Prevention of acute graft-vs.-host disease by targeting glycolysis and mTOR pathways in activated T cells.

Graft-versus-host disease (GvHD) is a common life-threatening complication that can occur following allogeneic hematopoietic stem cell transplantation. This occurs if donor T cells recognize the host as foreign. During acute GvHD (aGVHD), activated T cells utilize glycolysis as the main source of energy generation. Therefore, inhibition of T cell glycolysis is a potential treatment strategy for aGVHD. In the present study, the effects of the combination of the glycolysis inhibitor 3-bromopyruvate (3-BrPA) and the mTOR inhibitor rapamycin (RAPA) on a mode of aGVHD were explored. In vitro mixed lymphocyte culture model was established by using splenocytes from C57BL/6 (H-2b) mice as responder and inactivated splenocytes from BALB/c (H-2d) mice as stimulator. In this model, 3-BrPA treatment (0-100 µmol/l) was found to suppress cell viability, increase cell apoptosis and reduce IFN-γ secretion, in a concentration-dependent manner. 3-BrPA treatment (0-100 µmol/l) was found to suppress cell viability, increase cell apoptosis and reduce IFN-γ secretion, in a concentration-dependent manner. In addition, combined treatment with 3-BrPA (0-100 µmol/l) alongside RAPA (20 µmol/l) exhibited synergistic effects on inhibiting cell viability and IFN-γ production, compared with those following either treatment alone. An aGVHD model was established by injection of bone marrow cells and spleen cells from the donor-C57BL/6(H-2b) mice to the receptor-BALB/c(H-2d) mice which were underwent total body irradiation first. In the aGVHD model, 3-BrPA (10 mg/kg/day), RAPA (2.5 and 5 mg/kg/day) and both in combination (5 and 2.5 mg/kg/day for 3-BrPA and RAPA, respectively) were all found to alleviate the damage caused by aGVHD, in addition to prolonging the survival time of mice with acute GvHD. In particular, the combined 3-BrPA and RAPA treatment resulted in the highest median survival time among all groups tested. In addition, the effects induced by combined 3-BrPA and RAPA treatment were found to be comparable to those in the 5 mg/kg/day RAPA group but superior to the 3-BrPA group with regards to the cumulative survival profile, GvHD score and lung histological score. The 3-BrPA and RAPA combination group also exhibited the lowest IFN-γ levels among all groups. Therefore, the combination of inhibiting both glycolysis and mTOR activity is a promising strategy for acute GvHD prevention.

Basiliximab for steroid-refractory acute graft-versus-host disease: A real-world analysis.

Steroid-refractory (SR) acute graft-versus-host disease (aGVHD) is one of the leading causes of early mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We investigated the efficacy, safety, prognostic factors, and optimal therapeutic protocol for SR-aGVHD patients treated with basiliximab in a real-world setting. Nine hundred and forty SR-aGVHD patients were recruited from 36 hospitals in China, and 3683 doses of basiliximab were administered. Basiliximab was used as monotherapy (n = 642) or in combination with other second-line treatments (n = 298). The cumulative incidence of overall response rate (ORR) at day 28 after basiliximab treatment was 79.4% (95% confidence interval [CI] 76.5%-82.3%). The probabilities of nonrelapse mortality and overall survival at 3 years after basiliximab treatment were 26.8% (95% CI 24.0%-29.6%) and 64.3% (95% CI 61.2%-67.4%), respectively. A 1:1 propensity score matching was performed to compare the efficacy and safety between the monotherapy and combined therapy groups. Combined therapy did not increase the ORR; conversely, it increased the infection rates compared with monotherapy. The multivariate analysis showed that combined therapy, grade III-IV aGVHD, and high-risk refined Minnesota aGVHD risk score before basiliximab treatment were independently associated with the therapeutic response. Hence, we created a prognostic scoring system that could predict the risk of having a decreased likelihood of response after basiliximab treatment. Machine learning was used to develop a protocol that maximized the efficacy of basiliximab while maintaining acceptable levels of infection risk. Thus, real-world data suggest that basiliximab is safe and effective for treating SR-aGVHD.

