Internet for Pharmaceutical and Biotech Communities
| Newsletter | Advertising |
 
 
 

  

Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 
   



 

Title:  Method of diagnosing, monitoring, and staging lung cancer
United States Patent: 
7,160,679
Issued: 
January 9, 2007

Inventors: 
Yang; Fei (San Diego, CA), Macina; Roberto A. (San Jose, CA), Sun; Yongming (San Jose, CA)
Assignee: 
diaDexus, Inc. (South San Francisco, CA)
Appl. No.: 
09/700,770
Filed: 
May 12, 1999
PCT Filed: 
May 12, 1999
PCT No.: 
PCT/US99/10344
371(c)(1),(2),(4) Date: 
January 16, 2001
PCT Pub. No.: 
WO99/60160
PCT Pub. Date: 
November 25, 1999


 

Pharm Bus Intell & Healthcare Studies


Abstract

The present invention provides a new method for detecting, diagnosing, monitoring, staging, and prognosticating lung cancer.

Description of the Invention

FIELD OF THE INVENTION

This invention relates, in part, to newly developed assays for detecting, diagnosing, monitoring, staging, and prognosticating cancers, particularly lung cancer.

BACKGROUND OF THE INVENTION

Primary lung cancer is divided into three main types including small cell lung cancer, non-small cell lung cancer, and mesothelioma. Small cell lung cancer is also called "Oat Cell" lung cancer because the cancer cells are a distinctive oat shape. There are three types of non-small cell lung cancer which are grouped together based upon similar behavior patterns and response to treatment which is different from small cell lung cancer. The three types of non-small cell lung cancer are squamous cell carcinoma, adenocarcinoma and large cell carcinoma. Squamous cell cancer is the most common type of lung cancer. It develops from the cells that line the airways. Adenocarcinoma also develops from the cells that line the airways, but it develops from a particular type of cell that produces mucus (phlegm). In large cell lung cancer, the cells appear large and rounded when viewed under a microscope. Mesothelioma is a rare type of cancer which affects the covering of the lung, the pleura. It is often caused by exposure to asbestos.

Secondary lung cancer is cancer that has started somewhere else in the body (for example, the breast or bowel) and spread to the lungs. The choice of treatment depends on where the cancer began. For example, cancer that has spread from the breast should respond to breast cancer treatments and cancer that has spread from the bowel should respond to bowel cancer treatments. The stage of a cancer provides information regarding how far a cancer has spread. Staging is important because treatment of the cancer is often decided based upon its stage. Staging is different for non-small cell versus small cell cancers of the lung.

Non-small cell cancer is divided into four stages. Stage I is very localized cancer with no cancer in the lymph nodes. In stage II, cancer has spread to the lymph nodes at the top of the affected lung. In stage III, cancer has spread near to where the cancer started. This can be to the chest wall, the covering of the lung (pleura), the middle of the chest (mediastinum) or other lymph nodes. Stage IV cancer has spread to another part of the body.

Small cell lung cancers are divided into two groups. This is because small cell lung cancer often spreads quite early. Even if spreading of the cancer is not visible on scans, it is likely that some cancer cells will have broken away and traveled through the bloodstream or lymph system. Accordingly, it is often preferred to treat small cell lung cancers as if they have spread, whether or not any secondary cancer is seen.

The two stages of small cell lung cancers are limited disease, that is cancer that can only be seen in one lung and in nearby lymph nodes, and extensive disease, that is cancer that has spread outside the lung to the chest or to other parts of the body. Because surgery is not usually used to treat small cell cancer, except in very early cases, the staging is not as important as it is with some other types of cancer. Chemotherapy with or without radiotherapy is usually preferred for treatment of small cell lung cancers. Initial scans and tests are used for comparison with later scans and test to see how well a patient is responding to treatment.

Procedures used for detecting, diagnosing, monitoring, staging and prognosticating lung cancer are of critical importance to the outcome of the patient. For example, patients diagnosed with early lung cancer generally have a much greater five-year survival rate as compared to the survival rate for patients diagnosed with distant metastasized lung cancer. New diagnostic methods which are more sensitive and specific for detecting early lung cancer are clearly needed.

Lung cancer patients are also closely monitored following initial therapy and during adjuvant therapy to determine response to therapy and to detect persistent or recurrent disease of metastasis. There is clearly a need for a lung cancer marker which is more sensitive and specific in detecting lung cancer recurrence.

