[Chinese guideline for diagnosis and treatment of adult acute lymphoblastic leukemia (2024)].

Adult acute lymphoblastic leukemia (ALL) is one of the most common acute leukemia in adults. There are relatively unified diagnostic criteria and systematic treatment regimens reported by different research groups in the world. Since 2013, three versions of expert consensus/guidelines on the diagnosis and treatment of adult ALL in China have been published, which are of great significance for improving the level of diagnosis and treatment of this disease. In 2022, the classification of ALL (precursor lymphocyte neoplasms-lymphoblastic leukemias/lymphomas) had been updated in the WHO classification of haematolymphoid tumors, and some new concepts had been proposed. In recent years, the rapid development of immunotherapy has improved the efficacy of adult ALL, and commercial antibodies and CAR-T cell products have been available in China, the clinical practice is increasing. In order to promote the standardization of clinical diagnosis and treatment of adult ALL, by referring to the latest guidelines and literatures all over the world, this guideline may contribute to the better understanding of diagnosis, treatment and efficacy monitoring for adult ALL.

[Mutational spectrum and its prognostic significance in childhood acute lymphoblastic leukemia based on next-generation sequencing technology].

Objective: This study analyzed the correlation between genetic mutation and prognostic significance in childhood acute lymphoblastic leukemia (ALL) . Methods: Targeted exome by next-generation sequencing (NGS) technology was used to carry out molecular profiling of untreated 141 children with ALL in Fujian Medical University Union Hospital from November 2016 to December 2019. Correlation of genetic features and clinical features and outcomes was analyzed. Results: Among the 141 pediatric patients with ALL, 160 somatic mutations were detected in 83 patients (58.9% ) , including 37 grade Ⅰ mutations and 123 grade Ⅱ mutations. Single nucleotide variation was the most common type of mutation. KRAS was the most common mutant gene (12.5% ) , followed by NOTCH1 (11.9% ) , and NRAS (10.6% ) . RAS pathway (KRAS, FLT3, PTPN11) , PAX5 and TP53 mutations were only detected, and NRAS mutations was mainly found in B-ALL while FBXW7 and PTEN mutations were only found, and NOTCH1 mutation was mainly detected in T-ALL. The average number of mutations detected in each child with T-ALL was significantly higher than in children with B-ALL (4.16±1.33 vs 2.04±0.92, P=0.004) . The children were divided into mutation and non-mutation groups according to the presence or absence of genetic variation. There were no statistically significant differences in sex, age, newly diagnosed white blood cell count, minimal or measurable residual disease monitoring results, expected 3-year event-free survival (EFS) and overall survival (OS) between the two groups (P>0.05) . On the other hand, the proportion of T-ALL and fusion gene negative children in the mutant group was significantly higher than the non-mutation group (P=0.021 and 0.000, respectively) . Among the patients without fusion gene, the EFS of children with grade I mutation was significantly lower than children without grade I mutation (85.5% vs 100.0% , P=0.039) . Among children with B-ALL, the EFS of those with TP53 mutation was significantly lower than those without TP53 mutation (37.5% vs 91.2% , P<0.001) . Conclusion: Genetic variation is more common in childhood ALL and has a certain correlation with clinical phenotype and prognosis. Therefore, targeted exome by NGS can be used as an important supplement to the traditional morphology, immunology, cytogenetics, and molecular biology classification.

[The efficacy and safety of low-dose chemotherapy combined with tyrosine kinase inhibitors in the treatment of Philadelphia-chromosomal-positive acute lymphoblastic leukemia].

Objective: The study aims to explore the efficacy and safety of low-dose chemotherapy combined with tyrosine kinase inhibitor (TKI) as an induction therapy for Philadelphia-chromosomal-positive acute lymphoblastic leukemia (Ph(+) ALL) . Methods: The data of the consecutive newly diagnosed patients with Ph(+) ALL were reviewed. The efficacy and safety of low-dose chemotherapy and conventional-dose chemotherapy combined with TKI were compared. Results: A total of 217 patients with a median age of 38 (10-69) years old were included in this study. 78 patients were in the low-dose chemotherapy group, and 139 patients were in the conventional-dose chemotherapy group. There were no significant differences in the 4-week complete remission (CR) rate (98.7% vs 97.0%, P=0.766) and overall CR rate (100% vs 100%, P=1.000) between the two groups. Multivariate analyses showed that the chemotherapy intensity was not related to the disease-free survival rate and overall survival rate. However, the lower incidence of infection (P=0.017) , the shorter duration of neutropenia (P=0.001) and PLT<20 × 10(9)/L (P=0.057) , and the lower red blood cell transfusion volume (P=0.002) were more common in the low-dose chemotherapy group than in the conventional-dose chemotherapy group. Conclusions: The low-dose chemotherapy is superior to the conventional-dose chemotherapy combined with TKI as induction therapy in Ph(+) ALL with similar efficacy but is safer.

