Evaluation of TIM-3+ LSCs Is a Highly Sensitive MRD Monitoring Strategy to Predict AML Relapse after Allogenic Stem Cell Transplantation
Authors: Hayashi Masayasu , Sakoda Teppei , Kikushige Yoshikane , Irifune Hidetoshi , Yoshimoto Goichi , Miyamoto Toshihiro , Akashi Koichi 
Published: October 2019

Abstract
Relapse of acute myeloid leukemia (AML) is a critical problem in clinics. Particularly, the prognosis of patients who relapsed after allogeneic stem cell transplantation (allo-SCT) is still poor. Self-renewing leukemic stem cells (LSCs), which are mainly enriched within CD34+CD38- fraction, cause re-growth of leukemia and eventually lead to recurrence. Therefore, evaluation of residual LSCs is crucial to estimate the efficacy of therapies including allo-SCT. As we previously described, T-cell immunoglobulin mucin-3 (TIM-3) is a useful marker for discrimination of normal and malignant HSCs. We, therefore, prospectively evaluated the frequencies of LSCs at multiple time points and assessed the validity of TIM-3 as a minimal residual disease (MRD) marker in the setting of allo-SCT.
50 AML patients who had been detected CD34+CD38-TIM-3+ LSCs in BM sample at least once before SCT were analyzed. All 50 patients underwent allo-SCT at Fukuoka Blood and Marrow Transplantation Group (FBMTG)-related hospitals from July 2015 to May 2018. 43 out of 50 patients achieved complete donor chimerism confirmed by short tandem repeat PCR (STR-PCR) at the time of engraftment (day 15-36), and these 43 patients was prospectively evaluated the frequencies of residual CD34+CD38-TIM-3+ LSCs using multi-color flow cytometry. Median age of the evaluated 43 AML patients at SCT was 44.8 years (28-67). 26 patients underwent SCT on disease and 17 patients at hematological CR. Relapse-free survival (RFS) was calculated from the time of SCT until last follow-up or documentation of relapse.
In each timing (at diagnosis, relapse, engraftment, etc.), we tracked the frequencies of TIM-3+ cells within CD34+CD38- fraction in BM. Of note, the proportion of CD34+CD38- fraction was 0.016 [0.0056 - 0.0267] % of BMMNC at engraftment. Then, according to the frequency of TIM-3+ cells within CD34+CD38- fraction, we classified patients into 3 groups; 'high' (> 90 % of CD34+CD38- cells were positive for TIM-3, n=5), 'intermediate' (61-90 % of CD34+CD38- cells were positive for TIM-3, n=11), and 'low' (< 60 % of CD34+CD38- cells were positive for TIM-3, n=27). This classification revealed that the recurrence rate within the observation period (median 334.4 days) was 100 % (5 of 5) in 'high' group, 45.4 % (5 of 11) in 'intermediate' group and 14.8 % (4 of 27) in 'low' group. Relapse-free survival was 83 (72-92) days in 'high' group, 368 (56-869) days in 'intermediate' group and 482.6 (45-1029) days in 'low' group, respectively (p<0.01) (Figure 1). Of note, all of 5 patients in 'high' group exhibited hematological relapse within 100 days. Thus, our new classification using TIM-3 as LSC-specific marker could isolate the patients at high risk for early relapse in allo-SCT settings even if they were considered as hematological CR and maintained complete donor chimerism assessed by clinically available methods (three-color FACS, STR-PCR, FLT3-ITD status and fusion gene specific qPCR) at the same timepoint. It suggests that monitoring of LSCs using TIM-3 should be a more sensitive and versatile strategy to predict early relapse of AML than aberrant surface markers monitoring by multi-color FACS because conventional FACS did not detect MRD population at engraftment while our strategy detected (we could detect LSCs (CD34+CD38-TIM-3+ fraction) within BMMNC in 0.0053 [0 - 0.012] %), and, the cases whose aberrant markers could be traced were only 28 cases of 43 in this study.
We also confirmed that the identical driver mutations were detected at both the initial diagnosis and relapse using whole exome sequencing (WES) of purified LSCs. As a typical example, WES detected CEBPA mutation (c.11dupG, 72.7 % and 50.0 %) and WT1 mutation (c.1091_1092insTTGTACGGTC, 41.9 % and 39.5 %) in a single patient. Additionally, we also validated that purified TIM-3+ LSCs at engraftment harbored the identical mutations by amplicon sequencing. It indicates that CD34+CD38-TIM-3+ cells should represent LSCs throughout the clinical course, from diagnosis to relapse.
In summary, TIM-3 expression represents the clones of functional LSCs are involved in relapse. Evaluation of TIM-3+ LSCs by multi-color FACS should be a highly sensitive strategy to predict relapse in clinical allo-SCT settings, and might enable us to appropriately intervene to overcome the poor clinical outcomes of AML.
Figure 1. Figure 1.

Disclosures
Akashi: Taiho Pharmaceutical: Research Funding; Novartis pharma: Research Funding; Pfizer: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Eisai: Research Funding; Celgene: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; sanofi: Research Funding; MSD: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Chugai Pharma: Research Funding; Eli Lilly Japan: Research Funding; Ono Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi-kasei: Research Funding.