US20090291436A1 - Methods for detecting nucleic acids indicative of cancer - Google Patents

Methods for detecting nucleic acids indicative of cancer Download PDF

Info

Publication number
US20090291436A1
US20090291436A1 US11/960,313 US96031307A US2009291436A1 US 20090291436 A1 US20090291436 A1 US 20090291436A1 US 96031307 A US96031307 A US 96031307A US 2009291436 A1 US2009291436 A1 US 2009291436A1
Authority
US
United States
Prior art keywords
cancer
dna
sample
precancer
stool
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/960,313
Inventor
Anthony P. Shuber
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Exact Sciences Corp
Esoterix Genetic Laboratories LLC
Original Assignee
Genzyme Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genzyme Corp filed Critical Genzyme Corp
Priority to US11/960,313 priority Critical patent/US20090291436A1/en
Assigned to EXACT LABORATORIES, INC. reassignment EXACT LABORATORIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SHUBER, ANTHONY P.
Assigned to EXACT SCIENCES CORPORATION reassignment EXACT SCIENCES CORPORATION CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: EXACT LABORATORIES, INC.
Assigned to GENZYME CORPORATION reassignment GENZYME CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: EXACT SCIENCES CORPORATION
Publication of US20090291436A1 publication Critical patent/US20090291436A1/en
Priority to US12/729,051 priority patent/US20100173320A1/en
Assigned to ESOTERIX GENETIC LABORATORIES, LLC reassignment ESOTERIX GENETIC LABORATORIES, LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GENZYME CORPORATION
Priority to US13/919,622 priority patent/US20130280727A1/en
Assigned to EXACT SCIENCES DEVELOPMENT COMPANY, LLC reassignment EXACT SCIENCES DEVELOPMENT COMPANY, LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: EXACT SCIENCES CORPORATION
Assigned to EXACT SCIENCES CORPORATION reassignment EXACT SCIENCES CORPORATION MERGER (SEE DOCUMENT FOR DETAILS). Assignors: EXACT SCIENCES DEVELOPMENT COMPANY, LLC
Abandoned legal-status Critical Current

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57419Specifically defined cancers of colon
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57473Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA

Definitions

  • This invention relates to methods for the early detection of cancer in patients by screening for large DNA fragments. Methods of the invention are especially useful in the detection of colon cancer.
  • Genometics often are associated with disease or with the propensity for disease. For example, many cancers are thought to arise through a series of mutations in genomic DNA, resulting in genomic instability in the form of uncontrolled cellular growth.
  • damage to genomic DNA typically leads to expression of tumor suppressors, such as the cell-cycle regulator, p53.
  • p53 the cell-cycle regulator
  • damage to cellular DNA results in increased expression of p53 which arrests the cell cycle to allow repair of the damage. If the damaged DNA cannot be repaired, the cell undergoes apoptosis, thus preventing the accumulation of additional mutations in daughter cells.
  • apoptosis is important not only in the regulation of cellular metabolism, but also in inhibiting oncogenesis.
  • the nucleus becomes small and fragmented.
  • Nuclear DNA is digested into spindle fragments that are generally no larger than about 200 base pairs.
  • the process continues, usually through multiple pathways, the cell membrane breaks down, and cellular contents are metabolized. As a result, cells that have the potential to enter the multi-step pathway leading to cancer are eliminated.
  • colorectal cancers typically originate in the colonic epithelium, and are not extensively vascularized (and therefore not invasive) during early stages of development. The transition to a highly-vascularized, invasive and ultimately metastatic cancer commonly takes ten years or longer. If the presence of cancer is detected prior to extensive vascularization, surgical removal typically is an effective cure. However, colorectal cancer is often detected only upon manifestation of clinical symptoms, such as pain and black tarry stool. Generally, such symptoms are present only when the disease is well established, and often after metastasis has occurred. Early detection of colorectal cancer therefore is important in order to significantly reduce its morbidity.
  • Invasive diagnostic methods such as endoscopic examination, allow direct visual identification, removal, and biopsy of potentially-cancerous tissue. Endoscopy is expensive, uncomfortable, inherently risky, and not a practical tool for early diagnosis.
  • the present invention provides methods for identifying indicia of cancer in tissue or body fluid samples by identifying non-apoptotic DNA in those samples.
  • the invention also provides methods for identifying indicia of cancer or precancer in samples containing exfoliated epithelial cells. It has now been recognized that DNA obtained from exfoliated normal (non-cancerous) cells is different than DNA obtained from exfoliated cancer or precancer cells. Normal exfoliated cells typically have undergone apoptosis, and thus produce cells or cellular debris (depending upon the stage of apoptosis) comprising DNA that has been substantially degraded.
  • Exfoliated cancer or precancer cells typically have not undergone apoptosis, and such cells or their debris, while producing some very small fragments as a result of degradation in the sample, typically also contain a higher proportion of large DNA fragments (compared to those observed in cells or debris from exfoliated normal cells).
  • the difference in DNA integrity between normal and abnormal cells is a marker for the presence of cancer or precancer in a sample comprising exfoliated cells.
  • Stool is a good sample for exemplification of methods of the invention.
  • the colonic epithelium undergoes a continual process of exfoliation.
  • Normal epithelial cells undergo apoptosis, and are sloughed into the lumen of the colon, and onto forming stool.
  • Cells from polyps and tumors are also sloughed onto forming stool.
  • cells from polyps or tumors are, by definition, not apoptotic.
  • Methods of the invention take advantage of the different characteristics between apoptotic and non-apoptotic cells in order to screen patient samples for indicia of cancer or precancer.
  • non-cancerous (normal) cells undergo apoptosis at regular intervals, or in response to irreparable cell damage.
  • DNA from normal cells is cleaved into small fragments having about 200 or fewer base pairs, and typically 180 base pairs or less.
  • DNA obtained from cancer or precancer cells is much larger than the typical apoptotic fragments.
  • the presence of large DNA fragments in a sample indicates that there are or were cells in the sample (or the specimen from which it was obtained) that have avoided apoptosis, and its coincidental degradation of DNA.
  • the presence of large DNA fragments represents a positive screen for cancer or precancer.
  • methods of the invention comprise detecting the presence in a biological sample of species-specific nucleic acids indicative of cancer or precancer. Samples comprising such nucleic acids are identified as having indicia of cancer or precancer. In preferred methods, patients presenting samples having a high proportion of non-apoptotic nucleic acids as determined by methods of the invention are further evaluated for the presence of a tumor, adenoma, or other cancerous or precancerous lesion.
  • methods of the invention comprise detecting in a biological sample one or more DNA fragment(s) of a length that would not be substantially present in noncancerous cells or cellular debris.
  • such fragments are larger than a typical apoptotic spindle fragment, or larger than about 170 base pairs.
  • methods of the invention comprise detecting DNA fragments that are greater than about 200 base pairs, and preferably greater than about 500 base pairs. There is no upper limit on these fragments, as all that is necessary is that the fragment be larger than an apoptotic fragment.
  • fragments indicative of cancer or precancer cells are between about 200 and about 3500 base pairs, and ideally between about 500 and about 2500 base pairs.
  • methods of the invention comprise detecting in a tissue or body fluid sample the presence of nucleic acid fragments having greater than about 500 base pairs or having a molecular weight corresponding to greater than about 500 base pairs.
  • methods of the invention comprise detecting nucleic acid fragments having between about 200 and about 1000 base pairs, preferably between about 200 and about 600 base pairs, and most preferably about 500 base pairs.
  • methods of the invention comprise determining a ratio of large fragments (200-3500 bp) to small fragments (less than 200 bp), and determining whether the ratio exceeds an empirically-derived threshold.
  • the threshold is determined empirically by analyzing ratios of large-to-small fragments, and correlating those ratios with the disease state of a selected population of normal and cancer patients.
  • amounts of large and small fragments are determined by polymerase chain reaction amplification of sample DNA using primers selected to amplify long and short fragments. Alternatively, amounts of large and small fragments are determined using the same primer.
  • Preferred methods of the invention comprise amplifying nucleic acids in a representative stool sample using human-specific primers, and detecting amplicons having greater than about 200, and preferably about 500 or more base pairs.
  • amplification is accomplished by polymerase chain reaction (PCR) using forward and reverse primers directed against human-specific nucleic acid fragments, and spaced apart to provide a lower limit on the resulting amplicons.
  • primers for PCR are directed against human oncogene or tumor suppressor sequences.
  • Preferred target nucleic acids for PCR primers include p53, Kras, apc, dcc, and other genes known or suspected to be associated with cancer, and especially colorectal cancer.
  • Preferred biological samples include stool, pus and urine.
  • Method of the inventions are especially useful for the detection of large DNA fragments in samples comprising exfoliate.
  • Tissue e.g., colon, lungs, bladder
  • cells especially epithelial cells, are exfoliated are most preferred for screening methods of the invention.
  • continuing cellular renewal requires that cells are regularly sloughed after having undergone apoptosis.
  • Samples of the exfoliate tissue or body fluid containing the exfoliated cells predominantly comprise apoptotic DNA.
  • Preferred methods of the invention for use on a stool sample comprise obtaining a representative stool sample.
  • An especially-preferred method for preparing a stool sample is disclosed in U.S. Pat. No. 5,741,650, and in co-owned, co-pending U.S. patent application Ser. No. 09/059,713 (attorney docket No. EXT-015), each of which is incorporated by reference herein.
  • methods of the invention comprise homogenizing a representative stool sample in a solvent in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5 to 1.
  • An especially-preferred ratio of solvent volume to stool mass is about 20:1.
  • a preferred solvent for preparing stool samples according to the invention is a physiologically-compatible buffer comprising a detergent and a proteinase and optionally a DNase inhibitor, such as a buffer comprising Tris-EDTA-NaCl.
  • a preferred buffer is 50 mM Tris, 150 mM EDTA and 10 mM NaCl at pH 9.0.
  • Another preferred solvent is guanidine isothiocyanate (GITC).
  • Preferred methods of the invention further comprise enriching sample for human DNA.
  • Preferred enrichment methods for use in the invention include enriching a desired human target sequence using an affinity column, sequence-specific capture, or through the use of preferred buffers that bias isolation of human DNA.
  • a preferred enrichment method is based upon the capture of unique human nucleic acids using, for example, an affinity column. Details of such methods are provided below.
  • methods further comprise the step of extracting DNA from the homogenized sample mixture using sequence-specific nucleic acid probes.
  • probes hybridizing to human DNA.
  • the probes are preferably labeled.
  • Preferred labels include radioactive labels, fluorescent labels, molecular weight labels and enzymatic labels. Other labels are well known in the art.
  • gel electrophoresis, affinity chromatography, or mass spectrometry are used to detect large DNA fragments (fragments comprising greater than about 200 base pairs). The presence of large DNA fragments in the sample is indicative of colorectal cancer.
  • capture probes comprise DNA, RNA or PNA, and are detectably labeled using methods known in the art.
  • probes are labeled with radioactive isotopes such as 32 P, 33 P, 35 S, 125 I, or any other detectable isotope useful for labeling a hybridization probe.
  • probes are labeled with fluorescent molecules. Numerous fluorescent labels are known in the art, and any detectable fluorescent probe label is useful for practice of the invention.
  • probes are attached to moieties which increase their molecular weight. For example a probe may be directly attached to a glycoprotein, or a glass bead, or any compound which has a detectable effect on the molecular weight of the probe.
  • probes are labeled with a compound that is detectable due to specific interactions with an additional compound.
  • biotinylated probes are detectable via interaction with streptavidin.
  • the streptavidin moiety is attached to a detectable label such as a bead, a fluorescent tag, or an enzyme.
  • the probes are labeled with a hapten or other antigen which is specifically recognized by an antibody.
  • the antibody is made detectable using methods known in the art including radioactive isotopes, fluorescent tags, and enzyme reactions.
