Analysis of Factors Involved in Malignant Progression of Anaplastic Large Cell Lymphoma Kinase (ALK) -Negative Anaplastic Large Cell Lymphoma.
Authors: Nakamaki Tsuyoshi , Maeda Takashi , Nakashima Hidetoshi , Hattori Masamichi , Usui Takako , Tomoyasu Shigeru , Honma Yoshio 
Published: October 2019

Abstract
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.