Engraftment of heavily transfused patients with severe aplastic anemia with a fludarabine-based regimen.

We have developed a practical conditioning regimen without anti-thymocyte globulin (ATG), irradiation, or other myeloablative alkylating agent for low-income countries in which patients with severe aplastic anemia (SAA), who usually have heavily transfused and a prolonged disease history. The application of ATG, Busulphan, and/or irradiation to cyclophosphamide (Cy) to avoid graft rejection has many short- and long-term complications. In this study, we focused on evaluating a fludarabine-based conditioning regimen, among 83 patients with SAA. Patients were treated with fludarabine (40 mg/m(2) /d; day [-5 to -2]) and cyclophosphamide (50 mg/kg/d; day [-5 to -2]). Altogether, 81 patients indicated initial engraftment, whereas two cases showed primary graft failure. And four of the 81 cases indicated graft rejection during follow-up. Regardless of a high cumulative incidence of acute (55/83; 66.2% grade II-IV; 47/83; 56.6% III-IV) and chronic graft-versus-host disease (50/83; 60.2%), in total, 77 patients showed durable engraftment and transfusion independence, and 64 are alive at a median time of 49 months with an overall survival rate of 66%. In conclusion, this conditioning indicated well toleration, mild toxicity, durable engraftment, excellent survival as well as less cost. Its application might shed new light on SAA at high risk of graft rejection in resource-limited countries.

[Significance of Lymphocyte-to-Monocyte Ratio and Corrected Levels of Serum Calcium as Prognostic Markers in Patients with Newly Diagnosed Multiple Myeloma].

OBJECTIVE: To investigate the significance of peripheral blood lymphocyte to monocyte ratio (LMR) and corrected levels of serum calcium (cCa) as prognostic markers for the newly diagnosed multiple myeloma (MM) patients. METHODS: The clinical data of 114 newly diagnosed MM patients in the Second Affiliated Hospital of Kunming Medical University from January 2013 to March 2020 were retrospectively analyzed. Receiver operating characteristic (ROC) curve analysis was used to identify the optimal cutoff value, and the patients were divided into high LMR group and low LMR group (LMR≥3.35 and LMR < 3.35). Moreover, the patients were divided into four groups according to initial diagnosis LMR and LMR after four courses of treatment (LMR4): Group A (LMR≥3.35, LMR4≥3.35), Group B (LMR≥3.35, LMR4 < 3.35), Group C (LMR < 3.35, LMR4≥3.35), and group D (LMR < 3.35, LMR4 < 3.35). The simple prognosis model was established by combined with LMR and cCa, the patients were divided into Group a (no risk factor), group b (1 risk factor) and Group c (2 risk factors). Independent sample T-test, Pearson Chi-square test or Mann-Whitney U test were used to evaluate the differences between various parameters, and Kaplan-Meier method and Cox regression were used for survival analysis. RESULTS: The median follow-up time was 13.05（0.1-72.5）months. Survival analysis showed that the patients with low LMR predicted poor prognosis, the overall survival (OS) time of the patients with low LMR was significantly shorter (17 vs 50.5 months, P=0.006) than the patients with high LMR, the difference was also significant between group A and Group D (56.5 vs 30.5 months, P=0.043). The OS of the patients was also significantly shorter in the high cCa group (≥2.75 mmol/L) compared with normal group (8.5 vs 34 months, P=0.006). Multivariate survival analysis showed that LMR < 3.35 (P=0.028) and cCa≥2.75 mmol/L (P=0.036) were the independent risk factors affecting prognosis of MM patients. The comparison of risk factors showed that the median OS of Group a, b and c was 50, 20, and 8.5 months, respectively. The prognosis of the patients without risk factors was better than that of patients with 1-2 risk factors (Group a vs Group b, P < 0.0001; Group a vs Group c, P=0.002). CONCLUSION: LMR and cCa are the independent risk factors affecting the prognosis of newly diagnosed MM patients, and the development of a simple prognosis system combining them can quickly identify the prognosis of newly diagnosed MM patients.

Multicenter phase II study of a combination of cyclosporine a, methotrexate and mycophenolate mofetil for GVHD prophylaxis: results of the Chinese Bone Marrow Transplant Cooperative Group (CBMTCG).

