The fundamental interest of the Laboratory of Myeloid Malignancies is the detection, prevention and treatment of acute myeloid leukemia (AML) relapse. Our work has focused on clinical trials of novel biomarker and immunotherapy approaches, and the development of molecular and genomic laboratory methods (Measurable Residual Disease, MRD) to predict development or recurrence of myeloid malignancy. Foundational to our objective has been the development of high sensitivity biomarkers for residual AML in those patients who have been treated to apparent remission but remain at risk of clinical relapse. Previously we have demonstrated the ability to risk stratify AML patients into groups with either high and low leukemic relapse rates, based on pre-transplant a peripheral blood sample, prior to either allogeneic (PMID: 25665046, 27185839) or autologous hematopoietic stem cell transplantation (PMID: 27544285). This year we extended our prior work in this area by investigating the impact of conditioning intensity of allogeneic transplantation (alloHCT) for acute myeloid leukemia for patients in clinical complete remission but with genomic evidence of residual disease. It is currently unknown if modulating the intensity of the alloHCT conditioning regimen in AML patients testing positive for MRD can prevent relapse and improve survival rates. BMT CTN 0901 NCT NCT01339910 was a phase III randomized clinical trial comparing outcomes by conditioning intensity in adult patients with myeloid malignancy undergoing an alloHCT in morphological complete remission (ie: <5% marrow myeloblasts at the time of pre-transplant assessment, CR).
We aim ed to determine the impact of alloHCT conditioning intensity on post-transplant outcomes in AML patients in CR but with pre-transplant genomic evidence of residual disease. We used ultra-deep error-corrected DNA sequencing, using a custom 51kb anchored multiplex PCR panel with coverage of 13 commonly mutated genes in AML (ASXL1, DNMT3A, FLT3, IDH1, IDH2, JAK2, KIT, NPM1, NRAS, RUNX1, SF3B1, TET2 and TP53) on pre-conditioning blood samples of 190 AML patients from the BMT CTN 0901 study receiving either myeloablative (MAC) or reduced intensity conditioning (RIC). These subjects were well matched for other baseline characteristics. We showed that detection of an AML-associated variant using ultra-deep next-generation DNA sequencing in the blood of AML patients in CR prior to alloHCT was associated with increased relapse rate and inferior overall survival in those randomized to RIC. This study provides strong evidence that intervention for AML patients with MRD can result in improved survival. This work was presented, as a late-breaking abstract, at the Presidential Plenary Session of the European Hematology Association Annual Congress in Amsterdam (LBA2600, June 14th 2019) and is currently being prepared for publication. In addition, this year with Dr Gottesman of NCI we published a report of our collaboration investigating drug resistance in AML. Through transcriptomic analysis of 154 cases of treatment-naive AML, three chemorefractory patient groups with distinct expression profiles were identified. A classifier, trained based on the expression profile of the highest risk refractory patients, was validated in an independent cohort (n = 131), was prognostic for overall survival (OS) and refined an established 17-gene stemness score. Ex vivo drug sensitivity to 122 small-molecule inhibitors revealed effective potential group-specific targeting of pathways among these three refractory groups (Ref 2). We also reported long-term follow-up analysis of patients with Myelodysplastic Syndrome (MDS) treated on a phase 1/2 pilot clinical trial of alemtuzumab (05-H-0206, NCT00217594) which enrolled 39 patients at the NIH Clinical Center between 2005 and 2013. This report highlighted six exceptional responders who remained alive and in complete hematologic remission 5 years from enrollment (Ref 2). DNA-based sequencing is not capable of tracking the approved European LeukemiaNet molecular targets for AML MRD. We therefore developed, using iterative modifications and novel bioinformatics approaches, a novel RNA-sequencing assay with greater than 100-fold increase in performance compared with some commercially available targeted RNA-sequencing approaches and a limit of detection as low as one leukemic cell in 100,000 cells measured, which is comparable to gold-standard quantitative polymerase chain reaction analysis. This assay, which can be customized and expanded, is the first demonstrated use of high-sensitivity RNA-sequencing for measurable residual disease detection in acute myeloid leukemia and could serve as a broadly applicable standardized tool (Ref 3). Our systematic and comprehensive assessment of human bone marrow using genomic and immunophenotyping high dimensional single-cell analysis was also published during this period (Ref 4). We recruited a cohort of twenty healthy adult volunteers for collection of research samples from bone marrow aspiration for analysis by droplet-based single cell RNA sequencing, 13-color flow cytometry and mass cytometry. This unique dataset serves for a healthy control cohort across the full range of adulthood (age 24-84) providing not only cell population frequencies and characteristics, but also highlighting individual variation in human cohorts and reproducibility and correlation between these techniques. In conjunction with Drs McReynolds, Holland and Calvo we completed genomic analysis for recurrent AML somatic mutations in bone marrow samples from patients with germline GATA2 mutation and either progression to AML (Ref 5), or with MDS or a pre-MDS state (Ref 6). For AML MRD in the clinic, our analysis of our clinical trial for AML patients with relapsed or refractory disease, PEARL15: Personalized Early Assessment of Response During Salvage Chemotherapy in People with Relapsed or Refractory Acute Myeloid Leukemia (PEARL15, 15-H-0176, NCT02527447) was published. This trial sought to test the hypothesis that high-sensitivity assays for residual disease burden may allow much earlier assessment (day 4 in blood, compared with day 28-42 from cytomorphological examination of bone marrow conventionally) of the success of therapy for relapsed and refractory AML (Ref 7). Finally, in addition to those described above a number of impactful collaborations were completed and published this year; with the BeatAML group (Ref 9), with Dr. Neal Young in the Hematology Branch of NHLBI on high-sensitivity detection of telomere variants (Ref 10), with the group of Dr. Dan Larson of NCI on the U2AF1 splicing factor in AML (Ref 11), with Dr Edjah Ndoum of the Surgical Neurology Branch of NINDS (MDS/AML associated somatic mutations in patients with Glioblastoma, Ref 12) and with Drs. Daver, Sharma, Kantarjian and Allison of MD Anderson Cancer Center (Ref 13). In summary, the primary interest of the Myeloid Malignancies Section remains the detection, prevention and treatment of AML relapse, in particular the development of molecular and genomic laboratory methods to predict development or recurrence of myeloid malignancy. In the past year we have completed a large study involving ultra-deep error-corrected DNA sequencing of AML patients treated on a phase III randomized clinical trial and showed for the first time that intervention can change meaningful clinical outcomes for patients with AML MRD. Going forward we will continue to integrate genomic and immunophenotypic approaches on carefully annotated clinical samples to better understand myeloid malignancy.
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