- Email: [email protected]
BTK Inhibitor, Exerts Superior Potency against AML Cells Harboring ITD, TKD and Gatekeeper Mutated FLT3 or Wild-Type FLT3
Abstracts Houston, United States; 3Department of Hematopathology, The
involved in T-cell receptor signaling were differentially methylated and consequently differentially expressed between myeloid-T and myeloid-B. Unsupervised hierarchical clustering by promoter CpG methylation pattern of all MPAL, AML, B-ALL and T-ALL revealed that myeloid-T MPAL consistently showed similar methylation pattern with T-ALL, while myeloid-B showed random similarity with either B-ALL or AML. Conclusions: MPAL is genetically heterogeneous and myeloid-T and myeloid-B show distinct patterns of mutation landscapes, methylation and gene expressions. Myeloid-T MPAL resembles T-ALL based on mutation and methylation pattern, suggesting that myeloid-T MPAL patients may benefit from T-ALL type therapy. Treatment for myeloid-B MPAL may need to be tailored to either AML- or BALL-like regimen based on methylation profile.
University of Texas MD Anderson Cancer Center, Houston, United States; 4Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, United States; 5Department of Hematology and Oncology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
Background: Mixed phenotype acute leukemia (MPAL) is a rare subgroup of acute leukemia characterized by blasts that show immunophenotypes of both myeloid and lymphoid lineages. It poses significant diagnostic and therapeutic challenges in clinic, yet genetic basis of MPAL is not well understood. Methods: Bone marrow samples from 31 patients with MPAL were studied by targeted capture sequencing of 295 genes (median depth 393x), RNA sequencing and methylation array. Mutational landscape was compared to that of 194 AML, 71 B-ALL, and 14 T-ALL cases. Promoter CpG methylation pattern was compared to that of 194 AML, 505 B-ALL and 101 T-ALL cases. Seven cases had paired bone marrow samples at pre-treatment and remission assessed for mutation clearance. Results: Eighteen (58%) had myeloid-T and 13 (42%) had myeloid-B phenotype. Four had Philadelphia chromosome and 1 had 11q23 rearrangement. Table 1 shows mutational landscape of MPAL and its comparison to AML, B-ALL and TALL. MPAL shared both AML- and ALL-type mutations. However, myeloid-T and myeloid-B showed distinct patterns of mutations. Mutations in JAK-STAT and NOTCH pathways were specific to myeloid-T and T-ALL, whereas RUNX1, FLT3 and NRAS mutations were enriched in myeloid-B and AML. In longitudinal analysis, 5 cases showed mutation clearance at remission, while two cases showed persistence of mutations in RUNX1 and DNMT3A, respectively. Analysis of promoter CpG methylation revealed that myeloid-T and myeloid-B have distinct patterns of methylation. Overall, myeloid-T were more hypermethylated than myeloid-B. Promoter regions for genes
AML-045 CG0 806, a First-in-Class FLT3/BTK Inhibitor, Exerts Superior Potency against AML Cells Harboring ITD, TKD and Gatekeeper Mutated FLT3 or Wild-Type FLT3 Weiguo Zhang ,1 Hongying Zhang,2 Andrea Local,2 Wiliam Rice,2 Charlie Ly,1 Guopan Yu,1 Stephen Howell,3 Michael Andreeff4 1
Section of Molecular Hematology and Therapy, Department of
Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States; 2Aptose Biosciences, San Diego, United States; 3University of California, San Diego, United States; 4Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States
FLT3 is widely accepted as a prime target for acute myeloid leukemia (AML) therapy. However, targeted therapy using current
Table 1 Mutation Comparison between MPAL, AML, B-ALL and T-ALL AML (N [ 194), %
B-ALL (N [ 71), %
Myeloid/B (N [ 13), %
Myeloid/T (N [ 18), %
T-ALL (N [ 14), %
Median number of mutation
3
0
2
2
2
Pathway Chromatin
Gene ASXL1
9
0
23
0
0
STAG2 DNMT3A
7 22
0 7
0 7
5 33
0 14
IDH1 IDH2
4 16
1 0
15 7
0 33
0 7
TET2 IL7R
18 0
0 0
15 0
0 16
0 14
FBXW7 NOTCH1
0 1
0 0
0 0
5 38
14 42
Nucleophosmin RTK-RAS
NPM1 FLT3
28 15
0 0
0 23
0 11
0 0
Transcription Factor
NRAS RUNX1
13 10
5 2
23 46
16 5
7 0
WT1 CEBPA
6 11
0 0
7 0
0 5
14 0
