| Background and PurposeMultiple myeloma (MM) is a clonal plasma cell malignancy which is usually restricted to bone marrow. Extramedullary disease (EMD) is considered as one rare clinical manifestation, developing at diagnosis or relapse, either adjacent to bones or in distant organs, which is associated with poor prognosis. Yet the outcome in patients with EMD is greatly heterogenous. The pathogenesis and risk factors of EMD are still unclear. MYC is an old pro-oncogene. Its rearrangement is usually seen in highly aggressive malignancies. Recent data have shown that MYC activation plays an important role in the progression from MGUS to MM. Some upper stream genes including Mucl and Pyk2 stimulate MYC expression. However, the significance of MYC abnormality and Myc overexpression in the development of EMD are rarely reported. This study aims to:(1) To reveal the correlation between MYC activation and EMD, analyze the prognostic significance of MYC abnormality in MM patients. (2) To study cytogenetic aberrations including MYC rearrangement in extramedullary plasmacytoma to investigate the role of clonal evolution in the development of EMD. (3) To explore the possible molecular mechanism of MYC activation in EMD which is associated with Mucl and Pyk2.MethodsThis is a retrospective case-control study. Newly diagnosed MM patients were enrolled at Peking Union Medical College Hospital from Jan 2008 to Nov 2015. Clinical data of patients with EMD and nonEMD were collected. MYC and other cytogenetic abnormalities of marrow cells were studied by fluorescence in situ hybridization (FISH). The expression of Myc, as well as two relevant proteins Muc1 and Pyk2, were tested by immunohistochemistry (IHC). Formalin-fixed and paraffin-embedded (FFPE) tumor tissues of EMD patients were collected, with solitary plasmacytoma as control group. A panel of probes including MYC, Iq21,8p21, RBI, D13S319, P53 and IGH was used in FISH to screen cytogenetic aberrations. Myeloma cell lines including U266, RPM18226 and NCI-H929 were studied to reveal the effect of Myc and Pyk2 inhibitor on the biologic behavior of MM cells, such as proliferation apoptosis and invasiveness.Results1. Cytogenetic abnormalities and protein expression in newly diagnosed MM patients: There were 65 nonEMD and 42 EMD patients enrolled in this study, whose overall survival was significantly different (not reached vs 24m, p=0.021). FISH analysis of bone marrow cells showed that positive rates of MYC rearrangement in nonEMD and EMD groups were 20.0% and 33.3% respectively (p=0.121). The positive rate of MYC rearrangement in patients with distant organ EMD was 42.3%(p=0.029). Patients with positive MYC rearrangement had a significantly shorter median overall survival compared with MYC negative ones (15.4m vs not reached, p＜0.001). The incidence of concomitant 1 q21 amplification and MYC rearrangement in nonEMD and EMD were 6.2% and 23.8% respectively (p=0.025). Single either 1q21 amplification or 8p21 deletion was not associated with poor prognosis. However, concomitant 1q21 amplification and MYC rearrangement was an indicator of very short OS compared with 1q21 amplification alone (11.0m vs not reached, p=0.001). Multivariate analysis revealed that MYC rearrangement was an independent risk factor of poor prognosis in MM patients (p<0.001). Immunohistochemistry of proteins:The incidence of Myc high expression (>30%) in nonEMD and EMD patients were 15.7% and 48.2%(p< 0.001)respectively. The incidences of Muc1 high expression in nonEMD and EMD were 16.3% and 35.2%(p=0.030) respectively. Myc expression level was higher in Muc1 high expression group than that in Muc1 low expression group (34.2% vs 20.8%, p=0.010). Similarly, Myc expression level was higher in Pyk2 high expression group than that in Pyk2 low expression group (29.6% vs 18.0%, p=0.011). Myc protein level was not coincident with its gene abnormalities.2. FFPE tumor tissues of 32 EMD and 14 SP patients were tested for FISH analysis. The incidences of MYC rearrangement (46.7% vs 14.3%, p=0.038)、 1q21 amplification (65.5%vsl4.3%, p=0.002)、 8p21 deletion (37.9%vs0%.p=0.008)、RB1 deletion (65.5%vs14.3%, p=0.001)、 D13S319 deletion (58.6%vs21.4%, p=0.022). P53 deletion (37.9%vs0%.p=0.008)、 IGH translocation (48.3% vs7.1%,p=0.015) were significantly higher in EMD patients than those of SP patients. Extramedullary plasmacytoma had more aberrant plasma clones compared to the lesions in bone marrow. The rate of high Myc expression in EMD patients was higher than that of SP patients (43.3%vs 16.7%), but the discrepancy was not significant (P=0.103).One patient with MYC positive in SP group progressed into active myeloma within 2 years.3. In vitro, inhibition of Myc reduced the invasivness of MM cells. The level of Myc expression and transwell ability were both decreased after being treated with Pyk2 inhibitor.ConclusionsMM patients with EMD have shorter overall survival, yet the clinical outcome is greatly heterogenous. MYC rearrangement is more prevalent in patients with EMD, which is associated with the poor prognosis. Cytogenetic aberrations are more prevalent in EMD than those in SP. Plasma cells in extramedually tissue have more clones with abnormal cytogenetics. The activation of Myc that probably stimulated by Mucl and Pyk2 is associated with the invasion ability of MM cells. |
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