Long-term outcome of children with acute promyelocytic leukemia: a randomized study of oral versus intravenous arsenic by SCCLG-APL group.

Realgar-Indigo naturalis formula (RIF), an oral traditional Chinese medicine mainly containing Realgar (As4S4), is highly effective in treating adult acute promyelocytic leukemia (APL). However, the treatment efficacy and safety of RIF have not been verified in pediatric patients. SCCLG-APL group conducted a multicenter randomized non-inferiority trial to determine whether intravenous arsenic trioxide (ATO) can be substituted by oral RIF in treating pediatric APL. Of 176 eligible patients enrolled, 91 and 85 were randomized to ATO and RIF groups, respectively. Patients were treated with the risk-adapted protocol. Induction, consolidation, and 96-week maintenance treatment contained all-trans-retinoic acid and low-intensity chemotherapy, and either ATO or RIF. The primary endpoint was 5-year event-free survival (EFS). The secondary endpoints were adverse events and hospital days. After a median 6-year follow-up, the 5-year EFS was 97.6% in both groups. However, the RIF group had significantly shorter hospital stays and lower incidence of infection and tended to have less cardiac toxicity. All 4 relapses occurred within 1.5 years after completion of maintenance therapy. No long-term arsenic retentions were observed in either group. Substituting oral RIF for ATO maintains treatment efficacy while reducing hospitalization and adverse events in treating pediatric APL patients, which may be a future treatment strategy for APL.

Application of Gene Therapy in Hemophilia.

Gene therapy refers to introducing normal exogenous genes into target cells to correct or compensate for the diseases caused by defective and abnormal genes for the purpose of therapy. It holds out hope of a cure for single-gene genetic diseases such as thalassemia, hemophilia, etc. At present, gene therapy is performed in two ways: introducing exogenous genes, and gene editing. A great number of clinical trials of gene therapy in hemophilia have been carried out using viral vectors to introduce foreign genes into target cells. However, the production of neutralizing antibodies following injection and the inability to prepare viral vectors in large quantities limit their application. Although gene-editing methods like CRISPR avoid the above problems, the potential risks of off-target effects are still unknown. More trials and evidence are needed to elucidate the safety and accuracy of gene therapy. This paper will review the bench and clinical work of gene therapy in hemophilia in recent years, and summarize the challenges and prospects of gene therapy, so as to provide directions for future scientific research in this field.

Value of Contrast-enhanced Ultrasound in Evaluating Synovitis and Predicting Recurrent Joint Bleeding of Hemophilia.

OBJECTIVE: Contrast-enhanced ultrasound (CEUS) is advantageous for evaluating microcirculation, and has been applied to assess arthritis in previous studies. However, CEUS examinations have not been studied for hemophilia arthritis. Hemophilia arthritis is different from other arthritis, because it is induced by spontaneous joint bleeding. Hence, CEUS may have special value in evaluating hemophilia arthritis. The present study assessed the value of CEUS in evaluating synovial hypertrophy and predicting recurrent joint bleeding in severe hemophilia A patients. METHODS: From August 2016 to January 2017, 81 severe hemophilia A patients, who were referred to our hospital for ultrasound joint assessment with conventional ultrasound, were enrolled. Among these 81 patients, 46 patients consented for CEUS examinations on the same day. RESULTS: Compared to color Doppler flow imaging (CDFI), four more joints presented with a blood flow signal under CEUS mode. In addition, the synovial hypertrophy measured by CEUS was thicker than that measured by conventional ultrasound. The ultrasound scores (including the total grey-scale ultrasound score, joint effusion/hemarthrosis, synovial hypertrophy, CDFI semi-quantitative score, and CEUS semi-quantitative score) were significantly higher in the joint bleeding group than in the no joint bleeding group (P<0.05). Furthermore, these ultrasound scores were positively correlated with the joint bleeding frequency, and had the highest correlation with the CEUS score (r=0.620, P<0.05). CONCLUSION: CEUS can more accurately assess the degree of synovial hypertrophy and vascularization, and diagnose synovitis, when compared to conventional ultrasound. In addition, CEUS appears to be essential for evaluating the possibility of recurrent joint bleeding, and providing more reliable evidence for individualized treatment.

