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`WO 2005/113769
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`PCT/US2005/017046
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`METHOD FOR STABILIZING BIOLOGICAL SAMPLES FOR NUCLEIC ACID
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`ANALYSIS
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`RELATED APPLICATIONS
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`5
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`This application claims priority under 35 U.S.C. § I 19(e) to U.S. Provisional
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`Application Serial No. 60/571,120, entitled "Method for Stabilizing a Biological Sample for a
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`Nucleic Acid Integrity Assay" filed on May 14, 2004, the disclosure of which is herein
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`incorporated by reference in its entirety.
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`10
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`15
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`FIELD OF THE INVENTION
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`The invention relates generally to assays to detect nucleic acid markers indicative of
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`cancer and other diseases and more particularly to preparing nucleic acid-containing biological
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`samples for use in these assays.
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`BACKGROUND OF THE INVENTION
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`Tissue and body fluid samples, including stools, contain shed cellular debris. In healthy
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`patients, such debris is the result of apoptotic degradation as part of the normal cell cycle.
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`Apoptosis reduces the integrity (intactness) of nucleic acids, proteins, and other cellular
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`components in healthy individuals, so that only small fragments exist in the debris that results
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`20
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`from the apoptotic process (e.g., exfoliated cellular debris).
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`In diseases in which cell cycle mechanisms are destroyed or impaired, cellular debris
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`can include high-integrity cellular components, such as nucleic acids that have not been
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`degraded by apoptosis. One class of disease in which cell cycle mechanisms are disrupted is
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`cancer. An increased presence of high molecular weight nucleic acids in a biological sample
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`25
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`therefore can reveal the presence of cancer in a patient from whom the biological sample was
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`obtained. Disease detection assays known as nucleic acid integrity analysis assays have been
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`developed that are based on the increased levels of non-degraded nucleic acid in a cancerous
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`tissue or body fluid as compared to the level of non-degraded nucleic acid in a non-cancerous
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`tissue or body fluid.
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`30
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`Nucleic acids in patient samples tend to degrade after they have been removed from the
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`patient. This degradation can diminish the effectiveness of a nucleic acid integrity assay that
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`scores a sample as diseased (e.g., cancerous) based on the presence of intact nucleic acids; if
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`Geneoscopy Exhibit 1008, Page 2
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`the sample is excessively degraded, a sample that is actually positive may appear to be
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`negative.
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`SUMMARY OF THE INVENTION
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`5
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`The invention is based in part on the discovery of a method for stabilizing nucleic acids
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`in tissue and body fluid samples so as to facilitate analysis of the samples in a nucleic acid
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`integrity assay. It has been unexpectedly found that contacting a patient sample with a
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`stabilization solution stabilizes the DNA so that intact nucleic acids indicative of diseased cells
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`are more effectively detected in a nucleic acid integrity assay.
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`Accordingly, in one aspect, the invention provides a method for preparing a nucleic
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`acid sample for a nucleic acid integrity analysis assay. In one aspect, the invention provides a
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`method for preparing a nucleic acid sample for a nucleic acid integrity analysis assay. The
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`method includes providing a patient sample that includes shed cells or cellular debris and a
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`nucleic acid and contacting the patient sample with a stabilization solution under conditions
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`15
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`sufficient to stabilize the nucleic acid for nucleic acid integrity analysis. In one embodiment,
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`the stabilization solution includes a buffer, a salt, and a chelating agent.
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`In some embodiments, the conditions are sufficient to detect at least a three-fold
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`genomic equivalent (GE) increase in a nucleic acid integrity analysis of a patient sample
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`having adenoma or cancer as compared to the GE detected in a nucleic acid integrity analysis
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`20
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`of a sample from the patient that is not incubated with the stabilization solution. In one
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`embodiment, the integrity analysis is performed by determining an amount of nucleic acid
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`greater than about 200 bp in length using an assay that detects a nucleic acid (which can be a
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`wild-type or mutant nucleic acid). The nucleic acid is present in a patient sample that includes
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`shed cells or cellular debris. A patient is identified as having cancer or adenoma if the amount
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`of nucleic acid is greater than an amount of nucleic acid expected to be present in a sample
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`obtained from a patient who does not have cancer or adenoma.
