(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`20 August 2009 (20.08.2009) (10) International Publication Number
`
`(43) International Publication Date
`
`WO 2009/102788 A2
`
`
`(26)
`
`(30)
`
`(71)
`
`(71)
`(72)
`
`(51)
`
`International Patent Classification:
`
`GOIN 33/574 (2006.01)
`GOIN 33/68 (2006.01)
`
`C12@ 1/68 (2006.01)
`
`(21)
`
`International Application Number:
`PCT/US2009/033793
`
`(22)
`
`International Filing Date:
`
`(25)
`
`Filing Language:
`
`Publication Language:
`
`11 February 2009 (11.02.2009)
`
`English
`
`English
`
`Priority Data:
`61/029,221
`
`15 February 2008 (15.02.2008)
`
`US
`
`Applicant (for all designated States except US): MAYO
`FOUNDATION FOR MEDICAL EDUCATION AND
`
`RESEARCH[US/US]; 200 First Street S.W., Rochester,
`MN 55905 (US).
`
`Applicants and
`Inventors: TAYLOR,William R. [US/US]; 218 N. Gar-
`den Street, Lake City, MN 55041 (US). HARRINGTON,
`Jonathan J. [US/US]; 36705 County Road 72, Zumbro
`Falls, MN 55991 (US). QUINT,Patrick S. [US/US]; 805
`8th Street N.W., Kasson, MN 55944 (US). ZOU,
`Hongzhi
`[CN/US];
`2015 Woodcrest Lane
`S.W.,
`Rochester, MN 55902 (US). BERGENIII, Harold R.
`[US/US]; 18162 161st Avenue, Spring Valley, MN 55975
`(US). SMITH, David I. [US/US]; 1060 Foxcroft Circle
`S.W., Rochester, MN 55902 (US). AHLQUIST, David
`A. [US/US]; 6567 Buck Ridge Court N.E., Rochester,
`MN 55906 (US).
`
`(74)
`
`(81)
`
`Agents: ROBINSON,Lisbeth C. et al.; P.O. Box 1022,
`Minneapolis, MN 55440-1022 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ,
`EC, EE, EG, ES, FL GB, GD, GE, GH, GM, GT, HN,
`HR, HU,ID,IL, IN, IS, JP, KE, KG, KM, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO,
`NZ, OM, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG,
`SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, TZ, UA,
`UG,US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU,TJ,
`TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, FI, FR, GB, GR, HR, HU,IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, TR),
`OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML,
`MR, NE, SN, TD, TG).
`Published:
`
`without international search report and to be republished
`upon receipt of that report (Rule 48.2(g))
`
`with sequencelisting part ofdescription (Rule 5.2(a))
`
`(54) Title: DETECTING NEOPLASM
`
`(57) Abstract: This documentrelates to methods and materials for detecting premalignant and malignant neoplasms. For example,
`methods and materials for determining whetheror not a stool sample from a mammal contains nucleic acid markers or polypeptide
`markers of a neoplasm are provided.
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`2009/102788A2|IIITINNIIMIININNANIINTOINANATTAATAATM
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`DETECTING NEOPLASM
`
`BACKGROUND
`
`1. Technical Field
`
`This documentrelates to methods and materials involved in detecting premalignant
`
`and malignant neoplasms(e.g., colorectal and pancreatic cancer).
`
`2. Background Information
`
`About half of all cancer deaths in the United States result from aero-digestive cancer.
`
`For example, of the estimated annual cancer deaths, about 25 percent result from lung cancer;
`
`about 10 percent result from colorectal cancer; about 6 percent result from pancreas cancer;
`
`about 3 percent result from stomach cancer; and about 3 percent result from esophagus
`
`cancer. In addition, over 7 percent of the annual cancer deaths result from other aero-
`
`digestive cancers such as naso-oro-pharyngeal, bile duct, gall bladder, and small bowel
`
`cancers.
