Contributions
Abstract: PB1693
Type: Publication Only
Background
Despite high clinical remission rates (90%), pediatric acute myeloid leukemia (pAML) patients exhibit a high risk of relapse (30-40%). Recently, the leukemic stem cell (LSC) load at diagnosis, determined as CD34+/CD38−/CD45low cells expressing an aberrant marker, was shown to be an independent prognostic factor for relapse in pAML. Nevertheless, the flow cytometric aberrancies of LSCs remain poorly described. In adult AML, it was shown that the stem cell-associated antigen CLEC12A (CD371, CLL-1) strongly aids in discriminating normal hematopoietic stem cells (HSCs) from LSCs.
Aims
To evaluate the frequency and specificity of CLEC12A in the LSC fraction of pAML at diagnosis and relapse.
Methods
Bone marrow (BM, n=6) and/or peripheral blood (PB, n=4) of seven pAML patients were taken at diagnosis and relapse. For three patients, BM and PB was available at both time points. FCM data acquisition for CLEC12A (PE, clone 50C1) was performed together with backbone markers CD34 (PerCP-Cy5.5), CD38 (APC-H7) and CD45 (PacO or V500). All samples were analyzed on a 3-laser, 8-color FACSCanto II (BD) with instrument set-up performed according to EuroFlow guidelines. Within the CD34+/CD38- (103 cut-off for CD38) stem cell compartment, expression of CLEC12A was analysed using the lymphocytes as negative control. In addition, CD34+/CD38- cells from cord blood (CB, n=8) and normal pediatric bone marrow (NBM, n=6) were used to evaluate CLEC12A expression on normal HSC. Data analysis was performed using Infinicyt software v.1.8 (Cytognos, Salamanca, Spain) with a previously described gating strategy. Paired samples t-test was used to calculate statistical significance.
Results
The median blast percentage (% of white blood cells (WBC)), and CD34+ fraction within the blasts, was 40 % and 64 %, respectively, at diagnosis and 64 % and 62 %, respectively, at relapse. Total events measured ranged between 8575-1027340 (median 221579). Differences between the median CD34+CD38- load within the WBC compartment at diagnosis versus relapse were not significant in our cohort (BM: 0.08% versus 0.13%, PB: 0.07% versus 0.27%; P>0.05). At diagnosis, five out of seven patients showed aberrant CLEC12A expression, with a median CLEC12A pos/CLEC12A neg ratio within the CD34+CD38- compartment of 1.55 (range 0.46 – 20, BM=4, PB=3). The percentage of CLEC12A positive CD34+CD38- cells of ranged between 4 – 95 % (median 44%). At relapse, only one patient lost CLEC12A expression. CLEC12A pos/CLEC12A neg ratios were not significantly different at relapse (range 0 – 26, median 0.18) compared to diagnosis (range 0 – 20, median 0.49) in regard to the other six patients (BM=5, PB=3) (P>0.05). Analysis of PB was highly comparable to BM for the three patients with paired BM and PB samples. Importantly, no CLEC12A positive stem cell population (≥ 4 events and clustered on SSC/FSC) was observed within the CD34+/CD38- compartment of pediatric NBM or CB.
Conclusion
This report describes the frequency of aberrant CLEC12A expression in LSC in pAML. These data suggest that CLEC12A is a valuable and specific marker in children to discriminate leukemic from normal HSC.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): AML, Diagnosis, Stem cell marker
Abstract: PB1693
Type: Publication Only
Background
Despite high clinical remission rates (90%), pediatric acute myeloid leukemia (pAML) patients exhibit a high risk of relapse (30-40%). Recently, the leukemic stem cell (LSC) load at diagnosis, determined as CD34+/CD38−/CD45low cells expressing an aberrant marker, was shown to be an independent prognostic factor for relapse in pAML. Nevertheless, the flow cytometric aberrancies of LSCs remain poorly described. In adult AML, it was shown that the stem cell-associated antigen CLEC12A (CD371, CLL-1) strongly aids in discriminating normal hematopoietic stem cells (HSCs) from LSCs.
Aims
To evaluate the frequency and specificity of CLEC12A in the LSC fraction of pAML at diagnosis and relapse.
Methods
Bone marrow (BM, n=6) and/or peripheral blood (PB, n=4) of seven pAML patients were taken at diagnosis and relapse. For three patients, BM and PB was available at both time points. FCM data acquisition for CLEC12A (PE, clone 50C1) was performed together with backbone markers CD34 (PerCP-Cy5.5), CD38 (APC-H7) and CD45 (PacO or V500). All samples were analyzed on a 3-laser, 8-color FACSCanto II (BD) with instrument set-up performed according to EuroFlow guidelines. Within the CD34+/CD38- (103 cut-off for CD38) stem cell compartment, expression of CLEC12A was analysed using the lymphocytes as negative control. In addition, CD34+/CD38- cells from cord blood (CB, n=8) and normal pediatric bone marrow (NBM, n=6) were used to evaluate CLEC12A expression on normal HSC. Data analysis was performed using Infinicyt software v.1.8 (Cytognos, Salamanca, Spain) with a previously described gating strategy. Paired samples t-test was used to calculate statistical significance.
Results
The median blast percentage (% of white blood cells (WBC)), and CD34+ fraction within the blasts, was 40 % and 64 %, respectively, at diagnosis and 64 % and 62 %, respectively, at relapse. Total events measured ranged between 8575-1027340 (median 221579). Differences between the median CD34+CD38- load within the WBC compartment at diagnosis versus relapse were not significant in our cohort (BM: 0.08% versus 0.13%, PB: 0.07% versus 0.27%; P>0.05). At diagnosis, five out of seven patients showed aberrant CLEC12A expression, with a median CLEC12A pos/CLEC12A neg ratio within the CD34+CD38- compartment of 1.55 (range 0.46 – 20, BM=4, PB=3). The percentage of CLEC12A positive CD34+CD38- cells of ranged between 4 – 95 % (median 44%). At relapse, only one patient lost CLEC12A expression. CLEC12A pos/CLEC12A neg ratios were not significantly different at relapse (range 0 – 26, median 0.18) compared to diagnosis (range 0 – 20, median 0.49) in regard to the other six patients (BM=5, PB=3) (P>0.05). Analysis of PB was highly comparable to BM for the three patients with paired BM and PB samples. Importantly, no CLEC12A positive stem cell population (≥ 4 events and clustered on SSC/FSC) was observed within the CD34+/CD38- compartment of pediatric NBM or CB.
Conclusion
This report describes the frequency of aberrant CLEC12A expression in LSC in pAML. These data suggest that CLEC12A is a valuable and specific marker in children to discriminate leukemic from normal HSC.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): AML, Diagnosis, Stem cell marker