`
`IMPROVED METHODS OF DETECTING COLORECTAL CANCER
`
`
`FIELD OF THE INVENTION
`The presentinvention is concerned with the diagnosis, staging and treatment of disease,
`in particular cancer and more specifically colorectal cancer. The invention relates to
`methods andkits for diagnosing colorectal cancer based upon detecting epigenetic
`modifications, typically in specific genes. The methods and kits may also permit the
`detection of blood in a fecal sample, with the combined tests proving particularly
`advantageous.
`
`
`BACKGROUNDOF THE INVENTION
`
`Colorectal cancer (CRC)is a leading cause of cancer-related deaths worldwide, andis
`the second leading cause of cancer-related deaths in the United States. A patient's
`prognosis is goodif the cancer is caught early, when the site of the canceris confined to
`its site of origin. However, the cure rates fall once the cancer has spread. Most colon
`cancersarise from conventional adenomatous polyps (conventional adenoma-to-
`carcinoma sequence), while some colon cancers appearto arise from the recently
`recognized serrated adenomatouspolyp (serrated adenoma-to-carcinoma theory).
`Because conventional adenomasand serrated adenomasare usually asymptomatic,
`massscreening of asymptomatic patients has becomethe cornerstone for detecting and
`eliminating these precursor lesions to reduce the risk of colon cancer.
`
`A numberofdifferent 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. Other less invasive screening
`tests include fecal occult blood test (FOBT); fecal immunochemicaltest (FIT); barium
`enemawith 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 CRC becauseofits high
`sensitivity and specificity, and the ability to perform polypectomy. While sensitive and
`specific, the procedureis invasive, costly, has limited availability and includes certain
`risks such as induction of infection and perforation of the bowel.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`1001082
`
`
`
`
`Geneoscopy Exhibit 1014, Page 1
` Copyprovided by USPTO from the IFW Image Database on 02/02/2010
`
`
`
`Geneoscopy Exhibit 1014, Page 1
`
`

`

`A commonly used and less expensive way of screening for CRCis a fecal occult blood
`test (FOBT), which tests for the presence of blood in faeces. The presence of
`haemoglobin as a representative blood protein in faecesis an indicator ofintestinal
`bleeding, which is frequently associated with CRC. However, since occult in a fecal
`sample could beindicative of a variety of gastrointestinal disorders, further medical
`testing such as colonoscopy remains necessary to identify colorectal cancer.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Fecal occult blood tests fall primarily into two categories, tests based on the use of
`chromogenic chemical reagents such as gum guaiac and immunochemicaltests. The
`chemically based guaiac methods determine the presence of occult blood by the
`detection of the perioxidase activity of the hemoglobin in the blood presentin the faecal
`sample. They require catalysis of peroxide into oxygen and water, and the subsequent
`oxidation of a colorless dye (mostoften into a colored form). However, peroxidase
`activity 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
`substancesprior to and during the sample collection period. The sensitivity of the most
`commonly 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. Immunochemicaltests (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-basedtests. In addition, human hemoglobin in fecal samples
`degrades with time, resulting in a loss of antigenicity which can producefalse negative
`results. Reported sensitivity of these immunologic tests varies widely butis typically 60-
`80% depending on the population tested. Specificity is estimated to be ~98%. Because
`of the intermittent nature of colorectal bleeding, the sensitivity of FOBT and FITis directly
`proportional to the number of samples taken and the frequency of testing.
`
`Recent developments in testing look specifically for mutations in DNA characteristic of
`colorectal neoplasia that are detectable in exfoliated epithelial cells in the stool (Pignone,
`et al., 2002; Ahiquist, et al., 2002). While neoplastic bleeding is intermittent, epithelial
`shedding is continual, potentially making stool-based DNA testing (i.e., also known as
`fecal DNA [f-DNA] and stool DNA [sDNA]) testing more sensitive than other methods.
`
`1001082
`
`
`
`Geneoscopy Exhibit 1014, Page 2
` Copy provided by USPTO from the IFW Image Database on 02/02/2010
`
`Geneoscopy Exhibit 1014, Page 2
`
`

