CA2384368A1 - Method of detecting or screening cancer or precancer by determining the amount and integrity of nucleic acids - Google Patents
Method of detecting or screening cancer or precancer by determining the amount and integrity of nucleic acids Download PDFInfo
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- CA2384368A1 CA2384368A1 CA002384368A CA2384368A CA2384368A1 CA 2384368 A1 CA2384368 A1 CA 2384368A1 CA 002384368 A CA002384368 A CA 002384368A CA 2384368 A CA2384368 A CA 2384368A CA 2384368 A1 CA2384368 A1 CA 2384368A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
Abstract
The present invention provides methods for detecting disease, specially cancer, by analysis of a patient sample to determine the integrity of nucleic acids in the sample.
Description
METHODS FOR DISEASE DETECTION
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 s 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, ~o 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 i s 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 oligonucieotide fragments of characteristically short length. In fact, those characteristic DNA cleavage patterns have been proposed as an assay for apoptosis.
2o 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 2~ 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 s 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 1o 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 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 2o 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 2s 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 3o 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 s 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 io 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 is 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 2o 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 2s 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 3o 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.
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 s 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, ~o 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 i s 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 oligonucieotide fragments of characteristically short length. In fact, those characteristic DNA cleavage patterns have been proposed as an assay for apoptosis.
2o 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 2~ 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 s 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 1o 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 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 2o 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 2s 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 3o 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 s 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 io 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 is 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 2o 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 2s 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 3o 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) s 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 amplification io reaction is reverse transcriptase PCR. Primers are designed to amplify the locus or loci chosen for analysis. For purposes of the invention a "genomic locus" is any genetic element, including but not limited to a coding region of a gene, a non-coding nucleic acid region, a regulatory element of a gene, an intron or RNA. It is not required that the target genomic loci be associated with any specific disease, as an increase in is amplifiable nucleic acid is itself diagnostic.
In one preferred embodiment, the presence of a single high molecular weight amplicon is a positive screen. Preferably, a fragment of about 1.3 Kb or greater is measured as an indicator of high integrity nucleic acids in the patient sample.
In a highly-preferred embodiment, a profile of amplification products across a 2o range of nucleic acid fragments of different lengths is produced. In a preferred embodiment, a series of amplification reactions is conducted at a single genomic locus, each reaction being designed to amplify a fragment of unique length. If detectable amplicon is produced in each reaction, or in a number of reactions greater than expected in a sample obtained from a healthy patient, the sample is determined to be 2s positive. For example, attempts are made to amplify fragments of 200 bp, 400 bp, 800 bp, 1.3 Kb, 1.8 Kb, and 2.4 Kb at the same genomic locus. In a sample obtained from a healthy individual (a "normal" sample), it would be expected that little or no amplification product is observed, especially when the longer portions of the locus are used as the template. To the contrary, at least some proportion of cells and cellular debris in a 3o sample obtained from a diseased patient will contain intact fragments.
In another embodiment, a profile of amplification products across a range of nucleic acid fragments of different lengths is produced by a series of amplification reactions conducted on a series of different genomic loci, each reaction being designed to amplify a fragment of unique length. If detectable amplicon is produced in each reaction, or in a number of reactions greater than expected in a sample obtained from a patient not having the disease being screened, the sample is determined to be positive.
According to methods of the invention, normal samples do not produce significant amounts of detectable amplicon at any length significantly greater than the typical apoptotic fragment (about 175 bp). Accordingly, whether primers are spaced to amplify fragments of only one length at a given genomic locus, or whether a series of amplifications at the locus are conducted, differences are readily observable between normal and diseased samples.
to As detailed below, methods of the invention are useful to detect disease, preferably cancer or precancer, in biological samples comprising shed cells or cellular debris. For example, the presence in a patient stool sample of amounts of nucleic acid, preferably DNA, above a predetermined threshold for healthy patients is indicative that the patient has cancer. Follow-up analysis is used to determine where the disease is resides. However, the general disease screen is effective independent of the locus of the disease and the specimen taken for analysis. Thus, while the analysis of nucleic acids in stool is predictive of disease generally, it does not necessarily indicate that the disease is of gastrointestinal origin. However, follow-up screening based, for example, on mutational analysis, is adequate to identify the locus of disease. Numerous 2o mutational analyses are known in the art and include, for example, U.S.
Patent No.
5,670,325, incorporated by reference herein.
In an alternative embodiment, screening of patient samples by detecting amounts of nucleic acid in the sample is combined with an assay for apoptotic cell activity. Such assays may be combined with detecting amounts of nucleic acid in a patient sample as 2s a screen for disease status. A positive screen is one that produces both:
(1) an amount of nucleic acid that is greater than the amount expected to be present in a normal sample (e.g., one not having the disease being screened), and (2) an amount of apoptotic cell activity that is less than that expected to be present in a normal sample.
In a highly preferred embodiment, methods of the invention comprise analyzing a 3o plurality of genomic loci to determine an amount of amplifiable nucleic acid present at each locus. Analysis across multiple loci using methods of the invention may increase the sensitivity of the screening assay.
In one preferred embodiment, the presence of a single high molecular weight amplicon is a positive screen. Preferably, a fragment of about 1.3 Kb or greater is measured as an indicator of high integrity nucleic acids in the patient sample.
In a highly-preferred embodiment, a profile of amplification products across a 2o range of nucleic acid fragments of different lengths is produced. In a preferred embodiment, a series of amplification reactions is conducted at a single genomic locus, each reaction being designed to amplify a fragment of unique length. If detectable amplicon is produced in each reaction, or in a number of reactions greater than expected in a sample obtained from a healthy patient, the sample is determined to be 2s positive. For example, attempts are made to amplify fragments of 200 bp, 400 bp, 800 bp, 1.3 Kb, 1.8 Kb, and 2.4 Kb at the same genomic locus. In a sample obtained from a healthy individual (a "normal" sample), it would be expected that little or no amplification product is observed, especially when the longer portions of the locus are used as the template. To the contrary, at least some proportion of cells and cellular debris in a 3o sample obtained from a diseased patient will contain intact fragments.
In another embodiment, a profile of amplification products across a range of nucleic acid fragments of different lengths is produced by a series of amplification reactions conducted on a series of different genomic loci, each reaction being designed to amplify a fragment of unique length. If detectable amplicon is produced in each reaction, or in a number of reactions greater than expected in a sample obtained from a patient not having the disease being screened, the sample is determined to be positive.
According to methods of the invention, normal samples do not produce significant amounts of detectable amplicon at any length significantly greater than the typical apoptotic fragment (about 175 bp). Accordingly, whether primers are spaced to amplify fragments of only one length at a given genomic locus, or whether a series of amplifications at the locus are conducted, differences are readily observable between normal and diseased samples.
to As detailed below, methods of the invention are useful to detect disease, preferably cancer or precancer, in biological samples comprising shed cells or cellular debris. For example, the presence in a patient stool sample of amounts of nucleic acid, preferably DNA, above a predetermined threshold for healthy patients is indicative that the patient has cancer. Follow-up analysis is used to determine where the disease is resides. However, the general disease screen is effective independent of the locus of the disease and the specimen taken for analysis. Thus, while the analysis of nucleic acids in stool is predictive of disease generally, it does not necessarily indicate that the disease is of gastrointestinal origin. However, follow-up screening based, for example, on mutational analysis, is adequate to identify the locus of disease. Numerous 2o mutational analyses are known in the art and include, for example, U.S.
Patent No.
5,670,325, incorporated by reference herein.
In an alternative embodiment, screening of patient samples by detecting amounts of nucleic acid in the sample is combined with an assay for apoptotic cell activity. Such assays may be combined with detecting amounts of nucleic acid in a patient sample as 2s a screen for disease status. A positive screen is one that produces both:
(1) an amount of nucleic acid that is greater than the amount expected to be present in a normal sample (e.g., one not having the disease being screened), and (2) an amount of apoptotic cell activity that is less than that expected to be present in a normal sample.
In a highly preferred embodiment, methods of the invention comprise analyzing a 3o plurality of genomic loci to determine an amount of amplifiable nucleic acid present at each locus. Analysis across multiple loci using methods of the invention may increase the sensitivity of the screening assay.
As will be exemplified in detail below, methods of the invention comprise screening a biological sample for an abnormality in a nucleic acid by conducting an amplification reaction using as a template a nucleic acid suspected or expected to be in the sample; determining ari amount of amplification product obtained;
comparing the s amount of amplicon obtained to a standard amount of amplification product;
and identifying a sample as having an abnormality in a nucleic acid if the amount of amplification product differs from the standard amount. In a preferred embodiment, a standard amount of amplification product is determined by amplification of a locus, or portion thereof, being screened (e.g., an intact, wild-type nucleic acid) in a known to normal sample (one obtained from an individual known not to have the disease being screened). Also in preferred embodiments, a standard amount is determined by reference to the art. In certain embodiments of the invention, the standard amount is essentially no detectable amplicon due to the lack of high-integrity nucleic acids in the sample. Accordingly, any detectable amplicon in a patient sample is indicative of a ~s positive screen. That is the case especially when a large (e.g., 1.8 Kb or 2.4 Kb) fragment is being screened. Finally, the standard amount can be a molecular weight marker on, for example, an electrophoretic gel.
In a preferred embodiment of the invention, the sample is prepared from a specimen selected from the group consisting of stool, sputum, blood, urine, 2o cerebrospinal fluid, seminal fluid, saliva, breast nipple aspirate, and biopsy tissue.
However, any tissue or body fluid specimen may be used according to methods of the invention. Especially preferred are samples of luminal fluid because such samples are generally free of intact, healthy cells. Such samples include blood, urine, bile, pancreatic juice, stool, sputum, pus, and the like.
2s Also in a preferred embodiment, the nucleic acid or nucleic acids being interrogated is (are) DNA. In a more particular embodiment, the nucleic acid being analyzed is selected from a coding region of a gene, or portion thereof, a noncoding nucleic acid region, or portion thereof, a regulatory element of a gene or a portion thereof, and an unidentified fragment of genomic DNA. Also in a preferred embodiment, 3o the nucleic acid being interrogated is RNA. As is appreciated by the skilled artisan, any genomic locus is amenable to screening according to the invention. The particular locus or loci chosen for analysis depends, in part, on the disease being screened, and the convenience of the investigator. It is not necessary that the locus or loci chosen for analysis be correlated with any specific disease because methods of the invention contemplate measuring either the total nucleic acid in a sample or amplifiable nucleic acid in a sample as an indicator of overall disease status or the presence and/or extent of apoptosis in the sample. However, disease-associated loci (those in which a s mutation is indicative, causative, or otherwise evidence of a disease) can be used.
Preferred disease-associated loci include p53, apc, MSH-2, dcc, scr, c-myc, B-catnenin, mlh-1, pms-1, pms-2, pol-delta, and bax.
The amount of amplification product may be determined by any suitable or convenient means. Preferably, the amount of amplification product is determined by gel io electrophoresis. Labels, such as fluorescent or radioactive labels, may be used. The amounts of amplification product produced may be compared to standard amounts by any suitable or convenient means, including, but not limited to visual comparison, machine-driven optical comparison, densitometry, mass spectroscopy, hybrid capture, and other known means. The amplification reaction itself can be any means for is amplifying nucleic acid, including, but not limited to PCR, RT-PCR, OLA, rolling circle, single base extension, and others known in the art. The amplification product can also be measured by signal amplification techniques, such as branch chain amplification (Chiron). Methods of the invention are useful with any platform for the identification, amplification, sequencing, or other manipulation of nucleic acids. For example, zo methods of the invention can be applied to ligase chain reaction, strand displacement (Becton-Dickinson), and others.
Also in a preferred embodiment of the invention, a series of amplification reactions is conducted on a single genomic locus. Each amplification reaction in the series is designed to amplify a fragment of a different length. In a preferred 2s embodiment, the target fragment lengths are 200 bp, 400 bp, 800 bp, 1.3 Kb, 1.8 Kb, and 2.4 Kb. Primers for amplification are designed according to knowledge in the art in order to amplify template, if present, of the desired length at the desired locus. A
positive screen is one that produces amplicon in at least one, and preferably at least two of the series of amplification reactions. As noted above, a normal sample which 3o has undergone or which is undergoing apoptosis typically contains little or no fragments of significant length. Thus, a series of amplification reactions targeting fragments from about 200 by to about 2.4 Kb and longer reveals samples that contain nucleic acids that have avoided apoptosis as evidenced by the amplification of large fragments.
_$_ Preferred methods of the invention also comprise conducting amplification reactions on a series of different genomic loci. Preferably, from about 2 to about 7 loci are used. However, the precise number of interrogated loci is determined by the individual investigator based upon the disease to be detected or based upon s convenience. According to methods of the invention, primers are designed to amplify nucleic acid (preferably DNA) at each of the chosen loci. A sample in which at least one locus, preferably at least two loci, and most preferably at least three loci produce detectable amplification product is considered a positive sample. The lengths of fragments to be amplified in this assay may be varied, but are preferably at least about Io 180 by each in length. It is not necessary that the same length fragments be amplified at each of the chosen loci.
Methods of the invention also comprise conducting a series of amplification reactions at a series of different genomic loci. Each amplification reaction in the series is designed to amplify a fragment of a different length. Preferably, from about 2 to about Is 7 amplification reactions on about 2 to about 7 loci are used. However, the precise number of interrogated loci is determined by the individual investigator based upon the disease to be detected or based upon convenience. In a preferred embodiment, the target fragment lengths are 200 bp, 400 bp, 800 bp, 1.3 Kb, 1.8 Kb, and 2.4 Kb.
Primers for amplification are designed according to knowledge in the art in order to 2o amplify template if present. It is preferred, but not necessary, that the same length fragments be amplified at each of the chosen loci. A positive screen is one that produces amplicon in at least one, and preferably at least two of the series of amplification reactions and in which at least one locus, preferably at least two loci, and most preferably at least three loci produce detectable amplification product.
As noted 2s above, a normal sample which has undergone or which is undergoing apoptosis typically contains little or no fragments of significant length. Thus, a series of amplification reactions targeting fragments from about 200 by to about 2.4 Kb and longer reveals samples that contain nucleic acids that have avoided apoptosis as evidenced by the amplification of large fragments.
3o Methods of the invention may also be used to assess the integrity of DNA in a biological sample. Such methods comprise conducting an amplification reaction using at least two loci suspected to be in the sample as templates; determining which loci produce detectable amplicon; and assessing the integrity of DNA in the sample as a _g_ function of the number of loci producing amplicon. The integrity of DNA in the sample is high when amplicon is produced in one or more of the amplification reactions.
This method is especially useful for determining whether a heterogeneous sample has sufficient nucleic acid for measurement. Accordingly, such methods are used to screen s or to "qualify" samples for further analysis (e.g., genetic, biochemical, cytological, or other analyses).
Methods of the invention may also be used to assess fetal abnormalities by conducting amplification reactions on nucleic acids in maternal blood. Just as described above, the ability to amplify significant amounts of nucleic acid is an indicator of a to genomic instability. A baseline for comparison of the extent of nucleic acid amplification can be amounts of nucleic acids from known normal samples. The amount of amplification obtained from fetal samples is placed on a continuum, and the investigator must analyze any given sample in terms of the amount of fetal nucleic acid produced in various disease states and in normal samples.
is Methods of the invention are useful as diagnostic screening methods. Often it is desirable to perform follow-up testing on a patient in order to confirm a suspected disease state. Such follow-up procedures are determined based upon the disease state being interrogated. For example, a colonoscopy may be suggested in a case in which a stool sample is positively screened according to methods of the invention.
Such follow-2o up procedures are contemplated herein as part of the invention.
