United States Patent
`US 11,634,781 B2
`(10) Patent No.:
`(12)
`Louwagie
`(45) Date of Patent:
`*Apr. 25, 2023
`
`
`US011634781B2
`
`(54) FECAL SAMPLE PROCESSING AND
`ANALYSIS COMPRISING DETECTION OF
`BLOOD
`;
`;
`(71) Applicant: Exact Sciences Corporation, Madison,
`WI (US)
`
`(72)
`Inventor:
`(*) Notice:
`
`Joost Louwagie, Dornach (CH)
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`;
`;
`j
`}
`}
`This patent is subject to a terminal dis-
`claimer.
`
`.
`(21) Appl. No.: 17/936,335
`:
`Filed:
`
`Sep. 28, 2022
`
`(22)
`
`(65)
`
`Prior Publication Data
`US 2023/0048152 Al
`Feb. 16, 2023
`
`Related U.S. Application Data
`.
`.
`(63) 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 ofapplication 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.
`
`(51)
`
`(2018.01)
`(2006.01)
`
`Int. Cl.
`C12Q 1/6886
`GOIN 33/574
`(52) U.S. Cl
`.
`an
`CPC..... C12Q 1/6886 (2013.01), GOIN 33/57446
`(2013.01); C12Q 2600/106 (2013.01); C12Q
`2600/154 (2013.01); CI2Q 2600/158
`(2013.01); GOIN 2333/805 (2013.01); GOIN
`2800/50 (2013.01); GOIN 2800/52 (2013.01)
`Field of Classification
`S °
`h
`Classification
`Seare
`Field
`of
`CPC woeeeeeteee C12Q 1/68; C12Q 2600/154; C12Q
`2600/158; GOIN 33/57446; GOIN
`.
`. 2333/805
`See application file for complete search history.
`References Cited
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`FOREIGN PATENT DOCUMENTS
`0032782
`7/1981
`EP
`0308227
`3/1989
`EP
`0817968
`1/1998
`EP
`2919065
`1/2009
`FR
`WO 1990/06995
`6/1990
`WO
`WO 1997/46705
`12/1997
`WO
`WO soonocosae
`WO
`Sout
`we we sootlogeaoo
`5ood
`Wo WO 2004/0927209
`10/2004
`(Continued)
`
`OTHER PUBLICATIONS
`Ohlsson et al., British J. of Cancer 95 218-225 (Year: 2006).*
`Abbaszadegan et al., Stool-based DNAtesting, a new noninvasive
`method for colorectal cancer screening, the first report from Iran.
`World J Gastroenterol. Mar. 14, 2007;13(10):1528-33.
`Ahlquist et al., Cologuard Primed to Change Landscape of CRC
`Screenin, Mayo Clinic Clinical Updates, http://www.mayoclinic.
`org/medical-professionals/clinical-updates/digestivediseases/cologuard-
`primed-to-change-landscape-of-cre-screening, pp. 1-4, Dec. 3, 2014.
`Auerkari, Methylation of tumor suppressor genes p16(INK4a),
`p27(Kip1) and E-cadherin in carcinogenesis. Oral Oncol. Jan.
`2006;42(1):5-13.
`Chen et al., Detection in fecal DNA of colon cancer-specific
`methylation of the nonexpressed vimentin gene. J Natl CancerInst.
`Aug. 3, 2005:97(15):1124-32.
`(Continued)
`Primary Examiner — Ethan C Whisenant
`(74) Attorney, Agent, or Firm — Casimir Jones, $.C.;
`Mary
`Ann D. B
`ay
`row
`ABSTRACT
`(57)
`A methodof processing a fecal sample from a human subject
`comprising removing a portion of a collected fecal sample
`and adding the removedportion of the sample to a buffer that
`prevents denaturation or degradation of blood proteins found
`in the sample, and detecting the presence of human blood in
`the removedportion of the fecal sample. The method further
`comprises stabilizing the remaining portion of the fecal
`sample.
`
`20 Claims, No Drawings
`
`Specification includes a Sequence Listing.
