JP3524061B2 - Novel way to diagnose, monitor, stage, image and treat lung cancer - Google Patents

Description

Detailed Description of the Invention

[0001] TECHNICAL FIELD The present invention to which the invention pertains, in part, cancer, especially detection of lung cancer, diagnosis,
It relates to newly developed assays for monitoring, staging, prognosis, imaging and treatment. BACKGROUND OF THE INVENTION Lung cancer is the second most common type of cancer in the United States in both sexes and is the most common cause of cancer death in both sexes. Lung cancer can result from a primary tumor of lung origin or a secondary tumor that has spread from other organs such as the intestine or breast.
Primary lung cancer is roughly classified into three types: 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 have a unique oat shape. There are three types of non-small cell lung cancer, but they are grouped together because they behave similarly and have a different response to treatment than small cell lung cancer. These three types are squamous cell carcinoma, adenocarcinoma, and large cell cancer. Squamous cell carcinoma is the most common type of lung cancer. It arises from the cells that line the airways. Adenocarcinoma also begins in the cells that line the airways. However, adenocarcinoma arises from a special type of cell that produces mucus, phlegm. Large cell lung cancer is so named because the cells look like a large circle when viewed under a microscope. Mesothelioma is a rare type of cancer in which there is a lesion in the lining of the lung called the pleura. Mesothelioma is often caused by asbestos exposure.

Secondary lung cancer is a cancer that originates elsewhere in the body (eg breast or intestine) and has spread to the lungs. The choice of treatment for secondary lung cancer depends on where the cancer originated. In other words, cancer that has spread from the breast should respond to treatment of breast cancer, and cancer that spread from the intestine should respond to treatment of intestinal cancer.

The stage of cancer indicates how far the cancer has spread. Staging is important because treatment is often determined according to the stage of the cancer. The staging of non-small cell and small cell lung cancer is different.

Non-small cell cancer can be divided into four stages. Stage I is a highly localized cancer with no lymph node cancer. Stage II cancer has spread to the lymph nodes above the diseased lung. Stage III cancer has spread around the area where the cancer started. It can be the chest wall, the lung envelope (pleura), the middle part of the lung (mediastinum), or other lymph nodes. Stage IV cancer has spread to other parts of the body.

Since small cell lung cancer can spread very early in disease development, it can only be classified into two groups. That is, localized disease in which the cancer is found only in one lung and nearby lymph nodes; and spread disease in which the cancer has spread out of the lung and has spread to the chest or other parts of the body. Furthermore, some cancer cells may pop out and migrate through the bloodstream or lymphatic system, even if the spread is not apparent on the scan. Therefore, for the sake of safety, it is preferable to treat the small cell lung cancer as if the secondary cancer is invisible and has spread. Surgery is not commonly used to treat small cell cancers, except in very early cases, so staging is less important than other types of cancer. Chemotherapy with or without radiation therapy is often used. The scans and exams initially performed will be used at a later date to see how well the patient is responding to the procedure.

[0006] In WO98 / 56951 (published on December 17, 1998), adjacent and partially overlapping cDNA
Disclosed is a set of A sequences and polypeptides encoded thereby designated LS170. It is suggested that these sequences are useful for detection, diagnosis, staging, monitoring, prognosis, in vivo imaging, prevention or treatment, and determining an individual's predisposition to lung diseases and conditions such as lung cancer. Has been done. The disclosure of WO98 / 56951 teaches that the LS170-specific polynucleotide has at least 50% identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 1-9. SEQ ID NO: 1 taught in WO98 / 56951 is
It overlaps with the LSG of SEQ ID NO: 4 used in the present invention.

The present invention provides a method for detecting, diagnosing, monitoring, staging, predicting prognosis, in vivo imaging and treating lung cancer by using 5 kinds (5) of lung-specific genes (LSG). The five LSGs specifically refer to native proteins expressed by a gene comprising the polynucleotide sequence of any of SEQ ID NOs: 1, 2, 3, 4, or 5. Alternatively, as used herein, 5 LSGs refer to natural mRNAs encoded by a gene comprising the polynucleotide sequence of any of SEQ ID NOs: 1, 2, 3, 4, or 5, or SEQ ID NOs: 1, 2, 3, 4,
Alternatively, it may refer to the actual gene comprising any of the polynucleotide sequences of 5.

