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Background Maintenance therapy (MT) deepens response and prolongs progression free survival (PFS) in patients with newly diagnosed multiple myeloma (NDMM) after frontline regimens. Ixazomib, a 2 nd generation oral proteasome inhibitor (PI), has been approved for MT because of the convenience and tolerability. Aims We conducted this prospective multi-center study to compare the efficacy and safety of Ixazomib (I-MT) or Ixazomib plus Lenalidomide (IL-MT) to Lenalidomide (L-MT) as maintenance regimens in NDMM patients. Methods This study was approved by the Institutional Review Board of Peking Union Medical College Hospital and registered (NCT04217967). NDMM patients were enrolled from 10 centers of North China MM Registry since September 2019. After 4 cycles of front-line induction therapy, patients who reached partial response (PR) would receive autologous stem cell transplantation (ASCT) if eligible, or keep up to 5 cycles of front regimens if ineligible, then start maintenance therapy. Patients did not reach PR would switch to a 2nd-line induction for 2-5 cycles and start MT once PR was achieved. For MT, 4mg of Ixazomib was given on day 1,8,15, and 25mg of Lenalidomide every other day on days 1-21 of a 28-day cycle. Patients in dual drug group were administrated with both Ixazomib and Lenalidomide, dose as listed above. The primary endpoint was PFS from MT. Results A total of 149 patients were enrolled, including 54 in I-MT, 65 in L-MT and 30 in IL-MT. The demographic and clinical characteristics were comparable among three groups at baseline (Table 1), including gender ratio, age, paraprotein isotype, ISS, R-ISS, response status before MT, and ASCT rate. The proportions of patients with high-risk cytogenetic abnormalities (HRCAs), defined as amplification 1q21, deletion 17p, t(4,14) and t(14,16), were comparable. The median follow-up duration since MT was 6.1, 11.1, and 5.9 months in I-MT, L-MT and IL-MT group, respectively. Disease progression developed in 9.3%, 12.3% and 10% (N=5, 8, 3, respectively) patients. The median PFS was not reached (NR) in all groups. Only one death occurred in I-MT group. There were 84%, 72.3% and 83.3% of the patients reached very good partial response (VGPR) or better before MT, while the best response rates rose to 93%, 82.3% and 90% during maintenance. The prevalence of peripheral neuropathy was 18.5% on I-MT, 10.8% on L-MT and 30% on IL-MT. Grade 2 PN occurred in 3, 1, and 0 patients, respectively. The incidence of gastrointestinal events was 11.1%, 1.5% and 20%, respectively. Grade 3-4 hematologic toxicities was 3.7%, 4.6%, and 3.3%. Infection rates were 7.4%, 6.2% and 3.3%. Skin rashes were more common in lenalidomide containing regimens (3.7%, 7.3% and 6.7%). No drug withdrawal was related to adverse events. Conclusions Due to inadequate access to melphalan and low rate of ASCT in China, there is still a gap of PFS in NDMM patients with those in western countries. We herein design this multi-centered prospective study to evaluate if dual drug MT will further strengthen response and make up the gap. Though the primary endpoint--PFS has not been reached in all treatment groups, dual MT improves response most and is quite tolerable. Figure 1. Baseline Characteristics in three groups. Abbreviations: ISS: international staging system; R-ISS: revised-ISS; HRCAs: high risk cytogenetic abnormalities. sCR: stringent complete remission. CR: complete remission. VGPR: very good partial remission. PR: partial remission. * p ＜ 0.05. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

DNA (cytosine-5)-methyltransferase 3A (DNMT3A) mutations occur in ~20% of de novo acute myeloid leukemia (AML) patients, and >50% of these mutations in AML samples are heterozygous missense alterations within the methyltransferase domain at residue R882. DNMT3A R882 mutations in AML patients promote resistance to anthracycline chemotherapy and drive relapse. In this study, we performed high-throughput screening and identified that oridonin, an ent-kaurene diterpenoid extracted from the Chinese herb Rabdosia rubescens, inhibits DNMT3A R882 mutant leukemic cells at a low-micromolar concentration (IC50 = 2.1 µM) by activating both RIPK1-Caspase-8-Caspase-3-mediated apoptosis and RIPK1-RIPK3-MLKL-mediated necroptosis. The inhibitory effect of oridonin against DNMT3A R882 mutant leukemia cells can also be observed in vivo. Furthermore, oridonin inhibits clonal hematopoiesis of hematopoietic stem cells (HSCs) with Dnmt3a R878H mutation comparing to normal HSCs by inducing apoptosis and necroptosis. Overall, oridonin is a potential and promising drug candidate or lead compound targeting DNMT3A R882 mutation-driven clonal hematopoiesis and leukemia.

