Comparison of plasma and blood cell samples in metagenomic next-generation sequencing for identification of the causative pathogens of fever.

Background: Metagenomic next-generation sequencing (mNGS) of plasma DNA has become an attractive diagnostic method for infectious diseases; however, the rate of false-positive results is high. This study aims to evaluate the diagnostic accuracy of mNGS in plasma versus blood cell samples for immunocompromised children with febrile diseases. Methods: The results of conventional microbiological test (CMT) and mNGS using plasma and blood cells in 106 patients with 128 episodes of febrile diseases from the Department of Hematology/Oncology were analyzed and described. Results: The positivity rates for CMT and mNGS of plasma and blood cells were 35.9 %, 84.4 % and 46.9 %, respectively (P < 0.001). Notably, mNGS identified multiple pathogens in a single specimen in 68.5 % of plasma samples and 38.3 % of blood cell samples (P < 0.001). Furthermore, plasma and blood cell mNGS identified causative pathogens in 58 and 46 cases, accounting for 53.7 % and 76.7 % of the mNGS-positive cases for each sample type, respectively (P = 0.002). By integrating results from both plasma and blood cell samples, causative pathogens were identified in 77 cases (60.2 %), enhancing sensitivity to 87.5 % but reducing specificity to 15.0 %, compared to plasma (65.9 % sensitivity and 20.0 % specificity) and blood cell samples (52.3 % sensitivity and 80.0 % specificity). Conclusions: mNGS of plasma is sensitive but has a high false-positive rate, while mNGS of blood cells has low sensitivity but higher specificity.

The predictive utility of cytokines, procalcitonin and C-reactive protein among febrile pediatric hematology and oncology patients with severe sepsis or septic shock.

Severe sepsis and septic shock are life-threatening for pediatric hematology and oncology patient receiving chemotherapy. Th1/Th2 cytokines, C-reactive protein (CRP), and procalcitonin (PCT) are all thought to be associated with disease severity. The aim of this study was to prospectively verify the utility of Th1/Th2 cytokines and compare them with PCT and CRP in the prediction of adverse outcomes. Data on patients were collected from January 1, 2011, to December 31, 2020. Blood samples were taken for Th1/Th2 cytokine, CRP, and PCT measurements at the initial onset of infection. Severe infection (SI) was defined as severe sepsis or septic shock. Th1/Th2 cytokine levels were determined by using flow cytometric bead array technology. In total, 7,735 febrile episodes were included in this study. For SI prediction, the AUCs of IL-6, IL-10 and TNF-α were 0.814, 0.805 and 0.624, respectively, while IL-6 and IL-10 had high sensitivity and specificity. IL-6 > 220.85 pg/ml and IL-10 > 29.95 pg/ml had high odds ratio (OR) values of approximately 3.5 in the logistic regression. Within the subgroup analysis, for bloodstream infection (BSI) prediction, the AUCs of IL-10 and TNF-α were 0.757 and 0.694, respectively. For multiorgan dysfunction syndrome (MODS) prediction, the AUC of CRP was 0.606. The AUC of PCT for mortality prediction was 0.620. In conclusion, IL-6 and IL-10 provide good predictive value for the diagnosis of SI. For children with SI, IL-10 and TNF-α are associated with BSI, while CRP and PCT are associated with MODS and death, respectively.

The effect of the plasma methotrexate concentration during high-dose methotrexate therapy in childhood acute lymphoblastic leukemia.

Two hundred and thirty-one acute lymphoblastic leukemia (ALL) children with 1376 high-dose methotrexate (HD-MTX) courses (3-5 g/m2) were enrolled to analyze the influence of the plasma MTX concentration (CMTX) in ALL. The 24-h target peak CMTX (C24h) was set at 33 µmol/l for low-risk (LR) and 65 µmol/l for intermediate/high-risk (IR/HR) groups. The median C24h was 42.0 µmol/l and 69.7 µmol/l for LR and IR/HR groups, respectively. MTX excretion delay was observed in 14.6% of courses, which was more frequent in IR/HR groups (56.9% vs. LR group 40.2%, p = .014) and T-ALL patients (82.6% vs. B-ALL 47.1%, p = .001). MTX-related toxicities were more common in courses with MTX excretion delay. However, survival between the patients who failed to reach the target C24h or not, with or without MTX excretion delay, was comparable. These findings suggest that, owing to the effectiveness of risk stratification chemotherapy, CMTX does not exert an independent influence on the prognosis of childhood ALL.

