[Effect of donor and recipient HLA mismatched locus on the prognosis of childhood with leukemia after umbilical cord blood transplantation].

Objective: The aim of the study was to investigate the impact of the sites of high-resolution human leukocyte antigen (HLA) mismatch on the prognosis of children with leukemia undergoing umbilical cord blood transplantation (UCBT). Methods: Clinical data and high-resolution HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 locus gene information were collected in the children who underwent the UCBT for the first time at Children's Hospital of Soochow University between January 2016 and June 2023. In each locus, according to whether the two genes were compatible, they were divided into a compatible group (two genes were perfectly matched) and a non-compatible group (one gene was not matched). In different loci, the differences in occurrence, recurrence, non-recurrence death and survival of acute graft versus host disease (aGVHD) were compared between the two groups. Multivariate Cox regression was employed to analyzed the influencing factors for overall survival rate, and Fine-Gray proportional hazards model was employed to analyze the influencing factors of other outcome events. Results: A total of 100 patients were enrolled (55 males and 45 females), whose age [M (Q1, Q3)] at the time of transplantation was 3.9 (2.0, 6.5) years. There were 55 cases in the HLA-A matched group and 45 cases in the mismatched group. The 5-year non-recurrence mortality (NRM) in the HLA-A matched group was lower than that in the mismatched group (P=0.024). The cumulative incidence of aGVHD within 100 days after transplantation in the HLA-A matched group was lower than that in the mismatched group (P=0.017), and there were no statistically significant differences in other outcome events between the groups (all P>0.05). There were 70 cases in the HLA-B matched group and 30 cases in the mismatched group. The 5-year cumulative recurrence rate in the HLA-B matched group was higher than that in the mismatched group (P=0.027). There were 79 cases in the HLA-C matched group and 21 cases in the mismatched group, and there were no statistically difference in the outcome events between the groups (P>0.05). There were 73 cases in HLA-DRB1 matched group and 27 cases in mismatched group. The 5-year overall survival rate in HLA-DRB1 matched group was higher than that in mismatched group (P=0.036), the 5-year cumulative recurrence rate in HLA-DRB1 matched group was higher than that in mismatched group (P=0.028), and the 5-year NRM in HLA-DRB1 matched group was lower than that in mismatched group (P=0.008). The cumulative incidence of aGVHD within 100 days after transplantation in the matched group was lower than that in the mismatched group (P=0.010), and and there were no statistically significant difference in other outcome events between the groups (P>0.05). There were 68 cases in HLA-DQB1 matched group and 32 cases in mismatched group. There was no statistical difference in outcome events between the two groups (all P>0.05). The risk of aGVHD in HLA-A mismatched group was higher than that in HLA-A matched group (HR=1.25, 95%CI: 1.12-1.38). The risk of recurrence in HLA-B mismatched group was lower than that in HLA-B matched group (HR=0.77, 95%CI: 0.63-0.91). Mismatched group at HLA-DRB1 compared with matched group at HLA-DRB1, had a higher risk of aGVHD (HR=1.37, 95%CI: 1.26-1.48), a higher risk of non-recurrence death (HR=1.39, 95%CI: 1.28-1.50), and a higher risk of death (HR=1.27, 95%CI: 1.18-1.36). No association was found between HLA-C and HLA-DQB1 locus with the risk of aGVHD, recurrence, non-recurrence death, and survival (all P>0.05). Conclusions: In UCBT, the risk of aGVHD in children with matching HLA-A sites of donor and recipient is lower than that in children with incompatible HLA-A sites. Compared with children with incompatible HLA-DRB1 sites, children with HLA-DRB1 matched sites has a lower risk of acute GVHD, a lower 5-year NRM, and a higher risk of death. The recurrence rate of children with matching HLA-B loci is higher than that of children without matching HLA-B loci.

[Efficacy of decitabine combined with low dose chemotherapy on children with acute myeloid leukemia].

Objective: To evaluate the efficacy of decitabine combined with low dose chemotherapy (LDC) in the treatment of high-risk, refractory and relapsed pediatric acute myeloid leukemia (AML). Methods: Clinical data of 19 AML children treated with decitabine combined with LDC in the Department of Hematology, Children's Hospital of Soochow University from April 2017 to November 2019 were analyzed retrospectively. The therapeutic response, adverse effects and survival status were analyzed,and the outcomes of patients were followed up. Results: Among 19 AML cases, there were 10 males and 9 females. Five cases were high-risk AML, 7 cases were refractory AML, and 7 cases were relapsed AML. After one course of decitabine+LDC treatment, 15 cases achieved complete remission, 3 cases got partial remission, and only 1 case didn't get remission. All patients received allogeneic hematopoietic stem cell transplantation as consolidation therapy. The follow-up time of all cases was 46 (37, 58) months, 14 children had survived. The cumulative three-year overall survival rate was (79±9) %, events free survival rates was (68±11) %, and recurrence free survival rate was (81±10) %. The most common adverse effects related to the induction treatment were cytopenia (19 cases) and infection (16 cases).There were no treatment-related death during the therapy. Conclusion: Decitabine combined with LDC is a safe and effective option for high-risk, refractory and relapsed AML children, which provides an opportunity for HSCT.

