20q11 deletions in 10% of PMF cases and in none of the PV or ET cases. The 20q11 deletion region spanned 1,662 KB and encompassed 37 genes, of which ASXL1 was included. To test whether ASXL1 contained lesions in the MPN cohort at large, we directly sequenced key regions of the ASXL1 gene in 65 PMF, 11 PV and 14 ET cases, as well as 7 controls from the SNP-array cohort. Genomic DNA from neutrophils and in select cases, purified CD34+ cells was used for both SNP-A and direct sequencing. Clinical parameters were correlated with genomic findings and the quantitative JAK2 V617F neutrophil allele burden Molecular genetics: 26/65 (40%) of PMF cases had abnormalities in ASXL1 (4 deletions, 22 mutations) whereas none of the 32 PV, ET or control cases had such lesions. The majority of ASXL1 sequence variations were nonsense lesions including the previously reported 1934dupG which comprised 30% of all of the mutations. The residual ASXL1 allele in all 20q11 deletion cases containing the ASXL1 gene was intact. In three PMF cases, more than one distinct ASXL1 mutation was identified, and cloning experiments on two of those cases indicated that the lesions were biallelic. Using banked samples, we observed the acquisition of an ASXL1 lesion over time, and established that ASXL1 lesions detected in 2 post ET-MF cases were also detected at low levels in the ET phase of the MPN. Genotype/Phenotype Correlations: ASXL1 deletions and mutations were prevalent in de novo PMF (37%), post PV-PMF (20%) post ET-PMF (62%) and in PMF/AML (33%). ASXL1 mutations did not associate with chemotherapy exposure as the prevalence of hydroxyurea use was similar in patients with and without mutations, and ASXL1 –mutation positive cases were present in patients who had never received any form of chemotherapy. There was no dependence upon JAK2 status as the presence of ASXL1 mutations were identified in JAK2 V617F-negative cases (9/26); JAK2 V617F-heterozygous cases (10/26); and JAK2 V617F-homozygous cases (7/26). Based on results of SNP-A, patients with ASXL1 mutations were equally as likely to have uniparental disomy (involving 9p or other regions) and loss/gain abnormalities (>1MB) compared to those without ASXL1 mutations. There were no differences in sex, age, or disease duration between PMF patients with and without ASXL1 mutations. In the ASXL1-mutant group, there was a trend toward a lower median white blood cell count (8 vs. 12.5 k/cu mm; p=0.3) and hemoglobin (9.7 vs. 11 g/dl; p=0.3) compared to ASXL1-wild-type patients. Furthermore, those PMF patients with ASXL1 mutations were significantly more likely to have received anemia-directed therapy (transfusion, erythropoietin, immunomodulating agents, steroids) compared to those without mutations (15/26 (58%) vs. 11/39 (23%); p=0.02). Post ET-MF patients comprised 31% (8/26) of ASXL1-mutant cases, compared to only 10% (4/39) ASXL1- wild-type cases (p=0.03). However, the presence of an ASXL1 mutation did not associate with an accelerated transition rate from ET to MF; among the 12 post ET-MF cases in the cohort, the median time of transition from ET to MF was 15.5 years in those with ASXL1 mutations compared to 7 years in those with ASXL1 wild-type status (p=0.02). Conclusion: Disruption of the ASXL1 gene occurs in 40% of PMF cases. The association of ASXL1 lesions, due to either mutation or deletion, suggests that ASXL1 haplo-insufficiency is associated with a PMF phenotype in the context of other known and unknown lesions, and that disruption of ASXL1 function may directly contribute to the pathophysiology and clinical complications of primary and secondary myelofibrosis. These data support the concepts that cooperative lesions in addition to JAK2 V617F are critical in generating PMF, that PMF is molecularly more complex than either PV or ET, and that the transition of PV or ET to PMF is associated with the acquisition of genomic lesions, such as ASXL1, that are present in PMF at large. Disclosures: No relevant conflicts of interest to declare.