Prognostic Value and Outcome for ETV6/RUNX1-Positive Pediatric Acute Lymphoblastic Leukemia: A Report From the South China Children's Leukemia Group.

PURPOSE: To analyzed the outcome of ETV6/RUNX1-positive pediatric acute B lymphoblastic leukemia (B-ALL) with the aim of identifying prognostic value. METHOD: A total of 2,530 pediatric patients who were diagnosed with B-ALL were classified into two groups based on the ETV6/RUNX1 status by using a retrospective cohort study method from February 28, 2008, to June 30, 2020, at 22 participating ALL centers. RESULTS: In total, 461 (18.2%) cases were ETV6/RUNX1-positive. The proportion of patients with risk factors (age <1 year or ≥10 years, WB≥50×109/L) in ETV6/RUNX1-positive group was significantly lower than that in negative group (P<0.001), while the proportion of patients with good early response (good response to prednisone, D15 MRD < 0.1%, and D33 MRD < 0.01%) in ETV6/RUNX1-positive group was higher than that in the negative group (P<0.001, 0.788 and 0.004, respectively). Multivariate analysis of 2,530 patients found that age <1 or ≥10 years, SCCLG-ALL-2016 protocol, and MLL were independent predictor of outcome but not ETV6/RUNX1. The EFS and OS of the ETV6/RUNX1-positive group were significantly higher than those of the negative group (3-year EFS: 90.11 ± 4.21% vs 82 ± 2.36%, P<0.0001, 3-year OS: 91.99 ± 3.92% vs 88.79 ± 1.87%, P=0.017). Subgroup analysis showed that chemotherapy protocol, age, prednisone response, and D15 MRD were important factors affecting the prognosis of ETV6/RUNX1-positive children. CONCLUSIONS: ETV6/RUNX1-positive pediatric ALL showed an excellent outcome but lack of independent prognostic significance in South China. However, for older patients who have the ETV6/RUNX1 fusion and slow response to therapy, to opt for more intensive treatment.

[Clinical features of children with acute lymphoblastic leukemia complicated by pulmonary infection after chemotherapy].

OBJECTIVE: To examine the clinical features of children with acute lymphoblastic leukemia (ALL) complicated by pulmonary infection after chemotherapy. METHODS: The clinical data of 108 ALL children (115 case-times) with post-chemotherapy pulmonary infection were retrospectively reviewed. The risk factors for pulmonary infection and the relationship between pathogens and chest CT findings were evaluated. RESULTS: The highest incidence (77.4% ) of pulmonary infection occurred during remission induction, peaking at 31-60 days after chemotherapy. Patients with neutropenia had the highest incidence rate of pulmonary infection (67.0%). Bacteria (36%) and fungi (41%) were the two most common pathogens in the 41 patients who were etiologically suspected of or diagnosed with pulmonary infection. There was no significant difference in chest CT findings between patients with bacterial and fungal infections. CONCLUSIONS: The children with ALL are most susceptible to pulmonary infection during remission induction, especially when they are neutropenic. Bacteria and fungi are the main pathogens of pulmonary infections in these patients. However, the changes in chest CT images are poor indicators of the nature of pulmonary infection.

[Nosocomial septicemia in the early stage after stem cell transplantation in children with major ß-thalassemia].

OBJECTIVE: To explore the clinical characteristics of nosocomial septicemia in the early stage after hematopoietic stem cell transplantation (HSCT) in children with major ß-thalassemia. METHODS: The clinical data were retrospectively analyzed of 55 consecutive children with major ß-thalassemia who developed septicemia early after HSCT between January, 2011 and June, 2016. RESULTS: Among the total of 416 consecutive children with major ß-thalassemia undergoing allogeneic HSCT, the incidence of nosocomial infection early after transplantation was 77.16% (321/416), and 55 (17.13%) children showed positive findings in blood culture test. The infections occurred most commonly in the oral cavity (65.5%), followed by the respiratory tract, intestinal tract and skin. Gram-negative bacteria, including Escherichia coli (27.3%), Klebsiella pneumoniae (21.8%) and Pseudomonas aeruginosa (9.1%), were the most common causes of infections. Fungal (Candida tropicalis) infection caused septicemia in 1 case. Of all the pathogens, extended-spectrum ß-lactamase (ESBL)-producing bacteria were found in 6 cases, methicillin-resistant Staphylococcus aureus (MRSA) in 2 cases, and multidrug-resistant (MDR) bacteria in 2 cases. CONCLUSION: Gram-negative bacteria are the major pathogens causing septicemia in children early after HSCT for major ß-thalassemia, and the bacteria show a high level of drug resistance. Adequate preventive use of antibiotics and care of the oral cavity, the respiratory tract, and the perianal region following the transplantation are important measures to control nosoconial infection in these children.

