Hongqiang Shen

Absolute lymphocyte count is associated with minimal residual disease level in childhood B-cell precursor acute lymphoblastic leukemia.

The prognostic value of absolute lymphocyte count (ALC) has been a recent matter of debate in childhood acute lymphoblastic leukemia (ALL). In the current study, ALCs at the time of diagnosis (ALC-0), after 7 days of initial therapy (ALC-8) and at interim of the induction therapy (ALC-22) were examined in Chinese children with B-cell precursor (BCP) ALL and correlated with the level of minimal residual disease (MRD) at day 22 of induction therapy. Medical and laboratory records of 140 patients diagnosed with childhood BCP ALL were retrieved and analyzed. ALC-22 is significantly correlated with MRD level at day 22 of therapy and can be a good prognostic factor for childhood BCP-ALL. Furthermore, lymphocyte count at initial diagnosis is correlated with MRD level at day 22 in childhood BCP-ALL with the immnunophenotype of CD19(pos)/CD10(pos)/CD34(pos)/CD45(neg) and role as a new prognostic factor was determined.

Day 22 of induction therapy is important for minimal residual disease assessment by flow cytometry in childhood acute lymphoblastic leukemia

This study was aimed to illustrate the significance of minimal residual disease (MRD) assessment on day 22 in childhood acute lymphoblastic leukemia. MRD were measured on day 22, day 36, week 12, month 6 and month 12 by four-color flow cytometry. The 5-year cumulative incidence of relapse was significantly different for patients with MRD levels of <0.01%, 0.01-0.1%, 0.1-1.0% and ≥ 1.0% on day 22: 6.9 ± 2.6%, 16.7 ± 5.5%, 25.8 ± 6.2% and 58.4 ± 13.4% (P < 0.001). MRD on day 22 was more powerful than other parameters including NCI risk. However, other time points after induction, although predictive as well, were not accurate enough due to false positivity.

Targeting and internalization of sterically stabilized liposome modified with ZCH-4-2E8.

SummarySterically stabilized liposome (SL) modified with 2E8 monoclonal antibody (2E8-SL) was prepared in order to evaluate its targeting ability and internalization efficiency against tumor cells with high expression of CD19. 2E8 was coupled to the surface of SL using post-insertion technique. The shape of liposomes was observed under a transmission electronic microscope. The average size of liposomes was determined by using the Zetasizer instrument. The binding and internalization of 2E8-SL against tumor cells with higher expression of CD19 were tested by flow cytometry and confocal microscopy. The mean diameter of 2E8-SL was 121.25 nm. 2E8-SL was stable after up to 24 days in various buffers. 2E8-SL showed specific binding to tumor cells with high expression of CD19, including B cells in peripheral blood mononuclear cells (PBMCs). 2E8-SL could efficiently internalize into Nalm6 cells with CD19 high expression. It is suggested that 2E8-SL may serve as useful delivery carrier of anti-cancer drugs targeting to hematological malignant tumors with CD19 high expression.Sterically stabilized liposome (SL) modified with 2E8 monoclonal antibody (2E8-SL) was prepared in order to evaluate its targeting ability and internalization efficiency against tumor cells with high expression of CD19. 2E8 was coupled to the surface of SL using post-insertion technique. The shape of liposomes was observed under a transmission electronic microscope. The average size of liposomes was determined by using the Zetasizer instrument. The binding and internalization of 2E8-SL against tumor cells with higher expression of CD19 were tested by flow cytometry and confocal microscopy. The mean diameter of 2E8-SL was 121.25 nm. 2E8-SL was stable after up to 24 days in various buffers. 2E8-SL showed specific binding to tumor cells with high expression of CD19, including B cells in peripheral blood mononuclear cells (PBMCs). 2E8-SL could efficiently internalize into Nalm6 cells with CD19 high expression. It is suggested that 2E8-SL may serve as useful delivery carrier of anti-cancer drugs targeting to hematological malignant tumors with CD19 high expression.

