Evaluating Stem Cells to Predict Post-Transplant Relapse in the Myelodysplastic Syndromes

Malignant hematopoietic stem cells (HSCs) both initiate the myelodysplastic syndromes (MDS) and drive treatment failure through their resistance to frontline non-transplant therapies. Consequently, allogeneic transplant is the only curative therapy, but relapse can still occur. While post-transplant relapse is hypothesized to be driven by persistent MDS HSCs, their detection has proven challenging due to their limiting frequency in biospecimens. The accuracy of sequencing low-input specimens is limited by allelic drop-out and amplification bias. We adapted a hybridization capture-based targeted sequencing panel covering 260 myeloid malignancy associated genes that incorporates unique molecular identifiers capable of retaining read depths of >12x from as few as 19 sorted HSCs. We aimed to characterize residual disease after transplant with greater resolution than previously possible by sequencing sorted CD34+CD38-CD45RA- HSCs and multipotent progenitors (MPPs), CD34+CD38-CD45RA+ lymphoid-primed multipotent progenitors (LMPPs), and CD34+CD38+ progenitors.

We applied our technique to an MDS case characterized by bi-allelic TP53 mutations (Y220C and P75fs), assessing a bone marrow (BM) specimen D+168 after transplant, which was five months prior to clinical relapse. Both mutations were present at variant allelic frequencies (VAFs) of 8% and 4% in 258 sorted CD34+CD38- cells with 105x coverage (xc), but simultaneously undetectable in bulk BM (481xc) and CD34+CD38+ progenitor cells (239xc) despite greater cell number and read depth. We next evaluated a D+179 post-transplant BM from a case of MDS harboring ASXL1 (R713G), DNMT3A (R882H), and IDH2 (R140Q) mutations. All three mutations were detectable in 102 sorted HSCs/MPPs at VAFs of 20% (92xc), 13% (68xc), and 35% (89xc), respectively, while absent in bulk BM (369xc) and CD34+CD38+ progenitors (506xc). These findings preceded clinical relapse by nearly two years.

We additionally investigated pre- and post-transplant BM samples of a patient with a germline DDX41 M1I mutation treated for MDS. In such cases, a second somatic DDX41 mutation is often acquired, impairing ribosomal biogenesis. This mutation is always present at a low VAF, and it is unclear how it promotes disease. Prior to transplant, we identified a somatic DDX41 R525H mutation at a VAF of 2.6% in bulk BM. However, this mutation was present at a VAF of 45% in sorted HSCs/MPPs, revealing the pervasiveness of this "minor clone” at the apex of the hematopoietic hierarchy (Fig. 1). Additionally, two previously unknown TP53 clones were discovered at VAFs of 22% (K132R) and 1.8% (C275F) in HSCs/MPPs, and these were undetectable in bulk BM. At D+102 post-transplant, we detected persistence of both M1I (VAF 43%) and R52H (VAF 27%) DDX41 mutations in 19 sorted HSCs/MPPs (12xc) 78 days prior to relapse. These data suggest that while impaired ribosomal biogenesis in HSCs with bi-allelic DDX41 mutations prevents effective hematopoiesis, it also increases self-renewal and accumulation of HSCs.

Next, we sought to test if early identification of residual MDS HSCs post-transplant is predictive of disease relapse. We evaluated a cohort of patients who underwent myeloablative allogeneic transplant for MDS at UTSW and MSKCC (n=21). We evaluated BMs available from post-transplant dates ranging from D+30 to D+120, including any specimen from which we were able to sort at least 15 HSCs/MPPs and achieve an effective read depth of >10. We censored specimens with follow-up of less than one year. With a median follow-up of two years, detection of disease mutations in HSCs/MPPs was 100% specific and 88% sensitive for relapse (Fig. 2). The average time from detection of disease in HSCs to clinical relapse was 6.9 months. Detection in CD38+CD34+ progenitors was also 100% specific but only 67% sensitive for relapse. Assessment of mutations in bulk BM (mean 529xc) was only 22% sensitive and 50% specific for relapse.

In conclusion, our research shows for the first time that relapse of MDS after allogeneic transplant is driven by failure to eradicate MDS HSCs, and that detection of MDS HSCs early after transplant is highly predictive for relapse. Validation of this assay in larger cohorts promises to yield a tool to identify patients who may benefit from early post-transplant interventions to forestall relapse. We also demonstrate how this assay can reveal novel insights into human disease HSC biology.

Kroger:Takeda: Consultancy, Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; DKMS: Research Funding; Riemser: Research Funding; Neovii: Honoraria, Research Funding; Amgen: Honoraria; Kite: Honoraria; Jazz: Honoraria; Sanofi: Honoraria. Patel:Servier Pharmaceuticals: Current Employment. Anderson:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Beigene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Prothena: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Awan:Dava Oncology: Consultancy; Cellecter Bisosciences: Consultancy; ADCT Therapeutics: Consultancy; Cardinal Health: Consultancy; Johnson and Johnson: Consultancy; BeiGene: Consultancy; Caribou Biosciences: Consultancy; BMS: Consultancy; Epizyme: Consultancy; Merck: Consultancy; Incyte: Consultancy; Verastem: Consultancy; MEI Pharma: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Kite Pharma: Consultancy; Gilead Sciences: Consultancy; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; AbbVie: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy. Ramakrishnan:Kite: Consultancy; BMS: Consultancy; Rafael Pharma: Consultancy; Pharmacyclics LLC: Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Cellectar Biosciences: Membership on an entity's Board of Directors or advisory committees. Madanat:Sierra Oncology, Stemline Therapeutics and Novartis: Membership on an entity's Board of Directors or advisory committees; BluePrint Medicines, GERON, OncLiv: Consultancy, Honoraria.