Bispecific CAR T cell therapy targeting BCMA and CD19 in relapsed/refractory multiple myeloma: a phase I/II trial.

Despite the high therapeutic response achieved with B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T-cell therapy in relapsed and refractory multiple myeloma (R/R MM), primary resistance and relapse exist with single-target immunotherapy. Here, we design bispecific BC19 CAR T cells targeting BCMA/CD19 and evaluate antimyeloma activity in vitro and in vivo. Preclinical results indicate that BC19 CAR specifically recognize target antigens, and BC19 CAR T cells mediate selective killing of BCMA or CD19-positive cancer cells. BC19 CAR T cells also exhibit potent antigen-specific anti-tumor activity in xenograft mouse models. We conduct an open-label, single-arm, phase I/II study of BC19 CAR T cells in 50 patients with R/R MM (ChiCTR2000033567). The primary endpoint was safety. BC19 CAR T cells are well tolerated with grade 3 or higher cytokine release syndrome in 8% of patients and grade 1 neurotoxic events in 4% of patients, which meet the pre-specified primary endpoint. Secondary endpoints include overall response rate (92%), median progression-free survival (19.7 months), median overall survival (19.7 months) and median duration of response (not reached). Our study demonstrates that bispecific BC19 CAR T cells are feasible, safe and effective in treating patients with R/R MM.

Autoimmune hemolytic anemia and thrombocytopenia in a Chinese patient with heterozygous NBAS mutations: Case report.

RATIONALE: Neuroblastoma amplified sequence (NBAS)-associated disease is an autosomal recessive disorder and a broad spectrum of clinical symptoms has been reported. However, autoimmune mediated hemolytic anemia (AIHA) is rarely reported in NBAS disease. PATIENT CONCERNS: A now 21-year-old male harbors heterozygous variants of c.6840G>A and c.335 + 1G>A and was found had retarded growth, hypogammaglobulinemia, B lymphopenia, optic atrophy, horizontal nystagmus, slight splenomegaly and hepatomegaly since childhood. This case had normal hemoglobin level and platelet count in his childhood. He developed AIHA first in his adulthood and then thrombocytopenia during the treatment of AIHA. The mechanism underlying a case with pronounced hypogammaglobulinemia and B lymphopenia is elusive. In addition to biallelic NBAS mutations, a germline mutation in the ANKRD26 (c.2356C>T) gene was also detected. So either autoimmune or ANKRD26 mutation-mediated thrombocytopenia is possible in this case. INTERVENTION AND OUTCOME: He was initially managed with steroid and intermittent intravenous immunoglobulin supplement. After treatment, he responded well with a normalization of hemoglobin and serum bilirubin. But the patient subsequently experienced severe thrombocytopenia in addition to AIHA. He was then given daily avatrombopag in addition to steroid escalation. He responded again to new treatment, with the hemoglobin levels and platelet counts went back to the normal ranges. Now he was on de-escalated weekly avatrombopag and low-dose steroids maintenance. CONCLUSION: The phenotype of this case indicates that c.335 + 1G>A NBAS variant is probably a pathogenic one and c.2356C>T ANKRD26 variant is improbably a pathogenic one. AIHA may respond well to steroid even when happened in patients with NBAS disease.

Dysregulated CXCL12 expression in osteoblasts promotes B-lymphocytes preferentially homing to the bone marrow in MRL/lpr mice.

Objective: Todetect the abnormal distribution of B-lymphocytes between peripheral and bone marrow (BM) compartments and explore the mechanism of abnormal chemotaxis of B-lymphocytes in lupus subjects. Methods: The proportions of CXC chemokine receptor (CXCR)4+ B cells and CFDA-labeled MRL/lpr-derived B cells were detected by flow cytometry. The levels of CXC chemokine ligand (CXCL)12in peripheral blood (PB)were measured by ELISA. The migrated B cells to osteoblasts (OBs) was measured by transwell migration assay. The relative spatial position of B cells, OBs and CXCL12 was presented by Immunofluorescence assay. Results: Firstly, we found that the percentage of CXCR4+ B cells was lower in PB and higher in the BM from both MRL/lpr mice and patientswith Systemic lupus erythematosus (SLE). Secondly, OBs from MRL/lpr mice produced more CXCL12 than that from C57BL/6 mice. Besides, MRL/lpr-derived OBs demonstrated more potent chemotactic ability toward B-lymphocytes than control OBs by vitro an vivo. Additionally, more B-lymphocytes were found to co-localize with OBs within the periosteal zone of bone in MRL/lpr mice. Lastly, the percentages of CXCR4+B cells were found to be negatively correlated with serum Immunoglobulin (Ig) G concentration, moreover, BM CXCL12 levels were found to be positively correlated with SLE disease activity index Score and negatively correlated with serum Complement3 (C3) concentration. Conclusions: our results indicated that there is a shifted distribution of B-lymphocytes between BM and peripheral compartments in both SLE patients and MRL/lpr mice. Besides, the up-regulated levels of CXCL12 in OBs was indicated to contribute to the enhanced chemotactic migration and anchorage of B-lymphocytes to OBs.

