United States Patent 5
`Lapidusetal.
`
`00
`
`[11] Patent Number:
`[45] Date of Patent:
`
`5,741,650
`*Apr. 21, 1998
`
`[54]
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`[75]
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`[21]
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`[22]
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`[60]
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`[51]
`[52]
`[58]
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`[56]
`
`METHODS FOR DETECTING COLON
`CANCER FROM STOOL SAMPLES
`
`Inventors: Stanley N. Lapidus, Bedford, N.H.;
`Anthony P. Shuber, Milford; Kevin M.
`Ulmer, Cohasset, both of Mass.
`
`Appl. No.: 699,678
`
`Filed:
`
`Aug. 14, 1996
`
`Related U.S. Application Data
`
`Provisional application No. 60/010,856 Jan. 30, 1996,
`Frat, CUS oonccccecssssssscseccsececsesecececencseccanennens C120 68
`
`TUS. Ch.
`ceesessscsscscsssesssssssesscccsnseseess
`Field of Searle .....cccsscccsscccssssssccsscsssesssessees 435/6, 7.1
`
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`(List continued on next page.)
`
`Primary Examiner—Nancy Degen
`Assistant Examiner—Irem Yucel
`Attomey, Agent, or Firm—Testa, Hurwitz & Thibeault, LLP
`[37]
`ABSTRACT
`
`The present invention provides methods for screening for
`the presence of a subpopulation of cancerous or precancer-
`ous cells in a heterogencouscellular sample, such as a stool
`sample. The methods take advantage of the recognition that
`cellular debris from cancerous and precancerous cells is
`deposited onto only a longitudinal stripe of stool as the stool
`is forming in the colon. Accordingly, methods of the inven-
`tion comprise obtaining a representative sample, such as a
`cross-sectional sample of stool in order to ensure that any
`cellular debris that is shed by coloniccells is obtained in the
`sample.
`
`14 Claims, 4 Drawing Sheets
`
`Geneoscopy Exhibit 1068, Page 1
`
`Geneoscopy Exhibit 1068, Page 1
`
`

`

`5,741,650
`Page 2ee
`
`OTHER PUBLICATIONS
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`orectal Tumors” vol. 256 Science pp. 102-105.
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`of the National CancerInstitute pp. 1524-1529.
`Hasegawa, Y., et al., (1995) “Detection of K-ras mutations
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`10 Oncogene pp. 1441-1445.
`
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`
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`
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`ogy pp. 1346-1353.
`
`Nollau P., C. Moser, G. Weinland, and C. Wagener (1996)
`“Detection of K-ras Mutations in Stools of Patients with
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`Cancer pp. 332-336.
`
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`
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`of DNAfrom Stool and Bodily Fluids for PCR Amplication”
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`
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`
`Geneoscopy Exhibit 1068, Page 2
`
`Geneoscopy Exhibit 1068, Page 2
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`

`

`US. Patent
`
`Apr. 21, 1998
`
`Sheet 1 of 4
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`5,741,650
`
`
`
`Geneoscopy Exhibit 1068, Page 3
`
`Geneoscopy Exhibit 1068, Page 3
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`

`

`US. Patent
`
`Apr. 21, 1998
`
`Sheet 2 of 4
`
`5,741,650
`
`
`
`Geneoscopy Exhibit 1068, Page 4
`
`Geneoscopy Exhibit 1068, Page 4
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`

`

`U.S. Patent
`
`Apr. 21, 1998
`
`Sheet 3 of 4
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`5,741,650
`
`aq a
`nO Ie
`OOo
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`
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`Geneoscopy Exhibit 1068, Page 5
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`Geneoscopy Exhibit 1068, Page 5
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`