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`The patient sample can be obtained from a patient that is, e.g., a vertebrate, including a
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`mammal, a reptile, or an amphibian. A mammal can be, e.g., a human, a non-human primate
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`(such as a gorilla or monkey, including a chimpanzee), a rodent (such as a mouse, rat, guinea
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`pig, or gerbil) dog, cat, horse, pig, goat, sheep, or cow. The patient sample can be any body
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`tissue or fluid that is suspected of containing DNA from a diseased cell (such as a precancerous
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`or cancerous cell). In one embodiment, the patient sample is a stool sample.
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`Geneoscopy Exhibit 1008, Page 3
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`In some embodiments, the patient sample can be obtained as part of a screen for, e.g., a
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`disease or disease-associated condition that impairs, or could lead to impairment of, the proper
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`function of the gastrointestinal system. Gastrointestinal diseases can include, e.g., those
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`associated with the stomach, small intestine, and/or colon. The disease or condition can include
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`cancers or precancerous conditions such as an adenoma. Other conditions include
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`inflammatory bowel syndrome, inflammatory bowel disease, Crohn's disease, and others in
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`which a genomic instability is thought to play a role.
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`In some embodiments, the patient sample is frozen and thawed prior to incubation with
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`stabilization solution. In other embodiments, the patient sample is not frozen prior to
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`incubation with the stabilization solution.
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`In general, the stabilization solution is added to the patient sample at a ratio of about 1
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`ml/gram of patient sample to about 20 ml/gram of patient sample. In some embodiments, the
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`stabilization solution is provided at 1-15 ml/gram, 2-12 ml/gram, 3-11 ml/gram, or 4-7
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`ml/gram. However, higher or lower ratios may be used. When the patient sample is a stool
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`sample, and the stabilization solution is 10 mM Tris-Cl, 1 mM EDTA, and 150 mMNaCL, a
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`suitable ratio of stabilization solution to patient sample is 7 ml/gm.
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`In some embodiments, the patient sample and stabilization solution are incubated at
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`about 4 to 28 degrees Centigrade. In some embodiments the temperature is 17 to 27 degrees
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`Centigrade, e.g., about 20 to 25 degrees Centigrade. However, the sample and stabilization
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`solution may be exposed to higher or lower temperatures (e.g., the sample and stabilization
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`solution may be frozen). Also, a sample and buffer may be exposed to changing temperatures
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`during transport and/or storage.
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`In various embodiments, the patient sample and stabilization solution are incubated at
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`least 6 hours, e.g., at least 12 hours, at least 24 hours, or at least 36 hours.
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`25
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`In some embodiments, the buffer in the stabilization solution is 0.5 mM to 25 mM Tris,
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`e.g., 5 mM to 15 mM Tris, 8 mM to 13 mM Tris or about 10 mM Tris. However, other buffers
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`and/or concentrations may be used.
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`In some embodiments, the chelating agent in the stabilization solution is 0.01 to 2.5
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`mM EDTA, e.g., 0.75-1.25 mM EDTA, or lmM EDTA. However, other chelating agents
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`and/or concentrations may be used.
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`In some embodiments, the salt in the stabilization solution is 75 mM to 225 mM NaCl, e.g.,
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`100 mM to 175 mM NaCl, or 150 mM NaCL However, other salts (e.g., KCl etc.) and/or
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`concentrations may be used.
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`Geneoscopy Exhibit 1008, Page 4
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`In some embodiments, the stabilization solution is provided at pH 7.0 to 9.0, e.g., pH
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`7.5 to 8.5, or pH 8.0. However, higher or lower pHs may be used.
