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SERIAL SEQUENCING REVEALS CLONAL ORIGINS AND STRATEGIES FOR EARLY DETECTION OF POST-ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION (HCT) RELAPSE IN ACUTE MYELOID LEUKEMIA (AML)
Author(s): ,
Taehyung Kim
Affiliations:
Department of Computer Science,University of Toronto,Toronto,Canada;Donnelly Centre,University of Toronto,Toronto,Canada
,
Jae-Sook Ahn
Affiliations:
Department of Hematology-Oncology,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of;Genomic Research Center for Hematopoietic Diseases,Chonnam National University,Hwasun,Korea, Republic Of
,
Marc Tyndel
Affiliations:
Donnelly Centre,University of Toronto,Toronto,Canada;The Edward S. Rogers Sr. Department of Electrical and Computer Engineering,University of Toronto,Toronto,Canada
,
Hyeoung-Joon Kim
Affiliations:
Department of Hematology-Oncology,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of;Genomic Research Center for Hematopoietic Diseases,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of
,
Yeo-Kyeoung Kim
Affiliations:
Department of Hematology-Oncology,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of;Genomic Research Center for Hematopoietic Diseases,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of
,
Seung-Shin Lee
Affiliations:
Department of Hematology-Oncology,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of;Genomic Research Center for Hematopoietic Diseases,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of
,
Seo-Yeon Ahn
Affiliations:
Department of Hematology-Oncology,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of;Genomic Research Center for Hematopoietic Diseases,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of
,
Sung-Hoon Jung
Affiliations:
Department of Hematology-Oncology,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of;Genomic Research Center for Hematopoietic Diseases,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of
,
Deok-Hwan Yang
Affiliations:
Department of Hematology-Oncology,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of
,
Je-Jung Lee
Affiliations:
Department of Hematology-Oncology,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of;Genomic Research Center for Hematopoietic Diseases,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of
,
Seung Hyun Choi
Affiliations:
Genomic Research Center for Hematopoietic Diseases,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of
,
Ja-Yeon Lee
Affiliations:
Genomic Research Center for Hematopoietic Diseases,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of
,
Myung-Geun Shin
Affiliations:
Department of Laboratory Medicine,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of
,
Hoon Kook
Affiliations:
Department of Pediatrics,Chonnam National University Hwasun Hospital,Hwasun,Korea, Republic Of
,
Sung-Kyu Park
Affiliations:
Department of Hematology-Oncology,Soon Chun Hyang University Hospital,Bucheon,Korea, Republic Of
,
Sun Hee Kim
Affiliations:
Department of Laboratory Medicine,Samsung Medical Center,Seoul,Korea, Republic Of;Department of Laboratory Medicine,Sungkyunkwan University School of Medicine,Seoul,Korea, Republic Of
,
Zhaolei Zhang
Affiliations:
Department of Computer Science,University of Toronto,Toronto,Canada;Donnelly Centre,University of Toronto,Toronto,Canada;Department of Molecular Genetics,University of Toronto,Toronto,Canada
Dennis (Dong Hwan) Kim
Affiliations:
Princess Margaret Cancer Centre/University Health Network,University of Toronto,Toronto,Canada
(Abstract release date: 05/18/17) EHA Library. Kim T. 06/24/17; 181778; S491
Mr. Taehyung Kim
Mr. Taehyung Kim
Contributions
Abstract

Abstract: S491

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 16:00 - 16:15

Location: Room N103

Background
Clinical applications of next generation sequencing (NGS) in allogeneic hematopoietic stem cell transplantation are a topic of interest. Mutation dynamics post-HCT using longitudinal NGS have not been thoroughly examined. We hypothesized that serial sequencing of pre-HCT and post-HCT in AML patients could provide a much deeper and broader understanding of clonal origin/hierarchy of relapse after allogeneic HCT. The present study aimed to evaluate mutation dynamics in AML using serial samples from pre- and post-HCT with respect to transplant outcomes, particularly overall survival (OS) and relapse.