`
`SUMMARY
`
`This document relates to methods and materials for detecting premalignant and
`
`malignant neoplasms(e.g., colorectal and pancreatic cancer). For example, this document
`
`provides methods and materials that can be used to determine whether a sample(e.g., a stool
`
`sample) from a mammalcontains a marker for a premalignant and malignant neoplasm such
`
`as a marker from a colonic or supracolonic aero-digestive neoplasm located in the mammal.
`
`20
`The detection of such a marker in a sample from a mammalcan allowaclinician to diagnose
`
`cancerat an early stage. In addition, the analysis of a sample such as a stool sample can be
`
`muchless invasive than other types of diagnostic techniques such as endoscopy.
`
`This documentis based, in part, on the discovery of particular nucleic acid markers,
`
`polypeptide markers, and combinations of markers present in a biological sample(e.g., a
`
`25
`
`stool sample) that can be used to detect a neoplasm located, for example, in a mammal’s
`
`small intestine, gall bladder, bile duct, pancreas, liver, stomach, esophagus, lung, or naso-oro-
`
`pharyngeal airways. For example, as described herein, stool can be analyzedto identify
`
`mammals having cancer. Once a particular mammalis determined to have stool containing a
`
`neoplasm-specific markeror collection of markers, additional cancer screening techniques
`
`30
`
`can be usedto identify the location and nature of the neoplasm. For example, a stool sample
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`can be analyzed to determine that the patient has a neoplasm, while magnetic resonance
`
`imaging (MRI), endoscopic analysis, and tissue biopsy techniques can be usedto identify the
`
`location and nature of the neoplasm. In some cases, a combination of markers can be used to
`
`identify the location and nature of the neoplasm without additional cancer screening
`
`techniques such as MRI, endoscopic analysis, and tissue biopsy techniques.
`
`In general, one aspect of this documentfeatures a method of detecting pancreatic
`
`cancer ina mammal. The method comprises, or consists essentially of determining the ratio
`
`of an elastase 3A polypeptide to a pancreatic alpha-amylase polypeptide present within a
`
`stool sample. The presence ofa ratio greater than about 0.5 indicates that the mammal has
`
`pancreatic cancer. The presenceofa ratio less than about 0.5 indicates that the mammal does
`
`not have pancreatic cancer.
`
`In another aspect, this document features a method of detecting pancreatic cancer in a
`
`mammal. The method comprisesor consists essentially of determining the level of an
`
`elastase 3A polypeptide in a stool sample from the mammal. The presence of an increased
`
`level of an elastase 3A polypeptide, when compared to a normal control level, is indicative of
`
`pancreatic cancer in the mammal.
`
`In another aspect, this document features a method of detecting pancreatic cancer in a
`
`mammal. The method comprises, or consists essentially of, determining the level of a
`
`carboxypeptidase B polypeptide in a stool sample from the mammal. An increase in the level
`
`20
`
`of a carboxypeptidase B polypeptide, when compared to a normal controllevel, is indicative
`
`of pancreatic cancer in the mammal.
`
`In another aspect, this document features a method of detecting pancreatic cancer in a
`
`mammal. The method comprises, or consists essentially of, determining whether or not a
`
`stool sample from the mammal comprisesa ratio of a carboxypeptidase B polypeptide to a
`
`25
`
`carboxypeptidase A2 polypeptide that is greater than about 0.5. The presenceofthe ratio
`
`greater than about 0.5 indicates that the mammal has pancreatic cancer.
`
`In another aspect, this document features a method of detecting cancer or pre-cancer
`
`inamammal. The method comprises, or consists essentially of, determining whetheror not a
`
`stool sample from the mammalhasan increase in the number of DNA fragments less than
`
`30
`
`200 base pairs in length, as compared to a normal control. The presence ofthe increase in the
`
`number of DNA fragments less than 200 base pairs in length indicates that the mammal has
`
`cancer or pre-cancer. The DNA fragments can be less than 70 base pairs in length.