Clinical characteristics and prognosis analysis of children with acute lymphoblastic leukemia and TP53 mutation / 白血病·淋巴瘤

Objective:To investigate the clinical characteristics and efficacy of children with acute lymphoblastic leukemia (ALL) and TP53 mutation, and to explore the relationship between TP53 mutation and the prognosis of children with ALL.Methods:The clinical data of 141 children with newly diagnosed ALL from November 2016 to December 2019 in Fujian Medical University Union Hospital were collected, and the whole-exome gene assay was performed in bone marrow samples of the children by using next-generation sequencing technology. The clinical characteristics of children with TP53 mutation were retrospectively analyzed, and the Kaplan-Meier method was used to compare the overall survival (OS) and event-free survival (EFS) of children with or without TP53 mutation.Results:Among the 141 children with newly diagnosed ALL, TP53 mutations were detected in 5 children (3.5%), all of which were B-precursor acute lymphoblastic leukemia (B-ALL). No TP53 mutation was detected in T-cell acute lymphoblastic leukemia (T-ALL) children, and TP53 mutation accounted for 4.0% (5/126) of B-ALL children. The types of TP53 mutation were all single nucleotide variants. Five ALL children with TP53 mutation were male, with a median age of 60 months (16- 156 months). At the time of onset, all children had anemia and elevated lactate dehydrogenase, and 4 children had subcutaneous hemorrhage and hyperuricemia. The immunophenotypes of all children were precursor B-cell type, and 4 children had myeloid antigen expression. Among 4 ALL children with TP53 mutation who received standard treatment, 2 cases relapsed, and the recurrence time was 8.9 months and 12.1 months, respectively. The expected 15-month EFS rate and OS rate of ALL children with TP53 mutation were lower than those of ALL children without TP53 mutation (37.5% vs. 97.7%, χ2 = 29.90, P < 0.001; 37.5% vs.98.3%, χ2 = 24.90, P < 0.001). Conclusions:ALL children with TP53 mutation are more commonly found in male and B-cell type, with high early recurrence rate and poor efficacy. TP53 mutation may become a necessary supplement for prognostic assessment.

Status and significance of BCR-ABL1 kinase domain mutations in imatinib-resistant chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia patients from Northeast China / 白血病·淋巴瘤

Objective:To explore the characteristics of BCR-ABL1 kinase domain mutations in imatinib-resistant chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) patients from Northeast China and their impact on prognosis. Methods:The clinical data of 252 CML patients and 49 Ph + ALL patients who were admitted to the First Hospital of Jilin University from January 2013 to October 2018 were retrospectively analyzed. The samples of bone marrow or peripheral blood were collected from patients when imatinib treatment was not effective. Nested polymerase chain reaction (PCR) was used to amplify the BCR-ABL1 kinase domain, and Sequencing Analysis v5.4 software was used to analyze the mutation of BCR-ABL1 kinase domain. Patients were followed up for 6-48 months, and the survival analysis was performed. Results:Among 252 CML patients, the mutations in ABL1 kinase domain were found in 57 patients (22.6%), including 25 patients in the chronic phase, 21 patients in the accelerated phase and 11 patients in the blast crisis; 50 patients had 20 types of single point mutation, and the most common mutation types were E255K (16.0%, 8/50), T315I (14.0%, 7/50), M244V (8.0%, 4/50) and G250E (8.0%, 4/50), which were all concentrated in the P-loop and C-helix domains; 7 patients had double mutations; patients with multiple mutations had the worst prognosis, with a median overall survival (OS) time of 3.2 months. Among 49 Ph + ALL patients, 17 cases (34.7%) were positive for mutations in the BCR-ABL1 kinase domain, 14 patients had 12 types of single point mutation, and 3 patients had multiple mutations; the median OS time of patients with multiple mutations, mutations located in the P-loop and C-helix domains and mutations located in the other domains was 2.0, 8.0 and 18.0 months, and the difference in OS among the three groups was statistically significant ( P < 0.01). Conclusions:Among the imatinib-resistant CML and Ph + ALL patients from Northeast China, point mutations in the P-loop and C-helix domains are most commonly found. Multiple mutations, mutations in the P-loop and C-helix domains are related to the poor prognosis of the patients.

Mutational spectrum and its prognostic significance in childhood acute lymphoblastic leukemia based on next-generation sequencing technology / 中华血液学杂志