Minimal residual disease detection by next-generation sequencing of different immunoglobulin gene rearrangements in pediatric B-ALL.

While the prognostic role of immunoglobulin heavy chain locus (IGH) rearrangement in minimal residual disease (MRD) in pediatric B-acute lymphoblastic leukemia (B-ALL) has been reported, the contribution of light chain loci (IGK/IGL) remains elusive. This study is to evaluate the prognosis of IGH and IGK/IGL rearrangement-based MRD detected by next-generation sequencing in B-ALL at the end of induction (EOI) and end of consolidation (EOC). IGK/IGL rearrangements identify 5.5% of patients without trackable IGH clones. Concordance rates for IGH and IGK/IGL are 79.9% (cutoff 0.01%) at EOI and 81.0% (cutoff 0.0001%) at EOC, respectively. Patients with NGS-MRD < 0.01% at EOI or <0.0001% at EOC present excellent outcome, with 3-year event-free survival rates higher than 95%. IGH-MRD is prognostic at EOI/EOC, while IGK-MRD at EOI/EOC and IGL-MRD at EOI are not. At EOI, NGS identifies 26.2% of higher risk patients whose MRD < 0.01% by flow cytometry. However, analyzing IGK/IGL along with IGH fails to identify additional higher risk patients both at EOI and at EOC. In conclusion, IGH is crucial for MRD monitoring while IGK and IGL have relatively limited value.

Spectrum and clinical features of gene mutations in Chinese pediatric acute lymphoblastic leukemia.

PURPOSE: The 5-year survival rate of children with acute lymphoblastic leukemia (ALL) is 85-90%, with a 10-15% rate of treatment failure. Next-generation sequencing (NGS) identified recurrent mutated genes in ALL that might alter the diagnosis, classification, prognostic stratification, treatment, and response to ALL. Few studies on gene mutations in Chinese pediatric ALL have been identified. Thus, an in-depth understanding of the biological characteristics of these patients is essential. The present study aimed to characterize the spectrum and clinical features of recurrent driver gene mutations in a single-center cohort of Chinese pediatric ALL. METHODS: We enrolled 219 patients with pediatric ALL in our single center. Targeted sequencing based on NGS was used to detect gene mutations in patients. The correlation was analyzed between gene mutation and clinical features, including patient characteristics, cytogenetics, genetic subtypes, risk stratification and treatment outcomes using χ2-square test or Fisher's exact test for categorical variables. RESULTS: A total of 381 gene mutations were identified in 66 different genes in 152/219 patients. PIK3R1 mutation was more common in infants (P = 0.021). KRAS and FLT3 mutations were both more enriched in patients with hyperdiploidy (both P < 0.001). NRAS, PTPN11, FLT3, and KMT2D mutations were more common in patients who did not carry the fusion genes (all P < 0.050). PTEN mutation was significantly associated with high-risk ALL patients (P = 0.011), while NOTCH1 mutation was common in middle-risk ALL patients (P = 0.039). Patients with ETV6 or PHF6 mutations were less sensitive to steroid treatment (P = 0.033, P = 0.048, respectively). CONCLUSION: This study depicted the specific genomic landscape of Chinese pediatric ALL and revealed the relevance between mutational spectrum and clinical features of Chinese pediatric ALL, which highlights the need for molecular classification, risk stratification, and prognosis evaluation.