`

`Early studies of molecular feacal screening primarily focused on single mutations. Gene
`mutations in P53, K-ras, and BAT 26,for instance, have beenlinked to colorectal cancer
`and remain detectable in feacal samples. Colorectal neoplasmsare varied in nature and
`no single mutation has beenidentified as being expressed universally. For this reason,
`multiple target assay panels (MTAP) are preferably used. PreGen-Plus™ (EXACT
`Sciences Corporation, Maynard, MA; Laboratory Corporation 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 CRC are includedinthistest: APC, K-ras,
`and p53. This test is further designed to detect long DNA fragments, which have been
`specifically associated with cells called non-apoptotic colonocytes, which are commonin
`. CRC. While this test is more sensitive than fecal occult blood testing, it is not as sensitive
`as colonoscopyand will miss about half of cancers in an averagerisk 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 proposed as a potential tumor marker for CRC detection. Genes such as
`vimentin, which are transcriptionally silent in normal epithelium, have been considered as
`targets for cancer-associated aberrant methylation and for use as cancer markers (JNCI
`Journalof the National CancerInstitute 2005 97(15):1124-1132). A combined assay
`utilizing hypermethylated vimentin gene (hV) and a two site DNAintegrity assay (DY),
`demonstrated a sensitivity of 88% for CRC with a specificity of 82% (Am J Gastroenterol.
`2008 Nov; 103(11):2862-70). Further, ColoSure® is a single marker laboratory
`developed, stool based DNAtest. This method examines DNAin 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 averagerisk 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 S100 calcium binding protein A12 (S100A12) have
`been described. A diagnosis of colorectal cancer using a combination of fecal occult
`blood and novel fecal protein markers S100A12 and TIMP-1 has been describedin Clin
`
`.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`1001082
`
`
`
`Geneoscopy Exhibit 1014, Page 3
` Copy provided by USPTOfrom the IFW Image Database on 02/02/2010
`
`Geneoscopy Exhibit 1014, Page 3
`
`

`

`Gastroenterol Hepatol. 2008 Oct;6(10):1122-8. Dimeric isoenzymeof pyruvate kinase,
`M2-PK, expressed by tumorcells, hasas well been proposedasa screening toolfor
`CRC. The performanceof fecal M2-PK has been evaluated with IFOBT and colonoscopy
`in Am J Gastroenterol. 2008 Jun;103(6):1496-504. Compared to immunochemical
`FOBTs, TuM2-PK does not have supplemental value for screening for CRC because of a
`lowersensitivity and specificity (Eur J Gastroenterol Hepatol. 2007 Oct; 19(10):878-82)
`
`10
`
`15
`
`Although combined assays for detecting CRC have been described, their approach
`targets either multiple protein markers or either multiple DNA alterations. To date,
`immunochemical tests and DNA tests for CRC detection-have been evaluated and
`compared on a separatebasis only.
`
`EP0308227 describes a chemical fecal occult blood test employing a guaiac matrix.
`
`EP0032782 describes a method for the detection of haemoglobin or decomposition
`products of haemoglobin in feces by meansof an immunological reaction by using an
`antibody specific for human haemoglobin.
`
`US7288413 describes methods that combine a chemical fecal occult blood test and an
`
`20
`
`immunochemical fecal occult blood test.
`
`WO 04/092709 concernsa fecal blood test involving the dispersementof a dye in toilet
`water.
`
`25
`
`EP0817968 describes several suitable stool collecting and testing methods and devices.
`
`WO05/017207 discloses that the vimentin gene can be a commontarget for methylation
`and epigenetic gene silencing in colon neoplasia, and may function as a candidate tumor
`
`suppressor gene.
`
`30
`
`WO2008/084219 relates to detection of colorectal cancer based upon determining
`methylation of a numberof different genes, including panels of genes.
`
`
`
`1001082
`
`
`
` Copy provided by USPTOfrom the IFW Image Database on 02/02/2010
`
`Geneoscopy Exhibit 1014, Page 4
`
`Geneoscopy Exhibit 1014, Page 4
`
`