`
`In anotheraspect, this document features a method of detecting colorectal cancer or
`
`pre-cancer ina mammal. The method comprises, or consists essentially of, determining
`2
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`whetheror not a stool sample from the mammalhas an elevated K-ras (Kirsten rat sarcoma-2
`
`viral (v-Ki-ras2) oncogene homolog (GenBank accession no. NM_033360; gi|34485724]))
`
`mutation score, an elevated BMP3 (bone morphogenetic protein 3 (GenBank accession no.
`
`M22491; gi/179505)) methylation status, and an elevated level of human DNA as compared
`
`to anormal control. The presence of the elevated K-ras mutation score, elevated BMP3
`
`methylation status, and elevated level of human DNAlevelindicates that the mammal has
`
`colorectal cancer or pre-cancer. The K-ras mutation score can be measured bydigital melt
`
`curve analysis. The K-ras mutation score can be measured by quantitative allele specific
`
`PCR.
`
`In another aspect, this document features a method of detecting aero-digestive cancer
`
`or pre-cancer ina mammal. The method comprises, or consists essentially of, determining
`
`whetheror not a stool sample from the mammalhas an elevated K-ras mutation score, an
`
`elevated BMP3 methylation status, and an elevated level of human DNA as compared to a
`
`normal control. The presence of the elevated K-ras mutation score, elevated BMP3
`
`methylation status, and elevated level of human DNAlevelindicates that the mammal has
`
`aero-digestive cancer or pre-cancer. The K-ras mutation score can be measured bydigital
`
`melt curve analysis. The K-ras mutation score can be measured by quantitative allele-
`
`specific PCR. The method can further comprise determining whetheror not a stool sample
`
`from the mammalhas an elevated APC mutation score. The APC mutation score can be
`
`20
`
`measured by digital melt curve analysis.
`
`In another aspect, this document features a method of detecting aero-digestive cancer
`
`or pre-cancer ina mammal. The method comprises, or consists essentially of, determining
`
`whetheror not the mammalhasat least one mutation in six nucleic acids selected from the
`
`group consisting of p16, p53, k-ras, APC (adenomatosis polyposis coli tumor suppressor
`
`25
`
`(GenBank accession no. NM_000038; gi/189011564)), SMAD4 (SMAD family member 4
`
`(GenBank accession no. NM_005359; gi|195963400)), EGFR (epidermal growth factor
`
`receptor (GenBank accession no. NM_005228; gi/41327737|)), CTNNB1 (catenin (cadherin-
`
`associated protein), beta 1 (88kD) (GenBank accession no. X87838; gi|1154853])), and
`
`BRAF(B-Raf proto-oncogeneserine/threonine-protein kinase (p94) (GenBank accession no.
`
`30
`
`NM_004333; gij/187608632])) nucleic acids. The presenceofat least one mutation in each of
`
`the six nucleic acids indicates that the mammalhas aero-digestive cancer or pre-cancer. The
`
`method can further comprise determining whetheror not a stool sample from the mammal
`
`has an elevated level of a carboxypeptidase B polypeptide as compared to a normalcontrol.
`
`The presence of the elevated level of a caboxypeptidase B polypeptide indicates that the
`3
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`mammalhas aero-digestive cancer or pre-cancer in the mammal. The method can further
`
`comprise determining whether or not a stool sample from the mammalhas an elevated
`
`amount of DNA fragments less than 70 base pairs in length as compared to a normal control.
`
`The presence of the elevated amount of DNA fragments less than 70 base pairs in length
`
`indicates that the mammalhas aero-digestive cancer or pre-cancer. The method can further
`
`comprise determining whether or not a stool sample from the mammalhas an elevated
`
`amount of DNA fragments greater than 100 base pairs in length as compared to normal
`
`controls. The presence of the elevated amount of DNA fragments greater than 100 basepairs
`
`in length indicates that the mammal has aero-digestive cancer or pre-cancer. The method can
`
`further comprise determining whetheror not a stool sample from the mammal has an elevated
`
`BMP3 methylation status. The elevated BMP3 methylation status level indicates that the
`
`mammalhas aero-digestive cancer or pre-cancer. The determining step can comprise using
`
`digital melt curve analysis.