Clinical and Genetic Characteristics of IKZF1 Mutation in Chinese Children With B-Cell Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is a malignancy associated with altered lymphoid precursor hyperplasia and accompanied with different genetic mutations. Few studies have been reported on the association between gene mutations and clinical features of IKZF1 mutation in children with B-cell ALL (B-ALL). We investigated clinical and genetic characteristics in 200 newly diagnosed pediatric B-ALL through multiplex ligation-dependent probe amplification (MLPA) and targeted next-generation sequencing (NGS) method. We found that IKZF1 mutations, including large segment deletions, small insertions or deletions (InDels) and single nucleotide variations (SNVs), were detected in 22 patients with a positive mutation rate of 11.0%. IKZF1 mutation was significantly associated with higher WBC count (19.38 × 109/L vs. 5.80 × 109/L, p = 0.002). Compared with IKZF1 wild-type cases, a higher frequency of IL7R gene mutation was discovered in IKZF1 mutant cases (9.1% vs. 0.0%, p = 0.012). Patients with IKZF1 mutation were less sensitive to glucocorticoid induction than patients without IKZF1 mutation (63.6% vs. 9.0%, p < 0.001). On the 15th day of induction, minimal residual disease (MRD) > 10-3 level were higher in IKZF1 mutant patients than wild-type patients (45.5% vs. 22.3%, p = 0.018). In conclusion, our study reveals the association between genetic mutations and clinical features in Chinese children with B-ALL, which might contribute to molecular classification, risk stratification and prognosis evaluation, and provide new ideas for targeted therapy in ALL.

A Novel NUP98/RARG Gene Fusion in Pediatric Acute Myeloid Leukemia Resembling Acute Promyelocytic Leukemia.

Here, we introduced the first case of acute myeloid leukemia (AML) with RARG-NUP98 in a pediatric patient. The young male presented with structural and functional abnormalities similar to hypergranular acute promyelocytic leukemia, but was resistant to all transretinoic acids and arsenic trioxide. Till date, only 12 adult AML cases involving RARG rearrangement have been reported. At present, there is no standardized or optimal treatment option for this AML subtype. Disease management may typically require a joint treatment strategy involving chemotherapy, immunotherapy, and support therapy. In this study, we report the clinical manifestations and experimental results of a 10-year-old male and review other cases of RARG gene rearrangement reported in the literature.

Treatment of Relapsed and Refractory ALK-Positive Anaplastic Large Cell Lymphoma With ALK-Specific Tyrosine Kinase Inhibitor in Children: A Case Series.

Relapsed and refractory ALK-positive anaplastic large cell lymphoma (ALCL) has a poor prognosis. In this report, we present 3 relapsed/refractory pediatric ALCL patients, 1 of these with central nervous system involvement. All 3 patients were treated with ALK inhibitor and achieved complete response. Both crizotinib and alectinib have shown significant activity in pediatric patients with refractory ALK-positive ALCL.

Arsenic Combined With All-Trans Retinoic Acid for Pediatric Acute Promyelocytic Leukemia: Report From the CCLG-APL2016 Protocol Study.

PURPOSE: Arsenic combined with all-trans retinoic acid (ATRA) is the standard of care for adult acute promyelocytic leukemia (APL). However, the safety and effectiveness of this treatment in pediatric patients with APL have not been reported on the basis of larger sample sizes. METHODS: We conducted a multicenter trial at 38 hospitals in China. Patients with newly diagnosed APL were stratified into two risk groups according to baseline WBC count and FLT3-ITD mutation. ATRA plus arsenic trioxide or oral arsenic without chemotherapy were administered to the standard-risk group, whereas ATRA, arsenic trioxide, or oral arsenic plus reduced-dose anthracycline were administered to the high-risk group. Primary end points were event-free survival and overall survival at 2 years. RESULTS: We enrolled 193 patients with APL. After a median follow-up of 28.9 months, the 2-year overall survival rate was 99% (95% CI, 97 to 100) in the standard-risk group and 95% (95% CI, 90 to 100) in the high-risk group (P = .088). The 2-year event-free survival was 97% (95% CI, 93 to 100) in the standard-risk group and 90% (95% CI, 83 to 96) in the high-risk group (P = .252). The plasma levels of arsenic were significantly elevated after treatment, with a stable effective level ranging from 42.9 to 63.2 ng/mL during treatment. In addition, plasma, urine, hair, and nail arsenic levels rapidly decreased to normal 6 months after the end of treatment. CONCLUSION: Arsenic combined with ATRA is effective and safe in pediatric patients with APL, although long-term follow-up is still needed.