`

`
`
`
`
`
`
`
`
`
`
`WO 2006/113671 and WO 2008/010975 describe methylation markers relevant to
`colorectal cancer.
`
`
`SUMMARYOF THE INVENTION
`The invention provides a method of detecting a predisposition to, or the incidence of,
`colorectal cancer in a faecal sample comprising:
`(a)
`detecting the presence of blood in the faecal sample, wherein detection of
`the presenceof 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 predisposition to, or the incidence of, colorectal cancer.
`
`Also described herein is a method of sample processing, prior to carrying out a method
`of the invention, comprising removing a portion of a collected faecal sample and adding
`the removed portion of the sample to a buffer which prevents denaturation or
`degradation of blood proteins found in the sample.
`
`10
`
`15
`
`20
`
`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
`panelof at least two genes selected from PHACTR3, NDRG4 and FOXE1, wherein
`
`detection of the epigenetic modification in at least one of the genesin the panelis
`
`25
`
`indicative of a predisposition to, or the incidence of, colorectal cancer.
`
`The invention also provides a method of detecting a predisposition to, or the incidence
`of, cancer (and in particular colorectal cancer) in a sample comprising detecting an
`epigenetic modification in at least one gene selected from LAMA1 and CDO1, wherein
`detection of the epigenetic modification in the at least one geneis indicative of a
`predisposition to, or the incidence of, cancer (and in particular colorectal cancer).
`
`30
`
`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 epigenetic
`
`
`
`
`. 1001082
`
` Copy provided by USPTO from the IFW Image Database on 02/02/2010
`
`Geneoscopy Exhibit 1014, Page 5
`
`Geneoscopy Exhibit 1014, Page 5
`
`

`

`modification in at least one gene selected from GPNMB and MMP2, wherein detection of
`the epigenetic modification in the at least one geneis indicative of a predisposition to, or
`the incidence of, colorectal cancer.
`
`In related aspects, the invention provides
`-
`a methodfor predicting the likelihood of successful treatment of colorectal cancer
`with a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or HDAC
`inhibitor comprising detecting an epigenetic modification in:
`(a) a panelof at least two genes selected from PHACTR3, NDRG4 and 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 genesin the panel
`or in the at least one geneis indicative that the likelihood of successful treatmentis
`higherthan if the epigenetic modification is not detected.
`
`a methodfor predicting the likelihood of resistance to treatment of colorectal
`-
`cancerwith a DNA demethylating agent and/or DNA methyltransferase inhibitor and/or
`HDACinhibitor comprising detecting an epigenetic modification in
`(a) a panelof at least two genes selected from PHACTR3, NDRG4 and 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 genesin the panel
`or in the at least one geneis indicative that the likelihood of resistance to treatmentis
`lowerthan if the epigenetic modification is not detected.
`
`a method of selecting a suitable treatment regimen for colorectal cancer
`-
`comprising detecting an epigenetic modification in
`(a) a panelof at least two genes selected from PHACTR3, NDRG4 and 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 genesin the panel
`or 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
`
`10
`
`15
`
`20
`
`25
`
`30
`
`1001082
`
`
`
`Geneoscopy Exhibit 1014, Page 6
` Copy provided by USPTO from the IFW Image Database on 02/02/2010
`
`Geneoscopy Exhibit 1014, Page 6
`
`

