Prediction of Post-Transplant Relapse of the Myelodysplastic Syndromes Via Evaluation of Stem Cells

The myelodysplastic syndromes (MDS) are bone marrow failure disorders which lead to life-threatening cytopenias and an increased risk for acute myeloid leukemia. MDS is initiated and sustained by aberrant mutated hematopoietic stem cells (MDS HSCs). Although standard therapies such as hypomethylating agents and lenalidomide can induce complete clinical cytogenetic and molecular remissions, they uniformly fail to eradicate MDS HSCs. As a result, replacement of MDS HSCs by allogeneic hematopoietic cell transplantation (allo-HCT) is the only curative treatment for MDS. However, relapse after allo-HCT occurs in 40% of cases, and effective and clinically applicable biomarkers to predict relapse remain lacking.

Given that relapse after transplant likely arises from rare residual MDS HSCs, we sought to develop a method to longitudinally assess MDS HSC burden after allo-HCT. We thus developed a high-throughput targeted DNA sequencing assay capable of identifying mutated MDS HSCs from as few as 50 HSCs purified by fluorescence-activated cell sorting (FACS). We developed a workflow using a hybridization capture panel covering over 240 genes recurrently mutated in myeloid malignancies, capable of identifying somatic and germline variants, and utilizing unique dual index primer sequences to remove PCR duplicates. We performed limit of detection analyses on three pooled AML samples to test the ability of this assay to detect 17 pathologic and 442 germline mutations with variant allele frequencies (VAFs) ranging from 5% to 100% from limiting cell numbers. The assay demonstrated 75% sensitivity to detect mutations at VAFs as low as 5-10% from as few as 50 FACS-purified cells.

We next evaluated a cohort of 60 patients from four institutions who underwent allo-HCT for MDS, acquiring bone marrow (BM) samples from within 150 days after allo-SCT, at which point all evaluated patients were without clinical evidence for relapse. We applied our low-input sequencing assay to FACS-purified HSCs/Multipotent progenitors (MPPs, Lin-CD34+CD38-CD45RA-), lymphoid primed multipotent progenitors (LMPPs, Lin-CD34+CD38-CD45RA+), lineage committed progenitors (Prog, Lin-CD34+CD38+), and unfractionated BM. We excluded 12 cases from which we were unable to purify at least 50 HSC/MPPs or LMPPs, or for which we did not achieve a read depth of >10. Of the remaining 48 cases, disease-associated driver mutations were detected among HSCs/MPPs or LMPPs in 23, with 21 of such cases (88%) eventually relapsing. Thus, detection of persistent MDS HSCs is highly specific for post allo-HCT relapse. Additionally, detection of disease-associated driver mutations in either HSC/MPPs or LMPPs was more sensitive for predicting relapse than detection in unfractionated BM (68% vs 44%), despite much lower average read depths (HSC: 88, Bulk: 692) and cell counts (HSC: 338, Bulk: >1 million). Detection of disease-associated driver mutations exclusively among HSC/MPPs or LMPPs was associated with a longer median time to relapse (53 days) than when also detected in Prog (37 days, p=0.0415) or unfractionated BM (30 days, p=0.0614). Finally, to understand the impact of salvage therapies for post-transplant relapse on MDS HSCs, we performed longitudinal sequencing on four patients with relapsed MDS after allo-HCT who were treated with Venetoclax and Azacytidine (Ven/Aza). We observed that Ven/Aza, but not donor lymphocyte infusion, effectively depleted MDS HSCs.

Allo-HCT remains the only curative therapy for MDS, and thus biomarkers to predict its effectiveness remain sorely needed. Our data validate a novel high-throughput platform capable of detecting persistent MDS HSCs as a sensitive and specific predictor of post-transplant relapse. Furthermore, because of the expansive gene panel used, this assay allows de novo mutation calling, as well as chimerism assessment based on dozens of donor-specific germline polymorphisms, and it does not require patient-specific customization. The high specificity of MDS HSC detection for relapse suggests that residual clonal hematopoiesis is rare after allo-SCT, and that detection of mutations after allo-HCT is indicative of the presence of fully transformed disease. Furthermore, this assay provides an HSC-based biomarker to quantify treatment response to both frontline and salvage therapies, promising to improve the design and implementation of potentially curative therapeutic strategies.