High aldehyde dehydrogenase activity at diagnosis predicts relapse in patients with t(8;21) acute myeloid leukemia.

Acute myeloid leukemia (AML) with t(8;21) is a heterogeneous disease. Although the detection of minimal residual disease (MRD), which is indicated by RUNX1-RUNX1T1 transcript levels, plays a key role in directing treatment, risk stratification needs to be improved, and other markers need to be assessed. A total of 66 t(8;21) AML patients were tested for aldehyde dehydrogenase (ALDH) activity by flow cytometry at diagnosis, and 52 patients were followed up for a median of 20 (1-34) months. The median percentage of CD34+ALDH+, CD34+CD38-ALDH+, and CD34+CD38+ALDH+ cells among nucleated cells were 0.028%, 0.012%, and 0.0070%, respectively. The CD34+ALDH+-H, CD34+CD38-ALDH+-H, and CD34+CD38+ALDH+-H statuses (the percentage of cells that were higher than the individual cutoffs) were all significantly associated with a lower 2-year relapse-free survival (RFS) rate in both the whole cohort and adult patients (P = .015, .016, and .049; P = .014, .018, and .032). Patients with < 3-log reduction in the RUNX1-RUNX1T1 transcript level after the second consolidation therapy (defined as MRD-H) had a significantly lower 2-year RFS rate than patients with ≥ 3-log reduction (MRD-L) (P = .017). The CD34+ALDH+ status at diagnosis was then combined with the MRD status. CD34+ALDH+-L/MRD-H patients had similar 2-year RFS rates to both CD34+ALDH+-L/MRD-L and CD34+ALDH+-H/MRD-L patients (P = .50 and 1.0); and CD34+ALDH+-H/MRD-H patients had significantly lower 2-year RFS rate compared with CD34+ALDH+-L and/or MRD-L patients (P < .0001). Multivariate analysis showed that CD34+ALDH+-H/MRD-H was an independent adverse prognostic factor for relapse. In conclusion, ALDH status at diagnosis may improve MRD-based risk stratification in t(8;21) AML, and concurrent high levels of CD34+ALDH+ at diagnosis and MRD predict relapse.

[In Vitro Identification and Characteristics of CD34- Leukemia Stem Cell in t(8;21) Acute Myeloid Leukemia].

OBJECTIVE: To preliminarily identify the existence of CD34- leukemia stem cell (LSC) in t(8;21) acute myeloid leukemia (AML) by in vitro test. METHODS: Bone marrow samples collected from newly diagnosed t(8;21) AML patients were tested. Lin-CD34+ CD38-(abbreviation, CD34+CD38-), Lin-CD34+CD38+ (abbreviation, CD34+CD38+) and Lin-CD34-CD38-CD45dimSSClow(abbreviation, CD34-"LSC") cell fractions were gated by flow cytometry after staining with fluorescent antibodies. Cells in G0 phase were identified through Hoechst 33342 and pyronin Y staining. Aldefluor reagent was used to test aldehyde dehydrogenase (ALDH) activity. The above-mentioned 3 cell fractions were sorted, and mRNA levels of AML1-ETO and WT1 were measured by real-time quantitative PCR. RESULTS: The 3 tested samples displayed the same tendency in ratio of the cells in G0 phase: CD34-"LSC">CD34+ CD38->CD34+CD38+. The paired t-test of 53 patients showed that frequency of ALDHbr cells of both CD34+CD38- and CD34-"LSC" cell fractions was significantly higher than that of CD34+CD38+ (P<0.01), furthermore, the ALDHbr cell frequency was significantly higher in CD34-"LSC" than that in CD34+ CD38- (P<0.01). AML1-ETO mRNA levels of cells sorted from 3 patients were similar among the 3 cell fractions within each patient, whereas WT1 mRNA levels were significantly higher in CD34-"LSC" than that in other 2 cell fractions. CONCLUSION: CD34- LSC may exist in t(8;21) AML, and may be more primitive than CD34+ LSC. These results promote the necessity to perform in vivo xenogeneic transplantation mice.

PRAME Gene Copy Number Variation Is Related to Its Expression in Multiple Myeloma.

