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`WO 2008/084219
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`PCT/GB2008/000056
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`EPIGENETIC CHANGE IN SELECTED GENES AND CANCER
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`FIELD OF THE INVENTION
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`The present invention relates to methods and kits for
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`5
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`identifying and diagnosing cancer which include detecting an
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`epigenetic change, such as a change in the methylation
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`status, or the expression levels, or a combination thereof
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`of any one or more of a number of genes. Also described are
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`pharmacogenetic methods for determining suitable treatment
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`10
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`regimens for cancer and methods for treating cancer
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`patients, based around selection of the patients according
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`to the methods of the invention. The present invention is
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`also concerned with improved methods of collecting,
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`processing and analyzing samples, in particular body fluid
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`15
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`samples. More particularly, the invention relates to
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`methods for identifying epigenetic changes in body fluid
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`samples. These methods may be useful in diagnosing, staging
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`or otherwise characterizing various diseases. The invention
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`also relates to methods for identifying, diagnosing, staging
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`20
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`or otherwise characterizing cancers, in particular
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`gastrointestinal cancers such as colorectal cancers, gastric
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`cancers and oesophageal cancers. The methods of the
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`invention relate, inter alia, to isolating and analyzing the
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`human DNA component from faecal samples and blood-based
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`25
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`samples.
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`BACKGROUND OF THE INVENTION
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`In their earliest stages most cancers are clinically silent.
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`Patient diagnosis typically involves invasive procedures
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`30
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`that frequently lack sensitivity and accuracy. Highly
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`reliable, non-invasive screening methods would permit easier
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`patient screening, diagnosis and prognostic evaluation.
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`Tumour derived markers are biological substances that are
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`usually produced by malignant tumours. Ideally a tumour
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`derived marker should be tumour-specific, provide an
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`5
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`indication of tumour burden and should be produced in
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`sufficient amounts to allow the detection of minimal
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`disease. Most tumour derived markers used in clinical
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`practice are tumour antigens, enzymes, hormones, receptors
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`and growth factors that are detected by biochemical assays.
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`10
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`The detection of DNA alterations such as mutations,
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`deletions and epigenetic modifications (Baylin et al., 2000)
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`provide another means for identifying cancers.
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`An epigenetic modification can be described as a stable
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`15
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`alteration in gene expression potential that takes place
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`during development and cell proliferation, mediated by
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`mechanisms other than alterations in the primary nucleotide
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`sequence of a gene.
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`It is now general knowledge that both
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`genetic and epigenetic alterations can lead to gene
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`20
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`silencing and cellular dysfunction. Synergy between these
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`two processes drives tumor progression and malignancy.
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`Three related mechanisms that cause alteration in gene
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`expression are recognised: DNA methylation, histone code
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`changes and RNA interference.
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`25
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`DNA hypermethylation is an epigenetic modification whereby
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`the gene activity is controlled by adding methyl groups
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`(CH 3 ) to specific cytosines of the DNA. In particular,
`methylation occurs in the cytosine of the CpG dinucleotides
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`30
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`(CpG islands) which are concentrated in the promoter regions
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`and intrans in human genes (P.A. Jones et al., 2002; P.W.
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`Laird et al., 2003). Methylation is associated with gene
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`silencing. DNA hypermethylation is found to be involved in
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`a variety of cancers including lung, breast, ovarian,
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`kidney, cervical, prostate and also colorectal cancer.
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`Methylation patterns of DNA from cancer cells are
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`5
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`significantly different from those of normal cells.
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`Therefore, detection of methylation patterns in
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`appropriately selected genes of cancer cells can lead to
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`discrimination of cancer cells from normal cells, thereby
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`providing an approach to early detection of cancer.
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`10
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`DNA tumour markers, in particular DNA methylation markers,
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`offer certain advantages when compared to other biochemical
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`markers. An important advantage is that DNA alterations
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`often precede apparent malignant changes and thus may be of
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`15
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`use in early diagnosis of cancer. Since DNA is much more
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`stable and, unlike protein, can be amplified by powerful
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`amplification-based techniques for increased sensitivity, it
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`offers applicability for situations where sensitive
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`detection is necessary, such as when tumour DNA is scarce or
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`20 diluted by an excess of normal DNA (Sidransky et al.,1997).
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`Bodily fluids provide a cost-effective and early non(cid:173)
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`invasive procedure for cancer detection. In this context,
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`faecal-based cancer testing has been one area of
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`investigation.
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`25
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`Human colorectal cancer has provided a good model for
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`investigating whether DNA cancer markers can be adopted as
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`an optimal faecal-based diagnostic screening test. Central
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`to faecal-based colorectal cancer testing has been the
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`30
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`identification of specific and sensitive cancer derived
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`markers.