Methods of the invention are useful as screens for a wide range of disease states. In addition to colon cancers and adenomas, methods of the invention are useful to screen for other diseases, for example, as screens for lymphomas, or stomach, lung, liver, pancreas, prostate, kidney, testicular, bladder, uterus, or ovarian cancers or 2s adenomas. In addition to cancer, methods of the invention are useful, for example, as screens for diseases such as inflammatory bowel syndrome, inflammatory bowel disease, Crohn's disease, and others in which a genomic instability is thought to play a role. Methods of the invention are especially useful as screens for any disease that impairs the proper function of the gastrointestinal system; most especially diseases of 3o the colon. Methods of the invention are also useful to screen for apoptosis in a cellular sample. The profile of amplifiable DNA in a sample is correlated with proteins that have been associated with disease. For example up regulation of the apoptosis protein, survivin, is correlated with increased amounts of amplifiable DNA, as is the Ras oncogene, as well as other oncogenes and their gene products.
Methods of the invention are also useful as assays for apoptosis. The presence of high-integrity fragments or large quantities of nucleic acids in a sample indicates that s the sample was derived from cells that did not proceed through apoptosis.
The absence of such fragments or quantities indicates that cells that contributed to the sample did undergo apoptosis. Accordingly, an apoptotic activity assay of the invention, either alone or in combination with other assays for genomic instability, are useful as screens for disease.
io Finally, methods of the invention can be carried out by hybrid capture. For example, hybrid capture and subsequent analysis of the captured fragments can be used to determine the nucleic acid integrity of a sample.
The invention also provides a profile of nucleic acid fragments indicative of disease. A preferred profile is obtained through methods described above.
Preferred is profiles comprise nucleic acids having between about 200 by and about 2.4 Kb obtained in a patient sample comprising cellular debris according to methods described herein. A
highly preferred profile contains at least one nucleic acid of at least 1.3 Kb.
Other objects and advantages of the invention are apparent upon consideration of the following drawings and detailed description thereof.
2o Description of the Drawings Figure 1 is a gel photograph showing results of amplification of K-ras (exon 1 ) DNA isolated from stool using forward and reverse primers spaced about 200 by apart.
The band intensity relates to the amount of 200 by product or greater in the sample.
Lanes 1-4 are results from patients with cancer or adenoma, lane 5 is a positive control, Zs lanes 6-10 are from patients who did not have cancer or adenoma, lanes 11-12 are negative controls, and lanes 13-18 are standards at the approximate molecular weight indicated in the figure. Amplifications were graded A through C, A being the most intense band, C being the least.
Figures 2-4 are gel photographs showing results of amplification of apc (exon 15) 3o DNA isolated from stool using forward and reverse primers spaced about 200 by apart.
The band intensity relates to the amount of 200 by product or greater in the sample.
Lanes 1-4 are results from patients with cancer or adenoma, lane 5 is a positive control, lanes 6-10 are from patients who did not have cancer or adenoma, lanes 11-12 are negative controls, and lanes 13-18 are standards at the approximate molecular weight indicated in the figure. Amplifications were graded A through C, A being the most intense band, C being the least.
s Figure 5 is a gel photograph showing results of amplification of p53 (exon 5) DNA
isolated from stool using forward and reverse primers spaced about 200 by apart. The band intensity relates to the amount of 200 by product or greater in the sample. Lanes 1-4 are results from patients with cancer or adenoma, lane 5 is a positive control, lanes 6-10 are from patients who did not have cancer or adenoma, lanes 11-12 are negative io controls, and lanes 13-18 are standards at the approximate molecular weight indicated in the figure. Amplifications were graded A through C, A being the most intense band, C being the least.
Figure 6 is a gel photograph showing results of amplification of p53 (exon 7) DNA
isolated from stool using forward and reverse primers spaced about 200 by apart. The is band intensity relates to the amount of 200 by product or greater in the sample. Lanes 1-4 are results from patients with cancer or adenoma, lane 5 is a positive control, lanes 6-10 are from patients who did not have cancer or adenoma, lanes 11-12 are negative controls, and lanes 13-18 are standards at the approximate molecular weight indicated in the figure. Amplifications were graded A through C, A being the most intense band, 2o C being the least.
Figure 7 is a gel photograph showing results of amplification of p53 (exon 8) DNA
isolated from stool using forward and reverse primers spaced about 200 by apart. The band intensity relates to the amount of 200 by product or greater in the sample. Lanes 1-4 are results from patients with cancer or adenoma, lane 5 is a positive control, lanes 2s 6-10 are from patients who did not have cancer or adenoma, lanes 11-12 are negative controls, and lanes 13-18 are standards at the approximate molecular weight indicated in the figure. Amplifications were graded A through C, A being the most intense band, C being the least.
Figure 9-10 are gel photographs of results of amplification of DNA from stool 30 samples using forward and reverse primers spaced approximately 1.8 Kb apart. The band intensity shows the amount of 1.8 Kb or greater product. Lanes 1, 8, and 9 are negative controls, lanes 2, 3, and 5 are results from patients with cancer or adenoma, lanes 4, 6, and 7 are results from patients who did not have cancer or adenoma, and lanes 10-14 are molecular weight standards.
Figures 11 A and B are gel photographs of results of amplification of DNA in stool from a total of 30 patients and controls. The band intensity relates to the amount s of amplifiable DNA in the sample. Lanes N are negative controls, lanes 1, 3, 11, and 18 are results from patients which are indicative of the presence off cancer or adenoma, lanes 2, 4, 5-10, 12-17, and 19-30 are results from patients which are indicative of the absence of cancer or adenoma. The remaining lanes are markers or standards.
Figure 12 shows a schematic representation of the placement of the primers for to amplification in a method of the present invention. In this method, a single forward primer, F~, is used in conjunction with a series of reverse primers, R~ to R6, chosen to amplify progressively longer portions of the target.
Figure 13 shows a schematic representation of the placement of the primers for amplification in a method of the present invention. In this method, a series of forward is and reverse primer pairs, (F~ , R~) to (F3 , R3), are chosen to amplify portions of the target spaced at intervals along the target.
Detailed Description of the Invention The invention provides methods for the analysis of biological samples. Methods of the invention provide diagnostically-relevant information based upon the integrity of 2o nucleic acids in a biological sample. Normal biological samples (those not having indicia of the disease being screened), especially those comprising luminal tissue andlor fluid, typically comprise a majority of short-fragment, low-integrity nucleic acids (especially DNA) which are the result of degradation by apoptosis. When a mutation has caused genomic instability, the normal cell cycle may be disrupted and apoptotic 2s degradation may not occur at the rate expected in a normal sample. Methods of the invention screen for such disruptions.
Accordingly, preferred methods of the invention comprise determining an amount of amplifiable nucleic acid in a biological sample, and determining whether that amount is consistent with an amount expected in a normal sample. In many biological samples, 3o especially heterogeneous samples, there may be no detectable amplification product.
That is especially true when longer fragments are used as templates for amplification.
Generally, the probability that any given set of PCR primers will amplify a DNA fragment having a length exceeding the primer distance is expressed as of Fragments Amplified = (FL-PD)/(FL+PD) wherein FL is fragment length (in base pairs) and PD is primer distance (in base pairs). This equation assumes that sample DNA fragment lengths are uniformly distributed (i.e., there is no favored locus at which breaks occur).
s In a preferred embodiment, methods of the invention comprise amplifying sequences of different length in a sample, if present, in order to generate a profile of amplification products indicative of disease or the propensity for disease. In a preferred method, a sample is exposed to a set of PCR primers comprising a single forward primer, which may be a capture probe used to capture target fragments, and a plurality io of downstream reverse primers which hybridize to portions of a contiguous sequence (if present) in the sample. Amplifications using these primers will result in a series of amplification products, each having a different length, if the contiguous target sequence is present in the sample. The length of the amplification products are determined by the spacings between the forward primer and each of the downstream reverse primers. An is example is shown in Figure 12, which is a schematic representation showing placement of the primers for amplification.
If the target sequence, or a portion of it, is present in the sample, amplification will result in a series of fragments the length of which is dictated by the spacing of the primers. According to the principles adduced above, a sample from a diseased patient 2o will produce a profile of amplification products in the assay described above that differs from the profile obtained from a sample containing the smaller fragments expected to be produced as a result of normal apoptosis. In a preferred embodiment, the forward primer is designed to hybridize about 200 by upstream of the first reverse primer, and about 2.3 Kb upstream of the last reverse primer. Other reverse primers are designed 2s to hybridize at various locations between the first and last reverse primers. Preferred intervals between the forward primer and the various reverse primers are 200 by (F~-R~), 400 by (F~-R2), 800 by (F~-R3), 1.3 Kb, (F~-R4), 1.8 Kb (F~-R5), and 2.3 Kb (F~-R6).
The number and spacing of reverse primers is chosen at the convenience of the skilled artisan.
3o Also in a preferred embodiment, a hybrid capture probe is used to anchor a target sequence, preferably on a solid support (e.g., beads). A plurality of probes are then placed at various distances downstream of the capture probe. Those probes can be pairs of forward and reverse primers as discussed above, or they can be signal amplification probes, such as those used in Ligase Chain Reaction (LCR), and others used in the identification of sequences. The plurality of probes hybridize along the length of a target fragment if the target is present in the sample. Thus, by interrogating samples for the presence of the probes, one can determine the integrity of sequences s present in the sample. This can be done in numerous ways, including, but not limited to, hybrid capture, PCR, LCR, strand displacement, branched chain, or other assays known in the are that incorporate hybrid probes or primers in order to identify or quantitate sequence. A sample containing intact (high integrity) nucleic acids represents a positive screen according to the invention. In one embodiment, sample is ~o placed into wells (e.g., on a 96 well plate) containing support-bound capture probe. The capture probe immobilizes a target sequence, if present in the sample. Probes that hybridize to sequence downstream of the capture probe (downstream probes) are placed into each well, such that each downstream probe is spaced a unique distance apart from the common capture probe, and each well contains only one type of is downstream probe. Signal is then generated by, for example, amplification, or by standard ELISA procedure followed by amplification, or by LCR, or other methods mentioned above. The presence of signal in each well indicates the presence of sequence of at least the length between the capture probe and the downstream probe.
In an alternative embodiment, each well receives multiple different downstream probes, 2o which may be distinctly labeled, and the presence of labels) is correlated with the length of sequence presence in the sample.
A sample from a patient having, for example, cancer will produce amplicon between most or all of the primer pairs (depending, inter alia, on the length of the target fragments, on the spacing of the primers, and where on the target the primers are 2s spaced). Such a profile represents a positive screen for disease or the propensity for disease. A sample from a patient who does not have indicia of disease results in little or no amplification product in the assay described above. In a negative screen there may be amplification of small (e.g., 200 bp) fragments but there should be no amplification of larger fragments (i.e., fragments resulting from amplification between the forward primer 3o and spaced-apart reverse primers). In cancer diagnostics, the target fragment may optionally be an oncogene, a tumor suppressor, or any other marker associated with cancer. However, it is not necessary to use cancer-associated markers in methods of the invention, as such methods are based on the general recognition that samples indicative of disease contain a greater amount of intact nucleic acids and a greater amount of long fragment nucleic acids. Accordingly, any convenient target nucleic acid locus may be used in the methods of the invention.
The amplification reactions described above may be conducted according to any s suitable or convenient protocol and the fragment size of the resulting amplification products (if any) may be determined by any suitable or convenient means.
In an alternative embodiment, methods of the invention comprise conducting a series of amplification reactions on a contiguous nucleic acid target fragment, each application reaction comprising one forward primer and one reverse primer, such that to pairs of forward and reverse primers are spaced at intervals on a contiguous fragment suspected to be in the sample. An example of this arrangement is shown in Figure 13.
Preferably, the spacings between each forward and reverse primer pair are equivalent.
In a positive screen, the assay described above will result in a series of same-size fragments for most if not all of the primer pairs. Such an array of amplification products is evidences a contiguous target sequence indicative of disease (see above). A
sample from a disease-free patient should produce little or no amplification product, but in any case will not produce the contiguous array of amplification products expected from a sample containing a relatively intact diagnostic target sequence.
Each of the methods described above are based upon the principle that an intact 2o nucleic acid, or a segment of an intact nucleic acid, in a sample is diagnostic. Thus, variations on the methods described above are contemplated. Such variations include the placement of primers, the number of primers used, the target sequence, the method for identifying sequences, and others. For example, in the method depicted in Figure 13, and described above, it is not necessary that the numbers of forward and reverse 2s primers be equal. A forward primer may, for example, be used to amplify fragments between two reverse primers. Other variations in primer pair placement are within the skill in the art, as are details of the amplification reactions to be conducted. Finally, as represented in Figures 12 and 13, capture probes may be used in methods of the invention in order to isolate a chosen target sequence.
3o The following examples provide further details of methods according to the invention. For purposes of exemplification, the following examples provide details of the use of the method if the present invention in colon cancer detection.
Accordingly, while exemplified in the following manner, the invention is not so limited and the skilled artisan will appreciate its wide range of application upon consideration thereof.
Exemplary Method for the Detection of Colon Cancer The following example relates to screening for colon cancer in voided stool s samples. Based upon the principles upon which the invention is based (see above), the same analysis can be performed on other samples, such as those mentioned above, with the same results as shown herein.
For the analysis of stool samples, preferred methods of the invention comprise obtaining at least a cross-sectional or circumferential portion of a voided stool as taught to in U.S. patent number 5,741,650, and co-pending, co-owned U.S. patent application serial number 09/059,718, both of which are incorporated by reference herein.
While a cross-sectional or circumferential portion of stool is desirable, methods provided herein are conducted on random samples obtained from voided stool, which include smears or scrapings. Once obtained, the stool specimen is homogenized. A preferable buffer for is homogenization is one that contains at least 16 mM
ethylenediaminetetraacetic acid (EDTA). However, as taught in co-pending, co-owned U.S. patent application serial number 60/122,177, incorporated by reference herein, it has been discovered that the use of at least 150 mM EDTA greatly improves the yield of nucleic acid from stool.
Thus, a preferred buffer for stool homogenization comprises phosphate buffered saline, 20 20-100 mM NaCI or KCI, at least 150 mM EDTA, and optionally a detergent (such as SDS) and a proteinase (e.g., proteinase K).
After homogenization, nucleic acid is preferably isolated from the stool sample.
Isolation or extraction of nucleic acid is not required in all methods of the invention, as certain detection techniques can be adequately performed in homogenized stool without 2s isolation of nucleic acids. In a preferred embodiment, however, homogenized stool is spun to create a supernatant containing nucleic acids, proteins, lipids, and other cellular debris. The supernatant is treated with a detergent and proteinase to degrade protein, and the nucleic acid is phenol-chloroform extracted. The extracted nucleic acids are then precipitated with alcohol. Other techniques can be used to isolate nucleic acid 3o from the sample. Such techniques include hybrid capture, and amplification directly from the homogenized stool. Nucleic acids can be purified and/or isolated to the extent required by the screening assay to be employed. Total DNA is isolated using techniques known in the art.
Screening Assay Protocol The size of human DNA fragments obtained above can be determined by numerous means. For example, human DNA can be separated using gel electrophoresis. A 3% agarose gel is prepared using techniques known in the art. See s Ausubel et. al., Short Protocols in Molecular Biology, John Wiley & Sones, 1195, pgs. 2-23-2-24, incorporated by reference herein. The size of human DNA fragments is then determined by comparison to known standards. Fragments greater than about 200 by provide a positive screen. While a diagnosis can be made on the basis of the screen alone, patients presenting a positive screen are preferably advised to seek follow-up ~o testing to render a confirmed diagnosis.
A preferred means for determining human DNA fragment length uses PCR.