[Relationship between Muscle Mass of Limbs and aGVHD in Patients with Allogeneic Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To explore the correlation of limb muscle mass and acute graft-versus-host disease. METHODS: Clinical data from 144 patients treated by allo-HSCT in Guangzhou First People's Hospital were collected and analyzed retrospectively. The age, sex, diagnosis, donor age, sex of the donors, preparative regimen, ATG dose, HLA match, graft source, and number of infused stem cells of the patients were collected as baseline information. Meanwhile, bioelectrical impedance principle (BIA) was used to measure the limb muscle mass, body weight, body mass index (BMI), waist-to-hip ratio, upper arm muscle circumference, triceps skinfold thickness, and body fat rate of the patients before and after transplantation, so as to compare the changes of limb muscle mass and investigate its correlation with aGVHD. RESULTS: It was found that 61.11% of allo-HSCT patients showed muscle mass loss, and the proportion of male and female was 35.42% and 25.69%, respectively. There were reduction in the body weight, BMI, upper arm muscle circumference and muscle mass of limbs after transplantation as compared with those before transplantation (P<0.05). By comparing with the cumulative incidence of aGVHD between the patients in low muscle mass group and normal muscle mass group, it was found that the cumulative incidence of Ⅱ-Ⅳdegree aGVHD in patients with low muscle mass (30.38%) was higher than those with normal muscle mass (8.93%), which showed statistical difference (P<0.05). Univariate analysis showed that muscle mass, the sex of the donors, and preparative regimen were the influencing factors of aGVHD (P<0.05). Binary logistic regression showed that low muscle mass was the independent risk factor affecting aGVHD (P<0.05). CONCLUSION: Patients treated by allo-HSCT shows a decline in muscle mass after transplantation, and the incidence of aGVHD is high in patients with low muscle mass. Therefore, the assessment of muscle quality in early stage in patients with HSCT can facilitate earlier detection of aGVHD.

[Clinical Analysis of CMV Infection after Allogeneic Hematopoietic Stem Cell Transplantation in Severe Aplastic Anemia].

OBJECTIVE: To investigate the clinical characteristics and risk factors of cytomegalovirus (CMV) infection after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with severe aplastic anemia (SAA). METHODS: Clinical data from 270 SAA patients with allo-HSCT were retrospectively analyzed, including 108 sib congruence patients and 162 substitute donors (68 unrelated donor congruence patients and 94 related haploid patients). Different pretreatment schemes were selected for different transplantation modes. The HLA-identical sibling and haploid grafts were all bone marrow and peripheral blood stem cells, and the grafts from unrelated donors were peripheral blood stem cells. After granulocyte implantation, blood CMV-DNA was regularly monitored. Flow cytometry was also used to determine the absolute number of CD3+, CD4+T lymphocytes and CD19+B lymphocytes at 1, 2, 3, 6 and 12 months after transplantation. RESULTS: CMV infection occurred in 229 of 270 patients with an incidence of 84.8%. Among them, 18 patients developed giant cell disease. Univariate analysis showed that alternative donors (unrelated total and haploid donors), mycophenolate mofetil and acute graft-versus-host disease were statistically significantly associated with CMV infection (P<0.05). Multivariate analysis showed that alternative donors were associated with CMV infection. The recovery of CD3+ and CD4+ in 6 months in the substitute donors was delayed in comparison with that in the full sib group. CONCLUSION: After allo-HSCT, substitute donors are more easily to develop CMV infection than full-sibling donors, and the reconstruction of immune function is delayed after transplantation.

Comparable survival outcome between transplantation from haploidentical donor and matched related donor or unrelated donor for severe aplastic anemia patients aged 40 years and older: A retrospective multicenter cohort study.