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`In some embodiments, the method further includes determining in the incubated patient
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`sample an amount of nucleic acid greater than about 200 hp in length using an assay that
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`5
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`detects wild-type or mutant nucleic acid, and identifying the patient as having cancer or
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`adenoma if the amount is greater than an amount of nucleic acid expected to be present in a
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`sample obtained from a patient who does not have cancer or adenoma ( e.g., more than about
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`1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 50 fold, or more greater than an amount expected
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`in a normal individual). In one aspect, a DNA integrity assay may include interrogating a
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`sample for the presence of long DNA :fragments ( e.g., longer than 200 nucleotides, longer than
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`500 nucleotides, longer than 1,000 nucleotides, etc.) at two or more different loci. In one
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`embodiment, a patient is identified as having cancer or adenoma if two or more loci (e.g., 3, 4,
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`or more loci) are positive for the presence of abnormally high levels of long DNA.
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`In one embodiment, the invention provides a method for preparing a nucleic acid
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`15
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`sample for a nucleic acid integrity and/or multiple mutation analysis assays for diagnosing a
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`carcinoma or adenoma. The method may include providing a patient stool sample that
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`includes shed cells or cellular debris and a nucleic acid, and incubating the patient sample with
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`a stabilization solution under conditions sufficient to stabilize the nucleic acid for nucleic acid
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`integrity and/or multiple mutation analysis. The stabilization solution may be about pH 7.5 to
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`about pH 8.5 and may include 0.5 mM to 25 mM Tris, 0.01 to 2.5 mM EDTA and 100 mM to
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`200 mM NaCl. For example, the stabilization solution can be 10 mM Tris-Cl pH 8.0, 1 mM
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`EDTA, and 150mM NaCl. In one embodiment, the conditions are sufficient to detect at least a
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`three-fold genomic equivalent (GE) increase in a nucleic acid integrity analysis of a patient
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`sample having adenoma or cancer as compared to the GE detected in a nucleic acid integrity
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`25
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`analysis of a sample from the patient that is not incubated with the stabilization solution.
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`Aspects of the invention may be used for transporting or storing biological samples
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`after they are obtained and before they are processed for analysis. For example, methods of the
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`invention may be used to stabilize nucleic acid in biological samples (e.g., stool samples) for
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`about 12 hours, about 24 hours, about 36 hours, or longer (e.g., 4 days, 5, days, 6 days, 1 week,
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`or longer), even in the absence ofrefrigeration or freezing.
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`Aspects of the invention may be particularly useful for detecting indicia of adenomas,
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`early stage cancers, and/or other diseases that may be characterized by very low frequencies of
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`mutant nucleic acid in a sample.
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`Geneoscopy Exhibit 1008, Page 5
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`Aspects of the invention may be particularly useful for preserving biological samples
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`for nucleic acid integrity assays or for multi-panel screens that include one or more nucleic
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`acid integrity assays. Aspects of the invention may be particularly useful for detecting
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`mutations in samples that contain very little human DNA (e.g., DIA negative samples).
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`5 According to the invention, the amount of human DNA recovered from stool from different
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`subjects may vary and it may be particularly important to preserve nucleic acids in samples
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`that contain low amounts of human DNA.
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`Accordingly, aspects of the invention may be useful for screening a population to
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`identify individuals with indicia of disease, and for avoiding or reducing the number of false
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`10
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`negatives (subjects with a disease but who are identified as normal or healthy) in such screens.
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`In one embodiment, average-risk individuals may screened for one or more indicia of a
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`sporadic disease (e.g., adenoma, cancer, etc.). In one embodiment, high-risk individuals may
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`be screened for a sporadic disease or a disease that may be inherited. In one embodiment, a
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`screen may be performed on a population of individuals regardless of their risk profile.
