In Search of Drivers of CD34+/CD38-/CD26+ Leukemia Stem Cells Persistence in CML Patients

Introduction: In Chronic Myeloid Leukemia (CML) patients, we confirmed that the expression of CD26 discriminates CML Leukemic Stem Cells (LSCs) from normal Hematopoietic Stem Cells (HSCs) or from LSCs of other myeloid neoplasms. CD34+CD38-CD26+ cells are easily measurable in Peripheral Blood (PB) at diagnosis, and circulating residual CD26+LSCs persist in most patients during treatment with Tyrosine Kinase Inhibitors (TKIs) and after successful TKI discontinuation. The reason for LCSs persistence may be correlated to mechanisms helping their survival, such as resistance to apoptosis (via BCL-2 or CD47 expression) or immune checkpoint escaping (via PD-L1). Moreover, few is still known about the "activity state" or "quiescence" of CML LSCs.

Aim of the study: to unravel phenotypic characteristics that could help CML LSCs persistence, evaluating by flow-cytometry the expression level of BCL-2 protein, PD-L1, CD47 and Ki67 on CD26+ LSCs population.

Methods: Flow-cytometry assays were performed on PB from newly diagnosed CML patients by using lyophilized reagents (CD34-FITC, CD26-PE, CD38-APC, CD45-V500) and liquid reagents (CD47-V450, PD-L1-Cy7, BCL-2-FITC, Ki67-PerCP-Cy 5.5). Analysis have been done using a BD FACSLyric cytometer.

Results: 47 newly diagnosed CML patients entered this study. With regards to PD-L1 expression on CD26+LSCs, 22/47 (47%) scored negative while 25/47 (53%) resulted positive, albeit with a quite variable percentage of expression (median 28.5 % of CD26+ cells, range 12-82.8 %). BCL-2 protein was expressed in 47/47 (100%) CD26+ LSCs, and the level of expression evaluated comparing the Mean Fluorescent Index (MFI) was found much lower on CD26+ LSCs than lymphocytes (median 5.98, range 1.90-32.14; median 17.20, range 7-76, respectively, p value <0.05). Regarding CD47 expression, 47/47 (100%) patients scored positive, with an MFI that is double on lymphocytes compared to CD26+ LSCs (median 26.58, range 5.70-87.09; median 13.22, range 5.64-33.22, respectively). Lastly, all 47/47 (100%) CD26+ LSCs were found Ki-67 positive.

Conclusions: These preliminary results show that in newly diagnosed CML patients, Ki67 is highly expressed on circulating CD26+ LSCs, suggesting an unexpected "active proliferative state" of CD26+ LSCs, rather than a "quiescent" status. The latter may explain the rapid reduction of the bulk of CD26+LSCs we observe already at 3 months of TKI treatment. Both BCL-2 protein and CD47 are expressed at a higher level on lymphocytes than on CD26+ CML LSCs, indicating that the "don't eat me signal" that blocks apoptosis may not be relevant for CML LSCs persistence. On the other hand, CD26+/PD-L1+ LSCs (found in about 53% of CML patients) could theoretically escape the immune checkpoint controls, an aspect that could be taken in consideration for a future Treatment Free Survival (TFR) approach in CML patients as it may lead to a loss of molecular response compared to patients with CD26+ LSCs scoring negative for PD-L1. Additional studies need to be performed on a larger cohort of patients with a longer follow-up to support these hypotheses; however, these preliminary data may start to pave the way to understand the relationship between CD26+ LSC and immunity of CML patients.

Abruzzese:BMS, Incyte, Novartis, Pfizer: Consultancy. Crugnola:Novartis: Speakers Bureau; Amgen: Speakers Bureau. Galimberti:Abbvie Incyte, Novartis, Janssen, Astrazeneca, Pfizer: Honoraria.