OBJECTIVESTo evaluate the specificity of three-color flow cytometry in childhood acute lymphoblastic leukemia (ALL) immunophenotyping.

METHODSImmunophenotyping was performed by three-color flow cytometry analysis using CD(45)/SSC gating.

RESULTSThe percentage of blasts was correlated better with leukemic cell count compared with that of FSC/SSC, and the false positive results were low. Among eighty six cases of ALL, 95.3% was B-ALL, in which common-ALL and Pro-B-ALL were 76.8% and 6.1%, respectively, and 2.3% was T-ALL. CD(34)(+) and myeloid-associated antigen expression were observed in 57.0% and 34.9% of the cases, respectively, among which Pro-B-ALL was the commonest. CD(33) was more commonly expressed than CD(13) in Pro-B-ALL cases, but no difference in the expression between these two antigens in other subtypes.

CONCLUSIONGating of CD(45)/SSC eliminated effection of normal cells to blasts in bone marrow, with which the immunophenotyping results were more reliable.

Antigens, CD ; analysis ; Child ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology

B-Lymphocytes ; Child ; Humans ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; immunology ; Precursor Cells, B-Lymphoid ; pathology

Acute Disease ; Adult ; CD56 Antigen ; immunology ; Female ; Humans ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; complications ; immunology ; surgery ; Recurrence ; Stem Cell Transplantation

OBJECTIVETo probe into the occurrence rates of the effective antigen combinations which were used to detect the minimal residual disease (MRD) by flow cytometry in childhood B-lineage acute lymphoblastic leukemia (B-ALL), as well as the relationship between clinical-biologic factors and different combinations.

METHODSAmong the 327 B-ALL children enrolled in our study, 289 cases were identified with at least one antigen combination as MRD marker. Their bone marrow samples were monitored by using 9 combinations with 4 antigens each, analyzed the occurrence rates and compared them with international reports. Also the differences in the distribution of each antibody combination in the different clinical-biologic groups were compared by the chi-square test and Fisher's exact test.

RESULTS(1) Totally 327 cases of childhood B-ALL were screened for antibody combinations of interest and 88.4 percent of them (289 cases) were identified with effective antibody combinations. (2) The occurrence frequencies of antigen combinations were different. The highest frequency was seen with TdT/CD10/CD34/CD19 combination which was 70.59 percent. Expressions of antigen combinations in Chinese children were different from those in western countries. (3) Some antibody combinations presented different frequency among different clinical groups. CD38/CD10/CD34/CD19 was expressed more often in samples of relapsed patients (P = 0.045). CD66c/CD10/CD34/CD19 expression was significantly higher in BCR/ABL positive group (P = 0.037) and relapsed patients group (P = 0.047). TdT/CD10/CD34/CD19 was expressed more in MLL-AF4 negative group (P = 0.005) and Prednisone Good Response group (P = 0.002). CD58/CD10/CD34/CD19 was correlated with low relapse rate (P = 0.032).

CONCLUSION(1) The coverage rate of 9 antigen combinations in our study was 88.4%. The occurrences of frequency of different antibody combinations in B-ALL were different, and also different from that of western countries. The occurrence frequencies of antibody combinations CD21/CD10/CD34/CD19, CD22/CD10/CD34/CD19, CD10/CD56/CD34/-CD19 and TdT/Cu/CD34/CD19 were lower than those of the western report, while CD38/CD10/-CD34/CD19, CD45/CD19/CD10/CD34, CD58/CD10/CD34/CD19 and CD66c/CD10/CD34/CD19 were similar to those of the reports from western countries. (2) TdT/CD10/CD34/CD19 may work as a simplified method to detect MRD in Chinese population. (3) The occurrence frequency of CD38/CD10/CD34/CD19, CD45/CD19/CD10/CD34, CD58/CD10/CD34/CD19, TdT/CD10/CD34/CD19 could be effective remediation and evidence to evaluate the remission quality and guide the therapy, especially for those with no original MRD marker record. (4) CD58/CD10/CD34/CD19 and TdT/CD10/CD34/CD19 may correlate with good prognosis, but CD66c/CD10/CD34/CD19 and CD38/CD10/CD34/CD19 may predict poor prognosis. These results might contribute to individual risk evaluation and guide the therapy selection.