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`Unless otherwise defined, all technical and scientific terms used herein have the same
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`meaning as commonly understood by one of ordinary skill in the art to which this invention
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`belongs. Although methods and materials similar or equivalent to those described herein can be
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`used in the practice or testing of the invention, suitable methods and materials are described
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`below. All publications, patent applications, patents, and other references mentioned herein are
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`incorporated by reference in their entirety. In the case of conflict, the present Specification,
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`including definitions, will control. In addition, the materials, methods, and examples are
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`illustrative only and not intended to be limiting.
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`Other features and advantages of the invention will be apparent from the following
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`detailed description and claims.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`Figure 1 is a Table (Table 2) showing the results of DNA Integrity Assay (DIA)
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`analysis performed on stool samples under different experimental conditions.
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`Figure 2 is a Table (Table 5) showing the results of DNA Integrity Assay (DIA)
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`analysis performed on stool samples under different experimental conditions.
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`Geneoscopy Exhibit 1008, Page 6
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`DETAILED DESCRIPTION OF THE INVENTION
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`It has been unexpectedly found that a DNA integrity signal can be stabilized and/or
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`enhanced by mixing, or incubating, a patient sample known to or suspect of containing DNA
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`indicative of a disease with a stabilization solution prior to performing the DNA integrity
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`5
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`assay. The stabilization solution typically includes one or more buffers, and/or chelating
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`agents, and/or salts. Aspects of the invention are particularly useful for nucleic acid integrity
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`assays. However, mutation detection (for example, in a multiple mutation assay) and
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`hypermethylation analysis also can benefit from aspects of the invention.
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`According to the invention, an important challenge for colon cancer detection from
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`stool is to preserve the integrity of human DNA in the hostile stool environment, in order to
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`recover, amplify, and interrogate the DNA for known cancer related abnormalities. Nucleases
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`that are active in stool have the potential to rapidly degrade DNA, including the minor human
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`DNA component, and measures may be taken to minimize their negative impact. Typically,
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`clinical samples are frozen as quickly as possible after collection. However, in order to use
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`fecal DNA tests in population screens, it should be expected that there will be some variability
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`in the time between sample collection and shipping to testing labs, and furthermore, some
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`variability in the temperature at which stool samples are transported. In order to eliminate any
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`variables in sample handling that might have an impact on assay performance we have run
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`controlled sample incubation experiments and looked at how different markers in a multi-
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`target assay are affected.
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`Markers may be chosen that yield an acceptable clinical sensitivity for the intended
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`application such as screening a population for indicia of a disease. In addition, for stool
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`sample analysis, mutation detection methods should offer sufficient analytical sensitivity since
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`the human DNA recovered from stool is highly heterogeneous. Normal cells are sloughed into
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`the colonic lumen along with the mutant cells. Therefore, in one embodiment, analytical
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`methods should detect as little as 1 % (or less) mutant DNA in the presence of excess wild-type
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`DNA. Also, certain sample preparation methodologies may be used for maximum recovery of
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`human DNA from samples. The vast majority of DNA recovered from stool often is bacterial
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`in origin, with the human DNA component representing only a small minority. Certain
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`purification methodologies can efficiently select for the rare human component, and since the
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`mutant copies (when they exist) represent only a small percentage of the total human DNA
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`from stool it may be important to maximize the recovery of human DNA in order to maximize
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`the probability of amplifying mutant copies in the PCR reactions. In one embodiment, gel
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`electrophoresis methods for capturing human DNA may be used. However, according to the
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`invention, it may be particularly important to preserve san1ple DNA for purification, especially
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`when looking for early indicia of diseases ( e.g., indicia of adenomas or early stage cancers that
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`may be present in less than about 1 %, or about 0.1 % or less of human genomes isolated from a
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`stool sample). A common method to insure that DNA remains stable is to freeze stool samples
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`as quickly as possible after collection, or to receive samples in centralized testing labs as
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`quickly as possible. However, in order to provide the option of decentralized sample analysis
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`and still retain maximum sample integrity, it is desirable to use a more robust and standardized
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`sample handling method.