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`The N-Myc downstream-regulated gene (NDRG) family comprises
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`four family members: NDRGl
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`(NDRG-family member 1), NDRG2
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`(NDRG-family member 2), NDRG3
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`(NDRG-family member 3) and
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`NDRG4
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`(NDRG-family member 4). The human NDRGl and NDRG3
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`5
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`belong to one subfamily, and NDRG2 and NDRG4 to another. At
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`amino acid (aa) level, the four members share 53-65%
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`identity. The four proteins contain an alpha/beta hydrolase
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`fold as in human lysosomal acid lipase but are suggested to
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`display different specific functions in distinct tissues.
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`10
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`NDRGl codes for a cytoplasmic protein believed to be
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`involved in stress responses, hormone responses, cell
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`growth, and cell differentiation. NDRGl has been
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`demonstrated to be upregulated during cell differentiation,
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`15
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`repressed by N-myc and c-myc in embryonic cells, and
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`suppressed in several tumor cells (Qu X et al.,2002; Guan et
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`al.,2000).
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`NDRG3 is believed to play a role in spermatogenesis since it
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`20
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`is highly expressed in testis, prostate and ovary (Zhao Wet
`
`al., 2001). Its involvement in brain cancer development has
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`also been suggested (Qu X et al. 2002).
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`NDRG2 codes for a cytoplasmic protein that seems to be
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`25
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`involved in neurite outgrowth and in glioblastoma
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`carcinogenesis (Deng Yet al., 2003).
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`It is upregulated at
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`both the RNA and protein levels in Alzheimer's disease
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`brains (Mitchelmore C et al., 2004), and has also been
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`suggested to play an important role in the development of
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`30 brain cancer (Qu X et al. 2002), pancreatic cancer and liver
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`cancer (Hu XL et al., 2004).
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`The NDRG4 cytoplasmic protein is involved in the regulation
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`of mitogenic signalling in vascular smooth muscles cells
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`(Nishimoto Set al.). The NDRG4 gene contains 17 exons, and
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`several alternatively spliced transcript variants of this
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`5
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`gene have been described. NDRG4 may also be involved in
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`brain cancer development (Qu X et al. 2002).
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`Suppressed expression of NDRG-family genes has been
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`demonstrated in a number of tumours (Qu X et al. 2002) and
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`10
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`the involvement of DNA promoter hypermethylation is limited
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`to the reporting of NDRG2 methylation in brain tumors (Lusis
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`et al., 2005).
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`Initially, faecal-based DNA assays investigated the
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`15 usefulness of specific point mutations markers for detecting
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`colorectal cancer. Later, the DNA integrity in faecal
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`samples proved to be a useful marker (Boynton et al., 2003).
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`Finally, faecal testing based on DNA alterations gradually
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`evolved into the development of a multi-target DNA assay
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`20
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`using specific point mutation markers, a microsatellite
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`instability marker and a marker for DNA integrity.
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`Recently, the potential of faecal DNA testing targeting
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`epigenetic alterations has been investigated (Muller et al.,
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`2004, Chen et al., 2005) and ha~ been added to the multi-
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`25
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`target DNA assay. Genes having an altered methylation
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`status traceable in faecal DNA from colon cancer patients
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`versus control samples from healthy subjects have been
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`discovered (Belshaw et al., 2004; Petko et al.,2005; Lenhard
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`et al.,2005; Muller et al.,2004; Chen et al., 2005 and Lueng
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`30
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`et al., 2004).
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`Factors that may influence the sensitivity of the selected
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`markers are sampling processing procedures and DNA isolation
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`and extraction protocols. One challenge faced by
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`researchers investigating colorectal cancer is the diversity
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`5
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`of DNA present in stool samples. Most of the DNA recovered
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`from faecal samples is bacterial in origin, with the human
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`DNA component representing only a very small minority. Human
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`DNA from cells sloughed from the colonic mucosa represents
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`as little as 0.1 to 0.01% of the total DNA recoverable from
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`10
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`stool. Additionally, the human DNA recovered is highly
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`heterogeneous. Normal cells are sloughed into the colonic
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`lumen along with only a small amount of tumour cells
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`(approximately 1% of the cells sloughed). Thus, the DNA of
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`interest represents only a very small percentage of the
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`15
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`total DNA isolated from stool. Therefore, along with the
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`exploration of suitable DNA markers, techniques for improved
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`DNA isolation and enrichment of the human DNA component from
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`faecal samples have been developed for more sensitive cancer
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`detection.