Objective: This study analyzed the correlation between genetic mutation and prognostic significance in childhood acute lymphoblastic leukemia (ALL) . Methods: Targeted exome by next-generation sequencing (NGS) technology was used to carry out molecular profiling of untreated 141 children with ALL in Fujian Medical University Union Hospital from November 2016 to December 2019. Correlation of genetic features and clinical features and outcomes was analyzed. Results: Among the 141 pediatric patients with ALL, 160 somatic mutations were detected in 83 patients (58.9% ) , including 37 grade Ⅰ mutations and 123 grade Ⅱ mutations. Single nucleotide variation was the most common type of mutation. KRAS was the most common mutant gene (12.5% ) , followed by NOTCH1 (11.9% ) , and NRAS (10.6% ) . RAS pathway (KRAS, FLT3, PTPN11) , PAX5 and TP53 mutations were only detected, and NRAS mutations was mainly found in B-ALL while FBXW7 and PTEN mutations were only found, and NOTCH1 mutation was mainly detected in T-ALL. The average number of mutations detected in each child with T-ALL was significantly higher than in children with B-ALL (4.16±1.33 vs 2.04±0.92, P=0.004) . The children were divided into mutation and non-mutation groups according to the presence or absence of genetic variation. There were no statistically significant differences in sex, age, newly diagnosed white blood cell count, minimal or measurable residual disease monitoring results, expected 3-year event-free survival (EFS) and overall survival (OS) between the two groups (P>0.05) . On the other hand, the proportion of T-ALL and fusion gene negative children in the mutant group was significantly higher than the non-mutation group (P=0.021 and 0.000, respectively) . Among the patients without fusion gene, the EFS of children with grade I mutation was significantly lower than children without grade I mutation (85.5% vs 100.0% , P=0.039) . Among children with B-ALL, the EFS of those with TP53 mutation was significantly lower than those without TP53 mutation (37.5% vs 91.2% , P<0.001) . Conclusion: Genetic variation is more common in childhood ALL and has a certain correlation with clinical phenotype and prognosis. Therefore, targeted exome by NGS can be used as an important supplement to the traditional morphology, immunology, cytogenetics, and molecular biology classification.

[Dasatinib combined with multi-agent chemotherapy regimen in newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia: a prospective study from a single center].

Objective: This study evaluates the efficacy and safety of dasatinib combined with a multi-agent chemotherapy regimen of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) patients. Methods: This prospective, single-arm, and open clinical study enrolled 30 adult Ph(+) ALL patients who were newly diagnosed and treated from January 2016 to April 2018 in the center of this study. Standard induction chemotherapy was given for 4 weeks. However, dasatinib (100 mg/d) was continuously administered from day 8 until the end of the whole therapy in the induction therapy. Patients who are available for allogeneic or autologous stem cell transplantation (SCT) received transplantation when the disease was evaluated as complete remission. Results: All 30 patients achieved hematological complete remission (HCR) after the induction chemotherapy, and 70.0% (21/30) of them achieved the accumulated molecular complete remission (MCR) . The patients were followed up with a median follow-up time of 37.8 months (32.0-46.6) . The 3 year overall survival (OS) and 3 year hematological relapse-free survival (HRFS) were 68.1% and 61.6%, respectively. Moreover, 63.3% and 43.3% of the patients achieved molecular major remission and MCR, respectively. Consequently, 60.0% of the patients achieved MCR until 6 months. The patients who achieved MCR within 6 months had superior OS (P=0.004) , HRFS (P=0.049) , and event-free survival (EFS; P=0.001) . Fifteen patients (50.0%) received SCT at the first HCR. However, HRFS (P=0.030) and EFS (P=0.010) in the SCT group were better than those in the chemotherapy group. Conclusions: The regimen of dasatinib combined with a multi-agent chemotherapy was proven safe and effective in the treatment of newly diagnosed adult Ph(+) ALL patients. Clinical trial registration: ClinicalTrials.gov, NCT02523976.

[Preparation of a novel tri-specific T cell engager targeting CD19 antigen and its anti-leukemia effect exploration].

Objective: To prepare a novel tri-specific T cell engager (19TriTE) targeting CD19 antigen, and to investigate its immunotherapeutic effect on CD19-positive hematological malignancies. Methods: 19TriTE was constructed by molecular cloning technology and successfully expressed through the eukaryotic expressing system. The effects of 19TriTE on the proliferation and activation of T cells, as well as the specific cytotoxicity against CD19 positive tumor cell lines were verified. Results: ①19TriTE expressing plasmid was constructed and successfully expressed through the eukaryotic expressing system. ②19TriTE can specifically bind to T cells and Nalm6 cells, with equilibrium dissociation constants of 19.21 nmol/L and 11.67 nmol/L, respectively. ③The expression rates of CD69 positive T cells and CD25 positive T cells were 35.4% and 49.8% respectively, when 2 nmol/L 19TriTE were added in the co-culture system, which were significantly higher than those in the control group. ④19TriTE can significantly promote the proliferation of T cells. The absolute count of T cells expanded from the initial one million to 74 million with an 74 fold increase at the concentration of 1 nmol/L on day 12. ⑤19TriTE can significantly mediate T cells killing of CD19 positive target cells in a dose-dependent manner. At the concentration of 10 nmol/L, the target cells lysis reached 50%. ⑥Degranulation experiment verified that 19TriTE can activate T cells in the presence of CD19 positive target cells, and the activation of T cells positively correlated with the dose of 19TriTE. ⑦When 19TriTE fusion protein co-cultured with T cells and target cells overexpression RFP and luciferase genes respectively, 19TriTE can notably mediate T cells killing of CD19 positive target cells through fluorescent microscope or bioluminescence imaging technology. Conclusion: In this study, we successfully constructed and expressed 19TriTE fusion protein and verified that it can effectively activate T cells and promote their proliferation in vitro. At the same time, it can bind to CD19 positive target cells and T cells, as well as enhance T cells anti-leukemia effect in vitro, providing the foundation for further clinical research.