`
`In another aspect, this document features a method of detecting aero-digestive cancer
`
`or pre-cancer ina mammal. The method comprises, or consists essentially of, measuring
`
`mutations in a matrix marker panel in a stool sample. The marker panel can comprise
`
`measuring DNA mutations in p16, p53, k-ras, APC, SMAD4, EGFR, CTNNB1, and BRAF
`
`nucleic acids. The presence of a mutation in each ofthe nucleic acidsis indicative of the
`
`presence of aero-digestive cancer or pre-cancer in a mammal.
`
`20
`
`In another aspect, this document features a method of detecting aero-digestive cancer
`
`inamammal. The method comprises, or consists essentially of, determining whetheror not
`
`the methylation status of an ALX4(aristaless-like homeobox 4 (GenBank accession no.
`
`AF294629; gi/10863748])) nucleic acid in a stool sample from the mammalis elevated, as
`
`compared to a normal control. The presence of an elevated ALX4 methylation status
`
`25
`
`indicates the presence of aero-digestive cancer in the mammal.
`
`In another aspect, this document features a method of diagnosing pancreatic cancerin
`
`amammal. The method comprises, or consists essentially of, obtaining a stool sample from
`
`the mammal, determining the ratio of an elastase 3A polypeptide to a pancreatic alpha-
`
`amylase polypeptide present within a stool sample, and communicating a diagnosis of
`
`30
`
`pancreatic cancerif the ratio is greater than about 0.5, thereby diagnosing the mammalwith
`
`pancreatic cancer.
`
`In another aspect, this document features a method of diagnosing a mammal with
`
`pancreatic cancer. The method comprises, or consists essentially of, obtaining a stool sample
`
`from the mammal, measuring mutations in a matrix marker panel of nucleic acids present in
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`the sample, determining the ratio of a carboxypeptidase B polypeptide to a carboxypeptidase
`
`A2 polypeptide present within the sample, and communicating a diagnosis of pancreatic
`
`canceror pre-cancerif a mutation is detected in each of the marker panel nucleic acids and
`
`the ratio is greater than 0.5, thereby diagnosing the mammal. The matrix marker panel
`
`comprises or consists essentially of p16, p53, k-ras, APC, SMAD4, EGFR, CTNNBI, and
`
`BRAFnucleic acids.
`
`In another aspect, this document features method of diagnosing a mammal with
`
`colorectal cancer. The method comprises, or consists essentially of, obtaining a stool sample
`
`from the mammal, detecting mutations in a matrix marker panel comprising of p16, p53, k-
`
`ras, APC, SMAD4, EGFR, CTNNBI, and BRAFnucleic acids in DNApresent within the
`
`sample, measuring the level of a serotransferrin polypeptide present within the sample, and
`
`communicating a diagnosis of colorectal cancer or pre-cancerif a mutation is detected in each
`
`of the nucleic acids and the level of a serotransferrin polypeptide is elevated as comparedto a
`
`reference level, thereby diagnosing the mammal.
`
`Unless otherwise defined, all technical and scientific terms used herein have the same
`
`meaning as commonly understood by oneofordinary skill in the art to which this invention
`
`pertains. Although methods and materials similar or equivalent to those described herein can
`
`be used in the practice or testing of the present invention, suitable methods and materials are
`
`described below. All publications, patent applications, patents, and other references
`
`20
`
`mentioned herein are incorporated by reference in their entirety. In case of conflict, the
`
`present specification, including definitions, will control. In addition, the materials, methods,
`
`and examplesare illustrative only and not intendedto be limiting.
`
`The details of one or more embodiments of the invention are set forth in the accompa-
`
`nying drawings and the description below. Other features, objects, and advantages of the
`
`25
`
`invention will be apparent from the description and from the claims.
`
`DESCRIPTION OF THE DRAWINGS
`
`Figure 1: Adjusted Cut-off Levels with Quantitative Stool Markers to Achieve 95%
`
`Specificity across Age and Genderusing the Q-LEAD Model. Solid line for women,dotted
`
`30
`
`line for men.