`
`Geneoscopy Exhibit 1030, Page 1
`
`Geneoscopy Exhibit 1030, Page 1
`
`

`

`US 11,634,781 B2
`Page 2
`
`(56)
`
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`References Cited
`
`FOREIGN PATENT DOCUMENTS
`
`WO 2005/014154
`WO 2005/017207
`WO 2006/113671
`WO 2008/010975
`WO 2008/084219
`WO 2008/100913
`WO 2008/102002
`
`2/2005
`2/2005
`10/2006
`1/2008
`7/2008
`8/2008
`8/2008
`
`OTHER PUBLICATIONS
`
`Conroy et al., Exact Sciences Completes 40,000 Cologuard. Tests
`During First Quarter 2016, Exact Sciences Latest News, pp. 1-10,
`May 3, 2016.
`Exact Sciences, Cologuard(TM) sDNA-based Colorectal Cancer
`Screening Test—Instructions for Use, pp. 1-71, 2013.
`Hammer, Human hybirds. Sci Am. May 2013;308(5):66-71.
`Itzkowitz et al., A simplified, noninvasive stool DNA test for
`colorectal cancer detection. Am J Gastroenterol. Nov.
`2008; 103(11):2862-70.
`Karl et al., Improved diagnosis of colorectal cancer using a com-
`bination of fecal occult blood and novel fecal protein markers. Clin
`Gastroenterol Hepatol. Oct. 2008;6(10):1122-8.
`Kim et al., Noninvasive Molecular Biomarkers for the Detection of
`Colorectal Cancer. BMB Rep. Oct. 31, 2008;41(10):685-92.
`
`Kutzneret al., Non-invasive detection of colorectal tumours by the
`combined application of molecular diagnosis and the faecal occult
`blood test. Cancer Lett. Nov. 8, 2005;229(1):33-41.
`Laird, The power and the promise of DNA methylation markers. Nat
`Rev Cancer. Apr. 2003;3(4):253-66.
`Lenhard et al., Analysis of promoter methylation in stool: a novel
`method for the detection of colorectal cancer. Clin Gastroenterol
`Hepatol. Feb. 2005;3(2):142-9.
`Levi et al., A quantitative immunochemical faecal occult blood test
`is more efficient for detecting significant colorectal neoplasia than
`a sensitive guaiac test. Aliment Pharmacol Ther. May 1,
`2006;23(9): 1359-64.
`Mulder et al., Tumor pyruvate kinase isoenzyme type M2 and.
`immunochemical fecal occult blood test: performance in screening
`for colorectal cancer. Eur J Gastroenterol Hepatol. Oct. 2007; 19(10):878-
`82.
`Rein et al., Identifying 5-methylcytosine and related modifications
`in DNA genomes. Nucleic Acids Res. May 15, 1998;26(10):2255-
`64.
`Shastri et al., Comparison of an established simple office-based
`immunological FOBT with fecal tumor pyruvate kinase type M2
`(M2-PK) for colorectal cancer screening: prospective multicenter
`study. Am J Gastroenterol. Jun. 2008;103(6):1496-504.
`Sommeret al., Minimal homology requirements for PCR primers.
`Nucleic Acids Res. Aug. 25, 1989;17(16):6749.
`Turgeonet al., Fecal DNA-Based Detection of Colorectal Neoplasia.
`Curr Colorectal Cancer Rep. Oct. 2007;3(4): 171-177.
`Villar-Garea et al., DNA demethylating agents and chromatin-
`remodelling drugs: which, how and why? Curr Drug Metab. Feb.
`2003;4(1): 11-31.
`
`* cited by examiner
`
`Geneoscopy Exhibit 1030, Page 2
`
`Geneoscopy Exhibit 1030, Page 2
`
`

`

`This application is a continuation of U.S. patent applica-
`tion Ser. No. 15/634,607, filed Jun. 27, 2017, which is a
`continuation of U.S. application Ser. No. 15/010,436, filed
`Jan. 29, 2016, now abandoned, which is a continuation of
`USS. application Ser. No. 13/147,570, filed Mar. 12, 2012,
`now abandoned, which is the U.S. national stage of Inter-
`national Application PCT/GB2010/000180,
`filed Feb. 3,
`2010, which claimspriority 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 “35239-306_SEQUENCE_LISTING”,cre-
`ated Sep. 28, 2022, having a file size of 35,630 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%.
