Expression and Prognostic Value of a Novel B7-H3 (CD276) Antibody in Acute Myeloid Leukemia

Abstract

Despite recent advances in immunophenotyping, the prognosis of acute myeloid leukemia (AML) is still mainly estimated using age and genetic markers. As the genetic heterogeneity of AML patients is high, flow cytometry-based classification with appropriate biomarkers can efficiently complement risk stratification and treatment selection. An increased expression of B7-H3 (CD276), an immune checkpoint protein, has been reported and associated with poor prognosis. However, the available data are limited and heterogeneous. Here, we used a novel, proprietary murine anti-B7-H3 8H8 antibody for the flow cytometric analysis of B7-H3 expression in AML blasts from 77 patients. Our antibody reliably detected substantial B7-H3 expression in 62.3% of AML patients. B7-H3 expression was higher in the monocytic French-American-British (FAB) M5 group and in intermediate and poor risk patients according to the European Leukemia Network. Using receiver operating characteristics (ROCs), we identified a specific fluorescence intensity cut-off of 4.45 to discriminate between B7-H3^high and B7-H3^low expression. High B7-H3 expression was associated with shorter overall survival (OS) and progression-free survival (PFS). In conclusion, we have developed a novel B7-H3 antibody that serves as a new tool for the detection of B7-H3 expression in AML and may help to facilitate risk stratification and treatment selection in AML patients.

Keywords: B7-H3; acute myeloid leukemia; monoclonal antibody; prognostic biomarker.

Figure 1

B7-H3 expression on AML blasts. B7-H3 expression on AML blasts was analyzed by flow cytometry. (A) Gating strategy for two exemplary AML samples: singlets, viable (7-AAD⁻), blast marker (CD33⁺) and B7-H3 expression as a percentage. The histograms show the B7-H3-specific staining using the murine 8H8 mAb (shaded peaks) and the corresponding isotype control (empty peaks). (B,C) B7-H3 expression on blasts from AML patients (n = 77) is shown as percentage of B7-H3-positive blasts (B) and as SFI levels (C) (box plots with min/max whiskers). AML SFI levels ≥1.5 and blasts ≥10% B7-H3 expression were considered positive (dotted gray lines). (D) Percentage of B7-H3-positive samples according to SFI ≥1.5. (E) Spearman correlation of B7-H3 SFI with percentage of B7-H3-positive blasts (Spearman’s rank correlation coefficient (r_s) = 0.899, p < 0.001).

Figure 2

Association of B7-H3 with clinical and genetic parameters. B7-H3 SFI levels according to (A) primary (pAML) vs. secondary (sAML) AML and age < 60 and >60 years (single values with median and 25/75 percentile; Mann–Whitney test), (B) the different FAB subgroups (single values with median and 25/75 percentile, pairwise Mann–Whitney test), (C) FAB M0–2 vs. FAB M4–5 (single values with median and 25/75 percentile; Mann–Whitney test), (D) CD34− and CD34+ cell populations, (E) cytogenetic [karyotype aberration, t(15;17), inv(16)] and monogenetic [FLT3-ITD, NPM1, CEBPA] markers (single values with median and 25/75-percentile, pairwise Mann–Whitney test) and (F) ELN 2022 favorable vs. intermediate/poor risk groups (single values with median and 25/75-percentile; Mann–Whitney test). (G) Overall survival (OS) in all patients regardless of their therapy according to B7-H3 SFI quartiles in Kaplan–Meier analysis. Median OS was reached after 2.1 months in the 4th quartile (blue line) and after 18.4 months in the 1st quartile (grey line) (log-rank test, p = 0.022). (H) Distribution of patients separated by B7-H3^low and B7-H3^high expression levels among ELN risk groups and among (I) FAB subtypes.

Figure 3

Impact of B7-H3 expression on clinical outcome (A) Progression-free survival (PFS) in all patients regardless of their therapy according to B7-H3^low and B7-H3^high expression in Kaplan–Meier analysis. Median PFS was 2.7 months in B7-H3^high (blue line) and 16.8 months in B7-H3^low (grey line) (log-rank test, p = 0.016). (B) Overall survival (OS) in all patients regardless of their therapy according to B7-H3^low and B7-H3^high expression in Kaplan–Meier analysis. Median OS was reached after 2.1 months in B7-H3^high (blue line) and 25.1 months in B7-H3^low (grey line) (log-rank test, p = 0.021). (C) OS in patients with anthracycline-based 1st induction therapy according to B7-H3^low and B7-H3^high expression in Kaplan–Meier analysis. Median OS was reached after 16.8 months in B7-H3^high (blue line) and after 138.8 months in B7-H3^low (grey line) (log-rank test, p = 0.019). (D) OS in patients receiving non-intensive treatment only, according to B7-H3^low and B7-H3^high expression in Kaplan–Meier analysis. Median OS was 0.6 months in B7-H3^high (blue line) and 1.0 months in B7-H3^low (grey line) (log-rank test, p = 0.167).

Figure 4

Cox’s proportional hazard regression analysis. Whole model specifications: Chi-Square(5) = 13.18, p = 0.0218, n = 55. HR: hazard ratio; CI: confidence interval; ELN: European Leukemia Network; FAB: French–American–British; WBC: white blood cell.