`
`Figure 2: Sensitive and specific detection of pancreatic cancer by fecal ratio of
`
`carboxypeptidase B:carboxypeptidase A2. Note that ratio in stools from patients with
`
`colorectal cancer is no different from ratios with healthy controls.
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`Figure 3: Elastase levels quantified in stools from patients with pancreatic cancer,
`
`patients with colorectal cancer, and healthy controls.
`
`Figure 4: Ratio of elastase3A:pancreatic alpha amylase differentiates patients with
`
`pancreatic cancer from patients with colorectal cancer and from healthy controls.
`
`Figure 5: Digital Melt Curve to detect mutations by targeted gene scanning
`
`(temperature (x-axis) v. temperature-normalized fluorescence (y-axis)). Eight pairs of
`
`primers, which amplify 100-350 bp gene fragments, were used to scan K-ras and APC genes
`
`and detect mutations (substitution and deletion mutations, respectively) at 1% mutant/wild-
`
`typeratio.
`
`Figure 6: Quantitative detection of low abundance mutations by Digital Melt Curve
`
`Assay. Varying Mutant:Wild-type ratios of K-ras and APC gene mixtures were prepared and
`
`assayed blindly by Digital Melt Curve.
`
`Figure 7: High analytical sensitivity by Digital Melt Curve (temperature (x-axis) v.
`
`temperature-normalized fluorescence (y-axis)). To test the detection limit of digital melt
`
`curve (DMC)assay, mutant copies were spiked in wild-type copies at 0.1, 0.5, 1, 5, and 10%
`
`dilutions. DMC could detect up to 0.1% mutant/wild-type level when 1000 copies were
`
`dispersed to one 96-well plate. The numbersof positive wells increased proportionally when
`
`spiked mutant copies were increased. A pair of primers that amplify 248 bp K-ras gene
`
`fragment were used as an example here. Primers that amplify 119 bp K-ras gene, 162 bp
`
`20
`
`APC gene, and 346 bp APC geneswerealso used to test the detection limit and quantitative
`
`property of DMC.
`
`Figure 8: Superior screen detection of colorectal precancerous polyps by Digital Melt
`
`Curve (DMC). Histogram comparessensitivity by DMC with that by commonfecal occult
`
`blood tests (Hemoccult and HemoccultSENSA)and by the commercial stool DNAtest
`
`25
`
`(PreGenPlus, Exact Sciences). Detection by DMC wassignificantly better than by any other
`
`test (p<0.05).
`
`Figure 9: Distributions of short fragment human DNA (short DNA) and long fragment
`
`human DNA (long DNA)in stools from patients with normal colonoscopy,large
`
`precancerous adenomas, and colorectal cancer. Human DNA quantified by an assay of Alu
`
`30
`
`repeats. Short DNArepresents 45 bp fragment amplification products, and long DNA
`
`represents 245 bp amplification products.
`
`Figure 10: Stool distributions of short and long DNA in patients with pancreatic
`
`cancerandin healthy controls. Short DNA represents 45 bp fragment amplification products,
`
`and long DNArepresents 245 bp amplification products.
`6
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`Figure 11: Methylated BMP3 genein stool for detection of colorectal neoplasia.
`
`Methylated BMP3 wasblindly quantified in stools from patients with colorectal cancers,
`
`precancerous adenomas, and normalindividuals with real-time methylation-specific PCR.
`
`Eachcircle represents a stool sample.
`
`Figure 12: Frequency of Specific Base Changes in Colorectal Tumors.
`
`DETAILED DESCRIPTION
`
`This document provides methods and materials related to detecting a neoplasm in a
`
`mammal(e.g., a human). For example, this document provides methods and materials for
`
`using nucleic acid markers, polypeptide markers, and combinations of markers present in a
`
`biological sample (e.g., a stool sample) to detect a neoplasm in a mammal. Such a neoplasm
`
`can be a canceror precancerin the head and neck, lungs and airways, esophagus, stomach,
`
`pancreas, bile ducts, small bowel, or colorectum. It will be appreciated that the methods and
`
`materials provided herein can be used to detect neoplasm markers in a mammal having a
`
`combination of different neoplasms. For example, the methods and materials provided herein
`
`can be used to detect nucleic acid and polypeptide markers in a human having lung and
`
`stomach neoplasms.
