Expression of CRBN Signaling Pathway Proteins Assessed By Immunohistochemical Staining As Candidate Biomarkers for Outcome in Myeloma Patients Treated with IMiDs

Cereblon (CRBN), Ikaros (IKZF1), Aialos (IKZF3) and multiple myeloma oncogene 1 (MUM1) are important component of CRBN signaling pathway when treat MM cells with IMiDs. CRBN interacts with the DNA damage-binding protein-1 (DDB1), Cullin 4 (Cul4A or Cul4B) and regulator of Cullins 1 (ROC1) to form the functional E3 ubiquitin ligase complex (E₃ULC). CRBN increases the interaction between E₃ULC and IKZF1/3, leading to increased ubiquitination degradation of IKZF1/3, and then induce cytotoxicity in myeloma cells. Subsequently, degradation of IKZF1/3 induce depression of multiple myeloma oncogene 1 (MUM1), which is also called interferon regulatory factors (IRF4) and proved to be involved in the anti-MM activity of IMIDs in previous studies. Immunohistochemical (IHC) staining may be a convenient approach for researchers to differentiate the myeloma cells and non-myeloma cells in BM samples. In this study, we evaluated CRBN, IKZF1/3 and MUM1 expression level in bone marrow (BM) by immunohistochemical (IHC) staining and investigated the relationship between expression level and treatment outcome after IMiDs-based or bortezomib-based therapy in 123 newly diagnosed multiple myeloma (NDMM) patients. H-score method was applied according to both intensity and extent of staining. The intensity was graded from 0 to 3("0"for absent staining, "1" for weak expression, "2" for intermediate expression, and "3" for strong expression of the protein). The extent was graded from "0" to "100" to represent the percentage of MM cells with positive staining of any intensity. H-score was obtained by multiplying the intensity and extent score, ranging from 0 to 300, which reflected protein expression level in MM cells. The median H-score of CRBN, IKZF1, IKZF3 and MUM1 were 200, 0, 180 and 180, respectively. According to the median H-score, we classified the patients into high or low expression group. One hundred and twenty-three NDMM patients were enrolled in this study, including 64 males (52.0%) and 59 females (48.0%). The median age was 60 years (range 34-84). Fifty-one patients (41.5%) received IMiDs-containing regimen as the first-line therapy. The median follow-up time was 24.0 months (range, 10-76 months). After treated with IMiDs, patients with high level of CRBN and MUM1 achieved better overall response rate (ORR) than those expressed low level (CRBN, 88.0% vs. 42.3%, P=0.001; MUM1, 83.3% vs. 48.1%, P=0.009). Besides, patients with CRBN and MUM1 overexpression also had better overall survival (median OS, CRBN, not reached vs. 21.0 months, P=0.004; MUM1, not reached vs. not reached, P=0.021) and progression free survival (median PFS, CRBN, 28.0 vs. 12.0 months, P=0.002; MUM1, 32.0 vs. 12.0 months, P<0.001) than patients with low level, as well as 2-year OS rate (CRBN, 86% vs. 44%, P=0.005; MUM1, 81% vs. 51%, P=0.003) and PFS rate (CRBN, 66% vs. 17%, P=0.001; MUM1, 63% vs. 20%, P<0.001). In addition, in high CRBN and MUM1 expression group, patients treated with IMiDs had a higher 2-year PFS rate than Bortezomib presentation (CRBN, 66% vs. 45%, P=0.004; MUM1, 63% vs. 36%, P=0.003). In low CRBN and MUM1 expression group, patients treated with IMiDs had an inferior OS (median OS, CRBN, 21.0 vs. 57.0 months, P=0.001; MUM1, not reached vs. 57.0 months, P<0.001) and PFS (median PFS, CRBN, 12.0 vs. 31.0 months, P=0.003; MUM1, 12.0 vs. 32.0 months, P<0.001) than patients with Bortezomib, as well as 2-year OS rate (CRBN, 44% vs. 91%, P=0.002; MUM1, 51% vs. 100%, P<0.001) and PFS rate (CRBN, 17% vs. 59%, P=0.010; MUM1, 20% vs. 68%, P<0.001). This study identified high levels of CRBN pathway proteins CRBN and MUM1 were correlated with favorable ORR and survival in NDMM patients with IMiDs therapy, also suggested that expression levels of CRBN signaling pathway proteins in plasma cells assessed by IHC staining could better direct clinic individualized therapy.