[Risk Factors and the Effect of Antiviral Prophylaxis for Herpes Zoster in Multiple Myeloma Patients].

OBJECTIVE: To study the incidence and risk factors of herpes zoster in patients with multiple myeloma and to evaluate the preventive effect of antiviral therapy. METHODS: The clinical features of multiple myeloma patients with herpes zoster were retrospectively analyzed, the risk factors of herpes zoster and the effect of antiviral prophylaxis were analyzed. RESULTS: Among 180 patients with multiple myeloma, 23 cases developed herpes zoster (12.8%). The incidence of herpes zoster was 19.1% in patients with renal dysfunction and 23.5% after autologous hematopoietic stem cell transplantation (ASCT). The incidence of herpes zoster was higher in patients receiving bortezomib-containing regimens (21/137, 15.3%) than that in those without bortezomib (2/43, 4.7%), but there was no statistical difference (P =0.067). Antiviral prophylaxis was associated with fewer zoster infections, 8/111(7.2%) developed herpes zoster in patients who received antiviral prophylaxis, and 15/69 (21.7%) in those receiving no prophylaxis(P =0.005). 65.2% of patients with herpes zoster did not receive antiviral prophylaxis. Multivariate analysis showed that bortezomib treatment, AHSCT and renal dysfunction were independent risk factors for multiple myeloma with herpes zoster, while antiviral prophylaxis was independently associated with reducing the risk of herpes zoster. Herpes zoster had no effect on OS in patients with multiple myeloma. CONCLUSION: The risk of herpes zoster in multiple myeloma patients was increased. Antiviral prophylaxis can reduce the risk of herpes zoster in patients on bortezomib-based therapy.

Long-term analysis of cellular immunity in patients with RRMM treated with CAR-T cell therapy.

Chimeric antigen receptor T (CAR-T) cell therapy exhibits remarkable efficacy against refractory or relapsed multiple myeloma (RRMM); however, the immune deficiency following CAR-Ts infusion has not been well studied. In this study, 126 patients who achieved remission post-CAR-Ts infusion were evaluated for cellular immunity. Following lymphodepletion (LD) chemotherapy, the absolute lymphocyte count (ALC) and absolute counts of lymphocyte subsets were significantly lower than baseline at D0. Grade ≥ 3 lymphopenia occurred in 99% of patients within the first 30 days, with most being resolved by 180 days. The median CD4+ T-cell count was consistently below baseline and the lower limit of normal (LLN) levels at follow-up. Conversely, the median CD8+ T-cell count returned to the baseline and LLN levels by D30. The median B-cell count remained lower than baseline level at D60 and returned to baseline and LLN levels at D180. In the first 30 days, 27 (21.4%) patients had 29 infections, with the majority being mild to moderate in severity (21/29; 72.4%). After day 30, 44 (34.9%) patients had 56 infections, including 20 severe infections. One patient died from bacteremia at 3.8 months post-CAR-Ts infusion. In conclusion, most patients with RRMM experienced cellular immune deficiency caused by LD chemotherapy and CAR-Ts infusion. The ALC and most lymphocyte subsets gradually recovered after day 30 of CAR-Ts infusion, except for CD4+ T cells. Some patients experience prolonged CD4+ T-cell immunosuppression without severe infection.

