PF164 CD9 IS AN ADVERSE PROGNOSTIC MARKER AND PROMISING THERAPEUTIC TARGET IN HIGH‐RISK PEDIATRIC B‐PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA
Authors: Zhang C. , Cheung J.T. K. , Chan K.Y. Y. , Wang H. , Man T.K. F. , Li K. , Li C.K. , Leung K.T. 
Published: June 2019

Background:

B‐precursor acute lymphoblastic leukemia (B‐ALL) is the commonest childhood malignancy. Despite advances in chemotherapy‐based treatment protocols, specific subtypes of high‐risk and relapsed/refractory B‐ALL have been associated with dismal outcomes, underscoring the need for development of novel targeted agents. CD9, a tetraspanin family protein, has been implicated in tumor progression but its prognostic relevance and therapeutic value in pediatric B‐ALL remain largely unknown.

Aims:

(1) To characterize the expression of CD9 in a pediatric B‐ALL patient cohort and its association with long‐term survival outcomes; (2) to assess the efficacy of targeting CD9 by a neutralizing antibody for treatment of pediatric B‐ALL in the NOD/SCID mouse xenograft model; and (3) to evaluate the potential toxicity of anti‐CD9 using a hematopoietic stem cell transplantation model.

Methods:

Primary B‐ALL cells were isolated from bone marrow (BM) aspirates of patients consecutively enrolled in the HKALL97, IC‐BFM‐ALL2002 and CCLG2008 studies. Immunophenotypes of leukemic blasts were characterized with CD9, CD19, CD34 and CD45 antibodies by flow cytometry. Patients were stratified into CD9+ and CD9‐ subgroups for comparison of overall survival (OS) and relapse‐free survival (RFS). NOD/SCID mice were infused with B‐ALL cell lines, patient‐derived leukemic blasts or cord blood CD34+ cells, and treated with IgG control or anti‐CD9 as a single‐agent or combined with standard chemotherapy. Leukemic load and stem cell engraftment in hematopoietic organs and CNS were measured by flow cytometry, bioluminescence imaging and immunohistochemistry.

Results:

Among 136 pediatric B‐ALL cases, blasts of 107 patients (78.7%) were CD9+. The 5‐year RFS rate of CD9+ patients was significantly lower than that of CD9‐ patients (65.4% vs.86.2%; P = 0.038). Subgroup analysis revealed significantly reduced RFS (33.3% vs.80.0%; P = 0.022) and a trend of lower OS (55.6% vs.90.0%; P = 0.055) in CD9+ patients of the high‐risk group. Multivariate analysis revealed that CD9 positivity independently predicted inferior survival (HR = 5.2; P = 0.004), and could be applied with established prognostic features, including prednisone response and cytogenetic status, for refinement of patient stratification. In NOD/SCID mice xenografted with CD9+ B‐ALL cell lines (n = 3) and primary leukemic blasts from patients with high‐risk and refractory B‐ALL (n = 5), administration of CD9 antibody substantially reduced leukemic burden in BM, spleen, blood, liver and CNS by 78.4–99.9% (P < 0.01) and prolonged animal survival by 1.6–2.1‐fold (P < 0.01). Anti‐CD9 did not compromise cord blood stem cell engraftment (n = 3), indicating leukemia‐selective activities. Combined treatment with anti‐CD9 and conventional chemotherapy (vincristine, dexamethasone and L‐asparaginase) further reduced leukemic load and extended survival, when compared with animals receiving anti‐CD9 (P = 0.043) or chemotherapy alone (P < 0.001).

Summary/Conclusion:

Our study identified CD9 as a new prognostic marker for predicting survival outcomes in pediatric B‐ALL and validated the efficacy of anti‐CD9 for suppressing leukemia progression. We propose that inclusion of CD9 into the diagnostic marker panels would potentially identify patients who might benefit from targeted therapy. In addition, anti‐CD9 could be developed as an adjunct to standard chemotherapy for treatment of high‐risk or relapsed/refractory pediatric B‐ALL, and this strategy could be extended to adult B‐ALL and other malignancies that express the CD9 surface antigen.