  • the probes are directly attached to an enzyme which is detectable via a specific enzyme catalyzed reaction generating a detectable product.
  • methods of the invention allow one to approximate the position in the colon of a colorectal lesion based upon the relative amount of DNA fragments in a stool sample that are greater than 200 base pairs in length.
  • This aspect of the invention relies on the fact that the lytic properties of stool are greater in the proximal colon than they are in the distal colon.
  • stool In the proximal colon, stool is typically in liquid form. Therefore, the cell lysis and DNA degrading enzymes in the colon have greater access to exfoliated cells in the liquid mixture of the proximal colon as compared to their access to exfoliated cells sloughed onto formed or forming stool that is typical in the distal colon.
  • FIG. 1 provides an example of the progression of DNA sizes expected for cancer or precancer cells exfoliated into different regions of the colon.
  • the size of DNA fragments from non-cancerous or precancerous cells is the same throughout the colon due to the fact that the DNA from those cells is degraded primarily through apoptosis.
  • cell lysis and DNA degradation play only minor roles in determining the size of DNA fragments from most exfoliated normal cells throughout the colon.
  • FIG. 1 shows a schematic representation of the colon, and the representative (typical) DNA fragment length for DNA obtained from a cancer or precancer exfoliated cell over the representative (typical) apoptotic (normal) DNA fragment length for various regions of the colon.
  • FIG. 2 is a gel photograph showing results of amplification of Kras (exon 1) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart. The band intensity relates to the amount of 200 bp 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.
  • FIG. 3 is a gel photograph showing results of amplification of apc (exon 15) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart.
  • the band intensity relates to the amount of 200 bp 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
  • lanes 13-18 are standards at the approximate molecular weight indicated in the figure.
  • FIG. 4 is a gel photograph showing results of amplification of apc (exon 15) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart.
  • the band intensity relates to the amount of 200 bp 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
  • lanes 13-18 are standards at the approximate molecular weight indicated in the figure.
  • FIG. 5 is a gel photograph showing results of amplification of apc (exon 15) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart.
  • the band intensity relates to the amount of 200 bp 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 is negative controls
  • lanes 13-18 are standards at the approximate molecular weight indicated in the figure.
  • FIG. 6 is a gel photograph showing results of amplification of p53 (exon 5) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart.
  • the band intensity relates to the amount of 200 bp 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
  • lanes 13-18 are standards at the approximate molecular weight indicated in the figure.
  • FIG. 7 is a gel photograph showing results of amplification of p53 (exon 7) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart.
  • the band intensity relates to the amount of 200 bp 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
  • lanes 13-18 are standards at the approximate molecular weight indicated in the figure.
  • FIG. 8 is a gel photograph showing results of amplification of p53 (exon 8) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart.
  • the band intensity relates to the amount of 200 bp 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
  • lanes 13-18 are standards at the approximate molecular weight indicated in the figure.
  • FIG. 9 is a gel photograph of results of amplification of DNA from stool 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.
  • FIG. 10 is a gel photograph of results of amplification of DNA from stool 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.
  • FIG. 11 is a gel photograph of results of amplification of DNA from stool 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.
  • Methods of the invention are based upon the observation that samples comprising cells from patients with cancer or precancer contain a greater amount of high molecular weight (long sequence) DNA fragments as compared to corresponding samples obtained from individuals that are free of cancer/precancer. Accordingly, methods of the invention provide accurate screening and diagnostic procedures for cancer or precancer.
  • Methods of the invention are useful to detect nucleic acid indicia of cancer or precancer in any tissue or body fluid sample.
  • sputum samples are used to detect the presence of high molecular weight (long sequence) DNA as a marker for cancer.
  • the majority of cells exfoliated into sputum have undergone apoptosis and subsequent further enzymatic degradation.
  • the predominant DNA from those cells is small, apoptotic DNA.
  • Cancer cells produced by, for example, the lungs, the nasal passages, or the trachea will also be sloughed into sputum.
  • the DNA from those cells while being exposed to enzymatic processes, has not been affected by apoptosis. Accordingly, fragments from cancer or precancer cells found in sputum are larger than fragments expected to be produced by normal cells.
  • cells sloughed by cancerous or precancerous lesions in the bladder or kidney produce non-apoptotic DNA in urine
  • cancerous or precancerous lesions in the lymph nodes result in non-apoptotic DNA fragments in lymph
  • cancerous or precancerous cells in the breast slough non-apoptotic DNA-containing cells that can be harvested via aspiration.
  • methods of the invention are useful in any tissue or body fluid.
  • stool sample were used to predict the presence of colorectal cancer or precancer.
  • Stool is an excellent specimen for analysis due to the characteristic exfoliation of colonic epithelia as described above.
  • Methods of the invention are practiced by detecting the presence of DNA fragments having a sequence length that would not be expected to be present in significant amounts in a sample obtained from a healthy individual (i.e., an individual who does not have cancer or precancer).
  • a threshold amount of large fragments is an amount that exceeds a predetermined level expected or determined for non-cancerous/non-precancerous cells.
  • the predetermined level or standard can be determined by detecting the amount of a particular size of DNA fragment (preferably apoptotic fragments characteristic of normal cells) in a population or subpopulation of normal patients. Standards can be determined empirically, and, once determined, can be used as the basis for further screening.
  • the size of fragments to be used is chosen based upon the convenience of the individual performing the screen. Factors affecting the size of fragments used in screening or diagnostic methods of the invention include the availability and costs of probes and primers, the desired target of amplification, the type of cancer being screened, and the patient sample on which screening takes place.
  • the invention takes advantage of the recognition that large fragments exist in greater abundance in abnormal samples than in normal samples. Accordingly, the precise size of fragments used in methods of the invention does not matter.
  • a cutoff must be determined to distinguish between normal and abnormal samples. Preferably, the cutoff is determined empirically based upon known normal and abnormal sample, and then is used in future screenings.
  • preferred methods of the invention comprise obtaining at least a cross-section or circumfrential portion of a voided stool as taught in U.S. Pat. No. 5,741,650, and co-pending, co-owned U.S. patent application Ser. No. 09/059,718, both of which are incorporated by reference herein. While a cross-sectional or circumfrential 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 homogenization is one that contains at least 16 mM ethylenediaminetetraacetic acid (EDTA).
  • a preferred buffer for stool homogenization comprises phosphate buffered saline, 20-100 mM NaCl or KCl, at least 150 mM EDTA, and optionally a detergent (such as SDS) and a proteinase (e.g., proteinase K).
  • 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 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 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.
  • the sample preferably is enriched for human nucleic acids using sequence specific capture probes.
  • Pelletized DNA is resuspended in TE buffer. Guanidine isothiocyanatate (GITC) is then added. An excess of capture probes that target human DNA are added to the sample. The sample is heated to denature the DNA and then cooled. Finally, probe and target DNA are allowed to hybridize.
  • Steptavidin-coated magnetized beads are suspended in water and added to the mixture. After briefly mixing, the mixture is maintained at room temperature for approximately 30 minutes. Once the affinity binding is completed, a magnetic filed is applied to the sample to draw the magnetized isolation beads (both with and without hybridized complex).
  • the beads are then washed four (4) times in 1M GITC/0.1% Igepal (Sigma, St. Louis, Mo.) solution for 15 minutes, followed by two (2) washes with warm buffer (TE with 1M NaCl) for 15 minutes in order to isolate complexed streptavidin. Finally, distilled water is added to the beads and heated to elude the DNA. Gel electrophoresis can then be performed on the human DNA that has been captured.
  • human DNA fragments obtained above can be determined by numerous means.
  • human DNA can be separated using gel electrophoresis.
  • a 5% acrylamide gel is prepared using techniques known in the art. See 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 bp 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 testing to render a confirmed diagnosis.
  • a preferred means for determining human DNA fragment length is by using 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 bp produced by PCR represents a positive screen. Other amplification reactions 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.
  • 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 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 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 though and homogenized in 500 mM Tris, 16 mM EDTA, and 10 mM NaCl, pH 9.0 at a volume:to mass ratio of 3:1.
  • Samples 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 ⁇ g. 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 ⁇ TE), pH 7.4. Finally, the DNA was treated with Rnase.
  • apc-1309 was 5′TTCCAGCAGTGTCACAGCACCCTAGAACCAAATCCAG 3′ (SEQ ID NO: 2)
  • apc-1378 was 5′CAGATAGCCCTGGACAAACMTGCCACGAAGCAGAAG 3′ (SEQ ID NO: 3).
  • probe-bead complexes were isolated using streptavidin-coated beads (Dynal). After washing, probe-bead complexes were suspended at 25° C. for 1 hour in 0.1 ⁇ TE buffer, pH7.4. The suspension was then heated for 4 minutes at 85° C., and the beads were removed.
  • Captured DNA was then amplified using PCR, essentially as described in U.S. Pat. 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 (33 cycles each) were run in duplicate using primers directed to detect fragments in the sample having 200 base pairs or more.
  • FIGS. 2-8 Each Figure represents the results for all 9 patients (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.
  • 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 5% acrylamide gel. The results are shown in FIGS. 9-11 . 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 presence of high-molecular weight DNA was indicative of the disease status of the patient.

