Effects Of Metformin On B Cell Lymphoproliferative Disorder(BLPD)Study On MYD88L265P Mutations In Patients With B Cell Lymphoproliferative Disorder (BLPD)The Prognostic Value Of Serum IL-6in Newly Diagnosed Multiple Myeloma Patients

Metformin is a safe and cheap biguanide drug, widely used in the treatment of diabetes. Recent studies have revealed that metformin treatment was associated with a decreased incidence of cancers, such as breast, prostate, colon and pancreatic cancer, et al. Experiments have showed that metformin may inhibit the growth of those tumor cells. However, the efficacy of metformin on MCL or other BLPD has never been studied.Objective1. To study the effects of metformin in different doses on proliferation, cell cycle, apoptosis in MCL cell lines (Z138, Granta-519) and BLPD primary cells in vitro.2. To explore the mechanisms of metformin in some classic pathological pathways in vitro, including the NF-kb pathway, Cyclin Dl pathway and apoptosis pathway.3. To evaluate the response of treatment with metformin in a murine lymphoma subcutaneous xenograft model.Methods1. MCL cells (Granta-519and Z138) and BLPD primary cells were treated with metformin in vitro. The proliferation of MCL cells was assessed by CCK8assay at the time of0,24,48and72hours after treatment. The proliferation of BLPD primary cells was assessed by CCK8assay at the time of72hours after treatment.2. After treatment with metformin for72hours, apoptosis of Granta-519and Z138cells was examined by Flow cytometry. Apoptosis of BLPD primary cells was examined by Flow cytometry at the time of24,48and72hours after treatment with metformin.3. After treatment with metformin for48hours, cell cycle of Granta-519and Z138cells was examined by Flow cytometry.4. Western blotting was performed to detect the status of important pathways in proliferation, cell cycle, apoptosis of MCL and primary BLPD cells:MCL cells and primary BLPD cells were collected after treated with metformin for24h, Western blotting was performed to measure NF-kb pathway, Cyclin D1pathway and apoptosis pathway.5. MCL xenograft tumor model was established in NOD/SCID mouse and tumor growth was monitored during metformin treatment.6. The expression of cell cycle related proteins in xenograft tumor tissues were detected by immunohistochemistry. The apoptosis of xenograft tumor tissues was detected by TUNEL analysis.Results:1. Proliferation of MCL cells and BLPD primary cells was decreased after metformin treatment.2. After treatment for72hours, metformin induced Granta-519cells apoptosis, whereas it could not induce Z138cells apoptosis. Metformin (20mM) treatment induced BLPD primary cells apoptosis.3. After treatment for48hours, metformin induced significant increase of the G0/G1phases fraction of Granta-519and Z138cells in a dose-dependent manner.4. Metformin treatment inhibited the NF-kb pathway, Cyclin D1pathway, meanwhile activated the apoptosis pathway.5. In mouse xenograft model of Granta-519and Z138cells, intraperitoneal treatment of metformin for15days reduced tumor growth significantly(P<0.05).6. Immunohistochemical study showed a decreased positive rate of Cyclin D1in the metformin group than control, meanwhile metformin group showed an increased positive rate for TUNEL staining.Conclusion:1. Metformin inhibits the growth of MCL cells both in vitro and vivo. In our present experiments metformin can inhibit the proliferation of BLPD primary cells and induce these cells apoptosis in vitro.2. In MCL cells and BLPD primary cells NF-kb pathway, Cyclin D1pathway and apoptosis pathways are involved in the antitumor effect of metformin.3. Our study deepens the understanding of MCL and other BLPD, provides a new insight for therapy strategies with metformin. ObjectTo investigate the relationship between MYD88L265P mutation and B cell lymphoproliferative disorder (BLPD), especially Waldenstrom’s Macroglobulinemia/Lymphoplasmacytic lymphoma(WM/LPL).MethodA retrospective study on199BLPD cases was performed. MYD88L265P mutation was detected by direct sequencing. The clinical and laboratory features were compared between the patients with MYD88L265P mutation and those with wild type.ResultsMYD88L265P mutation was identified in43.33%(13/30) WM/LPL, while in2.7%(2/74) CLL,12.9%(4/31) BLPD-U,9.1%(1/11) highly suspected SMZL, but not identified in MCL, MZL, SMZL, MALT, HCL and FL. This mutation was not associated with TTT (time to treatment) in WM/LPL. Two CLL patients with MYD88L265P mutation were diagnosed with Binet stage B and C, both accompanied with IGHV mutation, while ZAP70and CD38of these two patients were both negative, and TTT of these two CLL patients were12and2months respectively. In four BLPD-U Patients with MYD88L265P mutation, abnormal Immunofixation Electrophoresis was detected in two cases, one exhibited an monoclonal IgM k, the other exhibited an monoclonal IgM k and monoclonal light chain k. These four patients all exhibited IGHV mutation, in which three cases were VH3predominance, one was VH4predominance. One highly suspected SMZL patient with MYD88L265P mutation also exhibited an monoclonal IgM k peak.Conclusion1. MYD88L265P mutation is more common in WM/LPL, suggesting a clinical significance in WM/LPL and other BLPD differentiation;2. MYD88L265P mutation may be associated with abnormal monoclonal IgM secretion;3. The detection of MYD88L265P mutation is conducive to guide the application of BTK inhibitors in BLPD. This study was aimed to evaluate the clinical significance of serum IL-6(S-IL-6levels) in newly diagnosed multiple myeloma patients. This study retrospectively investigated the S-IL-6levels in238newly diagnosed multiple myeloma patients, and analyzed the clinical characteristics and prognosis in different IL-6groups. The newly diagnosed patients with MM were divided into two groups:the low S-IL-6group(S-IL-6<100pg/ml) and the high S-IL-6group(S-IL-6≥100pg/ml). The results showed that high S-IL-6level was more common in patients with ECOG performance score>3, myeloma bone disease (MBD) between grade2to4, and high creatinine level, but there was no significant differences in age, abnormal karyotype percentage, chromosome13deletion percentage, CD138+/CD38+cells percentage and the level of calcium, phosphorus,(32-MG, hemoglobin, C-reactive protein, albumin, lactate dehydrogenase between the two groups at diagnosis, and also no significant difference in response to initial induction chemotherapy among the two groups. The overall survival is significantly different between the low and high IL-6groups (P=0.04,35m vs29m); but no difference in time to progress between the two groups (P=1.93,23m vs14m). In conclusion, the S-IL-6levels correlates with the clinical characteristics and prognosis. Serum IL-6levels measured by radioimmunoassay can be used as a routine examination in newly diagnosed multiple myeloma patients, which can effectively prompt prognosis.