Chunrui Li

Arsenic disulfide synergizes with the phosphoinositide 3-kinase inhibitor PI-103 to eradicate acute myeloid leukemia stem cells by inducing differentiation

Although dramatic clinical success has been achieved in acute promyelocytic leukemia (APL), the success of differentiating agents has not been reproduced in non-APL leukemia. A key barrier to the clinical success of arsenic is that it is not potent enough to achieve a clinical benefit at physiologically tolerable concentrations by targeting the leukemia cell differentiation pathway alone. We explored a novel combination approach to enhance the eradication of leukemia stem cells (LSCs) by arsenic in non-APL leukemia. In the present study, phosphatidylinositol 3-kinase /AKT/mammalian target of rapamycin (mTOR) phosphorylation was strengthened after As(2)S(2) exposure in leukemia cell lines and stem/progenitor cells, but not in cord blood mononuclear cells (CBMCs). propidium iodide-103, the dual PI3K/mTOR inhibitor, effectively inhibited the transient activation of the PI3K/AKT/mTOR pathway by As(2)S(2). The synergistic killing and differentiation induction effects on non-APL leukemia cells were examined both in vitro and in vivo. Eradication of non-APL LSCs was determined using the nonobese diabetic/severe combined immunodeficiency mouse model. We found that a combined As(2)S(2)/PI-103 treatment synergized strongly to kill non-APL leukemia cells and promote their differentiation in vitro. Furthermore, the combined As(2)S(2)/PI-103 treatment effectively reduced leukemia cell repopulation and eradicated non-APL LSCs partially via induction of differentiation while sparing normal hematopoietic stem cells. Taken together, these findings suggest that induction of the PI3K/AKT/mTOR pathway could provide a protective response to offset the antitumor efficacy of As(2)S(2). Targeting the PI3K/AKT/mTOR pathway in combination with As(2)S(2) could be exploited as a novel strategy to enhance the differentiation and killing of non-APL LSCs.

Abnormal immunophenotype provides a key diagnostic marker: a report of 29 cases of de novo aggressive natural killer cell leukemia

Aggressive natural killer (NK) cell leukemia (ANKL) is a systemic neoplastic proliferation of NK cells with an aggressive clinical course. Currently, the diagnosis of ANKL remains challenging. In the current study, we report the clinical, laboratory, immunophenotypic, and genetic findings from 29 cases of de novo ANKL in a single center and evaluate the relative contribution of these features to the diagnosis of ANKL. Clinical features, laboratory findings, morphologic, cytogenetic features, and Epstein-Barr virus status were important factors for diagnosing aggressive NK cell leukemia. On the other hand, ANKL displays a strikingly abnormal immunophenotype in contrast to nonneoplastic NK cells. The immunophenotype of ANKL cells may differ from reactive NK cells in 4 respects. First, the CD45/linear side scatter gating of flow cytometry allows the initial identification of neoplastic subpopulations for additional immunophenotypic analysis in half of ANKL cases. Second, unusual expression of surface antigens in ANKL cells was a prominent feature. Third, the clonality of ANKL cells could be identified using antibodies against CD158a/h, CD158b, or CD158e. Last, the positive rate of Ki-67 expression in ANKL cells was generally high. Based on these findings, we provide an objective marker based on clinical data for the definite diagnosis of ANKL.

Aberrant Methylation of Death-Associated Protein Kinase 1 CpG Islands in Myelodysplastic Syndromes

Death-associated protein kinase 1 (DAPK1), a proapoptotic serine/threonine kinase, is a candidate tumor suppressor gene. We studied the methylation status of the promoter region of the DAPK1 gene and the expression of the DAPK1 protein in 78 bone marrow samples from untreated patients with myelodysplastic syndrome (MDS) by PCR and Western blot analysis. Hypermethylation of DAPK1 was present in 42.3% (33 of 78) of MDS specimens and was significantly correlated with the loss of DAPK1 mRNA and protein expression (p < 0.01). There were no significant differences in methylation frequency among subgroup of MDS. DAPK1 hypermethylation in MDS was associated with the presence of cytogenetic abnormalities in the bone marrow at the time of the initial diagnosis. A higher frequency of DAPK1 hypermethylation was found in the unfavorable cytogenetic risk group (12 of 21 cases; 57.1%) compared to the favorable cytogenetic risk group (5 of 20 cases; 25.0%, p = 0.0368). These findings suggest that suppression of DAPK1 expression by DNA methylation may play a substantial role in the development of MDS.