Cohesin DNA methylation
JAK-STAT NOTCH
S274
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Clinical Lymphoma, Myeloma & Leukemia September 2017
Abstracts available FLT3 inhibitors only achieved limited clinical efficacy in mono-therapy. Acquired mutations of FLT3, including D835 or “gatekeeper” F691 mutations, have been identified in clinical patients who showed resistance/relapse to the inhibitors. CG’806 is a small molecule multi-kinase inhibitor against FLT3 and BTK kinases that is under development to treat FLT3-driven AML. CG’806 exerted potent picomolar IC50 anti-proliferative activity against human AML cells and against Ba/F3 mouse AML cells with FLT3-ITD mutations (about 50 to 250-fold higher activity compared to quizartinib or gilteritinib). Specifically, compared to second-generation FLT3 inhibitors quizartinib or gilteritinib, CG’806 showed much more pronounced anti-proliferative effects in leukemia cells with D835 mutations, the ITD plus F691L/Y842D/ D835 mutations, or in FLT3 wild-type cells (IC50s were 0.17, 0.82, 9.49, 0.30, 8.26, 9.72, and 0.43 nM for human ITD-mutated AML cells MV4-11, MOLM13, murine ITD mutated leukemia cells Ba/F3 WT, Ba/F3-ITD, Ba/F3-D835Y, Ba/FLT3ITD+D835Y, and “gatekeeper” mutation Ba/F3-ITD+F691L cells, respectively). Furthermore, CG’806 triggered profound apoptosis in cell lines and primary AML patient samples harboring FLT3-ITD mutations as well as WT. Mechanistically, CG’806 profoundly suppressed FLT3 and its downstream MAPK/AKT signaling, as well as phospho-Aurora, and/or phospho-BTK proteins, suggesting the ability of CG’806 to inhibit various kinases that function in AML signaling and appear to contribute to its effectiveness. Moreover, CG’806 demonstrated in vivo tumor eradication without toxicity when administered orally, once daily for 14 d as a single agent in the MV4:11 AML murine xenograft model, and demonstrated sustained micromolar plasma drug levels in mice after a single oral administration. CG’806 can be considered not only a pan-FLT3 inhibitor for targeting FLT3 wild type and ITD-mutant AML, but also a one-of-a-kind agent killing leukemia cells with TKD or dual TKD plus ITD mutations, which are frequently associated with resistance/relapse in FLT3-mutant AML patients. CG’806 exerts a robust therapeutic window in animal xenograft studies. Thus, CG’806 warrants further investigation for the treatment of newly diagnosed and relapsed/refractory patients with FLT3-mutated AML.
prior history of AHD. Pts were then classified into 4 groups based on somatic mutations as suggested by Lindsley et al (Blood, 26 Feb 2015 x Vol125): group 1 (pAML) with CBF rearrangements, 11q23/MLL gene rearrangements and NPM1 mutation (MT); group 2 (sAML) with SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 MT; group 3 with TP53 MT; and group 4 as not otherwise specified (NOS). Results: Based on clinical criteria, 95 pts were classified as pAML with a median OS of 11.2 mo and 82 pts were classified as sAML with a median OS 5.1 mo (p¼.21). Utilizing AML cytogenetic risk stratification only 4 pts met the criteria for good risk, 109 pts intermediate risk, and 57 pts poor risk. The median OS was 22.4,15.4, and 4 mo respectively (p<0.001). Based on the molecular signature proposed by Lindsley et al, 8 pts were classified as pAML, 72 pts sAML, 28 pts had TP53 MT, and 70 pts were classified as NOS. The median OS was 22.4,14, 2.8, and 11.2 mo respectively for the 4 proposed groups based on molecular signature (p <0.001). Clinical versus molecular classification was discordant where 25% of pts (n¼2) classified as pAML by molecular signature had a history of AHD while 44% of pts (n¼32) classified molecularly as sAML had no prior AHD. In TP53 MT and NOS categories, 37% (n¼28) and 43% (n¼70) of pts had AHD, respectively. Conclusions: Molecular annotation of elderly AML patients by NGS reclassifies a significant proportion of patients as sAML. Pts molecularly categorized as sAML have worse outcomes than pts categorized as pAML, regardless of AHD history. Pts with TP53 MT are a distinct group who have particularly poor outcomes.