[Outcome of childhood high-risk acute lymphoblastic leukemia treated with the ALL-BFM 95 protocol].

OBJECTIVE: To evaluate the effectiveness and the practicability of the Acute Lymphoblastic Leukemia Berlin-Frankfurt-Münster 95 (ALL-BFM 95) protocol in treating childhood high-risk acute lymphoblastic leukemia (HR-ALL). METHODS: A retrospective analysis of 47 children with newly diagnosed HR-ALL between July 2003 and August 2013 was performed. These children were treated by the ALL-BFM 95 protocol. Survival was evaluated by Kaplan Meier analysis and Log-Rank test. RESULTS: Relapse-related death occurred in 12 of 47 patients (26%), and 5 of 47 patients (11%) were treatment-related mortality. Five-year probability of event-free-survival (pEFS) was 62%. Children with hematopoietic stem cell transplantation (HSCT) after chemotherapy achieved significantly better pEFS than those with chemotherapy alone (77% vs 52%; P=0.035). The patients who were only poor responders to prednisone had a better outcome (5-year pEFS 80%) than the Days 15 and 33 bone marrow M3 subgroups (5-year pEFS 60% and 0 respectively). CONCLUSIONS: ALL-BFM 95 protocol can improve the outcome of children with high-risk ALL. The major cause of death is attributed to relapse. Chemotherapy plus HSCT can produce a better outcome than chemotherapy alone. The Days 15 and 33 bone marrow M3 subgroups have a poor prognosis.

[Comparison of clinical implications of p16 deletion in childhood and adult B-lineage acute lymphoblastic leukemia].

OBJECTIVE: To investigate and compare the clinical implications of p16 deletion in childhood and adult B-lineage acute lymphoblastic leukemia (B-ALL). METHODS: A total of 129 cases of de novo childhood (73 cases) and adult (56 cases) B-ALL were examined genetically and immunologically using G-banding techniqhe, interphase fluorescence in situ hybridization (I-FISH) and immunophenotyping by flow cytometry, and their clinical data were retrospectively analyzed. RESULTS: Of 73 childhood cases, the prevalences of homozygous deletion, hemizygous deletion and no deletion of p16 were 24.7% (18 cases), 6.8% (5 cases) and 68.5% (50 cases) respectively, and of 56 adult cases, the incidences as of 14.3% (8 cases), 8.9% (5 cases) and 76.8% (43 cases) respectively. The incidence of p16 deletion between the two groups had no significant difference (P = 0.338). In both groups, patients with or without p16 deletion had no significant difference in terms of white blood cells (WBC) count at diagnosis, BM blast percentage, chromosome karyotype, extra-infiltration and CR1 rate. Of note, there were 2 cases, each in childhood and adult, showed no deletion at the time of diagnosis, their p16 deletions occurred at relapse. The deletion of p16 was associated with poor overall survival and event-free survival (EFS) in both childhood and adults. According to the standard of NCI risk stratification, we divided patients of two groups into standard and high risk category respectively, and performed further analysis. The significance of different risk category in children and adults was disparity. The overall survival (OS) rates of deletion and no deletion of p16 were 45.3% and 79.8% (P = 0.006) in children, and 7.7% and 22.6% (P = 0.002) in adults, respectively. EFS rates of deletion and no deletion of p16 were 33.5% and 58.1% (P = 0.008) in children, and 0 and 10.9% (P < 0.01) in adults, respectively. Of the standard risk category in children, OS rates of deletion and no deletion of p16 were 46.8% and 89.3% (P = 0.015) respectively, and EFS rates of deletion and no deletion of p16 as of 40.9% and 82.1% (P = 0.007) respectively. Of the high risk category in children, OS rates of deletion and no deletion of p16 were 41.7% and 67.4% (P = 0.193) respectively, and EFS rates of deletion and no deletion of p16 were 25.0% and 25.6% (P = 0.305) respectively. Of the standard risk category in adults, OS rates of deletion and no deletion of p16 were 20.0% and 46.9% (P = 0.092) respectively, and EFS rates of deletion and no deletion of p16 were 0 and 25.0% (P = 0.062) respectively. Of the high risk category in adults, OS rates of deletion and no deletion of p16 were 0 and 12.4% (P < 0.001) respectively, and EFS rate of deletion and no deletion of p16 was 0 and 4.8%(P < 0.001), respectively. CONCLUSION: This study indicated that deletion of p16 was associated with poor prognosis in both childhood and adult B-ALL, which highlighted an important significance to define the status of p16 in both childhood and adult B-ALL for predicting prognosis and guiding clinical intervention.

