Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-02-02
2001-10-16
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06303304
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for disease diagnosis, including the early detection of colon cancer in patients, and more particularly to methods for preparing stool samples for disease diagnosis, including the detection of colon cancer, so as to assure or increase the likelihood that the sample will contain the diagnostically relevant information if the patient has a disease, for example a cancerous or precancerous lesion, and to methods for stool sample analysis.
BACKGROUND OF THE INVENTION
Stool samples frequently must be prepared for medical diagnostic analysis. Stool samples may be analyzed to help diagnose medical conditions ranging from parasitic, bacterial or viral infections to inflammatory bowel disease and colorectal cancer.
Colorectal cancer is a leading cause of death in Western society. However, if diagnosed early, it may be treated effectively by surgical removal of the cancerous tissue. Colorectal cancers originate in the colorectal epithelium and typically are not extensively vascularized (and therefore not invasive) during the early stages of development. Colorectal cancer is thought to result from the clonal expansion of a single mutant cell in the epithelial lining of the colon or rectum. The transition to a highly vascularized, invasive and ultimately metastatic cancer which spreads throughout the body commonly takes ten years or longer. If the cancer is detected prior to invasion, surgical removal of the cancerous tissue is an effective cure. However, colorectal cancer is often detected only upon manifestation of clinical symptoms, such as pain and black tarry stool. Generally, such symptoms are present only when the disease is well established, often after metastasis has occurred, and the prognosis for the patient is poor, even after surgical resection of the cancerous tissue. Early detection of colorectal cancer therefore is important in that detection may significantly reduce its morbidity.
Invasive diagnostic methods such as endoscopic examination allow for direct visual identification, removal, and biopsy of potentially cancerous growths such as polyps. Endoscopy is expensive, uncomfortable, inherently risky, and therefore not a practical tool for screening populations to identify those with colorectal cancer. Non-invasive analysis of stool samples for characteristics indicative of the presence of colorectal cancer or precancer is a preferred alternative for early diagnosis, but no known diagnostic method is available which reliably achieves this goal.
Current non-invasive diagnostic methods involve assaying stool samples for the presence of fecal occult blood or for elevated levels of carcinoembryonic antigen, both of which are suggestive of the presence of colorectal cancer. Additionally, recent developments in molecular biology provide methods of great potential for detecting the presence of a range of DNA mutations or alterations associated with and indicative of the presence of colorectal cancer. The presence of such mutations theoretically can be detected in DNA found in stool samples during the early stages of colorectal cancer. However, stool comprises cells and cellular debris from the patient, from microorganisms, and from food, resulting in a heterogeneous population of cells. This makes detection of a small, specific subpopulation impossible to detect reliably.
Stool diagnostic assays for colorectal cancer described in the art typically are performed on samples prepared from randomly sampled portions of voided stool. However, samples prepared according to such methods do not reproducibly yield characteristics indicative of the presence of colorectal cancer or precancer, even when prepared from stool voided by a patient with colorectal cancer or precancer. There is therefore a need in the art for methods for early diagnosis of colorectal cancer or precancer that will reproducibly detect characteristics indicative of the presence of cancerous or precancerous material in samples prepared from stool voided by a patient with colorectal cancer or precancer. Such methods are provided herein.
SUMMARY OP THE INVENTION
It has now been appreciated that cells and cellular debris are shed from colonic epithelial cells onto forming stool in a longitudinal “stripe” of material along the length of the stool. The shed material is confined to this longitudinal stripe, as shown in
FIG. 1
(designated “C”). Based upon this recognition, Applicants teach that stool sample preparation for diagnostic testing must include taking a representative sample in order to ensure that the sample will contain any cells or cellular debris that was shed into the stool as it passed through the colon. Accordingly, methods of the invention comprise obtaining at least a representative (e.g. a cross-section or circumferential surface) portion of stool voided by a patient, and performing an assay to detect in the sample the presence of cells or cellular debris shed from epithelial cells lining the colon that may be indicative of cancer or precancer. Most often, such cells will be derived from a polyp or a cancerous or precancerous lesion at a discrete location along the colon. For purposes of the present invention, a precancerous lesion comprises precancerous cells, and precancerous cells are cells that have a mutation that is associated with cancer and which renders such cells susceptible to becoming cancerous. As shown in
FIG. 1
, a cross-sectional sample is a sample that contains at least a circumferential surface of the stool (or portion of a stool comprising an entire cross-sectional portion), as, for example, in a coronal section or a sagittal section. A sample comprising the surface layer of a stool (or of a cross-section of a stool) also contains at least a circumferential surface of the stool. Both cross-sections and circumferential surfaces comprise longitudinal stripes of sloughed colonic epithelium, and are therefore representative samples.
In a preferred embodiment, methods of the invention comprise the steps of obtaining at least a circumferential surface or cross-sectional portion of a stool voided by a patient, and performing an assay to detect debris indicative of disease. For example, such debris may comprise a clonal subpopulation of cells having one or more mutations (for purposes of the present application, a mutation is a deletion, substitution, addition, modification, intercalation or rearrangement of DNA). Preferred methods of the invention comprise detection of characteristics of such transformed cells, including, for example, mutations, proteins expressed uniquely or in altered amounts in transformed cells, and blood. Particularly preferred methods of the invention comprise obtaining at least a circumferential surface or cross-sectional portion of a stool sample, and performing an assay to detect DNA characteristics indicative of the presence of a clonal subpopulation of cells in the sample. The clonal subpopulation may be, for example, a subpopulation of cancerous or precancerous cells, having a mutation in, for example, a p53 tumor suppressor gene. Clonal subpopulations of cells detected by methods according to the invention are often characterized by a massive loss of DNA, resulting in a loss of heterozygosity that renders ineffective the gene or genes encompassed by the deletion. Alternative methods of the invention comprise performing an assay to detect the presence of an infectious disease, for example debris from a microorganism.
Methods of the invention also comprise obtaining a representative (i.e., cross-sectional or circumferential) sample of stool and homogenizing the stool in a buffer, such as a buffer comprising a detergent and a proteinase and optionally a DNase inhibitor.
In methods according to the invention, an assay performed on at least a circumferential surface or cross-sectional portion of stool may be an assay to detect a disease when a diagnostic indicator of the disease is incorporated into stool. Methods of the invention are useful to detect cellular debris shed from the epithelial lining of the colon
Lapidus Stanley N.
Shuber Anthony P.
Exact Laboratories, Inc.
Ketter James
Testa Hurwitz & Thibeault LLP
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