BACKGROUND: Improvement of current GVHD prophylactic therapies remains an important goal in the allo-HSCT. We have described a novel prophylaxis regimen in a single institution trial. The Chinese Bone Marrow Transplant Cooperative Group (CBMTCG) initiated a phase II multicenter study. METHODS: The study was designed as a prospective, single arm phase II open-label, multicenter clinical trial. The primary endpoint was improvement of aGVHD by 25% over historical control (40%) in Chinese patients. 508 patients were enrolled. All of the patients received cyclosporine A (CsA), methotrexate (MTX) and mycophenolate mofetil (MMF) (0.5-1.0 g daily for 30 days) as GVHD prophylaxis regimen. RESULTS: The primary endpoint was met with cumulative incidences of grades 2 to 4 and grades 3 to 4 aGVHD of 23.2% and 10.3%, respectively. Incidence for cGVHD was 67.4%. The non-relapse mortality (NRM) rate was 18.4% at 2 years. The probabilities of leukemia free survival (LFS) for non-advanced stage and advanced stage patients at 2 years were 69.7% and 44.8% respectively (p = 0.000). Recipient age ≥ 40 years, advanced stage and Busulfan-Fludarabine(BuFlu) conditioning regimen were identified as major risk factors for aGVHD. Recipient age ≥ 40 years, BuFlu conditioning regimens, female donor/male recipient and prior aGVHD were associated with cGVHD. Despite lower RM (relapse mortality), patients with grade 2-4 aGVHD had higher NRM and worse OS and LFS compared to patients with grade 0-1 aGVHD. In contrast, patients with cGVHD had better OS and LFS and lower RM compared to patients without cGVHD. CONCLUSION: The novel GVHD regimen decreased the risk for aGVHD by 42% without improving the risk for cGVHD compared to historical controls. Development of aGVHD was associated with worse OS and LFS as well as higher NRM. In contrast, cGVHD was associated with improved OS and LFS likely attributed to a GVL effect.

[HLA haploidentical peripheral blood stem cells transplantation for ß thalassemia major].

OBJECTIVE: To evaluate the feasibility of HLA haploidentical peripheral blood hematopoietic stem cell transplantation (PBSCT) for patients with ß thalassemia major. METHODS: Sixteen patients with ß thalassemia major received HLA haploidentical PBSCT from parents. Two conditioning regimens were used. Regimen A was adopted before December 2007, which consisted of fludarabine (total 150 mg/m²), busulfex (total 520 mg/m²), cyclophosphamide (CTX, total 100 mg/kg), antithymocyte globulin (ATG, total 10 mg/kg) and total body irradiation of 3 Gy. Regimen B was adopted after December 2007, which consisted of fludarabine (total 240 mg/m²), busulfex (total 520 mg/m²), CTX (total 100 mg/kg), and ATG (total 10 mg/kg). Combination of cyclosporin (CsA), methotrexate (MTX) and mycophenolate mofetil (MMF) were used for prophylaxis of graft-versus-host disease (GVHD). RESULTS: Of 16 patients, 14 (87.5%) had sustained engraftment. The median days of neutrophil exceeding 0.5 × 109/L and platelet exceeding 20 × 109/L were 13 days (range 10 - 17 days) and 15 days (range 14 - 20 days) after PBSCT, respectively. Complete chimerism was achieved in all the 14 patients at one month after PBSCT. One patient lost his graft with autologous reconstitution 52 days after transplantation. Four patients had grade II-IV acute GVHD and one patient had chronic extensive GVHD. In the 49-month median follow-up duration, 13 of 16 patients were alive in disease-free situation. CONCLUSION: HLA haploidentical PBSCT, which could provide stable and sustained engraftment for thalassemia major patients with no HLA identical donor, is a promising treatment strategy.

[Tyrosine kinase JAK2V617F mutation in human myeloproliferative disorders].

This study was aimed to investigate the JAK2V617F mutation in myeloproliferative disorders (MPD) and to evaluate the significance of JAK2V617F in diagnosis and therapy of MPD. The bcr/abl fusion gene in 70 MPD patients was detected by reverse transcription polymerase chain reaction (PT-PCR). The JAK2V617F mutation was detected by allele-specific polymerase chain reaction (AS-PCR) and the results were confirmed by sequence analysis. The results indicated that the bcr/abl fusion gene could be detected in 38 patients with chronic myeloid leukemia (CML), but not in the 32 none-CML patients. The JAK2V617F mutation was detected in 12 out of 16 (75%) patients with polycythemia vera (PV), 3 out of 10 (30%) patients with essential thrombocythemia (ET), 3 out of 6 (50%) patients with idiopathic myelofibrosis (IMF), but not in any of the CML patients. The JAK2V617F mutation frequencies between CML and bcr/abl negative MPD patients were statistically significant (p < 0.05). It is concluded that the JAK2V617F may be a characteristic molecular event in PV, ET and IMF patients which may serve as an important molecular marker for the diagnosis and classification of the three diseases.

[Role of paternal veto cells in preventing graft-versus-host disease after HLA-haploidentical stem cell transplantation in mice].