Methods for implementing PCR are well-known. In the present invention, human DNA
fragments are amplified using human-specific primers. Amplicon of greater than about 200 by produced by PCR represents a positive screen. Other amplification reactions is and modifications of PCR, such as ligase chain reaction, reverse-phase PCR, Q-PCR, and others may be used to produce detectable levels of amplicon. Amplicon may be detected by coupling to a reporter (e.g. fluorescence, radioisotopes, and the like), by sequencing, by gel electrophoresis, by mass spectrometry, or by any other means known in the art, as long as the length, weight, or other characteristic of the amplicons 2o identifies them by size.
Examples Experiments were conducted to determine whether characteristics of amplifiable DNA in stool were predictive of cancer or precancer in patients from whom stools samples were obtained. In the first experiment, the amount of amplifiable DNA
was 2s measured in each of several stool samples using PCR amplification to detect DNA
fragments in the sample of at least 200 base pairs in length. The second experiment determined the amount of long fragments (greater than 200 base pair) in the same samples, and then determined ratios of long product to short product. The third experiment determined a profile of amplification products with nucleic acid fragment 30 lengths of 200 bp, 400 bp, 800 bp, 1.3 Kb, 1.8 Kb and 2.4 Kb. The fourth and fifth experiments were clinical studies correlating the integrity of nucleic acids in patient stool samples with overall patient disease status.
Stool samples were collected from 9 patients who presented with symptoms or a medical history that indicated that a colonoscopy should be performed. Each stool sample was frozen. Immediately after providing a stool sample, each patient was given s a colonoscopy in order to determine the patient's disease status. Based upon the colonoscopy results, and subsequent histological analysis of biopsy samples taken during colonoscopy, individuals were placed into one of two groups: normal or abnormal. The abnormal group consisted of patients with cancer or with an adenoma of at least 1 cm in diameter. Based upon these results, 4 of the 9 patients were placed to into the abnormal group.
The samples were screened by hybrid capturing human DNA, and determining the amount of amplifiable DNA having at least 200 base pairs. Each frozen stool specimen, weighing from 7-33 grams, was thawed and homogenized in 500 mM Tris, 16 mM EDTA, and 10 mM NaCI, pH 9.0 at a volume, to mass ratio of 3:1. Samples is were then rehomogenized in the same buffer to a final volume-to-mass ratio of 20:1, and spun in glass macro beads at 2356 xg. The supernatant was collected and treated with SDS and proteinase k. The DNA was then phenol-chloroform extracted and precipitated with alcohol. The precipitate was suspended in 10 mM Tris and 1 mM
EDTA (1 x TE), pH 7.4. Finally, the DNA was treated with Rnase.
2o Human DNA was isolated from the precipitate by sequence-specific hybrid capture. Biotynilated probes against portions of the p53, K-ras, and apc genes were used.
The K-ras probe was 5'GTGGAGTATTTGATAGTGTATTAACCTTATGTGTGAC
3' (SEQ ID NO: 1).
2s There were two apc probes: apc-1309 was 5'TTCCAGCAGTGTCACAGCACCCTAGAACCAAATCCAG 3' (SEQ ID NO: 2), and apc-1378 was 5'CAGATAGCCCTGGACAAACAATGCCACGAAGCAGAAG 3' (SEQ ID
NO: 3).
There were four probes against p53, the first (hybridizing to a portion of exon 5) 3o was 5'TACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGG3' (SEQ ID N0:4), the second (hybridizing to a portion of exon 7) was 5'ATTTCTTCCATACTACTACCCATCGACCTCTCATC3' (SEQ ID NO: 5), the third, also hybridizing to a portion of exon 7 was 5'ATGAGGCCAGTGCGCCTTGGGGAGACCTGTGGCAAGC3' (SEQ ID NO: 6); and finally, a probe against exon 8 had the sequence 5'GAAAGGACAAGGGTGGTTGGGAGTAGATGGAGCCTGG3' (SEQ ID NO: 7).
A 10 p.1 aliquot of each probe (20 pmol/capture) was added to a suspension s containing 300 p1 DNA in the presence of 310 ~I 6M GITC buffer for 2 hours at room temperature. Hybrid complexes were isolated using streptavidin-coated beads (Dynal).
After washing, probe-bead complexes were suspended at 25° C for 1 hour in 0.1x TE
buffer, pH7.4. The suspension was then heated for 4 minutes at 85° C, and the beads were removed.
lo Captured DNA was then amplified using PCR, essentially as described in U.S.
Patent No. 4,683,202, incorporated by reference herein. Each sample was amplified using forward and reverse primers through 7 loci (Kras, exon 1, APC exon 15 (3 separate loci), p53, exon 5, p53, exon 7, and p53, exon 8) in duplicate (for a total of 14 amplifications for each locus). Seven separate PCRs (40 cycles each) were run in is duplicate using primers directed to detect fragments in the sample having 200 base pairs or more. Amplified DNA was placed on a 4% Nusieve (FMC Biochemical) gel (3%
Nusieve, 1 % agarose), and stained with ethidium bromide (0.5 ~g/ml). The resulting amplified DNA was graded based upon the relative intensity of the stained gels. The results are shown in Figures 1-7. Each Figure represents the results for all 9 patients 20 (including standards) for the seven different loci that were amplified. As shown in the Figures, each sample from a patient with cancer or adenoma was detected as a band having significantly greater intensity than the bands associated with samples from patients who did not have cancer or precancer. All four cancer/adenoma patients identified using colonoscopy were correctly identified by determining the amount of 2s amplifiable DNA 200 base pairs or greater in length. As shown in Figures 1-7, the results were the same regardless of which locus was amplified. Accordingly, the amount of 200 by or greater DNA in a sample was predictive of patient disease status.
An experiment was conducted that was essentially identical to the one described 3o above in Example 1, but forward and reverse primers were placed such that fragments of about 1.8 Kb and above were amplified.
DNA was prepared as described above. Forward and reverse primers were spaced so as to hybridize approximately 1.8 Kb apart on three different loci (Kras, exon 1, APC, exon 15, and p53 exon 5). Thirty-three rounds of amplification were performed, and the resulting DNA was placed on a 3% agarose gel. The results are shown in s Figures 8-10. As shown in the Figures (which show results from three separate experiments to amplify and detect "long" product), samples from individuals having cancer or precancer produced large amounts of high-molecular weight (in this case 1.8 Kb and above) DNA; whereas samples from patients who did not have cancer or precancer produced no DNA in the range of about 1.8 Kb and higher. Thus, the io presence of high-molecular weight DNA was indicative of the disease status of the patient.
An experiment was conducted to determine the molecular weight profile of DNA
from samples collected and prepared as part of a blind study on 30 patients who is presented at the Mayo Clinic with suspected gastrointestinal disorders.
Stool samples were obtained, and DNA was isolated as described above.
Prior to amplification, DNA was isolated from the samples by hybrid capture.
Biotynilated probes against portions of the BRCA1, BRCA2, p53, APC genes were used.
2o The BRCA1 probe was 5'GATTCTGAAGAACCAACTTTGTCCTTAACTAGCTCTT3' (SEQ ID NO: 8).
The BRCA2 probe was 5'CTAAGTTTGAATCCATGCTTTGCTCTTCTTGATTATT3' (SEQ ID NO 9).
The APC1 probe was 2s 5'CAGATAGCCCTGGACAAACCATGCCACCAAGCAGAAG3' (SEQ ID NO 10).
The p53 probe, hybridizing to a portion of exon 5, was 5'TACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGG3' (SEQ ID N0:4).
The APC2 probe was 5'GAAGTTCCTGGATTTTCTGTTGCTGGATGGTAGTTGC3' (SEQ ID NO 11).
A 300 ~I aliquot of sample was placed in 300 ~I of 6 M guanidine isothiocyanate buffer with 10 p1 of each capture probe, and incubated overnight at 25 C.
Captured DNA was isolated using 100 ~I capture beads incubated for one hour at room temperature. The DNA was eluted off the beads and PCR amplified under standard PCR conditions.
According to methods of the invention, amplification reactions were conducted using forward and reverse primers through the 5 loci for each sample. Forward and s reverse primers were spaced to amplify fragments of 200 bp, 400 bp, 800 bp, 1.3 Kb, 1.8 Kb, and 2.4 Kb. Each of 30 PCR reactions was run for 36 cycles. Amplicon was run on a 3% Seakeam gel, and stained with ethidium bromide. The results are shown in Figures 11A and 11 B. Each figure represents the results for 15 of the 30 patients.
As shown in those figures, patients with cancer or adenoma have an increased Io yield of amplifiable DNA. That is especially true at the 1.8 Kb level and above. Thus, patients with cancer or adenoma not only produce more amplifiable DNA in their stool, but also produce larger DNA fragments than are produced in the stool of patients who do not have cancer. Thus, both an increased yield of amplifiable DNA and the presence of high molecular weight DNA, especially that at 1.8 Kb and above, were indicative of is patient disease status.
In this example, methods of the invention were correlated with clinical outcome in numerous patients who had a colorectal adenoma or colorectal cancer as diagnosed using colonoscopy, and 79 patients who were diagnosed as not having colorectal 2o cancer or adenoma. A stool sample was obtained from each of these patients and prepared as described above. Fragments of the 5 different loci referred to above were amplified using primers spaced 200, 400, 800, 1300, 1800, and 2400 base pairs apart using the protocol described above in Example 3. Each amplification was scored such that successful amplification of a fragment received a score of 1, and no amplification 2s received a score of 0. Since five loci were interrogated using 6 primer pairs each, the maximum score was 30 (successful amplification of all 6 fragments at all five loci). The cutoff for a positive screen was set at 21. The results are shown below.
Table 1 Table 2 Normals Adenomas Patient A a Score Patient A Score No No. a . P-003 29 Table 3 Carcinomas Patient A a Score No.
As shown above, methods of the invention are effective in screening for the presence of colorectal cancer and adenoma.
In this example, methods of the invention were used to detect non-colonic cancers in 28 patients.
A stool sample was obtained from each of the 28 patients. The sample was prepared as described above. Fragments of the 5 different loci referred to above were amplified using primers spaced 200, 400, 800, 1300, 1800, and 2400 base pairs apart using the protocol described above in Example 3. Each amplification was scored such that successful amplification of a fragment received a score of 1, and no amplification received a score of 0. Since five loci were interrogated using 6 primer pairs each, the maximum obtainable score was 30 (successful amplification of all 6 fragments at all five loci). A score of 21 was used as a cutoff between diseased and non-diseased patients.
The results are shown below.
Table 4 Supercolonic Cancers Patient SupercolonicAge Score No. Cancer P-145 Pancreas 68 30 P-164 Lun CA 68 30 P-166 Bile Duct 52 30 P-189 Bile Duct 43 30 P-190 Lun CA 50 30 P-019 Atypical 71 29 Findings in Stomach P-152 Lun CA 77 28 P-167 Pancreas 72 28 P-011 Lun CA 73 27 P-153 Pancreas 65 27 P-165 Lun CA 85 27 P-170 Duodenum 65 27 P-182 Barrett's 58 27 Eso ha us P-146 Bile Duct 63 26 P-081 Barrett's 74 26 Eso ha us P-151 Pancreas 49 25 P-155 Lun CA 60 25 P-156 Lun CA 57 25 P-150 Pancreas 78 23 P-149 Eso ha 59 19 us P-154 Eso ha 80 19 s P-169 Pancreas 71 19 P-168 Lun CA 63 18 P-180 Pancreas 67 13 P-144 Eso ha 59 9 us P-147 Stomach 57 7 P-148 Stomach 69 6 P-171 ~ Esophagus -76 0 As shown above, methods of the invention successfully screened 18 out of 27 patients who actually had non-colonic cancer. Only one patient was misdagnosed as having cancer when he did not. Thus, the methods of the invention are useful for non-invasive diagnosis of a non-specified cancerous disease state in a patient.
The threshold of 21 for a positive screen can be changed to accommodate desired sensitivities and specificities. For example, if 18 were the false negative results shown in Table 4 would be avoided. The skilled artisan knows how to set thresholds depending on the patient (e.g., a lower threshold for patients with symptoms than patients presenting with no symptoms), the disease being diagnosed, and the desired level of sensitivity and specificity. Regardless of the threshold, the principle of the invention remains that nucleic acid integrity is a viable marker for disease, and especially for cancer.
In addition, the propensity for disease may be measured using methods of the invention. For example, periodic molecular weight profiling in accordance with the methods of the invention may be used to monitor the disease state of a patient presenting no or minimal symptoms. Such longitudinal monitoring will determine whether a patient is progressing with increasing amounts of high integrity nucleic acids - indicating the desirability for follow-up examination.
What is claimed is:
SEQUENCE LISTING
<110> Shuber, Anthony P
<120> Methods for Disease Detection <130> EXT-034PC
<140>
<141>
<150> US 60/152,847 <151> 1999-09-08 <150> US 09/455,950 <151> 1999-12-7 <160> 11 <170> PatentIn Ver. 2.0 <210> 1 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:K-ras probe <400> 1 gtggagtatt tgatagtgta ttaaccttat gtgtgac 37 <210> 2 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:apc probe apc-1309 <400> 2 ttccagcagt gtcacagcac cctagaacca aatccag 37 <210> 3 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:apc probe apc-1378 <400> 3 cagatagccc tggacaaaca atgccacgaa gcagaag 37 <210> 4 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223>Description of ArtificialSequence:p53 exon 5 probe <400>4 tactcccctg caactgg 37 ccctcaacaa gatgttttgc <210>5 <211>35 <212>DNA
<213>Artificial Sequence <220>
<223>Description of ArtificialSequence:p53 exon 7 probe <400>5 atttcttcca tcatc 35 tactactacc catcgacctc <210>6 <211>37 <212>DNA
<213>Artificial Sequence <220>
<223>Description of ArtificialSequence:p53 exon 7 probe <400>6 atgaggccag ggcaagc 37 tgcgccttgg ggagacctgt <210>7 <211>37 <212>DNA
<213>Artificial Sequence <220>
<223>Description of ArtificialSequence:p53 exon 8 probe <400>7 gaaaggacaa agcctgg 37 gggtggttgg gagtagatgg <210>8 <211>37 <212>DNA
<213>Artificial Sequence <220>
<223>Description of ArtificialSequence:BRCAl probe <400>8 gattctgaag agctctt 37 aaccaacttt gtccttaact <210>9 <211>37 <212>DNA
<213>Artificial Sequence <220>
<223>Description of ArtificialSequence:BRCA2 probe <400> 9 ctaagtttga atccatgctt tgctcttctt gattatt 37 <210> 10 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:APCl probe <400> 10 cagatagccc tggacaaacc atgccaccaa gcagaag 37 <210> 11 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:APC2 probe <400> 11 gaagttcctg gattttctgt tgctggatgg tagttgc 37
comparing the s amount of amplicon obtained to a standard amount of amplification product;
and identifying a sample as having an abnormality in a nucleic acid if the amount of amplification product differs from the standard amount. In a preferred embodiment, a standard amount of amplification product is determined by amplification of a locus, or portion thereof, being screened (e.g., an intact, wild-type nucleic acid) in a known to normal sample (one obtained from an individual known not to have the disease being screened). Also in preferred embodiments, a standard amount is determined by reference to the art. In certain embodiments of the invention, the standard amount is essentially no detectable amplicon due to the lack of high-integrity nucleic acids in the sample. Accordingly, any detectable amplicon in a patient sample is indicative of a ~s positive screen. That is the case especially when a large (e.g., 1.8 Kb or 2.4 Kb) fragment is being screened. Finally, the standard amount can be a molecular weight marker on, for example, an electrophoretic gel.
In a preferred embodiment of the invention, the sample is prepared from a specimen selected from the group consisting of stool, sputum, blood, urine, 2o cerebrospinal fluid, seminal fluid, saliva, breast nipple aspirate, and biopsy tissue.
