Cancer is one of the deadliest diseases in the world today. Cancer generally refers to one of a group of more than 100 diseases that are caused by the uncontrolled growth and spread of abnormal cells. Unlike normal cells which reproduce until maturation is attained and then only reproduce as necessary to replace wounded cells, cancerous cells grow and divide endlessly, crowding out nearby cells and eventually spreading to other parts of the body.
The most common sites in which cancer develops include the skin, lungs, female breasts, colon, rectum, uterus, blood-forming tissues, and lymphatic system. Cancerous cells that have developed at one of these sites will grow rapidly into a malignant tumor, invading and destroying nearby tissues. Malignant cancerous tumors will eventually metastasize, or spread to other parts of the body, unless their progression is stopped.
Cancers are easier to treat and cure if they are discovered and treated prior to metastasis. Once cancerous cells metastasize by leaving a tumor, they will travel through the bloodstream or lymphatic system to other parts of the body, where the cells begin multiplying and developing into new tumors. It is this spreading of cancerous cells (also known as tumor progression) that makes cancer dangerously fatal. Although there have been great improvements in diagnosis, general patient care, surgical techniques, and local and systemic adjuvant therapies, most deaths from cancer are still due to metastases that are resistant to conventional therapies.
Despite the resistance of certain cancers to treatment, early detection methods have been utilized to identify cancerous cells so that current treatments can be used to slow and, in many cases, completely halt cancerous tumor progression. The chances of survival for patients with malignant forms of cancers increase greatly when the cancerous cells are detected at an early stage. Conventional techniques, such as surgical removal, chemotherapy, and radiation, can often be utilized to provide a full recovery if the cancer is caught at an early stage.
Most diagnostic methods depend on microscopic observation of tissue that has been removed from the body during a biopsy or other tissue removal procedure. For example, if cancer of the colon is suspected, a colonoscope is used to enter the large intestine and remove a sample of tissue. That sample is then sent to the pathology laboratory which will then determine the presence of abnormal cellular structure which would lead to a conclusion of cancerous cells.
Such current methods are very invasive in that they require surgical removal of tissue for analysis. It would be advantageous if less invasive tests could be developed. In addition, such analyses are, obviously, subjectively dependent upon the ability of the personnel performing the determinations. One major disadvantage of current methods is that the pathological analysis is sometimes initially equivocal, which results in performing additional surgical procedures to obtain sufficiently large samples of tissue for further diagnosis. It would also be advantageous if methods could be developed that are objective and that are based on relatively small tissue samples. Such methods could be used to either complement currently used subjective methods or vice versa.
Recently, various objective analyses that depend on DNA studies have been developed and are being pursued and approved for cancer detection. The detection of cancerous cells under such objective analyses often depends on an understanding of the molecular mechanisms involved in metastasis.
The following mechanisms are important to an understanding of the applicability of the present inventive diagnostic test which involves extracting RNA and then forming complementary DNA. The invasion by malignant cancerous cells of adjacent fibroconnective tissue and subsequent metastasis is a multi-stage process in which the degradation of the extracellular matrix surrounding the tumor is an essential step in allowing invasion of neoplastic cells. If the basement membrane integrity in cancerous tumors, such as colorectal and breast carcinomas is breached, then there is an increased probability of metastasis.
The secreted proteinases, including the matrix metalloproteinases, play a very important role in the progression of a number of malignant tumors in several tissues, including the colon, and are expressed at the initiation of invasion and at metastasis. The enzymes associated with invasive cancer are frequently localized to the stroma (which is quantitatively and qualitatively different around a cancer when compared to that beneath normal tissue). The enzymes degrade components of the extracellular matrix including those of the basement membrane. Understanding of this stage in the development of cancers is important as tumor cell interaction with the basement membrane is central to tumor invasion and is the step just preceding initiation of the metastatic cascade.
The matrix metalloproteinases are a group of endopeptidases involved in this process. They are subdivided into interstitial collagenases, stromelysins and type IV collagenases. A recent addition to the stromelysin group is metalloproteinase-11 (or stromelysin-3) which was first identified in the stromal cells of breast carcinoma, but which has now been found in the stroma of invasive basal cell carcinoma, peritumoral fibroblasts of squamous cell carcinomas of the head and neck, carcinoma of the lungs, and colon carcinoma. The expression of these identifiable metalloproteinases in the stromal cells of certain malignant tumors has been found to indicate a cancerous invasion of the surrounding tissue.
Previously, the expression of stromelysin-3 has been analyzed in a variety of carcinomas using primarily in-situ hybridization or Northern blotting techniques. In-situ hybridization involves the creation of complementary DNA clones synthesized after isolation of an individual gene or gene transcript. Northern blotting, on the other hand, involves the hybridization of a specific nucleic acid probe to messenger RNA which has been isolated from homogenized tissue, separated by electrophoretic methods, and transferred to a stable matrix such as nylon.
While Northern blotting and in-situ hybridization have been used to identify the presence of stromelysin-3 in cancerous cells, a reverse transcriptase-polymerase chain reaction technique has not been heretofore involved in such an identification. Reverse transcriptase-polymerase chain reaction has been used only as a means to clone stromelysin-3 in order to produce large amounts of the enzyme itself instead of being used as an identifying diagnostic test. In this particular prior use, the primers utilized were not those utilized here and the resulting product was not intended for identification purposes as in the present invention.
Polymerase chain reactions have also been utilized in the past to amplify the genomic DNA for stromelysin-3. However, no reverse transcriptase procedure was used that employed transcribing the complementary DNA. Instead, these previous uses employ RNA directly in hybridization processes such as Southern blotting.
The present invention overcomes some of the deficiencies of prior cancer detection methods by allowing an objective analysis to be conducted on cells taken from a relatively small sample of human tissue. In certain embodiments, the present invention allows for the non-invasive diagnosis of cancerous cells.