CLEARANCE OF PHENOTYPICALLY DISTINCT FLT3-ITD AND FLT3-TKD CLONES BY TREATMENT WITH CRENOLANIB AND CHEMOTHERAPY AS DETECTED BY LONGITUDINAL SINGLE-CELL DNA SEQUENCING ANALYSIS
Author(s): ,
Richard Stone
Affiliations:
Dana-Farber Cancer Institute,Boston,United States
,
Martha Wadleigh
Affiliations:
Dana-Farber Cancer Institute,Boston,United States
,
Ilene Galinksy
Affiliations:
Dana-Farber Cancer Institute,Boston,United States
Boo Messahel
Affiliations:
Arog Pharmaceuticals,Dallas,United States
EHA Library. Messahel B. 06/12/20; 294502; EP584
Dr. Boo Messahel
Dr. Boo Messahel
Contributions
Abstract

Abstract: EP584

Type: e-Poster

Presentation during EHA25: All e-Poster presentations will be made available on the on-demand Virtual Congress platform as of Friday, June 12 at 08:30 CEST and will be accessible until October 15, 2020.

Background
Single cell RNA (scRNA) sequencing analysis suggests that the presence of FLT3-ITD mutation is associated with a progenitor-like phenotype (1van Galen, et al. Cell, 2019: 176, 1265) while activating mutations in the FLT3 kinase domain are associated with monocytic differentiation. One of the patients analyzed by scRNA was a 54-year-old patient with cytogenetically normal AML. NGS showed multiple mutations: DNMT3A (41.9%), NPM1 (37.9%), CEBPA (42.9%), FLT3-ITD (13.5%) and two FLT3-TKD mutations (N841K 16.2% and A680V 29%).

Crenolanib is a FLT3 inhibitor with in vitro activity against FLT3-ITD and various FLT3-TKD mutations being evaluated for clinical activity. This patient was treated with cytarabine/daunorubicin/crenolanib induction, four cycles of HiDAC/crenolanib consolidation and one year of crenolanib maintenance as part a Phase II clinical trial (NCT02283177). We hypothesized that single-cell DNA (scDNA) sequencing technology can review the clearance of distinct clones during treatment by analyzing longitudinal samples.

Aims

To evaluate the clearance of distinct AML clones during treatment with crenolanib combination therapy.

Methods
Mononuclear cells were purified from bone marrow aspirates collected at diagnosis, at achievement of CR after induction (day 35), after four cycles of HIDAC/crenolanib consolidation, and during month two and six of maintenance crenolanib. The Tapestri (Mission Bio) platform was used to prepare single cell genetic libraries for 19 commonly mutated AML genes. scDNA libraries were sequenced using MiSeq, data analysis was performed via Tapestri Insight software.

Results

scDNA analysis of 2,920 cells, revealed 4 distinct FLT3 subclones at diagnosis, including FLT3-ITD, A680V, N841K, and D839G. All of the mutations in FLT3 were found with concurrent NPM1 and DNMT3A-R882C mutations. FLT3-ITD was primarily found in leukemic populations that also had an activating FLT3-A680V mutation; however, the FLT3 mutations N841K and D839G were found to be in clones separate from the A680V and FLT3-ITD clones. We also detected wild type FLT3 clones that contained either NRAS (1%) or KRAS (1%) mutations, with concurrent NPM1 and DNMT3A mutations.

After one cycle of induction chemotherapy plus crenolanib the patient achieved a morphological CR with full count recovery at day 26. scDNA sequencing of 387 cells revealed clearance of FLT3-A680V and N841K, but persistence of 3% FLT3-ITD with NPM1 and DNMT3A. A FLT3- D835E mutation (1%) was detected.

The patient subsequently completed four cycles of HiDAC consolidation with crenolanib. Of the previously detected FLT3 mutations, only FLT3-A680V (1% of 3457 cells) was detected. After two months of crenolanib maintenance, no FLT3 mutations were detected out of 4583 cells, and the patient has remained in remission 23 months after completing 12 months of crenolanib maintenance. The patient has a persistent DNMT3A-R882C clonal abnormality, but the significance of this is unknown.