`
`In somecases, the methods and materials provided herein can be used to quantify
`
`multiple markers in biological samples(e.g., stool) to yield high sensitivity for detection of
`
`20
`
`lesions (e.g., neoplasms), while preserving high specificity. Such methods can include, for
`
`example, a logistic model that adjusts specificity cut-offs based on age, gender, or other
`
`variables in a target population to be tested or screened.
`
`In somecases, the methods and materials provided herein can be used to determine
`
`whether a mammal(e.g., a human) has colorectal cancer or pancreatic cancer. For example,
`
`25
`
`serotransferin, methylated BMP3, and mutant BRAF markersin stool can be usedto identify
`
`a mammalaslikely having colorectal cancer, while mutant p16, carboxypeptidase B/A, and
`
`elastase 2A markers can be used to identify a mammalas likely having pancreatic cancer.
`
`Anysuitable method can be used to detect a nucleic acid marker in a mammalian
`
`stool sample. For example, such methods can involve isolating DNA from a stool sample,
`
`30
`
`separating out one or more particular DNAs from the total DNA, subjecting the DNAsto
`
`bisulfite treatment, and determining whether the separated DNAsare abnormally methylated
`
`(e.g., hypermethylated or hypomethylated). In some cases, such methods can involve
`
`isolating DNA from a stool sample and determining the presence or absence of DNA having
`
`a particular size (e.g., short DNA). It is noted that a single stool sample can be analyzed for
`7
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`one nucleic acid marker or for multiple nucleic acid markers. For example, a stool sample
`
`can be analyzed using assays that detect a panel of different nucleic acid markers. In
`
`addition, multiple stool samples can be collected from a single mammal and analyzed as
`
`described herein.
`
`Nucleic acid can be isolated from a stool sample using, for example, a kit such as the
`
`QIAamp DNAStool Mini Kit (Qiagen Inc., Valencia, CA). In addition, nucleic acid can be
`
`isolated from a stool sample using the following procedure: (1) homogenizing samples in an
`
`excess volume (>1:7 w:v) of a stool stability buffer (0.5M Tris pH 9.0, 150mM EDTA,
`
`10mM NaCl) by shaking or mechanical mixing; (2) centrifuging a 10 gram stool equivalent
`
`of each sample to removeall particulate matter; (3) adding 1 wL of 100 ug/L RNase A to the
`
`supernatant and incubating at 37°C for 1 hour; (4) precipitating total nucleic acid with 1/10
`
`volume 3M NaAcand an equal volume isopropanol; and (5) centrifuging and then
`
`resuspending the DNApellet in TE (0.01 M Tris pH 7.4, 0.001 M EDTA). U.S. Patents
`
`5,670,325; 5,741,650; 5,928,870; 5,952,178, and 6,020,137 also describe various methods
`
`that can be used to prepare and analyze stool samples.
`
`Oneor more specific nucleic acid fragments can be purified from a nucleic acid
`
`preparation using, for example, a modified sequence-specific hybrid capture technique (see,
`
`e.g., Ahlquist et al. (2000) Gastroenterology, 119:1219-1227). Such a protocol can involve:
`
`(1) adding 300 uL of sample preparation to an equal volume of a 6 M guanidine
`
`20
`
`isothiocyanate solution containing 20 pmolbiotinylated oligonucleotides (obtained from,for
`
`example, Midland Certified Reagent Co., Midland, TX) with sequences specific for the DNA
`
`fragments to be analyzed; (2) incubating for two hours at 25°C; (3) adding streptavidin coated
`
`magnetic beads to the solution and incubating for an additional hour at room temperature; (4)
`
`washing the bead/hybrid capture complexes four times with 1X B+W buffer (1 M NaCl, 0.01
`
`25
`
`M Tris-HCI pH 7.2, 0.001 M EDTA, 0.1% Tween 20); and (5) eluting the sequence specific
`
`captured DNAinto 35 uwL L-TE (1 mM Tris pH 7.4, 0.1 M EDTA) by heat denaturation of
`
`the bead/hybrid capture complexes. Any other suitable technique also can be usedto isolate
`
`specific nucleic acid fragments.