The methods used in the detection, diagnosis, monitoring, staging, and prognosis of lung cancer have significant implications for patient outcome. For example, the 5-year survival rate for patients diagnosed with early stage lung cancer is generally much greater than the survival rate for patients diagnosed with distant metastatic lung cancer. There is a clear need for newer, more sensitive and specific diagnostic methods for early lung cancer detection.

Patients with lung cancer are closely monitored after initial therapy and during adjunct therapy to determine response to therapy and to detect persistent metastatic or recurrent disease. Clearly, there is a need for more sensitive and specific lung cancer markers for detecting lung cancer, its recurrence and progression.

Another important step in the management of lung cancer is the staging of the patient's disease. Staging has prognostic value in prognosis and provides the basis for designing optimal therapy. Pathological staging of lung cancer is generally preferred over clinical staging. The former provides a more accurate prognosis. However, it would be preferable if clinical staging was at least as accurate as pathological staging. This is because clinical staging does not rely on invasive procedures to obtain the tissues required for pathological evaluation. Staging of lung cancer will be improved by detecting novel markers in cells, tissues, or body fluids that can distinguish different stages of invasiveness.

Other objects, features, advantages and aspects of the present invention will be apparent to those of skill in the art from the following description. It should be understood, however, that the following description and specific examples, while indicating preferred embodiments of the present invention, are provided for purposes of illustration only. Various modifications within the scope of the disclosed invention will be readily apparent to those skilled in the art from reading the following description as well as other parts of the disclosure. SUMMARY OF THE INVENTION To these and other ends, it is an object of the present invention to provide a method of diagnosing the presence of lung cancer.
The method determines the change in LSG concentration in cells, tissues or body fluids, preferably in the same type of cells, tissues of a normal human control,
Alternatively, the presence of lung cancer is diagnosed by analysis in comparison to LSG concentration in body fluids, where an increase in LSG concentration in a patient relative to a normal human control is associated with lung cancer.

The present invention further provides a method for diagnosing metastatic lung cancer in a patient having a cancer that has not been found to have metastasized. The method identifies human patients suspected of having metastasized lung cancer; cells of such patients,
A tissue or body fluid sample is analyzed for LSG; the concentration of LSG in such cells, tissue or body fluid is preferably the same type of cells, tissue or body fluid of a normal human control.
As compared to G levels, increased LSG levels in patients relative to normal human controls are associated with metastasized lung cancer.

The present invention further provides a method for staging lung cancer in a human having lung cancer. The method identifies a human patient with such cancer; analyzes a cell, tissue, or body fluid sample of such patient for LSG; normalizes LSG levels in such cell, tissue, or body fluid. An increase in LSG concentration in a patient relative to a normal human control is associated with ongoing cancer and a decrease in LSG concentration is compared to that of a human control, preferably in the same type of cell, tissue, or body fluid sample. Associated with regressed or remitted cancer.

The present invention further provides a method of monitoring the initiation of lung cancer metastasis in a human having lung cancer. The method identifies human patients with such cancers who are not known to have metastasized; periodically analyze cells, tissues, or body fluid samples of such patients for LSG; Increasing a patient's LSG concentration relative to a normal human control includes comparing the LSG concentration in the tissue or body fluid to the LSG concentration in a normal human control, preferably the same type of cell, tissue or body fluid sample, as compared to a normal human control. Related to.

Furthermore, the present invention provides a method for monitoring changes in the stage of lung cancer in humans having lung cancer by observing the LSG concentration in humans having such cancer. The method identifies a human patient having such cancer; periodically analyzes a cell, tissue or body fluid sample of such patient for LSG; LSG in such cell, tissue or body fluid Increasing the concentration of LSG in a patient relative to a normal human control is associated with an ongoing cancer, and the concentration of LSG in the normal human control is preferably in the same type of cell, tissue, or body fluid sample. Reduced concentrations are associated with regressing or remissioning cancer.

