Jine Zheng

Increased population of CD4+CD25high regulatory T cells with their higher apoptotic and proliferating status in peripheral blood of acute myeloid leukemia patients

Abstract:  Purpose: Regulatory T cells (T‐reg) that control harmful autoimmune T cells in the periphery may also suppress the immune response against cancer. In this study we investigated the possible involvement of CD4+CD25high T‐reg in the immune impairment of patients with acute myeloid leukemia (AML). Experimental design: The frequencies and phenotypes of CD4+CD25high T cells in the peripheral blood of AML patients were determined by flow cytometry. To assess the functional activity of CD4+CD25high T cells, CD4+CD25high, and CD4+CD25− T cells were sorted from peripheral blood mononuclear cells with FACS Vantage. The immunoregulatory properties of CD4+CD25high and CD4+CD25− T cells were characterized by proliferation assays and cytokine production assays. In addition, the frequency of apoptotic and proliferating cells in CD4+CD25high T cells were respectively evaluated by 7AAD and ki67 binding cells using flow cytometry. Results: Compared with healthy controls, AML patients had a higher proportion of CD4+CD25high T cells in peripheral blood. These cells were CD45‐RA(−), CD69(−), CD45‐RO(+), CD95(+), and intercellular CTLA‐4(+), and secreted low levels of TNF‐α and IL‐10, but no IL‐2, IL‐4, IL‐5, and IFN‐γ. They inhibited the proliferation and cytokine production (IL‐2, IFN‐γ) of CD4+CD25− T cells, but improved IL‐10 production under the co‐culture of both subsets with stimulation, thus behaving as T‐reg. Notably, CD4+CD25high T cells in AML patients presented significantly higher apoptosis and proliferation than that of healthy individuals. Conclusions: The frequency of CD4+CD25high T‐reg in peripheral blood in AML patients is significantly higher when compared with healthy individuals, likely due to the increasing proliferation of CD4+CD25high T cells.

A correlation study of immunophenotypic, cytogenetic, and clinical features of 180 AML patients in China

New WHO classification has been widely applied in the diagnosis of leukemia. To elucidate the immunophenotype of acute myeloid leukemia (AML) and characterize the correlation among morphological, immunological, cytogenetic, and clinical features, we studied the bone marrow immunophenotypes of 180 AML patients in China by flow cytometry. The results showed that CD34, CD2, CD14, CD19, CD56, and HLA‐DR were correlated with FAB subtypes. Amongst the 180 patients enrolled in this study, 122 cases were also subjected to karyotype analysis by G‐banding technology and abnormal karyotypes were detected in 69 out of 122 patients. Correlation assay showed that t(8;21) was only present in 16 AML‐M2 patients, and strongly associated with the individual or combinational expressions of CD15/CD19/CD34/CD56. As to M3, although lymphoid lineage antigens were observed in a considerable number of patients, they were never detected in t(15;17) positive patients. The expressions of CD22, CD56, and TdT showed significant correlation with the overall presence of abnormal karyotype. Additionally, the expressions of CD4, CD7, CD14, CD56, and TdT were positively correlated with clinical features such as white blood cell count, platelet count, and patients age. In conclusion, immunophenotype analysis was useful for AML diagnosis and classification. At the same time, the data also suggested that the karyotype abnormalities and clinical features were tightly linked with abnormal antigen expression characteristics in AML patients. As one of the largest correlative study performed in China, the results highlighted the importance of a morphological, immunological, and cytogenetic classification of AML that might constitute a working basis for future studies aimed at a better definition of clinicopathological features and optimal treatment strategy for these leukemias.

The insulin-like growth factor-1 receptor kinase inhibitor, NVP-ADW742, suppresses survival and resistance to chemotherapy in acute myeloid leukemia cells.

Deregulation of insulin-like growth factor-1 receptor (IGF-1R) is closely associated with malignant transformation and tumor cell survival in various cancers. We found that IGF-1R expression level in leukemia cells positively correlated with the percentage of blast in bone marrow from de novo acute myeloid leukemia (AML) patients. Moreover, we showed that NVP-ADW742, a novel small weight molecular inhibitor of IGF-IR, could induce apoptosis in both HL-60 cell line and primary AML blasts. However, no significant alteration of cell cycle was observed in HL-60 cells. Further studies revealed that NVP-ADW742 induced Akt dephosphorylation, which might subsequently induce p38 phosphorylation and decrease antiapoptotic protein Bcl-2 expression in HL-60 cells. Finally, we demonstrated that NVP-ADW742 could synergize with Ara-C to induce the kill in a subset of drug-resistant AML specimens. We suggested that IGF-lR targeting might be therapeutically beneficial for some AML patients.

Transcript level of nucleostemin in newly diagnosed acute myeloid leukemia patients.