Abstract

The invention provides methods for screening tissue or body fluid samples for nucleic acid indicia of cancer or precancer.

Description

  • This application claims benefit of U.S. provisional patent application Ser. No. 60/128,629, the disclosure of which is incorporated by reference herein.
  • FIELD OF THE INVENTION
  • This invention relates to methods for the early detection of cancer in patients by screening for large DNA fragments. Methods of the invention are especially useful in the detection of colon cancer.
  • BACKGROUND OF THE INVENTION
  • Alterations in genomic integrity often are associated with disease or with the propensity for disease. For example, many cancers are thought to arise through a series of mutations in genomic DNA, resulting in genomic instability in the form of uncontrolled cellular growth. In normal cells, damage to genomic DNA typically leads to expression of tumor suppressors, such as the cell-cycle regulator, p53. For example, damage to cellular DNA results in increased expression of p53 which arrests the cell cycle to allow repair of the damage. If the damaged DNA cannot be repaired, the cell undergoes apoptosis, thus preventing the accumulation of additional mutations in daughter cells. If however, there is a mutation in the p53 gene itself (or in another cell cycle regulator), damaged cells will proceed through the cell cycle, giving rise to progeny in which additional DNA mutations will go unchecked. It is the accumulation of these mutations that is the hallmark of many cancers.
  • The process of apoptosis is important not only in the regulation of cellular metabolism, but also in inhibiting oncogenesis. As cells undergo apoptosis, the nucleus becomes small and fragmented. Nuclear DNA is digested into spindle fragments that are generally no larger than about 200 base pairs. As the process continues, usually through multiple pathways, the cell membrane breaks down, and cellular contents are metabolized. As a result, cells that have the potential to enter the multi-step pathway leading to cancer are eliminated.
  • Many cancers are curable if detected early in their development. For example, colorectal cancers typically originate in the colonic epithelium, and are not extensively vascularized (and therefore not invasive) during early stages of development. The transition to a highly-vascularized, invasive and ultimately metastatic cancer commonly takes ten years or longer. If the presence of cancer is detected prior to extensive vascularization, surgical removal typically is an effective cure. However, colorectal cancer is often detected only upon manifestation of clinical symptoms, such as pain and black tarry stool. Generally, such symptoms are present only when the disease is well established, and often after metastasis has occurred. Early detection of colorectal cancer therefore is important in order to significantly reduce its morbidity.
  • Invasive diagnostic methods, such as endoscopic examination, allow direct visual identification, removal, and biopsy of potentially-cancerous tissue. Endoscopy is expensive, uncomfortable, inherently risky, and not a practical tool for early diagnosis.
  • Established non-invasive screening methods involve assaying stool samples for the presence of fecal occult blood or for elevated levels of carcinoembryonic antigen, both of which are suggestive of the presence of colorectal cancer. Additionally, recent developments in molecular biology provide methods of great potential for detecting the presence of a range of DNA mutations indicative of colorectal cancer. The presence of such mutations can be detected in DNA found in stool samples during various stages of colorectal cancer. However, stool comprises cells and cellular debris from the patient, from microorganisms, and from food, resulting in a heterogeneous population of cells. This makes detection of small, specific subpopulations difficult to detect reliably.
  • There is a need in the art for additional non-invasive methods for early diagnosis of cancer that will detect characteristics indicative of the presence of cancer.
  • SUMMARY OF THE INVENTION
  • The present invention provides methods for identifying indicia of cancer in tissue or body fluid samples by identifying non-apoptotic DNA in those samples. The invention also provides methods for identifying indicia of cancer or precancer in samples containing exfoliated epithelial cells. It has now been recognized that DNA obtained from exfoliated normal (non-cancerous) cells is different than DNA obtained from exfoliated cancer or precancer cells. Normal exfoliated cells typically have undergone apoptosis, and thus produce cells or cellular debris (depending upon the stage of apoptosis) comprising DNA that has been substantially degraded. Exfoliated cancer or precancer cells typically have not undergone apoptosis, and such cells or their debris, while producing some very small fragments as a result of degradation in the sample, typically also contain a higher proportion of large DNA fragments (compared to those observed in cells or debris from exfoliated normal cells). The difference in DNA integrity between normal and abnormal cells is a marker for the presence of cancer or precancer in a sample comprising exfoliated cells.
  • Stool is a good sample for exemplification of methods of the invention. The colonic epithelium undergoes a continual process of exfoliation. Normal epithelial cells undergo apoptosis, and are sloughed into the lumen of the colon, and onto forming stool. Cells from polyps and tumors are also sloughed onto forming stool. However, cells from polyps or tumors are, by definition, not apoptotic. Methods of the invention take advantage of the different characteristics between apoptotic and non-apoptotic cells in order to screen patient samples for indicia of cancer or precancer.
  • As noted above, non-cancerous (normal) cells undergo apoptosis at regular intervals, or in response to irreparable cell damage. As a result of apoptosis, DNA from normal cells is cleaved into small fragments having about 200 or fewer base pairs, and typically 180 base pairs or less. In contrast, DNA obtained from cancer or precancer cells is much larger than the typical apoptotic fragments. Thus, the presence of large DNA fragments in a sample (e.g., of sloughed colonic epithelium) indicates that there are or were cells in the sample (or the specimen from which it was obtained) that have avoided apoptosis, and its coincidental degradation of DNA. The presence of large DNA fragments represents a positive screen for cancer or precancer.
  • Accordingly, methods of the invention comprise detecting the presence in a biological sample of species-specific nucleic acids indicative of cancer or precancer. Samples comprising such nucleic acids are identified as having indicia of cancer or precancer. In preferred methods, patients presenting samples having a high proportion of non-apoptotic nucleic acids as determined by methods of the invention are further evaluated for the presence of a tumor, adenoma, or other cancerous or precancerous lesion.
  • In general, methods of the invention comprise detecting in a biological sample one or more DNA fragment(s) of a length that would not be substantially present in noncancerous cells or cellular debris. In a preferred embodiment, such fragments are larger than a typical apoptotic spindle fragment, or larger than about 170 base pairs. However, also in a preferred embodiment, methods of the invention comprise detecting DNA fragments that are greater than about 200 base pairs, and preferably greater than about 500 base pairs. There is no upper limit on these fragments, as all that is necessary is that the fragment be larger than an apoptotic fragment. Typically, however, fragments indicative of cancer or precancer cells are between about 200 and about 3500 base pairs, and ideally between about 500 and about 2500 base pairs.
  • Accordingly, in a preferred embodiment, methods of the invention comprise detecting in a tissue or body fluid sample the presence of nucleic acid fragments having greater than about 500 base pairs or having a molecular weight corresponding to greater than about 500 base pairs. In other preferred embodiments, methods of the invention comprise detecting nucleic acid fragments having between about 200 and about 1000 base pairs, preferably between about 200 and about 600 base pairs, and most preferably about 500 base pairs.
  • Also in a preferred embodiment, methods of the invention comprise determining a ratio of large fragments (200-3500 bp) to small fragments (less than 200 bp), and determining whether the ratio exceeds an empirically-derived threshold. The threshold is determined empirically by analyzing ratios of large-to-small fragments, and correlating those ratios with the disease state of a selected population of normal and cancer patients. In preferred embodiments, amounts of large and small fragments are determined by polymerase chain reaction amplification of sample DNA using primers selected to amplify long and short fragments. Alternatively, amounts of large and small fragments are determined using the same primer.
  • Preferred methods of the invention comprise amplifying nucleic acids in a representative stool sample using human-specific primers, and detecting amplicons having greater than about 200, and preferably about 500 or more base pairs. In a highly-preferred embodiment, amplification is accomplished by polymerase chain reaction (PCR) using forward and reverse primers directed against human-specific nucleic acid fragments, and spaced apart to provide a lower limit on the resulting amplicons. Also in a highly-preferred embodiment, primers for PCR are directed against human oncogene or tumor suppressor sequences. Preferred target nucleic acids for PCR primers include p53, Kras, apc, dcc, and other genes known or suspected to be associated with cancer, and especially colorectal cancer. Methods for conducting PCR are provided in U.S. Pat. No. 4,683,202, incorporated by reference herein. The presence of amplicon greater than about 200 base pairs in length is indicative of template nucleic acid in the sample of that length (or longer). According to methods of the invention such long sequences represent a positive screen, and are indicative of cancer or precancer.
  • Preferred biological samples include stool, pus and urine. Method of the inventions are especially useful for the detection of large DNA fragments in samples comprising exfoliate. Tissue (e.g., colon, lungs, bladder) in which cells, especially epithelial cells, are exfoliated are most preferred for screening methods of the invention. In such tissues, continuing cellular renewal requires that cells are regularly sloughed after having undergone apoptosis. Samples of the exfoliate (tissue or body fluid containing the exfoliated cells) predominantly comprise apoptotic DNA.
  • Preferred methods of the invention for use on a stool sample comprise obtaining a representative stool sample. An especially-preferred method for preparing a stool sample is disclosed in U.S. Pat. No. 5,741,650, and in co-owned, co-pending U.S. patent application Ser. No. 09/059,713 (attorney docket No. EXT-015), each of which is incorporated by reference herein.
  • In a preferred embodiment, methods of the invention comprise homogenizing a representative stool sample in a solvent in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5 to 1. An especially-preferred ratio of solvent volume to stool mass is about 20:1. A preferred solvent for preparing stool samples according to the invention is a physiologically-compatible buffer comprising a detergent and a proteinase and optionally a DNase inhibitor, such as a buffer comprising Tris-EDTA-NaCl. A preferred buffer is 50 mM Tris, 150 mM EDTA and 10 mM NaCl at pH 9.0. Another preferred solvent is guanidine isothiocyanate (GITC). Providing an optimal solvent volume to stool mass ratio increases the yield of nucleic acid generally from the sample. Further details regarding sample preparation are disclosed in co-owned, co-pending U.S. patent application Ser. No. 09/198,083 (Attorney Docket No. EXT-0028) incorporated herein by reference.
  • Preferred methods of the invention further comprise enriching sample for human DNA. Preferred enrichment methods for use in the invention include enriching a desired human target sequence using an affinity column, sequence-specific capture, or through the use of preferred buffers that bias isolation of human DNA. A preferred enrichment method is based upon the capture of unique human nucleic acids using, for example, an affinity column. Details of such methods are provided below.
  • In a preferred embodiment, methods further comprise the step of extracting DNA from the homogenized sample mixture using sequence-specific nucleic acid probes. Particularly preferred are probes hybridizing to human DNA. The probes are preferably labeled. Preferred labels include radioactive labels, fluorescent labels, molecular weight labels and enzymatic labels. Other labels are well known in the art.
  • In a preferred embodiment gel electrophoresis, affinity chromatography, or mass spectrometry are used to detect large DNA fragments (fragments comprising greater than about 200 base pairs). The presence of large DNA fragments in the sample is indicative of colorectal cancer.
  • In a preferred embodiment capture probes comprise DNA, RNA or PNA, and are detectably labeled using methods known in the art. In one embodiment probes are labeled with radioactive isotopes such as 32P, 33P, 35S, 125I, or any other detectable isotope useful for labeling a hybridization probe. In an another embodiment, probes are labeled with fluorescent molecules. Numerous fluorescent labels are known in the art, and any detectable fluorescent probe label is useful for practice of the invention. Alternatively, probes are attached to moieties which increase their molecular weight. For example a probe may be directly attached to a glycoprotein, or a glass bead, or any compound which has a detectable effect on the molecular weight of the probe. In a further embodiment, probes are labeled with a compound that is detectable due to specific interactions with an additional compound. For example, biotinylated probes are detectable via interaction with streptavidin. The streptavidin moiety is attached to a detectable label such as a bead, a fluorescent tag, or an enzyme. In another example, the probes are labeled with a hapten or other antigen which is specifically recognized by an antibody. The antibody is made detectable using methods known in the art including radioactive isotopes, fluorescent tags, and enzyme reactions. In a further example the probes are directly attached to an enzyme which is detectable via a specific enzyme catalyzed reaction generating a detectable product.
