United States Patent [19J
`Lapidus et al.
`
`[54] METHODS FOR DISEASE DIAGNOSIS
`FROM STOOL SAMPLES
`
`[75]
`
`Inventors: Stanley N. Lapidus, Bedford, N.H.;
`Anthony P. Shuber, Milford, Mass.
`
`[73] Assignee: Exact Laboratories, Maynard, Mass.
`
`[ *] Notice:
`
`This patent is subject to a terminal dis(cid:173)
`claimer.
`
`[21] Appl. No.: 09/059,713
`
`[22] Filed:
`
`Apr. 13, 1998
`
`Related U.S. Application Data
`
`[63] Continuation-in-part of application No. 08/699,678, Aug.
`14, 1996, Pat. No. 5,741,650.
`Int. Cl.6
`....................................................... C12Q 1/68
`[51]
`[52] U.S. Cl. ................................. 435/6; 435/7.1; 435/7.2;
`435/91.1; 435/91.2
`[58] Field of Search .................................. 435/6, 7.1, 7.2,
`435/91.1, 91.3
`
`[56]
`
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`I IIIII IIIIIIII Ill lllll lllll lllll lllll lllll lllll lllll lllll 111111111111111111
`US005952178A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,952,178
`*Sep.14,1999
`
`WO 92/13103
`WO 93/20233
`WO 94/00603
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`
`Primary Examiner-Nancy Degen
`Assistant Examiner---1rem Yucel
`Attorney, Agent, or Firm-Testa, Hurwitz, & Thibeault
`
`[57]
`
`ABSTRACT
`
`The present invention provides methods for preparing a
`stool sample in order to screen for the presence of indicators
`of a disease, for example a subpopulation of cancerous or
`precancerous cells. The methods take advantage of the
`recognition that cellular debris from cancerous and precan(cid:173)
`cerous cells is deposited onto only a longitudinal stripe of
`stool as the stool is forming in the colon. Accordingly,
`methods of the invention comprise obtaining a representa(cid:173)
`tive sample, such as a circumferential or cross-sectional
`sample of stool in order to ensure that any disease indicator,
`such as cellular debris that is shed by colonic cells, is
`obtained in the sample.
`
`18 Claims, 4 Drawing Sheets
`
`Geneoscopy Exhibit 1069, Page 1
`
`

`

`5,952,178
`Page 2
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`
`Geneoscopy Exhibit 1069, Page 2
`
`

`

`

`

`

`

`

`

`U.S. Patent
`
`Sep.14,1999
`
`Sheet 4 of 4
`
`5,952,178
`
`M
`
`F
`
`1:
`2:
`
`3:
`4:
`
`1:
`2:
`
`3:
`4:
`
`3'ACGCTACGG5'
`5' .... ATCGGCTTACTGCGATGCC .... 3'
`
`3 ' .... TAGCCGAATGACGCTACGG .... 5'
`5'ATCGGCTTA3'
`
`3'ACGCTACGG5'
`5' .... ATCGGCTTATTGCGATGCC .... 3'
`
`3' .... TAGCCGAATAACGCTACGG .... 5'
`5'ATCGGCTTA3'
`
`FIG. 4
`
`Geneoscopy Exhibit 1069, Page 6
`
`