AML-053 Survival Time is Problematic as an Indicator of an anti- AML Drug’s Effectiveness Carole Shaw ,1 Shelly Hager,1 Mary-Elizabeth Percival,2 Roland Walter,1 Pamela Becker,2 Kathleen Shannon-Dorcy,2 Elihu Estey,2 Kelda Gardner2 1
Fred Hutchinson Cancer Research Center, Seattle, United States;
AML-052 Defining Acute Myeloid Leukemia Ontogeny in Older Patients Megan Melody , David Sallman, Najla AlAli, Hanadi Ramadan, Ling Zhang, Eric Padron, Kendra Sweet, Martine Extermann, Alan List, Jeffery Lancet, Rami Komrokji H. Lee Moffitt Cancer Center and Research Institute, Tampa, United States
Objective: The primary objective of this study was to validate the use of somatic mutations to determine AML ontogeny in the elderly population. Methods: Utilizing the AML Database at Moffitt Cancer Center, we identified 178 elderly pts (>70yo) with AML with NexGen Sequencing (NGS) of up to 54 genes obtained at the time of diagnosis. Pts were divided into pAML or sAML based on
2
University of Washington, Seattle, United States
“Complete response” (CR) in AML was originally recognized as a distinct response when because people who met the criteria for CR (ANC > 1,000, platelet count > 100,000, marrow with <5% blasts) lived longer than those who did not, with the difference due to time spent in CR (Freireich et al. J Chron Dis 1961;14:593608). Essentially patients would soon die absent these blood count levels. However, in the intervening years there have been substantial improvements in supportive care, particularly anti-fungal therapy. These improvements prompted us to examine whether patients who entered CR and relapsed still spent the great majority of their life after achieving CR in CR. We compared time from CR to relapse with time to relapse to death in 159 patients who attained CR; 105 of these had no accompanying measurable residual disease (MRD). Because relapse and death had occurred in all patients there were no censored data. Data were as follows:
Clinical Lymphoma, Myeloma & Leukemia September 2017
- S275
involved in T-cell receptor signaling were differentially methylated and consequently differentially expressed between myeloid-T and myeloid-B. Unsupervised hierarchical clustering by promoter CpG methylation pattern of all MPAL, AML, B-ALL and T-ALL revealed that myeloid-T MPAL consistently showed similar methylation pattern with T-ALL, while myeloid-B showed random similarity with either B-ALL or AML. Conclusions: MPAL is genetically heterogeneous and myeloid-T and myeloid-B show distinct patterns of mutation landscapes, methylation and gene expressions. Myeloid-T MPAL resembles T-ALL based on mutation and methylation pattern, suggesting that myeloid-T MPAL patients may benefit from T-ALL type therapy. Treatment for myeloid-B MPAL may need to be tailored to either AML- or BALL-like regimen based on methylation profile.