Case report: Deep molecular remissions post two separate CD19-targeted chimeric antigen receptor T-cell therapies do not prevent disease from relapsing in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is an aggressive B-cell malignancy. The management of a relapsed Ph+ ALL patient is challenging. Currently, either allogeneic stem cell transplant (allo-SCT) or CD19-targeted chimeric antigen receptor T-cell (CAR T-cell) are usually employed as salvage modalities for a relapsed patient. However, there are few reports concerning cases that had both allo-SCT and multiple CAR T-cell therapies, and the optimal management of such patients is unclear. Here, we report a relapsed Ph+ ALL male who was first salvaged with autologous CAR T-cell therapy, followed by allo-SCT. Unfortunately, he had a second relapse even with complete molecular remission (CMR) response after the first CAR T and allo-SCT. This patient was then successfully salvaged by a second CAR T-cell product that is donor-derived. However, even with a CMR response once again following the second CAR T-cell therapy and prophylactic donor lymphocyte infusion, he experienced a molecular relapse; ponatinib was employed as the subsequent salvage treatment. He achieved a CMR response following ponatinib and was still in remission at the last follow-up. No ABL kinase mutation was detected during the whole course of the disease. This case indicated that a repeated CD19-targeted CAR T-cell treatment is feasible and may be effective in a relapsed Ph+ ALL patient that had previous CAR T-cell and allo-SCT, even though both CAR T-cell have the same construction. However, even with a deep response after each CAR T-cell therapy and allo-SCT, there is still a very small amount of undetectable leukemic cells. The optimal management of Ph+ ALL patients who have a deep response after a second CAR T-cell therapy deserves further exploration.

[Analysis of Related Factors of Autologous Peripheral Hematopoietic Stem Cell Mobilization in Patients with Lymphoma and Myeloma].

OBJECTIVE: To explore the related factors affecting of autologous peripheral hematopoietic stem cell mobilization in patients with single center lymphoma and multiple myeloma. METHODS: The clinical total of 30 patients with lymphoma or multiple myeloma who underwent autologous peripheral hematopoietic stem cell mobilization and transplantation in the Affiliated Hospital of Jiangsu University from March 2012 to December 2021 were retrospectively analyzed, including the patients' age, gender, disease type, chemotherapy course, mobilization scheme, collection times, CD34+ cell count, adverse events, days of neutrophil and platelet implantation after transplantation. The related factors affecting to the mobilization efficiency of peripheral blood stem cells was analyzed. RESULTS: The mobilization scheme had a significant effect on the mobilization success rate of CD34+ cells. The mobilization success rate and optimal mobilization rate of intermediate-dose VP-16+G-CSF were higher than that of high-dose VP-16+G-CSF (P<0.05); the mobilization success rate of patients with previous chemotherapy courses ≤4 was higher than that of patients with chemotherapy courses >4 (100% vs 72.22%, P<0.05); the mobilization success rate of lymphoma patients was lower than that of myeloma patients (66.67% vs 94.44%, P<0.05); the mobilization success rate of lymphoma patients who received intermediate-dose VP-16+G-CSF was higher than that received high-dose VP-16+G-CSF patients (100% vs 42.86%, P<0.05). Patients' gender, age, time from diagnosis to mobilization and disease status had no significant effect on the efficiency of stem cell mobilization. Fifteen patients (50%) had febrile neutropenia during stem cell mobilization. There was no statistical difference in the incidence of febrile neutropenia between the two mobilization schemes (P>0.05); the incidence of severe thrombocytopenia in intermediate-dose VP-16+G-CSF group was higher than that in high-dose VP-16+G-CSF group (P<0.05). There was no statistical difference in the time of granulocyte implantation and platelet implantation after stem cell transplantation in patients with different mobilization schemes (P>0.05). CONCLUSION: Mobilization regime, the number of previous chemotherapy course and disease type affect the mobilization efficiency of stem cells. Intermediate dose VP-16+G-CSF can improve the mobilization efficiency of stem cell in lymphoma patients, but should pay attention to the risk of bleeding.

[Effect of Doxycycline on Intrinsic Apoptosis of Myeloma Cell Line H929 and Its Mechanism].