Efficacy and safety of immunosuppressive therapy with or without eltrombopag in pediatric patients with acquired aplastic anemia: A Chinese retrospective study.

To determine the efficacy and safety of eltrombopag (E-PAG) combined with intensive immunosuppressive therapy (IST) for the treatment of pediatric patients with severe aplastic anemia (SAA). A total of 57 pediatric patients with newly diagnosed severe aplastic anemia were enrolled in this study. Thirty nine patients were treated with IST alone, consisting of porcine anti-human thymocyte globulin (30 mg/kg/day × 5 days) and cyclosporine A (CsA) (treated for 2 years, with a trough concentration maintained at 200-250 ng/mL), and 18 patients were treated with IST + E-PAG (12.5-50 mg/day, maintained for 6 months). We found no statistical difference between the response rates at 3 months for the two groups (CR: 12.8% vs. 22.2% p > 0.05, ORR: 56.4% vs. 77.7% p > 0.05). However, we found a statistical difference between the response rates at 6 months for the two groups (CR: 17.9% vs. 50% p < 0.05, ORR: 69.2% vs. 94.4% p < 0.05). The main side-effect during treatment with E-PAG was having a slightly to moderately elevated bilirubin level, which was temporary and controllable, accounting for approximately 66.6% (12/18) of patients in the IST + E-PAG group vs. 20.5% (8/39) of those in the IST group (p < 0.05). IST + E-PAG therapy appears to be more effective than IST alone for the treatment of pediatric SAA, with good tolerability and compliance. This approach deserves further exploration.

Pediatric blastic plasmacytoid dendritic cell neoplasm: report of four cases and review of literature.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematological malignancy with poor outcome. Four children with BPDCN treated at our hospital were enrolled. All the four cases presented with cutaneous lesions. Bone marrow and central nervous system was involved in 50% and 25% of patients, respectively. The whole exome sequencing analysis revealed that KMT2 family genes were the most frequently mutated (4/4, 100%), followed by IKZF2 (2/4, 50%). The point mutation p.D348N was found in three patients and one patient had p.C394Y mutation in the KMT2C gene. Translocation of KMT2A-MLLT3 was found in Case 2. Case 1 had complex karyotype, who was induced by acute myeloid leukemia-like regimens. Although he received allogeneic hematopoietic stem cell transplantation twice as well as CD123 chimeric antigen receptor T cell therapy, the disease still progressed and he died 37 months after diagnosis. The other three patients were treated with Interfant-99 protocol. They tolerated the therapy well without significant toxicities and now in complete remission so far with a median follow up time of 9 months. More studies are needed to address the question whether the complex karyotype and KMT2 family genes are the causes of the relapse and refractory in BPDCN.

Clinical assessment of the utility of metagenomic next-generation sequencing in pediatric patients of hematology department.

INTRODUCTION: Metagenomic Next-Generation Sequencing (mNGS) is an emerging technique for microbial identification and diagnosis of infectious diseases. The clinical utility of mNGS, especially its real-world impact on antimicrobial treatment and patient outcome has not been systematically evaluated. METHODS: We prospectively assessed the effectiveness of mNGS in 70 febrile inpatients with suspected infections at Hematology department of the Children's Hospital, National Clinical Research Center for Child Health. 69/70 patients were given empirical antibiotics prior to mNGS. A total of 104 samples (62 plasma, 34 throat swabs, 4 bone marrow, 4 bronchoalveolar lavage) were collected on day 1-28 (mean 6.9) following symptom onset and underwent mNGS testing. RESULTS: Traditional microbiological tests discovered causal microorganisms in 5/70 (7.14%) patients, which were also detected by mNGS. In addition, mNGS reported possible pathogens when routine tests were negative. Antibiotics were adjusted accordingly in 55/70 (78.6%) patients that led to improvement/relief of symptoms within 3 days. In contrast, mNGS results were considered irrelevant in 15/70 (21.4%) patients by a board of clinicians, based on biochemical, serological, imaging evidence, and experiences. CONCLUSION: mNGS expanded the capacity of pathogen detection and made a positive impact on clinical management of suspected infections through (a) differential diagnosis which may rule out infectious diseases and (b) adjustment or de-escalation of empirical antibiotics.

