[The safety and efficacy of blinatumomab in the treatment of B-cell acute lymphoblastic leukemia in children].

To explore the safety and efficacy of blinatumomab in the treatment of CD19 positive (CD19+) B-cell acute lymphoblastic leukemia (B-ALL) in children. A retrospective analysis was conducted on the clinical data of pediatric B-ALL patients who received blinatumomab treatment from Hematology & Blood Diseases Hospital of Chinese Academy of Medical Sciences from August 2021 to October 2023. Based on their disease status, the patients were divided into refractory/relapsed(RR) group, minimal residual disease clearance (MC) group, and chemotherapy intolerance (IC) group. Clinical data of the children were collected to evaluate the adverse drug reactions, therapeutic efficacy and survival of the children. In total, 35 patients were included, with 20 males and 15 females, aged from 0.6 to 16.4 (9.9±4.2) years old. There were 10 cases in the RR group, 20 cases in the MC group and 5 cases in the IC group. A total of 56 cycles of infusion were completed, with one cycle in 24 cases, two cycles in 5 cases, three cycles in 2 cases and four cycles in 4 cases. The median infusion time [M (Q1, Q3)] from the first to the fourth cycle was 14 (14, 28) days, 28 (28, 28) days, 28 (28, 28) days and 28 (26, 28) days, respectively. In terms of adverse reactions, the incidence of grade 1-2 cytokine release syndrome(CRS) was 57.1% (32/56), with grade 1 CRS accounting for 84.4% (27/32). The incidence rate of immune effector cell-associated neurotoxicity syndrome(ICANS) (grade 4) was 1.8% (1/56). In the RR group, 6 cases were treated effectively, and minimal residual disease(MRD) turned negative, before treatment, MRD levels were all less than 20%. Among them, 3 cases had MRD turning positive again 14 to 42 days after discontinuation of Belintoumab. Four cases were treated ineffectively, with MRD >20% before treatment. All MRD positive cases in MC group turned negative and all MRD negative cases in the IC group remained negative after treatment. The median follow-up time of RR group was 5.7 (3.8, 9.4) months, and 1 year median survival rate and event-free survival rate were 40.0%±21.9% and 33.3%±19.2%, respectively. The median follow-up time for MC and IC group patients was 6.7 (5.2, 12.5) months and 7.1 (5.1, 7.6) months, respectively, with an event free survival rate of 100%. The safety and efficacy of using belintoumab in partial RR, MRD clearance, and chemotherapy intolerance are good.

[Clinical features and prognostic analysis of testicular relapse in pediatric acute lymphoblastic leukemia].

Objective: To investigate the clinical features and prognosis of testicular relapse in pediatric acute lymphoblastic leukemia (ALL). Methods: Clinical data including the age, time from initial diagnosis to recurrence, relapse site, and therapeutic effect of 37 pediatric ALL with testicular relapse and treated in Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences between November 2011 and December 2022 were analyzed retrospectively. Patients were grouped according to different clinical data. Kaplan-Meier analysis was used to evaluate the overall survival (OS) rate and event free survival (EFS) rate for univariate analysis, and Cox proportional-hazards regression model was used to evaluate the influencing factors of OS rate and EFS rate for multivariate analysis. Results: The age at initial diagnosis of 37 pediatric testicular relapse patients was (5±3) years and the time from initial diagnosis to testicular recurrence was (37±15) months. The follow-up time was 43 (22, 56) months. Twenty-three patients (62%) were isolated testis relapse. The 5-year OS rate and EFS rate of the 37 relapsed children were (60±9) % and (50±9) % respectively. Univariate analysis showed that the 2-year EFS rate in the group of patients with time from initial diagnosis to testicular recurrence >28 months was significantly higher than those ≤28 months ((69±10)% vs. (11±11)%, P<0.05), 2-year EFS rate of the isolated testicular relapse group was significantly higher than combined relapse group ((66±11)% vs. (20±13) %, P<0.05), 2-year EFS rate of chimeric antigen receptor T (CAR-T) cell treatment after relapse group was significantly higher than without CAR-T cell treatment after relapse group ((78±10)% vs. (15±10)%, P<0.05). ETV6-RUNX1 was the most common genetic aberration in testicular relapsed ALL (38%, 14/37). The 4-year OS and EFS rate of patients with ETV6-RUNX1 positive were (80±13) % and (64±15) %, respectively. Multivariate analysis identified relapse occurred≤28 months after first diagnosis (HR=3.09, 95%CI 1.10-8.72), combined relapse (HR=4.26, 95%CI 1.34-13.52) and CAR-T cell therapy after relapse (HR=0.15,95%CI 0.05-0.51) were independent prognostic factors for 2-year EFS rate (all P<0.05). Conclusions: The outcome of testicular relapse in pediatric ALL was poor. They mainly occurred 3 years after initial diagnosis. ETV6-RUNX1 is the most common abnormal gene.Patients with ETV6-RUNX1 positive often have a favorable outcome. Early relapse and combined relapse indicate unfavorable prognosis, while CAR-T cell therapy could significantly improve the survival rate of children with testicular recurrence.

