Methods for disease detection

Details Methods for disease detection Methods for disease detection

1999-12-07

2003-07-01

Fredman, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S024300

Reexamination Certificate

active

06586177

ABSTRACT:

BACKGROUND OF THE INVENTION
Many diseases are associated with genomic instability. That is, a disruption in genomic stability, such as a mutation, has been linked to the onset or progression of certain diseases. Accordingly, various aspects of genomic instability have been proposed as reliable markers for disease. For example, mutations in the BRCA genes have been proposed as markers for breast cancer, and mutations in the p53 cell cycle regulator gene have been associated with numerous cancers, especially colorectal cancer. It has been suggested that specific mutations might be a basis for molecular screening assays for the early stages of certain types of cancer. See, e.g., Sidransky, et al., Science, 256: 102-105 (1992).
The search for genomic disease markers has been especially intense in the area of cancer detection. Cancer is characterized by uncontrolled cell growth which can be associated with one or more genetic mutations. Such mutations can cause the affected cells to avoid cell death. For example, a mutation in a tumor suppressor gene can cause cells to avoid apoptosis—a type of cell death thought to be under direct genetic control. During apoptosis, cells lose their membranes, the cytoplasm condenses, and nuclear chromatin is split into oligonucleotide fragments of characteristically short length. In fact, those characteristic DNA cleavage patterns have been proposed as an assay for apoptosis.
Attempts have been made to identify and use nucleic acid markers that are indicative of cancer. However, even when such markers are found, using them to screen patient samples, especially heterogeneous samples, has proven unsuccessful either due to an inability to obtain sufficient sample material, or due to the low sensitivity that results from measuring only a single marker. Simply obtaining an adequate amount of human DNA from one type of heterogeneous sample, stool, has proven difficult. See Villa, et al., Gastroenterol., 110: 1346-1353 (1996) (reporting that only 44.7% of all stool specimens, and only 32.6% of stools from healthy individuals produced sufficient DNA for mutation analysis). Other reports in which adequate DNA has been obtained have reported low sensitivity in identifying a patient's disease status based upon a single cancer-associated mutation. See Eguchi, et al., Cancer, 77: 1707-1710 (1996) (using a p53 mutation as a marker for cancer).
Investigators have attempted to analyze mutations in DNA of tumor cells shed into luminal areas, such as the colon, bile ducts, blood vessels and the like. Such attempts have only been successful when there is a known mutation and a relatively high concentration of cellular material has been found. See e.g., Mulcahy, et al., Ann. Oncol. 10 Suppl 4:114-117 (1999). No attempts have been made to correlate disease status with DNA integrity in shed cellular material.
SUMMARY OF THE INVENTION
The present invention provides that the integrity of nucleic acids in biological samples comprising shed cellular material is an indicator of the disease status of the patient from whom the sample was obtained. According to the invention, certain tissue or body fluid samples, especially those described below, contain debris from cells that have been shed from surrounding organs or tissue. In healthy patients, such debris is the result of apoptosis as part of the normal cell cycle. Apoptosis reduces nucleic acid integrity, so that only small-fragment nucleic acids exist in exfoliated cellular debris in healthy individuals. To the contrary, in diseases such as cancer in which cell cycle mechanisms are destroyed or impaired, cellular debris comprises high-integrity nucleic acids (i.e., nucleic acids that have not been degraded by apoptosis ). Thus, methods of the invention comprise using nucleic acid integrity as a measure of patient disease status. Integrity can be measured by any convenient means. Preferred means include the amount of nucleic acid in a sample, the length of nucleic acids in a sample, or the molecular weight of nucleic acids in a sample.
The invention provides methods for detecting disease in a patient based upon the integrity of patient nucleic acids present in a specimen or sample obtained from the patient. According to methods of the invention, a tissue or body fluid specimen containing sloughed cellular debris obtained from a patient having a disease contains an amount of intact nucleic acid that is greater than would be expected in such a specimen obtained from a healthy patient. Thus, a measure of intact nucleic acid in a patient sample is indicative of the overall disease status of the patient. As used herein, “intact” refers to nucleic acids that are longer than those expected to be present as a result of apoptosis. The invention is equally applicable to human and to veterinary uses. Accordingly, “patient” as defined herein means humans or other animals.
A healthy patient generally produces cellular debris through normal apoptotic degradation, resulting in relatively short nucleic acid fragments in samples derived from luminal tissue and fluids. Patients having a disease generally produce cells and cellular debris, a proportion of which has avoided normal cell cycle regulation, resulting in relatively long, intact nucleic acid fragments. Without being held to theory, the present invention takes advantage of this and other insights concerning the ways in which cells respond to diseases, especially diseases associated with genetic abnormalities (either induced or inherited). As a result, it has been discovered that the disease status of a patient is determined by analysis of patient nucleic acids produced in specimens obtained from the patient. Most preferably, such specimens are those most likely to contain sloughed cellular debris. Such specimens include, but are not limited to, stool, blood serum or plasma, sputum, pus, colostrum, and others. In diseases, such as cancer, in which genomic instabilities or abnormalities have interfered with normal cell cycle regulation, specimens such as those identified above contain relatively intact nucleic acid fragments. The presence of such fragments is a general diagnostic screen for disease.
Accordingly, methods of the invention comprise screening a patient for disease by analysis of the integrity of nucleic acids in a tissue or body fluid specimen obtained from the patient. Preferred specimens include those comprising shed cells or cellular debris. Thus, highly-preferred specimens are those not containing an abundance of intact (non-exfoliated) cells. Such preferred specimens comprise stool, sputum, urine, bile, pancreatic juice, and blood serum or plasma, all of which contain shed cells or cellular debris. Methods of the invention are especially useful as screens for cancer. Cancer is a disease thought to be associated with genomic instabilities, and specifically with the loss of control over the normal cell cycle. Thus, tumor cells are typically intact and routinely are shed into, for example, stool, sputum, urine, bile, pancreatic juice, and blood. Such shed cells and cellular debris contain higher integrity nucleic acids compared to those found in specimens obtained from a healthy patient. There are numerous ways in which the integrity of nucleic acids in a patient specimen are measured as a screen for disease.
In a preferred embodiment, nucleic acid integrity is measured by the ability to amplify nucleic acids in a sample. Thus, a preferred method comprises conducting in a tissue or body fluid sample an amplification reaction using as a template a nucleic acid locus suspected to be in the sample. If the amount of amplification product (amplicon) is greater than the amount of amplicon expected to be present in a normal sample (e.g., one not having the disease being screened), the sample is determined to be positive. In some cases, the presence of any amplification product is sufficient to justify a positive screen for disease. It is preferable that, in the case of DNA, the amplification reaction is a polymerase chain reaction (PCR) or, in the case of RNA, that the a

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