Child ; Child, Preschool ; Humans ; Infant ; Leukemia, B-Cell ; immunology ; therapy ; Neoplasm, Residual ; Neprilysin ; immunology ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; therapy

OBJECTIVETo analyze the incidence, clinical characteristics and prognosis of childhood T-cell acute lymphoblastic leukemia (T-ALL).

METHODSFrom January 1999 to April 2005, 305 patients with newly diagnosed ALL were enrolled in protocol ALL-XH-99. The clinical characteristics of these children were analysed.

RESULTSOf 305 childhood ALL patients, 43 were T-ALL. There were 34 males among the 43 T-ALLs. The mean age at diagnosis was 7.8 (2.2 to 16.4) years, 29 (67.4%) cases of them were older than 10 years, and 27 (62.8%) cases had initial WBC count more than 50 x 10(9)/L. In comparison with that of B cell ALL (B-ALL), the percentages of age older than 10 years, initial WBC count more than 50 x 10(9)/ L, prednisone poor response (PPR), and failed to achieve remission at day 19 of induction chemotherapy in the T-ALLs were all higher. No statistic difference was found in sex between them. The eight-year event-free survival (EFS), relapse-free survival (RFS) and overall survival (OS) were (40.2 +/- 10.1)%, (51.4 +/- 11.6)% and (49.8 +/- 9.9)% for T-ALL, and (72.1-3.0)%, (83.2 +/- 2.7)%, and (76.6 +/- 2.9)% for B-ALL, respectively, being differed significantly between the two types of ALL (P < 0.01).

CONCLUSIONThere were statistic differences between T-cell and B-cell childhood ALLs in age, initial WBC count, early response to therapy, and eight-year EFS and RFS. Childhood T-ALL was associated with a worse prognosis than other sub-types of childhood ALL.

Adolescent ; Child ; Child, Preschool ; Disease-Free Survival ; Female ; Humans ; Immunophenotyping ; Infant ; Karyotyping ; Male ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology ; pathology ; therapy ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology ; pathology ; therapy ; Prognosis

Chimeric antigen receptor (CAR) is a recombinant immunoreceptor combining an antibody-derived targeting fragment with signaling domains capable of activating cells, which endows T cells with the ability to recognize tumor-associated surface antigens independent of the expression of major histocompatibility complex (MHC) molecules. Recent early-phase clinical trials of CAR-modified T (CAR-T) cells for relapsed or refractory B cell malignancies have demonstrated promising results (that is, anti-CD19 CAR-T in B cell acute lymphoblastic leukemia (B-ALL)). Given this success, broadening the clinical experience of CAR-T cell therapy beyond hematological malignancies has been actively investigated. Here we discuss the basic design of CAR and review the clinical results from the studies of CAR-T cells in B cell leukemia and lymphoma, and several solid tumors. We additionally discuss the major challenges in the further development and strategies for increasing anti-tumor activity and safety, as well as for successful commercial translation.
Animals ; Humans ; Immunity, Cellular ; Immunotherapy ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; pathology ; therapy ; Receptors, Antigen, T-Cell ; immunology ; Recombinant Fusion Proteins ; immunology ; T-Lymphocytes ; immunology ; transplantation

OBJECTIVETo investigate the expression of CD19 on childhood acute leukemia (AL) and its significance, and to provide evidence for the diagnosis and differential diagnosis as well as monoclonal antibody-targeting treatment of leukemia.

METHODSThere were 210 cases of childhood AL, of which 130 cases were male and 80 were female with a mean age of 9 years old. Using a panel of 27 fluorochrome directly labeled monoclonal antibodies, 210 samples from the patients were analyzed with CD45/SSC double parameters and multi-color flow cytometry to determine the expression of CD19.

