Compositions for detecting mutant anaplastic lymphoma kinase in lung cancer

Patent metadata

Field	Value
Publication number	US-9988688-B2
Application number	US-201514870154-A
Country	US
Kind code	B2
Filing date	Sep 30, 2015
Priority date	Apr 14, 2006
Publication date	Jun 5, 2018
Grant date	Jun 5, 2018

Abstract

Novel gene deletions and translocations involving chromosome 2 resulting in fusion proteins combining part of Anaplastic Lymphoma Kinase (ALK) kinase with part of a secondary protein have been identified herein in human solid tumors, e.g. non-small cell lung carcinoma (NSCLC). Secondary proteins include Echinoderm Microtubule-Associated Protein-Like 4 (EML-4) and TRK-Fusion Gene (TFG). The EML4-ALK fusion protein, which retains ALK tyrosine kinase activity, was confirmed to drive the proliferation and survival of NSCLC characterized by this mutation. The invention therefore provides, in part, isolated polynucleotides and vectors encoding the disclosed mutant ALK kinase polypeptides, probes for detecting it, isolated mutant polypeptides, recombinant polypeptides, and reagents for detecting the fusion and truncated polypeptides. The disclosed identification of this new fusion protein enables methods for screening for compounds that inhibit the proteins, and methods for inhibiting the progression of a cancer characterized by the mutant polynucleotides or polypeptides.

First claim

What is claimed is:

1. A composition, comprising a nucleic acid reagent, a DNA polymerase, and a biological sample from a human having lung cancer, wherein the nucleic acid reagent comprises a primer pair that hybridizes to a polynucleotide encoding an Echinoderm Microtubule-Associated Protein-Like 4 (EML4)-Anaplastic Lymphoma Kinase (ALK) fusion polypeptide, wherein the EML4-ALK fusion polypeptide comprises SEQ ID NO: 1 or SEQ ID NO: 18, and wherein the primer pair amplifies at least a fragment of said polynucleotide comprising the fusion junction.

2. The composition of claim 1 , wherein said lung cancer is non-small cell lung cancer (NSCLC).

3. The composition of claim 1 , wherein said biological sample is a serum, pleural effusion, or tissue biopsy sample.

4. The composition of claim 1 , wherein the polynucleotide is mRNA.

5. The composition of claim 1 , wherein one primer of the primer pair hybridizes to exon 6 of an EML4 gene.

6. The composition of claim 1 , wherein one primer of the primer pair hybridizes to exon 13 of an EML4 gene.

7. The composition of claim 1 , wherein one primer of the primer pair hybridizes to exon 20 of an ALK gene.

8. A composition comprising fluorescent labeled break-apart probes that are specific to an ALK locus for use in a fluorescence in situ hybridization (FISH) assay, and a biological sample from a human having lung cancer, wherein the break apart probes hybridize to genomic DNAs on opposite sides of the breakpoint of a wild type ALK gene, wherein the coding DNA sequence of the wild type ALK gene comprises SEQ ID NO: 6, and wherein the breakpoint corresponds to nucleotide 3171 of SEQ ID NO: 6.

9. The composition of claim 8 , wherein the sample is a formalin-fixed, paraffin-embedded lung tissue sample comprising tumor cells.

10. The composition of claim 8 , wherein said lung cancer is non-small cell lung cancer (NSCLC).

11. The composition of claim 8 , wherein said biological sample is a serum, pleural effusion, or tissue biopsy sample.

12. A composition, comprising a nucleic acid reagent, a DNA polymerase, and a biological sample from a human having lung cancer, wherein the nucleic acid reagent comprises a primer pair that hybridizes to
(i) a region of a polynucleotide encoding a polypeptide with ALK kinase activity, wherein the region consists of a nucleotide sequence coding for amino acids 234-796 of SEQ ID NO: 1; or
(ii) nucleotides 701-2391 of SEQ ID NO: 2.

13. The composition of claim 12 , wherein said lung cancer is non-small cell lung cancer (NSCLC).

14. The composition of claim 12 , wherein said biological sample is a serum, pleural effusion, or tissue biopsy sample.

15. The composition of claim 12 , wherein the polynucleotide is mRNA.

Assignees

• Cell Signaling Technology Inc

Inventors

• Rikova Klarisa
• Haack Herbert
• Sullivan Laura
• Guo Ailan
• Possemato Anthony
• Macneill Joan
• Gu Ting-Lei
• Yu Jian

Classifications

• G01N33/6893

  related to diseases not provided for elsewhere · CPC title

• C12N9/12

  transferring phosphorus containing groups, e.g. kinases (2.7) · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q2600/112

  Disease subtyping, staging or classification · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title