The present invention further provides antibodies to LSGs or fragments of such antibodies. It can be used to detect or image the localization of LSGs in a patient for the purpose of detecting or diagnosing a disease or condition. Such antibodies can be polyclonal or monoclonal, or can be produced by molecular biology techniques.
As used herein and throughout the specification, the term "antibody" is meant to also include those referred to as SELEX and aptamers and single stranded oligonucleotides such as those obtained from laboratory evolution protocols well known to those of skill in the art. To do. The antibody can be labeled with various detectable labels. Examples include, but are not limited to, radioactive isotopes and paramagnetic metals. These antibodies or fragments thereof are
It can also be used as a therapeutic agent in the treatment of diseases characterized by the expression of LSG. In therapeutic applications, the antibody can be used with or without derivatization to a cytotoxin such as a radioisotope, enzyme, toxin, drug or prodrug.

Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. It should be understood, however, that the following description and specific examples, while indicating preferred embodiments of the present invention, are provided for purposes of illustration only. Various modifications within the scope of the disclosed invention will be readily apparent to those skilled in the art from reading the following description as well as other parts of the disclosure. DESCRIPTION OF THE INVENTION The present invention provides quantitative and qualitative methods for detecting, diagnosing, monitoring, staging, prognosing, in vivo imaging and treating cancer by comparing LSG levels to LSG levels in normal human controls. Diagnostic assay and diagnostic method.
As used herein, LSG concentration means the concentration of native protein expressed by the gene comprising the polynucleotide sequence of any of SEQ ID NOs: 1, 2, 3, 4, or 5. Alternatively, the LSG used here
The concentration means the concentration of natural mRNAs encoded by a gene containing the polynucleotide sequence of SEQ ID NO: 1, 2, 3, 4, or 5, or SEQ ID NO: 1, 2, 3,
It means the concentration of the gene containing the polynucleotide sequence of either 4 or 5. Such concentration is preferably measured in at least one of cells, tissues and / or body fluids,
It also includes determination of normal and abnormal concentrations. Thus, for example, diagnostic assays according to the present invention for diagnosing LSG protein overexpression as compared to normal control body fluids, cells, or tissue samples can be used to diagnose the presence of cancer, including lung cancer. In the method of the present invention, whether any of the five LSGs is measured alone or all together,
The measurement may be performed by arbitrarily combining the five types.

All of the methods of the present invention optionally use LSG
In addition to, the measurement of the concentration of other cancer markers may be included. Cancer markers other than LSG useful in the present invention include:
It depends on the cancer being tested and is well known to those skilled in the art. Diagnostic Assays The present invention provides a method of diagnosing the presence of lung cancer by analyzing changes in LSG levels in cells, tissues or fluids compared to LSG levels in cells, tissues or fluids, preferably of the same type, in normal human controls. I will provide a. Increased LSG levels in patients relative to normal human controls are associated with the presence of lung cancer.

Although not limiting to the present invention, generally with respect to quantitative diagnostic assays, a positive result indicating the presence of cancer in the tested patient is a cell, tissue, or cancer marker marker such as LSG. The body fluid concentration is at least 2 times that of a normal human control, preferably the same cell, tissue or body fluid concentration
It is twice as high, most preferably at least 5 times higher.

The present invention also provides a method of diagnosing the onset of metastatic lung cancer in a patient with lung cancer that has not yet metastasized. The methods of the invention identify human cancer patients suspected of having lung cancer that may have metastasized (but has not previously been known to have metastasized). 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 usually diagnosed with lung cancer as a result of conventional detection methods.

In the present invention, L in cells, tissues, or body fluids
Determining the presence of SG concentrations is particularly useful in distinguishing non-metastatic and metastatic lung cancer. Although existing technologies have difficulty distinguishing metastatic and non-metastatic lung cancer, the choice of appropriate treatment often depends on such knowledge.

In the present invention, the cancer marker concentration measured in such cells, tissues or body fluids is LSG.
Which is compared to LSG concentrations in normal human controls, preferably in the same type of cells, tissues, or body fluids. That is, if the only cancer marker being observed is LSG in serum, this concentration is preferably compared to the LSG concentration in serum of normal human patients. Increased LSG in patients as compared to normal human controls is associated with metastasized lung cancer.