[Clinical features and prognosis of ETV6-RUNX1-positive childhood B-precursor acute lymphocyte leukemia].

Objective: To investigate the clinical features and prognosis of ETV6-RUNX1-positive childhood B-precursor acute lymphocyte leukemia (B-ALL) . Methods: The clinical data of 927 newly diagnosed children with B-ALL admitted to the Fujian Medical University Union Hospital from April 2011 to May 2020 were retrospectively analyzed. According to the results of ETV6-RUNX1 gene, the patients were divided into ETV6-RUNX1(+) and ETV6-RUNX1(-) groups. The clinical features and prognosis between the two groups were compared. Among the 182 children with ETV6-RUNX1(+), 144 patients received the Chinese Childhood Leukemia Collaborative Group (CCLG) -ALL 2008 protocol (CCLG-ALL 2008 group) and 38 received the China Childhood Cancer Collaborative Group (CCCG) -ALL2015 protocol (CCCG-ALL 2015 group) . The efficacy, serious adverse effects (SAE) incidence, and treatment-related mortality (TRM) of the two groups were also compared. Results: Of the 927 B-ALL patients, 189 (20.4% ) were ETV6-RUNX1(+). The proportion of patients with risk factors (age ≥10 years or <1 year, white blood cell count ≥50×10(9)/L) in the ETV6-RUNX1(+) group was significantly lower than that in the ETV6-RUNX1(-) group (P=0.000, 0.001, respectively) , while the proportion of patients with good early response (good response to prednisone, d15 or d19 MRD <1% , and d33 or d46 MRD<0.01% in induction chemotherapy) in the ETV6-RUNX1(+) group was significantly higher than that in the ETV6-RUNX1(-) group (P=0.028, 0.004, respectively) . The 5-year EFS and OS of the ETV6-RUNX1(+) group were significantly higher than those of the ETV6-RUNX1(-) group (EFS: 89.8% vs 83.2% , P=0.003; OS: 90.2% vs 86.3% , P=0.030) . The incidence of infection-related SAE and TRM was significantly higher than that of CCCG-ALL 2015 group. A statistical difference was observed between the incidence of infection-related SAE of the two groups (27.1% vs 5.3% , P=0.004) , but no difference in TRM (4.9% vs 0, P=0.348) . Conclusion: ETV6-RUNX1(+)B-ALL children have fewer risk factors at diagnosis, better early response, lower recurrence rate, and good prognosis than that of ETV6-RUNX1(-)B-ALL children. Reducing the intensity of chemotherapy appropriately can lower the infection-related SAE and TRM and improve the long-term survival in this subtype.

[Efficacy and prognostic factors of the chemotherapy regimen of CCLG-ALL-2008 on pediatric acute lymphoblastic leukemia with ETV6-RUNX1 rearrangement].

Objective: To evaluate the predictive role of ETV6-RUNX1 fusion gene in protocol CCLG-ALL-2008 as well as identify the prognostic factors that influence the outcome of ALL with ETV6-RUNX1 fusion gene. Methods: One hundred and seventy-eight patients newly diagnosed with pediatric acute lymphoblastic leukemia with ETV6-RUNX1 rearrangement from April 2008 to April 2015 were enrolled in CCLG-ALL-2008. The follow up period ended in July 2018; we performed retrospective analyses of their data to determine the efficacy of the regimen and the prognostic factors. Results: The median age of the study population (178 pediatric patients) , including 100 boys and 78 girls was 4 (1-13) y, and the median white blood cell count at diagnosis was 9.46 (1.25-239.83) ×10(9)/L. Three patients died, and 1 was lost to follow up by the end of the first induction chemotherapy, resulting in an induced remission rate of 97.8% (174/178) . The cumulative incidence of relapse was 15.9% with a median follow up of 73.5 mon. Total 83.3% of the relapse cases were those of isolated bone marrow relapse, while 79.2% of the cases were those of late relapse. The median interval time between relapse and first complete remission was 35.5 mon (range, 1-62 months) . One of the 5 patients with early recurrence and 7 of the 19 with late recurrence cases survived. The 5-year-OS and 5-year-EFS of ETV6-RUNX1 positive children was (89.4±2.4) % and (82.1±6.9) %, respectively. The estimated 10-year-OS and 10-year-EFS of ETV6-RUNX1 positive children was (88.6±2.5) % and (77.3±4.0) %, respectively. The Kaplan-Meier method and Log-rank test were used to estimate and compare the survival. Univariate statistical analysis showed that poor prognostic factors that influenced survival included central nervous system state 2 at diagnosis, poor prednisone response, high risk, gene positivity after induction chemotherapy, as well as MRD positivity and gene positivity at the 12(th) week. In the multivariate analysis, only the central nervous system state 2 at diagnosis and MRD positivity at the 12(th) week were associated with the outcome. Conclusion: ETV6-RUNX1-positive ALL is a subgroup with a favorable prognosis as per the CCLG-ALL-2008 protocol. Patients with ETV6-RUNX1 should be given more intensive therapy, including hematopoietic stem cell transplantation when they are CNS2 at diagnosis or have high level of MRD at the 12(th) week after treatment.