However, any tissue or body fluid specimen may be used according to methods of the invention. Especially preferred are samples of luminal fluid because such samples are generally free of intact, healthy cells. Such samples include blood, urine, bile, pancreatic juice, stool, sputum, pus, and the like.
2s Also in a preferred embodiment, the nucleic acid or nucleic acids being interrogated is (are) DNA. In a more particular embodiment, the nucleic acid being analyzed is selected from a coding region of a gene, or portion thereof, a noncoding nucleic acid region, or portion thereof, a regulatory element of a gene or a portion thereof, and an unidentified fragment of genomic DNA. Also in a preferred embodiment, 3o the nucleic acid being interrogated is RNA. As is appreciated by the skilled artisan, any genomic locus is amenable to screening according to the invention. The particular locus or loci chosen for analysis depends, in part, on the disease being screened, and the convenience of the investigator. It is not necessary that the locus or loci chosen for analysis be correlated with any specific disease because methods of the invention contemplate measuring either the total nucleic acid in a sample or amplifiable nucleic acid in a sample as an indicator of overall disease status or the presence and/or extent of apoptosis in the sample. However, disease-associated loci (those in which a s mutation is indicative, causative, or otherwise evidence of a disease) can be used.
Preferred disease-associated loci include p53, apc, MSH-2, dcc, scr, c-myc, B-catnenin, mlh-1, pms-1, pms-2, pol-delta, and bax.
The amount of amplification product may be determined by any suitable or convenient means. Preferably, the amount of amplification product is determined by gel io electrophoresis. Labels, such as fluorescent or radioactive labels, may be used. The amounts of amplification product produced may be compared to standard amounts by any suitable or convenient means, including, but not limited to visual comparison, machine-driven optical comparison, densitometry, mass spectroscopy, hybrid capture, and other known means. The amplification reaction itself can be any means for is amplifying nucleic acid, including, but not limited to PCR, RT-PCR, OLA, rolling circle, single base extension, and others known in the art. The amplification product can also be measured by signal amplification techniques, such as branch chain amplification (Chiron). Methods of the invention are useful with any platform for the identification, amplification, sequencing, or other manipulation of nucleic acids. For example, zo methods of the invention can be applied to ligase chain reaction, strand displacement (Becton-Dickinson), and others.
Also in a preferred embodiment of the invention, a series of amplification reactions is conducted on a single genomic locus. Each amplification reaction in the series is designed to amplify a fragment of a different length. In a preferred 2s embodiment, the target fragment lengths are 200 bp, 400 bp, 800 bp, 1.3 Kb, 1.8 Kb, and 2.4 Kb. Primers for amplification are designed according to knowledge in the art in order to amplify template, if present, of the desired length at the desired locus. A
positive screen is one that produces amplicon in at least one, and preferably at least two of the series of amplification reactions. As noted above, a normal sample which 3o has undergone or which is undergoing apoptosis typically contains little or no fragments of significant length. Thus, a series of amplification reactions targeting fragments from about 200 by to about 2.4 Kb and longer reveals samples that contain nucleic acids that have avoided apoptosis as evidenced by the amplification of large fragments.
_$_ Preferred methods of the invention also comprise conducting amplification reactions on a series of different genomic loci. Preferably, from about 2 to about 7 loci are used. However, the precise number of interrogated loci is determined by the individual investigator based upon the disease to be detected or based upon s convenience. According to methods of the invention, primers are designed to amplify nucleic acid (preferably DNA) at each of the chosen loci. A sample in which at least one locus, preferably at least two loci, and most preferably at least three loci produce detectable amplification product is considered a positive sample. The lengths of fragments to be amplified in this assay may be varied, but are preferably at least about Io 180 by each in length. It is not necessary that the same length fragments be amplified at each of the chosen loci.
Methods of the invention also comprise conducting a series of amplification reactions at a series of different genomic loci. Each amplification reaction in the series is designed to amplify a fragment of a different length. Preferably, from about 2 to about Is 7 amplification reactions on about 2 to about 7 loci are used. However, the precise number of interrogated loci is determined by the individual investigator based upon the disease to be detected or based upon convenience. In a preferred embodiment, the target fragment lengths are 200 bp, 400 bp, 800 bp, 1.3 Kb, 1.8 Kb, and 2.4 Kb.
Primers for amplification are designed according to knowledge in the art in order to 2o amplify template if present. It is preferred, but not necessary, that the same length fragments be amplified at each of the chosen loci. A positive screen is one that produces amplicon in at least one, and preferably at least two of the series of amplification reactions and in which at least one locus, preferably at least two loci, and most preferably at least three loci produce detectable amplification product.
As noted 2s above, a normal sample which has undergone or which is undergoing apoptosis typically contains little or no fragments of significant length. Thus, a series of amplification reactions targeting fragments from about 200 by to about 2.4 Kb and longer reveals samples that contain nucleic acids that have avoided apoptosis as evidenced by the amplification of large fragments.
3o Methods of the invention may also be used to assess the integrity of DNA in a biological sample. Such methods comprise conducting an amplification reaction using at least two loci suspected to be in the sample as templates; determining which loci produce detectable amplicon; and assessing the integrity of DNA in the sample as a _g_ function of the number of loci producing amplicon. The integrity of DNA in the sample is high when amplicon is produced in one or more of the amplification reactions.
This method is especially useful for determining whether a heterogeneous sample has sufficient nucleic acid for measurement. Accordingly, such methods are used to screen s or to "qualify" samples for further analysis (e.g., genetic, biochemical, cytological, or other analyses).
Methods of the invention may also be used to assess fetal abnormalities by conducting amplification reactions on nucleic acids in maternal blood. Just as described above, the ability to amplify significant amounts of nucleic acid is an indicator of a to genomic instability. A baseline for comparison of the extent of nucleic acid amplification can be amounts of nucleic acids from known normal samples. The amount of amplification obtained from fetal samples is placed on a continuum, and the investigator must analyze any given sample in terms of the amount of fetal nucleic acid produced in various disease states and in normal samples.
is Methods of the invention are useful as diagnostic screening methods. Often it is desirable to perform follow-up testing on a patient in order to confirm a suspected disease state. Such follow-up procedures are determined based upon the disease state being interrogated. For example, a colonoscopy may be suggested in a case in which a stool sample is positively screened according to methods of the invention.
Such follow-2o up procedures are contemplated herein as part of the invention.
Methods of the invention are useful as screens for a wide range of disease states. In addition to colon cancers and adenomas, methods of the invention are useful to screen for other diseases, for example, as screens for lymphomas, or stomach, lung, liver, pancreas, prostate, kidney, testicular, bladder, uterus, or ovarian cancers or 2s adenomas. In addition to cancer, methods of the invention are useful, for example, as screens for diseases such as inflammatory bowel syndrome, inflammatory bowel disease, Crohn's disease, and others in which a genomic instability is thought to play a role. Methods of the invention are especially useful as screens for any disease that impairs the proper function of the gastrointestinal system; most especially diseases of 3o the colon. Methods of the invention are also useful to screen for apoptosis in a cellular sample. The profile of amplifiable DNA in a sample is correlated with proteins that have been associated with disease. For example up regulation of the apoptosis protein, survivin, is correlated with increased amounts of amplifiable DNA, as is the Ras oncogene, as well as other oncogenes and their gene products.
Methods of the invention are also useful as assays for apoptosis. The presence of high-integrity fragments or large quantities of nucleic acids in a sample indicates that s the sample was derived from cells that did not proceed through apoptosis.
The absence of such fragments or quantities indicates that cells that contributed to the sample did undergo apoptosis. Accordingly, an apoptotic activity assay of the invention, either alone or in combination with other assays for genomic instability, are useful as screens for disease.
io Finally, methods of the invention can be carried out by hybrid capture. For example, hybrid capture and subsequent analysis of the captured fragments can be used to determine the nucleic acid integrity of a sample.
The invention also provides a profile of nucleic acid fragments indicative of disease. A preferred profile is obtained through methods described above.
Preferred is profiles comprise nucleic acids having between about 200 by and about 2.4 Kb obtained in a patient sample comprising cellular debris according to methods described herein. A
highly preferred profile contains at least one nucleic acid of at least 1.3 Kb.
Other objects and advantages of the invention are apparent upon consideration of the following drawings and detailed description thereof.
2o Description of the Drawings Figure 1 is a gel photograph showing results of amplification of K-ras (exon 1 ) DNA isolated from stool using forward and reverse primers spaced about 200 by apart.
The band intensity relates to the amount of 200 by product or greater in the sample.
Lanes 1-4 are results from patients with cancer or adenoma, lane 5 is a positive control, Zs lanes 6-10 are from patients who did not have cancer or adenoma, lanes 11-12 are negative controls, and lanes 13-18 are standards at the approximate molecular weight indicated in the figure. Amplifications were graded A through C, A being the most intense band, C being the least.
Figures 2-4 are gel photographs showing results of amplification of apc (exon 15) 3o DNA isolated from stool using forward and reverse primers spaced about 200 by apart.
The band intensity relates to the amount of 200 by product or greater in the sample.
Lanes 1-4 are results from patients with cancer or adenoma, lane 5 is a positive control, lanes 6-10 are from patients who did not have cancer or adenoma, lanes 11-12 are negative controls, and lanes 13-18 are standards at the approximate molecular weight indicated in the figure. Amplifications were graded A through C, A being the most intense band, C being the least.
s Figure 5 is a gel photograph showing results of amplification of p53 (exon 5) DNA
isolated from stool using forward and reverse primers spaced about 200 by apart. The band intensity relates to the amount of 200 by product or greater in the sample. Lanes 1-4 are results from patients with cancer or adenoma, lane 5 is a positive control, lanes 6-10 are from patients who did not have cancer or adenoma, lanes 11-12 are negative io controls, and lanes 13-18 are standards at the approximate molecular weight indicated in the figure. Amplifications were graded A through C, A being the most intense band, C being the least.
Figure 6 is a gel photograph showing results of amplification of p53 (exon 7) DNA
isolated from stool using forward and reverse primers spaced about 200 by apart. The is band intensity relates to the amount of 200 by product or greater in the sample. Lanes 1-4 are results from patients with cancer or adenoma, lane 5 is a positive control, lanes 6-10 are from patients who did not have cancer or adenoma, lanes 11-12 are negative controls, and lanes 13-18 are standards at the approximate molecular weight indicated in the figure. Amplifications were graded A through C, A being the most intense band, 2o C being the least.
Figure 7 is a gel photograph showing results of amplification of p53 (exon 8) DNA
isolated from stool using forward and reverse primers spaced about 200 by apart. The band intensity relates to the amount of 200 by product or greater in the sample. Lanes 1-4 are results from patients with cancer or adenoma, lane 5 is a positive control, lanes 2s 6-10 are from patients who did not have cancer or adenoma, lanes 11-12 are negative controls, and lanes 13-18 are standards at the approximate molecular weight indicated in the figure. Amplifications were graded A through C, A being the most intense band, C being the least.
Figure 9-10 are gel photographs of results of amplification of DNA from stool 30 samples using forward and reverse primers spaced approximately 1.8 Kb apart. The band intensity shows the amount of 1.8 Kb or greater product. Lanes 1, 8, and 9 are negative controls, lanes 2, 3, and 5 are results from patients with cancer or adenoma, lanes 4, 6, and 7 are results from patients who did not have cancer or adenoma, and lanes 10-14 are molecular weight standards.
Figures 11 A and B are gel photographs of results of amplification of DNA in stool from a total of 30 patients and controls. The band intensity relates to the amount s of amplifiable DNA in the sample. Lanes N are negative controls, lanes 1, 3, 11, and 18 are results from patients which are indicative of the presence off cancer or adenoma, lanes 2, 4, 5-10, 12-17, and 19-30 are results from patients which are indicative of the absence of cancer or adenoma. The remaining lanes are markers or standards.
Figure 12 shows a schematic representation of the placement of the primers for to amplification in a method of the present invention. In this method, a single forward primer, F~, is used in conjunction with a series of reverse primers, R~ to R6, chosen to amplify progressively longer portions of the target.
Figure 13 shows a schematic representation of the placement of the primers for amplification in a method of the present invention. In this method, a series of forward is and reverse primer pairs, (F~ , R~) to (F3 , R3), are chosen to amplify portions of the target spaced at intervals along the target.
Detailed Description of the Invention The invention provides methods for the analysis of biological samples. Methods of the invention provide diagnostically-relevant information based upon the integrity of 2o nucleic acids in a biological sample. Normal biological samples (those not having indicia of the disease being screened), especially those comprising luminal tissue andlor fluid, typically comprise a majority of short-fragment, low-integrity nucleic acids (especially DNA) which are the result of degradation by apoptosis. When a mutation has caused genomic instability, the normal cell cycle may be disrupted and apoptotic 2s degradation may not occur at the rate expected in a normal sample. Methods of the invention screen for such disruptions.
Accordingly, preferred methods of the invention comprise determining an amount of amplifiable nucleic acid in a biological sample, and determining whether that amount is consistent with an amount expected in a normal sample. In many biological samples, 3o especially heterogeneous samples, there may be no detectable amplification product.
That is especially true when longer fragments are used as templates for amplification.
Generally, the probability that any given set of PCR primers will amplify a DNA fragment having a length exceeding the primer distance is expressed as of Fragments Amplified = (FL-PD)/(FL+PD) wherein FL is fragment length (in base pairs) and PD is primer distance (in base pairs). This equation assumes that sample DNA fragment lengths are uniformly distributed (i.e., there is no favored locus at which breaks occur).
s In a preferred embodiment, methods of the invention comprise amplifying sequences of different length in a sample, if present, in order to generate a profile of amplification products indicative of disease or the propensity for disease. In a preferred method, a sample is exposed to a set of PCR primers comprising a single forward primer, which may be a capture probe used to capture target fragments, and a plurality io of downstream reverse primers which hybridize to portions of a contiguous sequence (if present) in the sample. Amplifications using these primers will result in a series of amplification products, each having a different length, if the contiguous target sequence is present in the sample. The length of the amplification products are determined by the spacings between the forward primer and each of the downstream reverse primers. An is example is shown in Figure 12, which is a schematic representation showing placement of the primers for amplification.
If the target sequence, or a portion of it, is present in the sample, amplification will result in a series of fragments the length of which is dictated by the spacing of the primers. According to the principles adduced above, a sample from a diseased patient 2o will produce a profile of amplification products in the assay described above that differs from the profile obtained from a sample containing the smaller fragments expected to be produced as a result of normal apoptosis. In a preferred embodiment, the forward primer is designed to hybridize about 200 by upstream of the first reverse primer, and about 2.3 Kb upstream of the last reverse primer. Other reverse primers are designed 2s to hybridize at various locations between the first and last reverse primers. Preferred intervals between the forward primer and the various reverse primers are 200 by (F~-R~), 400 by (F~-R2), 800 by (F~-R3), 1.3 Kb, (F~-R4), 1.8 Kb (F~-R5), and 2.3 Kb (F~-R6).
The number and spacing of reverse primers is chosen at the convenience of the skilled artisan.
3o Also in a preferred embodiment, a hybrid capture probe is used to anchor a target sequence, preferably on a solid support (e.g., beads). A plurality of probes are then placed at various distances downstream of the capture probe. Those probes can be pairs of forward and reverse primers as discussed above, or they can be signal amplification probes, such as those used in Ligase Chain Reaction (LCR), and others used in the identification of sequences. The plurality of probes hybridize along the length of a target fragment if the target is present in the sample. Thus, by interrogating samples for the presence of the probes, one can determine the integrity of sequences s present in the sample. This can be done in numerous ways, including, but not limited to, hybrid capture, PCR, LCR, strand displacement, branched chain, or other assays known in the are that incorporate hybrid probes or primers in order to identify or quantitate sequence. A sample containing intact (high integrity) nucleic acids represents a positive screen according to the invention. In one embodiment, sample is ~o placed into wells (e.g., on a 96 well plate) containing support-bound capture probe. The capture probe immobilizes a target sequence, if present in the sample. Probes that hybridize to sequence downstream of the capture probe (downstream probes) are placed into each well, such that each downstream probe is spaced a unique distance apart from the common capture probe, and each well contains only one type of is downstream probe. Signal is then generated by, for example, amplification, or by standard ELISA procedure followed by amplification, or by LCR, or other methods mentioned above. The presence of signal in each well indicates the presence of sequence of at least the length between the capture probe and the downstream probe.