Although not limiting to the present invention, generally with respect to quantitative diagnostic assays, a positive result indicating that the cancer of the patient being tested or monitored has metastasized is LSG.
Cell, tissue, or body fluid concentrations of cancer markers such as
Preferably at least 2-fold higher than in the same cells, tissues, or body fluids of a normal patient, most preferably at least 5
It is twice as expensive.

As used herein, normal human controls include cancer free patients and / or non-cancerous samples from patients. In the method of diagnosing or monitoring metastasis, a normal human control is preferably a sample from a human patient determined to have non-metastatic lung cancer by a reliable method, for example a sample from the same patient before metastasis. including. Staging The present invention also provides a method of staging lung cancer in human patients.

The method comprises identifying a human patient having such a cancer and analyzing a sample of cells, tissue or body fluid obtained from such a patient for LSG. next,
The measured LSG concentration is compared to that of a normal human control, preferably in the same type of cell, tissue, or body fluid sample. Increased levels of LSG in patients relative to normal human controls are associated with ongoing cancer, and decreased levels of LSG are associated with regressing or remitted cancer. Monitoring Further provided is a method of monitoring the onset of cancer metastasis in humans with lung cancer. The method identifies human patients with such cancers who are not known to have metastasized; periodically analyze cell, tissue, or body fluid samples obtained from such patients for LSG; Comparing the LSG concentration in a cell, tissue, or body fluid with that of a normal human control, preferably in the same type of cell, tissue, or body fluid sample. Increased LSG levels in patients relative to normal human controls are associated with metastasized cancer.

Further provided by the present invention is a method of monitoring changes in cancer staging in humans with lung cancer. The method identifies a human patient having such cancer; periodically analyzes a cell, tissue or body fluid sample obtained from such patient for LSG; in such cell, tissue or body fluid. Comparing the LSG concentration of the LSG to the LSG concentration in a cell, tissue, or body fluid sample, preferably of the same type, of a normal human control. Increased LSG levels in patients relative to normal human controls are associated with advanced stage cancer, and decreased LSG levels are associated with stage-regressing or remitted cancer.

Monitoring of the onset of metastases in such patients is carried out regularly, preferably four times a year. However, it may be more frequent or less frequent depending on the cancer, the particular patient, and the stage of the cancer. Assay Technique Gene expression level in a sample obtained from a host, such as L of the present invention
Assay techniques that can be used to measure SG are well known to those of skill in the art. Such assay methods include radioimmunoassay, reverse transcription PCR (RT-PCR) method, immunohistochemistry method, in situ hybridization method, competition-binding assay method, Western blot analysis method, ELISA method and proteome method. . Among them, ELISAs are often preferred for diagnosing gene-expressed proteins in biological fluids.

The ELISA method first involves providing an antibody specific to LSG, preferably a monoclonal antibody, although not readily available from commercial sources. Further, it is general to prepare a reporter antibody that specifically binds to LSG. The reporter antibody is linked to a detectable reagent such as a radioactive, fluorescent or enzymatic reagent, eg horseradish peroxidase enzyme or alkaline phosphatase.

To carry out the ELISA, an antibody specific for LSG is incubated on a support which binds the antibody, for example a polystyrene dish. All free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. The sample to be analyzed is then incubated in the dish during which LSG binds to the specific antibody bound to the polystyrene dish. Unbound sample is washed out with buffer. Placing a reporter antibody specifically directed against LSG and conjugated with horseradish peroxidase into the dish results in the binding of the reporter antibody with all monoclonal antibodies bound to LSG. Then, the unbound reporter antibody is washed away. Next, a reagent for peroxidase activity containing a colorimetric substrate is added to the dish. Immobilized peroxidase bound to the LSG antibody produces a colored reaction product. The amount of color developed during a given time is proportional to the amount of LSG protein present in the sample. Quantitative results are usually obtained by reference to a standard curve.

Competition assays can also be used. An antibody specific to LSG is bound to the support, and the labeled LSG and the sample taken from the host are flown onto the support. The detected amount of label bound to the support can then be correlated with the amount of LSG in the sample.