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`20
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`The initial DNA isolation techniques typically recovered DNA
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`from 10g to 4g stool and more conveniently purified the
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`human DNA component using streptavidin-bound magnetic beads
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`(Dong et al., 2001; Ahlquist et al., 2000). Further
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`25
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`improvements in recovery of target human DNA from stool
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`comprised an electrophoresis-driven separation of target DNA
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`sequences, using oligonucleotide capture probes immobilized
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`in an acrylamide gel (Whitney et al., 2004). Later, when DNA
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`integrity proved to be a suitable marker it was also
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`30
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`important to prevent degradation during sample handling.
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`Improved results were obtained with stool samples frozen as
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`quickly as possible after collection. Alternatively,
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`stabilization buffer was added to the stool samples before
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`further transport (Olson et al., 2005). A recent
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`improvement involves the use of an MBD column to extract
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`methylated human DNA in a high background of fecal bacterial
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`5
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`DNA
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`(Zou et al., 2007). However, despite these advances,
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`current tools for cancer detection in faecal samples are
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`still unsatisfactory.
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`Cancer at its early stage may release its cells or free DNA
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`10
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`into blood through apoptosis, necrosis or local
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`angiogenesis, which establishes a basis for blood-based
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`cancer testing. The usefulness of DNA methylation markers
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`for detecting colorectal cancers in serum and plasma has
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`been demonstrated (Grady et al., 2001, Leung et al., 2005;
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`15 Nakayama et al., 2007). However, the potential use of serum
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`and plasma for cancer detection is hampered by the limited
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`level of methylated DNA present in the total DNA collected
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`from plasma and serum samples (Zou et al. (2002) Clin Cancer
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`Res 188-91). A further drawback is the partial degradation
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`20
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`of the methylated DNA due to bisulfite treatment, a
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`treatment step required by many techniques that monitor DNA
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`methylation.
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`Methods and compositions for detection of early colorectal
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`25
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`cancer or pre-cancer using blood and body fluids have been
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`described.
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`WO 2006/113770 describes methods in which samples are pooled
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`and concentrated in an attempt to maximize DNA input per
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`30
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`reaction. The initial processing of 45 ml of blood allowed
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`a median DNA recovery of 3.86 ng/ml plasma. This was shown
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`to result in a sensitivity of 57% and specificity of 96% for
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`detection of colorectal cancer using a specific real-time
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`assay for detecting whether The Septin 9 gene was
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`methylated. Bisulphite treatment was focused on large
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`volume treatment and achieving maximal conversion.
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`5
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`Lofton-Day et al. (AACR general meeting April 2007, Los
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`Angeles, USA) mention improved detection of colorectal
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`cancer, and obtained a 70% sensitivity and 90% specificity,
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`with the same marker (Septin 9). The proposed method
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`10 utilised four blood draws (40 ml blood), double
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`centrifugation for plasma recovery and required four PCR
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`reactions to be carried out for each sample tested. Three
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`out of the four reactions used input DNA equivalent to 2 ml
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`of plasma per PCR reaction. The fourth reaction used a 1/10
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`15 dilution of this input DNA. Thus, repeated assays were
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`required (at least 4) and an algorithm utilised to determine
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`the final result. A sample was deemed positive if either
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`two out of the three reactions with input DNA equivalent to
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`2 ml of plasma, or the diluted measurement, were positive
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`20
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`for the Septin 9 assay. The improved sensitivity by using
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`the diluted samples indicates the presence of inhibitors in
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`the methods, a phenomena also described by Nakayama et al.
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`(2007, Anticancer Res. 27(3B) :1459-63).
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`25
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`The processing of smaller amounts of blood have been
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`described as well (US 20070141582, Hong-Zhi Zou et al. ,
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`and Satoru Yamaguchi et al.) but all result in low level of
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`methylated modified DNA detection.
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`30 Thus, current blood-based screening methods lack
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`sensitivity.
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`SUMMARY OF THE INVENTION
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`The invention, as set out in the claims, is based around the
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`finding that NDRG4/2 subfamily genes, undergoe CpG island
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`promoter methylation-associated gene silencing in human
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`5
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`cancer cells, in particular colon cancer cells. The
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`hypermethylation of the NDRG family gene, such as NDRG4
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`and/or NDRG2, in particular in the promoter region leads to
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`its loss of expression. Importantly, the presence of
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`aberrant methylation at the NDRG4/2 subfamily gene promoter
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`10
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`has a prognostic value. The epigenetic loss of NDRG4/2
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`function can be rescued by the use of DNA demethylating
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`agents and thus provides for a method for treatment. These
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`findings underline the significance of the epigenetic
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`silencing of the NDRG4/2 subfamily genes as one key step in
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`15
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`cancer development and may have an important clinical impact
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`for the treatment of the patients.