In an alternative embodiment, each well receives multiple different downstream probes, 2o which may be distinctly labeled, and the presence of labels) is correlated with the length of sequence presence in the sample.
A sample from a patient having, for example, cancer will produce amplicon between most or all of the primer pairs (depending, inter alia, on the length of the target fragments, on the spacing of the primers, and where on the target the primers are 2s spaced). Such a profile represents a positive screen for disease or the propensity for disease. A sample from a patient who does not have indicia of disease results in little or no amplification product in the assay described above. In a negative screen there may be amplification of small (e.g., 200 bp) fragments but there should be no amplification of larger fragments (i.e., fragments resulting from amplification between the forward primer 3o and spaced-apart reverse primers). In cancer diagnostics, the target fragment may optionally be an oncogene, a tumor suppressor, or any other marker associated with cancer. However, it is not necessary to use cancer-associated markers in methods of the invention, as such methods are based on the general recognition that samples indicative of disease contain a greater amount of intact nucleic acids and a greater amount of long fragment nucleic acids. Accordingly, any convenient target nucleic acid locus may be used in the methods of the invention.
The amplification reactions described above may be conducted according to any s suitable or convenient protocol and the fragment size of the resulting amplification products (if any) may be determined by any suitable or convenient means.
In an alternative embodiment, methods of the invention comprise conducting a series of amplification reactions on a contiguous nucleic acid target fragment, each application reaction comprising one forward primer and one reverse primer, such that to pairs of forward and reverse primers are spaced at intervals on a contiguous fragment suspected to be in the sample. An example of this arrangement is shown in Figure 13.
Preferably, the spacings between each forward and reverse primer pair are equivalent.
In a positive screen, the assay described above will result in a series of same-size fragments for most if not all of the primer pairs. Such an array of amplification products is evidences a contiguous target sequence indicative of disease (see above). A
sample from a disease-free patient should produce little or no amplification product, but in any case will not produce the contiguous array of amplification products expected from a sample containing a relatively intact diagnostic target sequence.
Each of the methods described above are based upon the principle that an intact 2o nucleic acid, or a segment of an intact nucleic acid, in a sample is diagnostic. Thus, variations on the methods described above are contemplated. Such variations include the placement of primers, the number of primers used, the target sequence, the method for identifying sequences, and others. For example, in the method depicted in Figure 13, and described above, it is not necessary that the numbers of forward and reverse 2s primers be equal. A forward primer may, for example, be used to amplify fragments between two reverse primers. Other variations in primer pair placement are within the skill in the art, as are details of the amplification reactions to be conducted. Finally, as represented in Figures 12 and 13, capture probes may be used in methods of the invention in order to isolate a chosen target sequence.
3o The following examples provide further details of methods according to the invention. For purposes of exemplification, the following examples provide details of the use of the method if the present invention in colon cancer detection.
Accordingly, while exemplified in the following manner, the invention is not so limited and the skilled artisan will appreciate its wide range of application upon consideration thereof.
Exemplary Method for the Detection of Colon Cancer The following example relates to screening for colon cancer in voided stool s samples. Based upon the principles upon which the invention is based (see above), the same analysis can be performed on other samples, such as those mentioned above, with the same results as shown herein.
For the analysis of stool samples, preferred methods of the invention comprise obtaining at least a cross-sectional or circumferential portion of a voided stool as taught to in U.S. patent number 5,741,650, and co-pending, co-owned U.S. patent application serial number 09/059,718, both of which are incorporated by reference herein.
While a cross-sectional or circumferential portion of stool is desirable, methods provided herein are conducted on random samples obtained from voided stool, which include smears or scrapings. Once obtained, the stool specimen is homogenized. A preferable buffer for is homogenization is one that contains at least 16 mM
ethylenediaminetetraacetic acid (EDTA). However, as taught in co-pending, co-owned U.S. patent application serial number 60/122,177, incorporated by reference herein, it has been discovered that the use of at least 150 mM EDTA greatly improves the yield of nucleic acid from stool.
Thus, a preferred buffer for stool homogenization comprises phosphate buffered saline, 20 20-100 mM NaCI or KCI, at least 150 mM EDTA, and optionally a detergent (such as SDS) and a proteinase (e.g., proteinase K).
After homogenization, nucleic acid is preferably isolated from the stool sample.
Isolation or extraction of nucleic acid is not required in all methods of the invention, as certain detection techniques can be adequately performed in homogenized stool without 2s isolation of nucleic acids. In a preferred embodiment, however, homogenized stool is spun to create a supernatant containing nucleic acids, proteins, lipids, and other cellular debris. The supernatant is treated with a detergent and proteinase to degrade protein, and the nucleic acid is phenol-chloroform extracted. The extracted nucleic acids are then precipitated with alcohol. Other techniques can be used to isolate nucleic acid 3o from the sample. Such techniques include hybrid capture, and amplification directly from the homogenized stool. Nucleic acids can be purified and/or isolated to the extent required by the screening assay to be employed. Total DNA is isolated using techniques known in the art.
Screening Assay Protocol The size of human DNA fragments obtained above can be determined by numerous means. For example, human DNA can be separated using gel electrophoresis. A 3% agarose gel is prepared using techniques known in the art. See s Ausubel et. al., Short Protocols in Molecular Biology, John Wiley & Sones, 1195, pgs. 2-23-2-24, incorporated by reference herein. The size of human DNA fragments is then determined by comparison to known standards. Fragments greater than about 200 by provide a positive screen. While a diagnosis can be made on the basis of the screen alone, patients presenting a positive screen are preferably advised to seek follow-up ~o testing to render a confirmed diagnosis.
A preferred means for determining human DNA fragment length uses PCR.
Methods for implementing PCR are well-known. In the present invention, human DNA
fragments are amplified using human-specific primers. Amplicon of greater than about 200 by produced by PCR represents a positive screen. Other amplification reactions is and modifications of PCR, such as ligase chain reaction, reverse-phase PCR, Q-PCR, and others may be used to produce detectable levels of amplicon. Amplicon may be detected by coupling to a reporter (e.g. fluorescence, radioisotopes, and the like), by sequencing, by gel electrophoresis, by mass spectrometry, or by any other means known in the art, as long as the length, weight, or other characteristic of the amplicons 2o identifies them by size.
Examples Experiments were conducted to determine whether characteristics of amplifiable DNA in stool were predictive of cancer or precancer in patients from whom stools samples were obtained. In the first experiment, the amount of amplifiable DNA
was 2s measured in each of several stool samples using PCR amplification to detect DNA
fragments in the sample of at least 200 base pairs in length. The second experiment determined the amount of long fragments (greater than 200 base pair) in the same samples, and then determined ratios of long product to short product. The third experiment determined a profile of amplification products with nucleic acid fragment 30 lengths of 200 bp, 400 bp, 800 bp, 1.3 Kb, 1.8 Kb and 2.4 Kb. The fourth and fifth experiments were clinical studies correlating the integrity of nucleic acids in patient stool samples with overall patient disease status.
Stool samples were collected from 9 patients who presented with symptoms or a medical history that indicated that a colonoscopy should be performed. Each stool sample was frozen. Immediately after providing a stool sample, each patient was given s a colonoscopy in order to determine the patient's disease status. Based upon the colonoscopy results, and subsequent histological analysis of biopsy samples taken during colonoscopy, individuals were placed into one of two groups: normal or abnormal. The abnormal group consisted of patients with cancer or with an adenoma of at least 1 cm in diameter. Based upon these results, 4 of the 9 patients were placed to into the abnormal group.
The samples were screened by hybrid capturing human DNA, and determining the amount of amplifiable DNA having at least 200 base pairs. Each frozen stool specimen, weighing from 7-33 grams, was thawed and homogenized in 500 mM Tris, 16 mM EDTA, and 10 mM NaCI, pH 9.0 at a volume, to mass ratio of 3:1. Samples is were then rehomogenized in the same buffer to a final volume-to-mass ratio of 20:1, and spun in glass macro beads at 2356 xg. The supernatant was collected and treated with SDS and proteinase k. The DNA was then phenol-chloroform extracted and precipitated with alcohol. The precipitate was suspended in 10 mM Tris and 1 mM
EDTA (1 x TE), pH 7.4. Finally, the DNA was treated with Rnase.
2o Human DNA was isolated from the precipitate by sequence-specific hybrid capture. Biotynilated probes against portions of the p53, K-ras, and apc genes were used.
The K-ras probe was 5'GTGGAGTATTTGATAGTGTATTAACCTTATGTGTGAC
3' (SEQ ID NO: 1).
2s There were two apc probes: apc-1309 was 5'TTCCAGCAGTGTCACAGCACCCTAGAACCAAATCCAG 3' (SEQ ID NO: 2), and apc-1378 was 5'CAGATAGCCCTGGACAAACAATGCCACGAAGCAGAAG 3' (SEQ ID
NO: 3).
There were four probes against p53, the first (hybridizing to a portion of exon 5) 3o was 5'TACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGG3' (SEQ ID N0:4), the second (hybridizing to a portion of exon 7) was 5'ATTTCTTCCATACTACTACCCATCGACCTCTCATC3' (SEQ ID NO: 5), the third, also hybridizing to a portion of exon 7 was 5'ATGAGGCCAGTGCGCCTTGGGGAGACCTGTGGCAAGC3' (SEQ ID NO: 6); and finally, a probe against exon 8 had the sequence 5'GAAAGGACAAGGGTGGTTGGGAGTAGATGGAGCCTGG3' (SEQ ID NO: 7).
A 10 p.1 aliquot of each probe (20 pmol/capture) was added to a suspension s containing 300 p1 DNA in the presence of 310 ~I 6M GITC buffer for 2 hours at room temperature. Hybrid complexes were isolated using streptavidin-coated beads (Dynal).
After washing, probe-bead complexes were suspended at 25° C for 1 hour in 0.1x TE
buffer, pH7.4. The suspension was then heated for 4 minutes at 85° C, and the beads were removed.
lo Captured DNA was then amplified using PCR, essentially as described in U.S.
Patent No. 4,683,202, incorporated by reference herein. Each sample was amplified using forward and reverse primers through 7 loci (Kras, exon 1, APC exon 15 (3 separate loci), p53, exon 5, p53, exon 7, and p53, exon 8) in duplicate (for a total of 14 amplifications for each locus). Seven separate PCRs (40 cycles each) were run in is duplicate using primers directed to detect fragments in the sample having 200 base pairs or more. Amplified DNA was placed on a 4% Nusieve (FMC Biochemical) gel (3%
Nusieve, 1 % agarose), and stained with ethidium bromide (0.5 ~g/ml). The resulting amplified DNA was graded based upon the relative intensity of the stained gels. The results are shown in Figures 1-7. Each Figure represents the results for all 9 patients 20 (including standards) for the seven different loci that were amplified. As shown in the Figures, each sample from a patient with cancer or adenoma was detected as a band having significantly greater intensity than the bands associated with samples from patients who did not have cancer or precancer. All four cancer/adenoma patients identified using colonoscopy were correctly identified by determining the amount of 2s amplifiable DNA 200 base pairs or greater in length. As shown in Figures 1-7, the results were the same regardless of which locus was amplified. Accordingly, the amount of 200 by or greater DNA in a sample was predictive of patient disease status.
An experiment was conducted that was essentially identical to the one described 3o above in Example 1, but forward and reverse primers were placed such that fragments of about 1.8 Kb and above were amplified.
DNA was prepared as described above. Forward and reverse primers were spaced so as to hybridize approximately 1.8 Kb apart on three different loci (Kras, exon 1, APC, exon 15, and p53 exon 5). Thirty-three rounds of amplification were performed, and the resulting DNA was placed on a 3% agarose gel. The results are shown in s Figures 8-10. As shown in the Figures (which show results from three separate experiments to amplify and detect "long" product), samples from individuals having cancer or precancer produced large amounts of high-molecular weight (in this case 1.8 Kb and above) DNA; whereas samples from patients who did not have cancer or precancer produced no DNA in the range of about 1.8 Kb and higher. Thus, the io presence of high-molecular weight DNA was indicative of the disease status of the patient.
An experiment was conducted to determine the molecular weight profile of DNA
from samples collected and prepared as part of a blind study on 30 patients who is presented at the Mayo Clinic with suspected gastrointestinal disorders.
Stool samples were obtained, and DNA was isolated as described above.
Prior to amplification, DNA was isolated from the samples by hybrid capture.
Biotynilated probes against portions of the BRCA1, BRCA2, p53, APC genes were used.
2o The BRCA1 probe was 5'GATTCTGAAGAACCAACTTTGTCCTTAACTAGCTCTT3' (SEQ ID NO: 8).
The BRCA2 probe was 5'CTAAGTTTGAATCCATGCTTTGCTCTTCTTGATTATT3' (SEQ ID NO 9).
The APC1 probe was 2s 5'CAGATAGCCCTGGACAAACCATGCCACCAAGCAGAAG3' (SEQ ID NO 10).
The p53 probe, hybridizing to a portion of exon 5, was 5'TACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGG3' (SEQ ID N0:4).
The APC2 probe was 5'GAAGTTCCTGGATTTTCTGTTGCTGGATGGTAGTTGC3' (SEQ ID NO 11).
A 300 ~I aliquot of sample was placed in 300 ~I of 6 M guanidine isothiocyanate buffer with 10 p1 of each capture probe, and incubated overnight at 25 C.
Captured DNA was isolated using 100 ~I capture beads incubated for one hour at room temperature. The DNA was eluted off the beads and PCR amplified under standard PCR conditions.
According to methods of the invention, amplification reactions were conducted using forward and reverse primers through the 5 loci for each sample. Forward and s reverse primers were spaced to amplify fragments of 200 bp, 400 bp, 800 bp, 1.3 Kb, 1.8 Kb, and 2.4 Kb. Each of 30 PCR reactions was run for 36 cycles. Amplicon was run on a 3% Seakeam gel, and stained with ethidium bromide. The results are shown in Figures 11A and 11 B. Each figure represents the results for 15 of the 30 patients.
As shown in those figures, patients with cancer or adenoma have an increased Io yield of amplifiable DNA. That is especially true at the 1.8 Kb level and above. Thus, patients with cancer or adenoma not only produce more amplifiable DNA in their stool, but also produce larger DNA fragments than are produced in the stool of patients who do not have cancer. Thus, both an increased yield of amplifiable DNA and the presence of high molecular weight DNA, especially that at 1.8 Kb and above, were indicative of is patient disease status.
In this example, methods of the invention were correlated with clinical outcome in numerous patients who had a colorectal adenoma or colorectal cancer as diagnosed using colonoscopy, and 79 patients who were diagnosed as not having colorectal 2o cancer or adenoma. A stool sample was obtained from each of these patients and prepared as described above. Fragments of the 5 different loci referred to above were amplified using primers spaced 200, 400, 800, 1300, 1800, and 2400 base pairs apart using the protocol described above in Example 3. Each amplification was scored such that successful amplification of a fragment received a score of 1, and no amplification 2s received a score of 0. Since five loci were interrogated using 6 primer pairs each, the maximum score was 30 (successful amplification of all 6 fragments at all five loci). The cutoff for a positive screen was set at 21. The results are shown below.