The nucleic acid method can be used to detect LSG mRNA as a lung cancer marker. Polymerase chain reaction (PCR) and other nucleic acid methods such as ligase chain reaction (LCR) and nucleic acid sequence-based amplification
acid sequence based amplification) (NASAB
By using A), malignant cells can be detected, and various malignant diseases can be diagnosed and monitored. For example, reverse transcription PCR (RT-PCR) is a powerful technique that can be used to detect the presence of a particular mRNA population in a complex mixture of thousands of other mRNA species. In RT-PCR,
mRNA species first use reverse transcriptase to produce complementary DNA
(CDNA) is reverse transcribed. Next, use cDNA as standard PC
Amplify as done in the R reaction. RT-PCR can thus reveal the presence of a single mRNA species by amplification. Thus, RT-PCR can be used to distinguish the presence of a particular cell type if the mRNA is very specific to the cell producing it.

Hybridization (ie, gridding) with clones or oligonucleotides arrayed on a support can be used both for detecting gene expression and quantifying its expression level. In this method, cDNA encoding the LSG gene is immobilized on a substrate. The substrate may be of any suitable type, such as, but not limited to, glass, nitrocellulose, nylon or plastic. At least a portion of the DNA encoding the LSG gene is bound to the substrate and then incubated with the analyte. The subject is an RN isolated from the tissue in question
It can be a complementary DNA (cDNA) copy of A or RNA. Hybridization between DNA bound to the substrate and the analyte can be detected and quantified by several means. For example, but not limited to, a radioactive or fluorescent label of the analyte, or a secondary molecule designed for hybrid detection. Quantification of gene expression
This can be done by comparing the intensity of the signal from the analyte with that determined from known standards. The standard can be obtained by in vitro transcription of the target gene (cell-free transcription), quantification of yield, and then generation of a standard curve using the substance.

Of the proteome methods, 2D electrophoresis is a technique well known to those skilled in the art. Isolation of individual proteins from samples such as serum is usually accomplished on polyacrylamide gels with the sequential separation of proteins by different properties. First, the proteins are separated by size using an electric current. The electric current acts uniformly on all proteins, so smaller proteins travel farther on the gel than larger proteins. Application of a current in the second dimension perpendicular to the first separates the proteins based on the specific charge carried by each protein rather than its magnitude. Since no two proteins with different sequences are identical in both size and charge, the result of 2D separation is a square gel where each protein occupies its own spot. Analysis of spots using chemical or antibody probes, or subsequent protein microsequencing, can reveal the relative abundance and protein identification of a given protein in a sample.

The above tests can be carried out on samples obtained from cells, body fluids and / or tissue extracts (homogenates or solubilized tissues) such as from tissue biopsies and autopsy material of various patients. Body 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 blood derivative. Use of Antibodies In Vivo Antibodies to LSG can also be used in vivo in patients with lung disease. Specifically, antibodies against LSG can be infused for diagnostic and / or therapeutic purposes in patients suspected of having lung disease. The use of antibodies for in vivo diagnosis is well known in the art. For example,
It has been described that an antibody-chelator labeled with indium-111 is used in imaging a carcinoembryonic antigen-expressing tumor by radioimmunoscintigraphy (S
umerdon et al., Nucl. Med. Biol. 19
90 17: 247-254). In particular, these antibody-chelators have been used to detect tumors in patients suspected of having recurrent colorectal cancer (Griffin et al., J. Cl.
in. Onc. 1991 9: 631-640). Antibodies with paramagnetic ions as labels for use in magnetic resonance imaging are also described (Lauffer, R .;
B. Magnetic Resonance in Me
dicine 1991 22: 339-342). L
Antibodies directed against SGs can be used as well. Labeled antibodies against LSG can be infused into patients suspected of having lung disease, such as lung cancer, for the purpose of diagnosis or determining the stage of a patient's condition. The label used will be selected according to the imaging modality used. For example, radiolabels such as Indium-111, Technetium-99m or Iodine-131 can be used for planar scanning or single photon emission computed tomography (SPECT). Positron radiolabels such as Fluorine-19 can be used for positron emission tomography. Paramagnetic ions such as gadolinium (III) or manganese (II) can be used for magnetic resonance imaging (MRI). The localization of the label in the lung or outside the lung makes it possible to determine the spread of the disease. The amount of label in the lung also allows the determination of the presence or absence of cancer in the lung.