  • Finally, methods of the invention allow one to approximate the position in the colon of a colorectal lesion based upon the relative amount of DNA fragments in a stool sample that are greater than 200 base pairs in length. This aspect of the invention relies on the fact that the lytic properties of stool are greater in the proximal colon than they are in the distal colon. In the proximal colon, stool is typically in liquid form. Therefore, the cell lysis and DNA degrading enzymes in the colon have greater access to exfoliated cells in the liquid mixture of the proximal colon as compared to their access to exfoliated cells sloughed onto formed or forming stool that is typical in the distal colon. As a consequence of the differences between the environments of the proximal and distal colon, the present invention provides that typical DNA fragments from cells exfoliated into the proximal colon are smaller than DNA fragments from cells exfoliated into the distal colon. FIG. 1 provides an example of the progression of DNA sizes expected for cancer or precancer cells exfoliated into different regions of the colon. The size of DNA fragments from non-cancerous or precancerous cells is the same throughout the colon due to the fact that the DNA from those cells is degraded primarily through apoptosis. Thus, cell lysis and DNA degradation play only minor roles in determining the size of DNA fragments from most exfoliated normal cells throughout the colon. It is noted, however, that normal cells that are, for example, mechanically sheared from the colon undergo the same lytic and degradation cycle as the typical cancer or precancer cell. However, the contribution of such non-apoptotic normal cells to the overall level of DNA in the stool sample is small, and is controlled for by establishing standards as taught below.
  • Further aspects and advantages of the invention are contained in the following detailed description thereof.
  • DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows a schematic representation of the colon, and the representative (typical) DNA fragment length for DNA obtained from a cancer or precancer exfoliated cell over the representative (typical) apoptotic (normal) DNA fragment length for various regions of the colon.
  • FIG. 2 is a gel photograph showing results of amplification of Kras (exon 1) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart. The band intensity relates to the amount of 200 bp 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.
  • FIG. 3 is a gel photograph showing results of amplification of apc (exon 15) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart. The band intensity relates to the amount of 200 bp 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.
  • FIG. 4 is a gel photograph showing results of amplification of apc (exon 15) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart. The band intensity relates to the amount of 200 bp 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.
  • FIG. 5 is a gel photograph showing results of amplification of apc (exon 15) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart. The band intensity relates to the amount of 200 bp 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 is negative controls, and lanes 13-18 are standards at the approximate molecular weight indicated in the figure.
  • FIG. 6 is a gel photograph showing results of amplification of p53 (exon 5) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart. The band intensity relates to the amount of 200 bp 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.
  • FIG. 7 is a gel photograph showing results of amplification of p53 (exon 7) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart. The band intensity relates to the amount of 200 bp 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.
  • FIG. 8 is a gel photograph showing results of amplification of p53 (exon 8) DNA isolated from stool using forward and reverse primers spaced about 200 bp apart. The band intensity relates to the amount of 200 bp 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.
  • FIG. 9 is a gel photograph of results of amplification of DNA from stool 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.
  • FIG. 10 is a gel photograph of results of amplification of DNA from stool 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.
  • FIG. 11 is a gel photograph of results of amplification of DNA from stool 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.
  • DETAILED DESCRIPTION OF THE INVENTION
  • Methods of the invention are based upon the observation that samples comprising cells from patients with cancer or precancer contain a greater amount of high molecular weight (long sequence) DNA fragments as compared to corresponding samples obtained from individuals that are free of cancer/precancer. Accordingly, methods of the invention provide accurate screening and diagnostic procedures for cancer or precancer.
  • Methods of the invention are useful to detect nucleic acid indicia of cancer or precancer in any tissue or body fluid sample. For example, sputum samples are used to detect the presence of high molecular weight (long sequence) DNA as a marker for cancer. The majority of cells exfoliated into sputum have undergone apoptosis and subsequent further enzymatic degradation. The predominant DNA from those cells is small, apoptotic DNA. Cancer cells produced by, for example, the lungs, the nasal passages, or the trachea will also be sloughed into sputum. However, the DNA from those cells, while being exposed to enzymatic processes, has not been affected by apoptosis. Accordingly, fragments from cancer or precancer cells found in sputum are larger than fragments expected to be produced by normal cells.
  • Similarly, cells sloughed by cancerous or precancerous lesions in the bladder or kidney produce non-apoptotic DNA in urine, cancerous or precancerous lesions in the lymph nodes result in non-apoptotic DNA fragments in lymph, and cancerous or precancerous cells in the breast slough non-apoptotic DNA-containing cells that can be harvested via aspiration. Accordingly, methods of the invention are useful in any tissue or body fluid. However, for purposes of exemplification of the methods described herein, stool sample were used to predict the presence of colorectal cancer or precancer. Stool is an excellent specimen for analysis due to the characteristic exfoliation of colonic epithelia as described above.
  • Methods of the invention are practiced by detecting the presence of DNA fragments having a sequence length that would not be expected to be present in significant amounts in a sample obtained from a healthy individual (i.e., an individual who does not have cancer or precancer). A threshold amount of large fragments is an amount that exceeds a predetermined level expected or determined for non-cancerous/non-precancerous cells. The predetermined level or standard can be determined by detecting the amount of a particular size of DNA fragment (preferably apoptotic fragments characteristic of normal cells) in a population or subpopulation of normal patients. Standards can be determined empirically, and, once determined, can be used as the basis for further screening.
  • The size of fragments to be used is chosen based upon the convenience of the individual performing the screen. Factors affecting the size of fragments used in screening or diagnostic methods of the invention include the availability and costs of probes and primers, the desired target of amplification, the type of cancer being screened, and the patient sample on which screening takes place. The invention takes advantage of the recognition that large fragments exist in greater abundance in abnormal samples than in normal samples. Accordingly, the precise size of fragments used in methods of the invention does not matter. For any given size of fragments to be analyzed, a cutoff must be determined to distinguish between normal and abnormal samples. Preferably, the cutoff is determined empirically based upon known normal and abnormal sample, and then is used in future screenings.
  • 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
  • For the analysis of stool samples, preferred methods of the invention comprise obtaining at least a cross-section or circumfrential portion of a voided stool as taught in U.S. Pat. No. 5,741,650, and co-pending, co-owned U.S. patent application Ser. No. 09/059,718, both of which are incorporated by reference herein. While a cross-sectional or circumfrential 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 homogenization is one that contains at least 16 mM ethylenediaminetetraacetic acid (EDTA). However, as taught in co-pending, co-owned U.S. patent application Ser. No. 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, 20-100 mM NaCl or KCl, 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 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 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.
  • Once DNA is isolated, the sample preferably is enriched for human nucleic acids using sequence specific capture probes. Pelletized DNA is resuspended in TE buffer. Guanidine isothiocyanatate (GITC) is then added. An excess of capture probes that target human DNA are added to the sample. The sample is heated to denature the DNA and then cooled. Finally, probe and target DNA are allowed to hybridize. Steptavidin-coated magnetized beads are suspended in water and added to the mixture. After briefly mixing, the mixture is maintained at room temperature for approximately 30 minutes. Once the affinity binding is completed, a magnetic filed is applied to the sample to draw the magnetized isolation beads (both with and without hybridized complex). The beads are then washed four (4) times in 1M GITC/0.1% Igepal (Sigma, St. Louis, Mo.) solution for 15 minutes, followed by two (2) washes with warm buffer (TE with 1M NaCl) for 15 minutes in order to isolate complexed streptavidin. Finally, distilled water is added to the beads and heated to elude the DNA. Gel electrophoresis can then be performed on the human DNA that has been captured.
  • III. Determination of Fragment Length
  • 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 5% acrylamide gel is prepared using techniques known in the art. See 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 bp 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 testing to render a confirmed diagnosis.
  • A preferred means for determining human DNA fragment length is by using 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 bp produced by PCR represents a positive screen. Other amplification reactions 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. flouresence, 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 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 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 (greater than 200 base pair) fragments in the same samples, and then to determine ratios of long product to short product.
  • I. The Use of Amplifiable DNA as a Marker for Cancer or Precancer
  • 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 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 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 though and homogenized in 500 mM Tris, 16 mM EDTA, and 10 mM NaCl, pH 9.0 at a volume:to mass ratio of 3:1. Samples 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×g. 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×TE), pH 7.4. Finally, the DNA was treated with Rnase.
  • 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′GTGGAGTATTTGATAGTGTATTMCCTTATGTGTGAC 3′ (SEQ ID NO: 1). There were two apc probes: apc-1309 was 5′TTCCAGCAGTGTCACAGCACCCTAGAACCAAATCCAG 3′ (SEQ ID NO: 2), and apc-1378 was 5′CAGATAGCCCTGGACAAACMTGCCACGAAGCAGAAG 3′ (SEQ ID NO: 3). There were four probes against p53, the first (hybridizing to a portion of exon 5) was 5′TACTCCCCTGCCCTCAACMGATGTTTTGCCMCTGG3′ (SEQ ID NO: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′ATGAGGCCAGTGCGCCTTGGGGAGACCTGTGGCMGC3′ (SEQ ID NO: 6); and finally, a probe against exon 8 had the sequence 5′GAAAGGACAAGGGTGGTTGGGAGTAGATGGAGCCTGG3′ (SEQ ID NO: 7). A 10 ul aliquot of each probe (20 pmol/capture) was added to a suspension containing 300 ul DNA in the presence of 310 ul 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 0.1×TE buffer, pH7.4. The suspension was then heated for 4 minutes at 85° C., and the beads were removed.
  • Captured DNA was then amplified using PCR, essentially as described in U.S. Pat. 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 (33 cycles each) were run in 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 ug/ml). The resulting amplified DNA was graded based upon the relative intensity of the stained gels. The results are shown in FIGS. 2-8. Each Figure represents the results for all 9 patients (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 amplifiable DNA 200 base pairs or greater in length. As shown in FIGS. 2-8, the results were the same regardless of which locus was amplified. Accordingly, the amount of 200 bp or greater DNA in a sample was predictive of patient disease status.
  • Example 2
  • An experiment was conducted that was essentially identical to the one described 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 5% acrylamide gel. The results are shown in FIGS. 9-11. 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 presence of high-molecular weight DNA was indicative of the disease status of the patient.
  • The invention has been described in terms of its preferred embodiments. Alternative embodiments are apparent to the skilled artisan upon examination of the specification and claims.