[Diagnosis of adult Philadelphia chromosome-like acute lymphoblastic leukemia by fluorescence in situ hybridization].

Objective: To establish a screening system of adult Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) by fluorescence in situ hybridization (FISH) . Method: Based on the genetic characteristics of Ph-like ALL, FISH probes were designed for ABL1, ABL2, JAK2, EPOR, CRLF2, CSF1R, PDGFRB, and P2RY8 gene breakpoints, which were used to screen Ph-like ALL in B-ALL patients without BCR-ABL1, ETV6-RUNX1, MLL, and E2A gene arrangement. Furthermore, it was analyzed in combination with flow immunophenotype, next-generation sequencing for targeted gene mutations, and RNA sequencing (RNA-seq) . Results: A total of 189 adult B-ALL patients diagnosed in Nanfang Hospital from January 2016 to April 2019 were enrolled in this study. Using FISH and/or PCR, BCR-ABL1, ETV6-RUNX1, MLL, or E2A arrangement was detected in 83 of them, and Ph-like ALL was detected by FISH in the other 106, resulting in the presence of typical gene arrangements of Ph-like ALL in 12 patients (11.3% , 12/106) . Validated by RNA-seq, the sensitivity and specificity of FISH for Ph-like ALL were 71.4% and 95.8% , respectively. After further analysis with immunophenotype, targeted gene mutations, and RNA-seq, 14 (13.2% , 14/106) were diagnosed with Ph-like ALL. Conclusion: This data shows high specificity of FISH for identification of Ph-like ALL and combining immunophenotype and sequencing technology can improve the diagnostic system.

[Prognostic analysis of allogeneic hematopoietic stem cell transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia in complete remission in the era of tyrosine kinase inhibitors].

Objective: To study the clinical results and prognostic factors for allo-HSCT of Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL) in complete remission (CR) in the era of tyrosine kinase inhibitors (TKI) . Methods: We performed a retrospective analysis of the clinical characteristics of 116 patients with Ph(+)ALL who underwent allo-HSCT while in CR. Results: The study population included 72 men and 44 women. The median patient age was 20 years (4-64 years) . The patients received sibling-identical donor (n=21) , haplo (n=77) , and unrelated donor (n=18) HSCT. The overall survival (OS) rate at 5 years was 73.2% (95% CI 63.8% -80.5% ) . In particular, the 5-year OS can reach 87.5% when the time from diagnosis to transplant is <180 days. The 5-years DFS was 61.4% (95% CI 51.8% -69.7% ) , the 5-year molecular and morphology cumulative relapse incidence was 18.5% (95% CI 12.6% -27.3% ) , and the 5-year TRM was 19.9% (95% CI 13.8% -28.7% ) . A multivariate analysis showed that an age range of 15-39 years (HR=2.730, P=0.044) , time from diagnosis to HSCT ≥ 180 days (HR=4.534, P=0.010) , and Ⅲ-Ⅳgrade aGVHD (HR=7.558, P=0.000) were significantly associated with an inferior overall survival. Limited cGVHD subgroup had better OS (HR=0.300, P=0.034) . Sex, WBC count at diagnosis, type of BCR-ABL fusion genes, somatic gene mutations, CR(1) or >CR(1), MRD negative or positive, conditioning regimen based on TBI or Bu, conditioning intensity, donor source, GVHD prophylactic proposal using cyclosporine or tacrolimus, presence/absence of CMV viremia, and presence/absence of EBV viremia were not significantly different in terms of the OS and DFS. Conclusion: Factors influencing the overall survival of Ph(+) ALL patients who underwent allo-HSCT in CR in the TKI era include age, time form diagnosis to HSCT, and aGVHD severity.

[Maintenance therapy following CD19 CAR-T treatment for relapsed B-cell acute lymphoblastic leukemia after allogeneic hematopoietic stem cell transplantation].