`

`5,952,178
`
`1
`METHODS FOR DISEASE DIAGNOSIS
`FROM STOOL SAMPLES
`
`This patent application is a continuation-in-part of U.S.
`Ser. No. 08/699,678, filed Aug. 14, 1996(now U.S. Pat. No. 5
`5,741,650) the disclosure of which is incorporated herein by
`reference.
`
`2
`lular debris from the patient, from microorganisms, and
`from food, resulting in a heterogeneous population of cells.
`This makes detection of a small, specific subpopulation
`impossible to detect reliably.
`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(cid:173)
`ducibly yield characteristics indicative of the presence of
`10 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(cid:173)
`ducibly detect characteristics indicative of the presence of
`15 cancerous or precancerous material in samples prepared
`from stool voided by a patient with colorectal cancer or
`precancer. Such methods are provided herein.
`
`FIELD OF THE INVENTION
`
`This invention relates to methods for disease diagnosis,
`including the early detection of colon cancer in patients, and
`more particularly to methods for preparing stool samples for
`disease diagnosis, including the detection of colon cancer, so
`as to assure or increase the likelihood that the sample will
`contain the diagnostically relevant information if the patient
`has a disease, for example a cancerous or precancerous
`lesion, and to methods for stool sample analysis.
`
`BACKGROUND OF THE INVENTION
`
`Stool samples frequently must be prepared for medical
`diagnostic analysis. Stool samples may be analyzed to help
`diagnose medical conditions ranging from parasitic, bacte(cid:173)
`rial or viral infections to inflammatory bowel disease and
`colorectal cancer.
`Colorectal cancer is a leading cause of death in Western
`society. However, if diagnosed early, it may be treated
`effectively by surgical removal of the cancerous tissue.
`Colorectal cancers originate in the colorectal epithelium and
`typically are not extensively vascularized ( and therefore not
`invasive) during the early stages of development. Colorectal
`cancer is thought to result from the clonal expansion of a
`single mutant cell in the epithelial lining of the colon or
`rectum. The transition to a highly vascularized, invasive and
`ultimately metastatic cancer which spreads throughout the
`body commonly takes ten years or longer. If the cancer is
`detected prior to invasion, surgical removal of the cancerous
`tissue is an effective cure. However, colorectal cancer is
`often detected only upon manifestation of clinical
`symptoms, such as pain and black tarry stool. Generally,
`such symptoms are present only when the disease is well
`established, often after metastasis has occurred, and the
`prognosis for the patient is poor, even after surgical resection
`of the cancerous tissue. Early detection of colorectal cancer
`therefore is important in that detection may significantly 45
`reduce its morbidity.
`Invasive diagnostic methods such as endoscopic exami(cid:173)
`nation allow for direct visual identification, removal, and
`biopsy of potentially cancerous growths such as polyps.
`Endoscopy is expensive, uncomfortable, inherently risky,
`and therefore not a practical tool for screening populations
`to identify those with colorectal cancer. Non-invasive analy(cid:173)
`sis of stool samples for characteristics indicative of the
`presence of colorectal cancer or precancer is a preferred
`alternative for early diagnosis, but no known diagnostic
`method is available which reliably achieves this goal.
`Current non-invasive diagnostic methods involve assay(cid:173)
`ing stool samples for the presence of fecal occult blood or
`for elevated levels of carcinoembryonic antigen, both of
`which are suggestive of the presence of colorectal cancer.
`Additionally, recent developments in molecular biology
`provide methods of great potential for detecting the presence
`of a range of DNA mutations or alterations associated with
`and indicative of the presence of colorectal cancer. The
`presence of such mutations theoretically can be detected in
`DNA found in stool samples during the early stages of
`colorectal cancer. However, stool comprises cells and eel-
`
`20
`
`SUMMARY OF THE INVENTION
`It has now been appreciated that cells and cellular debris
`are shed from colonic epithelial cells onto forming stool in
`a longitudinal "stripe" of material along the length of the
`stool. The shed material is confined to this longitudinal
`stripe, as shown in FIG. 1 (designated "C"). Based upon this
`25 recognition, Applicants teach that stool sample preparation
`for diagnostic testing must include taking a representative
`sample in order to ensure that the sample will contain any
`cells or cellular debris that was shed into the stool as it
`passed through the colon. Accordingly, methods of the
`30 invention comprise obtaining at least a representative ( e.g. a
`cross-section or circumferential surface) portion of stool
`voided by a patient, and performing an assay to detect in the
`sample the presence of cells or cellular debris shed from
`epithelial cells lining the colon that may be indicative of
`35 cancer or precancer. Most often, such cells will be derived
`from a polyp or a cancerous or precancerous lesion at a
`discrete location along the colon. For purposes of the present
`invention, a precancerous lesion comprises precancerous
`cells, and precancerous cells are cells that have a mutation
`40 that is associated with cancer and which renders such cells
`susceptible to becoming cancerous. As shown in FIG. 1, a
`cross-sectional sample is a sample that contains at least a
`circumferential surface of the stool ( or portion of a stool
`comprising an entire cross-sectional portion), as, for
`example, in a coronal section or a sagittal section. A sample
`comprising the surface layer of a stool ( or of a cross-section
`of a stool) also contains at least a circumferential surface of
`the stool. Both cross-sections and circumferential surfaces
`comprise longitudinal stripes of sloughed colonic
`50 epithelium, and are therefore representative samples.
`In a preferred embodiment, methods of the invention
`comprise the steps of obtaining at least a circumferential
`surface or cross-sectional portion of a stool voided by a
`patient, and performing an assay to detect debris indicative
`55 of disease. For example, such debris may comprise a clonal
`subpopulation of cells having one or more mutations (for
`purposes of the present application, a mutation is a deletion,
`substitution, addition, modification, intercalation or rear(cid:173)
`rangement of DNA). Preferred methods of the invention
`60 comprise detection of characteristics of such transformed
`cells, including, for example, mutations, proteins expressed
`uniquely or in altered amounts in transformed cells, and
`blood. Particularly preferred methods of the invention com(cid:173)
`prise obtaining at least a circumferential surface or cross-
`65 sectional portion of a stool sample, and performing an assay
`to detect DNA characteristics indicative of the presence of a
`clonal subpopulation of cells in the sample. The clonal
`
`Geneoscopy Exhibit 1069, Page 7
`
`