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`10
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`In one aspect, the invention provides methods for stabilizing biological samples (e.g.,
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`stool samples) by adding a stabilization solution to a sample as soon as possible after the
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`sample is obtained. Methods of the invention do not require refrigeration or freezing. Aspects
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`of the invention are based, in part, on the surprising discovery that nucleic acids in certain
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`biological samples are stable at room temperature for hours, and even days ( e.g., 1 day, 2 days,
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`15
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`3 days, or longer). However, in certain embodiments, samples with stabilization solution may
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`be refrigerated or frozen. Aspects of the invention are particularly useful for preserving
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`samples for nucleic acid integrity analysis. However, methods of the invention may be used to
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`preserve samples for other assays including mutation detection and/or hypermethylation
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`assays. In certain embodiments, methods of the invention are used to preserve a sample for
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`20
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`analysis using a nucleic acid integrity assay along with a mutation detection assay ( e.g., a
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`multiple mutation panel assay), a hypermethylation assay, or both.
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`Nucleic acid integrity assays are known in the art and are described in, e.g., US Patent
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`Application No. 20040043467, US Patent Application No. 20040014104, US Patent No.
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`6,143,529, and Boynton et al., Clin. Chem. 49:1058-65, 2003. Nucleic acid integrity assays
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`25
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`are based on higher levels of intact nucleic acid that appear in debris from cells that lyse non(cid:173)
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`apoptoTically. Healthy patient generally produces cellular debris through normal apoptotic
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`degradation, resulting in relatively small fragments of cellular components in tissue and body
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`fluid samples, especially luminal samples. Patients having a disease generally produce cells
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`and cellular debris, a proportion of which has avoided normal cell cycle regulation, resulting in
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`30
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`relatively large cellular components. As a result, the disease status of a patient is determined by
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`analysis of patient cellular components produced in specimens obtained from the patient. The
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`presence of such fragments is a general diagnostic screen for disease.
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`Geneoscopy Exhibit 1008, Page 8
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`Nucleic acids in patient samples tend to degrade after they have been removed from the
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`patient. This degradation can diminish the effectiveness of a nucleic acid integrity assay that
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`scores a sample as diseased ( e.g., cancerous) based on the presence of intact nucleic acids; if
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`the sample is excessively degraded, a sample that is actually positive may appear to be
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`5
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`negative. While not wishing to be bound by theory, it is postulated that the stabilization buffer
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`of the invention inhibits the nucleases that degrade the nucleic acids present in the diseased
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`patient samples.
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`In some aspects of the invention, the addition of a stabilization solution to a biological
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`sample may be used to preserve nucleic acid molecules containing one or more mutations that
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`10 may be detected in a multiple mutation analysis ( e.g., an analysis that involves interrogating a
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`sample for the presence of a mutation at one or more loci, for example at about 2, about 3,
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`about 4, about 5, about 10, about 15, about 20, about 25, or more loci). In some embodiments,
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`the addition of a stabilization solution may be used to preserve nucleic acid for a methylation
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`specific analysis to detect the presence of hyper-methylated nucleic acid molecules at one or
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`15 more loci that may be indicative of cancer, adenoma, or other disease. In some embodiments,
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`the addition of a stabilization solution may be used to preserve nucleic acid for a combination
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`of a nucleic acid integrity assay and/or a multiple mutation analysis and/or a methylation
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`detection assay. Assays may be performed under conditions to detect a small amount of
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`mutant nucleic acid in a heterogeneous sample containing an excess of non-mutant nucleic acid
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`( e.g., where the mutant nucleic acid represents less than 10%, less than 5%, less than 1 %, or
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`about O .1 % or less of the nucleic acid at a particular locus). In some embodiments, a digital
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`assay may be performed on the preserved nucleic acid in order to detect rare genetic events. In
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`some aspects, stabilization methods of the invention may preserve more than 80%, 85%, 90%,
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`91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the nucleic acids indicative of a
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`disease ( e.g., long nucleic acid :fragments, nucleic acid molecules containing one or more
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`specific mutations, and/or hyper-methylated nucleic acid molecules).