`

`U.S. Patent
`
`Apr. 21, 1998
`
`Sheet 4 of 4
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`5,741,650
`
`3’ACGCTACGGS’
`5’...ATCGGCTTACTGCGATGCC....3’
`
`3’...TAGCCGAATGACGCTACGG....5”
`5’ATCGGCTTA3’
`
`3’ACGCTACGGS’
`5’...ATCGGCTTATTGCGATGCCseed”
`
`Wo
`
`3°....TAGCCGAATAACGCTACGG....5’
`S’ATCGGCTTA3?’
`
`FIG. 4
`
`Geneoscopy Exhibit 1068, Page 6
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`Geneoscopy Exhibit 1068, Page 6
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`

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`5,741,650
`
`1
`METHODS FOR DETECTING COLON
`CANCER FROM STOOL SAMPLES
`
`This application claims priority under 35 U.S.C. $119)
`to provisional application Ser. No. 60/10,856,filed on Jan.
`30, 1996.
`
`FIELD OF THE INVENTION
`
`This invention relates to methods for the early detection
`of colon cancer in patients and moreparticularly to methods
`for preparing stool samplesfor the detection of colon cancer
`so as to assure or increase the likelihoodthat the sample will
`contain the diagnostically relevant informationif the patient
`has a cancerous or precancerouslesion, and to methods for
`stool sample analysis.
`
`2
`This makes detection of a small, specific subpopulation
`impossible to detectreliably.
`Stool diagnostic assays for colorectal cancer described in
`the art typically are performed on samples prepared from
`randomly sampled portions of voided stool. However,
`samples prepared according to such methods do not repro-
`ducibly yield characteristics indicative of the presence of
`colorectal cancer or precancer, even when prepared from
`stool voided by a patient with colorectal cancer or precancer.
`There is therefore a need in the art for methods for early
`diagnosis of colorectal cancer or precancer that will repro-
`ducibly detect characteristics indicative of the presence of
`cancerous or precancerous material in samples prepared
`from stool voided by a patient with colorectal cancer or
`precancer. Such methods are provided herein.
`SUMMARY OF THE INVENTION
`BACKGROUND OF THE INVENTION
`It has now been appreciated that cells and cellular debris
`are shed from colonic epithelial cells onto forming stool in
`Stool samples frequently must be prepared for medical
`a longitudinal “stripe” of material along the length of the
`diagnostic analysis. Stool samples may be analyzed to help
`stool. The shed material is confined to this longitudinal
`diagnose medical conditions ranging from parasitic, bacte-
`stripe, as shownin FIG.1 (designated “C”). Based upon this
`rial or viral infections to inflammatory bowel disease and
`recognition, Applicants teach that stool sample preparation
`colorectal cancer.
`for diagnostic testing must include taking a representative
`Colorectal cancer is a leading cause of death in Western
`sample in order to ensure that the sample will contain any
`society. However, if diagnosed early,
`it may be treated
`cells or cellular debris that was shed into the stool asit
`effectively by surgical removal of the cancerous tissue.
`passed through the colon. Accordingly, methods of the
`Colorectal cancers originate in the colorectal epithelium and
`invention comprise obtaining at least a cross-sectional por-
`typically are not extensively vascularized (and therefore not
`tion of stool voided bya patient, and performing an assay to
`invasive) during the early stages of development. Colorectal
`detect in the sample the presenceofcells or cellular debris
`cancer is thought to result from the clonal expansion of a
`shed from epithelial cells lining the colon that may be
`single mutant cell in the epithelial lining of the colon or
`indicative of cancer or precancer. Mostoften such cells will
`rectum. Thetransition to a highly vascularized, invasive and
`be derived from a polyp or a cancerous or precancerous
`ultimately metastatic cancer which spreads throughoutthe
`lesion at a discrete location along the colon. For purposes of
`body commonly takes ten years or longer. If the cancer is
`the present invention, a precancerous lesion comprises pre-
`detected prior to invasion, surgical removal of the cancerous
`cancerouscells, and precancerouscells are cells that have a
`tissue is an effective cure. However, colorectal cancer is
`mutation that is associated with cancer and which renders
`often detected only upon manifestation of clinical
`suchcells susceptible to becoming cancerous. As shownin
`symptoms, such as pain and black tarry stool. Generally,