Claims (7)

1. A method for screening a patient for cancer or precancer, the method comprising the steps of:
detecting in a patient tissue or body fluid sample comprising exfoliated cells and cellular debris, nucleic acid fragments that are greater than 200 base pairs in length;
the presence of said fragments being a positive screen for cancer or precancer.
2. The method of claim 1, wherein said detecting step comprises conducting an amplification reaction designed to amplify only nucleic acids in said sample that are greater than 200 base pairs in length.
3. The method of claim 1, wherein said sample is selected from the group consisting of stool, pus, and urine.
4. The method of claim 1, further comprising the step of enriching said sample for human DNA.
5. The method of claim 1, further comprising the step of isolating human DNA from said sample.
6. A method for screening a patient for cancer or precancer, the method comprising the steps of:
determining in a patient tissue or body fluid sample a first amount of nucleic acid fragments greater than 200 base pairs in length;
determining in said sample a second amount of nucleic acid fragments less than about 200 base pairs in length;
determining a ratio between said first amount and said second amount; and
identifying a positive screen if said ratios exceeds a threshold ratio for patients who do not have cancer or precancer.
7. A method for screening a patient for cancer or precancer, the method comprising the step of
detecting in a patient tissue or body fluid sample comprising exfoliated cells a nucleic acid fragment of a length that is not expected to be present in said sample in a healthy patient;
the presence of said fragment being a positive screen.
US11/960,313 1999-04-09 2007-12-19 Methods for detecting nucleic acids indicative of cancer Abandoned US20090291436A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US11/960,313 US20090291436A1 (en) 1999-04-09 2007-12-19 Methods for detecting nucleic acids indicative of cancer
US12/729,051 US20100173320A1 (en) 1999-04-09 2010-03-22 Methods for Detecting Nucleic Acids Indicative of Cancer
US13/919,622 US20130280727A1 (en) 1999-04-09 2013-06-17 Methods for detecting nucleic acids indicative of cancer

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US12862999P 1999-04-09 1999-04-09
US09/545,162 US6964846B1 (en) 1999-04-09 2000-04-07 Methods for detecting nucleic acids indicative of cancer
US11/090,479 US20050260638A1 (en) 1999-04-09 2005-03-23 Methods for detecting nucleic acids indicative of cancer
US11/960,313 US20090291436A1 (en) 1999-04-09 2007-12-19 Methods for detecting nucleic acids indicative of cancer

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US11/090,479 Continuation US20050260638A1 (en) 1999-04-09 2005-03-23 Methods for detecting nucleic acids indicative of cancer

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/729,051 Continuation US20100173320A1 (en) 1999-04-09 2010-03-22 Methods for Detecting Nucleic Acids Indicative of Cancer

Publications (1)

Publication Number Publication Date
US20090291436A1 true US20090291436A1 (en) 2009-11-26

Family

ID=22436245

Family Applications (5)

Application Number Title Priority Date Filing Date
US09/545,162 Expired - Fee Related US6964846B1 (en) 1999-04-09 2000-04-07 Methods for detecting nucleic acids indicative of cancer
US11/090,479 Abandoned US20050260638A1 (en) 1999-04-09 2005-03-23 Methods for detecting nucleic acids indicative of cancer
US11/960,313 Abandoned US20090291436A1 (en) 1999-04-09 2007-12-19 Methods for detecting nucleic acids indicative of cancer
US12/729,051 Abandoned US20100173320A1 (en) 1999-04-09 2010-03-22 Methods for Detecting Nucleic Acids Indicative of Cancer
US13/919,622 Abandoned US20130280727A1 (en) 1999-04-09 2013-06-17 Methods for detecting nucleic acids indicative of cancer

Family Applications Before (2)

Application Number Title Priority Date Filing Date
US09/545,162 Expired - Fee Related US6964846B1 (en) 1999-04-09 2000-04-07 Methods for detecting nucleic acids indicative of cancer
US11/090,479 Abandoned US20050260638A1 (en) 1999-04-09 2005-03-23 Methods for detecting nucleic acids indicative of cancer

Family Applications After (2)

Application Number Title Priority Date Filing Date
US12/729,051 Abandoned US20100173320A1 (en) 1999-04-09 2010-03-22 Methods for Detecting Nucleic Acids Indicative of Cancer
US13/919,622 Abandoned US20130280727A1 (en) 1999-04-09 2013-06-17 Methods for detecting nucleic acids indicative of cancer

Country Status (9)

Country Link
US (5) US6964846B1 (en)
EP (1) EP1169479B1 (en)
JP (2) JP4794052B2 (en)
AT (1) ATE331811T1 (en)
AU (1) AU767983B2 (en)
CA (1) CA2366778C (en)
DE (1) DE60029092T2 (en)
ES (1) ES2269129T3 (en)
WO (1) WO2000061808A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110244461A1 (en) * 2008-12-05 2011-10-06 Olympus Corporation Method for preparing stool sample, solution for preparing stool sample and stool collection kit

Families Citing this family (52)