Negative Effects of GM-CSF Signaling In t(8;21)-Induced Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3163-3163
Author(s):  
Shinobu Matsuura ◽  
Ming Yan ◽  
Eun-Young Ahn ◽  
Miao-Chia Lo ◽  
David Dangoor ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Sex Chromosome ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Receptor Activation ◽  
Pseudoautosomal Region ◽  
Wild Type ◽  
Gm Csf

Abstract Abstract 3163 The t(8;21)(q22;q22) translocation is one of the most common chromosomal translocations in de novo acute myeloid leukemia (AML). The 8;21 translocation is often associated with additional cytogenetic abnormalities. The loss of the sex chromosome (LOS) is by far the most frequent abnormality found in association with the t(8;21) leukemia, accounting for 32–59% of patients, in contrast to other types of AML in which the LOS occurs in less than 5% of patients. To evaluate the role of sex chromosome deletion in t(8;21)-related leukemogenesis, hematopoietic cells from a mouse line with only one sex chromosome were used in retrovirus-mediated t(8;21) (AML1-ETO) expression and transplantation assays. The absence of leukemia in those animals suggested that a gene present in the pseudoautosomal region of sex chromosomes in humans but not in mice may be the target gene in LOS. The granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene is one such gene and is also known to be involved in myeloid cell survival, proliferation and differentiation. The GM-CSFRα gene is specifically down-regulated in AML patients with t(8;21), but not in other common translocations (Valk PJM et al, NEJM, 2004). The GM-CSFR complex is composed of α and βc subunits that assemble into a complex for receptor activation and signaling. To investigate the role of GM-CSFR signaling in t(8;21)-mediated leukemogenesis, GM-CSFR common β subunit knockout (GM-CSFRβc-/-) mice were used in our studies as a model for deficient GM-CSFR signaling. Transduction of AML1-ETO in hematopoietic cells from GM-CSFRβc-/- resulted in myeloid leukemia of a median survival time of 225 days, high percentage of blasts in peripheral blood and bone marrow, anemia, thrombocytopenia, hepatomegaly and splenomegaly. Comparison of wild-type and GM-CSFRβc-/- cells in the same transplantation resulted in development of AML1-ETO-induced leukemia at higher penetrance in GM-CSFRβc-/- cells (28.5% vs 100%). Moreover, the latency of leukemia was shorter in GM-CSFRβc-/- cells than in wild-type cells after transduction of AML1-ETO9a. Analysis of the hematopoietic compartment of healthy GM-CSFRβc-/- mice detected no significant abnormalities in the immature hematopoietic compartment (LSK, CMP, GMP, MEP), suggesting that AML1-ETO expression is required for leukemia to occur. In vitro, expression of AML1-ETO alone is sufficient for the immortalization of normal hematopoietic cells, as demonstrated by serial replating capacity of cells in methylcellulose colony assay. Addition of mGM-CSF to the basic cytokine cocktail (mIL-3, hIL-6, mSCF, hEPO) did not significantly affect number, type, size, and cell composition of colony cells. In contrast, the addition of mGM-CSF eliminated the replating capacity of AML1-ETO expressing cells, although they survived longer than control vector-infected cells. The results suggest that activation of GM-CSF signaling can specifically abrogate the self-renewal ability of potential leukemic stem cells in the early immortalization phase. These results support a possible tumor suppressor role of GM-CSF in leukemogeneis by AML1-ETO and may provide clues to understand how AML1-ETO corrupts normal GM-CSF signals to its own advantage for leukemogenic transformation. Disclosures: No relevant conflicts of interest to declare.

Loss of the Wild-Type Allele Contributes to Myeloid Expansion and Disease Aggressiveness in FLT3/ITD Knock-in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 866-866
Author(s):  
Li Li ◽  
Emily Bailey ◽  
Sarah M Greenblatt ◽  
David Huso ◽  
Donald Small
Keyword(s):  
Median Survival ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Point Mutations ◽  
Kinase Domain ◽  
Wild Type Allele ◽  
Wild Type ◽  
Type Allele ◽  
Lower Ratio ◽  
The Impact