`Colorectal cancer (CRC) is a leading cause of cancer-
`Becauseof the intermittent nature of colorectal bleeding, the
`related deaths worldwide, and is the second leading cause of
`sensitivity of FOBT and FIT is directly proportional to the
`cancer-related deaths in the United States. A patient’s prog-
`number of samples taken and the frequency oftesting.
`nosis is good if the cancer is caught early, when the site of
`Recent developments in testing look specifically for muta-
`the canceris confinedto its site of origin. However, the cure
`tions in DNA characteristic of colorectal neoplasia that are
`rates fall once the cancer has spread. Most colon cancers
`detectable in exfoliated epithelial cells in the stool (Pignone,
`arise from conventional adenomatous polyps (conventional
`et al., 2002; Ahlquist, et al., 2002). While neoplastic bleed-
`adenoma-to-carcinoma sequence), while some colon can-
`ing is intermittent, epithelial shedding is continual, poten-
`cers appear to arise from the recently recognized serrated
`tially making stool-based DNAtesting (i.e., also known as
`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 MM).
`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, N.C.) 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 11,634,781 B2
`
`1
`FECAL SAMPLE PROCESSING AND
`ANALYSIS COMPRISING DETECTION OF
`BLOOD
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`20
`
`25
`
`30
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`35
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`40
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`45
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`50
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`55
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`65
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`Geneoscopy Exhibit 1030, Page 3
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`US 11,634,781 B2
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`3
`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 immunochemical
`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
`commontarget for methylation and epigenetic gene silenc-
`ing in colon neoplasia, and may function as a candidate
`tumor suppressor gene.
`WO 2008/084219 relates to detection of colorectal cancer
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`based upon determining methylation of a numberofdiffer-
`ent 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 predisposition 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 predispositionto, 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 demethylating
`agent and/or a DNA methyltransferase inhibitor and/or
`HDACinhibitor comprising detecting an epigenetic modi-
`fication 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 orin the
`at least one geneis indicative that the likelihood of success-
`ful 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 demethyiating
`agent and/or DNA methyltransferase inhibitor and/or HDAC
`inhibitor comprising detecting an epigenetic 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
`
`Geneoscopy Exhibit 1030, Page 4
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`US 11,634,781 B2
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`5
`(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 panelor in the
`at least one gene is indicative that the likelihood ofresis-
`tance to treatment is lower than if the epigenetic modifica-
`tion is not detected.
`
`a method of selecting a suitable treatment regimen for
`colorectal cancer comprising detecting an epigenetic modi-
`fication in
`
`(a) a panelofat least two genes selected from PHACTR3,
`NDRG4and FOXE1,
`(b) at least one gene selected from LAMA] 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 panelor in the
`at least one generesults in selection of a DNA demethylating
`agent and/or a DNA methyltransferase inhibitor and/or a
`HDACinhibitor for treatment and wherein if the epigenetic
`modification is not detected, a DNA demethylating agent
`and/or a DNA methyltransferase inhibitor and/or a HDAC
`inhibitor is not selected for treatment.
`a method for monitoring treatment of colorectal cancer
`with a DNA demethylating agent and/or a DNA methyl-
`transferase inhibitor and/or HDAC inhibitor comprising
`detecting an epigenetic modification in
`(a) a panelofat least two genes selected from PHACTR3,
`NDRG4and FOXE1,
`(b) at least one gene selected from LAMA] and CDO1; or
`(c) at least one gene selected from GPNMB and MMP2
`(in a faecal sample) wherein detection of a reduction in the
`epigenetic modification in at least one of the genes in the
`panel or in the at least one gene as treatment progresses is
`indicative of successful treatment.
`Thus, the epigenetic modification may be measuredat the
`start of the treatment and then once or more following
`treatment, or as treatment progresses, in order to determine
`if the treatment is achieving the desired effect. A return to
`lower levels of methylation of the genes is considered
`indicative of effective treatment.
`
`(c) predicting the likelihood of successful treatment of
`colorectal cancer with a DNA demethylating agent and/or a
`DNA methyltransferase inhibitor and/or HDAC inhibitor
`(d) predicting the likelihood of resistance to treatment of
`colorectal cancer with a DNA demethylating agent and/or
`DNAmethyltransferase inhibitor and/or HDAC inhibitor; or
`(e) selecting a suitable treatment regimen for colorectal
`cancer comprising means for detecting an epigenetic modi-
`fication in a panel of at
`least two genes selected from
`PHACTR3, NDRG4 and FOXE1.
