US011970746B2
`
`a2) United States Patent
`US 11,970,746 B2
`(0) Patent No.:
`Louwagie
`*Apr. 30, 2024
`(45) Date of Patent:
`
`(54) FECAL SAMPLE PROCESSING AND
`ANALYSIS COMPRISING DETECTION OF
`BLOOD
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`(71) Applicant: Exact Sciences Corporation, Madison,
`WI (US)
`
`(72)
`
`Inventor:
`
`Joost Louwagie, Dornach (CH)
`
`(73) Assignee: Exact Sciences Corporation, Madison,
`WI (US)
`
`(*) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`This patent is subject to a terminal dis-
`claimer.
`
`(21) Appl. No.: 18/179,945
`
`(22)
`
`Filed:
`
`Mar. 7, 2023
`
`(65)
`
`Prior Publication Data
`
`US 2023/0313317 Al
`
`Oct. 5, 2023
`
`Related U.S. Application Data
`
`(63) Continuation of application No. 17/936,335, filed on
`Sep. 28, 2022, now Pat. No. 11,634,781, which is a
`continuation of application No. 15/634,607, filed on
`Jun. 27, 2017, which is a continuation of application
`No. 15/010,436,
`filed on Jan. 29, 2016, now
`abandoned, whichis a continuation of application No.
`13/147,570,
`filed
`as
`application
`No.
`PCT/GB2010/000180
`on Feb.
`3,
`2010,
`now
`abandoned.
`
`(60) Provisional application No. 61/149,581, filed on Feb.
`3, 2009.
`
`4,437,975 A
`4,445,235 A
`4,582,811 A
`4,683,197 A
`4,859,610 A
`4,889,818 A
`5,137,806 A
`5,196,167 A
`5,198,365 A
`5,527,676 A
`5,741,650 A
`5,952,178 A
`6,410,276 Bl
`6,761,702 B2
`7,195,878 B2
`7,288,413 B2
`7,371,527 Bl
`8,969,046 B2
`9,891,223 B2
`11,634,781 B2*
`
`3/1984 Gillespie et al.
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`4/2023 Louwagie........ GOIN 33/57446
`435/6.1
`11,845,991 B2* 12/2023 Louwagie............ C12Q 1/6886
`2002/0096469 Al
`7/2002 Faulkner
`2002/0187476 Al
`12/2002 Koroulis et al.
`2003/0086869 Al
`5/2003 Stallings
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`
`(Continued)
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`
`0032782
`0308227
`
`T/A981
`3/1989
`
`(Continued)
`
`OTHER PUBLICATIONS
`
`Nechvatal et al. Journal of Microbiological Methods 72: 124-132
`(Year: 2008).*
`
`(Continued)
`
`Primary Examiner — Ethan C Whisenant
`(74) Attorney, Agent, or Firm — Casimir Jones, S.C.;
`Mary Ann D. Brow
`Feb. 3, 2010=(WO) we. PCT/GB2010/000180
`
`Foreign Application Priority Data
`
`(30)
`
`(51)
`
`(2018.01)
`(2006.01)
`
`Int. Cl.
`C12Q 1/6886
`GOIN 33/574
`(52) U.S. Cl.
`CPC ..... C12Q 1/6886 (2013.01); GOIN 33/57446
`(2013.01); C12Q 2600/106 (2013.01); C12Q
`2600/154 (2013.01); C12Q 2600/158
`(2013.01); GOIN 2333/805 (2013.01); GOIN
`2800/50 (2013.01); GOIN 2800/52 (2013.01)
`(58) Field of Classification Search
`CPC .. C12Q 1/68; C12Q 1/6886; C12Q 2600/106;
`C12Q 2600/154; C12Q 2600/158; GOIN
`33/57446; GOIN 2333/805; GOIN
`2800/50
`
`(57)
`
`ABSTRACT
`
`A methodof processing a fecal sample from a human subject
`comprising combining a first portion of a collected fecal
`sample with a stabilizing buffer, combining a second portion
`of the sample with a solution that prevents denaturation or
`degradation of blood proteins found in a fecal sample.
`Embodiments comprise testing nucleic acid extracted from
`the first portion of the fecal sample for an amount of a human
`nucleic acid, and testing the second portion of the fecal
`sample for the presence of human blood.
`
`19 Claims, No Drawings
`
`See application file for complete search history.
`
`Specification includes a Sequence Listing.
`
`Geneoscopy Exhibit 1001, Page 1
`
`Geneoscopy Exhibit 1001, Page 1
`
`

`

`US 11,970,746 B2
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`Page 2
`
`(56)
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`Geneoscopy Exhibit 1001, Page 2
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`Geneoscopy Exhibit 1001, Page 2
`
`

