Expression and Clinical Significance of CD137L in Multiple Myeloma Cells

【Background】

Human CD137L molecule, a member of the TNF superfamily, was found to be expressed in a variety of malignant tumors, such as acute myeloid leukemia, non Hodgkin's lymphoma, associated with complete remission. Our previous experiments showed that high level of CD137L expressed on the surface of myeloma cell line RPMI-8226, U266, LP1, MY5 and KMS-11, as well as MM primary cell. However, we have no idea about the level of CD137L on MGUS (monoclonal gammapathy of undetermined significance) and the relation between the expression level and tumor stage, bionomics, prognosis of multiple myeloma.

【Objective】

(1) To determine expression and clinical significance of CD137L molecular in patients with MGUS and multiple myeloma cells;

(2)To explore function of CD137L in multiple myeloma cell lines.

【Methods】

(1) The expression of CD137L molecule on myeloma cells/normal plasma cell surface was detected by flow cytometry;

(2) Clinical significance of CD137L molecule expressed by multiple myeloma cells was accessed via rank sum test; expression level of high/low of CD137L on overall survival was evaluated through survival analysis and Log-Rank test;

(3) SiRNA, the customization of SiRNA for CD137L gene, transfected myeloma cell lines U266, RPMI-8226, KMS-11 by Lipo3000.Then the expression of CD137L was detected by RT-PCR; cell cycle distribution after inhibition of CD137L signal was detected by PI; cell proliferation was detected by CCK8.

【Results】

(1) Fresh bone marrow specimens of 28 patients with newly diagnosed multiple myeloma patients were collected. The expression of CD137L molecule on CD45-/CD38+/CD138+ cell group in bone marrow was detected, and the median expression level was 29 (7-94)%; the expression of CD137L molecule on 9 patients with MGUS was 7 (2-57)%;

(2) That the expression level of CD137L between patients with MGUS and newly diagnosed MM showed statistic difference indicated that it could be as a marker for differential diagnosis; the different expression level by rank sum test between those with newly diagnosed MM and post-treated MM, post-treated MM and RRMM indicated that it could be a marker for MRD;

(3) The follow-up of patients found that after the treatment CD137L level of 11/13 patients decreased, and these patients at least achieved PR;

(4) there is no related with the level of CD137L and type, ISS stage, DS stage, white blood cell count, hemoglobin concentration, platelet count, serum beta 2- microglobulin, lactate dehydrogenase, serum albumin, calcium concentration, the ratio of bone marrow plasma cell by correlation analysis;

(5) According to median values of CD137L expression level, all the newly diagnosed patients were divided into two groups, low level expression and high level and survival analysis showed no significant difference by Log-Rank test. The 2 years survival rate of low level group and the high one was 84.7%, 74.1%;

(6) KMS-11, RPMI 8226,U266 cells were transfected using Lipo3000 and only U266 cell line was inhibited obviously. Inhibition of CD137L induced cell proliferation by CCK8 test and a distribution change of G1 and S phase on cell cycle.

【Conclusions】

Multiple myeloma cell lines and primary myeloma cells had a high expression level of CD137L, MGUS cells had a low level while normal plasma cells surface without CD137L expression; CD137L can be a marker for diagnosis of MM and MGUS and for minimal residual disease; CD137L expression of MM patients had no correlation with clinical and biological features; In vitro the inhibition of CD137L signaling on U266 cells can induce cells proliferation.