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Title:  Gene markers for lung cancer
United States Patent: 
7,214,781
Issued: 
May 8, 2007

Inventors: 
Mitsuhashi; Masato (Irvine, CA), Matsunaga; Hiroko (Kodaira, JP), Kambara; Hideki (Tokyo, JP), Kawamura; Masafumi (Tokyo, JP)
Assignee:
 Hitachi Chemical Research Center, Inc. (Irvine, CA), Hitachi Chemical Co., Ltd. (Tokyo, JP)
Appl. No.: 
10/297,277
Filed: 
June 21, 2001
PCT Filed: 
June 21, 2001
PCT No.: 
PCT/US01/19980
371(c)(1),(2),(4) Date: 
April 04, 2003
PCT Pub. No.: 
WO01/98539
PCT Pub. Date: 
December 27, 2001


 

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Abstract

A method for the diagnosis and identification of new or residual lung cancer is disclosed which uses newly identified markers for lung cancer including syndecan 1, collagen 1 alpha 2, and two novel proteins, 7013 and 7018. The method involves identification of  the lung cancer markers is blood from a patient. It is envisioned that at least one marker may be used or any mixture of the four. The method may also include the identification of cytokeratin-19.

Description of the Invention

FIELD OF THE INVENTION

The invention relates to a method for the diagnosis and identification of residual lung cancer. The invention further relates to the use of newly identified cellular markers for lung cancer. These markers include syndecan 1, collagen 1 alpha 2, and two novel proteins, 7013 and 7018.

BACKGROUND OF THE INVENTION

Lung cancer is one of the most common cancers in industrial nations and has an extremely high mortality rate. Early diagnosis and effective treatments are not available at this time. A chest X-ray is frequently used for lung cancer screening, however, it is not useful for the detection of early stage cancer. CT scans may also be used and may allow detection of the cancer at an earlier stage, however, this procedure takes time and has a risk of exposure.

Cancer cells or micrometastases are frequently detected in the blood stream of patients with melanomas, thyroid cancers, and prostate cancers. Currently reverse transcription-based polymerase chain reaction (RT-PCR) is a powerful method capable of detecting a single cancer cell within millions of normal blood cells by amplifying a cancer specific gene or marker. This makes RT-PCR detection of micrometastases a promising diagnostic procedure for the prognosis, choice of appropriate treatments, and monitoring of the efficacy of each treatment. Furthermore, blood tests do not induce any health hazards, whereas methods such as X-ray or CT scan do. In addition, blood tests cause very minor physical discomfort as compared to endoscopic examination and biopsy. Lung cancer frequently induces blood-born metastasis even in the early stages--before symptomatic disease and many lung cancers relapse as distant metastases, such as in brain, bone, and liver. This is due to the lung cancer induced blood-borne metastasis. This knowledge can be used, however, because it suggests that the diagnosis and detection of relapse could be made on the basis of these blood borne metastases at a very early stage. However, there are no good markers available for the identification of the metastatic lung cancers cells in the blood. Currently, lung cancer markers such as cytokeratin-19 and CEA are used for the diagnosis of non-small cell lung cancer by RT-PCR (reverse transcriptase polymerase chain reaction) but lack specificity, and result in a high number of false positives and negatives.

RT-PCR of micrometastases, then, is especially advantageous for the detection of lung cancer due to the large patient population, high incidence of blood-borne metastasis, poor prognosis, and high medical cost for advanced cancer treatments. In addition, specific antibodies are not available as of yet for lung cancer.

Therefore, specific markers and a method of diagnosis of lung cancer by detecting blood-borne metastasis is needed.

SUMMARY OF THE INVENTION

One embodiment is a method for the identification of lung cancer by isolating blood or non-lung tissue from a patient, and identifying the presence of at least one marker from the following: syndecan 1, collagen 1 alpha 2, 7013, and 7018. The method may also include identifying the presence of the marker cytokeratin-19. In a further embodiment at least two markers are identified. In a further embodiment more than two markers are identified. The method of identification may be any known to one of skill in the art, but may also include RT-PCR and/or antibody binding. The patient may be any living thing, but in one embodiment is a mammal, particularly a human, dog, or cat.

A further embodiment is a method for the isolation or removal of metastatic cancer cells, by treating cells or a non-lung tissue containing cancer cells with antibodies specific for at least one marker selected from the group consisting of: syndecan 1, collagen 1 alpha 2, 7013, and 7018. The method may also include antibodies specific for the marker cytokeratin-18. In one embodiment, the antibodies are bound to a moiety selected from the group consisting of metallic particles, fluorescent particles, chromatography beads, a chromatography gel and a solid support. In a further embodiment, two markers are used. In a further embodiment more than two markers are used.

