Prospective Study of CLL1 and CD45RA As Biomarkers of Leukemic Stem Cells to Predict Therapeutic Response in Acute Myeloid Leukemia
Authors: Verghese Andrea Verghese , Raju Suguna , Li Luke , Alfano Jessica , Vacchi-Suzzi Caterina , Lee Christina Y. , Lin Hong , Liu Suhu 
Published: December 2024

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by the clonal expansion of immature myeloblasts from leukemic stem cells (LSCs) and the disruption of normal hematopoiesis by hematopoietic stem cells (HSCs). Currently, treatment response is evaluated at the end of induction therapy (Days 28 ± 7). Despite an overall remission rate of about 70% for newly diagnosed AML and 30-50% for relapsed/refractory AML, many patients endure toxic treatments without clinical benefit. There is a need for biomarkers that enable timely evaluation of treatment response and facilitate real-time therapy adjustments that optimize therapeutic benefit while minimizing unnecessary toxicity.
Our prior retrospective study at Stony Brook University Hospital (SBUH) evaluated the dynamic changes of LSC versus HSC in AML patients using flow cytometric markers. Among the five LSC markers studied, changes in LSC/HSC subsets identified by CLL1 and CD45RA showed the strongest correlation with treatment response. Moreover, the presence of CLL1+ or CD45RA+ LSC/HSC subsets in peripheral blood correlated highly with bone marrow aspirate findings, suggesting that peripheral blood monitoring of LSC/HSC subsets could be a timely, non-invasive method to track LSC/HSC dynamics during AML treatment.
Aims: The primary aims of this study are to assess: 1) the feasibility of tracking changes in LSC/HSC subsets using peripheral blood CLL1 and CD45RA as biomarkers on Days 3, 5, and 7 of induction therapy in AML patients, and 2) the feasibility of using LSC/HSC subsets to predict therapeutic response as evaluated by bone marrow examination at the end of induction cycle (Days 28 ± 7).
Methods: This pilot prospective study plans to recruit 20 AML patients from SBUH who are 18 years or older, with newly diagnosed or relapsed/refractory AML and pre-treatment LSC marker (CLL1 and/or CD45RA) expression ≥50% of total HSCs. Following informed consent, peripheral blood samples are collected and processed for multicolor flow cytometry (MFC) on Days 3, 5, and 7 of induction therapy. The HSC population is defined as CD45dim/SSC and CD34+CD38low/-. CLL1 or CD45RA positive HSCs are identified as LSCs. LSC/HSC subsets are calculated as the percentage of CLL1+ or CD45RA+ cells within the HSC population. Changes in LSC/HSC subsets from pre-treatment will be evaluated and compared between patients with persistent disease and those achieving complete remission after induction chemotherapy. This study is registered at ClinicalTrials.gov (NCT06297551).
Results: To date, 7 patients with newly diagnosed or relapsed AML have been enrolled. In the 4 patients who achieved complete remission, LSC/HSC subsets changed by an average of -14.22 ± 18.70% on Day 3, -65.19 ± 29.65% on Day 5, and -78.26 ± 20.76% on Day 7 compared to pre-treatment levels (normalized as 0% change). In the 3 patients with persistent disease after induction therapy, LSC/HSC subsets changed by an average of +19.30 ± 23.55% on Day 3, +11.60 ± 18.34% on Day 5, and -2.57 ± 5.11% on Day 7.
Conclusions: Our preliminary results indicate that measuring the change in peripheral blood CLL1+ or CD45RA+ LSC/HSC subsets on Days 5 to 7 of induction therapy is feasible and may predict therapeutic outcome in AML. A significant decrease in CLL1+ or CD45RA+ LSC/HSC subsets in peripheral blood was observed as early as Days 5 to 7 of induction therapy, and this decrease in LSC/HSC subset biomarkers appears to be associated with complete remission by the end of the treatment cycle. Conversely, a lack of reduction in these subsets was seen in patients with persistent disease post induction therapy. Recruitment is ongoing, and we hope that additional data will further refine our findings for this study. In future studies, we hope to determine the optimal threshold of CLL1+ and CD45RA+ LSC/HSC subset change on Days 5 to 7 of induction therapy that is associated with remission versus disease persistence.