Table 1 Table 2 Normals Adenomas Patient A a Score Patient A Score No No. a . P-003 29 Table 3 Carcinomas Patient A a Score No.
As shown above, methods of the invention are effective in screening for the presence of colorectal cancer and adenoma.
In this example, methods of the invention were used to detect non-colonic cancers in 28 patients.
A stool sample was obtained from each of the 28 patients. The sample was prepared as described above. Fragments of the 5 different loci referred to above were amplified using primers spaced 200, 400, 800, 1300, 1800, and 2400 base pairs apart using the protocol described above in Example 3. Each amplification was scored such that successful amplification of a fragment received a score of 1, and no amplification received a score of 0. Since five loci were interrogated using 6 primer pairs each, the maximum obtainable score was 30 (successful amplification of all 6 fragments at all five loci). A score of 21 was used as a cutoff between diseased and non-diseased patients.
The results are shown below.
Table 4 Supercolonic Cancers Patient SupercolonicAge Score No. Cancer P-145 Pancreas 68 30 P-164 Lun CA 68 30 P-166 Bile Duct 52 30 P-189 Bile Duct 43 30 P-190 Lun CA 50 30 P-019 Atypical 71 29 Findings in Stomach P-152 Lun CA 77 28 P-167 Pancreas 72 28 P-011 Lun CA 73 27 P-153 Pancreas 65 27 P-165 Lun CA 85 27 P-170 Duodenum 65 27 P-182 Barrett's 58 27 Eso ha us P-146 Bile Duct 63 26 P-081 Barrett's 74 26 Eso ha us P-151 Pancreas 49 25 P-155 Lun CA 60 25 P-156 Lun CA 57 25 P-150 Pancreas 78 23 P-149 Eso ha 59 19 us P-154 Eso ha 80 19 s P-169 Pancreas 71 19 P-168 Lun CA 63 18 P-180 Pancreas 67 13 P-144 Eso ha 59 9 us P-147 Stomach 57 7 P-148 Stomach 69 6 P-171 ~ Esophagus -76 0 As shown above, methods of the invention successfully screened 18 out of 27 patients who actually had non-colonic cancer. Only one patient was misdagnosed as having cancer when he did not. Thus, the methods of the invention are useful for non-invasive diagnosis of a non-specified cancerous disease state in a patient.
The threshold of 21 for a positive screen can be changed to accommodate desired sensitivities and specificities. For example, if 18 were the false negative results shown in Table 4 would be avoided. The skilled artisan knows how to set thresholds depending on the patient (e.g., a lower threshold for patients with symptoms than patients presenting with no symptoms), the disease being diagnosed, and the desired level of sensitivity and specificity. Regardless of the threshold, the principle of the invention remains that nucleic acid integrity is a viable marker for disease, and especially for cancer.
In addition, the propensity for disease may be measured using methods of the invention. For example, periodic molecular weight profiling in accordance with the methods of the invention may be used to monitor the disease state of a patient presenting no or minimal symptoms. Such longitudinal monitoring will determine whether a patient is progressing with increasing amounts of high integrity nucleic acids - indicating the desirability for follow-up examination.
What is claimed is:
SEQUENCE LISTING
<110> Shuber, Anthony P
<120> Methods for Disease Detection <130> EXT-034PC
<140>
<141>
<150> US 60/152,847 <151> 1999-09-08 <150> US 09/455,950 <151> 1999-12-7 <160> 11 <170> PatentIn Ver. 2.0 <210> 1 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:K-ras probe <400> 1 gtggagtatt tgatagtgta ttaaccttat gtgtgac 37 <210> 2 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:apc probe apc-1309 <400> 2 ttccagcagt gtcacagcac cctagaacca aatccag 37 <210> 3 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:apc probe apc-1378 <400> 3 cagatagccc tggacaaaca atgccacgaa gcagaag 37 <210> 4 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223>Description of ArtificialSequence:p53 exon 5 probe <400>4 tactcccctg caactgg 37 ccctcaacaa gatgttttgc <210>5 <211>35 <212>DNA
<213>Artificial Sequence <220>
<223>Description of ArtificialSequence:p53 exon 7 probe <400>5 atttcttcca tcatc 35 tactactacc catcgacctc <210>6 <211>37 <212>DNA
<213>Artificial Sequence <220>
<223>Description of ArtificialSequence:p53 exon 7 probe <400>6 atgaggccag ggcaagc 37 tgcgccttgg ggagacctgt <210>7 <211>37 <212>DNA
<213>Artificial Sequence <220>
<223>Description of ArtificialSequence:p53 exon 8 probe <400>7 gaaaggacaa agcctgg 37 gggtggttgg gagtagatgg <210>8 <211>37 <212>DNA
<213>Artificial Sequence <220>
<223>Description of ArtificialSequence:BRCAl probe <400>8 gattctgaag agctctt 37 aaccaacttt gtccttaact <210>9 <211>37 <212>DNA
<213>Artificial Sequence <220>
<223>Description of ArtificialSequence:BRCA2 probe <400> 9 ctaagtttga atccatgctt tgctcttctt gattatt 37 <210> 10 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:APCl probe <400> 10 cagatagccc tggacaaacc atgccaccaa gcagaag 37 <210> 11 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:APC2 probe <400> 11 gaagttcctg gattttctgt tgctggatgg tagttgc 37
Claims (13)
1. A method for determining the disease status of a patient, the method comprising the steps of:
determining the integrity of nucleic acids in a patient sample comprising shed cells or cellular debris; and identifying said patient as having disease if intact nucleic acids are present in said sample in an amount greater than a predetermined threshold.
determining the integrity of nucleic acids in a patient sample comprising shed cells or cellular debris; and identifying said patient as having disease if intact nucleic acids are present in said sample in an amount greater than a predetermined threshold.
2. A method for screening a patient for disease, the method comprising the steps of:
determining the integrity of nucleic acids in a patient sample comprising shed cellular debris; and Identifying a positive screen as a sample in which the integrity of nucleic acids exceeds a predetermined threshold.
determining the integrity of nucleic acids in a patient sample comprising shed cellular debris; and Identifying a positive screen as a sample in which the integrity of nucleic acids exceeds a predetermined threshold.
3. A method for detecting disease in a patient, the method comprising the steps of:
determining an amount of patient nucleic acid present in a patient sample;
comparing said amount with a standard amount of nucleic acid expected to be present in a disease-negative sample; and detecting disease in said patient as an amount of patient nucleic acid greater than said standard amount.
determining an amount of patient nucleic acid present in a patient sample;
comparing said amount with a standard amount of nucleic acid expected to be present in a disease-negative sample; and detecting disease in said patient as an amount of patient nucleic acid greater than said standard amount.
4. The method of claim 3, wherein said nucleic acid is a fragment having a molecular weight of at least 1.3 Kb.
5. The method of claim 1, wherein said disease is cancer or pre-cancer.
6. The Method of claim 3, wherein said cancer is selected from the group consisting of colon cancer, lung cancer, esophageal cancer, prostate cancer, stomach cancer, pancreatic cancer, liver cancer, and lymphoma.
7. The method of claim 1, wherein said nucleic acid is selected from the group consisting of K-ras, p53, apc, dcc, tumor suppressor genes, and oncogenes.
8. The method of claim 1, wherein said patient sample is selected from the group consisting of stool, sputum, pancreatic fluid, bile, lymph, blood, urine, cerebrospinal fluid, seminal fluid, saliva, breast nipple aspirate, and pus.
9. A method for screening a patient sample to determine the disease status of a patient, the method comprising the steps of:
(a) amplifying patient nucleic acid in a sample obtained from a patient;
(b) determining an amount of amplified nucleic acid obtained in Step (a); and (c) identifying a positive screen when said amount of amplified nucleic acid is greater than an amount of amplified nucleic acid expected in a disease-negative sample.
(a) amplifying patient nucleic acid in a sample obtained from a patient;
(b) determining an amount of amplified nucleic acid obtained in Step (a); and (c) identifying a positive screen when said amount of amplified nucleic acid is greater than an amount of amplified nucleic acid expected in a disease-negative sample.
10. A method for screening a biological sample for disease, the method comprising the steps of:
selecting a plurality of genomic loci;
conducting an amplification reaction at each of said loci;
identifying a positive screen as a sample in which the number of loci producing an amplification product exceeds a predetermined threshold.
selecting a plurality of genomic loci;
conducting an amplification reaction at each of said loci;
identifying a positive screen as a sample in which the number of loci producing an amplification product exceeds a predetermined threshold.
11. A method for screening a patient sample for disease, the method comprising the steps of:
selecting a plurality of genomic loci;
conducting an amplification reaction at each of said loci;
assigning a first numerical score to each locus in which amplification takes place;
assigning a second numerical score to each locus in which amplification does not take place;
determining whether a total of said first numerical score exceeds a total of said second numerical score by a threshold amount, thereby to screen said sample for disease.
selecting a plurality of genomic loci;
conducting an amplification reaction at each of said loci;
assigning a first numerical score to each locus in which amplification takes place;
assigning a second numerical score to each locus in which amplification does not take place;
determining whether a total of said first numerical score exceeds a total of said second numerical score by a threshold amount, thereby to screen said sample for disease.
12. A nucleic acid fragment isolated from a patient sample, said nucleic acid being at least 1.3 Kb and indicative of nucleic acid integrity associated with cancer.
13. An isolated nucleic acid obtained by the method of claim 1.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10435735B2 (en) | 2014-03-07 | 2019-10-08 | Dna Genotek Inc. | Composition and method for stabilizing nucleic acids in biological samples |
US11002646B2 (en) | 2011-06-19 | 2021-05-11 | DNA Genotek, Inc. | Devices, solutions and methods for sample collection |
US11572581B2 (en) | 2002-06-07 | 2023-02-07 | DNA Genotek, Inc. | Compositions and methods for obtaining nucleic acids from sputum |
Families Citing this family (64)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6818404B2 (en) | 1997-10-23 | 2004-11-16 | Exact Sciences Corporation | Methods for detecting hypermethylated nucleic acid in heterogeneous biological samples |
AU767983B2 (en) * | 1999-04-09 | 2003-11-27 | Esoterix Genetic Laboratories, Llc | Methods for detecting nucleic acids indicative of cancer |
US6586177B1 (en) * | 1999-09-08 | 2003-07-01 | Exact Sciences Corporation | Methods for disease detection |
US6919174B1 (en) * | 1999-12-07 | 2005-07-19 | Exact Sciences Corporation | Methods for disease detection |
EP1238106A2 (en) * | 1999-12-07 | 2002-09-11 | Exact Sciences Corporation | Apparatus and methods for drug screening based 0on nucleic acid analysis |
JP2003516138A (en) | 1999-12-07 | 2003-05-13 | エグザクト サイエンシーズ コーポレイション | Detection of aerobic digestive neoplasms in the colon |
US6911308B2 (en) * | 2001-01-05 | 2005-06-28 | Exact Sciences Corporation | Methods for detecting, grading or monitoring an H. pylori infection |
US6428964B1 (en) * | 2001-03-15 | 2002-08-06 | Exact Sciences Corporation | Method for alteration detection |
US8980568B2 (en) | 2001-10-11 | 2015-03-17 | Aviva Biosciences Corporation | Methods and compositions for detecting non-hematopoietic cells from a blood sample |
US8986944B2 (en) | 2001-10-11 | 2015-03-24 | Aviva Biosciences Corporation | Methods and compositions for separating rare cells from fluid samples |
AU2003213107A1 (en) | 2002-02-15 | 2003-09-09 | Exact Sciences Corporation | Methods for analysis of molecular events |
US20030186248A1 (en) * | 2002-03-29 | 2003-10-02 | Erlander Mark G. | Interpreting cytological specimens via molecular histological signatures |
US8275554B2 (en) * | 2002-12-20 | 2012-09-25 | Caliper Life Sciences, Inc. | System for differentiating the lengths of nucleic acids of interest in a sample |
JP4395133B2 (en) | 2002-12-20 | 2010-01-06 | カリパー・ライフ・サイエンシズ・インク. | Single molecule amplification and detection of DNA |
US20050042639A1 (en) * | 2002-12-20 | 2005-02-24 | Caliper Life Sciences, Inc. | Single molecule amplification and detection of DNA length |
ITMI20030434A1 (en) | 2003-03-07 | 2004-09-08 | Istituto Oncologico Romagnolo Coope Rativa Sociale | METHOD FOR THE DETECTION OF COLON-RECTUM TUMORS. |
US20040259101A1 (en) * | 2003-06-20 | 2004-12-23 | Shuber Anthony P. | Methods for disease screening |
US20050014165A1 (en) * | 2003-07-18 | 2005-01-20 | California Pacific Medical Center | Biomarker panel for colorectal cancer |
DE10345021A1 (en) * | 2003-09-23 | 2005-05-04 | Univ Potsdam | Method for the non-invasive early detection of colon cancer and / or colon cancer precursor cells |
WO2005111244A2 (en) * | 2004-05-10 | 2005-11-24 | Exact Sciences Corporation | Methods for detecting a mutant nucleic acid |
WO2005113769A1 (en) * | 2004-05-14 | 2005-12-01 | Exact Sciences Corporation | Method for stabilizing biological samples for nucleic acid analysis |
WO2006026654A2 (en) | 2004-08-27 | 2006-03-09 | Exact Sciences Corporation | Method for detecting a recombinant event |
EP2295974A1 (en) | 2004-09-08 | 2011-03-16 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES | Compositions and methods for the detection of HIV-1/HIV-2 infection |
WO2006047787A2 (en) | 2004-10-27 | 2006-05-04 | Exact Sciences Corporation | Method for monitoring disease progression or recurrence |
WO2006094149A2 (en) * | 2005-03-01 | 2006-09-08 | Exact Sciences Corporation | Methods and compositions for detecting adenoma |
DE602005009324D1 (en) | 2005-04-06 | 2008-10-09 | Maurice Stroun | Method for cancer diagnosis by detection of DNA and RNA in the circulation |
US9777314B2 (en) * | 2005-04-21 | 2017-10-03 | Esoterix Genetic Laboratories, Llc | Analysis of heterogeneous nucleic acid samples |
EP1888786A4 (en) * | 2005-05-27 | 2009-12-30 | Wayne John Cancer Inst | Use of free circulating dna for diagnosis, prognosis, and treatment of cancer |
US8768629B2 (en) * | 2009-02-11 | 2014-07-01 | Caris Mpi, Inc. | Molecular profiling of tumors |