`
`is based upon
`The invention, as set out in the claims,
`successful attempts to improve the detection of colorectal
`The invention also relates to a kit for detecting a predis-
`cancer. In particular,
`the invention aims to improve the
`position to, or the incidenceof, colorectal cancer in a faecal
`positive and negative predictive value andalso the sensitiv-
`sample comprising:
`ity and specificity of detection of colorectal cancer through
`(a) means for detecting an epigenetic modification in the
`non-invasive means. The methods of the invention may
`DNAcontained within the faecal sample, wherein detection
`permit effective detection of colorectal cancer without the
`of the epigenetic modification is indicative of a predisposi-
`tion to, or the incidence of, colorectal cancer, and
`requirement for relatively expensive, highly invasive and
`painful procedures such as digital rectal examination, colo-
`(b) meansfor detecting the presence of blood inthe faecal
`noscopy and sigmoidoscopyto be performed. The invention
`sample, wherein detection of the presence of blood is
`is based upon a combination oftests for detecting proteins
`indicative of a predisposition to, or the incidence of, col-
`orectal cancer.
`and epigenetic modification markers respectively in the
`same faecal sample, shown for the first time herein to
`Also provided is a kit for any of:
`provide a particularly useful overall test.
`(a) detecting a predisposition to, or the incidence of,
`
`colorectal cancer in a sample Thus, according toafirst aspect, the invention provides a
`55
`(b) monitoring treatmentof colorectal cancer with a DNA
`method of detecting a predisposition to, or the incidenceof,
`demethylating agent and/or a DNA methyltransferase inhibi-
`colorectal cancer in a faecal sample comprising, consisting
`tor and/or HDAC inhibitor
`essentially of or consisting of:
`(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 predisposition 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 predispositionto, or the
`incidence of, colorectal cancer.
`
`6
`Similarly, the invention also provides a kit for any of:
`(a) detecting a predisposition to, or the incidence of,
`colorectal cancer in a sample
`(b) predicting the likelihood of successful treatment of
`colorectal cancer with a DNA demethylating agent and/or a
`DNAmethyltransferase inhibitor and/or HDAC inhibitor
`(c) predicting the likelihood of resistance to treatment of
`colorectal cancer with a DNA demethylating agent and/or
`DNAmethyltransferase inhibitor and/or HDAC inhibitor; or
`(d) selecting a suitable treatment regimen for colorectal
`cancer comprising meansfor detecting an epigenetic modi-
`fication in at least one gene selected from LAM AJ and
`CDO1.
`The invention also provides a kit for any of:
`(a) detecting a predisposition to, or the incidence of,
`colorectal cancer in a sample
`(b) predicting the likelihood of successful treatment of
`colorectal cancer with a DNA demethylating agent and/or a
`DNAmethyltransferase inhibitor and/or HDAC inhibitor
`(c) predicting the likelihood of resistance to treatment of
`colorectal cancer with a DNA demethylating agent and/or
`DNAmethyltransferase inhibitor and/or HDAC inhibitor; or
`(d) selecting a suitable treatment regimen for colorectal
`cancer comprising meansfor detecting an epigenetic modi-
`fication in at least one gene selected from GPNMB and
`MMP2and means for processing a faecal sample.
`The invention also provides a method of detecting a
`predisposition to, or the incidence of, colorectal cancer in a
`faecal sample comprising detecting an epigenetic modifica-
`tion in the DNA contained within the faecal sample, wherein
`detection of the epigenetic modification is indicative of a
`predisposition to, or the incidence of, colorectal cancer,
`characterised in that the faecal sample has previously been
`stored for at least approximately 6 months, 1, 2, 3, 4, 5, 6 or
`more years and/or is less than approximately 4, 3, 2, or 1 g
`in weight.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
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`Geneoscopy Exhibit 1030, Page 5
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`Geneoscopy Exhibit 1030, Page 5
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`US 11,634,781 B2
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`7
`As shownherein, the combination of methylation marker
`assay and fecal occult blood test (FOBT) gives very specific
`and sensitive results.