This retrospective multicenter cohort study aimed to compare the outcome of haploidentical hematopoietic stem cell transplantation (HID-HSCT) with matched sibling donor (MSD) and unrelated donor (URD) transplantation in severe aplastic anemia (SAA) patients 40 years of age and older. With a median follow-up time of 17.6 months, 85 consecutive patients were enrolled in the study, and the median patient age was 45 years (40, 58). The cumulative engraftment rates of neutrophil and platelet were 98.8 ± 0.0% and 92.9 ± 0.1%. The cumulative incidences of Grade 2-4 acute graft-versus-host disease (aGvHD) and chronic graft-versus-host disease (cGvHD) at 3 years were 14.1 ± 0.1% and 17.3 ± 0.2%. The 3-year estimated overall survival (OS) and failure-free survival (FFS) were 91.2 ± 3.2% and 89.7 ± 3.5%. In multivariate analysis, the only factor associated with inferior survival was an ECOG score ≥2. HID-HSCT was associated with a higher incidence of GvHD, but the difference of 3-year estimated OS between HID group and the other two cohorts was not significant (86.7 ± 6.4% for HID vs 92.1% ± 4.4% for MSD and 100% for URD, P = .481). HID-HSCT might be a feasible alternative option for selected SAA patients aged 40 years and older without a matched donor.

Expression profile analysis of prognostic long non-coding RNA in adult acute myeloid leukemia by weighted gene co-expression network analysis (WGCNA).

Background: Long non-coding RNAs (lncRNAs), which are over 200 nt in length, have a key role in tumorigenesis and disease progression. To explore the role of prognostic lncRNAs in adult acute myeloid leukemia (AML), the expression profiles of lncRNAs and mRNAs in AML were analyzed. Methods: The RNAseq data of 167 adult AML patients and the corresponding clinical information were downloaded from The Cancer Genome Atlas (TCGA), which is a publicly available database. The RPKM values of the RNAseq data were subjected to weighted gene co-expression network analysis (WGCNA) in modularization. Results: We identified survival specific lncRNAs and mRNAs, which were divided into modules by coexpression analysis. The lncRNAs were mainly annotated into "Fc gamma R-mediated phagocytosis". The hub lncRNA and co-expressed mRNAs were further selected for analysis of risk stratification. LncRNA-LOC646762 may contribute to AML through the "endocytosis" signaling pathway. Finally, the expression levels of LOC646762 and co-expressed CCND3, CBR1, C10orf54, CD97 and BLOC1S1 in the adult AML patients and healthy volunteers were validated by qRT-PCR, and then their roles in prognosis and risk stratification were identified. Conclusions: Prognostic lncRNA-LOC646762, which may contribute to AML through the "endocytosis" signaling pathway, may act as a biomarker for predicting the survival of adult AML patients, as well as for risk stratification.

Who is the best haploidentical donor for acquired severe aplastic anemia? Experience from a multicenter study.

BACKGROUND: Haploidentical transplantation has been proposed as an effective treatment for severe aplastic anemia (SAA). The majority of patients have more than one HLA-haploidentical donor. Herein, we compared the outcomes between different donor-recipient relationships for optimal haploidentical donor selection in acquired SAA. METHODS: We conducted a multicenter study based on a registered database of 392 patients with SAA treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT) between 2006 and 2018. In total, 223 patients received grafts from father donors, 47 from mother donors, 91 from siblings, 29 from children, and 2 from collateral donors. RESULTS: Of the 381 patients who survived more than 28 days, 379 (99.5%) recipients were engrafted. The 2-year overall survival (OS) was 86.6 ± 2.5%, 87.1 ± 4.9%, 84.3 ± 3.9%, and 92.2 ± 5.1% for recipients of father, mother, sibling, and child grafts, respectively, (P = 0.706). The 2-year failure-free survival (FFS) was 82.8 ± 2.7%, 86.7 ± 5.1%, 80.8 ± 4.2%, and 92.5 ± 5.1% for recipients of father, mother, sibling, and child grafts, respectively, (P = 0.508). There was no difference in the incidence of either acute or chronic graft-versus-host disease (GVHD) among the different donor sources in multivariate analyses. There were also no differences in the OS or FFS among the different donor sources in the Cox regression analysis. However, OS was significantly better in the patients with a shorter history of aplastic anemia (< 12 months), better performance status (ECOG scores 0-1), or moderate graft mononuclear cell (MNC) counts (6-10 × 108/kg), and in female recipients with male donors. The FFS was also higher in patients with a shorter history of aplastic anemia (< 12 months) and better performance status (ECOG scores 0-1). CONCLUSIONS: Fathers, mothers, siblings, and children are all suitable haploidentical donors for patients with SAA.

