ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin's lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK)

Anaplastic Lymphoma Kinase (ALK) was originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases that acquires transforming capability when truncated and fused to nucleophosmin (NPM) in the t(2;5) chromosomal rearrangement associated with non-Hodgkin's lymphoma, but further insights into its normal structure and function are lacking. Here, we characterize a full-length normal human ALK cDNA and its product, and determine the pattern of expression of its murine homologue in embryonic and adult tissues as a first step toward the functional assessment of the receptor. Analysis of the 6226 bp ALK cDNA identified an open reading frame encoding a 1620-amino acid (aa) protein of predicted mass approximately 177 kDa that is most closely related to leukocyte tyrosine kinase (LTK), the two exhibiting 57% aa identity and 71% similarity over their region of overlap. Biochemical analysis demonstrated that the approximately 177 kDa ALK polypeptide core undergoes co-translational N-linked glycosylation, emerging in its mature form as a 200 kDa single chain receptor. Surface labeling studies indicated that the 200 kDa glycoprotein is exposed at the cell membrane, consistent with the prediction that ALK serves as the receptor for an unidentified ligand(s). In situ hybridization studies revealed Alk expression beginning on embryonic day 11 and persisting into the neonatal and adult periods of development. Alk transcripts were confined to the nervous system and included several thalamic and hypothalamic nuclei; the trigeminal, facial, and acoustic cranial ganglia; the anterior horns of the spinal cord in the region of the developing motor neurons; the sympathetic chain; and the ganglion cells of the gut. Thus, ALK is a novel orphan receptor tyrosine kinase that appears to play an important role in the normal development and function of the nervous system.

Detection of the t(2;5)(p23;q35) and NPM-ALK fusion in non-Hodgkin's lymphoma by two-color fluorescence in situ hybridization.

The non-Hodgkin's lymphoma (NHL) subset commonly referred to as large cell lymphoma (LCL) has historically been characterized by it's marked cytological, immunological, and clinical heterogeneity. One potential defining feature of these lymphomas, the t(2;5)(p23;q35), occurs in 25% to 30% of anaplastic LCLs and is also found in cases with diffuse large cell or immunoblastic morphology. We recently identified nucleophosmin (NPM) and anaplastic lymphoma kinase (ALK) as the genes on chromosomes 5 and 2, respectively, that are juxtaposed by this translocation. To provide a complementary approach to the use of classical cytogenetics or polymerase chain reaction-based methods for the detection of this abnormality, we have developed a two-color fluorescent in situ hybridization (FISH) assay for the t(2;5) that may be used for the analysis of both interphase nuclei and metaphase chromosomes. Three overlapping chromosome 5 cosmid clones located immediately centromeric to the NPM gene locus and an ALK P1 clone located telomeric to the chromosome 2 breakpoint were labeled with digoxigenin or biotin, respectively, and used to visualize the derivative chromosome 5 produced by the t(2;5), evident as juxtaposed or overlapping red and green fluorescent signals. This NPM-ALK FISH assay was initially validated by analysis of a series of cytogenetically characterized cell lines, with the presence of the der(5) chromosome showed specifically only in those lines known to contain the t(2;5). The assay was then applied in a blinded fashion to a series of eight cytogenetically t(2;5)-positive clinical specimens and seven known t(2;5)-negative cases, including three NHL and four Hodgkin's disease biopsy samples. Whereas the t(2;5)-negative cases were negative by FISH, all eight t(2;5)-positive cases were positive. One additional case, initially thought to be positive for the translocation by cytogenetics, was proven to not be a classic t(2;5) by interphase and metaphase FISH. These data indicate that the FISH assay described is a highly specific and rapid test that should prove to be a useful adjunct to the currently available methods for detection of the t(2;5).

Isolation and characterization of the human nucleophosmin/B23 (NPM) gene: identification of the YY1 binding site at the 5' enhancer region.

NPM (nucleophosmin/B23) is a major nucleolar protein which is 20 times more abundant in tumor or proliferating cells than in normal resting cells. Recently, it was found that NPM gene is located at the breakpoints of the t(2:5), t(3:5) and t(5:17) chromosome translocation. To understand the human NPM gene's structure and regulation, four genomic clones were isolated from the human chromosome 5 library and their DNA sequences analyzed. The human NPM gene has 12 exons of sizes ranging from 58 to 358 bp. The chromosome breakpoint for t(2:5) and t(5:17) translocation is within intron 4 and the breakpoint for t(3:5) translocation is within intron 6. The initiation site is located 96 bp upstream from the ATG site. A typical TATA box (at -25 nt) and a GC box (at -65 nt) were identified in the promoter region. We identified two gel-shift bands (A and B) with DNA fragment E (-741/-250 nt) by EMSA. A DNA footprint was observed at (-371/-344 nt) with the nuclear extract. A double stranded DNA with the footprint sequence (-371/-344 nt) competed the formation of gel-shift bands A and B in EMSA suggesting that proteins A and B bind to the footprint region. We confirmed that protein A is transcription factor YY1. These results suggest that YY1 may play a role in NPM gene expression. This is the first report on human NPM gene structure and sequence.