`

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`Geneoscopy Exhibit 1001, Page 3
`
`Geneoscopy Exhibit 1001, Page 3
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`

`

`US 11,970,746 B2
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`Page 4
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`(56)
`
`References Cited
`OTHER PUBLICATIONS
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`
`* cited by examiner
`
`Geneoscopy Exhibit 1001, Page 4
`
`Geneoscopy Exhibit 1001, Page 4
`
`

`

`This application is a continuation of U.S. patent applica-
`tion Ser. No. 17/936,335, filed Sep. 28, 2022, now allowed,
`which is a continuation of U.S. patent application Ser. No.
`15/634,607, filed Jun. 27, 2017, which is a continuation of
`USS. application Ser. No. 15/010,436, filed Jan. 29, 2016,
`now abandoned, whichis a continuation of U.S. application
`Ser. No. 13/147,570, filed Mar. 12, 2012, now abandoned,
`whichis the U.S. national stage of International Application
`PCT/GB2010/000180,
`filed Feb. 3, 2010, which claims
`priority to U.S. Provisional Application No. 61/149,581,
`filed Feb. 3, 2009. The contents of these applications are
`incorporated herein by reference in their entireties.
`
`SEQUENCELISTING
`
`The text of the computer readable sequencelisting filed
`herewith, titled “35239-307_SEQUENCE_LISTING”, cre-
`ated Mar. 6, 2023, having a file size of 35,640 bytes,
`is
`hereby incorporated by reference in its entirety.
`
`FIELD OF THE INVENTION
`
`The present invention relates to methods and kits for
`analysis of fecal samples comprising removing a portion of
`a collected fecal sample and adding the removedportion of
`the fecal sample to a buffer that prevents denaturation or
`degradation of blood proteins found in the sample, and
`comprising detection of blood in the removedportion of the
`fecal sample, e.g., by immunoassay.
`
`BACKGROUND OF THE INVENTION
`
`2
`cific, the procedure is invasive, costly, has limited availabil-
`ity and includescertain risks such as induction of infection
`and perforation of the bowel.
`A commonly used and less expensive way of screening
`for CRC is a fecal occult blood test (FOBT), which tests for
`the presence of blood in faeces. The presence of haemoglo-
`bin as a representative blood protein in faeces is an indicator
`of intestinal bleeding, which is frequently associated with
`CRC. However, since occult in a fecal sample could be
`indicative of a variety of gastrointestinal disorders, further
`medical testing such as colonoscopy remains necessary to
`identify colorectal cancer.
`Fecal occult bloodtests fall primarily into two categories,
`tests based on the use of chromogenic chemical reagents
`such as gum guaiac and immunochemicaltests. The chemi-
`cally based guaiac methods determine the presence of occult
`blood by the detection of the perioxidase activity of the
`hemoglobin in the blood present in the faecal sample. They
`require catalysis of peroxide into oxygen and water, and the
`subsequent oxidation of a colorless dye (most often into a
`colored form). However, peroxidaseactivity is also found in
`meats and vegetables. In order to produce accurate results,
`these tests require restriction of the intake of certain foods,
`drugs, vitamins, and other substances prior to and during the
`sample collection period. The sensitivity of the most com-
`monly used guaiac FOBT (Hemoccult) is approximately
`50%. Despite a specificity of 98%, the positive predictive
`value for FOBT is low. Methods of detecting occult blood
`based on porphyrin (heme and protpoporphyrin IX) analysis
`or immunologic tests using anti-hemoglobin antibodies
`improve on these results. Immunochemical tests (FIT or
`iFOBT) that use anti-hemoglobin antibodies specific for
`human blood in extracts from stool do not require dietary
`restrictions; however, they are more complicated and more
`expensive than peroxidase-based tests. In addition, human
`hemoglobin in fecal samples degrades with time, resulting in
`a loss of antigenicity which can produce false negative
`results. Reported sensitivity of these immunologic tests
`varies widely but is typically 60-80% depending on the
`population tested. Specificity is estimated to be -98%.
`Becauseof the intermittent nature of colorectal bleeding, the
`Colorectal cancer (CRC) is a leading cause of cancer-
`sensitivity of FOBT and FIT is directly proportional to the
`related deaths worldwide, and is the second leading cause of
`number of samples taken and the frequency oftesting.
`cancer-related deaths in the United States. A patient’s prog-
`Recent developments in testing look specifically for muta-
`nosis is good if the cancer is caught early, when the site of
`tions in DNA characteristic of colorectal neoplasia that are
`the canceris confinedto its site of origin. However, the cure
