ALBERT

Description: Anaplastic large cell lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is a aggressive T cell lymphoma. The 5-year survival rate of ALK-negative ALCL is less than those of ALK-positive ALCL. To address factor(s)involved in malignant progression of ALK-negative ALCL, we established two cell lines (N1, N2) at the different clinical stages from a 59-years-old male patient. He developed rapid progression and died five months after diagnosis. As shown in Table, N1 and N2 represent early and advanced stages of lymphoma phenotype, respectively. Both of them expressed CD2 and CD30 on the cell surface and showed identical TCR gene rearrangement. By Enzyme immunoassay, N2 showed constitutively higher activated, but not total, NFkappaB activity compared to N1. cDNA microroarray analysis compared two cell lines and revealed that expression of four chemokine receptors(CCR3, CCR6, CCR8, CXCR4) and six chemokines (CCL5, CCL8, CCL26, CXCL2, CXCL3, CXCL16) were down-regulated greater than two-fold in N2 compared to N1. Protein array analysis showed that N1, not N2, secreted significant amounts of CCL5, IL8 and VEGF in the culture medium. Moreover, conditioned medium from N1 (CMN1) showed cytotoxic effects on HL60 myeloid leukemia cell lines in culture,indicating CMN1 may contain additional cytotoxic factors not identified, because none of CCL5, IL8 and VEGF show cytotoxic effect on HL60 cells. cDNA microroarray analysis also demonstrated that six major histocompatibility complex (MHC) class II gene expression (HLA-DRβ5, HLA-DPα1, HLA-DPβ1, HLA-DMα, HLA-DMβ, HLA-DOβ) were down-regulated greater than two-fold in N2 compared to N1. Flow cytometric analysis also showed cell surface expression of HLA-DR were strongly positive for N1, but not detected for N2(Table). Fifteen genes were up-regulated greater than five-fold in N2 relative to N1. They include genes involved in apoptosis such as growth arrest -specific 2 and tumor protein p53 inducible nuclear protein 1. They also include recently cloned two genes involved in tumor progression, Neuronal cell death inducible putative kinase (41-fold) and Niban (10- fold). The differential expression of the two genes was also confirmed by real-time PCR analysis. Table Characteristics of ALCL cell lines ALCL cell line Stage of Disease Cell morphology Cell surface marker Doubling time (liquid culture) Colony formation (soft agar) total κ NF- B(pg/μg protein activated NF- κB(pg/μg protein *p=0.274 **p=0.003 N1 early round cell surface, single nucleolus CD2, CD30, HLADR 32.4 hrs 〈0.1% 298±25 * 565±91 ** N2 advanced irregular cell surface, multiple nucleoli CD2, CD30 22.8 hrs 10% 334±66 * 1067±96 ** Taken together,in addition to CD30-induced up-regulation of NFkappaB activity, present study suggests that following factors are possibly involved in the rapid progression of ALK-negative ALCL, (1) the loss of one or more of chemokine receptors involved in homing the ALCL cells to local lymphnode may trigger the potential of these cells to metastasize; (2) the loss of some types of chemokines and cytokines produced by ALCL cells which negatively regulate tumor progression through autocrine and paracrine manner; (3) the loss of one or more MHC class II expression may allow ALCL cells to escape immune system of host against tumors; (4)significant over-expression of gene(s) with oncogenic potential was induced after chemotherapy.

Description: The retinoic acid syndrome (RAS) is a most important complication in all-trans retinoic acid (ATRA)-based remission induction therapy in acute promelocytic leukemia (APL). Predictive factors associated with the development of RAS have not been fully established. Cyclin A1 is a tissue specific cyclin that is required for G2/M phase transition in spermatogenesis. Abnormal expression of cyclin A1 has association with tumorigenesis. Human cyclin A1 is highly expressed in acute myeloid leukemia, especially in APL. The close association between expression of cyclin A1 and pathogenesis of APL was suggested by previous study, which showed (1) the APL-associated aberrant fusion proteins(PML-RARαor PLZF-RARα) caused overexpression of cyclin A1 mRNA through the direct activation of it’s promoter, (2) the elevation of cyclin A1 expression was reversed by treatment with ATRA in APL cells. To elucidate the clinical relevance between expression of cyclin A1 and APL, we examined expression of cyclin A1 mRNA in 37 APL samples having PML-RARα fusion gene, and correlated the results of clinical features and disease outcomes of the APL patients treated with ATRA-based differentiation therapy. Twenty-nine patients out of 37 were treated with ATRA-based remission induction therapy, including (1) ATRA(45mg/m2) followed by chemotherapy (cytarabine plus idarubicin) if the initial leukocyte count was below 3x109/L or (2) ATRA combined chemotherapy if the initial leukocyte count exceeded 3x109/L. Levels of cyclin A1 transcripts were determined by quantitative real-time RT-PCR. Cyclin A1 mRNA expression index (A1 index)was defined as copy numbers of cyclin A1 in μg RNA which contains 107 copies of GAPDH. Genomic methylation status of cyclin A1 promoter was also analyzed quantitatively using real-time PCR-based method as described previously. All APL samples expressed detectable cyclin A1 mRNA, and A1 index ranged from 4.3 to 202 (A1 index 71.9± 59.5 [mean±SD]), while CD34 +cells isolated from 4 normal bone marrow samples showed below detection levels of cyclin A1 mRNA. In APL cells, initial white blood cell count and A1 index showed significant correlation (r=0.557,p=0.0014). A1 index in 6 APL samples, which developed RAS, was significantly higher compared with those in a group without developing RAS (A1 index 101.5± 65.8 for a group with RAS vs. 72.9±60.1 for a group without RAS, p=0.015). Difference of initial white blood cell count between these 2 groups was not statistically significant (5.0±4.8x 109/L for a group with RAS vs. 9.6±20.6x109/L for a group without RAS, p=0.118). Furthermore, in a group received ATRA-based differentiation therapy, survival probability of the group with high A1 index (A1 index 〉 130, 9 cases) was significantly shorter compared with the group with low A1 index (A1 index 〈 130, 20 cases) (p=0.035). Hypermethylation of cyclin A1 promoter was detected in 4 out of 37 APL samples and the degree of CpG methylation varied from 8.6 to 85.1%. In Hela cells, 96% of sequence was methylated. No detectable genomic methylation was found in normal CD34+ cells. However, expression levels of cyclin A1 mRNA did not have significant association with detectable methylation in APL cells. (p=0.3) In summary, the present study demonstrated that increased expression of cyclin A1 mRNA in APL cells is a novel predictive factor for the development of RAS and an adverse prognostic indicator for APL in differentiation therapy by ATRA.

Description: Anaplastic large cell lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is an aggressive T cell lymphoma. CD30 is a characteristic cell surface protein overexpressed in ALCL cells as well as in malignant cells of Hodgkin lymphoma. CD30-mediated signals are molecular target(s) in the therapy for CD30-positive lymphoma. We studied molecular pathway(s) involved in the disease progression in two ALK- negative CD30-positive ALCL cell lines (N1, N2), which are recently established. In vitro culture, N1 and N2 represent early and advanced stage lymphoma phenotype, respectively. N2, compared to N1, was characterized by the increase of colony formation in soft agar more than 100 fold, the increase of cell surface expression of CD30 protein (p