`

`epigenetic modification is not detected, a DNA demethylating agent and/or a DNA
`methyltransferase inhibitor and/or a HDACinhibitor is not selected for treatment.
`
`a method for monitoring treatment of colorectal cancer with a DNA demethylating
`-
`agent and/or a DNA methyltransferase inhibitor and/or HDAC inhibitor comprising
`detecting an epigenetic modification in
`(a) a panelof at least two genes selected from PHACTR3, NDRG4 and 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 a reduction in the epigenetic modification in at least one of the
`genesin the panelorin the at least one gene as treatment progressesis indicative of
`successful treatment. Thus, the epigenetic modification may be measuredatthestart of
`the treatment and then once or more following treatment, or as treatment progresses,in
`orderto determineif the treatment is achieving the desired effect. A return to lower
`levels of methylation of the genes is considered indicative of effective treatment.
`
`Theinvention also relates to a kit for detecting a predisposition to, or the incidence of,
`colorectal cancerin a faecal sample comprising:
`(a)
`meansfor detecting an epigenetic modification in the DNA contained
`within the faecal sample, wherein detection of the epigenetic modificationis indicative of
`a predisposition to, or the incidence of, colorectal cancer, and
`(b)
`meansfor detecting the presence of biood in the faecal sample, wherein
`detection of the presence of bloodis indicative of a predisposition to, or the incidence of,
`colorectal cancer.
`
`Also providedis a kit for anyof:
`(a)
`detecting a predisposition to, or the incidence of, colorectal cancerin a sample
`(b)
`monitoring treatment of colorectal cancer with a DNA demethylating agent and/or
`a DNA methyltransferase inhibitor and/or HDACinhibitor
`(c)
`predicting the likelihood of successful treatment of colorectal cancer with a DNA
`demethylating agent and/or a DNA methyltransferase inhibitor and/or HDACinhibitor
`(d)
`predicting the likelihood of resistance to treatment of colorectal cancer with a
`DNA demethylating agent and/or DNA methyltransferase inhibitor and/or HDAC inhibitor;
`or
`
`10
`
`15
`
`20
`
`25
`
`30
`
`1001082
`
`
`
` Copy provided by USPTO from the IFW Image Database on 02/02/2010
`
`Geneoscopy Exhibit 1014, Page 7
`
`Geneoscopy Exhibit 1014, Page 7
`
`

`

`selecting a suitable treatment regimen for colorectal cancer
`(e)
`comprising meansfor detecting an epigenetic modification in a panelof at least two
`genesselected from PHACTR3, NDRG4 and FOXE1.
`
`Similarly, the invention also provides a kit for anyof:
`(a)
`detecting a predisposition to, or the incidence of, colorectal cancerin a sample
`(b)
`predicting the likelihood of successful treatment of colorectal cancer with a DNA
`demethylating agent and/or a DNA methyltransferase inhibitor and/or HDACinhibitor
`(c)
`predicting the likelihood of resistance to treatment of colorectal cancer with a
`DNA demethylating agent and/or DNA methyltransferase inhibitor and/or HDAC inhibitor;
`or
`
`selecting a suitable treatment regimen for colorectal cancer
`(d)
`comprising means for detecting an epigenetic modification in at least one gene selected
`from LAMA1 and CDO1.
`
`The invention also provides a kit for any of:
`(a)
`detecting a predisposition to, or the incidence of, colorectal cancerin a sample
`(b)
`predicting the likelihood of successful treatment of colorectal cancer with a DNA
`demethylating agent and/or a DNA methyltransferase inhibitor and/or HDACinhibitor
`(c)
`predicting the likelihood of resistance to treatment of colorectal cancer with a
`DNA demethylating agent and/or DNA methyltransferase inhibitor and/or HDACinhibitor;
`or
`
`selecting a suitable treatment regimen for colorectal cancer
`(d)
`comprising means for detecting an epigenetic modification in at least one gene selected
`from GPNMB and MMP2 and meansfor processing a faecal sample.
`
`
`DETAILED DESCRIPTION OF THE INVENTION
`Theinvention, as set out in the claims, is based upon successful attempts to improve the
`dectection of colorectal cancer.
`In particular, the invention aims to improve the positive
`and negative predictive value and also the sensitivity and specificity of detection of
`colorectal cancer through non-invasive means. The methodsof the invention may permit
`effective detection of colorectal cancer without the requirementfor relatively expensive,
`highly invasive and painful procedures suchasdigital rectal examination, colonoscopy
`and sigmoidoscopyto be performed. The invention is based upon a combination of tests
`
`10
`
`15
`
`20
`
`25
`
`30
`
`1001082
`
`
`
` Copy provided by USPTO from the IFW Image Database on 02/02/2010
`
`Geneoscopy Exhibit 1014, Page 8
`
`Geneoscopy Exhibit 1014, Page 8
`
`