[CDR3 spectratyping and clonal expansion of peripheral blood T cells in a patient with beta-mediterranean anaemia following peripheral blood stem cell transplantation].

OBJECTIVE: To study the CDR3 spectratyping and clonal expansion of T cells in the peripheral blood mononuclear cells (PBMCs) of patients with beta-mediterranean anemia patient undergoing allogeneic peripheral blood stem cell (PBSC) transplantation. METHODS: The total RNA was isolated from the PBMCs of a nine-year-old boy with beta-mediterranean anemia before and after PBSC transplantation, and at the time of occurrence of graft-versus-host disease (GVHD). The CDR3 length was analyzed using immunoscope technique, and the characteristics of the T cell receptor (TCR) on the T cells with clonal expansion were examined by gene sequencing. RESULTS: The 24 TCR BV CDR3 repertoire showed Gaussian distribution in the PBMCs isolated before the transplantation, and some of the TCR BV family CDR3 showed skewing in the PBMCs isolated 23 days after transplantation and at the onset of GVHD (28 days after transplantation), suggesting the clonal expansion of the donor PBSCs. CONCLUSION: The host PBMCs show muti-clonal expansion 23 days after PBSC transplantation, and in the stage of GVHD, some of the TCR BV family T cells show significant monoclonal expansion. Analysis of TCR CDR3 spectratyping and the molecular characteristics of specific TCR may help evaluate the immune reconstitution following the transplantation and indicate specific treatment for potential GVHD.

[Effects of mouse bone marrow mesenchymal stem cells on the expression of chemokine receptors of spleenocytes after phytohemagglutinin stimulation].

OBJECTIVE: To explore the effect of mouse bone marrow mesenchymal stem cells (MSCs) on the expression of chemokine receptors in T lymphocytes in vitro. METHODS: Mouse bone marrow MSCs were separated with Percoll, cultured and expanded in low glucose DMEM. C57BL/6 mouse spleenocytes were cultured in the 24-hole flasks by the density of 1 x10(6)/hole. Phytohemagglutinin (PHA) was then added to the holes and cultured for 72 hrs. This study consisted of three groups. Groups A and B were co-cultured by adding MSCs as the ratio of 0.1 and 0.01 to spleenocytes respectively. The control group was cultured without MSCs. Three days later the suspended spleenocytes were harvested for detecting the expression of three chemokine receptors CXCR3, CCR5 and CCR7 in T lymphocytes by the flow cytometry. RESULTS: The expression of CD3(+)CCR5(+) and CD3(+)CCR7(+) were statistically different among the three groups. Group A had the strongest expression, followed by group B and the control group. The expression of CD3(+)CXCR3(+) in group A was statistically higher than that in group B and the control group. CONCLUSIONS: MSCs could up-regulate the expression of chemokine receptors CXCR3, CCR5 and CCR7 in T lymphocytes stimulated by PHA.