RESULTSIn the 93 cases of B lineage acute lymphoblastic leukemia (ALL), the positive rate (98.9%, 92/93) of CD19 was significantly higher than that of the other B cell related antigens, such as CD10 (88.2%, 82/93, P = 0.003), CD20 (24.7%, 23/93, P = 0.001) and CD22 (60.2%, 56/93, P = 0.001). CD19 was expressed on all 8 cases of B/myeloid (My) hybrid acute leukemia (HAL) and 1 case of B/T HAL, but was not expressed on all 24 cases of T lineage leukemia and 5 cases of T/My HAL. In the 79 cases of acute myeloid leukemia (AML), only 5 (6.3%) cases expressed CD19. The positive rate (6.3%) of CD19 on AML was significantly lower than that on B lineage ALL (98.9%, P = 0.001). The percentage of CD19 positive cells in B/My HAL (41.6% - 88.7% with a mean of 73.8%) was significant higher than that in CD19(+)-AML (21.4% - 50.4% with a mean of 24.2%; Run Sum test, P = 0.0084). Of the 210 cases, 102 were B lineage related AL including B lineage ALL, B/My HAL and B/T HAL. In B lineage related AL, the sensitivity and the specificity of CD19 was 99.0% (101/102) and 95.4% (13/108) while the positive predictive and the negative predictive values to B lineage were 95.3% (101/106) and 99.0% (103/104), respectively. Using CD19(+) as a single reagent to diagnose B lineage, the false positive rate was 4.6% (5/108) and the false negative rate was 1.0% (1/102) with a general diagnosis index (GDI) of 94.4% [GDI = 1-(false positive rate + false negative rate)].

CONCLUSIONCD19 is continuously and stably expressed on all stages of B lineage differentiation. It is a reliable cell membrane marker for diagnosing B lineage ALL and an ideal target for antibody-targeting treatment of leukemia as well; the expression degree of CD19 can be used to distinguish B/My HAL from CD19(+)-AML; CD19 didn't express on normal myeloid cells but did on some AML cells. Therefore it can be used to detect the minimal residual disease.

Adolescent ; Antigens, CD19 ; analysis ; Child ; Child, Preschool ; Female ; Flow Cytometry ; Humans ; Infant ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; classification ; immunology

OBJECTIVETo explore the characteristics of morphology, immunophenotype and cytogenetics (MIC) of myeloid surface antigen-expressing acute lymphoblastic leukemia (My+ ALL).

METHODSOne hundred and twenty untreated acute lymphoblastic leukemia (ALL) patients were diagnosed by standard bone marrow smear morphologic analysis and peroxidase staining. Flow cytometry and myeloid monoclonal antibodies (McAb) were used to analyze immunophenotype. Chromosome karyotypes were analyzed by R-band technique.

RESULTSOf 120 cases, 66 (55%) were My+ ALL, including 50 cases of My+ B-ALL (56.8% of B-ALL ), 14 cases of My T-ALL (50% of T-ALL) and 2 cases of My+ T and B-ALL (50% of T and BALL). Of 66 My+ ALL, 10 cases (15.1%) were misdiagnosed as acute non-lymphoblastic leukemia (ANLL), the other 54 My- ALL cases were correctly diagnosed. The inconsistent rate between morphological and immunophenotype classifications was higher in My+ ALL than in My- ALL , and there were more atypical morphology cases in My+ ALL than in My- ALL (P < 0.01). In My+ ALL cases 95.5% expressed CD13, 81.8% CD33, 77.3% CD13 and CD33 simultaneously, and 1.5% CD117, but none CD14, CD15 and MPO. CD34 expression rate in My+ ALL cases was significantly higher than that in My- ALL (P < 0.01 ). Cytogenetic abnormalities rates in My+ ALL and My- ALL were 72.3% and 66.7% (P > 0.05) respectively. t(9;22) and t(9;22) plus other cytogenetic abnormalities were detected more frequently in My+ LL cases than in My- B-ALL (P < 0. 01), and not in My+ T-ALL and My- T-ALL cases. The complete remission (CR) rates was 83.9% in My+ ALL and 79% in My- ALL(P > 0.05).

