Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2001-04-09
2004-02-24
Chan, Christina (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
Reexamination Certificate
active
06696548
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to the field of molecular genetics of cancer. Specifically, the present invention relates to human lymphomas in which a translocation between chromosomes 2 and 5 (referred to in the art as “t(2;5)”) has occurred. On a molecular level, the DNA rearrangement in t(2;5) results in the fusion of the known NPM gene with a novel gene named ALK (Anaplastic Lymphoma Kinase) that encodes a protein tyrosine kinase (PTK).
2. Related Art
Chromosomal abnormalities are frequently associated with malignant diseases. In a number of instances, specific chromosomal translocations have been characterized, which generate fusion genes encoding proteins with oncogenic properties (Sawyers et al.,
Cell
64:337-350 (1991)). Perhaps the best example of genetic characterization of a malignant disease is provided by the analysis of the chromosomal abnormalities unique to different subsets of non-Hodgkin's lymphoma (NHL). The NHL subset commonly referred to as large cell lymphoma (which comprises ~25% and 40% of NHL in children and adults, respectively) has historically been the most ill-defined because of its marked cytological, immunological and clinical heterogeneity.
Approximately one-third of large cell lymphomas (10% of all NHL) contain the t(2;5)(p23;q35), usually as the only cytogenetic abnormality (R. Rimokh et al.,
Br. J. Haematol.
71:31-36 (1989); D. Mason et al.,
Br. J. Haematol.
74:161-168 (1990); H. Stein and F. Dallenbach, in
Neoplastic Hematopathology,
D. M. Knowles, ed., Williams & Wilkins, Baltimore (1992), pp.675-714), suggesting that rearrangement of cellular proto-oncogenes on these chromosomes contributes to lymphomagenesis.
The majority oft(2;5)-positive lymphomas (70-75%) express T-lymphoid markers, although usually in the aberrant, incomplete fashion characteristic of T-cell malignancies (e.g., preservation of CD2 and CD4 with infrequent expression of CD3) (M. E. Kadin,
J. Clin. Oncol.
12:884-887 (1994); J. T. Sandlund et al.,
Blood
84:2467-2471 (1994)). Less commonly, these neoplasms bear B-cell markers (~15%) or have a null phenotype with neither B- nor T-antigen expression (10%). Lymphomas with the t(2;5) typically involve lymph nodes, skin, lung, soft tissue, bone and the gastrointestinal tract, and arise predominantly from activated T lymphocytes (Y. Kaneko et al.,
Blood
73:806-813 (1989); M. M. Le Beau et al.,
Leukemia
3:866-870 (1989); R. Rimokh et al.,
Br. J. Haematol.
71:31-36 (1989); D. Y. Mason et al.,
Br. J. Haematol.
74:161-168 (1990); M. A. Bitter
Am. J. Surg. Pathol.
14:305-316 (1990); M. E. Kadin,
J. Clin. Oncol.
9:533-536 (1991); J. P. Greer et al.,
J. Clin. Oncol.
9:539-547 (1991); V. Vecchi et al.,
Med. Pediatr. Oncol.
21:402-410 (1993)). The malignant cells express IL-2 receptors and CD30 (Ki-1) antigen, a receptor for a newly described member of the tumor necrosis factor ligand family (H. Durkop et al.,
Cell
68:421-427 (1992); C. A. Smith et al.,
Cell
73:1349-1360 (1993)). By the updated Kiel lymphoma classification, most tumors with the t(2;5) are classified as anaplastic large cell non-Hodgkin's lymphomas (A. G. Stansfeld et al.,
Lancet
1:292-293 (1988)). These tumors typically behave as aggressive, high-grade NHL with most patients having advanced stage disease at presentation. From 30% to 40% of patients eventually succumb to their disease despite aggressive therapeutic intervention.
SUMMARY OF THE INVENTION
Disclosed herein is the cloning and sequencing of human nucleic acid sequences which are rearranged in the t(2;5)(p23;q35) chromosomal translocation event which occurs in human t(2;5) lymphoma. The rearrangement was found to bring sequences from the nucleolar phosphoprotein gene (the NPM gene) on chromosome 5 q35 to those from a previously unidentified protein tyrosine kinase (PTK) gene (the ALK gene) on chromosome 2p23. The sequence of the novel ALK gene and ALK protein, as well as the sequence of the t(2;5) fusion gene and fusion protein (the NPM/ALK gene and NPM/ALK protein, respectively), are disclosed herein.
