CD105 (Endoglin) as negative prognostic factor in AML

Abstract

While several genetic and morphological markers are established and serve to guide therapy of acute myeloid leukaemia (AML), there is still profound need to identify additional markers to better stratify patients. CD105 (Endoglin) is a type I transmembrane protein reported to induce activation and proliferation of endothelial cells. In addition, CD105 is expressed in haematological malignancies and the vessels of solid tumours. Here, CD105 associates with unfavourable disease course, but so far no data are available on the prognostic relevance of CD105 in haematological malignancies. We here generated a novel CD105 antibody for analysis of expression and prognostic relevance of CD105 in a cohort of 62 AML patients. Flow cytometric analysis revealed substantial expression in the various AML FAB types, with FAB M3 type displaying significantly lower surface levels. Next we established a cut-off specific fluorescence level of 5.22 using receiver-operating characteristics, which allowed to group patients in cases with CD105^lo and CD105^hi surface expression and revealed that high CD105 expression correlated significantly with poor overall and progression free survival. In conclusion, we here identify CD105 expression as a novel prognostic marker in AML, which may serve to optimize follow up and treatment decisions for AML patients.

Figure 1

Generation and functional characterization of CD105 antibody K-ro23. The CD105 antibody K-ro23 was generated as described in the methods section. (A) Size exclusion chromatography after the initial purification with protein A affinity chromatography. (B) SDS-PAGE analysis under reducing (R) and non-reducing conditions (NR). (C) Flow cytometry based detection of specific binding of the CD105 antibody (shaded peaks) and an isotype control (open peaks) at 1 nM on CD105 transfected Sp2/0 and mock transfected Sp2/0, and (D) analysis of the binding capacity of the CD105 antibody on CD105 transfected and mock transfected Sp2/0 as negative control. (E) Comparison of the specific binding of K-ro23 and the commercial clones SN6 and 166707 (shaded peaks) compared to the respective isotype controls (open peaks) at 5 nM on acute leukaemia cell lines. (F) Titration of the different CD105 clones on acute leukaemia cell lines. Specific fluorescence intensity (SFI) levels are shown.

Figure 2

CD105 expression on malignant haematopoietic cells. (A) Immunohistochemistry of CD105 expression of two exemplary AML patients. (B) Exemplary gating for two AML samples with the respective isotype control and specific CD105 binding. Specific gating strategy and controls are shown in Fig. S2. (C–G) CD105 expression was analysed on AML cells by flow cytometry. SFI levels above 1.5 were considered as positive expression (dotted line) (C) CD105 expression on blasts of AML patients (n = 62) are depicted as percentage of CD105 positive blasts and SFI levels (boxplots with Tukey whiskers). (D–F) CD105 SFI levels are depicted for the different FAB types (single values, median; Kruskal-Wallis-test) (D), FAB M3 vs. others FAB (boxplots with Tukey whiskers; Mann-Whitney-test) (E) and according to favourable and unfavourable FAB classification (boxplots with Tukey whiskers; Mann-Whitney-test) (F) are shown. (G) Distribution of CD105 expression (SFI) throughout NCCN risk group (boxplots with Tukey whiskers; Kruskal-Wallis-test).

Figure 3

Impact of CD105 expression on clinical outcome. (A) Specific binding of the CD105 antibody (shaded peaks) and an isotype control (open peaks) at 5 nM on AML blasts from exemplary patients of quartiles 1–4 using flow cytometry. (B) Kaplan-Meier analysis of SFI levels of CD105. Overall survival (OS) of each SFI quartile (1^st: 1–2.42; 2^nd: 2.43–5.25; 3^rd:5.26–26.37; 4^th: 26.38–65.2) was plotted followed by statistical analysis with log-rank test. (C) ROC for SFI levels of CD105 and OS were plotted. The crossing of the dotted line and the ROC curve marks the Youden-index for the highest sensitivity and specificity. (D) Overall survival according to CD105^lo and CD105^hi expression in Kaplan-Meier analysis. Mean OS was reached in CD105^hi after 199 days (dotted line; log-rank test). (E) PFS after any AML specific treatment according to CD105^lo and CD105^hi expression in Kaplan-Meier analysis. In CD105^hi the mean PFS was 383 days (dotted line; log-rank test).