Prognostic significance and therapeutic potential of the activation of anaplastic lymphoma kinase/protein kinase B/mammalian target of rapamycin signaling pathway in anaplastic large cell lymphoma.

BACKGROUND: Activation of the protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway has been demonstrated to be involved in nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-mediated tumorigenesis in anaplastic large cell lymphoma (ALCL) and correlated with unfavorable outcome in certain types of other cancers. However, the prognostic value of AKT/mTOR activation in ALCL remains to be fully elucidated. In the present study, we aim to address this question from a clinical perspective by comparing the expressions of the AKT/mTOR signaling molecules in ALCL patients and exploring the therapeutic significance of targeting the AKT/mTOR pathway in ALCL. METHODS: A cohort of 103 patients with ALCL was enrolled in the study. Expression of ALK fusion proteins and the AKT/mTOR signaling phosphoproteins was studied by immunohistochemical (IHC) staining. The pathogenic role of ALK fusion proteins and the therapeutic significance of targeting the ATK/mTOR signaling pathway were further investigated in vitro study with an ALK + ALCL cell line and the NPM-ALK transformed BaF3 cells. RESULTS: ALK expression was detected in 60% of ALCLs, of which 79% exhibited the presence of NPM-ALK, whereas the remaining 21% expressed variant-ALK fusions. Phosphorylation of AKT, mTOR, 4E-binding protein-1 (4E-BP1), and 70 kDa ribosomal protein S6 kinase polypeptide 1 (p70S6K1) was detected in 76%, 80%, 91%, and 93% of ALCL patients, respectively. Both phospho-AKT (p-AKT) and p-mTOR were correlated to ALK expression, and p-mTOR was closely correlated to p-AKT. Both p-4E-BP1 and p-p70S6K1 were correlated to p-mTOR, but were not correlated to the expression of ALK and p-AKT. Clinically, ALK + ALCL occurred more commonly in younger patients, and ALK + ALCL patients had a much better prognosis than ALK-ALCL cases. However, expression of p-AKT, p-mTOR, p-4E-BP1, or p-p70S6K1 did not have an impact on the clinical outcome. Overexpression of NPM-ALK in a nonmalignant murine pro-B lymphoid cell line, BaF3, induced the cells to become cytokine-independent and resistant to glucocorticoids (GCs). Targeting AKT/mTOR inhibited growth and triggered the apoptotic cell death of ALK + ALCL cells and NPM-ALK transformed BaF3 cells, and also reversed GC resistance induced by overexpression of NPM-ALK. CONCLUSIONS: Overexpression of ALK due to chromosomal translocations is seen in the majority of ALCL patients and endows them with a much better prognosis. The AKT/mTOR signaling pathway is highly activated in ALK + ALCL patients and targeting the AKT/mTOR signaling pathway might confer a great therapeutic potential in ALCL.

[Clinical and Prognostic analysis of 43 Children with Mature B-cell Non-Hodgkin's Lymphoma/Acute Lymphoblastic Leukemia].

OBJECTIVE: To explore the clinical and prognostic features as well as treatment response of childhood B-cell non-Hodgkin's lymphoma/acute lymphoblastic leukemia (B-NHL/B-ALL), so as to better modify the treatment for further improving the prognosis. METHODS: The clinical data of 43 patients with newly-diagnosed childhood B-NHL/B-ALL from July 2005 to December 2013 in West China Second Hospital of Sichuan University were retrospectively analyzed with particular focus on clinical presentations, laboratory findings and histology. Among them 26 patients received B-NHL-2010 protocol and 17 patients received LMB-89 protocol treatment. Kaplan-Meier method was used to compare the survival rates between groups, while multiple factor logistic regression was used to identify the prognostic factors. RESULTS: (1) The median age at diagnosis was 7.58 (2.42-13.67) years. The male-to-female ratio was 2.9 : 1. No significant difference was found in the median age at diagnosis between male and female children with B-NHL/B-ALL (P = 0.837). (2) Burkitt's lymphoma was the most common (34/43, 79.07%), followed by diffuse large B cell lymphoma (4/43, 9.3%), ALL-L3 (3/43, 6.98%) and others (2/43, 4.65%) in decreasing frequency. (3) According to St. Jude staging classification, 4 patients (9.30%) were divided into stage I, 9 patients (20.93%) into stage II, 23 patients (53.49%) into stage III and 7 patients (16.28%) into stage IV; (4) Clinically, the common predilection sites were as following: ileocecus (11/43, 25.58%), nasopharynx (10/43, 23.26%), faciomaxillary (9/43, 20.93%), superficial lymphadenopathy (8/43, 18.60%), other sites such as mediastinum and bone marrow (5/43, 11.63%). (5) With a median follow up of 24 months (0.7-105 months), the 2-year overall survival (OS) rate and event-free survival (EFS) rate were 79.8% ± 6.5%% and 71.0% ± 7.2%, respectively. The 2-year OS and EFS rates in patients treated with B-NHL-2010 protocol were 79.1% ± 8.4% and 74.1% ± 8.4%, while those in patients treated with LMB-89 protocol were 87.5% ± 8.3% and 66.7% ± 12.4%, respectively, but there was no significant difference between them (P > 0.05). The 2-year EFS rate in patients with LDH > 2N and bone marrow infiltration were significantly lower than that of other groups (P < 0.05). (6) 8 patients (18.6%) relapsed. The median relapsed time was 6 months (2-9 months). 1 patient suffered progressive disease. Male, systemic symptom, elevated LDH, bone marrow and CNS infiltration and advanced stage (stage III and stage IV) were associated with relapse /progressive disease. Logistic regression analysis showed that LDH > 2N was an independent unfavorable prognostic factors (OR = 31.129, P = 0.02). CONCLUSION: Outcome of B-NHL/B-ALL is greatly improved by current intensive and short-time chemotherapy regimen. The 2-year event-free survival (EFS) rate is 71.0% ± 7.2%. There is no significant difference in EFS rate between patients treated with B-NHL-2010 protocol and LMB89 protocol. The long-term survival rate in patient with advanced disease need to be further improved.