Objective: This study aimed to evaluate the maintenance therapy following an anti-CD19-CAR T-cell therapy for a B-cell acute lymphoblastic leukemia (ALL) patient who relapsed after allogeneic hematopoietic cell transplantation (allo-HSCT) and investigate the effect of donor stem cells and donor T lymphocyte infusion on the amplification of CD19 CAR-T cells. Methods: One refractory B-ALL patient relapsed after murine CD19 CAR-T cell therapy followed by a sibling allo-HSCT. He underwent a humanized CD19 CAR-T cell therapy followed by donor stem cell and donor T lymphocytes infusions as maintenance therapy in our hospital. The level of cytokines, the proportion of CD19 CAR-T cell, the level of CAR19 DNA expression in the peripheral blood, and the proportion of leukemia cells and donor chimerism in the bone marrow were detected. Correspondingly, T lymphocytes from the C57 spleen were separated to modify the CD19 CAR lentivirus and refused into C57 mice, and after 14 days, the B lymphocytes from C57 mice were separated and refused into the same C57 mice. The CD19 CAR T cells, B cells, and CD19 CAR gene counts in the peripheral blood were evaluated at different time points. Results: ①The patient achieved a complete response (CR) 14 days after a humanized CD19 CAR-T therapy with grade 1 cytokine release syndrome (CRS) and restored a donor chimerism to 99.76%. ② Following the remission from humanized CD19 CAR-T therapy, the patient received a maintenance therapy of donor stem cell infusion. Mild graft-versus-host disease (GVHD) manifested 24 days after infusion with an increased proportion of CD19 CAR-T cells and an increased level of CAR19 DNA expression in the peripheral blood. It fell with the remission of GVHD. The patient maintained CR and 99.69% donor chimerism during this period. ③ Throughout the subsequent donor T lymphocytes maintenance therapy, mild GVHD surfaced12 days after infusion without an increased proportion of CD19 CAR-T cells and an increased level of CAR19 DNA expression in the peripheral blood. The patient maintained CR and 99.87% donor chimerism during this period. ④ In vivo experiments on C57 mice confirmed that the proportion of CD19 CAR-T cells and the level of CAR19 DNA expression were upregulated in mice following CAR-T cell infusion, accompanied by depletion of CD19(+) B lymphocyte. After infusion of CD19(+) B lymphocyte cells, an increased proportion of CD19 CAR-T cells and an increased level of CAR19 DNA expression in the peripheral blood were observed again. Conclusions: The infusion of donor stem cells and donor T lymphocytes could be used as a maintenance treatment after CD19 CAR-T cell therapy for B-ALL patients who relapsed after allo-HSCT. Infusion of donor stem cells induced an increased proportion of CD19 CAR-T cells and an increased level of CAR19 DNA expression with the occurrence of GVHD. It might lead to further elimination of minimal residual disease.

[A pediatric-inspired regimen for adolescents and young adults with Philadelphia chromosome-negative acute lymphoblastic leukemia: results from a single center].

Objective: To investigate the efficacy of using a pediatric-inspired regimen for adolescents and young adults (AYA) with Philadelphia chromosome-negative (Ph(-)) acute lymphoblastic leukemia/lymphoblastic lymphoma (ALL/LBL) at a single center in China. Methods: Clinical data of 71 consecutive newly diagnosed AYA patients with Ph(-) ALL/LBL on a pediatric-inspired regimen in Peking Union Medical College Hospital from January 2012 to November 2018 were retrospectively analyzed. Results: Median age at diagnosis was 20 years (range: 15-38) , and 46 patients (64.8%) were male. Forty-nine (69.0%) had B-ALL/LBL. Among 62 ALL patients, 22 (35.5%) were high-risk. Complete remission rate was 93.0%. At follow-up with a median time of 44 months, the estimated 5-year disease-free survival (DFS) and overall survival (OS) was 56.3% and 64.3%, respectively. There was no significant difference in 5-year OS between allogeneic hematopoietic stem cell transplantation group and the continuous chemotherapy group after completion of 4 courses of chemotherapy. The 5-year DFS and OS for the non-high-risk group was 63.1% and 73.7%, respectively, which were significantly higher than 32.0% and 44.4% for the high-risk group, respectively (P<0.001) . Conclusions: The use of pediatric-inspired regimen for AYAs with Ph(-) ALL/LBL was feasible and effective.

[Clinical characteristics and gene expression profiles in children with ETV6-RUNX1 acute lymphoblastic leukemia].

Objective: To evaluate the heterogeneity in pediatric ETV6-RUNX1 acute lymphoblastic leukemia (ALL) by gene expression profile and to study clinical characteristics in different clusters. Methods: An improved advanced fragment analysis (iAFA) technique was developed to detect 57 marker genes in 264 pediatric ALL patients treated in Beijing Children's Hospital from August 2016 to June 2019. The 56 ALL patients with ETV6-RUNX1 positive were evaluated by clinical characteristics in gene expression profile, immunophenotype and early response of chemotherapy in different clusters. Results: The 56 ETV6-RUNX1-positive patients were clustered into 2 groups of E/R-1 (45, 80.4%) and E/R-2 (11, 19.6%) . Spearman coefficient was 0.788 and 0.901 in E/R-2 and E/R-1, respectively. The median of initial platelet counts was 104 (27-644) and 50 (8-390) (P<0.01) in E/R-2 and E/R-1, respectively. The median of proportion of initial bone marrow immature cells was 0.830 (0.270-0.975) and 0.935 (0.445-0.990) (P<0.05) in E/R-2 and E/R-1, respectively. The most specific immunophenotype at initial diagnosis, CD22(+)CD34(+)CD20(-)CD117(-)CD56(-), mainly gathered in E/R-2 (P<0.001) . Patients negative of minimal residual disease detected by flow cytometry (MRD-FCM) at day 33 were 5 (55.6%) and 32 (88.9%) in E/R-2 and E/R-1, respectively. There was no significant difference in the original analysis (P=0.064) but difference in sensitivity analysis (P=0.035) . Nevertheless, patients negative of MRD detected by polymerase chain reaction (MRD-PCR) at day 33 were 7 (77.8%) and 36 (100%) in E/R-2 and E/R-1, respectively, with significant difference (P=0.047) . Conclusion: Gene expression profile shows heterogeneous in ETV6-RUNX1 ALL, and the E/R-2 profile indicates that these patients may have a less tendency to thrombocytopenia at the initial diagnosis but have poorer response to induction chemotherapy and may influence further outcome.