`

`5,952,178
`
`10
`
`3
`subpopulation may be, for example, a subpopulation of
`cancerous or precancerous cells, having a mutation in, for
`example, a p53 tumor suppressor gene. Clonal subpopula(cid:173)
`tions of cells detected by methods according to the invention
`are often characterized by a massive loss of DNA, resulting 5
`in a loss of heterozygosity that renders ineffective the gene
`or genes encompassed by the deletion. Alternative methods
`of the invention comprise performing an assay to detect the
`presence of an infectious disease, for example debris from a
`microorganism.
`Methods of the invention also comprise obtaining a
`representative (i.e., cross-sectional or circumferential)
`sample of stool and homogenizing the stool in a buffer, such
`as a buffer comprising a detergent and a proteinase and
`optionally a DNase inhibitor.
`In methods according to the invention, an assay per- 15
`formed on at least a circumferential surface or cross(cid:173)
`sectional portion of stool may be an assay to detect a disease
`when a diagnostic indicator of the disease is incorporated
`into stool. Methods of the invention are useful to detect
`cellular debris shed from the epithelial lining of the colon, 20
`and from other tissue sources that shed cellular material into
`the gastrointestinal tract and preferably into the colon. Shed
`material may be from colonic epithelial cells, pancreatic
`cells, or may be indicative of infection ( e.g. bacterial or viral
`nucleic acids or proteins). In methods according to the 25
`invention, an assay performed on at least a circumferential
`surface or cross-sectional portion of stool may be an assay
`to detect the presence of elevated levels of carcinoembry(cid:173)
`onic antigen shed from cells lining the colon. Such an assay
`may also comprise detecting the presence of occult blood. 30
`However, methods of the invention preferably comprise an
`assay wherein the sample is 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. 35
`Methods of the invention are especially and most prefer(cid:173)
`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 40
`instability, and others. An assay for DNA characteristics in
`a method of the invention may comprise the step of deter(cid:173)
`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 45
`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 50
`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(cid:173)
`phic nucleotide, is determined. A statistically significant
`difference between a number of 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. Another
`useful assay is one to detect an infectious disease.
`Methods of the invention typically include, following
`sample preparation and an assay for characteristics of cells
`
`4
`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, methods of the invention provide means for
`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(cid:173)
`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 precancerous cells shed debris
`only onto or into part of the surface of the forming stool. The
`present methods reliably 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(cid:173)
`tion thereof.
`
`DESCRIPTION OF THE DRAWINGS
`FIG. 1 is a diagram of a cylinder which represents a
`formed stool and shows various cross-sections which will
`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 cancerous tissue
`which is deposited in a longitudinal stripe.
`FIG. 2 is a schematic diagram of a receptacle for con(cid:173)
`taining a stool sample.
`FIG. 3 is a schematic diagram of a multi-orifice imped(cid:173)
`ance counter; wherein reference numeral 1 indicates the
`direction of flow through the column; reference numeral 2
`indicates a plunger means for 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(cid:173)
`cates an array of orifices for measuring differential imped(cid:173)
`ance; and reference numeral 7 is a collection chamber.
`FIG. 4 is a diagram showing primers useful for the
`detection of single base polymorphisms SEQ. ID NOs. 1-8.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`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 precancerous cells, 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(cid:173)
`teristics indicative of cancer in a highly-reproducible and
`accurate way. In preferred methods of the invention, a
`55 sample representative of the surface of a stool is obtained.
`Such methods provide an improvement over the art inas(cid:173)
`much as they teach removing the portion of stool onto which
`the colonic epithelium sheds debris. Two preferred methods
`for obtaining such a portion are cross-sectioning and extract-
`60 ing the surface of the stool. In a more preferred method, the
`surface of a cross-section is obtained. Without the recogni(cid:173)
`tion that at least a cross-sectional sample must be obtained,
`there is no means for reproducibly obtaining a sample that
`will contain a cancerous or precancerous subpopulation of
`65 cells, if one exists.
`Methods described in the art do not recognize that, unlike
`infection by parasites, bacteria and viruses, characteristics
`
`Geneoscopy Exhibit 1069, Page 8
`
`

