Recipient-Derived Hematopoietic Cells Are the Source of Hematologic Malignancies after Graft Failure and Mixed Chimerism in Adults with Sickle Cell Disease
Authors: Ali Mohamed , Limerick Emily , Hsieh Matthew , Upadhyaya Kalpana , Xu Xin , Phang Oswald , Mukendi Jean Pierre Kambala , Calvo Katherine R. , Lopez-Ocasio Maria , Dagur Pradeep , Fitzhugh Courtney 
Published: December 2024

Sickle cell disease (SCD) is a genetic disorder leading to subclinical and overt organ damage and early mortality. The only cure for SCD is a hematopoietic cell transplant (HCT). Unfortunately, adults with overt organ damage cannot tolerate myeloablative conditioning. Thus, we and others developed a non-myeloablative (NM) allogeneic regimen to achieve a state of mixed donor/recipient chimerism, given that 20% donor myeloid chimerism (DMC) is sufficient to reverse the SCD phenotype. Traditionally, one consequence of this approach, especially in the haploidentical setting, has been a high risk of graft failure.

Further, we recently reported that the risk of hematologic malignancies (HMs) is higher than expected in patients with mixed chimerism or graft failure following NM HCT. Since most of these HMs were diagnosed after graft failure, their origin was assumed to be recipient-derived; however, this has yet to be confirmed. Here, we report, for the first time, the origin of leukemic blasts and myelodysplastic syndrome (MDS) mononuclear cells (MNCs) in patients with SCD after HCT.

We utilized frozen biospecimens from patients diagnosed with the most aggressive HMs after HCT. All samples were collected under an NHLBI IRB-approved protocol. Chimerism analysis was performed by PCR amplification of polymorphic short tandem repeats on DNA extracted from peripheral blood (PB) or bone marrow (BM) cellular fractions.

Five patients were included in this study: two patients aged 37 years at HCT had MDS 2 and 2.5 years post-HCT (SCD-01 and SCD-02); two patients aged 20 and 34 years at HCT had acute myeloid leukemia (AML) 4 months and 5.5 years post-HCT, respectively (SCD-03 and SCD-04); and one patient aged 39 years at HCT had T cell acute lymphoblastic leukemia (T-cell ALL) 3 years post-HCT ( SCD-05). Three patients (SCD-01, 02, and 03) had graft failure with 0% PB DMC and 0% donor lymphoid chimerism (DLC) accompanied by the return of SCD. SCD-04 had impending graft failure with 16% PB DMC and 18% DLC. SCD-05 had mixed chimerism with 30% PB DMC and 25% DLC.

Although there were no available samples for SCD-01, whole BM chimerism was performed at the time of the diagnostic evaluation, revealing 100% recipient chimerism. Further, we confirmed the origin of MDS MNCs from SCD-02 by analyzing the chimerism of DNA extracted from PB MNCs at Y3 post-HCT; cells were 100% recipient-derived. Moreover, from a PB sample of SCD-03 at Y5 post-transplant, we sorted 3 PB cell populations: CD3+ T cells, CD19+ B cells, and CD3-CD19-CD13+CD33+CD34+ leukemic blasts, all showing a 100% recipient-derived origin. In addition, we sorted 2 populations from SCD-04 PB at D100 post-HCT: CD3+ T cells and CD3-CD19-CD13+CD33+CD34+ leukemic blasts. While we detected 16% donor-derived cells in the T cell compartment, consistent with the PB DLC, the blast population showed 99% recipient-derived cells. Lastly, we sorted 3 populations from SCD-05 BM at Y3 post-HCT: CD19+ B cells, CD11b+ Myeloid cells, and CD19-CD11b-CD2+CD5+CD7+CD38+ CD1a-, mainly leukemic blasts. While we detected 11% BM DMC and 22% BM B-cell chimerism, the blast population was 100% recipient-derived.

In conclusion, we showed that in patients who experienced MDS, AML, or T-cell ALL following graft failure or mixed chimerism, leukemic blasts or MDS MNCs originated from patient cells, with 99-100% recipient chimerism. While the reasons are unknown why the risk of HMs is higher when the therapeutic goal is mixed chimerism, the goal of our future protocols is full donor chimerism to eliminate the possibilities of evolving pre-existing leukemic clones, or transforming recipient, clonally expanded hematopoietic cells, as a result of exposure to conditioning regimens, erythropoietic stress, or alloreactivity after graft failure. Further evaluation is ongoing.