Construction and Expression of Single-Chain Antibody Derived from a New Clone of Monoclonal Antibody Against Human CD14 in CHO Cells

Our aim was to construct the eukaryotic expressional system of single-chain antibody (ScFv2F9) derived from a new clone of monoclonal antibody named ZCH-7-2F9 against human CD14 antigen, and to obtain the ScFv2F9 protein with a high biological activity for further studies on ScFv2F9 immunotoxin and other kinds of genetically engineered antibodies. Primers were synthesized according to the gene sequence of ScFv2F9, 4 tandem glysines and 1 serine (Gly4Ser)3 linker and multiple cloning site(MCS) of pSectag2A vector, which included SfiI and EcoRI enzyme cleaving site, 6 × His and stop code TGA sequences. ScFv2F9 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pSectag2A vectors. Positive recombinants (pSectag2A/ScFv2F9) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into Chinese hamster ovary (CHO) cells for expression. The relative molecular weight (MW) and the binding activity of the interesting protein were determined by SDS-PAGE, Western-blot and flow cytometry (FCM), respectively. The scFv2F9 VH is a member of the IgH gene family V1 subgroup and the scFv2F9 VL gene belongs to the Igκ gene family V10 subgroup. The recombined ScFv2F9 gene was identified to be functional by sequencing and expressing. The interesting protein could be detected in the culture supernatant of transformed CHO cells with a MW of 31 kDa. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.3%and 63.38%, respectively after blockage of ScFv2F9. The ScFv2F9 gene was cloned and the interest protein against human CD14 antigen has been successfully expressed in eukaryotic cells with a high biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.

Detection of the GD2+/CD56+/CD45- immunophenotype by flow cytometry in cerebrospinal fluids from a patient with retinoblastoma.

Triple-color flow cytometry with a panel of antibodies comprising GD2, CD56, and CD45 was performed to analyze cerebrospinal fluids (CSF) from a patient with retinoblastoma who was suspicious of meningeal metastasis based on clinical presentation. Our results showed that the cells in CSF demonstrated the immunophenotype positive for GD2 and CD56 but negative for CD45 antigen, which suggested the presence of CSF metastasis of retinoblastoma. At the end of eight cycles of intrathecal chemotherapy, CSF specimen was analyzed with Flow cytometry immunophenotyping (FCI) again and the result showed no detectable malignant cells with the same immunophenotype. Our conclusion is that FCI can be a quick and reliable method for the diagnosis of CSF metastasis of retinoblastoma and the immunophenotype (GD2+, CD56+, and CD45−) can be used to recognize residual retinoblastoma cells in CSF.

Rapid detection of neoplastic cells in serous cavity effusions in children with flow cytometry immunophenotyping.

Abstract The aim of the study was to evaluate the role of flow cytometric immunophenotyping (FCI) as a rapid diagnostic tool for pediatric malignancy in serous cavity effusions (SCEs). FCI results for 103 SCEs in a pediatric population were compared with retrospective clinical outcomes (RCOs). Among 41 patients assessed as having malignancies by RCO, 36 patients were diagnosed with lymphoma (n =25), acute myeloid leukemia (n =2), neuroblastoma (n =8) and retinoblastoma (n =1) by FCI, respectively. The sensitivity and specificity of FCI for detecting neoplastic cells were 87.8% and 98.4%, respectively. The concordance of FCI data with final diagnoses of the patients was 94.2%. FCI data for lymphoma was concordant with the final diagnosis in 89.3% of cases. When Hodgkin lymphoma was excluded, the overall correlation increased to 96.1%. FCI is a useful tool for rapid and reliable diagnosis of pediatric non-Hodgkin lymphoma in SCE samples and also to suggest the presence of non-lymphoid malignancies, especially neuroblastoma.

3A4, a new potential target for B and myeloid lineage leukemias

Antibody-targeting therapy has drawn great interests to the hematologists and oncologists. Many antibodies have been studied for their potential targeting for hematopoietic malignancies. A few have been proved to be very effective for patients with these diseases. However, more antibodies are needed for clinical use. CD45 and its isoforms may convey clinical potential in terms of targeting therapy. Zhejiang Children’s Hospital (ZCH)-6-3A4 (3A4), a novel antibody that can recognize an isoform of CD45 has been found to react with restricted cell components in hematopoietic system, which may have the potential for targeting therapy. Herein, we conducted an in vitro study of our newly prepared antibody 3A4 using various cellular and immunocytological methods. The results showed that the antibody 3A4 (murine IgG1κ) was a new clone of anti-CD45RA. It could block the binding to an epitope of CD45RA recognized by a standard anti-CD45RA antibody (Clone name L48). The reactivity of the 3A4 to both fresh leukemia cells from patients and well-defined leukemia cell lines was largely similar to those of L48, but the former recognized more leukemia cells than the latter. Cytometric analysis after papain treatment showed that the internalization rate of the 3A4 antibody to the target cells was as high as 71.3% after incubation at 37°C for 4 h, which was significantly higher than that of L48 (20.4%). The norcantharidin (NCTD)-conjugated immunotoxin (NCTD-3A4) was generated using an active ester method. The targeting inhibition rate on KG1a was as high as 61.10% after 96 h incubation in a dose-dependent manner, which was significantly higher than that (3.56%, P < 0.01) with 3A4-negative Nalm-6 cells. In conclusion, our new anti-CD45RA antibody 3A4 is probably a new target molecule of leukemia cells and holds a targeting therapeutic potential for hematopoietic malignancies, which warrants further development of this agent.