SIL-TAL1 rearrangement is related with poor outcome: a study from a Chinese institution.

SIL-TAL1 rearrangement is common in T-cell acute lymphoblastic leukemia (T-ALL), however its prognostic implication remains controversial. To investigate the clinical characteristics and outcome of this subtype in Chinese population, we systemically reviewed 62 patients with newly diagnosed T-ALL, including 15 patients with SIL-TAL1 rearrangement. We found that SIL-TAL1+ T-ALL was characterized by higher white blood cell count (P = 0.029) at diagnosis, predominant cortical T-ALL immunophenotype (P = 0.028) of the leukemic blasts, and a higher prevalence of tumor lysis syndrome (TLS, P<0.001) and disseminated intravascular coagulation (DIC, P<0.001), which led to a higher early mortality (P = 0.011). Compared with SIL-TAL1− patients, SIL-TAL1+ patients had shorter relapse free survival (P = 0.007) and overall survival (P = 0.002). Our NOD/SCID xenotransplantation model also demonstrated that SIL-TAL1+ mice models had earlier disease onset, higher leukemia cell load in peripheral blood and shorter overall survival (P<0.001). Moreover, the SIL-TAL1+ mice models exerted a tendency of TLS/DIC and seemed vulnerable towards chemotherapy, which further simulated our clinical settings. These data demonstrate that SIL-TAL1 rearrangement identifies a distinct subtype with inferior outcome which could allow for individual therapeutic stratification for T-ALL patients.

Integrative analysis of prognostic factors in Chinese core binding factor leukemia.

The characteristics of core binding factor (CBF) leukemia appear to differ between Chinese and Caucasian patients. In this study, we analyzed the biological and clinical characteristics of 76 Chinese CBF leukemia patients out of 425 newly diagnosed acute myeloid leukemia (AML) patients. The frequency of CBF AML was 17.9%. Patients harboring t(8;21) were predominant in CBF AML. The incidence of c-kit mutation in CBF AML was 28.9%. The N822K mutation appeared to be more prevalent in Chinese CBF AML patients. Multivariate analysis showed that c-kit mutation and high white blood cell count could negatively impact overall survival (OS) (HR=2.74 and 6.24, P=0.007 and 0.022, respectively) but did not affect relapse-free survival (RFS). Kaplan-Meier analysis showed a significant difference in both OS and RFS between wild-type and mutated c-kit patients. Although we had included recently reported prognostic indicators in our analysis, our results demonstrated that only c-kit mutation and high white blood cell count had prognostic impact on Chinese CBF AML patients.

TMTP1, a Novel Tumor-homing Peptide, Specifically Targets Hematological Malignancies and Their Metastases