`detectable in exfoliated epithelial cells in the stool (Pignone,
`rates fall once the cancer has spread. Most colon cancers
`et al., 2002; Ahlquist, et al., 2002). While neoplastic bleed-
`arise from conventional adenomatous polyps (conventional
`ing is intermittent, epithelial shedding is continual, poten-
`adenoma-to-carcinoma sequence), while some colon can-
`tially making stool-based DNAtesting (i.e., also known as
`cers appear to arise from the recently recognized serrated
`adenomatous
`polyp
`(serrated
`adenoma-to-carcinoma
`fecal DNA [f-DNA] and stool DNA [sDNA]) testing more
`
`theory). Because conventional adenomas and_serrated sensitive than other methods. Early studies of molecular
`adenomas are usually asymptomatic, mass screening of
`feacal screening primarily focused on single mutations.
`Gene mutations in P53, K-ras, and BAT 26, for instance,
`asymptomatic patients has become the cornerstone for
`have been linked to colorectal cancer and remain detectable
`detecting and eliminating these precursor lesions to reduce
`the risk of colon cancer.
`A numberof different screening methods for CRC are
`available. Procedures such as digital rectal examination
`(DRE); colonoscopy or sigmoidoscopyare highly invasive,
`painful and can cause a great deal of patient discomfort.
`Otherless invasive screening tests include fecal occult blood
`test (FOBT);
`fecal
`immunochemical
`test
`(FIT); barium
`enema with air contrast; virtual colonoscopy; biopsy (e.g.,
`CT guided needle biopsy); and imaging techniques (e.g.,
`ultrasound, CT scan, PET scan, and MRI).
`Colonoscopy has becomethe primary screening test for
`CRCbecause ofits high sensitivity and specificity, and the
`ability to perform polypectomy. While sensitive and spe-
`
`in feacal samples. Colorectal neoplasmsare varied in nature
`and no single mutation has been identified as being
`expressed universally. For this reason, multiple target assay
`panels (MTAP) are preferably used. PreGen-Plus™ (EX-
`ACTSciences Corporation, Maynard, MA; Laboratory Cor-
`poration of America, Burlington, NC) is a single test that
`identifies the presence of 23 different microsatellite (MSI)
`mutations known to be associated with CRC,
`including
`mutations in BAT-26. Additionally, 21 other point mutations
`in other genes associated with CRCare includedinthistest:
`APC,K-ras, and p53. This test is further designed to detect
`long DNA fragments, which have been specifically associ-
`ated with cells called non-apoptotic colonocytes, which are
`
`US 11,970,746 B2
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`FECAL SAMPLE PROCESSING AND
`ANALYSIS COMPRISING DETECTION OF
`BLOOD
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`CROSS-REFERENCE TO RELATED
`APPLICATIONS
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`common in CRC. While this test is more sensitive than fecal
`occult bloodtesting, it is not as sensitive as colonoscopy and
`will miss about half of cancers in an average risk group of
`people without symptoms.
`Increased DNA methylation is an epigenetic alteration
`that is common in human cancers and is often associated
`with transcriptional silencing. Aberrantly methylated DNA
`has also been proposedasa potential tumor marker for CRC
`detection. Genes such as vimentin, which are transcription-
`ally silent in normal epithelium, have been considered as
`targets for cancer-associated aberrant methylation and for
`use as cancer markers (JNCI Journal of the National Cancer
`Institute 2005 97(15):1124-1132). A combinedassay utiliz-
`ing hypermethylated vimentin gene (hV) and a twosite
`DNA integrity assay (DY), demonstrated a sensitivity of
`88% for CRC with a specificity of 82% (Am J Gastroenterol.
`2008 November; 103(11):2862-70). Further, ColoSure® is a
`single marker laboratory developed, stool based DNAtest.
`This method examines DNA in exfoliated colon cells for
`
`cancer-associated aberrant methylation of the vimentin gene
`and reaches a sensitivity range of 72-77% and a specificity
`range of 83-94% in average risk individuals.
`Protein tests provide an alternative method for detecting
`CRC. Tests assessing the presence of tumor-derived
`enzymes such as M2 pyruvate kinase (M2-PK), and/or
`proteins such as calprotectin, carcinoembryonic antigen
`(CEA), tissue inhibitor of metalloproteinase-1 (TIMP-1) and
`5100 calcium binding protein Al2 (S100A12) have been
`described. A diagnosis of colorectal cancer using a combi-
`nation of fecal occult blood and novel fecal protein markers
`S100A12 and TIMP-1 has been described in Clin Gastro-
`
`enterol Hepatol. 2008 October; 6(10):1122-8. Dimeric
`isoenzyme of pyruvate kinase, M2-PK, expressed by tumor
`cells, has as well been proposedas a screening tool for CRC.
`The performance of fecal M2-PK has been evaluated with