The role of mammalian target of rapamycin and its suppressor sirolimus in the regulation of hematopoietic stem and progenitor cells (HSPCs) is controversial. We show here that sirolimus enhanced regeneration of HSPCs in mice exposed to sublethal total body irradiation (TBI) and other regenerative stressors. Sorted Lin−CD150+ bone marrow cells from sirolimus‐treated TBI mice had increased expression of c‐Kit and other hematopoietic genes. HSPCs from sirolimus‐treated TBI mice were functionally competent when tested by competitive engraftment in vivo. Postradiation regeneration of HSPCs in mice treated with sirolimus was accompanied by decreased γ‐H2AX levels detected by flow cytometry and increased expression of DNA repair genes by quantitative polymerase chain reaction. Reduction of cell death and DNA damage post‐radiation by sirolimus was associated with enhanced clearance of cellular reactive oxygen species (ROS) in HSPCs. Increased HSPC recovery with sirolimus was also observed in mice injected with hematoxic agents, busulfan and 5‐fluorouracil. In contrast, sirolimus showed no effect on HSPCs in normal mice at steady state, but stimulated HSPC expansion in mice carrying the Wv mutation at the c‐Kit locus. In human to mouse xenotransplantation, sirolimus enhanced engraftment of irradiated human CD34+ cells. In summary, our results are consistent with sirolimus' acceleration of HSPC recovery in response to hematopoietic stress, associated with reduced DNA damage and ROS. Sirolimus might have clinical application for the treatment and prevention of hematopoietic injury., Sirolimus accelerates hematopoietic stem and progenitor cell recovery in response to hematopoietic stress, which is associated with reduced DNA damage and reactive oxygen species.

Pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF, pegfilgrastim) is a long-acting derivative of recombinant human granulocyte colony-stimulating factor with limited renal clearance and a longer half-life. It is used for the prevention of febrile neutropenia, owing to its capacity to promote neutrophil recovery. In this study, the pharmacokinetics, pharmacodynamics, safety, and immunogenicity of 2 formulations of PEG-rhG-CSF were evaluated in healthy Chinese subjects. Twenty-four male subjects who received a single dose of subcutaneous PEG-rhG-CSF 100 µg/kg were randomized to either treatment A (3 mg/mL) or treatment B (1 mg/mL). Noncompartmental pharmacokinetic parameters of PEG-rhG-CSF were derived from serum concentration-time data. In addition, absolute neutrophil count (ANC) as a pharmacodynamic maker, immunogenicity through antidrug antibody testing, and safety were evaluated. The mean area under the concentration-time curve from time zero to the last quantifiable concentration (AUC0-t ) and the mean maximum concentration (Cmax ) of PEG-rhG-CSF after treatment A were 5070 ng·h/mL and 125 ng/mL, respectively; these values were comparable to those measured after treatment B (5340 ng·h/mL and 123 ng/mL, respectively). The mean value of area under the △ANC (baseline-adjusted ANC)-time curve and the maximum △ANC values were 4380 × 109 h/L and 33.1 × 109 /L, respectively, in the treatment A group, and 5170 × 109 h/L and 38.6 × 109 /L, respectively, in the treatment B group. The pharmacokinetic and pharmacodynamic profiles were similar for the 2 PEG-rhG-CSF formulations following a single dose of 100 µg/kg. The safety and immunogenicity profiles were also similar, with no significant differences. The dose adjustment of PEG-rhG-CSF was not considered necessary for formulation transformation.