`

`for detecting proteins and epigenetic modification markers respectively in the same
`faecal sample, shownforthe first time herein to provide a particularly useful overall test.
`
`_Thus, accordingtoafirst aspect, the invention provides a method of detecting a
`Predisposition to, or the incidence of, colorectal cancerin a faecal sample comprising,
`consisting essentially of or consisting of:
`(a)
`detecting the presence of blood in the faecal sample, wherein detection of
`the presenceof bloodis indicative of a predisposition to, or the incidence of, colorectal
`cancer,
`
`
`
`detecting an epigenetic modification in the DNA contained within the
`(b)
`faecal sample, wherein detection of the epigenetic modification is indicative of a
`predisposition to, or the incidence of, colorectal cancer
`and based upona positive result obtainedin either (a) or (b) or in both (a) and (b)
`detecting a predisposition to, or the incidence of, colorectal cancer.
`
`10
`
`15
`
`As shownherein, the combination of methylation marker assay and fecal occult blood
`test (FOBT) gives very specific and sensitive results.
`
`- 20
`
`The combined methodsof the invention improve the negative predictive value of the
`existing single tests. By improving sensitivity, the numberoffalse negative results is
`decreased andthis improves negative predictive value.
`
`25
`
`30
`
`Step (a) of the methodsinvolves detecting the presence of blood in the faecal sample,
`wherein detection of the presence of blood is indicative of a predisposition to, or the
`incidence of, colorectal cancer. Blood in the faecesis an indicatorofintestinal 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 methodsof
`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,and includesall variants of fecal
`occult blood tests. The test may be chromogenic or immunologicalin certain
`embodiments. The test may rely upon peroxidase activity of hemoglobin. Chromogenic
`
`1001082
`
`Geneoscopy Exhibit 1014, Page 9
` Copy provided by USPTOfrom the IFW Image Database on 02/02/2010
`
`
`
`Geneoscopy Exhibit 1014, Page 9
`
`

`

`~ 10 -
`
`tests are well known and commercially 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 notbind, 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 monoclonalor 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 fragment or scFv etc. in certain embodiments. Numerous
`techniques are available for producing antibodies and their derivatized forms, as would
`be well knownto one skilled in the art.
`
`As mentioned above, the combination of techniques maximises sensitivity of detection,
`without significantly compromising specificity. Thus, the threshold detection
`concentrations for detection of blood/hemoglobin in step (a) may be thosetypically
`employedin fecal occult blood tests. 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.
`
`However, in other embodiments, the methods of the invention may be employed to
`improve the sensitivity of the step (a) method, whilst preventing a resultantlossin
`specificity. 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) methodis
`increased.
`In orderto retain specificity, the step (b) method is employed on those
`samplesin whichlow levels, that is to say lower than the typically used threshold, of
`blood were detected in step (a). A positive result from the step (b) methodis required to
`confirm the positive result in step (a) for the "low level” samples. For those samples
`having blood (especially hemaglobin) concentrations above the typically employed
`
`10
`
`15
`
`20
`
`25
`
`30
`
`1001082
`
`
`
`Geneoscopy Exhibit 1014, Page 10
` Copy provided by USPTO from the [FW Image Database on 02/02/2010
`
`Geneoscopy Exhibit 1014, Page 10
`
`