To clarify the role of nucleostemin (NS) in AML, its transcription levels in bone marrow (BM) samples obtained from 128 newly diagnosed AML patients were analyzed. We determined that the highest NS transcription level was in M1 patients, while the lowest NS transcription level was in M3 patients. NS mRNA expression is positively correlated with blast percentages (%) and CD34, CD117 and CD123 antigen expression in BM samples but is unrelated to the transcription level of WT1. A significant difference in NS expression between poor-risk and better-risk and between poor-risk and intermediate-risk AML patients was found. Our initial data indicated that NS can be used for tracking minimal residual disease (MRD) and is a helpful guide for treatment.

Analysis of hematogones in bone marrow from acute myeloid leukaemia cases posttherapy

Increased bone marrow (BM) hematogones (HGs) are often observed in patients with marrow regenerating status. Many studies have focused on the role of HGs in acute lymphoblastic leukaemia (ALL), but very little has been done to understand their effects on acute myeloid leukaemia (AML).

Chronic myeloid leukemia and BCR/ABL signal pathways are not associated with AKT1 pleckstrin homology domain (E17K) mutations.

To the Editor: The v-akt murine thymoma viral oncogene homologues (AKT) is the major downstream mediator of growth factor receptors that signal through phosphatidylinositol-3 kinase (PI3K) (1). Mounting evidence suggests that aberrant activation of AKT proteins is involved in the development of human malignancies. The activation of AKT proteins has frequently been attributed to the presence of activating PIK3CA mutation and the lack of PTEN (phosphatase and tensin homology deleted on chromosome 10) activity in tumor cells (2, 3). Recently, Carpten et al. (4) described a somatic missense mutation of AKT1 in human cancers. The substitution of an amino acid (E17K) in pleckstrin homology domain of AKT1 Lys 17 resulted in an increased level of AKT phosphorylation on Thr 308 and Ser 473. Moreover, E17K mutation induces leukemia in mice, suggesting the oncogenic potential of this AKT1 activating mutation. Previous screening studies found E17K mutation in lung, breast, colorectal, ovarian and urothelial cancers but not in acute myeloid leukemia, B-cell-derived lymphoid leukemia, glioblastomas or medulloblastomas, promoting further studies to investigate the presence of this mutation in other malignancies (5–9). Chronic myeloid leukemia (CML) is a malignant clonal hematopoietic stem cell disorder characterized by the translocation of 9q34 to 22q11 (Ph), generating the breakpoint cluster region (BCR)-Abelson (ABL) fusion gene (10, 11). The BCR-ABL oncogenic protein has constitutive tyrosine kinase activity to enhance proliferative and anti-apoptotic signals. Increasing evidences demonstrate that PI3K ⁄AKT is one of major downstream signal pathways of BCR-ABL oncogene and plays essential role in BCR-ABL-mediated transformation. More recently, it has been shown that increased PI3K ⁄AKT activity in CML contributes to the development of drug resistance of malignant cells and the combination of PI3K ⁄AKT pathway inhibitor and rapamycin can efficiently kill imatinib (IM)-resistant BCR-ABL-expressing cells, highlighting the potential of PI3K ⁄AKT as the new targets for CML therapy (12). To study the frequency of AKT1 E17K mutation in Chinese CML patients as well as its possible relation to IM sensitivity, genome DNA was extracted from the peripheral blood mononuclear cell (PBMNC) or bone marrow mononuclear cell of 86 CML patients whose karotype showed the presence of Philadelphia chromosome at diagnosis. At the molecular level, B2a3, B3a3, B2a2 and B3a2 were identified by RT-PCR in 2, 1, 41 and 42 cases, respectively. The complete cytogenetic response (CCyR), major molecular response (MMR) and complete molecular response (CMR) were achieved in 74, 39 and 27 cases at 3–12 months after IM administration (Table 1). Direct PCR product sequencing analysis was performed as previously described (9). No AKT1 E17K mutation was found in all patients. The absence of AKT1 E17K mutation in our study suggests the pre-existence of AKT1 E17K mutation plays, if any, minor role in determining the clinical response to IM as well as for the leukemogenesis of CML. Nevertheless, because of the low counts of leukemic cells in most patients, we did not exam the presence of AKT1 E17K mutant in patients after IM exposure. Thus, the possibility remains that the acquisition of AKT1 E17K mutation after IM administration may lead to the failure of achieving MMR in some patients. Further studies with more sensitive techniques are required to clarify this possibility. Acquisition of additional genetic alternations has been considered as a major reason for the occurrence of CML blast crisis. We tested the possibility that AKT1 E17K mutation may contribute to the blast crisis of CML because oncogenic AKT1 E17K mutation has been shown to induce the development acute leukemia in transgenic

Development of a NanoString assay to detect leukemogenic fusion transcripts in acute myeloid leukemia

Detection of leukemogenic fusion transcripts in acute myeloid leukemia (AML) is critical for AML diagnosis. NanoString nCounter system is a novel probe‐based gene expression platform capable of measuring up to 800 targets with advantages of reproducibility, accuracy, and sample type flexibility. To study the potential application of NanoString in leukemia at clinic, we used this technology to detect AML leukemogenic fusion transcripts and compared the performances with clinical molecular assays.