[The induction and function of dendritic cells from human cord blood with IFN-alpha and GM-CSF in vitro].

OBJECTIVE: To probe into the possibility of obtaining dendritic cells (DCs) from human cord blood with rhIFN-alpha and rhGM-CSF. METHODS: The plastic adherent cells (monocyte-rich cells) from cord blood were cultured with rhGM-CSF and rhIFN-alpha for 7 days. The morphological properties of DCs were observed by microscopy, the expression of CD83 and CD86 on DCs were determined by flow cytometry. The function of DCs stimulating allogeneic T lymphocyte proliferation was detected by MTT colorimetric method. RESULTS: After 7 days culture, some of the cultured cells acquired typical DC morphology and showed increased expression of CD83 and CD86. The yield of DCs attained its peak when the concentrations of rhIFN-alpha and rhGM-CSF were 600 U/ml and 2000 U/ml, respectively. When cultured cells (containing DCs) as stimulator cells were co-cultured with allogeneic T lymphocytes (effector cells) in different ratio, they could stimulate T cell proliferation remarkably, especially when the ratio of effector cells to stimulator cells was 20 to 1. CONCLUSION: Our findings suggest that cord blood adherent cells, when cultured with rhGM-CSF and rhIFN-alpha, can be induced into mature DCs with the function of stimulating allogeneic T cell proliferation.

[The effect of erythropoitin and thyroid hormone on K562 iron metabolism].

OBJECTIVE: To explore the effect of recombinant human erythropoitin (rhEPO) and T3 on the iron metabolism of K562 cells and its possible mechanism. METHODS: With the use of the bone marrow iron stain method, the positive stained cells rate was calculated. Flow cytometry was performed to detect the transferrin receptor (TfR) expression of K562 cells. RESULTS: rhEPO increased the positive cell rate of iron stain, which was especially noticeable in the rhEPO 5 U/ml group (P < 0.01), and the effect was time-dependant, i.e., the longer the incubating time, the higher the positive rate. T3 also elevated the positive cell rate of iron stain significantly at different concentrations, and the highest rate was seen when incubating K562 cells for 48 hours. The expression of TfR was increased significantly when incubating with rhEPO for 72 hours (P < 0.05), but there was no marked change when incubating with T3. CONCLUSION: The possible mechanism by which rhEPO increased the positive cell rate of iron stain might be due to the enhancement of TfR expression and hence the increase in iron intake of K562 cells.

Relapsed APL patient with variant NPM-RARalpha fusion responded to arsenic trioxide-based therapy and achieved long-term survival.

The t(5;17)/NPM-RARalpha is the second variant chromosomal translocation in acute promyelocytic leukemia (APL) to be characterized and also the second most plentiful variant translocation. So far, there is a lack of information on the effectiveness of arsenic trioxide (ATO) in relapsed APL with variant RARalpha chimera including t(5;17)/NPM-RARalpha. We report here a long-term survived APL patient with variant NPM-RARalpha fusion who relapsed four times and each time responded well to ATO or ATO-based re-induction therapy. The patient had received a total of more than 3,500 mg of ATO, but showed no obvious arsenic-related toxicities. This case illustrates the long-term efficiency and safety of ATO-based therapy not only in newly diagnosed APL, but also in relapsed APL including those with variant translocations.

[Blockage of mTOR signaling pathway by rapamycin contributes to inhibition of tumor cell proliferation in ALK-positive lymphoid cell strains].