Immunosuppressive therapy versus haploidentical transplantation in adults with acquired severe aplastic anemia.

Our study aimed to compare treatment outcomes between hematopoietic stem cell transplantation (HSCT) from haploidentical donors (HID) and immunosuppressive therapy (IST) in adults with acquired severe aplastic anemia (SAA). The medical records of 113 SAA adults who received IST, including rabbit ATG and cyclosporin (N = 37), or HID HSCT (N = 76) within 6 months of diagnosis at two institutions were retrospectively reviewed. Estimated 8-year overall survival (OS) was comparable between the IST and HID HSCT groups (75.6 vs. 83.7%, respectively, P = 0.328), but failure free survival (FFS) was significantly lower in IST group than HID HSCT group (38.5 vs. 83.7%, respectively, P = 0.001). Furthermore, a significant improvement in FFS was observed with HSCT over IST in patients under 40 years old. At the last follow-up, patients in HSCT group achieved better Karnofsky Performance Status (KPS) than those in IST group (100 [20-100] vs. 90 [20-100], P = 0.002). In terms of blood count, 83.1% (54/65) of patients in HSCT group showed complete recovery compared to only 38.2% (13/34) in IST group (P < 0.001). These data suggest that HID HSCT could be an effective alternative treatment option for SAA adults, and additional prospective studies are necessary.

[Value of CD3+CD4+ T Cell Count in Prediction of Viral Infection after Hematopoietic Stem Cell Transplantation in Patients Severe Aplastic Anemia].

OBJECTIVE: To explore the value of CD3+CD4+ T cell count in prediction of viral infections after allogeneic hematopoietic stem cell transplantation(allo-HSCT) in the patients with severe aplastic anemia(SAA). METHODS: A total of 78 SAA patients with allo-HSCT in Guangzhou First People's Hospital from January 2014 to July 2016 were enrolled in this study. The absolute numbers of CD3+CD4+T cells were measured by flow cytometry at 1,2,3,6, and 12 month after allo-HSCT. According to the cell counts, the patients were divided into 3 groups: i.e. <50/µl (n=120), 50-100/µl(n=48) and >100/µl(n=123)groups. The infection incidences of human cytomegalovirus (HCMV) and Epstein-Barr virus(EBV) within 2 weeks around each time point were compared between different groups. According the counts of CD3+CD4+T cells at 3 months after-transplant, these patients were divided into 2 groups, i.e.>100/µl (n=30) and ≤100/µl (n=48). The incidences and duration of HCMV and EBV infection, overall survival rate were compared between 2 groups. RESULTS: The incidences of CMV and EBV infection significantly decreased in CD3+CD4+ T cell >100/µl group as compared with <50/µl and 50-100/µl groups. At 3 months after-transplant, there was lower incidence rates of CMV disease, EBV infection, shorter durations of CMV infection and better survival in CD3+CD4+ T cell >100/µl group as compared with ≤100/µl group. CONCLUSION: CD3+CD4+ T cell count is a good predictor for CMV and EBV infection after allo-HSCT in SAA patients. There are low risk of infe-ctions from CMV and EBV when CD3+CD4+ T cell count >100/µl in any time after transplant, which means lower occurrence of CMV and EBV infection and better survival when CD3+CD4+ T cell counts is >100/µl in 3 months after transplant in SAA patients.

[Comparison and significance of DCDF probe and ES probe in the detection of BCR/ABL fusion gene].