The therapeutic efficacy of pediatric ALL patients with MLL gene rearrangement treated with CCLG-ALL2008 protocol.

OBJECTIVE: In this study, we retrospectively evaluated the therapeutic efficacy of China Children Leukemia Group-ALL2008 (CCLG-ALL 2008) protocol in pediatric patients with mixed-lineage leukaemia (MLL) gene rearrangement of acute lymphoblastic leukemia (ALL) to identify the prognostic factors. PATIENTS AND METHODS: Six hundred and thirty-four patients with ALL were enrolled in this study between June 2008 and Dec 2014. High-risk group (HR) consisted of 217 cases, of which 28 cases were MLL related positive (first group), 22 cases were BCR/ABL positive (second group), and 167 cases were negative with MLL related or BCR/ABL (third group). The therapeutic efficacy was evaluated at the time points of day 8 (TP1), day 15 (TP2), day 33 (TP3) and 12th week (TP4) with the protocol, respectively. Overall-survival (OS) and relapse-free-survival (RFS) and treatment-related mortality (TRD) were analyzed as well. RESULTS: The first group accounted for 4.4% of all patients. Compared with the second and third group, the first group had more cases younger than 2 years, with initial leukocytes ≥50×109/L, and poor response on TP2. Moreover, patients older than 2 years old had a good 5 years OS (84% ± 9% vs. 37% ± 20%, p<0.05) and RFS (84% ± 9% vs. 29% ± 17%, p<0.05). There were no significant differences in the recurrence rate, TRD, 5 years OS and RFS among three groups. For the first group, compared with good response to prednisone, patients with poor response to prednisone had a poor 5 years RFS (41% ± 17% vs. 81% ± 10%, p<0.05). Multivariate Cox regression analysis identified that RFS and OS were influenced by such factors as age, MLL fusion partners, and prednisone response (p<0.05). CONCLUSIONS: Such factors as younger age than 2 years old, MLL/AF4 fusion gene, poor response to prednisone, or no complete remission (CR) on TP3 were poor prognostic parameters in predicting the outcome in childhood ALL with MLL gene rearrangement treated with CCLG-ALL 2008 protocol.

Suppressed CCL2 expression inhibits the proliferation of leukemia cells via the cell cycle protein Cyclin D1: preliminary in vitro data.

OBJECTIVE: Chemokine (C-C motif) ligand 2 (CCL2) is a member of the CC subfamily, which displays chemotactic activity for monocytes and basophils. This molecule plays a very important role in many solid tumors and shows changes in the bone marrow microenvironment. However, its role in acute myeloid leukaemia (AML) is still unclear. MATERIALS AND METHODS: In this study, we established a HL-60 cell line with CCL2 knockdown to explore its effect on leukemogenesis. Lentivirus with CCL2-knockdown was successfully constructed after screening effective CCL2 short hairpin RNA (shRNA) sequences and was transfected into HL-60 cells, which was further validated at the mRNA and protein levels by real-time polymerase chain reaction (PCR) and Western blotting, respectively. RESULTS: Low expression of CCL2 significantly decreased HL-60 cell growth by increasing the cell arrest at G1 phase by 12% more than controls. We applied RNA sequencing technology to discriminate the gene expression profiles between the cells with CCL2 knockdown and the controls, and Cyclin D1 was selected for further experiments as its expression level was significantly downregulated, which was validated at the mRNA and protein levels. Cyclin D1 knockdown experiments showed that the cell proliferation rate was evidently decelerated, and cell cycle analysis also indicated a similar pattern for CCL2. CONCLUSIONS: Our study revealed that Cyclin D1 is an effector that mediates CCL2's function in cell proliferation by blocking cells at G1 phase.

[The safety of decitabine as bridging pretreatment regimen before hematopoietic stem cell transplantation in pediatric hematological malignancies].

