CD45dimCD34+CD38-CD133+ cells are present in high numbers in the bone marrow of patients with acute myeloid leukemia
The work flow of the four-color flow cytometry experiments using monoclonal antibodies (mAbs) is shown in Fig. 1. As shown in Fig. 1A and 1B, live BMCs were collected and SSClow/CD45dim cells were obtained. The BMCs were stained with various combinations of monoclonal antibodies for 30 min such as isotype 1, isotype 2, and sample (Fig. 1C). The CD133 positive cells in the R1, R2, R3-gated cells were measured using flow cytometry, and the results were expressed as percentage changes from the isotype 2 (Fig. 1D). A total of 40 AML patients were examined for the expression of the target antigens, CD45dimCD34+CD38-CD133+ on the surface of BMCs. These cells were present in high numbers in the bone marrow samples isolated from patients with AML, but not in those of healthy controls (Fig. 2). These results indicated that CD45dimCD34+CD38-CD133+ cells in bone marrow are potential AML stem cells.
Elevated IL-1β, IL-6, IL-17 and IL-23 cytokine production of plasma in patients with AML
Recently, Th17 related cytokines such as IL-1β, IL-6, IL-17, IL-21, IL-22, and IL-23 play crucial roles in the pathogenesis of many diseases, including inflammatory diseases, autoimmune diseases, and cancers . They have been shown related to Th17 cells. Especially, elevated frequencies of these cytokines in patients with AML have been associated with prognosis . Therefore, we examined the levels of IL-1β, IL-6, IL-17 and IL-23 in the bone marrow plasma samples, which were matched to BMCs in AML patients. Plasma samples from the AML patients exhibited higher levels of IL-1β, IL-6, IL-17, and IL-23 than those from healthy controls (Fig. 3).
The CD45dimCD34+CD38-CD133+ cells are prominently detected in the bone marrow of patients with AML and CML
As shown in Fig. 4, the CD45dimCD34+CD38-CD133+ cells were examined by four-color flow cytometry in diverse hematological malignancies including AML (n = 40), CML (n = 6), DLBCL (n = 19), MM (n = 10), MDS, (n = 5), HL (n = 4), ALL (n = 3), and CLL (n = 2). These cells are significantly detected in the bone marrow of patients with AML and CML, but not in those with DLBCL, MM, MDS, ALL, CLL, and HL. These results indicated that CD45dimCD34+CD38-CD133+ cells in bone marrow are potential of AML stem cells. In addition, these cells might be used for the detection of AML stem cells.
Clinical characteristics according to levels of the CD45dimCD34+CD38-CD133+ cells
CD34+ AML and CD34- AML among 36 AML patients evaluable for CD34 expression was noted in 30 patients and 6 patients, respectively. The proportion of CD45dimCD34+CD38-CD133+ cells in CD34- AML were significantly lower than CD34+ AML (median, 5.0% [range, 1-14%] vs. 13.5% [range, 1.8-58%], P=0.001, respectively). And CD34- AML showed tendency to have lower proportion of CD45dimCD34+CD38-CD133+ cells. FLT3-ITD mutation was rarely found in AML patients with higher counts of CD45dimCD34+CD38-CD133+ cells (≥10%) than in patients with fewer CD45dimCD34+CD38-CD133+ cells (< 10%) (0% vs. 23.1%, respectively, P = 0.031). In addition, higher counts of CD45dimCD34+CD38-CD133+ cells (≥ 20%) were significantly associated with lower levels of IL-17, as compared to lower CD45dimCD34+CD38-CD133+ cells (< 20%) (118.0 vs. 35.0 pg/ml, respectively, P = 0.028). However, there was no significant difference in IL-1β, L-6, and IL-23 levels based on the population of CD45dimCD34+CD38-CD133+ cells.
High proportion of the CD45dimCD34+CD38-CD133+ cells predicts poor survival in AML patients
When we divided AML patients into three groups based on the percentage of CD45dimCD34+CD38-CD133+ cells (<10%, 10‒40%, and ≥40%), univariate analysis revealed that the 2-year OS rate was 64.3%, 57.9%, and 0%, respectively (P < 0.001) and the 2-year EFS was 62.3%, 37.2%, and 0% (P = 0.002), respectively (Supplementary Table 3). Among the three groups (CD45dimCD34+CD38-CD133+ cell proportions <10%, 10‒40%, and ≥40%), no significant differences were observed in baseline clinical factors including age (P = 0.085), white blood cell count (P = 0.397), platelet count (P = 0.737), and chemotherapy intensity (P = 0.158). Univariate analyses for OS and EFS in patients with AML revealed that older age (> 60 years) was significantly associated with worse OS than younger age (32.8% vs. 75% at 2-year, respectively, P = 0.041) (Supplementary Table 3). In addition, patients with higher marrow blast % (≥ 60%) showed significantly lower OS rates than those with lower marrow blast % (< 60%) (36.7% vs. 66.7%, P = 0.038) (Supplementary Table 3). Patients who were treated with intensive chemotherapy showed significantly better OS than those treated with hypomethylating agents (57.8% vs. 30.0%, P = 0.012). When we took into consideration other clinical parameters in univariate and multivariate analyses, higher percentage of CD45dimCD34+CD38-CD133+ cells (≥ 40%) was found to be an independent prognostic factor for OS (hazard ratio [HR], 6.052, P = 0.005) and EFS (HR, 9.028, P = 0.002) (Fig. 5. and Table 1). In addition, higher BM blast (%) ≥60% (HR, 2.607< P = 0.049) and chemotherapy intensity (hypomethylating agents vs. intensive chemotherapy) (HR, 4.058, P = 0.010) were significant prognostic factors for OS in multivariate analysis.