Aims
To track origins of post-HCT relapse in AML using serial sequencing 

Methods
88 AML patients were enrolled and sequenced using an Illumina HiSeq 2000 sequencer (84 myeloid custom gene panel) on 419 bone marrow samples at diagnosis (n=88), pre-HCT (n=88), 21 days after HCT (n=88), and at relapse (n=20). Two patients relapsed by day 21. T-cell (n=80) and donor samples (n=57) were also sequenced. All computational and statistical analyses were performed using Python and R.

Results
The mean on-target coverage in 419 samples was 1773.7x. In total, we detected 217 mutations throughout the course of treatment in 79/88 patients (89.8%). NPM1 (26.1%), DNMT3A (26.1%), CEBPA (13.6%), IDH2 (13.6%), FLT3 (12.5%), and PTPN11 (11.4%) were commonly mutated at diagnosis. Unsurprisingly, most mutations appeared at initial diagnosis (200/217, 92.1%). Only 1, 2, and 14 mutations were acquired/selected at pre-HCT (0.5%), day 21 (0.9%), and relapse (6.5%), respectively. Most mutations were cleared at pre-HCT (mean mutation allele frequency (VAF) from 27.4% to 2.9%) and were further reduced after HCT (mean VAF from 2.9% to 0.7%) (Fig A).

Leveraging serial samples, we inferred the clonal relationships between original and relapse samples in 20 patients (Fig B). Mutations from initial diagnosis re-appeared in 17 patients. The relapse clone of 13 patients was identical to or clonally evolved from the initial AML clone (7 and 6 patients, respectively). Relapse clones of 4 patients evolved from an inferred ancestral clone, distinct from the initial AML clone. The remaining 3 patients’ relapse clones appear to be genetically distinct from the initial AML clone (Fig B). Among 61 mutations from relapsed patients, 37 were stable, whereas 9 were cleared and 15 acquired (or selected) at relapse. Overall, serial samples and donor samples de-convoluted origins of relapse clone from all 20 patients. Among the 13 patients whose donor samples were sequenced, no mutation that was transferred from donor to recipient expanded at relapse.
We then assessed whether the mutation status at pre- and post-HCT has any impact on OS and relapse after HCT. With a follow-up duration of 6.9 years, patients with VAF ≥ 0.2% at day 21 in any gene showed worse OS (HR 2.9, p = 0.006) as well as increased risk of relapse (HR 5.3, p = 0.0003) (Fig C-D). Multivariate analyses verified that VAF ≥ 0.2% at day 21 was a prognostic factor independent of age and cytogenetics risk group for OS (HR 3.2, p = 0.003). Non-relapse mortality did not show significant difference (p = 0.3). Thirteen patients carried 20 mutations at day 21 (≥ 0.2%), 16 of which originated from the initial AML clone. Noticeably, 9 of these mutations (from 9 patients) were in DNMT3A, where one of them was a transferred mutation from the donor through HCT (donor VAF: 8.18%, VAF at day 21: 3.42%, non-relapse case). Mutation status at any other time points in any gene, defined with a hard cut-off (VAF > 2%), was neither significantly associated with overall survival nor relapse incidence after HCT.

Conclusion
This study revealed origins of mutations detected at post-HCT relapse. It also revealed that the presence of mutations immediately after HCT, within 21 days, with low VAF (0.2%) can be used to predict relapse after HCT, illustrating the value of longitudinal NGS-based monitoring strategies for AML patients after allogeneic HCT. 

Session topic: 22. Stem cell transplantation - Clinical

Keyword(s): AML, Relapsed acute myeloid leukemia, mutation analysis, Hematopoietic cell transplantation

Abstract: S491

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 16:00 - 16:15

Location: Room N103

Background
Clinical applications of next generation sequencing (NGS) in allogeneic hematopoietic stem cell transplantation are a topic of interest. Mutation dynamics post-HCT using longitudinal NGS have not been thoroughly examined. We hypothesized that serial sequencing of pre-HCT and post-HCT in AML patients could provide a much deeper and broader understanding of clonal origin/hierarchy of relapse after allogeneic HCT. The present study aimed to evaluate mutation dynamics in AML using serial samples from pre- and post-HCT with respect to transplant outcomes, particularly overall survival (OS) and relapse.