Infusion of antibodies against LSG may also have therapeutic benefit for patients diagnosed with lung cancer. The antibody alone can exert its therapeutic effect. Alternatively, the antibody is conjugated to a cytotoxic agent such as a drug, toxin or radionuclide to enhance its therapeutic effect. The monoclonal antibody of the drug is described in the literature in this technical field.
For example, Garnett and Baldwin, Canc.
er Research 1986 46: 2407-
2412. The use of toxin conjugated to monoclonal antibodies for the treatment of various cancers is also
Et al., Cell 1986 47: 641-648. It has been described that a yttrium-90 labeled monoclonal antibody maximizes the dose delivered to the tumor while limiting its toxicity to normal tissues (Goo).
dwin and Mearse Cancer Suppl
element 1997 80: 2675-2680).
Other cytotoxic radionuclides such as, but not limited to, copper-67, iodine-131 and rhenium-186 can also be used to label antibodies to LSGs.

The antibodies that can be used in these in vivo methods are:
It includes polyclonal and monoclonal antibodies, as well as antibodies produced by molecular biology techniques. Antibody fragments can also be used. Examples The invention will be further described by the following examples. These examples are provided solely for the purpose of illustrating the invention by reference to specific embodiments. These illustrations, while indicating certain particular aspects of the invention, are not intended to represent limitations or limit the scope of the disclosed invention. Example 1 A search was conducted to include Incy, Palo Alto, California.
The following search tools were used to identify LSGs as part of the LIFESEQ database available from te Pharmaceuticals.

Library comparison (comparing one library with another) allows the identification of clones expressed in tumor and absent or expressed at low levels in normal tissue.

Subsetting is similar to library comparison, but allows identification of clones expressed in a pool of the library and not in the second pool of the library or expressed at low concentrations.

Transcriptional imaging lists all clones in a single library or pool of libraries based on abundance. Each clone can then be examined using electronic Northern to determine the tissue source of their constituent ESTs.

Protein Function: Incyte identified a subset of ESTs with potential protein function based on homology to known proteins. Some examples included in this database are transcription factors and proteases and the like. EST in this database
Several lead genes were identified by searching for clones in which s showed disease specificity.

Electronic subtraction, transcriptional imaging and protein functional search were used to identify clones in which the constituent ESTs were found only in or frequently in libraries derived from a particular tumor. Each candidate clone was examined in detail by checking the origin of each EST.

[0042]

[Table 1]

The following examples were carried out using standard techniques, which are well known and routine to those of skill in the art, unless otherwise specified. Routine molecular biology techniques in the following examples are described in standard laboratory manuals, such as Sambrook et al., MOLEC.
ULA CLONING: ALABORATORY
MANUAL, 2nd Edition; Cold Spring Ha
rbor Laboratory Press, Col
d Spring Harbor, N.D. Y. (198
It can be carried out as described in 9). Example 2: Relative Quantification of Gene Expression Real-time quantitative PCR using a fluorescent Taqman probe was performed using the 5'-
It is a quantitative detection system utilizing 3'nuclease activity. This method uses an internal fluorescent oligonucleotide probe (Tacman) labeled with a 5'reporter dye and a downstream 3'quencher dye. PCR
Reporter was released by the 5'-3 'nuclease activity of Tuck DNA polymerase during the reaction, and its fluorescence was detected by the Model 7700 Sequence Detection System (PE Applied Biosys, Foster City, CA, USA).
tems) laser detector.

Amplification of the endogenous control was used to normalize the amount of sample RNA added to the reaction and reverse transcriptase (R
Normalize the efficiency of T). Cyclophilin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Alternatively, either 18S ribosomal RNA (rRNA) was used as this endogenous control. To calculate the relative quantification between all tested samples, the amount of target RNA in one sample was used as a standard for comparison results (calibrator). Relative quantification to the "calibrator" is obtained using standard curve or comparison methods (User
Bulletin # 2: ABI PRISM 77
00 sequence detection system).