OBJECTIVE: To explore the role of paternal veto cells in preventing graft-versus-host disease (GVHD) after related HLA- haploidentical stem cell transplantation in mice. METHODS: MHC-haploidentical recipient B6CF1(H-2 b/d) mice pretreated with total body irradiation at 9.0 Gy for 4 h before transplantation. The recipient mice were divided into 4 groups, and in the irradiation group, only injection of 0.3 ml D-Hank's liquid was given through the tail vein; in the control group, the mice received injection through the tail vein of 4.5x10(6) bone marrow cells mixed with 3.0x10(7) spleen cells from C57BL/6 mice without the preventive measures of GVHD; the mice in the two experiment groups received cell transplantation in the same manner, and on day 4 after transplantation, 5.0x10(6) and 1.0x10(7)spleen cells from BALB/c mice were injected through tail vein, respectively. The hematopoietic recovery, engraftment and GVHD of the recipient mice were observed. RESULTS: Without any treatment, all mice in the control group developed GVHD and died after transplantation. In the 10 mice with injection of 5.0x10(6) spleen cells, GVHD occurred in 5 mice with a 30-day survival rate of 50%; the median survival time of the mice with GVHD was 20 days, significantly longer than that of the control mice (14 days, P<0.05). In the 10 mice injected with 1.0x10(7) spleen cells, 2 developed GVHD and the 30-day survival rate was 80% (8/10) with a median survival time of 30 days, significantly longer than that of mice with injection of 5.0x10(6) spleen cells and the control mice (P<0.05). CONCLUSION: Paternal veto cell transplantation can decrease the occurrence of GVHD after related HLA haploidentical stem cell transplantation in mice.

[Are leukemic patient bone marrow mesenchymal stem cells malignant?].

The study was aimed to explore whether there are leukemic characteristics in the bone marrow mesenchymal stem cells (BMMSC) from leukemic patients as compared with normal controls. The mesenchymal stem cells from bone marrow of normal volunteers and patients with APL and CML were isolated, then cultured and proliferated in vitro. The morphology, growth curve and cell surface markers of two different sources mesenchymal stem cells were investigated for detecting whether the bone marrow mesenchymal stem cells derived from leukemia patients have the specific abnormal fusion gene of leukemia cells through fluorescent in situ hybridization. The results indicated that there was no significant difference between the mesenchymal stem cells derived from different subjects, the bone marrow mesenchymal stem cells derived from leukemia patients did not have the clonal malignant fusion gene as seen in the leukemia cells. Taken altogether, mesenchymal stem cells derived from leukemia patients had no biological differences as compared with those from normal volunteers, and no malignant clonal abnormality was found. It is concluded that mesenchymal stem cells derived from leukemia patients as an alternative vehicle may be used for assistant of autologous hematopoietic stem cell transplantation or cell therapy and gene therapy.

[Role of TJU103 in prevention of graft-versus-host disease after allogeneic stem cell transplantation in mice].

OBJECTIVE: To explore the role of TJU103 in preventing graft-versus-host disease (GVHD) after allogeneic stem cell transplantation in mice. METHODS: BALB/c mouse splenic lymphocytes were collected and treated by mitomycin as the activating cells and the C57BL/6 mouse splenic lymphocytes as the reacting cells. In the experimental groups, the effect of TJU103 on the proliferative response of T cells was observed. BALB/c(H-2d) and CB6F1(H-2d/b) mice were used as the MHC-full-mismatched recipients and MHC-haplo-identical recipients, respectively, and pretreated by total body irradiation at 9.0 Gy before transplantation. For the recipients of the irradiation group, 0.3 ml D-Hank's solution was injected through the tail vein without cell transplantation, the recipients of the control group received injection of 4.5x10(6) bone marrow cells mixed with 3.0x10(7) spleen cells from C57BL/6 mice through the tail vein, and those in the experimental group received cell transplantation in the same manner with also injection via the tail vein of 25 microg/ml TJU103, which was subsequently injected intraperitoneally for 7 consecutive days at daily dose of 50 microg. The hematopoietic recovery, engraftment and GVHD of the recipients were observed. RESULTS: TJU103 resulted in a dose-dependent inhibition of T cell proliferation in mixed lymphocyte reaction (MLR), and nearly 83% inhibition of the proliferative response was observed with the addition of 25 microg/ml of TJU103. Without any treatment, the occurrence of GVHD and death rate in the control group was both 10/10. Daily injection of TJU103 at 50 microg for the initial post-transplantation week protected the mice from GVHD. In the MHC-full-mismatched model, the incidence of GVHD and survival rate on day 30 of the experiment group was 2/10 and 8/10, showing significant difference from those in the control group (P<0.01). The median survival time (MST) was 30 days in the experimental group versus 15 days in the control group (P<0.05). In the MHC-haplo-identical model, the incidence of GVHD and the survival rate on day 30 of the experimental group was 1/10 and 9/10, which were significantly different from the control group (P<0.01). The MST was 30 days in the experimental group versus 14 days in the control group (P<0.05). CONCLUSION: TJU103 is capable of markedly inhibiting T cell proliferative response in vitro and can decrease GVHD incidence after allogeneic stem cell transplantation in mice.