`

`- 11 -
`
`thresholdin step(a), it is not necessary to perform the methodof step (b), since for these
`samples the step (a) methodis sufficiently specific for this not to be necessary. This has
`the advantagethat the step (b) test is not requiredforall samples, thus reducing costs
`and increasing throughput. Thus,in certain embodiments, the result in step (a)is
`consideredpositive if the concentration of hemoglobin detected is lower than is typically
`employed as the threshold concentration of hemoglobinin hemoglobin detection tests,
`but for those samples in which a "lower than typical threshold" concentration of
`hemoglobin is detected, step (b) is performed on these samples. The detection of the
`epigenetic modification in step (b) is then used to confirm the positive result in step (a).
`Thestep (b) test is not employed for those samples in which the concentration of
`hemoglobin detected is higher than the threshold typically employed in hemoglobin
`detection tests.
`
`In specific embodiments, the result in step (a) is considered positive if the concentration
`of hemoglobin detected is more than orat least (about) 5 to (about) 50 ng/ml, more
`specifically more than orat least (about) 5 to (about) 20 ng/ml and moreparticularly
`more than or at least (about) 10 ng/ml. By lowering the threshold, the sensitivity of the
`test is increased.
`In such embodiments, step (b) is performed only in the eventthat the
`concentration of hemoglobin detected is between (about) 5 ng/ml and (about) 250 ng/ml,
`more specifically between (about) 10 ng/ml and (about) 200 ng/ml. The detection of the
`epigenetic modification in step (b) is then used to confirm the positive result in step(a).
`Thus, a positive result in step (b) confirms the result in step (a) as positive.
`If no
`epigenetic modification of the DNAis detected, the result of step (a) is considered
`negative. For samples in which the concentration of hemoglobin detected is more than
`or at least (about) 200 ng/mi(or (about) 250 ng/ml), it is not necessary to perform step
`(b), since the result in step (a) will be of high specificity (i.e. is unlikely to be a false
`positive).
`,
`
`Step (b) involves 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. Thus, detection of the
`epigenetic modification is considered a "positive" result in step (b).
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`1001082
`
`Copy provided by USPTOtrom the IFW Image Database on 02/02/2010
`
`Geneoscopy Exhibit 1014, Page 11
`
`
`
`
`Geneoscopy Exhibit 1014, Page 11
`
`

`

`- 12 -
`
`In some embodiments, the epigenetic modification is detected in at least one gene
`selected from PHACTR3, NDRG4, FOXE1, GATA4, GPNMB,TFPI2, SOX17, SYNE1,
`LAMA1, MMP2, OSMR, SFRP2 and CDO1, with detection of the epigenetic modification
`in at least one of the genes providing an indication of a predisposition to, or incidenceof,
`colorectal cancer.
`
`In certain embodiments, the epigenetic modification is detected in at least one gene
`selected from PHACTR3, NDRG4 and FOXE1, with detection of the epigenetic
`modification in at least one of the genes providing an indication of a predisposition to, or
`incidence of, colorectal cancer.
`
`PHACTR3is the gene symbol approved by the HUGO Gene Nomenclature Committee.
`The geneis located on chromosome20 (location 20q13.32-q13.33) and the gene
`sequenceislisted under the accession numbers AJ311122 and NM_080672. The gene
`encodes the phosphatase and actin regulator 3.
`
`NDRG4is the gene symbol approved by the HUGO Gene Nomenclature Committee.
`The geneis located on chromosome16 (location q21-q22.3) and the gene sequenceis
`listed under the accession number AB044947. The gene encodes NDRG family member
`4.
`
`FOXE‘1is the gene symbol approved by the HUGO Gene Nomenclature Committee. The
`geneis located on chromosome9 (location 9q22) and the gene sequence is listed under
`the accession number U89995. The gene encodesthe forkhead box E1 (thyroid
`transcription factor 2).
`
`GATA4is the gene symbol approved by the HUGO Gene Nomenclature Committee. The
`geneis located on chromosome8 (location 8p23.1-p22) and the gene sequenceis listed
`under the accession numbers AK097060 and NM_002052. The gene encodes the GATA
`binding protein 4.
`
`GPNMEBis the gene symbol approved by the HUGO Gene Nomenclature Committee.
`The geneis located on chromosome7 (location 7p) and the gene sequenceis listed
`
`10
`
`15
`
`20
`
`25
`
`30
`
`1001082
`
`Copy provided by USPTO from the IFW Image Database on 02/02/2010
`
`Geneoscopy Exhibit 1014, Page 12
`
`
`
`
`
`Geneoscopy Exhibit 1014, Page 12
`
`