The tissue distribution and amount of the target gene was evaluated for each sample in normal and cancer tissues. Total RNA was extracted from these tissues and the corresponding matched adjacent tissues. Next, the first cDNA strand was produced using reverse transcriptase, and a polymerase chain reaction was performed using a primer and a TaqMan probe specific to each target gene. Results were analyzed using an ABI PRISM 7700 Sequence Detector. The anonymous number is the relative expression of the target gene in a particular tissue compared to the calibrator tissue. Comparative Example For comparison, a similar mRNA expression analysis was performed for genes encoding the diagnostic markers PSA (prostate specific antigen) and PLA2 (phospholipase A2). P
SA is the only cancer screening marker available in clinical laboratories. An analysis of a population of normal pooled tissue showed that PSA was highly expressed in the prostate but very little in the breast and testis. Over 55 matched samples from 14 different tissues
The data confirmed the tissue specificity seen in normal tissue samples. The expression of PSA was compared in cancer and normal adjacent tissues for 12 matched samples of prostate tissue. The relative expression level of PSA was high in 10 cancer samples (83%). Recent clinical data support the use of PLA2 as a staging marker for end-stage prostate cancer. The mRNA expression data showed mRNA overexpression in 8 out of 12 (66%) matched samples analyzed. The tissue specificity of PLA2 was not as good as that described for PSA. In addition to the prostate, the small intestine, liver, and pancreas also showed high mRNA expression levels for PLA2. SEQ ID NO: 5; Clone number 3117390; Gene number
Measurement of 7387 (Lng109) The anonymous number shown in Table 2 is the relative expression level of Lng109 (SEQ ID NO: 5) in 12 different normal tissues. All values are compared to normal small intestine (calibrator). These RNA samples are commercially available pools prepared by pooling samples of specific tissues obtained from different individuals.

[0046]

[Table 2]

The relative expression levels in Table 2 show that Lng109 (SEQ ID NO: 5) mRNA expression is higher in lung compared to all other normal tissues analyzed (46.6).
The testes with relative expression of 12.1 and the brain (26.6) are the only other tissues expressing significant mRNA for Lng109. From these results, the mR of Lng109
It is established that NA expression is highly lung specific.

The anonymous numbers in Table 2 were obtained by analyzing pools of samples of specific tissues from different individuals. These are not comparable to the unnamed numbers derived from RNA from one individual tissue sample in Table 3.

The anonymous numbers listed in Table 3 are the relative expression levels of Lng109 (SEQ ID NO: 5) in 57 sets of corresponding samples. All values are compared to normal small intestine (calibrator). The corresponding pair is m obtained from a cancer sample of a specific tissue.
It is formed by RNA and mRNA obtained from a normal adjacent sample of the same tissue from the same individual.

[0050]

[Table 3]

[0051]

[Table 4]

[0052]

[Table 5]

High levels of expression were found in lungs in the analysis of corresponding samples, indicating a high degree of tissue specificity for lung tissue. Of all non-lung samples analyzed, only one sample (liver 2 cancer sample, 48.6) showed expression comparable to mRNA expression in lung. These results confirmed the tissue specificity results (Table 2) obtained with the population of normal pool samples.

Furthermore, the mRNA expression levels in the cancer samples and the normal adjacent tissues of the same line obtained from the same individual were compared.
This comparison shows specificity with respect to the stage of the cancer (eg higher mRNA expression in cancer samples compared to normal neighbors). Table 3 shows Lng in 16 primary lung cancer tissues.
109 overexpression is shown in comparison to their corresponding normal flanks (lung samples # 1, 3, 4, 5, 7, 8, 9, 10,
11, 13, 14, 15, 18, 19, 20, and 2
1). There was overexpression in cancer tissue in 70% of the lung-corresponding samples tested (23 lung-corresponding samples in total).

Overall, in addition to a high degree of tissue specificity, mRNA in 70% of the primary lung cancer matched samples tested
Overexpression indicates that Lng109 is a diagnostic marker for lung cancer. SEQ ID NO: 4; clone number 1355520 (19817)
52); measurement of gene number 236760 (Lng110)
The anonymous number shown in Table 4 is the relative expression level of Lng110 in 12 different normal tissues. All values are compared to normal testis (calibrator). These R
NA samples are commercial pools made by pooling samples of specific tissues from different individuals.