`FIG. 1, a cross-sectional sample is a sample that containsat
`such symptoms are present only when the disease is well
`least an entire circumference of the stool (or portion of a
`established, often after metastasis has occurred, and the
`40
`stool comprising an entire cross-sectional portion), as, for
`prognosisfor the patient is poor, even after surgical resection
`example, in a coronal sectionorasagittal section.
`of the canceroustissue. Early detection of colorectal cancer
`In a preferred embodiment, methods of the invention
`therefore is important in that detection may significantly
`comprise the steps of obtaining at least a cross-sectional
`reduce its morbidity.
`portion of a stool voided by a patient, and performing an
`Invasive diagnostic methods such as endoscopic exami-
`assay to detect debris from a clonal population of trans-
`nation allow for direct visual identification, removal, and
`formedcells. The transformed cells comprise, for example,
`biopsy of potentially cancerous growths such as polyps.
`a clonal subpopulation of cells having one or more muta-
`Endoscopy is expensive, uncomfortable, inherently risky,
`tions (for purposes of the present application, a mutation is
`and therefore not a practical tool for screening populations
`a deletion, substitution, addition, modification, intercalation
`to identify those with colorectal cancer. Non-invasive analy-
`or rearrangement of DNA). Preferred methodsof the inven-
`sis of stool samples for characteristics indicative of the
`tion comprise detection of characteristics of such trans-
`presence of colorectal cancer or precancer is a preferred
`formed cells, including, for example, mutations, proteins
`alternative for early diagnosis, but no known diagnostic
`expressed uniquely or in altered amounts in transformed
`method is available which reliably achieves this goal.
`cells, and blood. Particularly preferred methods of the
`Current non-invasive diagnostic methods involve assay-
`invention comprise obtaining at least a cross-sectional por-
`ing stool samples for the presence of fecal occult blood or
`tion of a stool sample, and performing an assay to detect
`for elevated levels of carcinoembryonic antigen, both of
`DNAcharacteristics indicative of the presence of a clonal
`which are suggestive of the presence of colorectal cancer.
`subpopulation of cells in the sample. The clonal subpopu-
`Additionally, recent developments in molecular biology
`lation may be, for example, a subpopulation of cancerous or
`provide methodsof great potential for detecting the presence
`precancerouscells, having a mutation in, for example, a p53
`of a range of DNA mutations oralterations associated with
`tumor suppressor gene. Clonal subpopulations of cells
`and indicative of the presence of colorectal cancer. The
`detected by methods according to the invention are often
`presence of such mutations theoretically can be detected in
`characterized by a massive loss of DNA,resulting in a loss
`DNA found in stool samples during the early stages of
`of heterozygosity that renders ineffective the gene or genes
`colorectal cancer. However, stool comprises cells and cel-
`encompassed by the deletion.
`lular debris from the patient, from microorganisms, and
`Methods of the invention also comprise obtaining a
`from food,resulting in a heterogeneous populationof cells.
`representative (i.e., cross-sectional) sample of stool and
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55.
`
`60
`
`65
`
`Geneoscopy Exhibit 1068, Page 7
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`5,741,650
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`4
`contain material from the entire circumference of a stool.
`The Section labeled “A” is a typical coronal section and the
`section labeled “B” is a typical sagittal section. The strip
`labeled “C” represents material shed from canceroustissue
`which is deposited in a longitudinal stripe.
`FIG. 2 is a schematic diagram of a receptacle for con-
`taining a stool sample.
`FIG. 3 is a schematic diagram of a multi-orifice imped-
`ance counter; wherein reference numeral 1 indicates the
`direction of flow through the column; reference numeral 2
`indicates a plunger meansfor forcing material downward in
`the column; reference numerals 3 and 4 are different-sized
`hybridization beads; reference numeral 5 is an optional filter
`for extracting unwanted particles; reference numeral 6 indi-
`cates an array of orifices for measuring differential imped-
`ance; and reference numeral 7 is a collection chamber.