University of Texas MD Anderson Cancer Center, Houston, United States; 4Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, United States; 5Department of Hematology and Oncology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
Background: Mixed phenotype acute leukemia (MPAL) is a rare subgroup of acute leukemia characterized by blasts that show immunophenotypes of both myeloid and lymphoid lineages. It poses significant diagnostic and therapeutic challenges in clinic, yet genetic basis of MPAL is not well understood. Methods: Bone marrow samples from 31 patients with MPAL were studied by targeted capture sequencing of 295 genes (median depth 393x), RNA sequencing and methylation array. Mutational landscape was compared to that of 194 AML, 71 B-ALL, and 14 T-ALL cases. Promoter CpG methylation pattern was compared to that of 194 AML, 505 B-ALL and 101 T-ALL cases. Seven cases had paired bone marrow samples at pre-treatment and remission assessed for mutation clearance. Results: Eighteen (58%) had myeloid-T and 13 (42%) had myeloid-B phenotype. Four had Philadelphia chromosome and 1 had 11q23 rearrangement. Table 1 shows mutational landscape of MPAL and its comparison to AML, B-ALL and TALL. MPAL shared both AML- and ALL-type mutations. However, myeloid-T and myeloid-B showed distinct patterns of mutations. Mutations in JAK-STAT and NOTCH pathways were specific to myeloid-T and T-ALL, whereas RUNX1, FLT3 and NRAS mutations were enriched in myeloid-B and AML. In longitudinal analysis, 5 cases showed mutation clearance at remission, while two cases showed persistence of mutations in RUNX1 and DNMT3A, respectively. Analysis of promoter CpG methylation revealed that myeloid-T and myeloid-B have distinct patterns of methylation. Overall, myeloid-T were more hypermethylated than myeloid-B. Promoter regions for genes
AML-045 CG0 806, a First-in-Class FLT3/BTK Inhibitor, Exerts Superior Potency against AML Cells Harboring ITD, TKD and Gatekeeper Mutated FLT3 or Wild-Type FLT3 Weiguo Zhang ,1 Hongying Zhang,2 Andrea Local,2 Wiliam Rice,2 Charlie Ly,1 Guopan Yu,1 Stephen Howell,3 Michael Andreeff4 1
Section of Molecular Hematology and Therapy, Department of
Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States; 2Aptose Biosciences, San Diego, United States; 3University of California, San Diego, United States; 4Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States
FLT3 is widely accepted as a prime target for acute myeloid leukemia (AML) therapy. However, targeted therapy using current
Table 1 Mutation Comparison between MPAL, AML, B-ALL and T-ALL AML (N [ 194), %
B-ALL (N [ 71), %
Myeloid/B (N [ 13), %
Myeloid/T (N [ 18), %
T-ALL (N [ 14), %
Median number of mutation
3
0
2
2
2
Pathway Chromatin
Gene ASXL1
9
0
23
0
0
STAG2 DNMT3A
7 22
0 7
0 7
5 33
0 14
IDH1 IDH2
4 16
1 0
15 7
0 33
0 7
TET2 IL7R
18 0
0 0
15 0
0 16
0 14
FBXW7 NOTCH1
0 1
0 0
0 0
5 38
14 42
Nucleophosmin RTK-RAS
NPM1 FLT3
28 15
0 0
0 23
0 11
0 0
Transcription Factor
NRAS RUNX1
13 10
5 2
23 46
16 5
7 0
WT1 CEBPA
6 11
0 0
7 0
0 5
14 0
Cohesin DNA methylation
JAK-STAT NOTCH
S274
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Clinical Lymphoma, Myeloma & Leukemia September 2017
Abstracts available FLT3 inhibitors only achieved limited clinical efficacy in mono-therapy. Acquired mutations of FLT3, including D835 or “gatekeeper” F691 mutations, have been identified in clinical patients who showed resistance/relapse to the inhibitors. CG’806 is a small molecule multi-kinase inhibitor against FLT3 and BTK kinases that is under development to treat FLT3-driven AML. CG’806 exerted potent picomolar IC50 anti-proliferative activity against human AML cells and against Ba/F3 mouse AML cells with FLT3-ITD mutations (about 50 to 250-fold higher activity compared to quizartinib or gilteritinib). Specifically, compared to second-generation FLT3 inhibitors quizartinib or gilteritinib, CG’806 showed much more pronounced anti-proliferative effects in leukemia cells with D835 mutations, the ITD plus F691L/Y842D/ D835 mutations, or in FLT3 wild-type cells (IC50s were 0.17, 0.82, 9.49, 0.30, 8.26, 9.72, and 0.43 nM for human ITD-mutated AML cells MV4-11, MOLM13, murine ITD mutated leukemia cells Ba/F3 WT, Ba/F3-ITD, Ba/F3-D835Y, Ba/FLT3ITD+D835Y, and “gatekeeper” mutation Ba/F3-ITD+F691L cells, respectively). Furthermore, CG’806 triggered profound apoptosis in cell lines and primary AML patient samples harboring FLT3-ITD mutations as well as WT. Mechanistically, CG’806 profoundly suppressed FLT3 and its downstream MAPK/AKT signaling, as well as phospho-Aurora, and/or phospho-BTK proteins, suggesting the ability of CG’806 to inhibit various kinases that function in AML signaling and appear to contribute to its effectiveness. Moreover, CG’806 demonstrated in vivo tumor eradication without toxicity when administered orally, once daily for 14 d as a single agent in the MV4:11 AML murine xenograft model, and demonstrated sustained micromolar plasma drug levels in mice after a single oral administration. CG’806 can be considered not only a pan-FLT3 inhibitor for targeting FLT3 wild type and ITD-mutant AML, but also a one-of-a-kind agent killing leukemia cells with TKD or dual TKD plus ITD mutations, which are frequently associated with resistance/relapse in FLT3-mutant AML patients. CG’806 exerts a robust therapeutic window in animal xenograft studies. Thus, CG’806 warrants further investigation for the treatment of newly diagnosed and relapsed/refractory patients with FLT3-mutated AML.