`
`The combined methods of the invention improve the
`negative predictive value of the existing single tests. By
`improving sensitivity, the numberoffalse negative results is
`decreased and this improves negative predictive value.
`Step (a) of the methods involves detecting the presence of
`bloodin the faecal sample, wherein detection of the presence
`of bloodis indicative of a predisposition to, or the incidence
`of, colorectal cancer. Blood in the faeces is an indicator of
`intestinal bleeding, which is frequently associated with
`colorectal cancer. Thus, detection of blood in the faecal
`sample is considered a “positive” result in step (a). Any
`suitable method for detecting the presence of blood in the
`sample may be employed. Often, the methods of detecting
`blood will rely upon detecting a representative blood protein
`in the faecal sample. In certain embodiments, detecting the
`presence of blood in the faecal sample comprises, consists
`essentially of or consists of detection of haemoglobin in the
`faecal sample. Detection may be through any suitable
`means, andincludesall variants of fecal occult bloodtests.
`The test may be chromogenic or immunological in certain
`embodiments. The test may rely upon peroxidaseactivity of
`hemoglobin. Chromogenic tests are well known and com-
`mercially available and may rely upon chemical reagents
`such as gum guaiac. In specific embodiments, haemoglobin
`in the faecal sample is detected through immunochemical
`means. This may involve anti-hemoglobin antibodies in
`certain embodiments. The term “antibody” or “antibodies”
`herein refers to an antibody or antibodies, or a derivative
`thereof that retains specific binding activity. By specific
`binding activity is meant the ability to specifically bind to
`hemoglobin. Thus, such a reagent does not bind, or does not
`bind to a significant degree, to unrelated proteins found in
`the faecal sample. Any antibody or derivative may be
`employed. Thus,
`the antibody may be a monoclonal or
`polyclonal antibody. The derivative of the antibody that
`retains specific binding activity may comprise, consist
`essentially of or consist of a humanized version of a non-
`human antibody, a heavy chain antibody, a single domain
`antibody, a nanobody, a Fab fragmentor scFv etc. in certain
`embodiments. Numerous techniques are available for pro-
`ducing antibodies and their derivatized forms, as would be
`well known to one skilled in the art.
`
`the combination of techniques
`As mentioned above,
`maximises sensitivity of detection, without significantly
`compromising specificity. Thus, the threshold detection con-
`centrations for detection of blood/hemoglobin in step (a)
`maybe those typically employed in fecal occult bloodtests.
`Adding in the step (b) test improves overall sensitivity by
`picking up additional positive samples. For example,
`in
`some embodiments,
`the result
`in step (a)
`is considered
`positive if the concentration of hemoglobin detected is more
`than between (about) 50 to (about) 150 ng/ml. In more
`specific embodiments, the result in step (a) is considered
`positive if the concentration of hemoglobin detected is more
`than (about) 100 ng/ml.
`the methods of the
`However,
`in other embodiments,
`invention may be employed to improvethe sensitivity of the
`step (a) method, whilst preventing a resultant loss in speci-
`ficity. By lowering the threshold concentration of blood to be
`detected in the faecal sample to give a positive result in step
`(a), the sensitivity of the step (a) method is increased. In
`order to retain specificity, the step (b) method is employed
`on those samples in which low levels, that is to say lower
`than the typically used threshold, of blood were detected in
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`step (a). A positive result from the step (b) method is
`required to confirm the positive result in step (a) for the “low
`level” samples. For those samples having blood (especially
`hemoglobin) concentrations above the typically employed
`threshold in step (a),
`it is not necessary to perform the
`method of step (b), since for these samples the step (a)
`methodis sufficiently specific for this not to be necessary.
`This has the advantage that the step (b) test is not required
`for all samples, thus reducing costs and increasing through-
`put. Thus, in certain embodiments, the result in step (a) is
`considered positive if the concentration of hemoglobin
`detected is lower than is typically employedas the threshold
`concentration of hemoglobin in hemoglobin detectiontests,
`but for those samples in which a “lowerthan typical thresh-
`old” concentration of hemoglobin is detected, step (b) is
`performed on these samples. T