`
`Nucleic acid can be subjected to bisulfite treatment to convert unmethylated cytosine
`
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`residues to uracil residues, while leaving any 5-methylcytosine residues unchanged. A
`
`bisulfite reaction can be performed using, for example, standard techniques: (1) denaturing
`
`approximately 1 ug of genomic DNA (the amount of DNA can be less when using micro-
`
`dissected DNA specimens) for 15 minutes at 45°C with 2 N NaOH;(2) incubating with 0.1
`
`M hydroquinone and 3.6 M sodium bisulfite (pH 5.0) at 55°C for 4-12 hours; (3) purifying
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`the DNA from the reaction mixture using standard (e.g., commercially-available) DNA
`
`miniprep columnsorother standard techniques for DNA purification; (4) resuspending the
`
`purified DNA sample in 55 wL water and adding 5 wl 3 N NaOHfora desulfonation reaction
`
`that typically is performed at 40°C for 5-10 minutes; (5) precipitating the DNA sample with
`
`ethanol, washing the DNA,and resuspending the DNA in an appropriate volume of water.
`
`Bisulfite conversion of cytosine residues to uracil also can be achieved using other methods
`(e.g., the CpGenome'™™ DNA Modification Kit from Serologicals Corp., Norcross, GA).
`
`Any appropriate method can be used to determine whether a particular DNAis
`
`hypermethylated or hypomethylated. Standard PCR techniques, for example, can be used to
`
`determine whichresidues are methylated, since unmethylated cytosines converted to uracil
`
`are replaced by thymidine residues during PCR. PCRreactions can contain, for example, 10
`
`uL of captured DNAthateither has or has not been treated with sodium bisulfite, 1X PCR
`
`buffer, 0.2 mM dNTPs, 0.5 uM sequencespecific primers(e.g., primers flanking a CpG
`
`island within the captured DNA), and 5 units DNA polymerase (e.g., Amplitaq DNA
`
`polymerase from PE Applied Biosystems, Norwalk, CT) in a total volume of 50 ul. A typical
`
`PCRprotocol can include, for example, an initial denaturation step at 94°C for 5 min, 40
`
`amplification cycles consisting of 1 minute at 94°C, 1 minute at 60°C, and 1 minute at 72°C,
`
`and a final extension step at 72°C for 5 minutes.
`
`To analyze which residues within a captured DNA are methylated, the sequences of
`
`20
`
`PCRproducts corresponding to samples treated with and without sodium bisulfite can be
`
`compared. The sequence from the untreated DNA will reveal the positions ofall cytosine
`
`residues within the PCR product. Cytosines that were methylated will be converted to
`
`thymidine residues in the sequence ofthe bisulfite-treated DNA, while residues that were not
`
`methylated will be unaffected by bisulfite treatment.
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`
`Purified nucleic acid fragments from a stool sample or samples can be analyzed to
`
`determine the presence or absence of one or more somatic mutations. Mutations can be
`
`single base changes, short insertion/deletions, or combinations thereof. Methods of analysis
`
`can include conventional Sanger based sequencing, pyrosequencing, next generation
`
`sequencing, single molecule sequencing, and sequencing by synthesis. In somecases,
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`30
`
`mutational status can be determined by digital PCR followed by high resolution melting
`
`curve analysis. In other cases, allele specific primers or probes in conjunction with
`
`amplification methods can be used to detect specific mutations in stool DNA. The mutational
`
`signature can comprise not only the event of a base or sequence changein a specific gene, but
`
`also the location of the change within the gene, whether it is coding, non-coding,
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`synonymousor non-synonymous, a transversion or transition, and the dinucleotide sequence
`
`upstream and downstream from the alteration.