Abstract Abstract 866 Activating mutations of FLT3, either in the form of internal tandem duplication (ITD) mutations in the juxtamembrane domain or point mutations in the kinase domain, are one of the most frequent mutations in acute myeloid leukemia (AML). AML patients with FLT3/ITD mutations have poor prognosis. Loss of the wild-type FLT3 allele is associated with even worse prognosis when compared to those FLT3/ITD AML patients with the wild-type FLT3 allele still present. Also, FLT3/ITD patients with a high mutant-to-wild-type ratio have a significantly worse outcome than FLT3/ITD patients with a lower ratio. We have previously reported that heterozygous FLT3wt/ITD “knock-in” mice develop a slowly fatal MPN. In order to study the roles wild-type FLT3 play in the development of leukemia associated with FLT3/ITD mutations, we crossed FLT3wt/ITD mice with themselves or with FLT3 “knockout” (FLT3−/−) mice to obtain hemizygous (FLT3−/ITD) or homozygous (FLT3ITD/ITD) FLT3/ITD mice. Investigating phenotypic differences among them reveals the impact of wild-type FLT3 on the development of MPN resulting from FLT3/ITD mutations, and by extension, the effect on acute leukemia. FLT3−/ITD mice, with the loss of the wild-type allele, displayed a more severe MPN, as evidenced by even larger spleen, higher white blood counts and shorter survival, compared to FLT3wt/ITD mice. FLT3ITD/ITD mice had an even severe MPN compared to the FLT3−/ITD and FLT3wt/ITD mice. Fully transformed leukemia developed in some of the FLT3ITD/ITD (7%, 9/129), but not FLT3wt/ITD or FLT3−/ITD mice, with latency ranging from 139 to 304 days. Compared to FLT3wt/ITD mice, FLT3−/ITD and FLT3ITD/ITD mice displayed a further increase in the fraction of primitive hematopoietic cells, with notable increases in ST-HSCs and MPPs. Phosphorylation of STAT5, one of the key downstream targets for constitutively activated FLT3, was increased in FLT3wt/ITD, FLT3−/ITD and FLT3ITD/ITD mice compared to the wild-type control. FLT3wt/ITD, FLT3−/ITD and FLT3ITD/ITD BM also showed increased PU.1 expression and decreased GATA-1 expression, resulting in the subsequent expansion of granulocytic/monocytic/lymphocytic progenitors and a decrease in megakaryocytic/erythrocytic progenitors. It appears that the extent of myeloproliferation in FLT3/ITD mice correlates with loss of the wild-type allele (FLT3wt/ITD vs. FLT3−/ITD) and with the dose of mutant allele (FLT3−/ITD vs. FLT3ITD/ITD). In order to further explore the potential moderating effect of wild-type FLT3 expression on FLT3/ITD-associated MPN, we transduced wild-type FLT3 (wtFLT3, with the lentiviral vector co-expressing GFP) into lineage-depleted FLT3−/ITD CD45.2 BM cells and injected them into lethally irradiated CD45.1 recipients. When injected with sorted (GFP+) BM, vector alone-transduced GFP+FLT3−/ITD BM recipients died of MPN, with a median survival of 62 days. 100% of the recipients in the other three groups, i.e., those injected with vector alone-transduced GFP+ wild-type BM, wtFLT3-transduced GFP+ wild-type BM or wtFLT3-transduced GFP+ FLT3−/ITD BM, remained viable even after the point in time at which all of the recipients in the vector alone-transduced GFP+FLT3−/ITD group died. Similarly, recipients transplanted with unselected (including GFP+ and GFP− populations) vector alone-transduced FLT3−/ITD BM also died early, with a median survival of 73 days and overt signs of MPN. The percentages of GFP+ and GFP− cells in the BM of the dying recipients were comparable to those shortly after transplantation, indicative of the similar expansion ability of the GFP+ and GFP− populations in the BM. In contrast to the wtFLT3-transduced GFP+FLT3−/ITD BM recipients, which have a very prolonged survival, recipients injected with unselected wtFLT3-transduced FLT3−/ITD BM died of MPN, with a median survival of 91 days. Interestingly, 99% of the BM cells in the BM of the dying recipients were GFP−, demonstrating a proliferative/survival advantage for the FLT3−/ITD cells that had not been successfully transduced with wild-type FLT3. These results suggest that the presence of wild-type FLT3 delays and moderates the development of MPN caused by FLT3/ITD mutations. These results suggest that loss of the wild-type allele contributes to the development of a more severe phenotype. Thus, the wild-type FLT3 allele seemingly functions as a “tumor suppressor” in leukemia harboring FLT3/ITD mutations. Disclosures: No relevant conflicts of interest to declare.