A further embodiment is a polynucleotide comprising at least 17 nucleotides of SEQ ID NO: 16 (deposited as ATCC PTA-3471, Jun. 21, 2001) also identified herein as marker 7013. A further embodiment is at least 17 nucleotides of the polynucleotide deposited as ATCC PTA-3473, Jun. 21, 2001.

A further embodiment is a polynucleotide comprising at least 17 nucleotides of SEQ ID NO: 17 (deposited as ATCC PTA-3472, Jun. 21, 2001) also identified herein as marker 7018.

A further embodiment is a method for the identification of metastases of a solid tumor in a patient by, isolating blood or bone marrow from said patient; and identifying the presence of at least one marker selected from the group consisting of: syndecan 1, collagen 1 alpha 2, 7013, and 7018. The method may also include identifying cytokeratin-18. In one embodiment, the solid tumor is selected from the group consisting of bile duct, colon, breast, uterus, esophagus, and larynx.

A further embodiment is a method for the identification of a carcinoma, by obtaining a cancer cell; and identifying the presence of the markers selected from the group consisting of: 7013, 7018, and both.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Differential display was used to identify mRNA's specifically expressed by lung cancer cells which circulate in the blood and are not expressed by normal blood cells. The differential display method was modified from the classical procedure to allow a more comprehensive representation of the genes expressed in a cell.

In order to identify candidate genes, three major technologies are presently available: subtraction libraries, differential display, and DNA microchip arrays. However, subtraction libraries, though being potentially useful, may also identify genes which exhibit partial similarity in gene sequences and is a very laborious and time intensive technique. DNA microchip arrays, though quick and easy to do, are not sensitive enough to detect rare genes in blood samples. This is particularly important because the method needs to be able to detect rare micrometastases which express specific markers within a large background (millions of white blood cells) of normal cells which do not. Differential display (dd-PCR) allows for the amplification of all existing genes using combinations of multiple degenerate primers, thus increasing the likelihood that a rare marker or gene will be detected.

However, in order to improve the differential display technology to represent as many genes as possible, selective Amplified Fragment Length Polymorphism (s-AFLP) was used in Example 1. This approach has the advantages of 1) amplifying only the 3' end of the gene, 2) producing more reproducible gel patterns, 3) identifying fewer redundant genes, and 4) using more selective PCR conditions. The method resulted in the identification of 4 markers: Synd, Col, 7013 and 7018.

These markers may be used to identify lung cancer cells in the blood of a patient at any stage of disease. Any method known to one of skill in the art may be used to identify the markers in the blood or in any metastatic tissue. For example, the mRNA expressed by the cells which is specific to the markers may be identified.

Alternatively, the proteins themselves may be identified using immuno-techniques, such as Western blot, FACS technology, ELISA, and other methods known to one of skill in the art. The antibodies or functional antibody parts may be purchased, isolated, or produced using known methods.

In one embodiment, the gene expression of the marker is identified. Any method which allows for identification of expression of the gene associated with the marker can be used. Typically, the method amplifies the mRNA resulting from expression of the gene. In one embodiment, RT-PCR of the mRNA from blood or tissue is used. In a further embodiment, antibodies are used to identify cells in the blood containing these markers. For example, cell sorting can be used to identify cells which have been fluorescently labeled with antibodies to these markers.

In one embodiment, the method is used to identify the presence of lung cancer cells in the blood or bone marrow. However, it can be envisioned that mRNA from any tissue which does not normally produce these markers may be used. For example, the mRNA from an organ which is typically the site of metastases can be used. Therefore, in a further embodiment, the method is used to identify lung cancer cells in an organ such as liver or brain.

In one embodiment, the method is used to identify the presence of lung cancer cells at a very early stage in the disease, in a further embodiment, the lung cancer cells are identified after remission or to identify a relapse.

In a further embodiment, the markers are used to target the lung cancer cells in vivo or in vitro. In one embodiment, the body is cleared of cancer cells using affinity techniques which allow the cancer cells to be targeted and removed from the blood.

In a further embodiment, the markers are used to identify metastatic cells in the blood or bone marrow or a metastatic organ or tissue which are produced by such cancers as bile duct, colon, breast, uterus, esophagus, and larynx. The markers may alternatively be used to remove the metastatic cells from blood.

In a further embodiment, the markers, 7013 and 7018 are used to identify whether a cancer cell or other cell is of epithelial origin. For example, if the cell expresses one or both markers, it is likely of epithelial origin.

The patient may be any animal which is capable of having cancer. In one embodiment, the patient is a mammal. In a further embodiment, the mammal is a pet, such as a dog or cat. In a further embodiment, the patient is a human.
 


Claim 1 of 11 Claims

1. An isolated or purified polynucleotide consisting of SEQ ID NO: 16.
 

 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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