prior history of AHD. Pts were then classified into 4 groups based on somatic mutations as suggested by Lindsley et al (Blood, 26 Feb 2015 x Vol125): group 1 (pAML) with CBF rearrangements, 11q23/MLL gene rearrangements and NPM1 mutation (MT); group 2 (sAML) with SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 MT; group 3 with TP53 MT; and group 4 as not otherwise specified (NOS). Results: Based on clinical criteria, 95 pts were classified as pAML with a median OS of 11.2 mo and 82 pts were classified as sAML with a median OS 5.1 mo (p¼.21). Utilizing AML cytogenetic risk stratification only 4 pts met the criteria for good risk, 109 pts intermediate risk, and 57 pts poor risk. The median OS was 22.4,15.4, and 4 mo respectively (p<0.001). Based on the molecular signature proposed by Lindsley et al, 8 pts were classified as pAML, 72 pts sAML, 28 pts had TP53 MT, and 70 pts were classified as NOS. The median OS was 22.4,14, 2.8, and 11.2 mo respectively for the 4 proposed groups based on molecular signature (p <0.001). Clinical versus molecular classification was discordant where 25% of pts (n¼2) classified as pAML by molecular signature had a history of AHD while 44% of pts (n¼32) classified molecularly as sAML had no prior AHD. In TP53 MT and NOS categories, 37% (n¼28) and 43% (n¼70) of pts had AHD, respectively. Conclusions: Molecular annotation of elderly AML patients by NGS reclassifies a significant proportion of patients as sAML. Pts molecularly categorized as sAML have worse outcomes than pts categorized as pAML, regardless of AHD history. Pts with TP53 MT are a distinct group who have particularly poor outcomes.
AML-053 Survival Time is Problematic as an Indicator of an anti- AML Drug’s Effectiveness Carole Shaw ,1 Shelly Hager,1 Mary-Elizabeth Percival,2 Roland Walter,1 Pamela Becker,2 Kathleen Shannon-Dorcy,2 Elihu Estey,2 Kelda Gardner2 1
Fred Hutchinson Cancer Research Center, Seattle, United States;
AML-052 Defining Acute Myeloid Leukemia Ontogeny in Older Patients Megan Melody , David Sallman, Najla AlAli, Hanadi Ramadan, Ling Zhang, Eric Padron, Kendra Sweet, Martine Extermann, Alan List, Jeffery Lancet, Rami Komrokji H. Lee Moffitt Cancer Center and Research Institute, Tampa, United States
Objective: The primary objective of this study was to validate the use of somatic mutations to determine AML ontogeny in the elderly population. Methods: Utilizing the AML Database at Moffitt Cancer Center, we identified 178 elderly pts (>70yo) with AML with NexGen Sequencing (NGS) of up to 54 genes obtained at the time of diagnosis. Pts were divided into pAML or sAML based on
2
University of Washington, Seattle, United States
“Complete response” (CR) in AML was originally recognized as a distinct response when because people who met the criteria for CR (ANC > 1,000, platelet count > 100,000, marrow with <5% blasts) lived longer than those who did not, with the difference due to time spent in CR (Freireich et al. J Chron Dis 1961;14:593608). Essentially patients would soon die absent these blood count levels. However, in the intervening years there have been substantial improvements in supportive care, particularly anti-fungal therapy. These improvements prompted us to examine whether patients who entered CR and relapsed still spent the great majority of their life after achieving CR in CR. We compared time from CR to relapse with time to relapse to death in 159 patients who attained CR; 105 of these had no accompanying measurable residual disease (MRD). Because relapse and death had occurred in all patients there were no censored data. Data were as follows:
Clinical Lymphoma, Myeloma & Leukemia September 2017
- S275