`

`- 13 -
`
`under the accession numbers X76534 and NM_001005340. The gene encodesthe
`glycoprotein (transmembrane) nmb.
`
`TFPI2 is the gene symbol approved by the HUGO Gene Nomenclature Committee. The
`geneis located on chromosome7 (location 7q) and the gene sequenceis listed under
`the accession numbers L27624 and NM_006528. The gene encodestissue factor
`pathwayinhibitor 2.
`
`SOX17 is the gene symbol approved by the HUGO Gene Nomenclature Committee. The
`geneis located on chromosome8 (location 8q11.23) and the gene sequenceis listed
`under the accession number ABO73988. The gene encodes SRY (sex determining
`region Y)-box 17.
`
`SYNE1 is the gene symbol approved by the HUGO Gene Nomenclature Committee. The
`geneis located on chromosome6 (location 6q24.2-q25.3) and the gene sequenceis
`listed under the accession number AB018339. The gene encodes spectrin repeat
`containing, nuclear envelope 1.
`
`LAMA‘1is the gene symbol approved by the HUGO Gene Nomenclature Committee. The
`geneis located on chromosome18 (location 18p11.3) and the gene sequenceis listed
`under the accession numbers X58531 and NM_005559. The gene encodes laminin,
`alpha 1.
`
`MMP2is the gene symbol approved by the HUGO Gene Nomenclature Committee. The
`geneis located on chromosome16 (location 16q13-q21) and the gene sequenceis listed
`underthe accession number NM_001127891. The gene encodes matrix
`metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase).
`
`OSMRis the gene symbol approved by the HUGO Gene Nomenclature Committee. The
`geneis located on chromosome5 (location 5p13.2) and the gene sequenceis listed
`under the accession number U60805 and NM_003999. The gene encodesthe
`oncostatin M receptor.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`1001082
`
`
`
`Geneoscopy Exhibit 1014, Page 13
` Copy provided by USPTO from the IFW Image Database on 02/02/2010
`
`Geneoscopy Exhibit 1014, Page 13
`
`

`

`
`
`- 14 -
`
`SFRP2is the gene symbol approved by the HUGO Gene Nomenclature Committee. The
`geneis located on chromosome4 (location 4q31.3) and the gene sequenceislisted
`under the accession number AF017986. The gene encodesthe secreted frizzled-related
`protein 2.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`CD01is the gene symbol approved by the HUGO Gene Nomenclature Committee. The
`geneis located on chromosome5 (location 5q23.2) and the gene sequenceis listed
`under the accession number NM_001801. The gene encodesthe cysteine dioxygenase,
`typeI.
`
`It mayalsorelate to any gene
`By “gene" is meant the specific known genein question.
`whichis taken from the family to which the named “gene” belongs, in certain
`circumstances, and includes accordingto all aspects of the invention not only the
`particular sequencesfound in the publicly available database entries, but also
`encompassestranscript and nucleotide variants of these sequences,with the proviso
`that methylation or another epigenetic modification of the gene is linked to the incidence
`of colorectal cancer. Variant sequences mayhaveatleast 90%,at least 91 %, at least
`92%, at least 93%,at least 94%, at least 95%,at least 96%, at least 97%, at least 98%,
`or at least 99% nucleotide sequenceidentity with the nucleotide sequencesin the
`database entries. Computer programs for determining percentage nucleotide sequence
`identity are availablein the art, including the Basic Local Alignment Search Tool (BLAST)
`available from the National Center for Biotechnology Informat