OBJECTIVE: To investigate the mechanism of the in vitro toxicity of doxycycline to myeloma cell line H929 and also the possible pathway involved its toxicity. METHODS: Myeloma cell line H929 was treated with DOX, MEK inhibitor U0126 or RAS agonist ML-098, either alone or in combination. Then, the expression of p-MEK, caspase-3, caspase-9 and c-Jun in H929 were used to detected by Western blot; the cells proliferation and apoptosis were detected by CCK-8 assay and flow cytometry, respectively. RESULTS: DOX significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK in H929 (P<0.05). MEK antagonist U0126 significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK (P<0.05). After Dox combined with ML-098 treatment of H929 cells, the apoptosis rate of H929 cells was lower than that of DOX alone treatment group(P<0.05). Compared with DOX alone treatment group, the expressions of p-MEK and p-ERK1/2 in DOX+ML-098 combined treatment group were increased, and the levels of cleaved caspase-3,9 in H929 cells were decreased (P<0.05). The levels of c-Jun mRNA and protein increased in H929 when treated by DOX alone (P<0.05). CONCLUSION: DOX can induce apoptosis of H929 via intrinsic apoptosis pathway, and MEK/ERK pathway and c-Jun possibly play a role in this process.

[The Effect of Doxycycline on the Expression of MMP-2 and MMP-9 in Multiple Myeloma].

OBJECTIVE: To investigate the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in multiple myeloma (MM) patients, and analyze the effect of doxycycline (DOX) on the expression of MMP-2 and MMP-9 in MM cells. METHODS: The peripheral blood and bone marrow samples of MM patients were collected, and the patients were divided into three groups: newly diagnosed group, remission group and relapsed/refractory group, while the peripheral blood samples of 34 health people and the bone marrow samples of 17 IDA patients were selected as normal control and control group. The levels of MMP-2 and MMP-9 were detected by ELISA. The protein levels of MMP-2 and MMP-9 in H929 cells treated by different concentrations of DOX were analyzed by Western blot. After H929 cells was treated by Akt inhibitor MK-2206 2HCl in combination with DOX, Western blot was used to detect the levels of MMP-2 and MMP-9. RESULTS: The levels of MMP-2 and MMP-9 in newly diagnosed MM patients were higher than those in control (P<0.05), while for the patients in the remission group were decreased, but still higher than those in control. The levels of MMP-2 and MMP-9 were increased again for the patients in relapsed/refractory group, and showed no significant difference as compared with those in newly diagnosed group. The levels of MMP-2 and MMP-9 could be inhibited by 10 mg/L and 15 mg/L DOX treated by H929 cell. The protein levels of MMP-2 and MMP-9 showed no altered in H929 cells treated by 5 nmol/L MK-2206 2HCl alone. DOX exerted more profound inhibitory effect to MMP-2 and MMP-9 expression in H929 cells when Akt inhibitor MK-2206 2HCl was combined with DOX. CONCLUSION: The levels of MMP-2 and MMP-9 are increased in MM patients and related to the disease status of MM. DOX can inhibit the expression of MMP-2 and MMP-9 in MM cells, and antagonizing its activation of Akt signaling pathway can further enhance the inhibitory effect.

Long-Term Follow-Up of Combination of B-Cell Maturation Antigen and CD19 Chimeric Antigen Receptor T Cells in Multiple Myeloma.

PURPOSE: A combination of anti-B-cell maturation antigen (BCMA) and anti-CD19 chimeric antigen receptor (CAR) T cells induced high response rates in patients with relapsed or refractory (R/R) multiple myeloma (MM), but long-term outcomes have not been assessed yet. PATIENTS AND METHODS: In this single-arm, phase II trial, patients with R/R MM received a combination of anti-BCMA CAR T cells and anti-CD19 CAR T cells at a dose of 1 × 106 cells/kg, after receiving a conditioning chemotherapy consisting of cyclophosphamide and fludarabine. The overall response, long-term outcomes, and safety were assessed, as were their associations with clinical and disease characteristics. RESULTS: Of 69 enrolled patients, 62 received the combined infusion of anti-BCMA and anti-CD19 CAR T cells with a median follow-up of 21.3 months. The overall response rate was 92% (57/62), and complete response or better was observed in 37 patients (60%). Minimal residual disease-negativity was confirmed in 77% (43/56) of the patients with available minimal residual disease detection. The estimated median duration of response was 20.3 months (95% CI, 9.1 to 31.5). The median progression-free survival was 18.3 months (95% CI, 9.9 to 26.7), and the median overall survival was not reached. Patients with extramedullary disease had significantly inferior survival. Fifty-nine patients (95%) had cytokine release syndrome, with 10% grade 3 or higher. Neurotoxic events occurred in seven patients (11%), including 3% grade 3 or higher. Late adverse effects were rare, except for B-cell aplasia, hypogammaglobulinemia, and infections. CONCLUSION: The combination of anti-BCMA and anti-CD19 CAR T cells induced durable response in patients with R/R MM, with a median progression-free survival of 18.3 months and a manageable long-term safety profile.

