Targeted positron emission tomography imaging of CXCR4 expression in patients with acute myeloid leukemia

Abstract

Acute myeloid leukemia originates from leukemia-initiating cells that reside in the protective bone marrow niche. CXCR4/CXCL12 interaction is crucially involved in recruitment and retention of leukemia-initiating cells within this niche. Various drugs targeting this pathway have entered clinical trials. To evaluate CXCR4 imaging in acute myeloid leukemia, we first tested CXCR4 expression in patient-derived primary blasts. Flow cytometry revealed that high blast counts in patients with acute myeloid leukemia correlate with high CXCR4 expression. The wide range of CXCR4 surface expression in patients was reflected in cell lines of acute myeloid leukemia. Next, we evaluated the CXCR4-specific peptide Pentixafor by positron emission tomography imaging in mice harboring CXCR4 positive and CXCR4 negative leukemia xenografts, and in 10 patients with active disease. [(68)Ga]Pentixafor-positron emission tomography showed specific measurable disease in murine CXCR4 positive xenografts, but not when CXCR4 was knocked out with CRISPR/Cas9 gene editing. Five of 10 patients showed tracer uptake correlating well with leukemia infiltration assessed by magnetic resonance imaging. The mean maximal standard uptake value was significantly higher in visually CXCR4 positive patients compared to CXCR4 negative patients. In summary, in vivo molecular CXCR4 imaging by means of positron emission tomography is feasible in acute myeloid leukemia. These data provide a framework for future diagnostic and theranostic approaches targeting the CXCR4/CXCL12-defined leukemia-initiating cell niche.

Figure 1.

CXCR4 expression in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). (A) Flow cytometric evaluation of CXCR4 surface expression using an anti-CXCR4 antibody. Blasts were gated as CD45^low cell population. Anti-CD117 antibody was used for back-gating. Representative data of CXCR4 positive (upper panels) and CXCR4 negative (lower panels) patients are shown. (B and C) Median fluorescence intensity of surface CXCR4 expression relative to isotype control (n=67 patients). Horizontal bars indicate the mean of all individual patient values±SEM; Student’s t-test was used to compare mean relative blast CXCR4 expression. *Statistically significant differences between the groups. (B) MDS versus AML; P=0.062. (C) CXCR4 expression in patients with less than 30% blasts versus CXCR4 expression in patients with at least 30% blasts; P=0.004.

Figure 2.

Surface CXCR4 expression of acute myeloid leukemia (AML) cell lines correlates with transcript levels. (A) Flow cytometric evaluation of CXCR4 surface expression of the indicated AML cell lines using an anti-CXCR4 antibody. An isotype control antibody was used as a control. (B) Mean fluorescence intensity of surface CXCR4 expression relative to isotype control. Three replicates for each cell line were used. (C) CXCR4 transcript levels measured by qRT-PCR. Mean relative expression±SEM is shown (n=3 independent experiments). ΔΔCt values relative to ubiquitin (Ub) were normalized to those of peripheral blood mononuclear cells (PBMC) of 3 healthy individuals. (D) Correlation analysis between relative CXCR4 transcript and relative CXCR4 surface expression levels.

Figure 3.

In vivo Pentixafor PET imaging in acute myeloid leukemia (AML) correlates with CXCR4 surface expression and migration towards a CXCL12 gradient. (A) [⁶⁸Ga]Pentixafor-PET imaging of AML xenografts. The indicated cell lines were injected into immunodeficient mice to generate xenograft tumors. CXCR4 expression was then analyzed using in vivo [⁶⁸Ga]Pentixafor-PET (upper panels). CXCR4 surface expression was analyzed by flow cytometry (lower panels). N=2 tumors/cell line; n=1 mouse/cell line. (B) [⁶⁸Ga]Pentixafor-PET imaging of control and CXCR4 knock-out (sg2) OCI-AML3 xenografts (upper panel). The lower panel shows CXCR4 surface expression as assessed by CD184 flow cytometry. A representative image and histogram is shown. (C) CRISPR/Cas9-mediated CXCR4 knock-out results in significantly reduced migration towards a CXCL12 gradient. OCI-AML3 cells were assessed using a transwell chamber migration assay. N=3 independent experiments. Mean±SEM is shown. *P=0.002 (Student’s t-test). (D) Images of the explanted tumor shown in (B) and (C) (left panel). Tumor weight (right panel). Mean±SEM, no significant difference. (E) Quantification of [68Ga]Pentixafor uptake. Xenograft tumors were analyzed by means of voxel intensity measurement. Mean±SEM is shown, n=3 tumors for control and sg2, n=3 mice; *P=0.049 (Student’s t-test).

Figure 4.

[⁶⁸Ga]Pentixafor-PET/magnetic resonance (MR) imaging in acute myeloid leukemia (AML) patients. (A–F) Shown are 2 AML patients (#2 and #1) with visually positive [⁶⁸Ga]Pentixafor-PET/MR imaging. (G–I)[⁶⁸Ga]Pentixafor-PET/MR images of a visually negative AML patient. (J–L) Control patient without BM malignancy who underwent [⁶⁸Ga]Pentixafor-PET/MR imaging. (A, D, G, J) Maximum intensity projections of [⁶⁸Ga]Pentixafor uptake. (B, E, H, K) T1w MR imaging coronal sections. (C, F, I, L) Coronal PET/MR imaging fusion. (M) (Left) Schematic graph of locations assessed for SUV quantification. 1: cervical vertebra (7); 2: thoracic vertebra (12); 3: right os ilium; 4: lumbal vertebra (5); 5: left os ilium. (Right) Heatmap of SUV values in the 5 visually positive (AML⁺), 5 visually negative (AML⁻), and 5 control patients with non-hematologic disease (control). *Patient #5 was scored positive because of a [⁶⁸Ga]Pentixafor-PET positive extramedullary lesion. (N) Quantification of SUV values from (m). *P=0.036 for AML⁺ versus AML⁻ and P=0.040 for AML⁺ versus control. Error Bars represent the SEM. Patient #5 was excluded due to the lack of bone marrow involvement (extramedullary AML).

Figure 5.

CXCR4 expression in bone marrow of acute myeloid leukemia (AML) patients undergoing [⁶⁸Ga]Pentixafor imaging. (A–C) Representative H&E stains of 3 AML patients show hypercellular bone marrow (BM) with blast infiltration; embedded are the PET images of the corresponding patients; (A) and (B) are visually positive for CXCR4-directed PET and (C) is negative. (D–F) IHC for patient specific myeloid/blast markers; stained markers are shown in white. (G–I) IHC for CXCR4 in the corresponding BM samples. (A, D, G) Patient #1. (B, E, H) Patient #4. (C, F, I) Patient #10.