`FIG. 4 is a diagram showing primers useful for the
`detection of single base polymorphisms.
`‘DETAILED DESCRIPTION OF THE
`INVENTION
`
`10
`
`15
`
`20
`
`25
`
`3
`homogenizing the stool in a buffer, such as a buffer com-
`prising a detergent and a proteinase and optionally a DNase
`inhibitor.
`In methods according to the invention, an assay per-
`formedon at least a cross-sectional portion of stool may be
`an assay to detect the presence of elevated levels of carci-
`noembryonic antigen shed from cells lining the colon. Such
`an assay may also comprise detecting the presence of occult
`blood. However, methodsof the invention preferably com-
`prise an assay wherein the sampleis exposed to an antibody
`that specifically binds to a molecule characteristic of cellular
`debris shed from cells comprising a subpopulation of cells
`having a mutation that is potentially associated with cancer.
`Methods of the invention are especially and most prefer-
`ably useful for detecting DNA characteristics indicative of a
`subpopulation of transformed cells in a representative stool
`sample. The DNA characteristics may be, for example,
`mutations, including loss of heterozygosity, microsatellite
`instability, and others. An assay for DNA characteristics in
`a method of the invention may comprise the step of deter-
`mining whether a difference exists in a number X of a first
`allele known or suspected to be mutated in a subpopulation
`of cells in a representative stool sample, and a number Y of
`an allele known or suspected not to be mutated in the
`sample,a statistically-significant difference being indicative
`of a mutation and the possible presence of cancer in a
`subpopulation of cells in the sample. In an embodiment of
`the invention, the difference between a number of a tumor
`suppressor gene and a number of a non-cancer-associated
`gene are compared, a statistically-significant difference in
`the numbers being indicative of a mutation in the tumor
`suppressor gene.
`Assays useful in the practice of methods according to the
`invention also include an assay to detect the presence of a
`deletion or other mutation in a region encompassing a
`polymorphic nucleotide. In such an assay, a number of a
`polymorphic nucleotide present at maternal and paternal
`alleles, wherein the patient is heterozygous for the polymor-
`phic nucleotide,
`is determined. A statistically significant
`difference between a numberof a polymorphic nucleotide in
`a maternal allele and a paternal allele is indicative of the
`presence of a deletion in one of the two alleles.
`Methods of the invention typically include, following
`sample preparation and an assay for characteristics of cells
`or cellular debris, a visual examination of the colon in order
`to determine if a polyp or other lesion is, in fact, present.
`Finally, surgical resection of abnormal tissue may be done in
`order to prevent the spread of cancerous or precancerous
`tissue.
`Accordingly, methodsof the invention provide meansfor
`screening for the presence of a cancerous or precancerous
`subpopulation of cells in a heterogeneous sample, such as a
`stool sample. Methods of the invention reduce morbidity
`and mortality associated with lesions of the colonic epithe-
`lium. Moreover, methods of the invention comprise more
`accurate screening methods than are currently available in
`the art, because current methods take advantage of the
`observation that cancerous or precancerouscells shed debris
`only ontoor into part of the surface of the forming stool. The
`present methodsreliably assay over the entire circumference
`of the stool, thereby increasing the likelihood of detecting an
`abnormality if one exists. Further aspects and advantages of
`the invention are contained in the following detailed descrip-
`tion thereof.
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`Methods according to the present invention are useful for
`the preparation of stool samples that will reproducibly
`contain cells or cellular debris shed from a clonal population
`of cancerous or precancerouscells, if such a population is
`present at any site along the colon of a patient. These
`samples are then used to perform assays to detect charac-
`teristics indicative of cancer in a highly-reproducible and
`accurate way. Such methods provide an improvement over
`the art inasmuch as they teach removing at least a cross-
`sectional sample from a stool voided by a patient. Without
`the recognition thatat least a cross-sectional sample must be