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`In general any body organ, tissue, or fluid known to or suspected of containing nucleic
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`acid that can be characterized in a nucleic acid integrity analysis, multiple mutation assay, or
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`methylation study may be used. Suitable patient samples include those likely to contain
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`sloughed cellular debris. Such specimens include, but are not limited to, stool, blood serum or
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`plasma, sputum, pus, colostrum, and others. In diseases, such as cancer, in which genomic
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`instabilities or abnormalities have interfered with normal cell cycle regulation, specimens such
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`as those identified above contain relatively intact fragments of cellular components.
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`Geneoscopy Exhibit 1008, Page 9
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`The stabilization solution can be applied to a biological sample that is isolated directly
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`from a patient, i.e., a freshly isolated biological sample. Alternatively, the method can be used
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`on a biological sample that has been frozen (e.g., at -20 °C or -80 °C).
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`In aspects of the invention, stabilization solution may be added to a biological sample
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`5
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`at any suitable ratio of sample to buffer. Ratios may be determined as a weight to volume
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`(w/v) ratio. In some embodiments, the ratio may be about 1:1, about 1:2, about 1:3, about 1:4,
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`about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9 or about 1: 10 (w/v) sample to stabilization
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`solution. However, higher or lower ratios may be used.
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`In aspects of the invention, a biological sample may be weighed before a buffer is
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`10
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`added. In some embodiments, a sample (e.g., a stool sample) may weigh about 5 g, about 10 g,
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`about 15 g, about 20 g, about 25 g, about 30 g, about 35 g, about 40 g, about 45 g, about 50 g,
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`about 55 g, about 60 g, about 65 g, about 70 g, about 75 g, about 80 g, about 85 g, about 90 g,
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`about 95 g, about 100 g, or more. It should be appreciated that a large amount of sample ( e.g.,
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`over 25 g, 30 g, 50 g, 100 g, or more of stool) may be required in order to detect conditions
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`15
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`such as adenomas or early stage cancers where a very small amount of mutant or abnormal
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`nucleic acid may be present in a sample. According to the invention, it may be particularly
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`important to immediately stabilize biological samples with a stabilization solution when the
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`samples are to be interrogated for indicia of adenoma or early stage cancer. However, it also
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`may be useful to stabilize biological samples for detecting indicia of later stage diseases.
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`20
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`In general, a stabilization solution may include one or more buffers and/or one or more
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`chelating agents and/or one or more salts, or any combination of two or more thereof. The
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`choices of buffer, chelating agent, and salt can be determined by the artisan. The suitability of
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`a particular stabilization solution can be determined by comparing a nucleic acid integrity
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`assay on samples that have been incubated with the stabilization solution to a parallel
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`25
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`biological sample that has not been incubated with the stabilization solution. A suitable
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`stabilization solution is a solution that shows a significant average fold increase in genome
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`equivalents (GE) in a nucleic acid integrity assay compared to a GE determination made on
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`parallel samples that have not been treated with the stabilization solution. Methods of
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`calculating genome equivalents (GE) are known in the art (see, e.g., e.g., US Patent
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`30 Application No. 20040043467, US Patent Application No. 20040014104, US Patent No.
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`6,143,529, and Boynton et al., Clin. Chem. 49:1058-65, 2003) and are illustrated in the
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`Examples.
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`Geneoscopy Exhibit 1008, Page 10
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`The temperature and pH optimum can also be determined empirically and optimized
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`according to the combination of buffer, chelating agent and salt in the stabilization solution.
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`While room temperature has been found to be a suitable temperature for incubating the patient
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`sample and the stabilization solution, higher or lower temperatures ( e.g., 4 °C to 16 °C or 25
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`5
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`°C to about 37 °C) can also be used, provided they do not undermine the effectiveness of the
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`stabilization solution. The mixed patient sample and stabilization solution is preferably
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`subjected to a minimum of agitation. However, according to the invention, the addition of a
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`stabilization solution with little or no agitation is surprisingly effective at preserving nucleic
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`acids for subsequent analysis.