[Clinical and prognostic characteristics of pediatric acute myeloid leukemia with myelodysplasia-related changes under different diagnostic criteria].

Objective: To evaluate the clinical and prognostic differences in acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) children under different diagnostic criteria (World Health Organization (WHO) 2016 and WHO 2022 criteria). Methods: In this retrospective cohort study, clinical characteristics and prognosis information of 260 acute myeloid leukemia (AML) children admitted to Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences from August 2017 to August 2021 were analyzed retrospectively. According to WHO 2016 and WHO 2022 diagnostic criteria, patients were divided into AML-MRC group and non-AML-MRC group, the prognostic and genetic differences between two groups were compared respectively. Meanwhile, the characteristics of children with 8 MRC-related genes defined in WHO 2022 diagnostic criteria were described. Mann-Whitney U test, chi-square test were used for comparison between groups. Survival curve was plotted by Kaplan-Meier method, and comparison between groups was performed by Log-Rank method. Results: Among the 260 children, there were 148 males and 112 females. The follow-up time was 26 (16, 38) months. A total of 28 children (10.8%) were diagnosed with AML-MRC according to the WHO 2016 diagnostic criteria. Compared with non-AML-MRC children, the frequency of PTPN11, RUNX11, SH2B3, MPL and STAG2 mutations was higher in AML-MRC children (25.0% (7/28) vs. 4.3% (10/232), 14.3% (4/28) vs. 3.9% (9/232), 10.7% (3/28) vs. 2.2% (5/232), 10.7% (3/28) vs. 2.2% (5/232), 10.7% (3/28) vs. 0.9% (2/232), all P<0.05). The 2-year overall survival (OS) and events free survival (EFS) rate of 28 AML-MRC children under WHO 2016 diagnostic criteria were worse than those of 232 non-AML-MRC children ((62.1±10.8)% vs. (94.5±1.6)%, χ2=22.1,P<0.001;(48.0±10.6)% vs. (70.9±3.2)%, χ2=6.33,P=0.012). Twenty-seven children (10.4%) were eventually diagnosed with AML-MRC according to WHO 2022 criteria, their 2-year OS rate were worse than 233 non-AML-MRC children ((60.8±11.1)% vs. (94.5±1.6)%, χ2=24.49,P<0.001), and there was no statistically significant difference in EFS rate between two groups at 2 years ((55.1±10.8)% vs. (70.1±3.2)%, χ2=2.44, P=0.119). Conclusions: Compared with the 2022 WHO diagnostic criteria, the survival rates of children with AML-MRC under the 2016 WHO diagnostic criteria were worse than that of children without MRC.The new version of the AML-MRC diagnostic criteria emphasizes the importance of genes.

[Analysis of factors affecting the quality of donor corneal endothelial cells].