Background The current situation of maintenance (MT) after front-line therapy in non-transplant patients with multiple myeloma (MM) is not adequate in China. Hence, we conducted this multi-centered retrospective real-world study on efficacy and safety of the mainstream maintenance regimens, thalidomide (T-MT), lenalidomide (L-MT) and bortezomib (B-MT). Clinical data of newly diagnosed MM patients were collected from 9 centers of North China MM Registry from January 2010 to December 2020. The progression-free (PFS) and overall survival (OS) from maintenance, and adverse events were recorded. Method A total of 316 patients were enrolled including 156 in T-MT, 113 in L-MT and 47 in B-MT. At baseline, the gender ratio, paraprotein isotype, ISS, R-ISS, and response evaluation before MT were comparable. Patients on L-MT were older. Greater proportions of patients in L-MT and B-MT had high-risk cytogenetic abnormalities (HRCA), defined as amplification 1q21 (1q21+), deletion 17p (17p-), t(4,14), t(14,16). Results The median follow-up duration was 41.0, 21.9 and 20.7 months (m) in T-MT, L-MT and B-MT, respectively. Disease progression rate was 63.5%, 46% and 31.9%. Mortality rate was 29.5%, 19.5% and 8.5%. The median PFS was 23.7m in T-MT, compared with 23.5m in L-MT and 30.8m in B-MT (p=0.48). Median OS was 79.5m in T-MT, whereas not reached (NR) in the others (p=0.21). Patients reaching complete response (CR) or stringent CR (sCR) before MT had prolonged PFS compared to those with very good partial response (VGPR) or less in T-MT (28.7m vs 17.2m, p=0.048) and L-MT (29.9m vs 14.8m, p=0.01), while comparable in B-MT (NR vs 30.8, p=0.15). OS was similar despite of different response before MT. Patients with 1q21+ on T-MT had shorter PFS compared to those without (16.3m vs 22.8m, p=0.18), so did impaired median OS (53.1m vs 80.6m, p= 0.003). 1q21+ did not affect PFS in L-MT group (23.5m vs 27.4m, p=0.69), or B-MT (NR vs 30.8m, p=0.73). OS was not reached. Only a few patients with 17p-, yet also presented remarkably inferior PFS (6.8m vs 22.8m, p=0.007) and OS (32.3m vs 72.6m, p=0.02) in T-MT. PFS (15.6m vs 39.2 m, p=0.33) and OS (NR vs NR, p=0.83) were not of discrepancy in L-MT. In patients with any HRCA, T-MT resulted in shorter PFS (12.1m vs 22.8m, p=0.06) and OS (53.1m vs NR, p Conclusion In this multi-centered real-world study, thalidomide, a response of CR or better predicts superior PFS in non-transplant NDMM. HRCAs drag down survival in thalidomide, while lenalidomide and bortezomib mostly reverse the negative effects. Though patients on L-MT and B-MT have greater proportion of HRCAs, PFS in three groups are comparable. Clinicians in the real practice prefer lenalidomide or bortezomib as maintenance in high-risk patients.

It is unknown whether adding stanozolol to decitabine for maintenance can further improve progression-free survival (PFS) and overall survival (OS) after effective decitabine treatment in patients with high-risk myelodysplastic syndrome (MDS). Patients newly diagnosed with high-risk MDS who achieved at least partial remission after 4 cycles of decitabine (20 mg/m2 days 1–5) were selected. In total, 62 patients (median age 66 years) were enrolled, of whom 21 were treated with stanozolol and decitabine for maintenance, and 41 were treated with decitabine alone. The median number of cycles for maintenance treatment was 6 (2–11) and 5 (2–12) for the stanozolol and control groups, respectively (p > 0.05). PFS in the stanozolol group was significantly longer than in the control group (15.0 vs 9.0 months, hazard ratio [HR] = 0.35, 95%CI: 0.19–0.63, p = 0.0005), whereas OS was not significantly prolonged in the stanozolol group (21.0 vs 15.0 months, HR = 0.73, 95%CI: 0.39–1.37, p = 0.33). The proportion of patients with severe neutropenia during maintenance treatment in the stanozolol group was lower than in the control group (76.2% vs 95.1%, p = 0.039). In conclusion, adding stanozolol to decitabine after effective decitabine treatment can prolong PFS and reduce the severity of neutropenia for patients with high-risk MDS.

Interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) have been implicated historically in the immune pathophysiology of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. We recently defined the essential roles of IFN-γ produced by donor T cells and the IFN-γ receptor in the host in murine immune-mediated BM failure models. TNF-α has been assumed to function similarly to IFN-γ. We used our murine models and mice genetically deficient in TNF-α or TNF-α receptors (TNF-αRs) to establish an analogous mechanism. Unexpectedly, infusion of TNF-α−/− donor lymph node (LN) cells into CByB6F1 recipients or injection of FVB LN cells into TNF-αR−/− recipients both induced BM failure, with concurrent marked increases in plasma IFN-γ and TNF-α levels. Surprisingly, in TNF-α−/− recipients, BM damage was attenuated, suggesting that TNF-α of host origin was essential for immune destruction of hematopoiesis. Depletion of host macrophages before LN injection reduced T-cell IFN-γ levels and reduced BM damage, whereas injection of recombinant TNF-α into FVB-LN cell-infused TNF-α−/− recipients increased T-cell IFN-γ expression and accelerated BM damage. Furthermore, infusion of TNF-αR−/− donor LN cells into CByB6F1 recipients reduced BM T-cell infiltration, suppressed T-cell IFN-γ production, and alleviated BM destruction. Thus, TNF-α from host macrophages and TNF-αR expressed on donor effector T cells were critical in the pathogenesis of murine immune-mediated BM failure, acting by modulation of IFN-γ secretion. In AA patients, TNF-α–producing macrophages in the BM were more frequent than in healthy controls, suggesting the involvement of this cytokine and these cells in human disease.

Background Maintenance (MT) after front-line therapy is the current standard for multiple myeloma (MM). However, no adequate data has shown the present situation of maintenance treatment in China yet, and autologous stem cell transplantation (ASCT) rate is still low in eligible newly diagnosed MM (NDMM) patients. Hence, we conducted this retrospective real-world study on efficacy and safety of the mainstream maintenance regimens in non-transplant NDMM patients-thalidomide (T-MT), lenalidomide (L-MT) and bortezomib (B-MT). Methods Clinical data were collected from 9 centers of North China MM Registry, during January 2010 to December 2020. The progression-free (PFS) and overall survival (OS) from maintenance, and drug toxicities were compared in T-MT, L-MT and B-MT groups. Thalidomide 75-150mg/day was administrated in T-MT group. L-MT group received lenalidomide as 25mg every other day or 10mg daily, on day 1-21 of a 28-day cycle. Bortezomib (1.3 mg/m 2 s.c.) was administered every other week or 4 vials every 3 months. Dexamethasone was given along with L or B in some patients. Results A total of 355 patients were enrolled including 159 in T-MT, 143 in L-MT and 53 in B-MT. At baseline, the gender ratio, paraprotein isotype, ISS and R-ISS stage, as well as response status before MT were comparable. Patients on L-MT were significantly older than the other groups. Meanwhile, greater proportions of patients in L-MT and B-MT groups had high-risk cytogenetic abnormalities (HRCA), defined as amplification 1q21 (1q21+), deletion 17p (17p-), t(4,14), t(14,16). The median follow-up duration since maintenance was 40.1, 19.6 and 22.2 months (m) in T-MT, L-MT and B-MT groups, respectively. There were 67.9%, 61.5% and 60.4% patients with T-MT, L-MT and B-MT achieving very good partial response (VGPR) or better before maintenance. Disease progression was recorded in 101 patients (63.5%) with T-MT, 54 (37.5%) with L-MT and 19 (35.8%) with B-MT. While mortality was 46 (28.9%), 22 (15.3%) and 4 (7.5%), respectively. The median PFS was 23m in T-MT, as compared with 26.9m in L-MT and 37.0m in B-MT (p=0.59). Median OS was 91.0M in T-MT, whereas not reached (NR) in the others (p=0.50). Patients reached complete response (CR) or stringent CR (sCR) before MT had prolonged PFS compared to those with VGPR or less in T-MT (28.0m vs 17.0m, p=0.06) and L-MT group (27.4m vs 18.2m, p=0.02), while comparable in B-MT (NR vs 30.8m, p=0.25). Meanwhile, patients in each group had similar OS despite of different responses before MT. Patients with 1q21+ on T-MT had shorter median PFS compared to those without (12.2m vs 21.0m, p=0.08), as well as impaired median OS (53.1m vs 81.0m, p= 0.004), despite various second line therapies. While the PFS of L-MT was 26.9m for patients with 1q21+ and 27.4months for those without (p=0.99). In B-MT group, the PFS was 43.5m and 30.2m (p=0.64), respectively. Median OS was not reached in both L-MT and B-MT. Only a few patients with 17p- in T-MT and B-MT, yet also presented remarkably inferior PFS (7.0m vs 21.0m, p=0.011) and OS (32.0m vs 81.0m, p=0.001) with thalidomide. As for L-MT, PFS (22.2m vs 27.4 m, p=0.19) and OS (NR vs NR, p=0.55) were not of discrepancy between 17p- or