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`10
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`In one aspect of the invention, a stabilization solution may be particularly useful when
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`samples are not refrigerated or frozen or when there is a risk that a sample may not be
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`maintained at a sufficiently low temperature to preserve indicia of disease. For example, a
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`stabilization solution may be particularly useful if a sample is obtained at a remote location and
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`mailed or delivered to a testing center. However, stabilization solutions also may be useful to
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`15
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`preserve samples that are being processed on-site at a medical center.
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`Buffers
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`Suitable buffers include, e.g. tris(hydroxymethyl)aminomethane, sodium phosphate,
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`sodium acetate, MOPS, and other buffering agents as long as a buffer has the capacity to resist
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`a 0.1 to 1 molar tris(hydroxymethyl)aminomethane or 0.1 to 1 molar phosphate ion. A
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`combination of buffering agents can be used, so long as the solution has the required buffering
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`capacity. Methods for determining the buffering capacity of a solution are well known in the
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`art.
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`The comparison of buffering capacity is preferably carried out in the presence of the
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`salt and chelating agent to be used in the stabilization solution, at the salt concentration to be
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`25
`
`used, and with the solutions being compared at about the same temperature, preferably at a
`
`temperature within the range of about 15° C to about 25° C.
`
`Chelating Agents
`
`Table 1 provides a representative list of chelating agents that can be used in the
`
`stabilization solution. The list provided in Table 1 is not meant to be exhaustive. In some
`
`30
`
`embodiments, chelating agents are those which bind trace metal ions with a binding constant
`
`ranging from 101 to 10100; in some embodiments, chelating agents bind trace metal ions with
`
`Geneoscopy Exhibit 1008, Page 11
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`WO 2005/113769
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`PCT/US2005/017046
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`- 11 -
`
`a binding constant ranging from 1010 to 1080 ; in some embodiments, the chelators bind trace
`
`metal ions with a binding constant ranging from 1015 to 1060.
`
`Table 1. Examples of Chelating Agents
`
`ABB RE VIA TION
`
`EDT A free acid
`EDTA2Na
`EDTA 3Na
`
`CHELATORS
`
`FULL NAME
`
`Ethylenediamine-N ,N ,N' ,N' ,-tetraacetic acid
`Ethylenediamine-N,N,N',N',-tetraacetic acid, disodium salt, dihydrate
`
`Ethylenediamine-N,N,N',N',-tetraacetic acid, trisodium salt, trihydrate
`
`EDTA4Na
`
`IEthylenediamine-N,N,N',N'-tetraacetic acid, Tetrasodium salt, tetrahydrate
`
`IEDTA 2K
`
`IEthylenefisminr-N,N,N',N'-tetraacetic acid, dipotassium salt, dihydrate
`IEthylenefisminr-N,N,N',N'-tetraacetic acid, dipotassium salt, dihydrate
`
`IEDTA 2Li
`
`IEthylenediamine-N,N,N',N'-tetraacetic acid, dilithium salt, monhydrate
`
`IEDTA 2NH4