Aims
To track origins of post-HCT relapse in AML using serial sequencing 

Methods
88 AML patients were enrolled and sequenced using an Illumina HiSeq 2000 sequencer (84 myeloid custom gene panel) on 419 bone marrow samples at diagnosis (n=88), pre-HCT (n=88), 21 days after HCT (n=88), and at relapse (n=20). Two patients relapsed by day 21. T-cell (n=80) and donor samples (n=57) were also sequenced. All computational and statistical analyses were performed using Python and R.

Results
The mean on-target coverage in 419 samples was 1773.7x. In total, we detected 217 mutations throughout the course of treatment in 79/88 patients (89.8%). NPM1 (26.1%), DNMT3A (26.1%), CEBPA (13.6%), IDH2 (13.6%), FLT3 (12.5%), and PTPN11 (11.4%) were commonly mutated at diagnosis. Unsurprisingly, most mutations appeared at initial diagnosis (200/217, 92.1%). Only 1, 2, and 14 mutations were acquired/selected at pre-HCT (0.5%), day 21 (0.9%), and relapse (6.5%), respectively. Most mutations were cleared at pre-HCT (mean mutation allele frequency (VAF) from 27.4% to 2.9%) and were further reduced after HCT (mean VAF from 2.9% to 0.7%) (Fig A).

Leveraging serial samples, we inferred the clonal relationships between original and relapse samples in 20 patients (Fig B). Mutations from initial diagnosis re-appeared in 17 patients. The relapse clone of 13 patients was identical to or clonally evolved from the initial AML clone (7 and 6 patients, respectively). Relapse clones of 4 patients evolved from an inferred ancestral clone, distinct from the initial AML clone. The remaining 3 patients’ relapse clones appear to be genetically distinct from the initial AML clone (Fig B). Among 61 mutations from relapsed patients, 37 were stable, whereas 9 were cleared and 15 acquired (or selected) at relapse. Overall, serial samples and donor samples de-convoluted origins of relapse clone from all 20 patients. Among the 13 patients whose donor samples were sequenced, no mutation that was transferred from donor to recipient expanded at relapse.
We then assessed whether the mutation status at pre- and post-HCT has any impact on OS and relapse after HCT. With a follow-up duration of 6.9 years, patients with VAF ≥ 0.2% at day 21 in any gene showed worse OS (HR 2.9, p = 0.006) as well as increased risk of relapse (HR 5.3, p = 0.0003) (Fig C-D). Multivariate analyses verified that VAF ≥ 0.2% at day 21 was a prognostic factor independent of age and cytogenetics risk group for OS (HR 3.2, p = 0.003). Non-relapse mortality did not show significant difference (p = 0.3). Thirteen patients carried 20 mutations at day 21 (≥ 0.2%), 16 of which originated from the initial AML clone. Noticeably, 9 of these mutations (from 9 patients) were in DNMT3A, where one of them was a transferred mutation from the donor through HCT (donor VAF: 8.18%, VAF at day 21: 3.42%, non-relapse case). Mutation status at any other time points in any gene, defined with a hard cut-off (VAF > 2%), was neither significantly associated with overall survival nor relapse incidence after HCT.

Conclusion
This study revealed origins of mutations detected at post-HCT relapse. It also revealed that the presence of mutations immediately after HCT, within 21 days, with low VAF (0.2%) can be used to predict relapse after HCT, illustrating the value of longitudinal NGS-based monitoring strategies for AML patients after allogeneic HCT. 

Session topic: 22. Stem cell transplantation - Clinical

Keyword(s): AML, Relapsed acute myeloid leukemia, mutation analysis, Hematopoietic cell transplantation

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