SummaryTMTP1, a 5-amino acid peptide NVVRQ, obtained by using the flagella peptide library screening in our previous studies, can be used for the labeling of malignant in situ and metastatic lesions, and even micro-metastases. In this study, TMTP1 was assessed for its ability to specifically target the malignant hematopoietic cells and metastatic lesions of hematological malignancies. FITC-TMTP1 was chemically synthesized. Immunofluorescence assay and competitive test were carried out to determine the specific binding capacity of TMTPl to hematological malignant cell lines, including HL60, k562, SHI-1, Jurkat, Raji, El–4 and umbilical cord blood mononuclear cells. Mononuclear cells were isolated from the bone marrow of healthy subjects and patients with chronic myeloid leukemia. Then the cells were co-clutured with TMTP1 or scrambled peptides and the binding and affinity of TMTP1 peptide to the primary cells of hematological malignancies were flow cytometrically analyzed. The binding specificity of TMTP1 to target hematological malignancies was measured in vivo by intravenous injection of FITC-conjugated TMTP1 into El-4 lymphoma-bearing mice. The results showed that TMTP1 specifically bound to the cells of a series of hematological malignancies, including HL60, k562, Jurkat, Raji, El–4 and chronic myeloid leukemia primary cells but not to bone marrow mononuclear cells from healthy subjects. By contrast, TMTP1 could bind to the metastatic foci of lymphoma originating from the EL-4 cell line while the scrambled peptide failed to do so. Moreover, the occult metastases could be identified, with high specificity, by detecting FITC-TMTP1. We are led to conclude that TMTP1, as a novel tumor-homing peptide, can serve as a marker for primary malignant and metastatic lesions for the early diagnosis of hematological malignances and a carrier of anticancer drugs for cancer treatment.TMTP1, a 5-amino acid peptide NVVRQ, obtained by using the flagella peptide library screening in our previous studies, can be used for the labeling of malignant in situ and metastatic lesions, and even micro-metastases. In this study, TMTP1 was assessed for its ability to specifically target the malignant hematopoietic cells and metastatic lesions of hematological malignancies. FITC-TMTP1 was chemically synthesized. Immunofluorescence assay and competitive test were carried out to determine the specific binding capacity of TMTPl to hematological malignant cell lines, including HL60, k562, SHI-1, Jurkat, Raji, El–4 and umbilical cord blood mononuclear cells. Mononuclear cells were isolated from the bone marrow of healthy subjects and patients with chronic myeloid leukemia. Then the cells were co-clutured with TMTP1 or scrambled peptides and the binding and affinity of TMTP1 peptide to the primary cells of hematological malignancies were flow cytometrically analyzed. The binding specificity of TMTP1 to target hematological malignancies was measured in vivo by intravenous injection of FITC-conjugated TMTP1 into El-4 lymphoma-bearing mice. The results showed that TMTP1 specifically bound to the cells of a series of hematological malignancies, including HL60, k562, Jurkat, Raji, El–4 and chronic myeloid leukemia primary cells but not to bone marrow mononuclear cells from healthy subjects. By contrast, TMTP1 could bind to the metastatic foci of lymphoma originating from the EL-4 cell line while the scrambled peptide failed to do so. Moreover, the occult metastases could be identified, with high specificity, by detecting FITC-TMTP1. We are led to conclude that TMTP1, as a novel tumor-homing peptide, can serve as a marker for primary malignant and metastatic lesions for the early diagnosis of hematological malignances and a carrier of anticancer drugs for cancer treatment.

Aggressive Natural Killer Cell Leukemia Secondary to HodgkinLymphoma: a Case Report and Review of the Literature

A 32-year old Chinese woman presented with high fever (39C), lymphadenopathy, hepatosplenomegaly, shortness of breath and general fatigue after initial remission of Hodgkin lymphoma (HL). The immuno-phenotype of blast cells found in her peripheral blood and pleural fluid suggested abnormal Natural Killer (NK) cells (CD2+CD3- CD56+CD45RO+HLADR+CD94+ with high Ki67). Her serum EB virus DNA was over 1.0×107IU/mL. Combining her clinical symptom and laboratory data, the patient was diagnosed with Hodgkin Lymphoma, secondary NK cell leukemia and haemophagocytic lymphohistocytosis (HLH). To date, this is the first reported case that developed secondary ANKL after the treatment of Hodgkin Lymphoma. Secondary treatment-related cancers are a major problem in HL survivors. Thus, novel treatment strategies for HL should aim at a reduction of chemotherapy and radiation therapy in order to reduce the risk for development of therapy-related malignancies. In conclusion, improvements of combined therapy as well as close monitoring during clinical follow up could warrant the improved overall survival of HL and prevent fatal complications.

Induction of myelogenous leukemia cells with histone deacetylase inhibitors through down-regulating the Daxx protein expression