* Cited by examiner, † Cited by third party
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
ATE331811T1 (en) * 1999-04-09 2006-07-15 Exact Sciences Corp METHOD FOR DETECTING NUCLEIC ACIDS INdicative of CANCER
US6586177B1 (en) * 1999-09-08 2003-07-01 Exact Sciences Corporation Methods for disease detection
US7368233B2 (en) 1999-12-07 2008-05-06 Exact Sciences Corporation Methods of screening for lung neoplasm based on stool samples containing a nucleic acid marker indicative of a neoplasm
US6919174B1 (en) * 1999-12-07 2005-07-19 Exact Sciences Corporation Methods for disease detection
GB0130821D0 (en) * 2001-12-24 2002-02-06 Tayside University Hospitals N DNA screening
US7776524B2 (en) 2002-02-15 2010-08-17 Genzyme Corporation Methods for analysis of molecular events
US9353405B2 (en) 2002-03-12 2016-05-31 Enzo Life Sciences, Inc. Optimized real time nucleic acid detection processes
US7041481B2 (en) 2003-03-14 2006-05-09 The Regents Of The University Of California Chemical amplification based on fluid partitioning
US20050014165A1 (en) * 2003-07-18 2005-01-20 California Pacific Medical Center Biomarker panel for colorectal cancer
WO2005113769A1 (en) * 2004-05-14 2005-12-01 Exact Sciences Corporation Method for stabilizing biological samples for nucleic acid analysis
WO2006047787A2 (en) 2004-10-27 2006-05-04 Exact Sciences Corporation Method for monitoring disease progression or recurrence
WO2007044071A2 (en) 2005-04-21 2007-04-19 Exact Sciences Corporation Analysis of heterogeneous nucleic acid samples
US8249814B2 (en) 2005-10-21 2012-08-21 Genenews Inc. Method, computer readable medium, and system for determining a probability of colorectal cancer in a test subject
EP2167683A4 (en) 2007-05-31 2010-10-20 California Pacific Med 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
US9764322B2 (en) 2008-09-23 2017-09-19 Bio-Rad Laboratories, Inc. System for generating droplets with pressure monitoring
US8633015B2 (en) 2008-09-23 2014-01-21 Bio-Rad Laboratories, Inc. Flow-based thermocycling system with thermoelectric cooler
US9417190B2 (en) 2008-09-23 2016-08-16 Bio-Rad Laboratories, Inc. Calibrations and controls for droplet-based assays
US10512910B2 (en) 2008-09-23 2019-12-24 Bio-Rad Laboratories, Inc. Droplet-based analysis method
US8951939B2 (en) 2011-07-12 2015-02-10 Bio-Rad Laboratories, Inc. Digital assays with multiplexed detection of two or more targets in the same optical channel
US8709762B2 (en) 2010-03-02 2014-04-29 Bio-Rad Laboratories, Inc. System for hot-start amplification via a multiple emulsion
US9492797B2 (en) 2008-09-23 2016-11-15 Bio-Rad Laboratories, Inc. System for detection of spaced droplets
US9132394B2 (en) 2008-09-23 2015-09-15 Bio-Rad Laboratories, Inc. System for detection of spaced droplets
US11130128B2 (en) 2008-09-23 2021-09-28 Bio-Rad Laboratories, Inc. Detection method for a target nucleic acid
US9598725B2 (en) 2010-03-02 2017-03-21 Bio-Rad Laboratories, Inc. Emulsion chemistry for encapsulated droplets
US9156010B2 (en) 2008-09-23 2015-10-13 Bio-Rad Laboratories, Inc. Droplet-based assay system
US8663920B2 (en) 2011-07-29 2014-03-04 Bio-Rad Laboratories, Inc. Library characterization by digital assay
EP2473618B1 (en) 2009-09-02 2015-03-04 Bio-Rad Laboratories, Inc. System for mixing fluids by coalescence of multiple emulsions
EP2556170A4 (en) 2010-03-25 2014-01-01 Quantalife Inc Droplet transport system for detection
CA2767113A1 (en) 2010-03-25 2011-09-29 Bio-Rad Laboratories, Inc. Detection system for droplet-based assays
JP2013524171A (en) 2010-03-25 2013-06-17 クァンタライフ・インコーポレーテッド Droplet generation for drop-based assays
EP4016086A1 (en) 2010-11-01 2022-06-22 Bio-Rad Laboratories, Inc. System for forming emulsions
WO2012129187A1 (en) 2011-03-18 2012-09-27 Bio-Rad Laboratories, Inc. Multiplexed digital assays with combinatorial use of signals
JP2014512826A (en) 2011-04-25 2014-05-29 バイオ−ラド ラボラトリーズ インコーポレイテッド Methods and compositions for nucleic acid 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
WO2013155531A2 (en) 2012-04-13 2013-10-17 Bio-Rad Laboratories, Inc. Sample holder with a well having a wicking promoter
IL269097B2 (en) 2012-09-04 2024-01-01 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
US20160040229A1 (en) 2013-08-16 2016-02-11 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
WO2015066695A1 (en) 2013-11-04 2015-05-07 Exact Sciences Corporation Multiple-control calibrators for dna quantitation
EP3084004A4 (en) 2013-12-19 2017-08-16 Exact Sciences Corporation Synthetic nucleic acid control molecules
EP3378952B1 (en) 2013-12-28 2020-02-05 Guardant Health, Inc. Methods and systems for detecting genetic variants
EP3390668A4 (en) 2015-12-17 2020-04-01 Guardant Health, Inc. Methods to determine tumor gene copy number by analysis of cell-free dna
US11345949B2 (en) 2016-07-19 2022-05-31 Exact Sciences Corporation Methylated control DNA
EP3488003B1 (en) 2016-07-19 2023-10-25 Exact Sciences Corporation 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
WO2018064629A1 (en) 2016-09-30 2018-04-05 Guardant Health, Inc. Methods for multi-resolution analysis of cell-free nucleic acids
US10907211B1 (en) 2017-02-16 2021-02-02 Quantgene Inc. Methods and compositions for detecting cancer biomarkers in bodily fluids
CN108949750A (en) * 2018-08-17 2018-12-07 上海锐翌生物科技有限公司 Extract the method and kit of faeces DNA
AU2020216438A1 (en) 2019-01-31 2021-07-29 Guardant Health, Inc. Compositions and methods for isolating cell-free DNA