without. In B-MT, PFS (30.8m vs NR, p=0.99) was similar, though median OS was not reached, inferior tendency was observed (p= 0.04). As for patients with any adverse CA, T-MT resulted in impaired PFS (12.0m vs 23.0m, p=0.02) and OS (53.0m vs NR, p＜0.01). In contrast, PFS and OS were both comparable in patients with L-MT or B-MT no matter with HRCA or not. The main reason of maintenance withdrawal was disease progression. Among patients with detailed records of adverse effects, adverse event related discontinuation was seen in 5.3%(n=5), 6.1%(n=3) and 0 patients in T-MT, L-MT and B-MT, respectively. Conclusions In this multi-centered real-world maintenance study, thalidomide, lenalidomide or bortezomib after front-line therapy in non-transplant NDMM patients has similar PFS and OS. However, patients on L-MT and B-MT have greater proportion of HRCAs, which drags down survival in T-MT especially with 1q21+ and 17p-, while L-MT and B-MT mostly reverses the negative effects. Clinicians in the real practice prefer to select lenalidomide or bortezomib as maintenance in patients with HRCAs, while thalidomide is still an option for patients with standard risk. Figure 1. A) Baseline characteristics in three groups. * p＜ 0.05. B) PFS of the three groups. C) OS of the three groups. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Interleukin-18 (IL-18), also known as interferon-gamma (IFN-γ)-inducing factor, is involved in Th1 responses and regulation of immunity. Accumulating evidence implicates IL-18 in autoimmune diseases, but little is known of its role in acquired aplastic anemia (AA), the immune-mediated destruction of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). IL-18 protein levels were significantly elevated in sera of severe AA (SAA) patients, including both responders and nonresponders assayed before treatment, and decreased after treatment. IL-18 receptor (IL-18R) was expressed on HSPCs. Co-culture of human BM CD34+ cells from healthy donors with IL-18 upregulated genes in the helper T-cell and Notch signaling pathways and downregulated genes in the cell cycle regulation, telomerase, and IL-6 signaling pathways. Plasma IL-18 levels were also elevated in murine models of immune-mediated BM failure. However, deletion of IL-18 in donor lymph node cells or deletions of either IL-18 or IL-18R in recipients did not attenuate elevations of circulating IFN-γ, tumor necrosis factor-alpha, or IL-6, nor did they alleviate BM failure. In summary, our findings suggest that, although increased circulating IL-18 is a feature of SAA, it may reflect an aberrant immune response but be dispensable to the pathogenesis of AA.

Tumor necrosis factor alpha (TNF-α) is a critical cytokine in systemic inflammation and a potent inducer of apoptotic cell death. Type 1 cytokines are involved in the pathogenesis of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. Overproduction of interferon gamma (IFN-γ) and TNF-α from activated T cells or bone marrow cells has been proposed as a mechanism of BM failure. We hypothesized that inhibiting the production of TNF-α or blockade of the TNF-α signaling pathway would attenuate hematopoietic cell destruction in murine models of immune BM failure. Therefore, we tested the role of TNF-α in several mouse models of human AA, using C57BL/6 (B6) mice carrying germline deletions of TNF-α (TNF-α-/-) or its receptors including TNF-α receptor superfamily member 1a (TNF-rsf1a-/-), TNF-rsf1b-/-, and TNF-rsf1a-/-1b-/-as donors or recipients, respectively. Lymph node (LN) cells from both TNF-α-/- and TNF-α receptor knock out mice induced fatal BM failure with efficacy similar to LN cells from wild-type B6 donors when infused into CByB6F1 recipients that had received 5 Gy of total body irradiation (TBI), indicating that expression of TNF-α or TNF-α receptors on effector cells did not affect the development of BM failure. However, TNF-α-/- mice as recipients unexpectedly showed resistance to immune-mediated BM failure. Infusion of 5 million LN cells from MHC-mismatched FVB donors to 6.5 Gy TBI pre-conditioned TNF-α-/- recipients did not induce AA: the animals had normal or increased levels of WBC, RBC, platelets, and total BM cells relative to TBI-only TNF-α-/- mice; control wild-type B6 recipient mice developed severe pancytopenia and BM failure after LN infusion at 10 days (Fig. 1A&B). This result suggests that TNF-α production in the recipients had a role in immune attack by effector cells. When residual BM cells were cultured in vitro in cytokine-supplemented methylcellulose media, cells from wild-type BM failure mice, but not from TNF-α-/- recipient mice, showed a significant reduction in colony forming units relative to their TBI controls. By flow cytometry, Fas+ cells and Annexin V-binding cells were increased in BM of wild-type marrow failure mice relative to wild-type TBI controls, but no increase in Fas+ cells and Annexin