Egr1 and Apc Haploinsufficiency Cooperate in the Pathogenesis of Myeloid Neoplasia: A Mouse Model for Therapy-Related Myeloid Neoplasms with a Del(5q)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 117-117 ◽  
Author(s):  
Angela Stoddart ◽  
Jianghong Wang ◽  
Anthony Fernald ◽  
John Anastasi ◽  
Michelle Lebeau
Keyword(s):  
Bone Marrow ◽  
Mouse Models ◽  
Median Survival ◽  
De Novo ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Macrocytic Anemia ◽  
Complete Inactivation ◽  
Myeloid Neoplasms ◽  
Single Allele

Abstract Abstract 117 Heterozygous deletions of the long arm of chromosome 5 are among the most common abnormalities in de novo (∼15% of patients) and therapy-related myeloid neoplasms (t-MN) (∼40% of patients). Two minimally deleted segments have been identified - the minimally deleted segment within 5q31.2 is associated with de novo AML and t-MNs, whereas the other spans 5q33.1 and is associated with MDS with an isolated del(5q). Current studies support a haploinsufficiency model, in which loss of a single allele of more than one gene on 5q contributes to the development of myeloid neoplasms. Using mouse models, we previously showed that haploinsufficiency of Egr1 (5q31.2) or Apc (5q22-frequently deleted in t-MN) independently recapitulates some features of human myelodysplastic syndromes (MDS). To test the hypothesis that reduced levels of EGR1 and APC cooperate in the pathogenesis of MDS/AML, we generated mice expressing a single allele of Egr1 and Apc: Mx1-Cre+Apcfl/+Egr1+/−(Apcdel/+Egr1+/−). At 2 mos of age, we induced deletion of a single allele of Apc by injection of 3 doses of pI-pC. Survival curves clearly show that Egr1 and Apc haploinsufficiency cooperate in the development of disease with a median survival of 129 days for Apcdel/+Egr1+/− mice and 296 days for Apcdel/+mice (P<0.0001). Although disease latency was significantly shorter for Apcdel/+Egr1+/− mice, their phenotype was similar to Apcdel/+ mice, with only two exceptions. For both cohorts, mice typically developed splenomegaly and a lethal macrocytic anemia with monocytosis. Anemic mice had an increased proportion of CD71+Ter119+ erythroblasts, indicating a block in erythroid development between the early and late basophilic erythroblast stage. Two mice displayed anemia and leukocytosis (WBC 51–72 k/mL) with an increased proportion of Mac1+ cells in the spleen and Kit+ cells in the bone marrow (1 mouse). As anticipated, mice with wild type levels of Apc (Mx1-Cre-Apcfl/+) or with loss of one allele of Egr1 showed no signs of anemia. Mutations in TP53 are commonly found in t-MNs with a del(5q) and loss of Tp53 in mouse models has been shown to promote AML by enabling aberrant self renewal. To test the hypothesis that loss of TP53 may adversely advance disease development, we crossed Tp53+/− to Egr1+/− and Apcdel/+ mice. Similar to Apcdel/+Egr1+/− mice, Apcdel/+Tp53+/− mice rapidly developed macrocytic anemia with a median survival of 144 days, suggesting that partial loss of TP53 function accelerates the Apcdel/+ -induced macrocytic anemia. Triple heterozygous mice (Apcdel/+Tp53+/−Egr1+/−) had a median survival of 178 days, but survival was not statistically different than Apcdel/+Egr1+/− mice (P=0.35) suggesting that Egr1 and Tp53 loss play redundant roles in the development of disease in Apcdel/+ mice. Thus, in the context of Apc haploinsufficiency, loss of Egr1 or Tp53 function promotes erythroid failure. These results are in contrast to the setting of ribosomal protein haploinsufficiency, as is the case in MDS with an isolated del(5q), where induction of TP53 is essential for erythroid failure. It has been proposed that inactivation of TP53 (through additional TP53 mutations) would be required for progression to AML, in the setting of a 5q deletion. To this end we transduced Egr1+/−Apcdel/+ bone marrow cells with a Tp53-specific shRNA, known to reduce Tp53 transcripts by ∼90%, and transplanted them into lethally irradiated C57BL/6 mice. Although penetrance of disease was low, 2 out of 13 mice (15%) developed an aggressive AML, as compared to 0 of 12 mice transplanted with Egr1+/−Apcdel/+ cells transduced with control shRNA. These data suggest that EGR1 and APC haploinsufficiency cooperate in the development of myeloid disorders, characterized by ineffective erythropoiesis, and that further mutations, such as that achieved by complete inactivation of TP53, are required for progression to AML. Disclosures: No relevant conflicts of interest to declare.

Adverse Prognostic Impact of GAS6 Expression in De Novo Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) (CALGB 8461, 9665, 20202; Alliance)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1293-1293
Author(s):  
Susan Whitman ◽  
Jessica Kohlschmidt ◽  
Kati Maharry ◽  
Deedra Nicolet ◽  
Sebastian Schwind ◽  
...  