Chimeric antigen receptor T cell therapy can be administered safely under the real-time monitoring of Th1/Th2 cytokine pattern using the cytometric bead array technology for relapsed and refractory acute lymphoblastic leukemia in children.

CD19 chimeric antigen receptor T (CD19CAR-T) cell therapy has shown striking response in treating relapsed and refractory B-lineage acute lymphoblastic leukemia (r/r B-ALL). However, side-effects including cytokine release syndrome (CRS) and neurotoxicity can be fatal to patients. In this report, five patients with r/r B-ALL were treated with CD19CAR-T cells. Cytokine release syndrome experienced by four patients who achieved complete remission (CR) with minimal residual disease (MRD) negative. One patient who did not response to the treatment had no CRS. Acute toxicities including fever, hypotension and other neurological toxicities occurred in responding patients within 2 weeks post infusion and managed properly with tocilizumab and/or steroids according to the "real-time" monitoring of a simple 6 Th1/Th2 cytokine pattern. In conclusion, our study demonstrates that CD19CAR-T cell therapy can be safely administered for patients with relapsed and refractory leukemia under the "real-time" monitoring of a simple 6-cytokine pattern.

High CD38 expression in childhood T-cell acute lymphoblastic leukemia is not associated with prognosis.

BACKGROUND: Prognostic factors are not well exploited in childhood T-cell acute lymphoblastic leukemia (T-ALL). OBJECTIVE: The aim of this study was to analyze the prognostic role of CD38 as well as minimal residual disease (MRD) and other biological factors in T-ALL. METHODS: Immunophenotyping of bone marrow (BM) at diagnosis and MRD levels were determined using a standard panel of antibodies by 4-colour flow cytometry. A total of 96 children with T-ALL were enrolled. RESULTS: The results showed that 97.9% of T-ALL patients were positive for CD38 with a median level of 85.3%. CD38-high group had a worse early treatment response than the CD38-low group. However, CD38 levels were not associated with prognosis, albeit CD38-high group had a worse 5-year event free survival rate (55.1% vs. 66.6%, P> 0.05) and a higher 5-year cumulative incidence of relapse (35.6% vs. 19.8%, P> 0.05). Very high MRD levels (> 10%) were related to the worse survival. Neither flow cytometry based minimal residual disease (MRD) levels nor CD38 expression levels showed significant relation to the hazard of relapse (P> 0.05). CONCLUSIONS: We conclude that T-ALL has a high level of CD38 expression which is not associated with prognosis. Very high MRD level (> 10%) is related to the worse survival, however, FCM based MRD detection does not convey a significant prognostic value.

Minimal Residual Disease-guided Risk Restratification and Therapy Improves the Survival of Childhood Acute Lymphoblastic Leukemia: Experience From a Tertiary Children's Hospital in China.