Objective: To explore the factors influencing the quality of donor corneal endothelium. Methods: A retrospective case series study was conducted. Data from 568 donor corneas obtained from the Shandong Eye Bank between July 1, 2020, and June 30, 2021, were collected for analysis. The corneal endothelium of the donor corneas was observed using corneal endothelial microscopy to assess corneal endothelial cell density (ECD), coefficient of variation, and hexagonal cell ratio (HEX). Relevant factors of corneal donors were collected, including gender, age, cause of death, season of death, time from death to corneal retrieval, and methods of corpse preservation, to investigate their impact on the quality of donor corneal endothelium. The age factor was divided into five age groups: 0-20 years, 21-40 years, 41-60 years, 61-80 years, and >80 years. The time of corneal retrieval was divided into three periods based on the time elapsed since the donor's death: <6 hours, 6-12 hours, and >12 hours. The relationship between these factors and corneal endothelial conditions was analyzed. Results: The 568 donor corneas were obtained from 288 donors, including 225 males (78.13%) and 63 females (21.87%). The mean age was 51.77±18.48 years. The causes of death among donors were as follows: cardiovascular diseases 54.58% (275 individuals), cancer 17.96% (74 individuals), organ failure 14.26% (49 individuals), and accidents 13.20% (64 individuals). The mean time of corneal retrieval after donor death was 140 (76, 400) minutes (ranging from 30 minutes to 45 hours). Among the 145 corneas (25.53%) that had their initial corneal endothelial microscopy examination, the images were not clear, and after thorough rewarming, 106 corneas (18.7%) still had unclear images and could not be analyzed. Among the 462 corneas (81.3%) with clear images, the ECD was (2 602.23±318.40) cells/mm², the coefficient of variation was 36.61%±4.81%, and the HEX was 52.73%±7.15%. The ECD of corneas from older donors was lower compared to younger donors, and the differences between age groups were statistically significant (P<0.001). Corneas from donors who died due to accidents had a higher ECD [(2 829.88±313.90) cells/mm²] compared to those who died from cancer, cardiovascular diseases, and organ failure, and the differences were statistically significant (P<0.001). The ECD was highest when corneas were retrieved within 6 hours after death, and the difference was statistically significant (P<0.001). Older donors had higher coefficients of variation but lower HEX values (both P<0.05). Corneas retrieved after a longer time from death had higher coefficients of variation, and the difference was statistically significant (P<0.05), but there was no statistically significant difference in HEX (P>0.05). Organ failure, cryopreservation, and corneal retrieval time >12 hours were risk factors for unclear corneal endothelial imaging (all P<0.001). Among the 136 corneal endothelial images (23.94%), circular, oval, or band-shaped dark areas were observed, and corneas with dark areas had lower ECD (P<0.05). The longer the time elapsed from death to corneal retrieval, the more dark areas were observed (P<0.001). The presence of dark areas did not affect the coefficient of variation and HEX (P>0.05). Conclusion: Advanced donor age, death due to chronic diseases, longer time elapsed from death to corneal retrieval, and cryopreservation of the body lead to a decrease in the quality of donor corneal endothelium.

[Genomics of next generation sequencing in pediatric B-acute lymphoblastic leukemia and its impact on minimal residual disease].

Objective: To describe the gene mutation profile of newly diagnosed pediatric B-acute lymphoblastic leukemia (B-ALL) and analyze its effect on minimal residual disease (MRD). Methods: A total of 506 newly diagnosed B-ALL children treated in Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences from September 2018 to July 2021 were enrolled in this retrospective cohort study. The enrolled children were divided into MRD ≥1.00% group and <1.00% group according to MRD results on the 19th day since chemotherapy, and MRD ≥0.01% group and <0.01% group according to MRD results on the 46th day. Clinical characteristics and gene mutations of two groups were compared. Comparisons between groups were performed with chi-square test or Fisher's exact test. Independent risk factors of MRD results on the 19th day and the 46th day were analyzed by Logistic regression model. Results: Among all 506 patients, there were 318 males and 188 females. On the 19th day, there were 114 patients in the MRD ≥1.00% group and 392 patients in the MRD <1.00% group. On the 46th day, there were 76 patients in the MRD ≥0.01% group and 430 patients in the MRD <0.01% group. A total of 187 gene mutations were detected in 487 (96.2%) of 506 children. The most common gene mutations were signal transduction-related KRAS gene mutations in 111 cases (22.8%) and NRAS gene mutations in 99 cases (20.3%). Multivariate analysis showed that PTPN11 (OR=1.92, 95%CI 1.00-3.63), KMT2A (OR=3.51, 95%CI 1.07-11.50) gene mutations and TEL-AML1 (OR=0.48, 95%CI 0.27-0.87), BCR-ABL1 (OR=0.27, 95%CI 0.08-0.92) fusion genes and age >10 years (OR=1.91, 95%CI 1.12-3.24) were independent influencing factors for MRD ≥1.00% on the 19th day. BCORL1 (OR=2.96, 95%CI 1.18-7.44), JAK2 (OR=2.99, 95%CI 1.07-8.42) and JAK3 (OR=4.83, 95%CI 1.50-15.60) gene mutations and TEL-AML1 (OR=0.43, 95%CI 0.21-0.87) fusion gene were independent influencing factors for MRD ≥0.01% on the 46th day. Conclusions: Children with B-ALL are prone to genetic mutations, with abnormalities in the RAS signaling pathway being the most common. Signal transduction related PTPN11, JAK2 and JAK3 gene mutations, epigenetic related KMT2A gene mutation and transcription factor related BCORL1 gene mutation are independent risk factors for MRD.

The important role of TFEB in autophagy-lysosomal pathway and autophagy-related diseases: a systematic review.