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
White Blood Count ◽  
Categorical Variables ◽  
Prognostic Impact ◽  
Age Group ◽  
Wild Type ◽  
Aberrant Expression ◽  
Expression Status

Abstract Abstract 1293 Receptor tyrosine kinases (RTKs) constitutively activated by gene mutation, overexpression and/or autocrine activation via ligand expression have been shown to negatively impact on outcomes of AML patients (pts). AXL, a member of the TAM (TYRO3, AXL, MERTK) RTK gene family was reported to be overexpressed and associated with poor survival in AML (Rochlitz, et al, Leukemia, 1999: 13:1352–8). No AXL mutations have been described, suggesting its activation may occur via aberrant expression in leukemic blasts of a TAM RTK ligand, GAS6. GAS6 was shown to be overexpressed in AML (Dirks, et al Leuk. Res. 23:643–51); yet its prognostic relevance is unknown. We report clinical and molecular associations and prognostic impact of aberrant GAS6 expression, in the context of TAM RTKs and known prognostic markers in de novo CN-AML pts (n=270; aged 18–83 y) treated with cytarabine/anthracycline-based therapies. Sixty-nine (26%) pts expressed GAS6 (>background signal; derived from microarray gene expression profiles of AML samples). TYRO3 expression status [positive (+) vs negative (–)] was similar in GAS6+ and GAS6– pts (P=.74), while AXL+ (P<.001) and low expression MERTK (P=.02) were more frequent in GAS6+ pts. Compared to GAS6– pts, GAS6+ pts were older (P=.02), had more platelets (P=.03), lower % blood blasts (P=.01) and increased frequency of hepatomegaly (P=.006); were more often NPM1 (P<.001) and CEBPA (P=.02) wild-type, and RUNX1 (P<.001) and ASXL1 (P=.002) mutated and expressed higher MN1 levels (P=.05). In univariable analyses, none of the TAM RTKs associated with complete remission (CR) and only TYRO3+ associated with reduced disease-free (DFS; P=.005), overall (OS; P=.005) and event-free survival (EFS; P=.008). GAS6+ vs GAS6– pts had lower CR rates (P<.001), shorter DFS (P=.03), OS (P=.004) and EFS (P<.001). While no TAM RTK entered the CR multivariable (MVA) model, GAS6+ expression status remained an independent marker for lower CR rate after adjusting for NPM1 status, white blood count (WBC) and age group (Table). In the DFS, OS and EFS models (Table), there was an interaction between GAS6 and the combined dual receptor (TYRO3/AXL) variable. GAS6 independently associated with shorter survival in TYRO3–/AXL– pts but not TYRO3+/AXL+ pts after adjusting for other variables. We show for the 1st time that GAS6 expression is an independent prognostic marker in CN-AML; negatively impacting on CR attainment, independent of TAM RTKs and on survival endpoints in pts lacking TYRO3 and AXL expression, regardless of MERTK expression. Our results suggest that GAS6 expressed by AML blasts plays a role in chemotherapy resistance. As GAS6 is expressed but not its RTKs in a subgroup of pts with poor outcome, this may lead to the hypothesis that the prognostic impact of GAS6 in those patients is mediated by the encoded ligand acting on cells other than AML blasts including, for example, natural killer cells, where activation of AXL RTK is reported to suppress innate immunity. Table. MVA models Variable CR DFS OS EFS P OR (95% CI) P HR (95% CI) P HR (95% CI) P HR (95% CI) GAS6 expression, + v – .02 0.46 (0.24, 0.88) .03* 1.78 (1.07, 2.96) .05* 1.59 (1.00, 2.52) .03* 1.59 (1.06, 2.41) NPM1, mut v wt .001 2.97 (1.53, 5.77) – – – – .006 0.64 (0.46, 0.88) FLT3-ITD, present v absent – – .003 1.66 (1.19, 2.33) – – .005 1.55 (1.14, 2.11) WT1, mut v wt – – – – <.001 3.42 (1.97, 5.96) .03 1.84 (1.06, 3.18) RUNX1, mut v wt – – – – .002 2.00 (1.29, 3.10) – – ASXL1, mut v wt – – – – – – .003 1.66 (1.05, 2.60) DNMT3A
 R882 mut v wt
 Non-R882 v wt .006 .21 1.65 (1.15, 2.36) 1.33 (0.85, 2.08) WBC, continuous, 50 unit increase <.001 0.56 (0.41, 0.76) – – – – <.001 1.25 (1.12, 1.39) Age group, ≥ 60 y v < 60 y .01 0.40 (0.19, 0.83) <.001 2.09 (1.44, 3.03) <.001 2.64 (1.80, 3.87) <.001 2.16 (1.54, 3.02) OR, odds ratio; HR, hazard ratio; CI, confidence interval; mutated, mut; wild-type, wt. ORs > (<) 1.0 mean higher (lower) CR rate, and HRs > (<) 1.0 mean higher (lower) risk for relapse or death (DFS, EFS), respectively, for the higher values of the continuous variables and the first category listed for the categorical variables. Variables significant at α =.20 in univariable models were considered, although all considered variables are not shown. *There are interactions between GAS6 and TYRO3/AXL dual receptor status for DFS (P=.16), OS (P=.06) and EFS (P=.12). The P-values, HRs and CIs are for comparisons of GAS6+ v GAS6– pts within the TYRO3–/AXL– subset. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document