[Analysis of therapeutic effectiveness of Nanfang ALL 99 protocol in childhood acute lymphoblastic leukemia patients].

OBJECTIVE: With more precise diagnostic criteria and risk classifications, more effective therapy administered in clinical trials, and better supportive care, the outcome of children with acute lymphoblastic leukemia (ALL) has been improved dramatically. Today, approximately 80% of children treated for this disease in developed countries enjoy long-term event free survival (EFS) and in most instances, would be cured. In this study, treatment outcome of 82 childhood ALL patients in the hospital were analyzed, and ways for how to improve the EFS rate in childhood ALL were explored. METHODS: Eighty-two patients with ALL were enrolled into the Nanfang ALL 99 protocol which derived from German BFM ALL 95 and Hong Kong-Singapore acute lymphoblastic leukemia 97 (HK-SG ALL 97). Dexamethasone instead of hydrocortisone was used for triple intrathecal therapy. Standard at risk patients who had been irregularly treated in other hospitals for short periods of time were classified as at intermediate risk. When ANC was > or = 1.0 x 10(9)/L and platelet > or = 100 x 10(9)/L, chemotherapy was started. Life table method was used to estimate survival rate and statistical analysis was done by using software SPSS for Windows. RESULTS: From March 1999 to September 2003, 82 childhood ALL patients were treated with the Nanfang ALL 99 protocol and 78 (95.1%) patients attained complete remission (CR) in a median time of 33 days. Out of 82 patients, 13 patients dropped out of the the Nanfang ALL 99 protocol because of financial difficulty or other reasons. Sixty nine patients were consecutively treated with the Nanfang ALL 99 protocol. The overall EFS rate at 2 years, 3 years and 5 years were 91.3%, 85.9% and 75.2%, respectively, with a median observation duration of 34 months. Three patients died of complications (4.3%). The disease relapsed in 6 patients and they died finally. CONCLUSION: The outcome of patients treated with the Nanfang ALL 99 protocol was favorable, and the mortality rate of this chemotherapeutic protocol was low. This protocol was well tolerated by Chinese patients and therefore the protocol is worthy of application in China.

[Animal model of rat-to-mouse xenogeneic bone marrow transplantation with graft-versus-host disease].

To observe the graft-versus-host disease (GVHD) in rat-to-mouse model of bone marrow transplantation to build a GVHD model, BALB/c mice were conditioned with 8.5 Gy lethal total body irradiation and divided into two groups. One group of mice was infused with 4 x 10(7) bone marrow cells (BMC) from SD rats. The other group of mice was infused with 4 x 10(7) bone marrow cell and 2 x 10(7) spleen cells from SD rats. GVHD in mice of two groups were observed for 60 days. The results showed that mice in the group infused with only BMC mostly (80%) survived more than 60 days, but in the other group infused with mixed BMC and spleen cells, all mice died within 14 days and showed GVHD with pathologic evidence. In conclusion, to induce GVHD in rat-to-mouse bone marrow transplantation needs additional rat spleen cells.

[Construction of the recombinant plasmid FasAD-pTYB2 capable of Fas activation domain fusion expression].

OBJECTIVE: To construct the recombinant plasmid FasAD-pTYB2 for the fusion expression of Fas activation domain (FasAD). METHODS: FasAD cDNA was cloned by semi-nested reverse transcriptase-polymerase chain reaction (RT-PCR), and then inserted into the universal vector of pGEM-T for the identification of its DNA sequence. The target DNA fragment was inserted into the prokaryote vector pTYB2 that expressed intein to construct the plasmid recombinant FasAD-pTYB2 which was induced by isopropylthio-beta-D-galactoside for the expression of the fusion protein. RESULTS: DNA sequence analysis confirmed sequence of FasAD cDNA previously cloned, and expression of the fusion protein by the recombinant plasmid was achieved, the product recognizable by rabbit anti-human Fas polyclonal antibody as demonstrated by Western blotting analysis. CONCLUSION: The recombinant plasmid FasAD-pTYB2 constructed in this study is capable of expressing the fusion protein of FasAD and intein, which may be significant for further study of the epitope and function of Fas antigen.