Another important step in managing lung cancer is determination of the stage of the disease. Stage determination has potential prognostic value and provides criteria for designing optimal therapy. Generally, pathological staging of lung cancer is preferable over clinical staging because the former gives a more accurate prognosis. However, clinical staging would be preferred were it at least as accurate as pathological staging because it does not depend on an invasive procedure to obtain tissue for pathological evaluation. Staging of lung cancer would be improved by detecting new markers in cells, tissues or bodily fluids which could differentiate between different stages of invasion.

In the present invention, methods are provided for detecting, diagnosing, monitoring, staging and prognosticating lung cancer via six (6) Lung Specific Genes (LSGs). The six LSGs refer, among other things, to native proteins expressed by the genes comprising the polynucleotide sequences of any of SEQ ID NO: 1, 2, 3, 4, 5 or 6. In the alternative, what is meant by the six LSGs as used herein, means the native mRNAs encoded by the genes comprising any of the polynucleotide sequences of SEQ ID NO: 1, 2, 3, 4, 5 or 6 or levels of the genes comprising any of the polynucleotide sequences of SEQ ID NO: 1, 2, 3, 4, 5 or 6.

Other objects, features, advantages and aspects of the present invention will become apparent to those of skill in the art from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present invention to provide a method for diagnosing the presence of lung cancer in a patient which comprises measuring levels of LSG in a sample of cells, tissue or bodily fluid from the patient and comparing the measured levels of LSG with levels of LSG in preferably the same cells, tissue, or bodily fluid type of a control, wherein an increase in the measured LSG levels in the patient versus levels of LSG in the control is associated with lung cancer.

Another object of the present invention is to provide a method of diagnosing metastatic lung cancer in a patient which comprises measuring LSG levels in a sample of cells, tissue, or bodily fluid from the patient and comparing the measured LSG levels with levels of LSG in preferably the same cells, tissue, or bodily fluid type of a control, wherein an increase in measured LSG levels in the patient versus levels of LSG in the control is associated with a cancer which has metastasized.

Another object of the present invention is to provide a method of staging lung cancer in a patient which comprises identifying a patient having lung cancer, measuring levels of LSG in a sample of cells, tissues, or bodily fluid obtained from the patient, and comparing the measured LSG levels with levels of LSG in preferably the same cells, tissue or bodily fluid type of a control. An increase in measured LSG levels in the patient versus LSG levels in the control can be associated with a cancer which is progressing while a decrease or equivalent level of LSG measured in the patient versus the control can be associated with a cancer which is regressing or in remission.

Another object of the present invention is to provide a method of monitoring lung cancer in a patient for the onset of metastasis. The method comprises identifying a patient having lung cancer that is not known to have metastasized, periodically measuring levels of LSG in a sample of cells, tissues, or bodily fluid obtained from the patient, and comparing the measured LSG levels with levels of LSG in preferably the same cells, tissue, or bodily fluid type of a control, wherein an increase in measured LSG levels versus control LSG levels is associated with a cancer which has metastasized.

Yet another object of the present invention is to provide a method of monitoring the change in stage of lung cancer in a patient which comprises identifying a patient having lung cancer, periodically measuring levels of LSG in a sample of cells, tissue, or bodily fluid obtained from the patient, and comparing the measured LSG levels with levels of LSG in preferably the same cells, tissues, or bodily fluid type of a control wherein an increase in measured LSG levels versus the control LSG levels is associated with a cancer which is progressing and a decrease in the measured LSG levels versus the control LSG levels is associated with a cancer which is regressing or in remission.

Other objects, features, advantages and aspects of the present invention will become apparent to those of skill in the art from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.

DESCRIPTION OF THE INVENTION

The present invention relates to diagnostic assays and methods, both quantitative and qualitative for detecting, diagnosing, monitoring, staging, and prognosticating cancers by comparing levels of LSG with those of LSG in a normal human control. What is meant by "levels of LSG" as used herein, means levels of the native protein expressed by the gene comprising the polynucleotide sequence of any of SEQ ID NO: 1, 2, 3, 4, 5, or 6. In the alternative, what is meant by "levels of LSG" as used herein, means levels of the native mRNA encoded by the gene comprising any of the polynucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or levels of the gene comprising any of the polynucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6. Such levels are preferably measured in at least one of, cells, tissues and/or bodily fluids, including determination of normal and abnormal levels. Thus, for instance, a diagnostic assay in accordance with the invention for diagnosing over-expression of LSG protein compared to normal control bodily fluids, cells, or tissue samples may be used to diagnose the presence of cancers, including lung cancer. Any of the six LSGs may be measured alone in the methods of the invention, or all together or any combination of the six.

