Kategorie: Maligní lymfomy a leukémie
Číslo abstraktu: E1076
Autoři: PharmDr. Martin Čulen, PhD; Mgr. Marek Borský; Mgr. Veronika Némethová; Mgr. Jiří Šmejkal; Mgr. Tomáš Jurček; Ing. Dana Dvořáková, CSc.; MUDr. Barbora Weinbergerová (Wágnerová); MUDr. Lukáš Semerád; MUDr. Daniela Žáčková; prof. MUDr. Jiří Mayer, CSc.; Prof. MUDr. Zdeněk Ráčil, Ph.D.
Putative leukemic stem cells (LSC) persist in chronic myeloid leukemia (CML) patients during treatment and constitute risk of relapse. To study LSC and develop a targeted therapy, it is necessary to define these cells. Herrmann et al. (Blood 2014) recently proposed that CML LSC are defined by expression of CD26. We therefore set to further confirm these results on a larger group of patients.
To analyze the stem cell population in a wider cohort of chronic phase CML patients based on CD26 expression and to analyze the disease burden in sorted CD26- and CD26+ stem cells.
For 25 consecutive de novo patient samples, we FACS sorted CD45+34+38-/dim population into CD26- and CD26+ cells fractions and analyzed the Ph burden in both fractions by FISH. Additionally, we re-analyzed the FACS data and CD26 expression on SC using a more stringent CD45+34+38- gate. Detection of BCR-ABL1 in sorted sub-fractions was performed using single-cell nested RT-PCR.
WeFACS sorted CD26- and 26+ SC populations and analyzed the percentage of Ph+ cells in both fractions by FISH. This showed 96.2±9.3% (mean±SD) Ph+ cells in the supposed CD26+ LSC fraction, and 40,0±42,0% (mean±SD) Ph+ cells in the CD26- fraction, regarded as HSC, which, however, did not correspond with the results by Herrmann et al. We suspected that the Ph positivity in CD26- fraction originated from cells with slightly higher CD38 expression (thus being CD38dim non-SC), included in the sorting to provide enough cells for FISH analysis. Therefore, we performed a re-analysis of all our FACS data with more stringent CD38- gate and re-analyzed the CD26 expression in the pure CD38- SC population. This revealed three clear CD26 SC expression profiles among the patients: 1 – dominant CD26- population; 2 – equal CD26- and + populations; 3 – missing CD26- population. The re-analysis results therefore supported our previous theory; where, for example, the patients analyzed with almost 100 % FISH Ph+ cells in sorted CD26- fraction had no or very few (0-11%) HSC after the re-analysis (Profile 3). This confirmed that the FISH positivity in sorted CD26- fraction originated from the CD38dim/26- non-stem cells and proved the concept that CD26 expression reflects Ph positivity only in the true hematopoietic CD34+38- compartment.
To provide a further proof, we analyzed 3 samples using a nested RT-PCR protocol for single cell analysis. Here, we sorted 3-5 consecutive sub-fractions (each constituting 5-20 cells) for both CD26- and + populations, ranging from most CD38neg to CD38dim. CD26+ population proved positive in all sub-fractions, whereas in CD26- population, a weak BCR-ABL1 positivity could be detected only for sub-fractions previously defined for re-analysis as CD38dim.
We have confirmed that CD26 based differentiation of CML HSC and LSC (CD45+34+38-) is applicable irrespective of CD26 expression profiles, showing different HSC/LSC ratios, and proved the concept that CD26 expression reflects Ph positivity only in strictly gated true hematopoietic CD34+38- compartment. The results also show that simultaneous sorting of pure CD26+ LSC and CD26- HSC fractions from one patient will often be unfeasible due to low cell number in either population. These findings extend our knowledge on SC in CML and are important for further study and eradication attempts of LSC.
Datum přednesení příspěvku: 12. 6. 2015