The NPM gene encodes a highly conserved nonribosomal RNA-binding protein that shuttles ribosomal ribonucleoproteins (rRNPs) between the nucleolus and the cytoplasm; rRNPs associate with NPM in the nucleolus, are carried to the cytoplasm, and are released at the maturing ribosomes (M. S. Schmidt-Zachmann et al.,
EMBO J.
6:1881-1890 (1987); M. S. Schmidt-Zachmann et al.,
Chromosoma.
96:417-426(1988)). Bidirectional movement of NPM between the nucleolus and cytoplasm has been elegantly demonstrated in studies monitoring the equilibration of the protein between nuclei present in chicken-mouse heterokaryons (R. A. Borer et al.,
Cell
56:379-390 (1989)). Several groups have shown that NPM can exist in the cell as either a monomer or a homo-oligomeric hexamer, associated in a head-to-head/tail-to-tail fashion; it is not clear which form of the protein moves between the nucleolus and cytoplasm (M. S. Schmidt-Zachmann et al.,
Chromosoma.
96:417-426 (1988); B.Y. Yung et al.,
Biochim. Biophys. Acta.
925:74-82 (1989); Q. R. Liu et al.,
Eur. J. Biochem.
200:715-721 (1991)).
The NPM/ALK fusion gene was initially identified in anaplastic large cell lymphomas. However, its presence has since been observed in a significant number of diffuse and immunoblastic large cell cases as well. Expression of the NPM/ALK fusion gene in lymphoid cell lines which are otherwise dependent on IL-3 for growth results in transformed cells which proliferate in an IL-3-independent manner. This result suggests a means by which the NPM/ALK fusion acts to promote tumorigenesis and provides for methods of culturing lymphoid cells in vitro in the absence of IL-3.
Utilizing the sequences of the NPM/ALK fusion gene, the present invention provides methods of identifying the presence of nucleic acids containing the NPA/ALK fusion by means such as nucleic acid hybridization and detection methods (e.g., “Southems,” “Northerns” and the like) fluorescence in situ hybridization (FISH) and detection methods, or polymerase chain reaction (PCR) amplification and detection methods. Such methods can be used in, inter alia, to determine if particular cells or tissues express ALK or NPM/ALK coding sequences, or diagnostic assays designed to determine, for example, if a mammal has cancer or a genetic predisposition to (i.e., is at an increased risk of developing) cancer.
Detection methods utilizing the ALK sequences of the invention as probes are further used to isolate and clone ALK sequences and genes from a variety of mammalian species, and the ALK sequences and genes so prepared provide the foundation for further embodiments of the invention. For example, a “Southern” assay is used to identify clones from a library of murine cDNA sequences using human ALKDNA sequences as a radiolabeled probe. These ALK-positive clones are isolated, and the nucleotide sequence of the mouse cDNA inserted into the vector in these clones is determined using any standard method of nucleotide sequencing known to those of skill in the art. The human and murine ALK nucleotide sequences are aligned by computer program or by hand, and regions of identical or, at least well-conserved, nucleotide sequence between the ALK genes of the two species are identified. These identical/conserved sequences are used to design oligonucleotides which function as ALK-specific primers to be used in PCR amplifications in which the template DNA is genomic DNA or cDNA from a third mammal (i.e., one which is neither a mouse nor a human) in order to obtain ALK DNA, that can be cloned and sequenced, from said third mammal. Alternatively, human or murine ALK sequences, or oligonucleotides derived therefrom, are labeled and used as probes to identify ALK-positive clones in libraries prepared from genomic DNA or cDNA from a third mammal. In like fashion, ALK sequences from any mammal can be prepared and tested for its use in any appropriate embodiment of the present invention. Furthermore, cDNAs derived from a
Look A. Thomas
Morris Stephan W.
Belyavskyi Michail
Chan Christina
St. Jude Children's Research Hospital
Sterne Kessler Goldstein & Fox PLLC
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