Identification of DSG-3 as a biomarker for the detection of metastasis in lymph nodes
Inventors
Gutkind, J. Silvio • Patel, Vyomesh • Molinolo, Alfredo • Veenstra, Timothy D.
Assignees
US Department of Health and Human Services
Publication Number
US-9151762-B2
Publication Date
2015-10-06
Expiration Date
2030-06-11
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Abstract
Disclosed herein is a method of detecting metastasis of a tumor in a subject, such as metastasis to a lymph node. The method includes directly determining an amount of desmoglein-3 (DSG-3) protein in a sample from the subject (such as a lymph node sample) and comparing the amount of DSG-3 protein to a control, wherein an increase in the amount of DSG-3 protein in the sample relative to the control indicates metastasis of the tumor to the lymph node. The disclosed methods further include selecting a therapy (for example, surgery, lymph node dissection, radiation therapy, chemotherapy, or a combination of two or more thereof) for the subject based on the amount of DSG-3 protein in the sample from the subject.
Core Innovation
This invention provides a method for detecting metastasis of a tumor in a subject by directly determining the amount of desmoglein-3 (DSG-3) protein in a sample, such as a lymph node sample. An increase in DSG-3 protein amount relative to a control indicates metastasis of the tumor to the lymph node. The method may include the provision of test results to a user and selection of therapy based on DSG-3 levels.
The disclosed methods focus on the direct detection of DSG-3 protein, for example using immunoassays like ELISA or electrochemical immunoassays, rather than indirect detection such as quantification of mRNA. This direct detection is stated to be surprisingly more sensitive, offering about 100-fold greater sensitivity compared to RNA detection methods, and can detect DSG-3 protein in very few lymph node cells.
The problem addressed is the difficulty in detecting occult metastasis to lymph nodes, which complicates treatment decisions. Existing pre-operative clinical methods often fail to accurately diagnose the presence or absence of nodal metastasis, sometimes leading to unnecessary removal of lymph nodes or failure to remove nodes containing tumor cells. There is a need for molecular markers that more accurately detect lymph node metastasis, particularly in cancers like head and neck squamous cell carcinoma (HNSCC) which frequently metastasize to regional lymph nodes and have poor prognosis when metastasis is present.
Claims Coverage
The patent includes one independent claim presenting a method for detecting metastasis of squamous cell carcinoma to lymph nodes using an electrochemical immunoassay.
Detection of desmoglein-3 protein by electrochemical immunoassay
The method involves directly determining the amount of desmoglein-3 protein in a lymph node sample by electrochemical immunoassay using at least one anti-desmoglein-3 antibody that specifically binds an epitope in the desmoglein-3 extracellular domain.
Comparison of DSG-3 protein amount to control for metastasis detection
An increase in DSG-3 protein detected in the lymph node sample relative to a control indicates metastasis of the squamous cell carcinoma to the lymph node; detection is sensitive enough to identify increases in samples containing 1-100 cells expressing increased DSG-3 protein.
Therapeutic intervention based on DSG-3 detection
If increased DSG-3 protein is detected indicating metastasis, the subject is treated with lymph node dissection.
Application to specific squamous cell carcinomas
The squamous cell carcinoma comprises head and neck squamous cell carcinoma or lung squamous cell carcinoma, both expressing DSG-3 protein.
Selection of relevant lymph nodes
The lymph node is one to which metastatic spread of the squamous cell carcinoma would be expected, including regional lymph nodes or sentinel lymph nodes.
Use of specific anti-DSG-3 antibodies
The anti-desmoglein-3 antibody used in the assay includes BAF1720 or MAB1720 antibodies.
Specific method steps in electrochemical immunoassay
The electrochemical immunoassay comprises: contacting the sample with a gold nanoparticle electrode conjugated to the anti-DSG-3 antibody, incubating with magnetic beads conjugated with a secondary antibody and horseradish peroxidase, and detecting DSG-3 protein amount by measuring amperometric current in presence of an HRP substrate.
Lymph node dissection based on degree of DSG-3 increase
If DSG-3 protein increase is more than 10-fold, radical neck dissection (for HNSCC) or systemic mediastinal lymph node dissection (for lung SCC) is selected; if increase is about 3-10 fold, modified radical or limited dissection; if about 3-fold or less, selective neck dissection.
Provision of test output to user
The method further comprises providing a test output to a user concerning the detected amount of DSG-3 protein.
The claims define a sensitive method employing electrochemical immunoassay with specific antibodies to directly detect DSG-3 protein in lymph node samples to diagnose squamous cell carcinoma metastasis and guide lymph node dissection treatment accordingly.
Stated Advantages
Direct detection of DSG-3 protein is more sensitive than indirect detection via mRNA, providing about 100-fold greater sensitivity.
The method allows detection of a small number of tumor cells expressing DSG-3 protein among many non-expressing cells, increasing diagnostic accuracy.
Direct protein detection avoids amplification bias and is more stable and practical than RNA detection in clinical and laboratory settings.
Documented Applications
Detecting metastasis of tumors, such as head and neck squamous cell carcinoma and lung squamous cell carcinoma, to regional or sentinel lymph nodes.
Selecting therapies for cancer patients, including lymph node dissection (radical, modified, or selective), radiation therapy, and chemotherapy, based on detected DSG-3 protein levels in lymph nodes.
Diagnostic testing in clinical settings providing quantitative or qualitative outputs of DSG-3 protein levels to inform prognosis and treatment decisions.
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