[CD19 antigen loss after treatment of Bispecific T-cell Engager and effective response to salvage bispecific CAR-T therapy in B cell acute lymphoblastic leukemia: a case report and literature review].

Objective: To analyze the influence of CD19 isoforms to the efficacy of CD19/CD3 Bispecific T-cell Engager (BiTE) antibody, and explore the resistance mechanism of BiTE immunotherapy. Methods: Semi-quantitative RT-PCR (qRT-PCR) was used to detect the expression of CD19 mRNA isoforms before and after BiTE treatment in a patient with CD19(+) B cell acute lymphoblastic leukemia (ALL) . CD19 isoforms were analyzed by Sanger sequencing. Flow cytometry and transcriptome sequencing were performed to analyze the expression of cell lineage specific molecules before and after BiTE treatment. Results: The expression of CD19 isoform with exon 2 deletion was identified at diagnosis. After relapsed and treatment of BiTE antibody, the patient did not achieve remission and CD19 antigen on leukemic cells turned negative detected by flow cytometry after BiTE treatment. However the expression ratio of CD19 isoform with exon 2 deletion was not increased. Flow cytometry phenotype and transcriptome sequencing confirmed that no linage switching developed, which suggested the expression of CD19 isoform caused by exon alternative splicing and lineage switching was not related to CD19 epitope loss in this patient. This patient achieved complete remission by sequential administration of self-developed CD22 CAR-T and CD19 CAR-T after disease progression. Conclusion: Targeting or combining an alternative antigen specific CAR-T may be a promising treatment option after losing CD19 expression in relapsed ALL.

[Comparison of clinical efficacy between first-generation and second-generation tyrosine kinase inhibitors based regimen in the treatment of patients with BCR-ABL positive acute lymphoblastic leukemia].

Objective: To explore the clinical efficacy and prognostic factors of first-generation and second-generation tyrosine kinase inhibitors (TKI) based regimen in the treatment of patients with BCR-ABL positive acute lymphoblastic leukemia (ALL) . Methods: Retrospectively analyze the clinical characteristics and prognostic factors of 89 patients with BCR-ABL positive ALL from April 2012 to June 2018 in our hospital, the clinical efficacy of first-generation and second-generation TKI was compared. Results: 60 patients were classified into the first-generation TKI (imatinib) group, and 29 patients were in the second-generation TKI (dasatinib) group. There were no significant differences in gender, age, WBC, hemoglobin concentration, PLT, chromosomal karyotype, the types of fusion genes, allogeneic hematopoietic stem cell transplantation (allo-HSCT) and TKI initiation time between the two groups. The first-generation and second-generation TKI groups, for which the complete remission (CR) rate at the fourth week of induction therapy was 83.3% and 89.7% (P=0.637) , respectively, and the complete molecular remission (CMR) was 48.3%and 58.6% (P=0.363) , respectively, the difference was not statistically significant. The 2-year overall survival (OS) rate of first-generation and second-generation TKI group was 34.9% and 64.0% (χ(2)=4.743, P=0.029) , the 2-year relapse free survival (RFS) rate was 17.2% and 55.0% (χ(2)=8.801, P=0.003) , respectively. Multivariate analysis showed that complete molecular remission (HR=0.281, 95%CI 0.151-0.523, P<0.001) was independent favorable prognostic factor for overall survival (OS) , complete molecular remission (HR=0.209, 95%CI 0.112-0.390, P<0.001) and second-generation TKI (HR=0.318, 95%CI 0.158-0.641, P=0.001) were independent favorable prognostic factors for RFS. Conclusion: For TKI-based regimen of BCR-ABL positive ALL, second-generation TKI is superior to first-generation TKI in OS and RFS time.

[Efficacy of Hyper-CVAD/MA and CHALL-01 regimens in the treatment of Philadelphia chromosome-positive adult acute lymphoblastic leukemia patients under 60 years old].