`

`5,952,178
`
`5
`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 not include
`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.
`Methods of the invention are also useful to detect the
`presence of bacteria, virus or parasites, as they will also be
`present on the surface of formed stool.
`Sloughed cells 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. 15
`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 happens not 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 20
`anatomy and physiology of the colon will aid in an under(cid:173)
`standing of this phenomenon.
`A typical adult colon is approximately six feet in length,
`with a diameter of about two to three inches. Numerous
`bends and folds are present throughout its length. The colon 25
`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
`lumen of the colon, and the lumenal surface is organized into
`microscopic crypts. Colorectal epithelial cells are replaced 30
`every four to five days. The epithelial cells divide rapidly at
`the base of the crypts and migrate to the apeces, where cells
`appear to undergo apoptosis (programmed cell death), and
`cellular debris is shed into the lumen. The lining of the
`colorectal lumen is elastic and the diameter of the lumen is 35
`determined by the volume of stool 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. Shed epithelial cells
`(which may or may not have undergone apoptosis) and 40
`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 45
`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 50
`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 throughout the stool. As stool
`passes over a cancerous or precancerous growth, material 55
`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 precancerous tissue compris(cid:173)
`ing the lesion. Stool voided by a patient with colorectal
`cancer or precancer is therefore characterized by a longitu- 60
`dinal "stripe" of diagnostically relevant material derived
`from the cancerous or precancerous tissue.
`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 65
`derived from the cancerous or precancerous tissue.
`Currently, random, non-cross-sectional samples ("smears")
`
`6
`of voided stool are analyzed in clinical settings. In these,
`sloughed cancerous or precancerous cells and cellular debris
`have no possibility of detection unless the sample happens
`by chance to contain the portion of stool which made contact
`5 with the region of the colon from which cells were sloughed.
`Furthermore, cancers typically develop by clonal expan(cid:173)
`sion of a single mutant cell, and in the early stages of the
`disease, i.e., when surgical removal is an effective cure, the
`cancerous lesion will be very small and may lie on a small
`10 arc of the circumference of the 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 by a patient with early stage
`colorectal cancer or precancer only by chance will contain
`material indicative of the presence of the early stage can(cid:173)
`cerous or precancerous condition. However, early detection
`of colorectal cancer is very important for e