`obtained, there is no means for reproducibly obtaining a
`sample that will contain a cancerous or precancerous sub-
`population ofcells, if one exists.
`Methods described in the art do not recognizethat, unlike
`infection by parasites, bacteria and viruses, characteristics
`indicative of the presence of colon cancer, especially early
`stage colon cancer, are found only in a specific portion of
`voided stool. If the sampled portion of stool does notinclude
`the portion that happens to contain cells and cellular debris
`shed from early-stage cancer tissue, the diagnostic assay
`necessarily will fail to detect the characteristics indicative of
`the presence of colorectal cancer in a reliable manner even
`if homogenized, i.e., will produce a false-negative result.
`Sloughedcells from, for example, a polyp forming in the
`epithelial lining of the colon, or on early stage cancerous
`lesions, are sloughed onto only the portion of the forming
`stool that comes into contact with the polyp or lesion.
`Accordingly, in early stage disease, only a small portion of
`the surface layer of the forming stool will contain sloughed
`cells, and if that portion happensnot to be taken as part of
`the sample, an assay for indicia of colon cancer necessarily
`will produce a false-negative result. A brief review of the
`anatomy and physiology of the colon will aid in an under-
`standing of this phenomenon.
`A typical adult colon is approximately six feet in length,
`with a diameter of about two to three inches. Numerous
`bendsandfolds are present throughoutits length. The colon
`removes water from liquid or semi-liquid waste material that
`enters the colon, and relatively solid stool begins to form in
`the proximal third of the colon. Epithelial cells line the
`lumenofthe colon,andthe lumenal surface is organized into
`microscopic crypts. Colorectal epithelial cells are replaced
`every four to five days. The epithelial cells divide rapidly at
`the base of the crypts and migrate to the apeces, wherecells
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`Geneoscopy Exhibit 1068, Page 8
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`DESCRIPTION OF THE DRAWINGS
`FIG. 1 is a diagram of a cylinder which represents a
`formed stool and shows various cross-sections which will
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`Geneoscopy Exhibit 1068, Page 8
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`5,741,650
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`appear to undergo apoptosis (programmedcell death), and
`cellular debris is shed into the lumen. The lining of the
`colorectal lumenis elastic and the diameter of the lumenis
`determined by the volumeofstool that is passing through the
`colon at any given time. As a result, the surface of the
`forming stool passing through the colon is in direct contact
`with the epithelial lining of the lumen. Shedepithelial cells
`(which may or may not have undergone apoptosis) and
`cellular debris therefore are incorporated onto the surface of
`stool as it passes through the colon.
`Cells and cellular debris from colorectal epithelial cancers
`therefore also are shed onto forming stool. Most colorectal
`cancers develop in regions of the colon where stool is
`relatively solid, indeed approximately one third of such
`cancers develop in the rectum. Markers indicative of the
`presence of cancer, including cells, cellular debris, DNA,
`blood, and carcinoembryonic antigen, are shed onto the
`portion of the forming stool that contacts the cancerous
`tissue as the stool passes through the colon. Since the stool
`is relatively solid, these markers will remain on or near the
`surface of the stool where they were deposited and will not
`be homogeneously dispersed throughoutthe stool. As stool
`passes over a cancerous or precancerous growth, material
`from the growth will be deposited along the stool, but only
`on the part of the stool circumference that comes into direct
`contact with the cancerous or precanceroustissue compris-
`ing the lesion. Stool voided by a patient with colorectal
`cancer or precancer is therefore characterized by a longitu-
`dinal “stripe” of diagnostically relevant material derived
`from the cancerous or precanceroustissue.
`A sample that does not include material from the entire
`circumference of a stool voided by a patient with colorectal
`cancer or precancer will not reproducibly contain material
`derived from the cancerous or precancerous tissue.
`Currently, random, non-cross-sectional samples (“smears”)