Autophagy is a main metabolic process in which eukaryotic cells use lysosomes to eliminate abnormal proteins and damaged organelles to maintain cell homeostasis. Studies have revealed that neurodegenerative diseases, tumor, hepatic diseases, etc. are related to abnormal autophagy processes in recent years. Recent studies have shown that TFEB is a major transcription regulator of autophagy-lysosomal pathway (ALP) transcriptional regulation, which positively regulates the expression of autophagy and lysosomal biogenesis-related genes, thereby promoting autophagosome formation, autophagosome-lysosome fusion, and degradation of autophagy substrates. It has also been found that TFEB promotes clearance of intracellular substrates through lysosomal exocytosis. Therefore, the study of biological functions and related regulatory mechanisms of TFEB will provide important clues and theoretical basis for further explaining its physiological pathogenesis and the treatment of related diseases.

Effects of miR-18a on proliferation and apoptosis of gastric cancer cells by regulating RUNX1.

OBJECTIVE: The aim of this study was to explore the effects of micro ribonucleic acid (miR)-18a on the proliferation and apoptosis of gastric cancer (GC) cells, and to elucidate the possible underlying mechanism. PATIENTS AND METHODS: In this study, the expression of miR-18a in GC tissues and para-cancer tissues was verified by in situ hybridization (ISH) of GC tissue microarray (TMA). Meanwhile, the effect of miR-18a expression on the prognosis of GC patients was evaluated. GC AGS cell line was selected and transfected with miR-18a mimic and mimic control (NC) to up-regulate miR-18a expression in vitro. Thereafter, changes in cell proliferation, apoptosis and migration after transfection were detected by biological functional assays. Luciferase reporter gene assay was carried out to verify the target gene Runt-related transcription factor 1 (RUNX1) modulated by miR-18a. Finally, the Spearman's grade correlation coefficient was calculated to explore the correlation between the expressions of miR-18a and RUNX1. RESULTS: ISH results of TMA showed that overexpression of miR-18a in GC tissues was significantly associated with low survival rate of patients (p<0.001). High expression of miR-18a remarkably enhanced the proliferation, migration and invasion of GC cells (p<0.05). Besides, it has been predicted in biology that RUNX1 is one of the target genes of miR-18a. Luciferase reporter gene assay showed that Luciferase activity in cells transfected with wild-type (WT) RUNX1 3' untranslated region (3'UTR) was significantly reduced (p<0.05). Moreover, the protein expression of RUNX1 decreased remarkably in GC cells with over-expression of miR-18a (p<0.05). All these findings indicated that the expression of miR-18a was negatively correlated with RUNX1 in GC cells (p<0.001, r=0.86). CONCLUSIONS: MiR-18a exerts a high predictive value for the prognosis of GC patients by directly targeting the transcription factor RUNX1. All our findings may provide therapeutic candidates for GC identification.

[A special on epidemic prevention and control: analysis on expression of 2019-nCoV related ACE2 and TMPRSS2 in eye tissues].

This article was published ahead of print on the official website of Chinese Journal of Ophthalmolog on Apirl 22,2020. Objective: Angiotensin converting enzyme 2 (ACE2) and Transmembrane serine protease 2 (TMPRSS2) are the key proteins for 2019-nCoV entry into host cells. To evaluate the potential infection risk of 2019-nCoV on ocular surface, we compared ACE2 and TMPRSS2 expression among different eye tissues. Methods: Experimental study. Thirty mice were assigned to male, female, aged, diabetic and non-diabetic groups, with 6 mice in each group. Real-time PCR was performed to quantify ACE2 and TMPRSS2 gene expression in conjunctiva, cornea, lacrimal gland, iris, lens, retina, lung, heart, kidney, and liver from male mice. Immunohistochemistry staining was applied to visualize the distribution of the two proteins in different mice tissues, and in human corneal and conjunctival sections. Published transcriptome datasets were extracted to generate the expression comparasion of ACE2 and TMPRSS2 between human conjunctival and corneal tissues, and results were analyzed using Mann-Whitney U test. Female mice, aged mice, STZ-induced diabetic mice, diabetic group control mice were also subjected to ACE2 expression analysis. Results were analyzed using Student's t-test. Results: The expression of ACE2 and TMPRSS2 genes were the highest in conjunctiva among all the six mice eye tissues explored. The expression of these two genes in conjunctiva were lower than that in kidney and lung. ACE2 and TMPRSS2 shared similar expression pattern with the staining concentrated in corneal epithelium, conjunctival epithelium and lacrimal gland serous cells. The expression levels of ACE2 showed gender difference. Female mice had lower ACE2 in conjunctiva and cornea than male mice, with the expression levels being only 43% (t=3.269, P=0.031) and 63% (t=4.080, P=0.015) of that in the male conjunctiva and cornea, respectively. Diabetic mice expressed more ACE2 in conjunctiva (1.21-fold, P>0.05) and lacrimal gland (1.10-fold, P>0.05) compared with the control group. No significant difference on ACE2 expression was found between the aged and young adult mice. The expression level of human conjunctiva ACE2 and TMPRSS2 were significantly higher than that in the cornea (P=0.007), with 5.74-fold and 12.84-fold higher in the conjunctiva than in the corneal epithelium cells, which resembled the situation in mice. Conclusion: The observation of high-level ACE2 and TMPRSS2 expression in conjunctiva among the 6 eye tissues examined suggests that conjunctiva serves as an infection target tissue of 2019-nCoV. (Chin J Ophthalmol, 2020, 56:438-446).