US8700335B2 (en) | 2006-05-18 | 2014-04-15 | Caris Mpi, Inc. | System and method for determining individualized medical intervention for a disease state |
WO2008008515A2 (en) * | 2006-07-14 | 2008-01-17 | Aviva Biosciences Corporation | Methods and compositions for detecting rare cells from a biological sample |
AU2008260075A1 (en) | 2007-05-31 | 2008-12-11 | California Pacific Medical Center | Method to predict or diagnose a gastrointestinal disorder or disease |
WO2009015299A1 (en) | 2007-07-26 | 2009-01-29 | California Pacific Medical Center | Method to predict or diagnose a gastrointestinal disorder or disease |
WO2009051842A2 (en) * | 2007-10-18 | 2009-04-23 | The Johns Hopkins University | Detection of cancer by measuring genomic copy number and strand length in cell-free dna |
EP2474626B1 (en) | 2008-02-15 | 2014-08-13 | Mayo Foundation For Medical Education And Research | Detecting neoplasm from a stool sample |
US20100112713A1 (en) * | 2008-11-05 | 2010-05-06 | The Texas A&M University System | Methods For Detecting Colorectal Diseases And Disorders |
JP2012508577A (en) | 2008-11-12 | 2012-04-12 | カリス ライフ サイエンシズ ルクセンブルク ホールディングス | Method and system for using exosomes to determine phenotype |
WO2011044541A2 (en) * | 2009-10-09 | 2011-04-14 | Baylor Research Institute | Identification of micrornas (mirnas) in fecal samples as biomarkers for gastroenterological cancers |
WO2011066476A1 (en) * | 2009-11-25 | 2011-06-03 | Quantalife, Inc. | Methods and compositions for detecting genetic material |
CA2791905A1 (en) | 2010-03-01 | 2011-09-09 | Caris Life Sciences Luxembourg Holdings, S.A.R.L. | Biomarkers for theranostics |
EP2556172A4 (en) | 2010-04-06 | 2013-10-30 | Caris Life Sciences Luxembourg Holdings | Circulating biomarkers for disease |
EP2619329B1 (en) | 2010-09-24 | 2019-05-22 | The Board of Trustees of The Leland Stanford Junior University | Direct capture, amplification and sequencing of target dna using immobilized primers |
EP3940084A1 (en) | 2011-02-09 | 2022-01-19 | Bio-Rad Laboratories, Inc. | Analysis of nucleic acids |
WO2012129363A2 (en) | 2011-03-24 | 2012-09-27 | President And Fellows Of Harvard College | Single cell nucleic acid detection and analysis |
GB201107466D0 (en) | 2011-05-05 | 2011-06-15 | Loktionov Alexandre | Device and method for non-invasive collection of colorectal mucocellular layer and disease detection |
US8751261B2 (en) | 2011-11-15 | 2014-06-10 | Robert Bosch Gmbh | Method and system for selection of patients to receive a medical device |
US20160040229A1 (en) | 2013-08-16 | 2016-02-11 | Guardant Health, Inc. | Systems and methods to detect rare mutations and copy number variation |
US10876152B2 (en) | 2012-09-04 | 2020-12-29 | Guardant Health, Inc. | Systems and methods to detect rare mutations and copy number variation |
MX367963B (en) | 2012-09-04 | 2019-09-11 | Guardant Health Inc | Systems and methods to detect rare mutations and copy number variation. |
US11913065B2 (en) | 2012-09-04 | 2024-02-27 | Guardent Health, Inc. | Systems and methods to detect rare mutations and copy number variation |
US9944998B2 (en) | 2013-07-25 | 2018-04-17 | Bio-Rad Laboratories, Inc. | Genetic assays |
US10253358B2 (en) | 2013-11-04 | 2019-04-09 | Exact Sciences Development Company, Llc | Multiple-control calibrators for DNA quantitation |
WO2015095689A1 (en) | 2013-12-19 | 2015-06-25 | Exact Sciences Corporation | Synthetic nucleic acid control molecules |
EP3087204B1 (en) | 2013-12-28 | 2018-02-14 | Guardant Health, Inc. | Methods and systems for detecting genetic variants |
PL3250710T3 (en) | 2015-01-30 | 2019-02-28 | Enterome | Host dna as a biomarker of crohn's disease |
US20180300449A1 (en) * | 2015-10-10 | 2018-10-18 | Guardant Health, Inc. | Methods and applications of gene fusion detection in cell-free dna analysis |
CA3008651A1 (en) | 2015-12-17 | 2017-06-22 | Guardant Health, Inc. | Methods to determine tumor gene copy number by analysis of cell-free dna |
EP3978624A1 (en) | 2016-07-19 | 2022-04-06 | Exact Sciences Corporation | Methylated control dna |
CA3029838A1 (en) | 2016-07-19 | 2018-01-25 | Exact Sciences Development Company, Llc | Nucleic acid control molecules from non-human organisms |
US9850523B1 (en) | 2016-09-30 | 2017-12-26 | Guardant Health, Inc. | Methods for multi-resolution analysis of cell-free nucleic acids |
CN109642250A (en) | 2016-09-30 | 2019-04-16 | 夸登特健康公司 | The method of multiresolution analysis for cell-free nucleic acid |
US11746381B2 (en) | 2017-03-10 | 2023-09-05 | Cancer Diagnostics Research Innvovations, LLC | Methods for diagnosing and treating gastric cancer using miRNA expression |
CN113661249A (en) | 2019-01-31 | 2021-11-16 | 夸登特健康公司 | Compositions and methods for isolating cell-free DNA |
WO2023200404A2 (en) * | 2022-04-13 | 2023-10-19 | Lucence Life Sciences Pte. Ltd. | Method for determining cfdna fragment size ratio and fragment size distribution |
Family Cites Families (162)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US59718A (en) | 1866-11-13 | Improvement in knob-holes for carriage-curtains | ||
US3413464A (en) | 1965-04-29 | 1968-11-26 | Ibm | Method for measuring the nucleic acid in biological cells after enhancement in an acidic solution |
US4101279A (en) | 1977-04-06 | 1978-07-18 | Muhammed Javed Aslam | Device for the collection and processing of stool specimens |
US4333734A (en) | 1980-01-18 | 1982-06-08 | Sloan-Kettering Institute For Cancer Research | Diagnostic device for fecal occult blood and method of use |
US4535058A (en) | 1982-10-01 | 1985-08-13 | Massachusetts Institute Of Technology | Characterization of oncogenes and assays based thereon |
US4309782A (en) | 1980-09-11 | 1982-01-12 | Esteban Paulin | Device for collecting fecal specimens |
US4358535A (en) | 1980-12-08 | 1982-11-09 | Board Of Regents Of The University Of Washington | Specific DNA probes in diagnostic microbiology |
US5482834A (en) | 1982-05-17 | 1996-01-09 | Hahnemann University | Evaluation of nucleic acids in a biological sample hybridization in a solution of chaotrophic salt solubilized cells |
US4445235A (en) | 1982-09-13 | 1984-05-01 | Pearl Slover | Stool specimen collector |
EP0113656B1 (en) * | 1983-01-08 | 1990-02-07 | Fuji Photo Film Co., Ltd. | process for the preparation of a radiation image storage panel |
US4578358A (en) | 1983-05-03 | 1986-03-25 | Warner-Lambert Company | Collection of specimens and detection of occult blood therein |
US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
US4683195A (en) | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
US4871838A (en) | 1985-07-23 | 1989-10-03 | The Board Of Rijks Universiteit Leiden | Probes and methods for detecting activated ras oncogenes |
US4705050A (en) | 1985-10-02 | 1987-11-10 | Markham Charles W | Moisture-activated floatation device |
US5348855A (en) | 1986-03-05 | 1994-09-20 | Miles Inc. | Assay for nucleic acid sequences in an unpurified sample |
CA1284931C (en) | 1986-03-13 | 1991-06-18 | Henry A. Erlich | Process for detecting specific nucleotide variations and genetic polymorphisms present in nucleic acids |
US4981783A (en) | 1986-04-16 | 1991-01-01 | Montefiore Medical Center | Method for detecting pathological conditions |
CA1341576C (en) | 1986-08-11 | 2008-07-08 | Thaddeus P. Dryja | Diagnosis of retinoblastoma |
US4735905A (en) | 1986-08-15 | 1988-04-05 | V-Tech, Inc. | Specimen-gathering apparatus and method |
US4935342A (en) | 1986-12-01 | 1990-06-19 | Syngene, Inc. | Method of isolating and purifying nucleic acids from biological samples |
AU625169B2 (en) | 1987-03-23 | 1992-07-02 | Imperial Chemical Industries Plc | Molecular markers |
US4857300A (en) | 1987-07-27 | 1989-08-15 | Cytocorp, Inc. | Cytological and histological fixative formulation and methods for using same |
AU631788B2 (en) | 1988-04-15 | 1992-12-10 | Montefiore Medical Center | Method for determining state of malignant and pre-malignant progression |
FR2630000A1 (en) | 1988-04-18 | 1989-10-20 | Sultan Bernard | BOTTLE FOR COLLECTING AN URINARY BIOLOGICAL SAMPLE FOR CYTOBACTERIOLOGICAL EXAMINATION |
US5272057A (en) | 1988-10-14 | 1993-12-21 | Georgetown University | Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase |
US5087617A (en) | 1989-02-15 | 1992-02-11 | Board Of Regents, The University Of Texas System | Methods and compositions for treatment of cancer using oligonucleotides |
US5248671A (en) | 1989-02-15 | 1993-09-28 | Board Of Regents, The University Of Texas System | Methods and compositions for treatment of cancer using oligonucleotides |
US5380645A (en) | 1989-03-16 | 1995-01-10 | The Johns Hopkins University | Generalized method for assessment of colorectal carcinoma |
US5527676A (en) * | 1989-03-29 | 1996-06-18 | The Johns Hopkins University | Detection of loss of the wild-type P53 gene and kits therefor |
DE69032864T3 (en) | 1989-03-29 | 2013-01-31 | The Johns Hopkins University | Evidence of failure of the wild-type p53 gene |
US5362623A (en) | 1991-06-14 | 1994-11-08 | The John Hopkins University | Sequence specific DNA binding by p53 |
US5192501A (en) | 1989-04-04 | 1993-03-09 | Helena Laboratories Corporation | Method of formulating a test ink for a fecal occult blood test product |
US5196167A (en) | 1989-04-04 | 1993-03-23 | Helena Laboratories Corporation | Fecal occult blood test product with positive and negative controls |
US5589335A (en) | 1989-04-05 | 1996-12-31 | Amoco Corporation | Hybridization promotion reagents |
ES2059895T3 (en) | 1989-06-23 | 1994-11-16 | Canon Kk | METHOD FOR THE DETECTION OF NUCLEIC ACID. |
US5302509A (en) | 1989-08-14 | 1994-04-12 | Beckman Instruments, Inc. | Method for sequencing polynucleotides |
US5213961A (en) | 1989-08-31 | 1993-05-25 | Brigham And Women's Hospital | Accurate quantitation of RNA and DNA by competetitive polymerase chain reaction |
CA2029219A1 (en) | 1989-11-08 | 1991-05-09 | Mary K. Estes | Methods and reagents to detect and characterize norwalk and related viruses |
US5641628A (en) | 1989-11-13 | 1997-06-24 | Children's Medical Center Corporation | Non-invasive method for isolation and detection of fetal DNA |
US5137806A (en) | 1989-12-11 | 1992-08-11 | Board Of Regents, The University Of Texas System | Methods and compositions for the detection of sequences in selected DNA molecules |
JPH05508307A (en) | 1990-01-04 | 1993-11-25 | ザ・ジョーンズ・ホプキンス・ユニバーシティ | Genes deleted in human colorectal cancer |
US5126239A (en) | 1990-03-14 | 1992-06-30 | E. I. Du Pont De Nemours And Company | Process for detecting polymorphisms on the basis of nucleotide differences |
US5200314A (en) | 1990-03-23 | 1993-04-06 | Chiron Corporation | Polynucleotide capture assay employing in vitro amplification |
EP0489149B1 (en) | 1990-06-27 | 2001-10-17 | Princeton University | Probes for detecting mutant p53 |
JPH06510363A (en) | 1990-10-29 | 1994-11-17 | ディカルブ プラント ジェネティクス | Isolation of biological materials using magnetic particles |
US5846710A (en) | 1990-11-02 | 1998-12-08 | St. Louis University | Method for the detection of genetic diseases and gene sequence variations by single nucleotide primer extension |
US5352775A (en) | 1991-01-16 | 1994-10-04 | The Johns Hopkins Univ. | APC gene and nucleic acid probes derived therefrom |
CA2101943A1 (en) | 1991-02-05 | 1992-08-06 | Kamal Bahar | Simple test for detecting carcinoembryonic antigen |
EP0570497B1 (en) | 1991-02-05 | 1996-05-08 | Lifecodes Corporation | Molecular genetic identification using probes that recognize polymorphic loci |
US5330892A (en) | 1991-03-13 | 1994-07-19 | The Johns Hopkins University | MCC gene (mutated in colorectal cancer) used for diagnosis of cancer in humans |
US5378602A (en) | 1991-05-29 | 1995-01-03 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Highly informative microsatellite repeat polymorphic DNA markers twenty-[seven]six |
US5468610A (en) | 1991-05-29 | 1995-11-21 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Three highly informative microsatellite repeat polymorphic DNA markers |
US5149506A (en) | 1991-08-09 | 1992-09-22 | Sage Products, Inc. | Stool collection and transport device |
US5506105A (en) | 1991-12-10 | 1996-04-09 | Dade International Inc. | In situ assay of amplified intracellular mRNA targets |
WO1993020235A1 (en) | 1992-04-01 | 1993-10-14 | The Johns Hopkins University School Of Medicine | Methods of detecting mammalian nucleic acids isolated from stool specimen and reagents therefor |
US5489508A (en) | 1992-05-13 | 1996-02-06 | University Of Texas System Board Of Regents | Therapy and diagnosis of conditions related to telomere length and/or telomerase activity |
EP0654092B1 (en) | 1992-06-26 | 2003-09-10 | The Trustees Of Princeton University | Method for detecting pre-cancerous or cancerous cells using p90 and p53 antibodies or probes |
JPH08507198A (en) | 1992-07-02 | 1996-08-06 | エリフィーレ ビー.フェ. | Single nucleotide primer extension method for the detection of specific alleles and a kit therefor |
US5710028A (en) | 1992-07-02 | 1998-01-20 | Eyal; Nurit | Method of quick screening and identification of specific DNA sequences by single nucleotide primer extension and kits therefor |
US5804383A (en) | 1992-08-21 | 1998-09-08 | The Regents Of The University Of California | Method and assay for detection of the expression of allele-specific mutations by allele-specific in situ reverse transcriptase polymerase chain reaction |
US5409586A (en) | 1992-08-26 | 1995-04-25 | Hitachi, Ltd. | Method for analyzing nucleic acid or protein and apparatus therefor |
US5547838A (en) | 1992-10-01 | 1996-08-20 | Life Technologies, Inc. | Method for the rapid and ultra-sensitive detection of leukemic cells |
US5331973A (en) | 1993-03-15 | 1994-07-26 | Fiedler Paul N | Method for obtaining stool samples for gastrointestinal cancer testing |
CA2092115C (en) | 1993-03-22 | 1998-12-15 | Janet L. Taylor | Testing for infestation of rapeseed and other cruciferae by the fungus leptosphaeria maculans (blackleg infestation) |
US5518901A (en) | 1993-04-19 | 1996-05-21 | Murtagh; James J. | Methods for adapting nucleic acid for detection, sequencing, and cloning using exonuclease |
US5492808A (en) | 1993-05-05 | 1996-02-20 | The Johns Hopkins University | Means for detecting familial colon cancer (FCC) |
SE501439C2 (en) | 1993-06-22 | 1995-02-13 | Pharmacia Lkb Biotech | Method and apparatus for analyzing polynucleotide sequences |
US5466576A (en) | 1993-07-02 | 1995-11-14 | Fred Hutchinson Cancer Research Center | Modulation of PIF-1-type helicases |
EP0648845B1 (en) | 1993-07-08 | 2002-10-09 | Johnson & Johnson Clinical Diagnostics, Inc. | Method for coamplification of two different nucleic acid sequences using polymerase chain reaction |