V-binding cells was observed in TNF-α-/- mice that had received LN infusion. In parallel experiments, injection of FVB LN cells into sub-lethally-irradiated TNF-rsf1a-/-, TNF-rsf1b-/-, or TNF-rsf1a-/-1b-/- mice resulted in BM failure with declines in WBC and platelets as observed in wild-type B6 mice after FVB LN infusion. Plasma levels of both IFN-γ and TNF-α were markedly increased in all the wild-type B6 and TNF-α receptor knock out mice that developed BM failure, but not in TNF-α-/- recipient mice. These data suggest that depletion of TNF-α receptors could not attenuate immune-mediated BM destruction and that TNF-α did not exert its destructive effect on hematopooetic targets through the TNF-α/TNF-rsf1a and 1b pathways. We hypothesized that TNF-α might modulate T cell IFN-γ production to affect immune-mediated BM destruction. TNF-α is mainly produced by activated macrophages, while IFN-γ is mainly secreted by activated T cells. Blockade of either IFN-γ in effector cells or IFN-γ receptor in target cells ameliorates the development of BM failure (Chen J, Blood.2015;126:2621). To test this hypothesis, we first depleted macrophages from CByB6F1 recipient mice with liposomal clodronate before BM failure induction with TBI and B6 LN cell injection. Indeed, removal of macrophages attenuated BM destruction relative to BM failure mice pre-injected with PBS-loaded liposomes without macrophage depletion (Figure 1C). When we injected recombinant TNF-α daily i.v. for 1 week into TNF-α-/- recipients following TBI and FVB LN cell infusion, TNF-α-injected mice had decreased BM cellularity relative to mice that received TBI and LN infusion without TNF-α (Figure 1D). Remarkably, TNF-α injection increased intracellular IFN-γ expression in BM T cells as determined by flow cytometry, indicating that TNF-α was capable of stimulating T cells to secrete IFN-γ. In summary, our study reveals a critical role of TNF-α in target cells but not in effector cells, in the pathogenesis of immune-mediated BM failure by cell-cell "crosstalk" modulation of IFN-γ secretion. Disclosures Young: Novartis/GSK to institute: Research Funding.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by intravascular hemolysis, venous thrombosis, and bone marrow (BM) failure. Until now, allogeneic hematopoietic stem cell transplantation is still the only way to cure PNH. Eculizumab, although very promising, is not the eradication of the disease because of raising the possibility of severe intravascular hemolysis if therapy is interrupted. Here we enriched the residual bone marrow normal progenitor cells (marked by CD34+CD59+) from PNH patients, tried to find an effective way of expanding the progenitors cells used for autologous bone marrow transplantation (ABMT). Objective To expand CD34+CD59+ cells isolated from patients with PNH and observe the long-term hemaotopoietic reconstruction ability of the expanded cells both ex vivo and in vivo. Methods CD34+CD59+ cells from 13 patients with PNH and CD34+ cells from 11 normal controls were separated from the bone marrow monouclear cells first by immunomagnetic microbead and then by flow cytometry autoclone sorting. The selected cells were then cultivated under different conditions for two weeks to find out the optimal expansion factors. The long-term hematopoietic supporting ability of expanded CD34+CD59+ cells was evaluated by long-term culture in semi-solid medium in vitro and long-term engraftment in irradiated severe combined immunodeficiency(SCID) mice in vivo. Results The best combination of hematopoietic growth factors for ex vivo expansion was SCF+IL-3+IL-6+FL+Tpo+Epo, and the most suitable time for harvest was on day 7. Although the CD34+CD59+ PNH cells had impaired ex vivo increase compared with normal CD34+ cells (the biggest expansion was 23.49±3.52 fold in CD34+CD59+ PNH cells and 38.82±4.32 fold in CD34+ normal cells, P0.05). According to the above data, 11/13(84.3%) patients with PNH can get enough CD34+CD59+cells for ABMT after expansion. The survival rate and human CD45 expression in different organs was similar between the irradiated SCID mice transplanted with expanded CD34+CD59+ PNH cells and those with normal CD34+ cells (P>0.05). The peripheral blood cell count recovered on day 90 in mice transplanted with PNH cells, which was compatible with those transplanted with normal cells (P>0.05). On secondary transplantation, the peripheral blood cell count returned to almost normal on day 30 in mice transplanted with either PNH cells or normal cells. Lower CD45 percentage was found in secondary transplantation compared with primary transplantation but no difference between mice transplanted with different cells. Conclusion Isolated CD34+CD59+ cells from patients with PNH can be effectively expanded ex vivo and can support lasting hematopoiesis both ex vivo and in vivo. These data provide a new potential way of managing PNH with ABMT.