By "control" it is meant a human patient without cancer and/or non cancerous samples from the patient, also referred to herein as a normal human control; in the methods for diagnosing or monitoring for metastasis, control may also include samples from a human patient that is determined by reliable methods to have lung cancer which has not metastasized.

All the methods of the present invention may optionally include measuring the levels of other cancer markers as well as LSG. Other cancer markers, in addition to LSG, useful in the present invention will depend on the cancer being tested and are known to those of skill in the art.

Diagnostic Assays

The present invention provides methods for diagnosing the presence of lung cancer by analyzing for changes in levels of LSG in cells, tissues or bodily fluids compared with levels of LSG in cells, tissues or bodily fluids of preferably the same type from a normal human control, wherein an increase in levels of LSG in the patient versus the normal human control is associated with the presence of lung cancer.

Without limiting the instant invention, typically, for a quantitative diagnostic assay a positive result indicating the patient being tested has cancer is one in which cells, tissues, or bodily fluid levels of the cancer marker, such as LSG, are at least two times higher, and most preferably are at least five times higher, than in preferably the same cells, tissues, or bodily fluid of a normal human control.

The present invention also provides a method of diagnosing metastatic lung cancer in a patient having lung cancer which has not yet metastasized for the onset of metastasis. In the method of the present invention, a human cancer patient suspected of having lung cancer which may have metastasized (but which was not previously known to have metastasized) is identified. This is accomplished by a variety of means known to those of skill in the art. For example, in the case of lung cancer, patients are typically diagnosed with lung cancer following traditional detection methods.

In the present invention, determining the presence of LSG level in cells, tissues, or bodily fluid, is particularly useful for discriminating between lung cancer which has not metastasized and lung cancer which has metastasized. Existing techniques have difficulty discriminating between lung cancer which has metastasized and lung cancer which has not metastasized and proper treatment selection is often dependent upon such knowledge.

In the present invention, the cancer marker levels measured in such cells, tissues, or bodily fluid is LSG, and are compared with levels of LSG in preferably the same cells, tissue, or bodily fluid type of a normal human control. That is, if the cancer marker being observed is just LSG in serum, this level is preferably compared with the level of LSG in serum of a normal human patient. An increase in the LSG in the patient versus the normal human control is associated with lung cancer which has metastasized.

Without limiting the instant invention, typically, for a quantitative diagnostic assay a positive result indicating the cancer in the patient being tested or monitored has metastasized is one in which cells, tissues, or bodily fluid levels of the cancer marker, such as LSG, are at least two times higher, and most preferable are at least five times higher, than in preferably the same cells, tissues, or bodily fluid of a normal patient.

Staging

The invention also provides a method of staging lung cancer in a human patient.

The method comprises identifying a human patient having such cancer; analyzing a sample of cells, tissues, or bodily fluid from such patient for LSG. Then, the method compares LSG levels in such cells, tissues, or bodily fluid with levels of LSG in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in LSG levels in the patient versus the normal human control is associated with a cancer which is progressing and a decrease in the levels of LSG is associated with a cancer which is regressing or in remission.

Monitoring

Further provided is a method of monitoring lung cancer in a human having such cancer for the onset of metastasis. The method comprises identifying a human patient having such cancer that is not known to have metastasized; periodically analyzing a sample of cells, tissues, or bodily fluid from such patient for LSG; comparing the LSG levels in such cells, tissue, or bodily fluid with levels of LSG in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in LSG levels in the patient versus the normal human control is associated with a cancer which has metastasized.

Further provided by this inventions is a method of monitoring the change in stage of lung cancer in a human having such cancer. The method comprises identifying a human patient having such cancer; periodically analyzing a sample of cells, tissues, or bodily fluid from such patient for LSG; comparing the LSG levels in such cells, tissue, or bodily fluid with levels of LSG in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in LSG levels in the patient versus the normal human control is associated with a cancer which is progressing in stage and a decrease in the levels of LSG is associated with a cancer which is regressing in stage or in remission.