Safety and efficacy of a humanized CD19 chimeric antigen receptor T cells for relapsed/refractory acute lymphoblastic leukemia.

CD19-targeted chimeric antigen receptor T (CAR-T) cells using murine single-chain variable fragment (scFv) has shown substantial clinical efficacy in treating relapsed/refractory acute lymphoblastic leukemia (R/R ALL). However, potential immunogenicity of the murine scFv domain may limit the persistence of CAR-T cells. In this study, we treated 52 consecutive subjects with R/R ALL with humanized CD19-specific CAR-T cells (hCART19s). Forty-six subjects achieved complete remission (CR) (N = 43) or CR with incomplete count recovery (CRi) (N = 3) within 1 month post infusion. During the follow-up with a median time of 20 months, the 1-year cumulative incidence of relapse was 25% (95% confidence interval [CI] 13-46), and 1-year event-free survival was 45% (95% CI 29-60). To the cutoff date, 20 patients presented CD19+ relapse and 2 had CD19- relapse. Among the 22 relapsed patients, 14 had treatment-mediated and treatment-boosted antidrug antibodies (ADA) as detected in a sensitive and specific cell-based assay. ADA positivity was correlated with the disease relapse risk. ADA-positive patients had a significantly lower CAR copy number than ADA-negative patients at the time of recurrence (p < .001). In conclusion, hCART19s therapy is safe and highly active in R/R ALL patients, and the hCART19s treatment could induce the emergence of ADA, which is related to the recurrence of the primary disease.

Bilateral anterior cerebral artery occlusion following CD19- and BCMA-targeted chimeric antigen receptor T-cell therapy for a myeloma patient.

Chimeric antigen receptor T (CAR-T)-cell therapy is a promising treatment for relapsed/refractory multiple myeloma (RRMM). In our previous report, CD19- and BCMA-targeted CAR-T co-administration was associated with a high response rate. Although cytokine release syndrome (CRS) and neurotoxicity are frequent complications following CAR-T treatment, cerebral infarction is rarely reported as a CAR-T-related complication. We reported a 73-year-old female MM patient who received CD19- and BCMA-targeted CAR-T for refractory disease. Her disease responded to CAR-T therapy, but she developed neurological symptoms following CRS. Cranial CT and MRI demonstrated multiple cerebral infarctions and bilateral anterior cerebral artery (ACA) occlusion. We suggest that cerebral infarction other than CAR-T-related neurotoxicity is the underlying cause of abnormal neuropsychological symptoms, and diagnostic imaging tests should be actively performed to exclude ischemic cerebrovascular events.

[Up-Regulation of PI3K/Akt Signaling Pathway in H929 Cell Line by Doxycycline Not Beneficial to Its Anti-Myeloma Effect].

OBJECTIVE: To investigate the effect and possible mechanism of up-regulation of p-Akt by doxycycline (DOX) on myeloma cell line H929. METHODS: Multiple myeloma cell line H929 was treated with DOX at different concentrations for different times, and cell proliferation rate was measured by CCK-8 assay. The protein expression level of p-Akt, PTEN, p-PDK1, p-mTOR, p-GSK-3ß, and p-BAD was analyzed by Western blot. The mRNA levels of mTOR, BCL-2, and NF-κB was analyzed by RT-PCR. PI3K inhibitor Wortmannin was used to antagonize the up-regulation of p-Akt, and the cell proliferation and p-Akt protein expression level were analyzed by CCK-8 assay and Western blot respectively. RESULTS: DOX could inhibit the proliferation of H929 cells and up-regulate the expression of p-Akt at the same time. The protein levels of both p-PDK1 and PTEN in H929 cells did not alter significantly during DOX treatment. The expressions of p-BAD and p-GSK-3ß were up-regulated in H929 cells after treated with DOX, but the expression of p-mTOR was not altered. The mRNA levels of mTOR, BCL-2, and NF-κB in H929 were all down-regulated in H929 cells during DOX treatment. The effect up-regulating p-Akt level by DOX was suppressed when DOX combined with PI3K inhibitor Wortmannin and Wortmannin could enhance the inhibitory effect of DOX in H929 cells. CONCLUSION: DOX can activate PI3K/Akt signaling pathway in H929 cells, and antagonizing this effect of DOX may enhance its cytotoxicity to myeloma cells.