Objective: Observe the effect of normal and myeloma bone marrow stromal cells (BMSC) to the growth of myeloma cells. Evaluate the function of adhesion molecules in the mutual action of BMSC and myeloma cells. Methods: Immunomagnetic beads and anti-CD138 antibody were used to separate myeloma cells from patients’ bone marrow of multiple myeloma (MM). U266 and freshly purified myeloma cells respectively co-cultured with normal BMSC and myeloma BMSC. Single U266 or fresh myeloma cells were cultured as control group. Anti-CD11a, anti-CD29, anti-CD44 and anti-CD49d antibodies were respectively added in the co-cultured system and single myeloma cells. After culture for 72 hours, cell cycle of myeloma cells and the ratio of apoptotic cells were tested by flowcytometry. The level of interleukin 6 (IL-6) in the supernate was measured. Results: Normal BMSC inhibited the growth of myeloma cells while myeloma BMSC improved it. As to U266, S+G2% of myeloma cells in normal BMSC group and myeloma BMSC group were 55.5±4.0% and 65.4±4.5%(p Conclusion: We firstly proved that normal BMSC inhibited the growth of myeloma cells. Anti-CD29 and anti-CD44 can block the adhesion of myeloma cells and BMSC, resulting in the growth inhibition of myeloma cells. Figure Figure

A new human myeloma cell line WuS1 was established from the bone marrow of a 45-year-old Chinese male patient with IgGλ type multiple myeloma (stage IIIB). The growth of WuS1 cells is constitutively independent of exogenous growth factors of feeder cells. The WuS1 cell line proliferated consistently as free-floating single cells in suspension, sometimes in small clusters or slightly adherent on the bottom of the plastic culture flask, without forming clumps. The cell line has been maintained without any external growth factors for over a year, and cells frozen in liquid nitrogen can be revived successfully. The doubling time of the cells was about 11 hours and the colony-forming rate was 55.56±6.33%. WuS1 displayed immature plasma cell features with an obvious heterogeneity in size and a high nuclear-cytoplasmic ratio in Wright-Giemsa staining. Figure Figure They were positive for ALP, CE, ACP(not inhibited by tartrate) and PAS stainings and negative for POX and NBE. By transmission electron microscopy, the cytoplasm of WuS1 contained abundant mitochondria, and parallel endoplasmic reticulum or Golgi apparatus in some cells. The monoclonal immunoglobulin G and λ light chain were positive in cell lysate and not in cell culture supernatants by immnuoelectrophoresis. The cell surface antigens were positive for CD3, CD59, CD106 and CD138, and negative for CD4, CD5, CD8, CD10, CD13, CD14, CD19, CD20, CD22, CD29, CD31, CD33, CD34, CD38, CD44, CD49d, CD45, CD54, CD56 and HLA-DR by flow cytometry. Chromosomal analysis revealed a hypodiploidy and complex karyotype. WuS1 cells were negative for Epstein-Barr virus by PCR using EBV nuclear antigen-1 specific primers. Twelve SCID mice were injected with WuS1 cells intravenously or subcutaneously, and obvious tumor infiltration in bone marrow, liver, spleen, lung, kidney and injection site (subcutaneously group) were observed by pathologic examination. Figure Figure The novel WuS1 cell line will be useful in the study of the biology, etiology and treatment of multiple myeloma.