The minimal residual disease (MRD) has been shown to be very important to evaluate the prognostic significance in childhood acute lymphoblastic leukemia (ALL), but the impact under the current treatment protocol in China has not been fully elucidated. The aim of this study was to investigate the efficacy of MRD-guided risk restratification of ALL. A total of 676 children with ALL were enrolled. In the predictive study group, 476 patients were enrolled with 5-year cumulative incidence of relapse rates of the low-risk (LR), intermediate-risk (IR), and high-risk groups being 11.0%±2.3%, 12.6%±3.3%, and 32.7%±4.9%, respectively. In the intervention study group, 19/200 patients enrolled were reclassified into risk groups according to the MRD levels. The 3-year event-free survival and overall survival were 85.2%±2.9% and 90.6%±2.1%, respectively, which were higher than those of the predictive study group (79.1%±1.9% and 84.7%±1.7%, respectively; P<0.05). The 3-year cumulative incidence of relapse in the LR and IR groups of the intervention study group were 4.2%±3.1% and 6.4%±3.1%, respectively, which were significantly lower than those in the predictive study group (7.2%±1.8% and 11.8%±3.2%, respectively; P<0.05). We conclude that the risk of relapse in the LR and IR groups can be significantly reduced after MRD-guided risk restratification.

Construction, Expression, and Characterization of a Novel Human-Mouse Chimeric Antibody, Hm3A4: A Potential Therapeutic Agent for B and Myeloid Lineage Leukemias.

Antibody-targeting therapy has drawn great interests to the hematologists and oncologists. 3A4, a novel antibody recognizing human CD45RA antigen, is a new target molecule for leukemias and holds a therapeutic potential for myeloid lineage leukemias. However, murine antibodies cannot be safely used in patients because of their strong immune reaction, humanization of the antibodies interested will be an important development step for therapeutic purpose. The aim of this study was to engineer the mouse 3A4 and to investigate the biological activity of its chimeric form. The humanized antibody composed of the 3A4 single-chain fragment of variable region and the human IgG1 Fc region, which was named human-mouse chimeric antibody 3A4 (Hm3A4). The function and biological activities of Hm3A4 were characterized using a variety of biological approaches. The results showed that Hm3A4 retained a strong binding activity to its antigen and could significantly block the binding of parental 3A4 to the antigen. In vitro experiments revealed that Hm3A4 could kill the target cells through complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity function. In vivo, Hm3A4 showed efficient antileukemia activity outperforming the nontreated mice. In conclusion, the chimeric antibody has an excellent biological activity after humanization and holds targeting therapeutic potential for myeloid leukemia, which warrants further development of this agent.

The targeting effect of Hm2E8b-NCTD-liposomes on B-lineage leukaemia stem cells is associated with the HLF-SLUG axis.

To identify an agent with specific activity against B-lineage leukaemia stem cells (B-LSCs), we generated norcantharidin (NCTD)-encapsulated liposomes modified with a novel humanised anti-human CD19 monoclonal antibody, Hm2E8b (Hm2E8b-NCTD-liposomes). These liposomes were specially designed to recognise and kill B-LSCs in vitro, and to decrease non-specific cytotoxicity to untargeted cells. Hm2E8b-NCTD-liposomes selectively ablated B-LSCs through targeting hepatic leukaemia factor (HLF), which is implicated in haematopoietic stem cell regulation and is overexpressed in LSCs. Hm2E8b-NCTD-liposomes decreased HLF protein levels and induced apoptosis in the HAL-01 cell line harbouring the oncoprotein E2A-HLF. This resulted in modulation of the expression of several molecules that govern survival pathways, including HLF, SLUG, NFIL3 and C-Myc, thereby causing the induction of p53 and the mitochondrial caspase cascade. Therefore, the potent in vitro effect of Hm2E8b-NCTD-liposomes on B-LSC activity and survival pathways have the potential to be exploited clinically with appropriate drug combinations.