Multiple myeloma (MM) patients commonly present abnormal expression of cancer-testis antigens, which may serve as immunotherapeutic targets and prognostic factors. We previously reported that preferentially expressed antigen of melanoma (PRAME) overexpression in bone marrow mononuclear cells is related to progression in MM patients treated with non-bortezomib-containing regimens. The mechanism underlying variations in PRAME expression remains unknown. To investigate the impact of gene copy number variation (CNV) on PRAME expression, plasma cells were sorted from 50 newly diagnosed patients and 8 healthy volunteers to measure PRAME transcript levels and gene copy numbers by real-time quantitative polymerase chain reaction. A total of 14 (28.0%), 7 (14.0%), and 29 (58.0%) patients exhibited overexpression, expression within the normal range, and low expression, respectively. PRAME overexpression was significantly related to a lower 1-year progression-free survival rate compared with PRAME low expression (20.0% vs. 88.9%, p = 0.043). The mean PRAME gene copy number relative to albumin (ALB) in normal samples was ∼1.0, whereas 4.0%, 24.0%, 70.0%, and 2.0% of patients had PRAME gene relative copy numbers of approximately 0, 0.5, 1.0, and 2.0, respectively. Patients with PRAME gene deletion (relative copy number of 0 or 0.5) had significantly higher frequency of PRAME nonoverexpression and lambda light chain expression than those with no deletion (p = 0.011 and 0.003). Thus, PRAME gene CNV occurs in MM. Gene deletion may be one mechanism leading to PRAME nonoverexpression and related to immunoglobulin lambda light chain locus rearrangement. PRAME overexpression in plasma cells might be an adverse prognostic factor for progression in MM.

Methylation pattern of preferentially expressed antigen of melanoma in acute myeloid leukemia and myelodysplastic syndromes.

Preferentially expressed antigen of melanoma (PRAME), a tumor-associated antigen, is overexpressed in a variety of hematologic malignancies with a great variation in expression. The majority of patients with acute myeloid leukemia (AML) 1-eight-twenty one (ETO)+ AML and a certain number of myelodysplastic syndromes (MDS) have an abnormally high increase in PRAME expression level. The landscape of PRAME methylation requires evaluation in order to determine the most relevant sites and the exact association of its methylation with expression level and type of disease. In the present study, bone marrow samples collected from 8 AML1-ETO+ AML, 4 MDS, 3 AML1-ETO- AML and 2 normal volunteers underwent bisulfate sequencing to analyze the methylation status of all four 5'-C-phosphate-G-3' (CpG) regions within the entire PRAME gene. The median PRAME transcript level of 15 patients was 204.5% (range, 0.02-710.3%). PRAME transcript levels were inversely associated with the degree of methylation of the -389 to -146 CpG sites (r=-0.69; P=0.002) in the 3' part of the promoter region and the +132 to +363 CpG sites (r=-0.69; P=0.006) in the exon 1b region. However, not every sample strictly followed this correlation: Certain samples with high degrees of methylation demonstrated abnormally high expression levels, and vice versa. The methylation ratios of CpG sites in exon 1a were low for all samples (range, 0.0-13.8%), and those in exon 2 were similar in 16 samples (range, 72.4-93.4%), with the exception of one patient with high expression (425.2%) and significantly low degree of methylation in the PRAME gene (22.2%). MDS patients revealed similar methylation ratios in the 3' section of the promoter region, but tended to have lower methylation ratios in the exon 1b region (P=0.62 and P=0.09, respectively) compared with those observed in AML1-ETO+ patients with AML and similar degree of PRAME overexpression. Therefore, the hypomethylation of CpG sites in the 3' part of the promoter region and in exon 1b was typically found with PRAME overexpression in AML and MDS. Methylation of other CpG islands, epigenetic and genetic mechanisms, and type of disease may also be involved.

PRAME overexpression predicted good outcome in pediatric B-cell acute lymphoblastic leukemia patients receiving chemotherapy.

To investigate the prognostic value of PRAME expression in pediatric acute lymphoblastic leukemia(ALL), we measured PRAME transcript levels at diagnosis in 191 patients(146 B-ALL; 45T-ALL)receiving chemotherapy only. PRAME overexpression was defined as transcript levels higher than 0.30%, which is the upper limit of normal bone marrow and the optimal cutoff value derived from ROC curve analysis. PRAME overexpression was identified in 45.5% of patients. In B-ALL, PRAME overexpression was significantly associated with lower CIR(cumulative incidence of relapse), higher DFS (disease-freesurvival), and OS(overall survival) rates at 3 years, respectively (5.8% vs. 14.9%, P=0.014; 94.2% vs. 85.1%, P=0.014; 96.0% vs. 87.4%, P=0.039). PRAME overexpression had no impact on outcome in T-ALL patients. Among B-ALL patients with non-poor cytogenetic risk, those with PRAME overexpression showed significantly lower CIR, higher DFS and OS rates at 3 years, respectively (8.47% vs. 14.5%, P=0.009; 96.5% vs. 85.5%, P=0.009; 98.4% vs. 88.0%, P=0.023). Furthermore, PRAME overexpression was an independent good prognostic factor for relapse in all B-ALL patients and B-ALL patients with non-poor cytogenetic risk. Therefore, the prognostic significance of PRAME overexpression differed by ALL subtype; It predicted good outcome in pediatric B-ALL receiving chemotherapy.