A combination of humanised anti-CD19 and anti-BCMA CAR T cells in patients with relapsed or refractory multiple myeloma: a single-arm, phase 2 trial.

BACKGROUND: Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapy has been shown to have activity in patients with relapsed or refractory multiple myeloma. Reports have suggested that a small subgroup of less differentiated myeloma clones express CD19 and anti-CD19 CAR T-cell therapy has shown activity in some of these patients. We aimed to assess the activity and safety of a combination of humanised anti-CD19 and anti-BCMA CAR T cells in patients with relapsed or refractory multiple myeloma. METHODS: We did a single-centre, single-arm, phase 2 trial at the Affiliated Hospital of Xuzhou Medical University in China. Patients were eligible if they were aged 18-69 years, had histologically confirmed multiple myeloma, a Karnofsky Performance Score of 50 points or more, and met the International Myeloma Working Group diagnostic criteria for relapsed or refractory disease. Fludarabine (three daily doses of 30mg/m2) and cyclophosphamide (one daily dose of 750 mg/m2) were used to deplete lymphocytes before infusion of humanised anti-CD19 CAR T cells (1 × 106 cells per kg) and murine anti-BCMA CAR T cells (1 × 106 cells per kg). The primary outcome was the proportion of patients who achieved an overall response. Responses were assessed according to the International Myeloma Working Group criteria. This study is registered with the Chinese Clinical Trial Registration Center, number ChiCTR-OIC-17011272. FINDINGS: From May 1, 2017, to Jan 20, 2019, 22 patients were enrolled and 21 received an infusion of CAR T cells and were evaluable for safety and activity analyses. At a median follow-up of 179 days (IQR 72-295), 20 (95%) of 21 patients had an overall response, including nine (43%) stringent complete responses, three (14%) complete responses, five (24%) very good partial responses, and three (14%) partial responses. The most common adverse events included cytokine release syndrome (19 [90%] of 21), including 18 patients (86%) with grade 1-2 cytokine release syndrome. The most common serious adverse events were haematological toxicities, which occurred in 20 (95%) of 21 patients. Common grade 3 or higher adverse events included neutropenia (18 [86%]), anaemia (13 [62%]), and thrombocytopenia (13 [62%]). One patient died due to cerebral hemorrhage, which was considered related to sustained thrombocytopenia. No deaths were judged to be treatment-related. INTERPRETATION: Our results confirm that combined infusion of humanised anti-CD19 and anti-BCMA CAR T cells is feasible in patients with relapsed or refractory multiple myeloma, and the preliminary activity observed warrants further investigation in randomised trials. This dual CAR-T cell combinations might be a promising treatment option for relapsed or refractory multiple myeloma. FUNDING: National Natural Science Foundation of China, Natural Science Foundation, Key Research and Development Plan of Jiangsu.

[Increment of N-cadherin Expression in Bone Marrow Mesenchymal Stem Cells Accompanies by Imparment of Bone Marrow B Lymphocyte Production in MRL/lpr Lupus Mice].

OBJECTIVE: To investigate the hematological changes in MRL/lpr lupus mice and detect N-cadherin expression in their bone marrow mesenchymal stem cell (BMMSC). METHODS: Peripheral blood cell count was analyzed. The ratio of each lineage in bone marrow was analyzed by flow cytometry. Bone marrow CFU-pre-B, BFU-E and CFU-GM were detected by colony formation assay. Expression of N-cadherin in BMMSC was analyzed by Western blot before and after treatment with the BMP/Smad pathway aganist BMP-2 and inhibitor Noggin. RESULTS: Hemoglobin, red blood cell count and hematocrit decreased in lupus mice, compared with C57BL/6 mice. The ratio of B220+ lymphocyte and the number of CFU-pre-B in bone marrow of lupus mice decreased. Expression of N-cadherin in BMMSC of lupus mice was higher than that in the control group. Expression of N-cadherin decreased in BMP-2-treated BMMSC and increased after Noggin treatment. CONCLUSION: Hematological changes in lupus mice include anemia and impairment of bone marrow B cell production. The expression of N-cadherin in BMMSC of lupus mice increases which maybe involved in abnormal hemogenesis in MRI/Ipr lupus mice.