[0056]

[Table 6]

The relative expression level in Table 4 is the m of Lng110.
RNA expression is shown to be more than 300-fold higher in pools of normal lung compared to all other tissues analyzed (3
92.1). From these results, Lng110 mRNA
Is shown to be highly specific to the lungs.

The unnamed numbers in Table 4 were obtained by analyzing pools of samples of specific tissues from different individuals. These are not comparable to the unnamed numbers derived from RNA from one individual tissue sample in Table 5.

The unnamed numbers listed in Table 5 are the relative expression levels of Lng110 in 60 sets of corresponding samples. All values are compared to normal testis (calibrator). A matched pair is formed by mRNA from a cancer sample of a particular tissue and mRNA from a normal adjacent sample of the same tissue of the same individual.

[0060]

[Table 7]

[0061]

[Table 8]

[0062]

[Table 9]

[0063]

[Table 10]

High levels of expression were found in the lungs in the analysis of corresponding samples, indicating a high degree of tissue specificity for lung tissue. These results support the tissue specificity results (Table 4) obtained with normal pool samples.

Furthermore, the expression levels of mRNA in cancer samples and normal adjacent tissues of the same strain obtained from the same individual were compared.
This comparison shows specificity with respect to the stage of the cancer (eg higher mRNA expression in cancer samples compared to normal neighbors). Table 5 shows Lng in 18 primary lung cancer samples.
110 overexpression is shown relative to their corresponding normal flanks (lung samples # 1, 4, 5, 6, 7, 8, 9, 10,
13, 14, 15, 16, 17, 18, 19, 21, 2
3 and 24). There is overexpression in cancer tissue in 75% of the lung-corresponding samples tested (total of 24 primary lung cancer-corresponding samples).

Overall, in addition to a high degree of tissue specificity, the overexpression of mRNA in 75% of the lung-corresponding samples tested is an indication that Lng110 is a diagnostic marker for lung cancer. The amino acid sequence encoded by the open reading frame of Lng110 is shown in SEQ ID NO: 6.

[Sequence list]

Front page continuation (51) Int.Cl. ⁷ Identification code FI C12Q 1/02 G01N 33/53 M G01N 33/53 33/574 A C12N 15/00 A 33/574 A61K 49/02 C (72) Inventor Sun, Yongmin California 95128, San Jose, Winchester Boulevard 869, Apartment 260 (72) Inventor Recipon, Harve California 94115, San Francisco, Fortuna Avenue 85 (72) Inventor Masina Roberto・ A. USA, California 95136, San José, 4118 (56) References: Special Tables: 10-503087 (JP, A) International Publication 98/56951 (WO, A1) International Publication 99/60160 (WO, A1) ) WO 98/20143 (WO, A1) (58 ) investigated the field (Int.Cl. ^7, DB name) C12N 15/09 C12Q 1/02 C12Q 1/68 C07K 16/18 G01N 33/574 BIOSIS / PI (DIALOG) GenBank / EMBL / DDBJ / G eneSeq SwissProt / PIR / GeneS eq

Claims (4)

(57) [Claims] 1. A method for detecting the presence of lung cancer in a sample of relevant cells, tissues or body fluids from a patient, comprising: (a) an array in a sample of relevant cells, tissues or body fluids from a patient. Measuring the level of the polynucleotide comprising SEQ ID NO: 5 or the level of the protein expressed by the polynucleotide comprising SEQ ID NO: 5; and (b) the measured level of the polynucleotide comprising SEQ ID NO: 5 or SEQ ID NO: The measured level of a protein expressed by a polynucleotide comprising 5 comprises the level of a polynucleotide comprising SEQ ID NO: 5 in a sample of relevant cells, tissues or body fluids from a normal human control or a polynucleotide comprising SEQ ID NO: 5. Comparing with the level of the protein expressed by the nucleotide, SEQ ID NO: 5 in the sample obtained from the patient No polynucleotide of the measured level or SEQ ID NO: method of measuring levels of protein expressed by a polynucleotide comprising the 5, increase to normal human control, comprising associated with the presence of lung cancer. 2. An antibody against the protein expressed by the polynucleotide comprising SEQ ID NO: 5. 3. The antibody according to claim 2, which is labeled with a paramagnetic ion or a radioisotope. 4. The antibody according to claim 2, which is conjugated with a cytotoxin.