Effects of propofol on proliferation and apoptosis of cardia cancer cells via MAPK/ERK signaling pathway.

OBJECTIVE: To explore the influences of propofol on the proliferation and apoptosis of cardia cancer cells via mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway. PATIENTS AND METHODS: A total of 65 surgical resection specimens of cardia cancer were selected as research objects and divided into control group and with low (12.5 µmol/L), medium (25 µmol/L), and high (50 µmol/L) propofol concentration groups. The apoptosis of cancer cells, ERK1/2 phosphorylation level, expressions of Caspase-3, B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (Bax) in each group were detected. RESULTS: Propofol in different concentrations could all effectively inhibit the proliferation of cardia cancer cells in a dose-dependent manner. Different concentrations of propofol promoted the apoptosis of cardia cancer cells, and the apoptosis rate constantly increased with the rising concentration of propofol (p<0.05). Propofol could repress the expression of Bcl-2 and up-regulate the expression levels of Caspase-3, Bax, and phosphorylated ERK1/2. CONCLUSIONS: Propofol can inhibit the proliferation and induce the apoptosis of cardia cancer cells, and the action mechanism may be correlated with the inhibition on the MAPK/ERK signaling pathway.

Netrin-1 prolongs skin graft survival by inducing the transformation of mesenchymal stem cells from pro-rejection to immune-tolerant phenotype.

OBJECTIVE: Mesenchymal stem cells (MSCs) induce allograft immune tolerance, but low efficacy severely limits their wide application. In this work, Netrin-1 was used to maintain MSC function in an IR environment to study its role in the immune tolerance induction of the allograft. MATERIALS AND METHODS: The experiments were divided into three groups: the control group, the IR group and the Netrin-1 group (Netrin-1 was added to MSC medium and then cultured for 48 h). After digestion, MSCs were mixed with TLR4 and TLR3 antibodies (BD), incubated for 20 min, and washed with Phosphate-Buffered Saline (PBS) three times. The mean fluorescence intensity (MFI) of TLR4 and TLR3 was detected by flow cytometry. Isolated lymphocytes were divided into four groups: the control group (no treatment), the MSC group (lymphocytes were co-cultured with MSCs in the control group), the rejection group (lymphocytes were co-cultured with MSCs in the IR group), and the Netrin-1 group (MSCs in the IR group) was stimulated by Netrin-1 for 48h. RESULTS: Our study found that compared with control mice, toll-like receptor (TLR3) expression in bone marrow MSCs decreased as the expression of TLR4 increased, the secretion of transforming growth factor-ß (TGF-ß) and interleukin-10 (IL-10) was reduced, while the secretion of IL-6 significantly increased in immune rejection (IR) mice. MSCs in IR mice promoted T-cell proliferation and reduced the ratio of Treg cells. Netrin-1 inhibited the pro-rejection effect of these MSCs, further inhibited T-cell proliferation and facilitated an increase in the ratio of Treg cells. The animal experiment results showed that MSC transplantation in the rejection group would shorten the mean survival time of the skin graft and induce the infiltration of lymphocytes. Netrin-1 prolonged the mean survival time of the skin graft by enhancing MSC function. The immunohistochemistry results showed that, compared with the rejection group, the T cell number in the skin graft significantly decreased in the Netrin-1 group. CONCLUSIONS: MSC can be divided into immune-tolerant and pro-rejection types in organ transplantation and Netrin-1 can induce the transformation of MSC from the pro-rejection to immune-tolerant type and markedly prolong the skin graft survival time.