`of voided stool are analyzed in clinical settings. In these,
`sloughed cancerous or precancerouscells and cellular debris
`have no possibility of detection unless the sample happens
`by chance to contain the portion of stool which made contact
`with the regionof the colon from which cells were sloughed.
`Furthermore, cancers typically develop by clonal expan-
`sion of a single mutant cell, and in the early stages of the
`disease, i.e., when surgical removal is an effective cure, the
`cancerouslesion will be very small and maylie on a small
`arc of the circumference ofthe colon. Material derived from
`such an early stage cancer therefore will be shed onto or into
`stool
`in a very narrow stripe (labeled C in FIG. 1).
`Consequently, a sample that does not contain the entire
`circumference of a stool voided bya patient with early stage
`colorectal cancer or precancer only by chance will contain
`material indicative of the presence of the early stage can-
`cerous or precancerous condition. However, early detection
`of colorectal cancer is very important for effective surgical
`intervention. The present invention provides methods for
`reproducible early detection of characteristics indicative of
`the presence of cancer or precancer in a patient.
`Analysis of at least a cross-sectional sample of stool
`(including a whole stool), as shown in FIG. 1, ensures that
`at least a portion of cells and cellular debris shed from any
`existing cancerous or pre-cancerouscells (even if shed from
`small early stage cancerous or pre-canceroustissue, e.g..
`small polyps) will be present in the portion of the stool
`sample to be analyzed. Indeed takingat least a cross-section
`of the stool sample avoids the possibility of analyzing stool
`portions that will not contain sloughed cancerous or precan-
`cerous cells even when the patient has colorectal cancer or
`precancer.
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`Once a cross-sectional stool sample is obtained,it may be
`homogenized by known methods to distribute cells and
`cellular debris throughout the sample. An assay then is
`performed on the homogenate, or an extract of the
`homogenate, to detect the presence of cells and/or cellular
`debris in the sample. The assay may be any one or a
`combination of histological cellular assays, antibody based
`immunoassays (or other formats) designed to detect the
`presence of a molecule characteristic of transformation such
`as a protein, or DNA-basedassays for detecting mutations or
`genetic characteristics indicative of colorectal cancer.
`Knownassayprotocols, those disclosed herein or in copend-
`ing application serial number Co-owned U.S. patent
`application, Ser. No. 08/700,583 or assays hereafter devel-
`oped may be used in the practice of the invention. Non-
`limiting examples of useful known assay protocols include
`those disclosed in U.S. Pat. No. 5,137,806 (detection of
`sequencesin selected DNA molecules), U.S. Pat. No. 5,348,
`855 (assay for nucleic acid sequences), U.S. Pat. No. 5,512,
`441 (detection of mutant alleles), U.S. Pat. No. 5,272,057
`and U.S. Pat. No. 5.380,645 (RFLP analysis), U.S. Pat. No.
`5,527,676 (detection of p53 gene sequences), U.S. Pat. No.
`5,330,892 (detection of MCC gene sequences), U.S. Pat. No.
`5,352,775 (detection of APC gene sequences), U.S. Pat. No.
`5.532,108 (detection of DCC gene sequences), and in
`W096/08514 (monoclonal antibodies against human colon
`carcinoma-associated antigens), the disclosures of each of
`which are incorporated by reference herein. Alternatively, or
`additionally, an assay for fecal occult blood may be per-
`formed as reported in U.S. Pat. Nos. 4,333,734 and 5,196,
`167, incorporated by reference herein. Assays useful in the
`context of the present invention also include an assay for
`carcinoembryonic antigen as reported in U.S. Pat. No.
`5,380,647, incorporated by reference herein. Finally, the
`sample may be prepared, as reported in U.S. Pat. No.
`4,857,300, incorporated by reference herein,for histological
`examination to detect characteristics indicative of the pres-
`ence of cancerous or precancerouscells.
`The purpose of any assay protocol used in connection
`with obtaining at least a cross-sectional sampleis to identify
`candidates for subsequent invasive diagnostic procedure
`such as colonoscopy or sigmoidoscopy. The assay accord-
`ingly need notdefinitively detect the presence of a cancerous
`or precancerouslesion, although false negatives obviously
`are