US5688643A (en) | 1993-07-09 | 1997-11-18 | Wakunaga Seiyaku Kabushiki Kaisha | Method of nucleic acid-differentiation and assay kit for nucleic acid differentiation |
CA2126391C (en) | 1993-09-13 | 2002-01-08 | Nobuko Yamamoto | Determination of nucleic acid by pcr, measurement of number of microbial cells, genes, or gene-copies by pcr, and measuring-kit employed for the same |
GB9322795D0 (en) | 1993-11-05 | 1993-12-22 | Wellcome Found | Novel compounds |
US5925517A (en) | 1993-11-12 | 1999-07-20 | The Public Health Research Institute Of The City Of New York, Inc. | Detectably labeled dual conformation oligonucleotide probes, assays and kits |
US5416025A (en) | 1993-11-29 | 1995-05-16 | Krepinsky; Jiri J. | Screening test for early detection of colorectal cancer |
US5681697A (en) | 1993-12-08 | 1997-10-28 | Chiron Corporation | Solution phase nucleic acid sandwich assays having reduced background noise and kits therefor |
US5709998A (en) | 1993-12-15 | 1998-01-20 | The Johns Hopkins University | Molecular diagnosis of familial adenomatous polyposis |
US5538851A (en) | 1993-12-22 | 1996-07-23 | Institut Pasteur And Cneva | Primers for the amplification of genes coding for the enterotoxin and the lecithinase of Clostridium perfringens and their application to the determination of the presence and numeration of these bacteriae |
US5496470A (en) | 1994-05-27 | 1996-03-05 | Barnes International, Inc. | Magnetic separator |
US6037465A (en) | 1994-06-14 | 2000-03-14 | Invitek Gmbh | Universal process for isolating and purifying nucleic acids from extremely small amounts of highly contaminated various starting materials |
US5512441A (en) | 1994-11-15 | 1996-04-30 | American Health Foundation | Quantative method for early detection of mutant alleles and diagnostic kits for carrying out the method |
US5463782A (en) | 1994-11-21 | 1995-11-07 | Eric V. Carlson | Foldable stool sample collection device |
CA2220951A1 (en) | 1995-02-01 | 1996-08-08 | Research Development Foundation | Detecting dna damage, measuring dna repair rates, and monitoring dna repair enzyme activity |
US5599662A (en) | 1995-02-17 | 1997-02-04 | Hoffmann-La Roche Inc. | Oliconucleotide primers and probes for the detection of HIV-1 |
EP1505159B1 (en) | 1995-03-17 | 2008-05-28 | John Wayne Cancer Institute | Detection of breast metastases with a multiple marker assay |
AU5296496A (en) | 1995-03-24 | 1996-10-16 | Mitokor | Mutation detection by differential primer extension of mutant and wildtype target sequences |
DE19530132C2 (en) | 1995-08-16 | 1998-07-16 | Max Planck Gesellschaft | Process for the purification, stabilization or isolation of nucleic acids from biological materials |
US5800347A (en) | 1995-11-03 | 1998-09-01 | The General Hospital Corporation | ROC method for early detection of disease |
US6083698A (en) * | 1995-09-25 | 2000-07-04 | Oncormed, Inc. | Cancer susceptibility mutations of BRCA1 |
NO954667D0 (en) | 1995-11-17 | 1995-11-17 | Dagfinn Oegreid | Method for detecting Ki-ras mutations |
US5670325A (en) | 1996-08-14 | 1997-09-23 | Exact Laboratories, Inc. | Method for the detection of clonal populations of transformed cells in a genomically heterogeneous cellular sample |
AU711754B2 (en) | 1995-12-22 | 1999-10-21 | Esoterix Genetic Laboratories, Llc | Methods for the detection of clonal populations of transformed cells in a genomically heterogeneous cellular sample |
US5612473A (en) | 1996-01-16 | 1997-03-18 | Gull Laboratories | Methods, kits and solutions for preparing sample material for nucleic acid amplification |
WO1997028280A1 (en) * | 1996-01-17 | 1997-08-07 | David John Grainger | Diagnostic method and apparatus |
AU720489B2 (en) | 1996-01-30 | 2000-06-01 | Exact Sciences Corporation | Methods for detecting colon cancer from stool samples |
US5741650A (en) | 1996-01-30 | 1998-04-21 | Exact Laboratories, Inc. | Methods for detecting colon cancer from stool samples |
EP0929694A4 (en) * | 1996-03-15 | 2002-05-02 | Penn State Res Found | Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays |
US5897999A (en) | 1996-03-22 | 1999-04-27 | The Johns Hopkins University | Cancer drug screen based on cell cycle uncoupling |
US5645995A (en) | 1996-04-12 | 1997-07-08 | Baylor College Of Medicine | Methods for diagnosing an increased risk for breast or ovarian cancer |
US5736333A (en) | 1996-06-04 | 1998-04-07 | The Perkin-Elmer Corporation | Passive internal references for the detection of nucleic acid amplification products |
US5976800A (en) | 1996-06-28 | 1999-11-02 | The Regents Of The University Of California | Enhancement of cancer cell death |
US6203993B1 (en) | 1996-08-14 | 2001-03-20 | Exact Science Corp. | Methods for the detection of nucleic acids |
US6100029A (en) | 1996-08-14 | 2000-08-08 | Exact Laboratories, Inc. | Methods for the detection of chromosomal aberrations |
US6020137A (en) | 1996-08-14 | 2000-02-01 | Exact Laboratories, Inc. | Methods for the detection of loss of heterozygosity |
US5928870A (en) | 1997-06-16 | 1999-07-27 | Exact Laboratories, Inc. | Methods for the detection of loss of heterozygosity |
US5952178A (en) | 1996-08-14 | 1999-09-14 | Exact Laboratories | Methods for disease diagnosis from stool samples |
US6143529A (en) | 1996-08-14 | 2000-11-07 | Exact Laboratories, Inc. | Methods for improving sensitivity and specificity of screening assays |
US6146828A (en) | 1996-08-14 | 2000-11-14 | Exact Laboratories, Inc. | Methods for detecting differences in RNA expression levels and uses therefor |
US6300077B1 (en) | 1996-08-14 | 2001-10-09 | Exact Sciences Corporation | Methods for the detection of nucleic acids |
ATE378422T1 (en) | 1996-08-26 | 2007-11-15 | Invitek Biotechnik & Biodesign | METHOD FOR DETECTING CLINICALLY RELEVANT CHANGES IN THE DNA SEQUENCE OF THE KI-RAS ONCOGENE, ITS USE AND TEST KIT FOR EARLY DETECTION OF TUMORS |
CA2410120A1 (en) | 1996-08-28 | 1998-03-05 | The Johns Hopkins University School Of Medicine | Method for detecting cell proliferative disorders |
US5856104A (en) | 1996-10-28 | 1999-01-05 | Affymetrix, Inc. | Polymorphisms in the glucose-6 phosphate dehydrogenase locus |
US6235474B1 (en) | 1996-12-30 | 2001-05-22 | The Johns Hopkins University | Methods and kits for diagnosing and determination of the predisposition for diseases |
US5830665A (en) | 1997-03-03 | 1998-11-03 | Exact Laboratories, Inc. | Contiguous genomic sequence scanning |
GB9704444D0 (en) * | 1997-03-04 | 1997-04-23 | Isis Innovation | Non-invasive prenatal diagnosis |
DE19712332A1 (en) | 1997-03-25 | 1998-10-01 | Boehringer Mannheim Gmbh | Method for the detection of microsatellite instability for tumor diagnosis |
US6143496A (en) | 1997-04-17 | 2000-11-07 | Cytonix Corporation | Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly |
US6406857B1 (en) | 1997-06-16 | 2002-06-18 | Exact Sciences Corporation | Methods for stool sample preparation |
US5888778A (en) | 1997-06-16 | 1999-03-30 | Exact Laboratories, Inc. | High-throughput screening method for identification of genetic mutations or disease-causing microorganisms using segmented primers |
US6268136B1 (en) | 1997-06-16 | 2001-07-31 | Exact Science Corporation | Methods for stool sample preparation |
US6566101B1 (en) | 1997-06-16 | 2003-05-20 | Anthony P. Shuber | Primer extension methods for detecting nucleic acids |
US20020004201A1 (en) | 1997-06-16 | 2002-01-10 | Lapidus Stanley N. | Methods for the detection of loss of heterozygosity |
GB9715034D0 (en) | 1997-07-18 | 1997-09-24 | Zeneca Ltd | Assay |
US6013442A (en) | 1997-08-05 | 2000-01-11 | Wisconsin Alumni Res Found | Direct quantitation of low copy number RNA |
BR9809556A (en) * | 1997-08-06 | 2000-10-17 | Ludwig Inst Cancer Res | "methods for the quantification of nucleic acid molecules". |
US5942396A (en) | 1997-08-19 | 1999-08-24 | The Rockefeller University | Method for identifying individuals at risk for colorectal neoplasia by quantifying normal colonic mucosal epithelial cell apoptosis |
DE19736691A1 (en) | 1997-08-22 | 1999-02-25 | Michael Prof Dr Med Giesing | Characterising and identifying disseminated metastatic cancer cells |
CA2301997A1 (en) * | 1997-09-29 | 1999-04-08 | City Of Hope | Disease association by locus stratification |
CA2307177C (en) | 1997-10-23 | 2004-06-29 | Exact Laboratories, Inc. | Methods for detecting contamination in molecular diagnostics using pcr |
US6818404B2 (en) | 1997-10-23 | 2004-11-16 | Exact Sciences Corporation | Methods for detecting hypermethylated nucleic acid in heterogeneous biological samples |
US6251660B1 (en) | 1997-11-25 | 2001-06-26 | Mosaic Technologies, Inc. | Devices and methods for detecting target molecules in biological samples |
CA2321223A1 (en) * | 1998-02-25 | 1999-09-02 | National University Of Ireland, Cork | Hla linked pre-eclampsia and miscarriage susceptibility gene |
JP2002506204A (en) | 1998-03-03 | 2002-02-26 | モザイク テクノロジーズ | Purification and detection process using reversible affinity electrophoresis |
EP1066408A4 (en) | 1998-03-05 | 2002-09-25 | Diadexus Inc | A novel method of detecting and monitoring endometrial and uterine cancers |
WO1999066077A2 (en) | 1998-06-16 | 1999-12-23 | Exact Laboratories, Inc. | Methods for the detection of nucleic acids |
CA2331767A1 (en) | 1998-06-18 | 1999-12-23 | Mosaic Technologies | Denaturing gradient affinity electrophoresis and methods of use thereof |
AU762049B2 (en) | 1998-06-19 | 2003-06-19 | Exact Sciences Corporation | Detection of non-viral organisms with SRP RNA |
US6037130A (en) | 1998-07-28 | 2000-03-14 | The Public Health Institute Of The City Of New York, Inc. | Wavelength-shifting probes and primers and their use in assays and kits |
AU1704000A (en) | 1998-08-14 | 2000-03-14 | Exact Laboratories, Inc. | Method for diagnostic screening |
US20020001800A1 (en) | 1998-08-14 | 2002-01-03 | Stanley N. Lapidus | Diagnostic methods using serial testing of polymorphic loci |
AU6412799A (en) | 1998-10-05 | 2000-04-26 | Mosaic Technologies | Reverse displacement assay for detection of nucleic acid sequences |
AU2473900A (en) | 1998-11-23 | 2000-06-13 | Exact Sciences Corporation | Primer extension methods utilizing donor and acceptor molecules for detecting nucleic acids |
AU1526400A (en) | 1998-11-23 | 2000-06-19 | Exact Laboratories, Inc. | Method for detecting target sequences in small proportions in heterogeneous samples |
WO2000031298A1 (en) | 1998-11-23 | 2000-06-02 | Exact Laboratories, Inc. | Methods for detecting lower-frequency molecules |
US6503718B2 (en) | 1999-01-10 | 2003-01-07 | Exact Sciences Corporation | Methods for detecting mutations using primer extension for detecting disease |
US6280947B1 (en) | 1999-08-11 | 2001-08-28 | Exact Sciences Corporation | Methods for detecting nucleotide insertion or deletion using primer extension |
CA2362251C (en) | 1999-02-25 | 2008-06-17 | Exact Sciences Corporation | Methods for preserving dna integrity |
EP1157264B1 (en) | 1999-02-26 | 2004-04-21 | EXACT Sciences Corporation | Biochemical purification devices with immobilized capture probes and their uses |
JP2003508015A (en) | 1999-04-02 | 2003-03-04 | モザイク テクノロジーズ インコーポレーテッド | Electrophoretic analysis of target molecules using adapter molecules |
AU767983B2 (en) | 1999-04-09 | 2003-11-27 | Esoterix Genetic Laboratories, Llc | Methods for detecting nucleic acids indicative of cancer |
WO2000066005A1 (en) | 1999-05-03 | 2000-11-09 | Exact Laboratories, Inc. | Stool specimen collector |
US20010039012A1 (en) | 1999-06-14 | 2001-11-08 | Lapidus Stanley N. | Methods for diagnostic screening |
US6440706B1 (en) | 1999-08-02 | 2002-08-27 | Johns Hopkins University | Digital amplification |
US6849403B1 (en) | 1999-09-08 | 2005-02-01 | Exact Sciences Corporation | Apparatus and method for drug screening |
US6586177B1 (en) * | 1999-09-08 | 2003-07-01 | Exact Sciences Corporation | Methods for disease detection |
US6919174B1 (en) | 1999-12-07 | 2005-07-19 | Exact Sciences Corporation | Methods for disease detection |
JP2003516138A (en) | 1999-12-07 | 2003-05-13 | エグザクト サイエンシーズ コーポレイション | Detection of aerobic digestive neoplasms in the colon |
US6911308B2 (en) | 2001-01-05 | 2005-06-28 | Exact Sciences Corporation | Methods for detecting, grading or monitoring an H. pylori infection |
US6428964B1 (en) | 2001-03-15 | 2002-08-06 | Exact Sciences Corporation | Method for alteration detection |
US20030049659A1 (en) | 2001-05-29 | 2003-03-13 | Lapidus Stanley N. | Devices and methods for isolating samples into subsamples for analysis |
US20030039012A1 (en) * | 2001-08-21 | 2003-02-27 | Pezzaniti Joseph L. | Communication system and method to avoid laser-pulse broadening by multi-path effects |
US20070020513A1 (en) * | 2001-10-04 | 2007-01-25 | Ise Corporation | Energy Storage Cell Support Separator and Cooling System for a Multiple Cell Module |
-
1999
- 1999-12-07 US US09/455,950 patent/US6586177B1/en not_active Expired - Lifetime
-
2000
- 2000-09-08 EP EP00968342A patent/EP1212468B1/en not_active Expired - Lifetime
- 2000-09-08 CA CA002384368A patent/CA2384368A1/en not_active Abandoned
- 2000-09-08 WO PCT/US2000/024639 patent/WO2001018252A2/en active Application Filing
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- 2000-09-08 AT AT00968342T patent/ATE438737T1/en not_active IP Right Cessation
- 2000-09-08 DE DE60042695T patent/DE60042695D1/en not_active Expired - Lifetime
- 2000-09-08 EP EP09167115A patent/EP2110442A1/en not_active Ceased
- 2000-09-08 AU AU78276/00A patent/AU7827600A/en not_active Abandoned
- 2000-09-08 JP JP2001521786A patent/JP2003508083A/en active Pending
-
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- 2003-05-07 US US10/434,482 patent/US20040014104A1/en not_active Abandoned
-
2006
- 2006-09-07 US US11/517,808 patent/US7811757B2/en not_active Expired - Fee Related
-
2010
- 2010-02-23 US US12/710,798 patent/US20100151478A1/en not_active Abandoned
- 2010-09-29 JP JP2010219841A patent/JP2010279394A/en active Pending
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EP2110442A1 (en) | 2009-10-21 |
US20070202513A1 (en) | 2007-08-30 |
DE60042695D1 (en) | 2009-09-17 |
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US6586177B1 (en) | 2003-07-01 |
US20040014104A1 (en) | 2004-01-22 |
EP1212468B1 (en) | 2009-08-05 |
JP2003508083A (en) | 2003-03-04 |
EP1212468A2 (en) | 2002-06-12 |
US7811757B2 (en) | 2010-10-12 |
WO2001018252A3 (en) | 2001-10-25 |
US20100151478A1 (en) | 2010-06-17 |
ES2329543T3 (en) | 2009-11-27 |
ATE438737T1 (en) | 2009-08-15 |
AU7827600A (en) | 2001-04-10 |
WO2001018252A2 (en) | 2001-03-15 |
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