Introduction: FLT3/CD135 (also known as FLK2) is a membrane-bound surface protein encoded by the FLT3 gene and part of the class III receptor tyrosine kinase family. It binds to FLT3 ligand (FLT3L) to activate signaling pathways that regulate proliferation, survival and differentiation of both lymphoid and myeloid lineages. CD135 is normally expressed on both progenitor and monocytic cells and is frequently observed in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). As part of COG protocol AAML1031 for de novo pediatric AML, which included an arm evaluating treatment with the FLT3 inhibitor sorafenib, we prospectively evaluated CD135 expression using difference from normal (ΔN) flow cytometry and correlated its intensity with clinical characteristics and outcome. We hypothesized that high CD135 expression would be associated with differences in outcome in the context of exposure to FLT3 directed therapy.

Methods: In the AAML1031 phase 3 trial pediatric patients with de novo AML were randomized to receive standard chemotherapy (Arm A) or standard chemotherapy with bortezomib (Arm B); patients with high allelic ratio FLT3-ITD were eligible for standard chemotherapy plus sorafenib (Arm C) if they signed second consent and the treatment arm was open. Specifics of risk stratification and treatment protocols for each arm/risk group have been previously published. Patients who: (1) submitted a bone marrow aspirate for ΔN at diagnosis and (2) provided consent for correlative biology studies were included in this analysis. All diagnostic specimens were centrally and prospectively evaluated for the expression of CD135 by ΔN.

Results: Surface CD135 expression was available for 1024 patients and showed wide variability. For analysis, the study population was divided into quartiles based on CD135 expression levels. By flow cytometry, the top quartile (n = 256) was associated with expression of monocytic markers: most notably increased CD45 (P<0.0001), CD11b (P=0.002), CD14 (p<0.0001), CD36 (P<0.0001), and CD64 (P<0.0001) compared to patients in quartiles 1-3, and the top quartile was inversely associated with CD34 expression (p = 0.001).

Comparison to clinical characteristics showed that patients in the top quartile of CD135 expression had a significantly higher rate of KMT2A gene fusions (33.3% vs 19.6%, P<0.001) and NPM1 mutations (12.9% vs 8.6%, p=0.045). No significant associations were observed with other clinical parameters including age, risk group allocation, CR rate, MRD status at EOI1, or other genetic markers. Quartile 4 was not associated with FLT3 mutations or allocation to Arm C.

There was no difference in 5-year event-free survival (EFS) or overall survival (OS) between quartile 4 and quartile 1-3, either among all patients (45.4% vs. 45.6%, p=0.88 and 62.1% vs. 65.0%, p=0.46) or just those with or without FLT3 mutations (data not shown, p>0.46 for all). However, patients in the top quartile allocated to Arm C (standard chemotherapy plus sorafenib) had lower 5-year EFS and OS compared to Arm C quartile 1-3 patients (22.2% vs 54.2%, p=0.019 and 33.3% vs 66.9%, p=0.016) as well as quartile 4 patients in Arm A/B (22.2% vs 46.2%, p=0.021 and 33.3% vs 63.2%, p=0.009). In contrast, patients in quartile 1-3 treated on Arm C had no significant difference in 5-year EFS (54.2% vs 45.1%, p=0.399) and OS (66.9% vs 64.9%, p=0.990) from quartile 1-3 patients in Arm A/B.

Conclusions: These data demonstrate that high levels of FLT3 (CD135) expression is associated with monocytic immunophenotypes, KMT2A fusions, and NPM1 mutations. Patients in Arm C (those who received chemotherapy and sorafenib) with the highest CD135 expression had inferior outcomes, both compared to all other patients in Arm C and high expressing patients who did not receive sorafenib. This suggests that elevated CD135 expression may mechanistically contribute to resistance or reduced efficacy of sorafenib-based therapy. For example, overexpression of CD135 may activate downstream signaling pathways that counteract the inhibitory effects of sorafenib, upregulating leukemic cell survival and proliferation. These findings highlight the potential role of CD135 as a biomarker for treatment response in this context and provide a possible mechanistic role in FLT3 inhibitor resistance, highlighting potential benefit of combination strategies targeting additional downstream pathways alongside FLT3 inhibition.

This content is only available as a PDF.
2025
Sign in via your Institution