SummaryThe effects of two different histone deacetylase (HDAC) inhibitors, sodium butyrate (NaB) and trichostatin A (TSA),on apoptosis of human leukemic cells in vitro and the molecular mechanisms were investigated. The experiments were divided up 5 groups: control group, NaB group, TSA group, NaB+Z-VAD-FMK group and TSA+Z-VAD-FMK group. The apoptosis rate was determined by morphological analysis and flow cytomytry. The expression of Daxx, Bcl-2, and Bcl-xl proteins was detected by Western blot. NaB and TSA could induce the apoptosis of HL-60 and K562 cells, and Z-VAD-FMK caused a marked decrease in apoptosis induced by HDAC inhibitors. HDAC inhibitors could down-regulate the expression of Daxx protein, but had no significant influence on the expression of Bcl-2 and Bcl-xl proteins. The results suggested that NaB and TSA induce distinct caspase-dependent apoptosis of human leukemic cells through down-regulating the expression of Daxx protein in vitro.The effects of two different histone deacetylase (HDAC) inhibitors, sodium butyrate (NaB) and trichostatin A (TSA),on apoptosis of human leukemic cells in vitro and the molecular mechanisms were investigated. The experiments were divided up 5 groups: control group, NaB group, TSA group, NaB+Z-VAD-FMK group and TSA+Z-VAD-FMK group. The apoptosis rate was determined by morphological analysis and flow cytomytry. The expression of Daxx, Bcl-2, and Bcl-xl proteins was detected by Western blot. NaB and TSA could induce the apoptosis of HL-60 and K562 cells, and Z-VAD-FMK caused a marked decrease in apoptosis induced by HDAC inhibitors. HDAC inhibitors could down-regulate the expression of Daxx protein, but had no significant influence on the expression of Bcl-2 and Bcl-xl proteins. The results suggested that NaB and TSA induce distinct caspase-dependent apoptosis of human leukemic cells through down-regulating the expression of Daxx protein in vitro.

Clonal cytogenetic abnormalities are predictor in developing non-Hodgkin lymphomas?

Pathological analysis is the cornerstone for diagnosing malignant lymphoma. Status of cytogenetic abnormalities is frequently left unexamined if no evidence of malignancy is found in pathological analysis. In this study, we presented 3 cases in which clonal cytogenetic abnormalities were detected but morphological alterations of the same tissue did not support malignant non Hodgkin lymphoma at the first lymph node biopsy. Case 1 is a 55-year-old female with lymphadenopathy neoplastic process confirmed by flow cytometry and polymerase chain reaction (PCR). Chromosome analysis revealed 47,XX,t(3;22)(q27;q11),+del(9)(p12)[16]/46,XX[4]. The pathological analysis of subsequent lymph node biopsy indicated diffuse large B-cell lymphoma (DLBCL). Case 2, a 74-year-old female, for whom the pathological analysis, molecular studies and flow cytometric analysis of the first lymph node biopsy found no evidence of clonal cell. Cytogenetic analysis demonstrated a terminal deletion of chromosome 7 and 1, and the patient received a second lymph node biopsy and splenectomy. A pathological diagnosis of splenic marginal zone lymphoma (SMZL) was made. In Case 3 who was a 66-year-old female with right cervical and axillary lymph node enlargement. Cytogenetic analysis showed clonal karyotypic abnormalities: 48,XX, t(14;18)(q32;q21) [13]/46, XY [7]. The diagnosis of follicular lymphoma was rendered by the second biopsy of axillary lymph node according to the analysis of morphology and immunohistochemistry. We propose that clonal cytogenetic abnormalities may be a high potential risk for developing non-Hodgkin lymphomas. Follow-up and rebiopsy must be performed in patients who are cytogenetically abnormal but morphologically benign.

A clinical observation of Chinese chronic myelogenous leukemia patients after discontinuation of tyrosine kinase inhibitors

Whether tyrosine kinase inhibitors (TKIs) can be safely discontinued is a key focus of chronic myelogenous leukemia (CML) at present. We report a clinical observation of TKIs cessation in Chinese CML patients and a probable connection between CML leukemia stem cells (LSCs) and relapse. In all, 22 of 1057 patients consented to participate in this observation. The average time of complete molecular response was 12.73 months after TKI withdrawal. LSCs could be flow cytometrically detected in most of the patients. However, the number of LSCs did not differ between the relapsers and non-relapsers. We evaluated the leukemogenetic ability of the LSCs by transplanting bone marrow into irradiated NOD/SCID mice. The results indicated that part of the bone marrow from the relapsers lead to leukemogensis in the mice. Besides, we found that LSCs-derived microvesicles might serve as a novel factor for the stratification of undetectable minimal residual disease and an early warning sign of relapse. In summary, post-TKI cessation relapse seems to show none association with the number of LSCs. A mouse xenograft model would provide a novel and useful method of analyzing LSCs function and predicting relapse. Microvesicles may provide important information about optimal molecular monitoring schedules in TKI discontinuation strategies.