`
`In somecases, a sample can be assessed for the presence or absence of a polypeptide
`
`marker. For example, any appropriate method can be usedto assess a stool sample for a
`
`polypeptide marker indicative of a neoplasm. For example, a stool sample can be used in
`
`assays designed to detect one or more polypeptide markers. Appropriate methods such as
`
`those described elsewhere (Aebersold and Mann, Nature, 422:198-207 (2003) and McDonald
`
`and Yates, Dis. Markers, 18:99-105 (2002)) can be adapted or designed to detect
`
`polypeptides in a stool. For example, single-reaction monitoring using a TSQ mass
`
`spectrometer can specifically target polypeptides in a stool sample. High resolution
`
`instruments like the LTQ-FT or LTQ orbitrap can be used to detect polypeptides present in a
`
`stool sample.
`
`The term “increased level” as used herein with respect to the level of an elastase 3A
`
`polypeptide is any level that is above a median elastase 3A polypeptide level in a stool
`
`sample from a random population of mammals(e.g., a random population of 10, 20, 30, 40,
`
`50, 100, or 500 mammals) that do not have an aero-digestive cancer. Elevated polypeptide
`
`levels of an elastase 3A polypeptide can be any level provided that the level is greater than a
`
`corresponding reference level. For example, an elevated level of an elastase 3A polypeptide
`
`can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold greater than the reference level of elastase
`
`20
`
`3A polypeptide in a normal sample. It is noted that a reference level can be any amount. For
`
`example, a reference level for an elastase 3A polypeptide can be zero. In some cases, an
`
`increased level of an elastase 3A polypeptide can be any detectable level of an elastase 3A
`
`polypeptide in a stool sample.
`
`The term “increased level” as used herein with respect to the level of an
`
`25
`
`carboxypeptidase B polypeptide level is any level that is above a median carboxypeptidase B
`
`polypeptide level in a stool sample from a random population of mammals (e.g., a random
`
`population of 10, 20, 30, 40, 50, 100, or 500 mammals) that do not have an aero-digestive
`
`cancer. Elevated polypeptide levels of carboxypeptidase B polypeptide can be any level
`
`provided that the level is greater than a corresponding reference level. For example, an
`
`30
`
`elevated level of carboxypeptidase B polypeptide can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or
`
`more fold greater than the reference level carboxypeptidase B polypeptide observedin a
`
`normal stool sample. It is noted that a reference level can be any amount. For example, a
`
`reference level for a carboxypeptidase B polypeptide can be zero. In some cases, an
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`increased level of a carboxypeptidase B polypeptide can be any detectable level of a
`
`carboxypeptidase B polypeptide in a stool sample.
`
`The term “increased level” as used herein with respect to the level of DNA fragments
`
`less than about 200 or less than about 70 basepairs in length is any level that is above a
`
`median level of DNA fragments less than about 200 or less than about 70 base pairs in length
`
`in a stool sample from a random population of mammals(e.g., a random population of 10, 20,
`
`30, 40, 50, 100, or 500 mammals) that do not have an aero-digestive cancer. In some cases,
`
`an increased level of DNA fragments less than about 200 or less than about 70 base pairs in
`
`length can be any detectable level of DNA fragments less than about 200 or less than about
`
`70 base pairs in length in a stool sample.
`
`The term “elevated methylation” as used herein with respect to the methylation status
`
`of a BMP3 or ALX nucleic acid is any methylation level that is above a median methylation
`
`level in a stool sample from a random population of mammals(e.g., a random population of
`
`10, 20, 30, 40, 50, 100, or 500 mammals) that do not have an aero-digestive cancer. Elevated
`
`levels of BMP3 or ALX methylation can be any level provided that the level is greater than a
`
`corresponding reference level. For example, an elevated level of BMP3 or ALX methylation
`
`can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold greater than the reference