[IL-1ß-Treated Bone Marrow Mesenchymal Stem Cells Enhances Osteogenetic Potential via NF-κB Pathway].

Obsjective:To investigate the effects of differentaction time of IL-1ß on the osteogenic capacity of bone marrow mensenchymal cells(BMMSC) and the role of nuclear factor-κB (NF-kB) pathway. METHODS: BMMSC isolated from normal donors was treated with IL-1ß for 1 or 7 days, respectively. Alkaline phosphatase (ALP) and alizarin red(AR) stainings were used to detect the osteogenic differentiation potential of BMMSC. The mRNA expression of EphB4, IGF-1 and OPG in BMMSC was measured by real-time PCR. The immunohistochemistry was employed to measure the expression of bone morphgenetic protein-2(BMP-2) and p-Smad1/5/8 in BMMSC. Furthermore, the Western blot was used for the detection of iκBα and phospho-iκBα (p-ikBα) in IL-1ß-treated BMMSC. And the results of IL-1ß-treated BMMSC were compared with control group. RESULTS: Compared with control group, the osteogenetic potential of IL-1ß-treated BMMSC was enhanced, but the pro-osteogenic differentiation effect of IL-1ß was remarkedly inhibited in the presence of NF-kB pathway inhibitor PDTC. The total ikBα level of IL-1ß-treated BMMSC was lower (P<0.05), and phospho-iκBα (p-iκBα) level was higher (P<0.05). Besides, BMP-2 expression was higher (P<0.05) in the IL-1ß-treated BMMSC, however, p-Smad1/5/8 protien level was not significantly different among IL-1ß-treated for 1 d, 7 d and control groups (P<0.05). And the mRNA expression levels of IGF-1, EphB4 and OPG in BMMSC were up-regulated after IL-1ß treatment (P<0.05). In addition, the osteoblastogenesis of BMMSC treated with IL-1ß for 7 days was significantly different from those treated only for 1 day. CONCLUSION: Prolonging IL-1ß treatment can enhance the osteogenetic differentiation of BMMSC more significantly. And this osteogenetic alteration of BMMSC occurs via its NF-κB pathway, but not via BMP-2/Smad pathway.

[Clinical Efficacy of Decitabine Combined with Modified CAG Regimen for Relapse/Refractory Acute Myeloid Leukemia with AML1-ETO].

OBJECTIVE: To investigate the clinical characteristics of patients with relapse-refractory acute myeloid leukemia(AML) with AML1-ETO+, and therapeutic effcacy and side effects of decitabine combined with modified CAG regimen. METHODS: Clinical data of 8 cases of AML with AML1-ETO+ from June 2015 to January 2016 were analyzed retrospectively, including age, sex, initial symptoms, peripheral blood and bone marrow characteristics and so on. at the same time, the therapeutic effcacy and side effects of decitabine combined with modified CAG regimen were evaluated. The 8 patient were with median age of 44.5(16-59) years. RESULTS: Among these 8 patients, 1 patients were relapsed and other 7 patients were relapse/refractory patients, their median white blood cell count was 23.57(7.5-65.29)×109/L, median platelet count was 40(19-69)×109/L, median hemoglobin 1evel was 107(79-131) g/L, median lactate dehydrogenase level was 313.5(124.1-865.9) U/L at the initial diagnosis. The results showed that after treatment with decitabine combined with modified CAG, 7 patients achieved complete remission, 1 patient did not achieve remission, the overall remission rate was 87.5%(7/8). The main side effects of this regimen was myelosupp-ression, there were no new lung infection and other serious complications, 1 case without complete remission was treated with FLAG, and died of heart failure. CONCLUSION: According to preliminary results of decitabine combined with modified CAG regimen for treatment of relapse/refractory AML patients with AMLl-ETO+ displays higer remission rate and lower side effects.

Predicting early post-chemotherapy adverse events in patients with hematological malignancies: a retrospective study.