Associations between inflammatory cytokines and organ damage in pediatric patients with hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal disease characterized by overwhelming inflammation response and multiple organ damage. Most of the clinical and laboratory manifestations of HLH are thought to be related to hypercytokinemia and organ infiltration with lymphocytes and histiocytes. The aim of this study was to investigate the associations between cytokines and various manifestations of HLH. A total of 105 patients diagnosed with HLH were enrolled in this retrospective study. The information including the patients' demographic characteristics, clinical and laboratory findings at presentation and cytokine data were collected. The median age at diagnosis was 2.8years, with 74 patients (70.4%) documented Epstein-Barr virus infection. Hepatomegaly (88.6%), splenomegaly (81.9%), cytopenia (68.6%), elevated ferritin level (93.3%), hypofibrinogenemia (61.9%) and hemophagocytosis (77.3%) were found in more than half of the patients. Interleukin (IL)-6, IL-10 and interferon (IFN)-γ were found to be moderately or significantly elevated in most patients. In the correlation analysis, IFN-γ was closely related to the concentration of alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, lactate dehydrase (LDH), triglyceride and fibrinogen, while IL-10 was associated with platelet count. When split the patients into two groups according to the cytokine levels, patients with high IFN-γ presented higher level of ALT, AST, bilirubin, LDH, triglyceride, and fibrinogen, while patients with high IL-10 presented much lower hemoglobin and platelet count. In conclusion, the present study put forward clinical evidence that hypercytokinemia is related to organ damage in HLH. IFN-γ may contribute to liver impairment and coagulation disease, while IL-10 is a cytokine related to cytopenias.

Methotrexate Associated Renal Impairment Is Related to Delayed Elimination of High-Dose Methotrexate.

Although Methotrexate (MTX) is an effective drug for the treatment of acute lymphoblastic leukemia (ALL), the toxicity remains a significant problem. In this prospective study, fifty-four patients with ALL were enrolled. 3 g or 5 g MTX/m(2) was administered over 24 hours. Serum MTX concentrations were determined in 24, 48, and 96 hours after MTX infusion. Serum creatinine concentrations and creatinine clearance rate (CCR) were determined before and 24 and 48 hours after MTX infusion. A total of 173 courses of MTX infusion were administered. The serum creatinine concentrations did not change much after MTX infusion while the CCR was gradually decreased. MTX clearance status was independently related to CCR decrease, with the risk of 8.07 to develop renal impairment in patients with delayed MTX elimination. Serum creatinine concentration, serum creatinine ratio, CCR, and CCR ratio at 24 hours were all related to MTX elimination delay. Patients with serum creatinine level >35.0 µmol/L, creatinine ratio >1.129, or CCR <100.0 mL/min were more likely to undergo MTX elimination delay. In conclusion, MTX could induce transient renal impairment and compromised renal function will delay MTX clearance. The serum creatinine concentration and the ratio and CCR are useful tools for evaluating MTX elimination status.

Successful construction and massive expression of a novel Anti-CD19 human-mouse chimeric antibody Hm2E8b.

CD19 antigen is a major target for human B cell malignancies. Many studies have shown that the antibodies recognizing this antigen hold clinical therapeutic potential, while CD19 antibody of mouse origin requires genetic engineering to reduce the potential side effects of the antibody for their clinical use. There are many clones of CD19 antibodies available with different subclasses of immunoglobulin. IgM type antibody holds a high affinity and high complement activating capacities facilitating the targeting efficacy when it is used in targeting therapy. However, engineering the murine IgM antibody into a functional humanized antibody remains a challenge. The aim of this study was to construct a chimeric antibody composed of a CD19 specific murine IgM antibody 2E8 single-chain antibody fragment (scFv) and human IgG1 Fc region, which was named 2E8scFv-Fc or Hm2E8b. The function and the biological activities of this engineered antibody were characterized using a variety of approaches including cellular, immunological, flow cytometric, and molecular biological approaches. After switching from IgM- to IgG-like type antibody, Hm2E8b retained full antigen-binding activity to membrane CD19 antigen as its parental antibody 2E8, and the immune effector function analysis revealed that it could mediate complement-dependent cytotoxicity (CDC) to kill the target cells via IgG1 Fc domain. The yield of the engineered antibody Hm2E8b in the supernatant was 13.3 µg/mL expressed and secreted in the CHO cell system, which reached the secretory quantity of a regular mouse hybridoma cells. Our conclusion is that the IgM type of CD19 mouse antibody can be successfully engineered into an IgG1 type human-mouse chimeric antibody with similar affinity and biological activity. The yield of the Hm2E8b expression and secretion in CHO cell system was adequate to facilitate further development for therapeutic purpose.