The safety of decitabine as bridging treatment before allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children with refractory hematological malignancies was evaluated. All 11 cases succeeded in hematopoietic reconstitution. The main adverse reaction was hematological toxicity. Neither did infections occur, nor drug-induced liver damage and renal impairment during decitabine administration. Most cases showed grade Ⅰ-Ⅱgastrointestinal adverse events. One case was diagnosed as severe acute graft versus host disease and died of intracranial hemorrhage on day 61 after allo-HSCT. The other 10 patients survived. Decitabine bridge is a safe regimen before allo-HSCT in children with refractory hematological malignancies.

[Treatment of four cases of Fanconi anemia by allogeneic hematopoietic stem cell transplantation with low intensity conditional regimen].

Objective: To evaluate the efficiency and safety of low intensity conditional regimen for children with Fanconi anemia (FA) receiving allogenic hematopoietic stem cells transplantation (allo-HSCT). Methods: Four patients diagnosed as Fanconi anemia were enrolled in this study. One patient received HLA-identical sibling donor hematopoietic stem cell transplantation, two patients underwent unrelated donor matched (UD) HSCT, and one patient received unrelated cord blood transplantation. The conditional regimen consisted of Busulfan with low dose of cyclophosphamide. Results: All 4 cases succeeded in allo-HSCT. The median time for neutrophils engraftment was 11(9-15) day, median time to platelets (PLT) engraftment was 12 (8-28) day. One case occurred with grade I of aGVHD, 1 case with hemorrhagic cystitis. No patient happened with hepatic veno-occlusive disease (VOD). Conclusion: Low intensity of conditional regimen is efficient and safe which should be recommended for FA patients with HSCT.

A prospective evaluation of minimal residual disease as risk stratification for CCLG-ALL-2008 treatment protocol in pediatric B precursor acute lymphoblastic leukemia.

OBJECTIVE: The aim of this prospective study was to evaluate the cut-off value of minimal residual disease (MRD) in predicting the efficacy of CCLG-ALL-2008 or CCLG-2008 treatment protocol on pediatric B-precursor ALL (BP-ALL). PATIENTS AND METHODS: Three hundred and seventy-nine Chinese pediatric BP-ALL were enrolled in this study between Dec 2008 and Sep 2013 in two stratified cohorts. One hundred and fifty-three patients enrolled between Dec 2008 and Oct 2010 as the first cohort, and 196 patients enrolled from Nov 2010 to Sep 2013 as the second cohort. Clinical and biological characteristics and 5 years EFS, RFS, and OS were analyzed. RESULTS: Patients with E2A-PBX1 showed a favorable treatment response with a lower minimal residual disease (MRD) level (< 10-4) at the time point 1 (TP1, p = 0.039) and the highest proportion of the 5-year EFS, RFS, and OS. A high level of MRD was associated with high WBC counts, increased age, BCR-ABL1 fusion gene, MLL rearrangements and adverse karyotypes. In comparison with the first cohort, the second cohort with the MRD assay incorporated prospectively, the standard risk (SR) and the intermediate risk (IR) patients showed a better RFS, EFS, and OS while the high-risk (HR) patients displayed worse RFS, EFS, and OS than those of the first cohort, respectively. Patients with MRD level at either 10-4 or 10-3 showed a similar OS at TP1 or TP2, and patients with MRD level above 10-2 had the worst OS. CONCLUSIONS: This study indicated that the levels of MRD to be an adequate guide in risk-adapted treatment under the CCLG-ALL-2008 protocol and can be adapted to the future development of advanced clinical protocols.

Combinational synthesis of oligonucleotides and assembly fabrication of oligonucleotide array.

In this paper, a simple, reliable and flexible method, which integrated in situ synthesis with the spotting technique, was reported to fabricate oligonucleotide array. Different oligonucleotide sequences are synthesized on their relative code glass slides through combinational chemistry, thus the slides are broken into smaller pieces, in which the same code pieces have the same probe sequences. An oligonucleotide array is fabricated by arbitrarily assembling these different code pieces onto another solid substrate. In principle experimentation, four different sequences of P16 gene were synthesized and a 5 x 5 array including these four sequences and the control black was fabricated. The analysis results indicated that the hybridization fluorescence intensity of the same sequences locating different sets on the array gave the approximate values, and the fluorescence intensity ratio of matched sequence to one middle location base mismatched, two base mismatched, three middle base mismatched is (1.000+/-0.080):(0.4991+/-0.0671):(0.2360+/-0.0044):(0.0493+/-0.0033). Their relative accuracies were from 6.64 to 10.2%. This result might be used to rapidly screen single-nucleotide polymorphisms (SNPs).