Prognostic significance of flow cytometric minimal residual disease assessment after the first induction course in Chinese childhood acute myeloid leukemia.

Flow cytometry based minimal residual disease (MRD) was evaluated for outcome prediction in childhood acute myeloid leukemia (AML). The median levels of MRD in relapsed and nonrelapsed patients were different after the first induction (0.64% vs. 0.18%, P=0.030). A cutoff level of ≥ 0.25% after the first course of induction was correlated with a high risk of relapse in both univariate analysis (5-year cumulative incidence of relapse: 66.8% vs. 21.2%, P=0.002) and multivariate analyses (hazard ratio: 3.70, 95% CI, 1.23-11.08, P=0.020). Our results showed that MRD level after the first induction therapy provides important information for risk assessment in childhood AML.

Staging and monitoring of childhood rhabdomyosarcoma with flow cytometry

Patients with metastatic rhabdomyosarcoma (RMS) have a poor prognosis. The detection of contaminating RMS cells in the bone marrow (BM) is important in clinical staging and risk assessment. The cytological examination of the BM remains the gold standard for the diagnosis of RMS, but has a limited sensitivity. In the present study, 32 BM and two cerebrospinal fluid (CSF) samples from 11 patients with suspected metastasis were analyzed by flow cytometry (FCM) with ganglioside D2 (GD2) conjugated with fluorescein isothiocyanate, cluster of differentiation (CD)90-phycoerythrin, CD45-peridinin chlorophyll protein and CD56-allophycocyanin monoclonal antibody cocktail in parallel to morphological examination at diagnosis or during treatment. Five samples (14.7%) were positive for RMS onup morphological examination. By FCM, 16 samples (47.1%) were positive for RMS. A significant difference was identified between the two methods. The four-color FCM assay successfully detected RMS cells in BM samples to a level of 0.01% (1 per 104 cells). RMS cells demonstrated a phenotype with CD56+/CD90+/CD45−/GD2− expression, which is different from the CD56+/CD90+/CD45−/GD2+ expression phenotype in neuroblastoma cells. The follow-up of four patients by FCM demonstrated that two patients became minimal residual disease-negative following two and four cycles of chemotherapy, respectively, and survived. The other two cases remained FCM-positive despite receiving four courses of chemotherapy and consequently succumbed to progressive disease. In addition, FCM analysis of the CSF samples from one patient confirmed a diagnosis of CSF metastasis with RMS. In conclusion, FCM may have a role not only in staging and monitoring the effects of therapy, but also in providing diagnostic confirmation of CSF metastasis with RMS.

Simultaneous cytomorphological and multiparameter flow cytometric analysis of ALK-positive anaplastic large cell lymphoma in children.

The aim of this study was to investigate the pathological features of anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) in children and to establish the effectiveness of screening and diagnosing ALCL with multiparameter flow cytometry immunophenotyping (FCI) of lymphoid tissue samples. A total of 121 lymph node tissue specimens obtained from 121 patients with a suspected diagnosis of lymphoma were analyzed with cytomorphological and FCI analysis. Fifteen cases were diagnosed as ALK-positive ALCL based on the pathological features and immunohistochemical results. Of these, there were 3 different types, common type (10 cases), lymphohistiocytic type (4 cases) and neutrophil-rich type (1 case). Thirteen cases (10 common, 2 lymphohistiocytic and 1 neutrophil-rich type) were diagnosed as ALCL using FCI. These cases were CD30-positive and aberrantly expressed at least two T-cell antigens, including CD4 (84.6%), CD2 (76.9%), CD7 (61.5%), CD3 (53.8%) and CD5 (38.4%). Neoplastic cells accounted for only a small proportion of the total cells in FCI, with a median of 19.3% (range, 7.9–31.8%), which was significantly higher than those in the control groups (all <1.0%). The sensitivity of FCI for diagnosing ALCL in lymph node samples was 86.7% with a specificity of 100%. The majority of neoplastic cells demonstrated high light forward and high light side scatter, similar to monocytes or granulocytes in dot plots. FCI may be used as an adjunct to histopathological examination for rapid and reliable diagnosis of pediatric ALCL. Flexible gating strategies and careful analysis are required to identify neoplastic cells with FCI.