PURPOSE: The purpose of this study was to develop a mathematical model that predicts the definite adverse events following chemotherapy in patients with hematological malignancies (HMs). METHODS: This is a retrospective cohort study including 1157 cases with HMs. Firstly, we screened and verified the independent risk factors associated with post-chemotherapy adverse events by both univariate and multivariate logistic regression analysis using 70 % of randomly selected cases (training set). Secondly, we proposed a mathematical model based on those selected factors. The calibration and discrimination of the model were assessed by Hosmer-Lemeshow (H-L) test and area under the receiver operating characteristic (ROC) curve, respectively. Lastly, the predicative power of this model was further tested in the remaining 30 % of cases (validation set). RESULTS: Our statistical analysis indicated that liver dysfunction (OR = 2.164), active infection (OR = 3.619), coagulation abnormalities (OR = 4.614), intensity of chemotherapy (OR = 10.001), acute leukemia (OR = 2.185), and obesity (OR = 1.604) were independent risk factors for post-chemotherapy adverse events in HM patients (all P < 0.05). Based on the verified risk factors, a predictive model was proposed. This model had good discrimination and calibration. When 0.648 was selected as the cutoff point, the sensitivity and specificity of this predictive model in validation sets was 72.7 and 87.4 %, respectively. Furthermore, this proposed model's positive predictive value, negative predictive value, and consistency rate were 87.3, 73.0 and 80.0 %, respectively. CONCLUSIONS: Our study indicated that this six risk factor-based mathematical model is accurate and sufficient enough to predict definite post-chemotherapy adverse events in a HM patient and it may aid clinicians to optimize treatment for a HM patient.

Clinicopathologic analysis of POEMS syndrome and related diseases.

BACKGROUND: POEMS syndrome, a rare paraneoplastic disease, is related to multiple organs, multiple systems, and multiple disciplines and can be mistaken for other disorders. Consequently, the diagnoses are often delayed. In this work we studied the clinicopathologic characteristics of the POEMS syndrome to improve early diagnosis to prevent irreversible damage. PATIENTS AND METHODS: We conducted a clinicopathologic analysis of 9 cases of POEMS and made a differential diagnosis with related diseases. RESULTS: The patients with POEMS syndrome were shown to have complicated clinical characteristics, including peripheral neuropathy, organomegaly, endocrinopathy, monoclonal plasma proliferative disorder, skin changes, extravascular volume overload, sclerotic bone lesions, thrombocytosis, and Castleman disease. POEMS syndrome shared many elements with other diseases and the key way to differentiate them was to determine whether there were other fundamental POEMS syndrome symptoms or signs. The level of M-protein in serum and plasma cells in bone marrow of POEMS patients was lower than that of patients with multiple myeloma (MM). Sclerotic bone lesions were a distinctive feature in patients with POEMS, compared with in those with MM. CONCLUSION: Some unique clinicopathologic characteristics of POEMS syndrome can be used for differential diagnosis. This study provides increased awareness of POEMS syndrome.

In vitro migratory aberrancies of mesenchymal stem cells derived from multiple myeloma patients only partially modulated by bortezomib.

Recent studies indicated that bone marrow mesenchymal stem cells (BM-MSCs) derived from multiple myeloma (MM) patients were different from those of normal subjects in a variety of aspects. However, it is largely unknown whether BM-MSCs derived from MM patients display any aberrant chemotactic migration. To this aim, we compared the chemotactic migration of BM-MSCs derived from MM patients with those from normal subjects. Our results showed that BM-MSCs derived from MM patients migrated more vigorously to myeloma cell line. Furthermore, proteasome inhibitor bortezomib was showed to suppress chemotactic migration of BM-MSCs whatever their origins. However, although the chemotactic migration of BM-MSCs derived from MM patients to myeloma cell line was more significantly suppressed by bortezomib treatment, migration to SDF-1 or FBS of BM-MSCs was less compromised. Both SDF-1 and TNF-α enhanced phosphorylation of iκ-Bα in BM-MSCs. Although bortezomib significantly inhibited the iκ-Bα phosphorylation by SDF-1, it had little effect on iκ-Bα phosphorylation by TNF-α. Collectively, our results suggested that aberrant chemotactic migration of BM-MSCs derived from MM patients and the possible migration-regulatory role of bortezomib treatment.