OBJECTIVE: To investigate the signal patterns of dual color dual fusion (DCDF) probe and extra signal (ES) probe in the detection of BCR/ABL fusion gene, and illustrate the relation between the fluorescence in situ hybridization (FISH) pattern and the karyotype. METHODS: Sixty-five cases of chronic myelocytic leukemia (CML) and 50 cases of acute lymphoblastic leukemia (ALL) were detected by FISH with DCDF probe, the BCR/ABL positive samples were detected by FISH with ES probe. Among these cases, 47 cases of CML and 40 cases of ALL perform conventional cytogenetics simultaneously. RESULTS: All 65 cases of CML were all BCR/ABL positive by FISH. 17 cases showed the atypical pattern by DCDF-FISH, and 12 cases showed the atypical pattern by ES-FISH. There were 7 cases of BCR/ABL positive in 50 cases of ALL by FISH. By ES-FISH, there were 5 cases in which the break-point of BCR gene was located in m-bcr, 2 cases in which the break-point of BCR gene was located in M-bcr. Conventional cytogenetics demonstrated that 43/44(98%) cases of CML and 7/32(22%) cases of ALL were Ph positive. CONCLUSION: The features of DCDF-FISH, ES-FISH and conventional cytogenetic are different from each other. According to the features of these method, it can increase the precision of the adjustment of genetic feature to analyze these results comprehensively.

[Effect of different cytokine combinations on the expression of CD49d and CXCR4 and ex vivo expansion of umbilical cord blood mononuclear cells].

This study was purposed to explore the effect of different cytokine combinations on the expansion of the mononuclear cells drived from umbilical cord blood (CB) ex vivo and expression of CXCR4 and CD49d on CD34+ cells after expansion. Human fresh CB mononuclear cells were cultured in serum-free and stroma-free medium containing different combinations of cytokine for 7 days. At day o and 7, the total cells were counted, CD34+ cells and CD34+CXCR4+, CD34+CD49d+ cells were assayed by flow cytometry, and CFU were determined. According to the different combinations of cytokine, experiments were divided into four groups: control, SF group (SCF + FL), SFT group (SCF + FL + TPO) and SFT6 group (SCF + FL + TPO + IL-6). The results showed that the SF (SF group) combination supported only low expansion of total cells, CD34+ cells and CFU. The addition of TPO in SF group restored UCB stem/progenitors expansion to a higher level than that in SF group, while there was no difference between groups SFT and SFT6 (P > 0.05). The cytokine combinations in groups SF, SFT and SFT6 all could upregulate the expression levels of CD49d and CXCR4 on expanded cord blood CD34+ cells, but there were no significant differences between groups SF, SFT and SFT6 (P > 0.05). It is concluded that SCF + FL has no strong synergistic effects on primitive hematopoietic cells. TPO plays an important role in enhancing expansion of umbilical cord blood hematopoietic cells, while IL-6 only shows a neutral effect on it. SCF + FL + TPO combination not only promotes progenitor cells expansion but also upregulates the expression of CD49d and CXCR4 on CD34+ cells from cord blood.

[Ex vivo expansion of megakaryocyte progenitor cells from human cord blood in serum-free culture].

Prolonged thrombocytopenia is a puzzling problem following umbilical cord blood (CB) transplantation. It might be the result of inadequate megakaryocyte progenitors and the arrest in the megakaryocyte maturation. It is an important method to solve the problem by transfusing ex-vivo expanded CB megakaryocyte progenitor cells into the patients to shorten the duration of platelet recovery. However, the most optimal condition of expansion has not been established so far. In the study, cord blood mononuclear cells (MNC) were cultured in serum-free medium with TPO, IL-3, SCF and IL-6. The numbers of MNC, CFU-MK and CD41(+) cells were measured at 0, 6, 10 and 14 days, in order to find the best cytokine combination and optimal harvest time point for clinical use. The results showed that the megakaryocyte progenitors most efficiently expanded with the cytokine combination including TPO, IL-3, SCF and IL-6. The time point of maximal CFU-MK growth is day 10. At 10 days, the numbers of CFU-MK and CD41(+) cells expanded by 6.8- and 8.8-fold respectively. In conclusion, in vitro, the cytokine cocktail including TPO, IL-3, SCF and IL-6 was the most optimal cytokine combination which stimulates the ex vivo expansion of megakaryocyte progenitors in CB MNC and serum-free medium. The maximal CFU-MK colonies were harvested at 10 days, that may be an optimal harvest time for clinical transfusion.