OBJECTIVE: To investigate the relationship between mTOR signaling pathway and ALK-positive lymphoid cell lines. METHODS: The expression of the downstream effector proteins of mTOR were analyzed by Western blot before and after Karpas299, BaF3/NPM-ALK and BaF3 cell lines treated with rapamycin. Effect of rapamycin on cell proliferation was detected by MTT assay. FACS was used to analyze apoptosis and cell cycles. RESULTS: mTOR signaling phosphoproteins, p-p70S6K and p-4E-BP1 were highly expressed in ALK(+) Karpas299, BaF3/NPM-ALK and parental BaF3 cell lines, and they were dephosphorylated after 1 h withdrawal of IL-3 in BaF3 cells. After 48 h exposure to 10 nmol/L rapamycin, p-p70S6K and p-4E-BP1 proteins expression were decreased, and mainly for the former. The relative inhibitory rate to its control cells was 24.4% in Karpas299, 37.8% in BaF3/NPM-ALK and 61.6% in BaF3. The apoptotic ratio was increased from (11.97 +/- 0.11)% to (15.87 +/- 0.62)% in Karpas299 (P < 0.05), from (3.23 +/- 0.11)% to (7.67 +/- 0.49)% in BaF3 (P < 0.05) and from (1.90 +/- 0.47)% to (2.80 +/- 0.27)% in BaF3/NPM-ALK (P > 0.05). The fraction of G(1) phase cells increased from (37.63 +/- 1.91)% to (69.77 +/- 5.44)% in BaF3/NPM-ALK, from (31.13 +/- 2.51)% to (40.70 +/- 1.47)% in Karpas299 and (53.57 +/- 2.22)% to (63.70 +/- 1.20)% in BaF3 (P < 0.05). CONCLUSION: NPM-ALK kinase can activate mTOR signaling pathway. Rapamycin can inhibit the proliferation of ALK(+) lymphoid cells by blocking mTOR signaling pathway and inducing cell cycling arrest at G(1) phase.

[Effect of T3 on the expression of transferrin receptor and ferritin in K562 cells and its possible mechanism].

OBJECTIVE: To explore the effect of T(3) on the expression of transferrin receptor (TfR) and ferritin (Fn) in K562 cells and its possible mechanism. METHODS: Flow cytometry was used for the detection of TfR expression, radioimmunoassay for Fn expression, RNA/protein band shift assay for the binding activity of iron regulatory protein (IRP) and iron responsive elements (IRE), and RT-PCR for TfR and Fn mRNA levels. RESULTS: Different concentration of T(3) significantly increased Fn expression of K562 cells, especially at 100 nmol/L and 200 nmol/L (p < 0.05). However, T(3) had no effect on TfR expression. T(3) decreased the binding activity between IRP and IRE, particularly at concentration of 50 nmol/L. Different concentration of T(3) increased Fn-H mRNA level at different time point while it had no effect on TfR mRNA level. CONCLUSION: T(3) increased Fn expression of K562 cells through the possible mechanisms of either the post-transcriptional regulation or transcriptional modulation.

[Erythropoietin increases transferrin receptor expression and the impact of erythropoietin on K562 leukemic cell cycle].

OBJECTIVE: Functionally, erythropoietin (EPO) can promote the proliferation and growth of erythroid progenitor cells, and it is widely used in the treatment of anemia in chronic diseases caused by tumor and inflammation. However, it is unclear whether EPO has any effect on tumor cell iron metabolism and tumor cell proliferation. The purpose of this study was to explore the effects of recombinant human EPO (rhEPO) on the expression of transferrin receptor (TfR, CD(71) antigen) of leukemic cell K562 and its relation to cell cycle. METHODS: In vitro culture of K562 cell was performed with additions of various concentrations of rhEPO and Fe. Treatments were terminated at 24 h and 72 h, respectively. Then each group of cells was incubated with FITC-IgG antibody to CD(71) or PI, a kind of DNA dye. And TfR expression and DNA synthesis status were analyzed by flow-cytometry. RESULTS: (1) The expression of TfR by K562 cells increased significantly when incubated for 72 h with different concentrations of rhEPO. The measurement values of 5 U/ml, 10 U/ml and 20 U/ml groups were 12.2 +/- 1.40, 10.7 +/- 0.99 and 11.1 +/- 0.90, respectively. They were markedly increased when compared with that of control group (6.27 +/- 0.11, P < 0.05). (2) When incubated with rhEPO (5 u/ml) alone or combined with FeCl(3) (100 micro mol/L), the percentages of cells in S phase were 51.1% and 59.6%, respectively. They significantly increased when compared with that of control group (42.9%, P < 0.05). CONCLUSIONS: Iron is very important for the proliferation of both normal cells and leukemic cells. It is essential to the activity of ribonucleotide reductase (RR). The authors hypothesized that rhEPO would increase the expression of TfR and intracellular iron content of leukemic cells, which would enhance the DNA synthesis and cell proliferation. Therefore, the clinical application of rhEPO to promote erythropoiesis of cancer patients should be cautious.