Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia

Abstract

Owing to the more recent positive results with the anti-CD33 immunotoxin gemtuzumab ozogamicin, therapy against acute myeloid leukemias (AMLs) targeting CD33 holds many promises. Here, CD33 and CD123 expression on AML blasts was studied by flow cytometry in a cohort of 319 patients with detailed information on French-American-British/World Health Organization (FAB/WHO) classification, cytogenetics and molecular aberrations. AMLs of 87.8% express CD33 and would therefore be targetable with anti-CD33 therapies. Additionally, 9.4% of AMLs express CD123 without concomitant CD33 expression. Thus, nearly all AMLs could be either targeted via CD33 or CD123. Simultaneous presence of both antigens was observed in 69.5% of patients. Most importantly, even AMLs with adverse cytogenetics express CD33 and CD123 levels comparable to those with favorable and intermediate subtypes. Some patient groups with unfavorable alterations, such as FMS-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations, high FLT3-ITD mutant/wild-type ratios and monosomy 5 are even characterized by high expression of CD33 and CD123. In addition, blasts of patients with mutant nucleophosmin (NPM1) revealed significantly higher CD33 and CD123 expression pointing toward the possibility of minimal residual disease-guided interventions in mutated NPM1-positive AMLs. These results stimulate the development of novel concepts to redirect immune effector cells toward CD33- and CD123-expressing blasts using bi-specific antibodies or engineered T cells expressing chimeric antigen receptors.

Figure 1

Gating scheme of flow cytrometric analysis of CD33 and CD123 expression in AML blasts. (a) After excluding doublets and debris, lymphocytes and blasts were selected based on their SSC and CD45 expression profiles. If present, CD34 expression was used to further characterize the blast population. Thereafter, CD33 and CD123 expression was analyzed. (b) The geometric mean fluorescence intensities (MFIs) of CD33 and CD123 on blasts were normalized to the MFI of lymphocytes, which are negative for both surface markers. GeoMean ratios of 5, 10, 25, 50 and 100 are displayed in the graph together with representative FACS plots. FCS, forward scatter; SSC, side scatter.

Figure 2

The majority of AMLs are positive for CD33 and CD123. (a) Pie chart showing distribution of CD33 positivity among 319 samples analyzed (GeoMean ratio blasts/lymphocytes ⩾10 was considered positive). (b) Pie chart depicting distribution of CD123 positivity among 298 samples analyzed. (c) Pie chart showing distribution of CD33 and CD123 positivity among 298 samples analyzed. (d) Scatter plot depicting distribution of CD33 and CD123 expression (GeoMean ratios blasts/lymphocytes) among 298 samples analyzed. (e) Box plots showing CD33 and CD123 expression by normal myeloid progenitors and AML blasts. P-values are based on unpaired two-tailed t-test.

Figure 3

Expression of CD33 and CD123 by FAB/WHO subtype, cytogenetics and mutations. Box plots showing distribution of CD33 expression (a) or CD123 expression by FAB/WHO subtype (b). For further details see also Table 1. Box plots visualizing distribution of CD33 expression (c and d) or CD123 expression (e and f) based on cytogenetic features and common mutations. P-values are based on unpaired two-tailed t-test. For further details see also Table 2.

Figure 4

Expression of CD33 and CD123 by risk group. Box plots showing expression of CD33 (a) and CD123 (b) based on FLT3 status (FLT3 wt, a mutant/wild-type ratio <0.78 and a ratio >0.78). Box plots depicting expression of CD33 (c) and CD123 (d) in AML blasts grouped by prognosis. P-values are based on one-way analysis of variance followed by Tukey's multiple comparison test.

Figure 5

The majority of CD34⁺ AMLs express CD33 and CD123 in their CD34⁺ blast population. (a) Pie chart showing expression of CD33 in the CD34⁺ blast population of CD34⁺ leukemias (samples with a GeoMean ratio CD34⁺ blasts/lymphocytes ⩾10 were considered positive). (b) Pie chart depicting expression of CD123 in the CD34⁺ blast population of CD34⁺ leukemias. (c) Pie chart visualizing expression of CD33 and CD123 in the CD34⁺ blast population of CD34⁺ leukemias.