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, diammonium salt
`
`IEDTA 3K
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, Tripotassium salt, dihydrate
`
`Ba(ll)-EDTA
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, barium chelate
`
`Ca(II)-EDTA
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, calcium chelate
`
`Ce(III)-EDTA
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, cerium chelate
`
`Co(Il)-EDT A
`Cu( 11 )-EDT A
`
`Dy(Ill)-EDT A
`Eu(Ill)-EDTA
`Fe(III)-EDTA
`
`In(III)-EDTA
`La(lll)-EDTA
`
`Mg(II)-EDT A
`Mn( [1)-EDTA
`
`Ni(II)-EDTA
`Sm(IIl)-EDTA
`Sr(ll)-EDT A
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, cobalt chelate
`Ethylenediamine-N,N,N',N'-tetraacetic acid, copper chelate
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, dysprosium chelate
`Ethylenediamine-N,N,N',N'-tetraacetic acid, europium chelate
`Ethylenediamine-N,N,N',N'-tetraacetic acid, iron chelate
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, indium chelate
`Ethylenediamine-N,N,N',N'-tetraacetic acid, lanthanum chelate
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, magnesium chelate
`Ethylenediamine-N,N,N',N'-tetraacetic acid, manganese chelate
`Ethylenediamine-N,N,N ,N -tetraacetic acid, nickel chelate
`
`IEthylenediamine-N,N,N' ,N'-tetraacetic acid, samarium chelate
`IEthylenediamine-N,N,N',N'-tetraacetic acid, strontium chelate
`
`Z:n(Il)-EDTA
`CyDTA
`
`IEthylenediamine-N,N,N',N'-tetraacetic acid, zinc chelate
`~rans- I ,2-Diaminocyclohexane-N ,N ,N' ,N'-tetraaceticacid,monohydrate
`
`Geneoscopy Exhibit 1008, Page 12
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`WO 2005/113769
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`DREG
`
`DTPA-OH
`DTPA
`EDDA
`EDDP
`
`EDDPO
`
`EDTA-OH
`
`EDTPO
`
`IEGTA
`IHBED
`IHDTA
`HIDA
`
`IDA
`Methyl- EDTA
`
`NTA
`NTP
`NTPO
`0-Bistren
`
`TTHA
`
`- 12 -
`
`N,N-Bis(2-hydroxyethyl)glycine
`
`1,3-Diamino-2-hydroxypropane-N,N,N',N'-tetraacetic acid
`1,3-Di aminopropane-N,N,N',N'-tetraacetic acid
`
`Ethylenediamine-N,N'-diacetic acid
`Ethylenediamine-N,N'-dipropionic acid dihydrochioride
`
`Ethylenediamine-N,N'-bis(methylenephosphonic Acid), hemihydrate
`
`N-(2-Hydroxyethy l)ethy lenediamine-N ,N' ,N'-triacetic acid
`Ethylenediamine-N,N,N',N'-tetrakis(methylenephosponic acid)
`
`O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid
`IN,N-bis(2-hydroxybenzyl)ethylenediamine- N,N-diacetic acid
`1,6-H ex am ethyl enediamine-N,N,N',N'- tetraacetic acid
`~ -(2-Hydroxyethy l)irninodiacetic acid
`
`Jminodiacetic acid
`
`1,2-Diaminopropane-N, N ,N' ,N'-tetraacetic acid
`
`Nitrilotriacetic acid
`Nitrilotripropionic acid
`
`Nitrilotris(methylenephosphoric acid), trisodium salt
`7,19,30-Trioxa-1,4,10,13,16,22,27,33 -octaabicyclo [ 11, 11, 11] pentatriacontane,
`Triethylenetetramine-N,N,N',N",N"',N"'- hexaacetic acid
`
`Salts
`
`Candidate salts include, e.g, Nal, NaBr, NaCl, LiCl, KCl, KI, KBr, CsCl, GNHCl and
`
`GNSCN. In some embodiments, the salt is chaotropic and has an anion such as perchlorate,
`
`iodide, thiocyanate, acetate, trichloroacetate, hexafluorosilicate, tetrafluoroborate and the like.
`
`5 Cations for a chaotropic salt can include, e.g., the elements lithium, sodium, potassium,
`
`cesium, rubidium, guanidine and the like. More than one salt can be present in the buffered
`
`aqueous salt solution.
`
`Patient samples that have been treated with a stabilization solution can be subject to the
`
`nucleic acid integrity assay. For example the treated sample can be used in method that
`
`10
`
`includes determining an amount of nucleic acid greater than about 200 bp in length using an
`
`assay that detects wild-type or mutant nucleic acid, wherein said nucleic acid is present in a
`
`patient sample comprising shed cells or cellular debris; and identifying