Conclusion

This study presents kinetics of clearance of mutations by crenolanib plus standard chemotherapy. Using scDNA sequencing we show that the patient has no FLT3 mutations remaining. Despite known adverse risk factors, this patient remains free of disease 35 months after the initial diagnosis. The use of the Tapestri Platform allowed detection of rare populations of distinct clones that have not been identified by other sequencing techniques. This case highlights the clonal dynamics of FLT3 AML and provides insights on strategies to optimize treatment.

Session topic: 04. Acute myeloid leukemia - Clinical

Keyword(s): Acute myeloid leukemia, Mutation analysis

Abstract: EP584

Type: e-Poster

Presentation during EHA25: All e-Poster presentations will be made available on the on-demand Virtual Congress platform as of Friday, June 12 at 08:30 CEST and will be accessible until October 15, 2020.

Background
Single cell RNA (scRNA) sequencing analysis suggests that the presence of FLT3-ITD mutation is associated with a progenitor-like phenotype (1van Galen, et al. Cell, 2019: 176, 1265) while activating mutations in the FLT3 kinase domain are associated with monocytic differentiation. One of the patients analyzed by scRNA was a 54-year-old patient with cytogenetically normal AML. NGS showed multiple mutations: DNMT3A (41.9%), NPM1 (37.9%), CEBPA (42.9%), FLT3-ITD (13.5%) and two FLT3-TKD mutations (N841K 16.2% and A680V 29%).

Crenolanib is a FLT3 inhibitor with in vitro activity against FLT3-ITD and various FLT3-TKD mutations being evaluated for clinical activity. This patient was treated with cytarabine/daunorubicin/crenolanib induction, four cycles of HiDAC/crenolanib consolidation and one year of crenolanib maintenance as part a Phase II clinical trial (NCT02283177). We hypothesized that single-cell DNA (scDNA) sequencing technology can review the clearance of distinct clones during treatment by analyzing longitudinal samples.

Aims

To evaluate the clearance of distinct AML clones during treatment with crenolanib combination therapy.

Methods
Mononuclear cells were purified from bone marrow aspirates collected at diagnosis, at achievement of CR after induction (day 35), after four cycles of HIDAC/crenolanib consolidation, and during month two and six of maintenance crenolanib. The Tapestri (Mission Bio) platform was used to prepare single cell genetic libraries for 19 commonly mutated AML genes. scDNA libraries were sequenced using MiSeq, data analysis was performed via Tapestri Insight software.

Results

scDNA analysis of 2,920 cells, revealed 4 distinct FLT3 subclones at diagnosis, including FLT3-ITD, A680V, N841K, and D839G. All of the mutations in FLT3 were found with concurrent NPM1 and DNMT3A-R882C mutations. FLT3-ITD was primarily found in leukemic populations that also had an activating FLT3-A680V mutation; however, the FLT3 mutations N841K and D839G were found to be in clones separate from the A680V and FLT3-ITD clones. We also detected wild type FLT3 clones that contained either NRAS (1%) or KRAS (1%) mutations, with concurrent NPM1 and DNMT3A mutations.

After one cycle of induction chemotherapy plus crenolanib the patient achieved a morphological CR with full count recovery at day 26. scDNA sequencing of 387 cells revealed clearance of FLT3-A680V and N841K, but persistence of 3% FLT3-ITD with NPM1 and DNMT3A. A FLT3- D835E mutation (1%) was detected.

The patient subsequently completed four cycles of HiDAC consolidation with crenolanib. Of the previously detected FLT3 mutations, only FLT3-A680V (1% of 3457 cells) was detected. After two months of crenolanib maintenance, no FLT3 mutations were detected out of 4583 cells, and the patient has remained in remission 23 months after completing 12 months of crenolanib maintenance. The patient has a persistent DNMT3A-R882C clonal abnormality, but the significance of this is unknown.

Conclusion

This study presents kinetics of clearance of mutations by crenolanib plus standard chemotherapy. Using scDNA sequencing we show that the patient has no FLT3 mutations remaining. Despite known adverse risk factors, this patient remains free of disease 35 months after the initial diagnosis. The use of the Tapestri Platform allowed detection of rare populations of distinct clones that have not been identified by other sequencing techniques. This case highlights the clonal dynamics of FLT3 AML and provides insights on strategies to optimize treatment.

Session topic: 04. Acute myeloid leukemia - Clinical

Keyword(s): Acute myeloid leukemia, Mutation analysis

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