Effects of dendritic cell-activated and cytokine-induced killer cell therapy on 22 children with acute myeloid leukemia after chemotherapy

SummaryThe efficiency of dendritic cell-activated and cytokine-induced killer cell (DC-CIK) therapy on children with acute myeloid leukemia (AML) after chemotherapy was investigated. Mononuclear cells were collected from children achieving complete remission after chemotherapy, cultured in vitro and transfused back into the same patient. Interleukin-2 (IL-2) was injected subcutaneously every other day 10 times at the dose of 1×106 units. Peripheral blood lymphocyte subsets and minimal residual disease (MRD) were detected by flow cytometry. Function of bone marrow was monitored by methods of morphology, immunology, cytogenetics and molecular biology. The side effects were also observed during the treatment. The average follow-up period for all the 22 patients was 71 months and relapse occurred in two AML patients (9.1%). The percentage of CD3+/CD8+ cells in peripheral blood of 15 patients at the 3rd month after DC-CIK treatment (36.73%±12.51%) was dramatically higher than that before treatment (29.20%±8.34%, P<0.05). The MRD rate was >0.1% in 5 patients before the treatment, and became lower than 0.1% 3 months after the treatment. During the transfusion of DC-CIK, side effects including fever, chills and hives appeared in 7 out of 22 (31.82%) cases but disappeared quickly after symptomatic treatments. There were no changes in electrocardiography and liver-renal functions after the treatment. MRD in children with AML can be eliminated by DC-CIK therapy which is safe and has fewer side effects.The efficiency of dendritic cell-activated and cytokine-induced killer cell (DC-CIK) therapy on children with acute myeloid leukemia (AML) after chemotherapy was investigated. Mononuclear cells were collected from children achieving complete remission after chemotherapy, cultured in vitro and transfused back into the same patient. Interleukin-2 (IL-2) was injected subcutaneously every other day 10 times at the dose of 1×106 units. Peripheral blood lymphocyte subsets and minimal residual disease (MRD) were detected by flow cytometry. Function of bone marrow was monitored by methods of morphology, immunology, cytogenetics and molecular biology. The side effects were also observed during the treatment. The average follow-up period for all the 22 patients was 71 months and relapse occurred in two AML patients (9.1%). The percentage of CD3+/CD8+ cells in peripheral blood of 15 patients at the 3rd month after DC-CIK treatment (36.73%±12.51%) was dramatically higher than that before treatment (29.20%±8.34%, P<0.05). The MRD rate was >0.1% in 5 patients before the treatment, and became lower than 0.1% 3 months after the treatment. During the transfusion of DC-CIK, side effects including fever, chills and hives appeared in 7 out of 22 (31.82%) cases but disappeared quickly after symptomatic treatments. There were no changes in electrocardiography and liver-renal functions after the treatment. MRD in children with AML can be eliminated by DC-CIK therapy which is safe and has fewer side effects.

Characterisation of clonal Philadelphia‐negative cytogenetic abnormalities in a large cohort of chronic myeloid leukaemia

Clonal Philadelphia (Ph)‐negative cytogenetic abnormalities (CPCA) have been reported in chronic myeloid leukaemia (CML) patients treated with either interferon or tyrosine kinase inhibitor (TKI). However, the incidences and types of these cytogenetic abnormalities after treatment vary due to the limited populations enroled.

A Pediatric Acute Promyelocytic Leukemia With a Rare Karyotype of ider(17)(q10)t(15;17) and Favorable Outcome: A Case Report.

AbstractAcute promyelocytic leukemia (APL) is a specific malignant hematological disorder with a diagnostic hallmark of chromosome translocation t(15;17)(q22;q21). As a very rare secondary cytogenetic aberration in pediatric APL, ider(17q) (q10)t(15;17) was suggested to be a poor prognostic factor based on previous case reports.Here, we report a pediatric APL case with a rare karyotype of ider(17)(q10)t(15;17). Bone marrow aspiration, immunophenotyping, molecular biology, cytogenetic, and fluorescence in situ hybridization (FISH) analyses were performed at initial diagnosis and during the treatment.A 6-year-old boy was brought to our hospital with the chief complaint of bleeding gums twice and intermittent fever for 3 days in January 2013. He was diagnosed as low-risk APL according to the 2012 NCCN guideline on APL, with the expression of PML-RARA (bcr3 subtype) and the karyotype of 46,XY, der(15)t(15;17)(q22;q21),ider(17)(q10)t(15;17), which was further verified by FISH. The patient was treated through combination all-trans retinoic acid (ATRA) and arsenic with daunorubicin according to the 2012 NCCN guideline for APL. Continuous hematological completed remission (HCR) and major molecular remission (MMR) were achieved with normal karyotype for >28 months after induction chemotherapy.Different from previously reported cases, this pediatric APL patient with ider(17)(q10)t(15;17) displays favorable clinical outcomes, which might be related to the low-risk classification and arsenic treatment during the treatment. It suggests that ider(17)(q10)t(15;17) may not be the sole determinant for worse outcomes in pediatric APL and implies that more contributed factors should be considered for pediatric APL prognosis.