`IFOBTand colonoscopy in Am J Gastroenterol. 2008 June;
`103(6):1496-504. Compared to immunochemical FOBTs,
`TuM2-PK does not have supplemental value for screening
`for CRC because of a lower sensitivity and specificity (Eur
`J Gastroenterol Hepatol. 2007 October; 19(10):878-82)
`Although combined assays for detecting CRC have been
`described,
`their approach targets either multiple protein
`markers or either multiple DNAalterations. To date, immu-
`nochemical tests and DNA tests for CRC detection have
`been evaluated and compared on a separate basis only.
`EP0308227 describes a chemical fecal occult blood test
`employing a guaiac matrix.
`EP0032782 describes a method for the detection of hae-
`moglobin or decomposition products of haemoglobin
`in feces by means of an immunological reaction by
`using an antibody specific for human haemoglobin.
`USS. Pat. No. 7,288,413 describes methods that combine
`a chemical fecal occult blood test and an immuno-
`chemical fecal occult bloodtest.
`
`WO 04/092709 concerns a fecal blood test involving the
`dispersement of a dye in toilet water.
`EP0817968 describes several suitable stool collecting and
`testing methods and devices.
`WO 05/017207 discloses that the vimentin gene can be a
`common target for methylation and epigenetic gene
`silencing in colon neoplasia, and may function as a
`candidate tumor suppressor gene.
`WO 2008/084219 relates to detection of colorectal cancer
`
`based upon determining methylation of a number of
`different genes, including panels of genes.
`WO 2006/113671 and WO 2008/010975 describe meth-
`
`65
`
`ylation markers relevant to colorectal cancer.
`
`4
`SUMMARY OF THE INVENTION
`
`The invention provides a method of detecting a predis-
`position to, or the incidenceof, colorectal cancer in a faecal
`sample comprising:
`(a) detecting the presence of blood in the faecal sample,
`wherein detection of the presence of bloodis indicative
`of a predisposition to, or the incidence of, colorectal
`cancer,
`(b) detecting an epigenetic modification in the DNA
`contained within the faecal sample, wherein detection
`of the epigenetic modification is indicative of a predis-
`position to, or the incidence of, colorectal cancer
`and based upon a positive result obtained in either (a) or
`(b) or in both (a) and (b) detecting a predisposition to,
`or the incidence of, colorectal cancer.
`Also described herein is a method of sample processing,
`prior to carrying out a methodofthe invention, comprising
`removing a portion of a collected faecal sample and adding
`the removedportion of the sample to a buffer which prevents
`denaturation or degradation of blood proteins found in the
`sample.
`The invention also provides a method of detecting a
`predisposition to, or the incidence of, colorectal cancer in a
`sample comprising detecting an epigenetic modification in a
`panel of at
`least
`two genes selected from PHACTR3,
`NDRG4 and FOXE1, wherein detection of the epigenetic
`modification in at least one of the genes in the panel is
`indicative of a predisposition to, or the incidence of, col-
`orectal cancer.
`
`The invention also provides a method of detecting a
`predisposition to, or the incidence of, cancer (and in par-
`ticular colorectal cancer) in a sample comprising detecting
`an epigenetic modification in at least one gene selected from
`LAMAI and CDO1, wherein detection of the epigenetic
`modification in the at least one gene is indicative of a
`predisposition to, or the incidence of, cancer (and in par-
`ticular colorectal cancer).
`The invention also relates to a method of detecting a
`predisposition to, or the incidence of, colorectal cancer (in
`particular in a faecal sample) comprising detecting an epi-
`genetic modification in at
`least one gene selected from
`GPNMB and MMP2, wherein detection of the epigenetic
`modification in the at least one gene is indicative of a
`predisposition to, or the incidence of, colorectal cancer.
`In related aspects, the invention provides
`a method for predicting the likelihood of successful
`treatment of colorectal cancer with a DNA demethyl-
`ating agent and/or a DNA methyltransferase inhibitor
`and/or HDAC inhibitor comprising detecting an epi-
`genetic modification in:
`(a) a panel of at least two genes selected from PHACTR3,
`NDRG4and FOXE1,
`(b) at least one gene selected from LAMA1 and CDO1; or
`(c) at least one gene selected from GPNMB and MMP2
`(in a faecal sample) wherein detection of the epigenetic
`modification in at least one of the genes in the panel or
`in the at least one geneis indicative thatthe likelihood
`of successful treatment is higher than if the epigenetic
`modification is not detected.
`a method for predicting the likelihood of resistance to
`treatment of colorectal cancer with a DNA demethyi-
`ating agent and/or DNA methyltransfe