Expression of Annexin 2 in Pediatric B-Acute Lymphoblastic Leukemia: A Marker of Aggressiveness and A Potential Therapeutic Target.

Annexin II (ANXA2) is a member of a peripheral membrane-binding protein family acting in a calcium-dependent manner, is involved in many cellular mechanisms, as cell proliferation and membrane physiology and is related to cancer progression. The aim of this study was to assess the ANXA2 expression in B cell precursor acute lymphoblastic leukemia (Bcp-ALL), in the attempt to finally evaluate it as a new potential therapeutic target.

The ANXA2 expression was tested in 77 newly diagnosed pediatric Bcp-ALL diagnosed and treated in our centers, according with LLA-2000 protocol of Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP). Diagnostic samples and 3 B-ALL cell lines (REH, SEM, 697) were studied by reverse phase protein array (RPPA), western blot and real-time PCR (RQ-PCR) analyses. Furthermore, immunofluorescence on bone marrow smears and cytofluorimetric studies were performed, in order to visualize the protein subcellular location. The associations between the ANXA2 expression, molecular features and prognosis were evaluated. For statistical purpose, multivariate analyses with Wilcoxon test and t-Test with conservative Bonferroni corrections and Kaplan-Mayer analysis were performed. Pearson correlation was used to compare mRNA and protein levels.

Our analyses demonstrated a positive correlation between mRNA and protein ANXA2 expression (Pearson correlation or index 0.6). Comparing ANX2 expression and molecular features, we found a statistically significant difference between patients with unfavourable [t(9;22), t(4;11)] and favourable translocations [t(12;21)], showing a higher level of ANXA2 in the former group (p-value <0.05). Additionally ANXA2 resulted upregulated at both mRNA and protein levels in 24 out of 77 patients included in the study, and in the group presenting with high ANXA2 expression, 8 (33%) patients relapsed; in contrast, in the group with low ANXA2 expression only 8 out of 53 cases (15%) suffered from a relapse. Interestingly, 5 patients (21%) with high ANXA2 expression died of progressive disease, while with only one case (2%) in the group with low ANXA2 expression. A multivariate analysis also showed that ANXA2 is an independent predictor of disease's aggressiveness. Due to the heterogeneity of response to treatment among our patients, which imply a stratification based on detection of minimal residual disease (MRD), the correlation between high expression level of ANXA2 with prognosis resulted not statistically significant (Kaplan-Mayer p-value >0.05). However, our data strongly suggested a correlation with a worst prognosis in those cases with high ANXA2 expression. Furthermore, immunofluorescence and cytofluorimetric analyses performed on SEM and 697 cell lines showed that ANXA2 is localized on the cellular membrane's surface, where the protein is usually involved in many cell functions.

To date, our study reports on ANXA2 expression and location in pediatric ALL. Our findings suggest that ANXA2 expression represents a marker of aggressiveness in Bcp-ALL, confirmed by the correlation with unfavourable molecular rearrangement such as MLL/AF4. Although the prognostic impact of ANXA2 expression needs to be evaluated with a further retrospective study including a larger and selected population, our data already strongly suggest that ANXA2 expression could be considered as a new potential therapeutic target in pediatric Bcp-ALL.

No relevant conflicts of interest to declare.