Monitoring such patient for onset of metastasis is periodic and preferably done on a quarterly basis. However, this may be more or less frequent depending on the cancer, the particular patient, and the stage of the cancer.

Assay Techniques

Assay techniques that can be used to determine levels of gene expression, such as LSG of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays, in situ hybridization says, competitive-binding assays, Western Blot analyses and ELISA assays. Among these, ELISAs are frequently preferred to diagnose a gene's expressed protein in biological fluids.

An ELISA assay initially comprises preparing an antibody, if not readily available from a commercial source, specific to LSG, preferably a monoclonal antibody. In addition a reporter antibody generally is prepared which binds specifically to LSG. The reporter antibody is attached to a detectable reagent such as radioactive, fluorescent or enzymatic reagent, for example horseradish peroxidase enzyme or alkaline phosphatase.

To carry out the ELISA, antibody specific to LSG is incubated on a solid support, e.g., a polystyrene dish, that binds the antibody. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the sample to be analyzed is incubated in the dish, during which time LSG binds to the specific antibody attached to the polystyrene dish. Unbound sample is washed out with buffer. A reporter antibody specifically directed to LSG and linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to LSG. Unattached reporter antibody is then washed out. Reagents for peroxidase activity, including a calorimetric substrate are then added to the dish. Immobilized peroxidase, linked to LSG antibodies, produces a colored reaction product. The amount of color developed in a given time period is proportional to the amount of LSG protein present in the sample. Quantitative results typically are obtained by reference to a standard curve.

A competition assay may be employed wherein antibodies specific to LSG attached to a solid support and labeled LSG and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of LSG in the sample.

Nucleic acid methods may be used to detect LSG mRNA as a marker for lung cancer. Polymerase chain reaction (PCR) and other nucleic acid methods, such as ligase chain reaction (LCR) and nucleic acid sequence based amplification (NASABA), can be used to detect malignant cells for diagnosis and monitoring of various malignancies. For example, reverse-transcriptase PCR (RT-PCR) is a powerful technique which can be used to detect the presence of a specific mRNA population in a complex mixture of thousands of other mRNA species. In RT-PCR, an mRNA species is first reverse transcribed to complementary DNA (cDNA) with use of the enzyme reverse transcriptase; the cDNA is then amplified as in a standard PCR reaction. RT-PCR can thus reveal by amplification the presence of a single species of mRNA. Accordingly, if the mRNA is highly specific for the cell that produces it, RT-PCR can be used to identify the presence of a specific type of cell.

Hybridization to clones or oligonucleotides arrayed on a solid support (i.e., gridding) can be used to both detect the expression of and quantitate the level of expression of that gene. In this approach, a cDNA encoding the LSG gene is fixed to a substrate. The substrate may be of any suitable type including but not limited to glass, nitrocellulose, nylon or plastic. At least a portion of the DNA encoding the LSG gene is attached to the substrate and then incubated with the analyte, which may be RNA or a complementary DNA (cDNA) copy of the RNA, isolated from the tissue of interest. Hybridization between the substrate bound DNA and the analyte can be detected and quantitated by several means including but not limited to radioactive labeling or fluorescence labeling of the analyte or a secondary molecule designed to detect the hybrid. Quantitation of the level of gene expression can be done by comparison of the intensity of the signal from the analyte compared with that determined from known standards. The standards can be obtained by in vitro transcription of the target gene, quantitating the yield, and then using that material to generate a standard curve.

The above tests can be carried out on samples derived from a variety of patients' cells, bodily fluids and/or tissue extracts (homogenates or solubilized tissue) such as from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, urine, saliva, or any other bodily secretion or derivative thereof. Blood can include whole blood, plasma, serum, or any derivative of blood.
 


Claim 1 of 17 Claims

1. A method for detecting the presence of lung cancer in a patient comprising: (a) measuring levels of a polynucleotide comprising SEQ ID NO: 2, 4, 5 or 6 in a sample of cells, tissue or bodily fluid obtained from the patient; and (b) comparing the measured levels of the polynucleotide in the patient with levels of the polynucleotide in a sample of cells, tissue or bodily fluid obtained from a normal control, wherein an increase in levels of the polynucleotide in the patient as compared to levels of the polynucleotide in the normal control is associated with the presence of lung cancer.

 

____________________________________________
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.

 

 

     
[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]