Objective: To compare the difference of efficacy between traditional Hyper-CVAD/MA regimen and the adolescents inspired chemotherapy regimen, CH ALL-01, in treatment of adult Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) . Methods: In this study we retrospectively analyzed 158 Ph(+) ALL patients receiving Hyper-CVAD/MA regimen (n=63) or CHALL-01 regimen (n=95) in our center and Changzheng hospital from January 2007 to December 2017, excluding patients with chronic myeloid leukemia in blast crisis. Tyrosine kinase inhibitor (TKI) was administered during induction and consolidation chemotherapy. Patients who underwent hematopoietic stem cell transplantation received TKI as maintenance therapy. Results: Of them, 91.1% (144/158) patients achieved complete remission (CR) after 1-2 courses of induction. CR rate was 90.5% (57/63) for patients in Hyper-CVAD/MA group and 91.6% (87/95) for patients in CHALL-01 group. There was no difference in CR rates between the two groups (χ(2)=0.057, P=0.811) . The last follow-up was June 2018. A cohort of 134 CR patients could be used for further analysis, among them, 53 patients received Hyper-CVAD/MA regimen and other 81 patients received CHALL-01 regimen. The molecular remission rates were significantly higher in CHALL-01 group (complete molecular response: 44.4%vs 22.6%; major molecular response: 9.9% vs 18.9%) (χ(2)=7.216, P=0.027) . For the patients in Hyper-CVAD/MA group, the 4-year overall survival (OS) was 44.81% (95%CI: 30.80%-57.86%) and the 4-year disease free survival (DFS) was 37.95% (95%CI: 24.87%-50.93%) . For patients received CHALL-01 regimen, the 4-year OS was 55.63% (95%CI: 39.07%-69.36%) (P=0.037) and 4 year DFS was 49.06% (95%CI: 34.24%-62.29%) (P=0.015) , while there was no significant difference in 4 year cumulative incidence of relapse (CIR) (P=0.328) or cumulative incidence of nonrelapse mortality (CI-NRM) (P=0.138) . The rate of pulmonary infection was lower in patients received CHALL-01 regimen compared with patients received Hyper-CVAD regimen (43.4% vs 67.9%, χ(2)=7.908, P=0.005) . Conclusions: Outcome with CHALL-01 regimen appeared better than that with the Hyper-CVAD/MA regimen in Ph(+) ALL, which has lower incidence of pulmonary infection, higher molecular remission rate and better OS and DFS.

Efficacy of Hyper-CVAD/MA and CHALL-01 regimens in the treatment of Philadelphia chromosome-positive adult acute lymphoblastic leukemia patients under 60 years old / 中华血液学杂志

Objective@#To compare the difference of efficacy between traditional Hyper-CVAD/MA regimen and the adolescents inspired chemotherapy regimen, CH ALL-01, in treatment of adult Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) .@*Methods@#In this study we retrospectively analyzed 158 Ph+ ALL patients receiving Hyper-CVAD/MA regimen (n=63) or CHALL-01 regimen (n=95) in our center and Changzheng hospital from January 2007 to December 2017, excluding patients with chronic myeloid leukemia in blast crisis. Tyrosine kinase inhibitor (TKI) was administered during induction and consolidation chemotherapy. Patients who underwent hematopoietic stem cell transplantation received TKI as maintenance therapy.@*Results@#Of them, 91.1% (144/158) patients achieved complete remission (CR) after 1-2 courses of induction. CR rate was 90.5% (57/63) for patients in Hyper-CVAD/MA group and 91.6% (87/95) for patients in CHALL-01 group. There was no difference in CR rates between the two groups (χ2=0.057, P=0.811) . The last follow-up was June 2018. A cohort of 134 CR patients could be used for further analysis, among them, 53 patients received Hyper-CVAD/MA regimen and other 81 patients received CHALL-01 regimen. The molecular remission rates were significantly higher in CHALL-01 group (complete molecular response: 44.4%vs 22.6%; major molecular response: 9.9% vs 18.9%) (χ2=7.216, P=0.027) . For the patients in Hyper-CVAD/MA group, the 4-year overall survival (OS) was 44.81% (95%CI: 30.80%-57.86%) and the 4-year disease free survival (DFS) was 37.95% (95%CI: 24.87%-50.93%) . For patients received CHALL-01 regimen, the 4-year OS was 55.63% (95%CI: 39.07%-69.36%) (P=0.037) and 4 year DFS was 49.06% (95%CI: 34.24%-62.29%) (P=0.015) , while there was no significant difference in 4 year cumulative incidence of relapse (CIR) (P=0.328) or cumulative incidence of nonrelapse mortality (CI-NRM) (P=0.138) . The rate of pulmonary infection was lower in patients received CHALL-01 regimen compared with patients received Hyper-CVAD regimen (43.4% vs 67.9%, χ2=7.908, P=0.005) .@*Conclusions@#Outcome with CHALL-01 regimen appeared better than that with the Hyper-CVAD/MA regimen in Ph+ ALL, which has lower incidence of pulmonary infection, higher molecular remission rate and better OS and DFS.

Progress of the diagnosis and treatment of acute leukemia in China: work review of Institute of Hematology&Blood Diseases Hospital, Chinese Academy of Medical Sciences / 白血病·淋巴瘤

Acute leukemia is a kind of malignant proliferative disease originating from hematopoietic stem cells, which is characterized with molecular and clinical heterogeneity. Chemotherapy and hematopoietic stem cell transplantation are the major treatments for acute leukemia. In recent years, the development of targeted therapy has significantly improved the prognosis of acute leukemia patients. This article reviews the part of the work of Institute of Hematology&Blood Diseases Hospital, Chinese Academy of Medical Sciences on the research of pathogenesis, diagnosis and treatment of acute leukemia.