CONCLUSIONMy+ ALL had a specific characteristics in morphology, immunophenotype and cytogenetics. Some cases have a myeloid morphologic appearance and might be misdiagnosed as acute myeloid leukemia (AML). My+ ALL have a higher CD34 expression rate than My- ALL. t(9;22) abnormality was more frequently observed in My B-ALL than in My- B-ALL. There was no significant difference in CR rate between My+ ALL and My- ALL.

Adolescent ; Adult ; Aged ; Child ; Female ; Humans ; Immunophenotyping ; Karyotyping ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; classification ; genetics ; immunology

OBJECTIVETo study the immunophenotype and its relationship with clinical characteristics in children with acute lymphoblastic leukemia (ALL).

METHODSBone marrow or blood samples (2-3 mL) with heparin anticoagulation from 139 children with ALL were obtained, and immunophenotypes were identified by flow cytometry.

RESULTSIn 139 ALL children, there were 103 cases (74.1%) of B-ALL, 24 cases (17.3%) of T-ALL, 12 cases of T/B biphenotypic (8.6% of T/BALL). In the 103 children with B-ALL, CD19 (90.3%), CD10 (83.5%) and CD20 (27.2%) were expressed as major antigens. In the 24 children with T-ALL, the major antigens were CD3 (79.2%), CD7 (66.7%) and CD5 (33.3%). In the 12 children with B/T-ALL, T-lymphoid antigens included CD7 (50.0%) and CD5 (41.7%), while the B-lymphoid antigens included CD19 (50.0%) and CD10 (33.3%). Of the 139 children with ALL, 32 cases (23.0%) showed myeloid antigen expression (My+ ALL) and the main expression antigens were CD13, CD33, CD14 and MPO. CD34 was expressed in 31 cases. CD34-positive expression (15.6%) in My+ ALL children was significantly lower than in My-ALL children (24.3%). HLA-DR was expressed in 82 of the 139 ALL children. The expression of CD10, CD34 and HLA-DR in the standard-risk, medium risk, high-risk ALL children was significantly different. There were significant differences in gender and incidence of bleeding between the My+ ALL and My-ALL groups (P<0.05).

CONCLUSIONSImmunetyping can differentiate the sources of leukemic cells. The expression of CD10, CD34 and HLA-DR antigen is related to the clinical classification of ALL.

Adolescent ; Child ; Child, Preschool ; Female ; HLA-DR Antigens ; analysis ; Humans ; Immunophenotyping ; Infant ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology

OBJECTIVETo elevate the prognostic value of minimal residual disease (MRD) detection by four-color flow cytometry with the antibody panel in childhood B-cell acute lymphoblastic leukemia (B-ALL).

METHODSThe clinical data of 183 children with newly-diagnosed acute B-ALL and who accepted MRD detection between October 2010 and March 2012 was retrospectively reviewed. According to the detection time and result of MRD, the 183 children were classified into four groups: MRD negative (n=37) and positive (n=18) in the induction chemotherapy and MRD negative (n=113) and positive (n=15) in the maintenance chemotherapy.

RESULTSDuring both induction and maintenance chemotherapy, the percentage of patients at high and median risk in the MRD positive group was higher than in the MRD positive group (P<0.05). In the maintenance chemotherapy group, the 3- year cumulative incidence of relapse in MRD positive patients was higher than negative patients (P=0.04). The Cox's proportional hazards regression analysis showed that insensitive reaction for prednisone (RR=1.005, 95%CI: 0.864-1.170, P=0.032), bone marrow morphology that did not meet M1 on the 15th day (RR=6.454, 95%CI: 2.191-19.01, P=0.002) and MRD≥0.01% (RR=1.923, 95%CI: 0.750-4.933, P=0.043) were risk factors for relapse in children with B-ALL.

CONCLUSIONSThe four-color flow cytometry with the antibody panel can distinguish from MRD positive patients from negative patients with B-ALL. The result of MRD detection, as prednisone sensitivity and bone marrow morphology on the 15th day, is also a independent prognostic factor in children with B-ALL.

Child ; Child, Preschool ; Female ; Flow Cytometry ; methods ; Humans ; Infant ; Male ; Neoplasm, Residual ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; immunology