Leukemic Stem Cells (LSCs) As Peri-Transplant MRD Assessment in AML Patients Undergoing Allogeneic Stem Cell Transplantation in CR
Authors: Klyuchnikov Evgeny , Badbaran Anita , Massoud Radwan , Freiberger Petra , Wolschke Christine , Ayuk Francis A. , Bacher Ulrike , Kroeger Nicolaus 
Published: December 2023

Background: Several studies have shown that the leukemic stem cells (LSCs: CD34 +CD38 -CD45RA +/Combi-6 +) play a crucial role in the development of relapses in AML patients (pts). LSCs are less sensitive to conventional therapy; moreover, their phenotypical characteristics may allow immune evasion promoting relapses after allo-SCT. Although ELN guidelines recommend including LSCs into MRD assessment, this approach has not yet been studied extensively in the allogeneic setting. Here we suggested that peri-transplant LSCs detection may provide additional prognostic information to conventional (c)MRD in AML pts allografted in CR.
Methods: 90 pts (male, n=46; median, 60.5 years, 19-76) with AML in CR and available pre-transplant cMRD data (multicolor flow cytometry, “different from normal” approach, according to ELN guidelines), who received allografts (matched, n=73; mismatched, n=17) during 2019-2022 years at the Department of Stem Cell Transplantation at University Medical Centre Hamburg were included. LSCs were assessed according to Zeijlemaker et al. 2019 before and after transplant at days +30 and +100. Both methods (cMRD and LSCs detection) were investigated solely and combined for their prognostic impact.
Results: Majority of pts had de novo AML (76%), adverse ELN risk (48%), received matched grafts (related, n=73, 81%; unrelated, n=17, 19%) and MAC regimen (n=55, 61%). ATG was used for GvHD prophylaxis in 70 pts (78%). There were 47 cMRD neg (52%) and 43 cMRD pos (48%) pts.
LSCs were detected in 39 (56%) pts with a median proportion of 0.01% (0.005-0.37%) of WBC. There was no correlation between LSCs proportion and cMRD status. The cMRD pos pts were more frequently LSCs pos (67% vs 33%), and cMRD neg were more frequently LSCs neg (33% vs 67%, p=0.002). Before allo-SCT, 34 pts (38%) were cMRD neg/LSCs neg, 26 pts (29%) experienced cMRD pos/LSCs pos, 17 pts (19%) had cMRD neg/LSCs pos and 13 pts (14%) had cMRD pos/LSCs neg. The conformity between both assays was 67%.
During a median follow up of 10 months (1-29) there were 17 mortalities, 13 relapses and 11 NRM events. The 1-year OS and LFS were significantly better in LSCs neg than in LSCs pos pts: 91% (77-97%) vs 73% (55-86%, p=0.012) and 88% (72-96%) vs 52% (35-68%, p=0.001), respectively. This was due to a higher relapse rate in LSCs pos group: 33% (18-52%) vs 4% (1-20%, p=0.002). The median time to relapse was 180 days (40-395). The difference in NRM was not significant. The area under the ROC curve for relapses was 0.74 (0.61-0.88, p=0.006).
Regarding the combined used of LSCs/cMRD, the best 1-year OS (p=0.038) and LFS (p=0.002) were observed for the double negative group: 94% (77-99%) and 88% (65-97%); followed by cMRD pos/LSCs neg: 86% (59-96%) and 86% (61-97%); cMRD neg/LSCs pos: 77% (43-94%) and 71% (39-90%); and the double positive group: 63% (43-79%) and 42% (25-61%)., respectively ( Figure 1). This was due to in the differing relapse rate that was highest in cMRD neg/LSCs pos group: 47% (17-79%), followed by double positive: 29% (14-50%), double negative: 7% (1-32%), and cMRD pos/LSCs neg group: 0% (p=0.02).
Post-transplant cMRD/LSCs data on days +30 and +100 were available in 70 consecutive pts. The post-transplant conversion rates (positive &gt; negative) for LSCs, cMRD and cMRD/LSCs were 49%, 61%, and 63%, respectively. The majority of pts converted at day +30. We observed a higher conversion rate to LSC neg status after MAC vs RIC (12/16, 75% vs 5/11, 45%, p=0.12). Of nine pts who were MRD pos at day +30, six were LSC neg and three were LSC pos. One MRD pos/LSC neg pts died due to NRM while remaining five pts survived without relapse. All three MRD pos/LSC pos pts developed relapses with two mortalities.
Conclusion: Peri-transplant detection of LSCs has a strong predictive value for early post-transplant relapses in AML pts. This approach can improve pre-transplant risk assessment for cMRD pos patients by defining a population with better survival and low relapses (cMRD pos/LSCs neg) and one with worse survival and more relapses (cMRD pos/LSCs pos). The post-transplant clearance of residual disease is associated with better survival regardless of the flow strategy being used (cMRD, LSCs, or both). Pts with persisting cMRD early after allo-SCT may be subdivided into different risk groups according to their LSCs status.
Figure 1. Leukemia-free survival according to pre-transplant cMRD/LSCs status in 90 AML patients.