Family Cites Families (111)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US4578358A (en) 1983-05-03 1986-03-25 Warner-Lambert Company Collection of specimens and detection of occult blood therein
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying 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
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
US4857300A (en) 1987-07-27 1989-08-15 Cytocorp, Inc. Cytological and histological fixative formulation and methods for using same
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
US5362623A (en) 1991-06-14 1994-11-08 The John Hopkins University Sequence specific DNA binding by p53
US5527676A (en) 1989-03-29 1996-06-18 The Johns Hopkins University Detection of loss of the wild-type P53 gene and kits therefor
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
US5302509A (en) 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides
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
DE69032209T2 (en) 1990-01-04 1998-12-03 Univ Johns Hopkins GENE LACKING IN HUMAN COLORECTAL CARCINOMAS
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
DE69133528T2 (en) 1990-06-27 2006-09-07 Princeton University Protein complex p53 / p90
AU8951191A (en) 1990-10-29 1992-05-26 Dekalb Plant Genetics 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
DE69210588T2 (en) 1991-02-05 1996-09-19 Lifecodes Corp Molecular genetic identification using probes that recognize polymorphic sites
JPH07500411A (en) 1991-02-05 1995-01-12 ファロク セィディ Simple test method for detecting carcinoembryonic antigen
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
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
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
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
ATE253645T1 (en) 1992-04-01 2003-11-15 Univ Johns Hopkins Med METHOD FOR DETERMINING MAMMAL NUCLEIC ACIDS FROM Stool SAMPLES AND REAGENTS REQUIRED 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
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
US5409586A (en) 1992-08-26 1995-04-25 Hitachi, Ltd. Method for analyzing nucleic acid or protein and apparatus therefor
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)
US5492808A (en) 1993-05-05 1996-02-20 The Johns Hopkins University Means for detecting familial colon cancer (FCC)
US5466576A (en) 1993-07-02 1995-11-14 Fred Hutchinson Cancer Research Center Modulation of PIF-1-type helicases
US5688643A (en) 1993-07-09 1997-11-18 Wakunaga Seiyaku Kabushiki Kaisha Method of nucleic acid-differentiation and assay kit for nucleic acid differentiation
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
US5599662A (en) 1995-02-17 1997-02-04 Hoffmann-La Roche Inc. Oliconucleotide primers and probes for the detection of HIV-1
DE19530132C2 (en) 1995-08-16 1998-07-16 Max Planck Gesellschaft Process for the purification, stabilization or isolation of nucleic acids from biological materials
GB9518156D0 (en) * 1995-09-06 1995-11-08 Medical Res Council Method of isolating cells
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
US5612473A (en) 1996-01-16 1997-03-18 Gull Laboratories Methods, kits and solutions for preparing sample material for nucleic acid amplification
US5741650A (en) 1996-01-30 1998-04-21 Exact Laboratories, Inc. Methods for detecting colon cancer from stool samples
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
US6607946B1 (en) * 1996-05-22 2003-08-19 Micron Technology, Inc. Process for growing a dielectric layer on a silicon-containing surface using a mixture of N2O and O3
US5976800A (en) 1996-06-28 1999-11-02 The Regents Of The University Of California Enhancement of cancer cell death
US6100029A (en) 1996-08-14 2000-08-08 Exact Laboratories, Inc. Methods for the detection of chromosomal aberrations
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
US5928870A (en) 1997-06-16 1999-07-27 Exact Laboratories, Inc. Methods for the detection of loss of heterozygosity
US6300077B1 (en) * 1996-08-14 2001-10-09 Exact Sciences Corporation Methods for the detection of nucleic acids
US6203993B1 (en) 1996-08-14 2001-03-20 Exact Science Corp. Methods for the detection of nucleic acids
US6020137A (en) 1996-08-14 2000-02-01 Exact Laboratories, Inc. Methods for the detection of loss of heterozygosity
US6146828A (en) 1996-08-14 2000-11-14 Exact Laboratories, Inc. Methods for detecting differences in RNA expression levels and uses therefor
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
US5856104A (en) 1996-10-28 1999-01-05 Affymetrix, Inc. Polymorphisms in the glucose-6 phosphate dehydrogenase locus
US5830665A (en) 1997-03-03 1998-11-03 Exact Laboratories, Inc. Contiguous genomic sequence scanning
DE19712332A1 (en) 1997-03-25 1998-10-01 Boehringer Mannheim Gmbh Method for the detection of microsatellite instability for tumor diagnosis
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
US6406857B1 (en) 1997-06-16 2002-06-18 Exact Sciences Corporation Methods for stool sample preparation
WO1998058081A1 (en) * 1997-06-16 1998-12-23 Exact Laboratories, Inc. Methods for stool sample preparation
US20020004201A1 (en) * 1997-06-16 2002-01-10 Lapidus Stanley N. Methods for the detection of loss of heterozygosity
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
US6818404B2 (en) * 1997-10-23 2004-11-16 Exact Sciences Corporation Methods for detecting hypermethylated nucleic acid in heterogeneous biological samples
AU753732B2 (en) * 1997-10-23 2002-10-24 Genzyme Corporation Methods for detecting contamination in molecular diagnostics using PCR
US6251660B1 (en) 1997-11-25 2001-06-26 Mosaic Technologies, Inc. Devices and methods for detecting target molecules in biological samples
EP1066408A4 (en) 1998-03-05 2002-09-25 Diadexus Inc A novel method of detecting and monitoring endometrial and uterine cancers
WO1999066078A1 (en) 1998-06-18 1999-12-23 Mosaic Technologies Denaturing gradient affinity electrophoresis and methods of use thereof
US20020001800A1 (en) * 1998-08-14 2002-01-03 Stanley N. Lapidus Diagnostic methods using serial testing of polymorphic loci
WO2000020643A1 (en) 1998-10-05 2000-04-13 Mosaic Technologies Reverse displacement assay for detection of nucleic acid sequences
JP2002530120A (en) * 1998-11-23 2002-09-17 エグザクト サイエンシーズ コーポレイション Primer extension method using donor molecule and acceptor molecule for detecting nucleic acid
US20020048752A1 (en) * 1998-11-23 2002-04-25 Stanley N. Lapidus 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
ES2269102T3 (en) * 1999-02-25 2007-04-01 Exact Sciences Corporation PROCEDURES TO PRESERVE THE INTEGRITY OF DNA.
ATE331811T1 (en) * 1999-04-09 2006-07-15 Exact Sciences Corp METHOD FOR DETECTING NUCLEIC ACIDS INdicative of CANCER
WO2000066005A1 (en) 1999-05-03 2000-11-09 Exact Laboratories, Inc. Stool specimen collector
US6586177B1 (en) * 1999-09-08 2003-07-01 Exact Sciences Corporation Methods for disease detection
US7368233B2 (en) * 1999-12-07 2008-05-06 Exact Sciences Corporation Methods of screening for lung neoplasm based on stool samples containing a nucleic acid marker indicative of a neoplasm
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

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110244461A1 (en) * 2008-12-05 2011-10-06 Olympus Corporation Method for preparing stool sample, solution for preparing stool sample and stool collection kit

Also Published As

Publication number Publication date
JP2002541824A (en) 2002-12-10
ATE331811T1 (en) 2006-07-15
US20130280727A1 (en) 2013-10-24
AU4210500A (en) 2000-11-14
WO2000061808A9 (en) 2002-04-04
US20050260638A1 (en) 2005-11-24
DE60029092T2 (en) 2007-02-01
US6964846B1 (en) 2005-11-15
US20100173320A1 (en) 2010-07-08
WO2000061808A3 (en) 2001-08-02
EP1169479A2 (en) 2002-01-09
JP4794052B2 (en) 2011-10-12
CA2366778A1 (en) 2000-10-19
ES2269129T3 (en) 2007-04-01
DE60029092D1 (en) 2006-08-10
JP2010187701A (en) 2010-09-02
EP1169479B1 (en) 2006-06-28
AU767983B2 (en) 2003-11-27
CA2366778C (en) 2008-07-22
WO2000061808A2 (en) 2000-10-19

Similar Documents

Publication Publication Date Title
US6964846B1 (en) Methods for detecting nucleic acids indicative of cancer
US6406857B1 (en) Methods for stool sample preparation
ES2886923T3 (en) Methods and composition for generating single-sequence DNA probes, labeling of DNA probes, and the use of these probes
US6268136B1 (en) Methods for stool sample preparation
US20080145852A1 (en) Methods and compositions for detecting adenoma
CN109112216B (en) Triple qPCR (quantitative polymerase chain reaction) detection kit and method for DNA methylation
CN109825586B (en) DNA methylation qPCR kit for lung cancer detection and use method
AU767833B2 (en) Methods of detecting colorectal disease
CN110484621B (en) Early warning method for liver cancer
CN111235238A (en) DNA methylation detection method and related application
WO2021180106A1 (en) Probe composition for detecting five tumors of digestive tract
WO1998058081A1 (en) Methods for stool sample preparation
US20020004206A1 (en) Methods of screening for disease
WO2021185274A1 (en) Probe composition for detecting 6 cancers with high incidence in china
WO2021180105A1 (en) Probe composition for detecting common cancers of both sexes
JP5865241B2 (en) Prognostic molecular signature of sarcoma and its use
WO2021185275A1 (en) Probe composition for detecting 11 cancers
US7592137B2 (en) Genetic testing kits and a method of bladder cancer
WO2001086288A2 (en) Early diagnosis of bladder tumor in urine samples
JP2016198027A (en) Method for diagnosing ovarian cancer

Legal Events

Date Code Title Description
AS Assignment

Owner name: EXACT LABORATORIES, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SHUBER, ANTHONY P.;REEL/FRAME:020743/0582

Effective date: 20000713

AS Assignment

Owner name: EXACT SCIENCES CORPORATION, MASSACHUSETTS

Free format text: CHANGE OF NAME;ASSIGNOR:EXACT LABORATORIES, INC.;REEL/FRAME:020826/0887

Effective date: 20001201

AS Assignment

Owner name: GENZYME CORPORATION, MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:EXACT SCIENCES CORPORATION;REEL/FRAME:022460/0934

Effective date: 20090127

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: ESOTERIX GENETIC LABORATORIES, LLC, NORTH CAROLINA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:GENZYME CORPORATION;REEL/FRAME:025656/0581

Effective date: 20101130

AS Assignment

Owner name: EXACT SCIENCES DEVELOPMENT COMPANY, LLC, WISCONSIN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:EXACT SCIENCES CORPORATION;REEL/FRAME:044119/0001

Effective date: 20171001

AS Assignment

Owner name: EXACT SCIENCES CORPORATION, WISCONSIN

Free format text: MERGER;ASSIGNOR:EXACT SCIENCES DEVELOPMENT COMPANY, LLC;REEL/FRAME:058738/0465

Effective date: 20220101