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`The present invention is also based upon the discovery of
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`specific genes and panels of genes whose methylation status
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`20
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`is linked to the incidence of, or predisposition to,
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`gastrointestinal cancers such as colorectal cancer. Use of
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`these genes for detecting gastrointestinal cancers such as
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`colorectal cancer, in particular in the context of
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`appropriate tissue or faecal (stool) samples or of
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`25
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`appropriate blood samples (or derivatives thereof)
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`respectively, has been shown to produce highly sensitive and
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`specific results. The invention provides also for a method
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`for isolating increased amount of DNA from faecal samples,
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`which results in improved sensitivity of detection of
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`30
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`colorectal cancer in faecal samples.
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`The invention also provides a method for determining the
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`methylation status of a gene of interest in a blood based
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`sample, which requires only low volumes of blood sample
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`equivalent to generate specific and sensitive results. This
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`5
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`is advantageous since it permits smaller blood samples to be
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`obtained from the subject under test.
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`Accordingly, in a first aspect, the invention provides a
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`method of detecting a predisposition to, or the incidence
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`10
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`of, cancer in a sample comprising detecting an epigenetic
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`change in at least one gene selected from an NDRG4/NDRG2
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`subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5,
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`SFRPl, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXEl,
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`SYNEl, S0Xl7, PHACTR3 and JAM3, wherein detection of the
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`15
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`epigenetic change is indicative of a predisposition to, or
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`the incidence of, cancer.
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`Subsets of genes for all aspects and embodiments of the
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`invention include an NDRG4/NDRG2 subfamily gene (in
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`20 particular NDRG4), GATA4, OSMR, GATA5, SFRPl, ADAM23, JPH3,
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`SFRP2, APC and MGMT and TFPI2, BNIP3, FOXEl, SYNEl, S0Xl7,
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`PHACTR3 and JAM3 respectively. Each subset may be
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`particularly applicable to bodily fluid samples, such as
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`stool and plasma samples as discussed herein.
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`25
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`By "epigenetic change" is meant a modification in the gene
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`caused by an epigenetic mechanism, such as a change in
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`methylation status or histone acetylation for example.
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`Frequently, the epigenetic change will result in an
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`30
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`alteration in the levels of expression of the gene which may
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`be detected (at the RNA or protein level as appropriate) as
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`an indication of the epigenetic change. Often the
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`epigenetic change results in silencing or down regulation of
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`the gene, referred to herein as "epigenetic silencing''. The
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`most frequently investigated epigenetic change in the
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`methods of the invention involves determining the
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`5 methylation status of the gene, where an increased level of
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`methylation is typically associated with the relevant cancer
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`(since it may cause down regulation of gene expression).
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`In a related aspect, the invention provides a method of
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`10
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`diagnosing cancer or predisposition to cancer comprising
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`detecting epigenetic silencing of the NDRG4/NDRG2 subfamily
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`gene, wherein epigenetic silencing of the gene is indicative
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`for cancer or predisposition to cancer.
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`15 The NDRG family genes have been characterised in the art
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`(see, for example, Qu X et al., 2002 and references cited
`
`therein) and their epigenetic silencing can be assessed in
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`terms of DNA methylation status or expression levels as
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`determined by their methylation status.
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`20
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`In on~ embodiment, the invention provides for a method of
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`diagnosing cancer or predisposition to cancer comprising
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`detecting epigenetic silencing of the NDRG4/NDRG2 subfamily
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`gene, wherein epigenetic silencing of the NDRG2/NDRG4-family
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`25
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`gene is detected by determination of the methylation status
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`of the NDRG4/2 family gene and wherein methylation of the
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`gene is indicative for cancer or predisposition to cancer.
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`Since methylation of the NDRG4/NDRG2 subfamily gene
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`30 manifests itself in reduced expression of the gene the
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`invention also provides for a method of diagnosing cancer or
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`predisposition to cancer comprising detecting epigenetic
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`silencing of the NDRG4/NDRG2 subfamily gene, wherein
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`epigenetic silencing of the NDRG2/NDRG4-family gene is
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`determined by measurement of expression levels of the gene,
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`wherein reduced expression of the gene is indicative for
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`5
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`cancer or predisposition to cancer.
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`In a related aspect, the invention provides method of
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`prognosis to cancer or predisposition to cancer comprising
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`detecting epigenetic silencing of the NDRG4/NDRG2 subfamily
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`10
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`gene, wherein epigenetic silencing of the gene is indicative
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`for cancer development or predisposition to cancer.
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`Preferably, epigenetic silencing is detected by
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`determination of the methylation status and/or measurement
`
`of expression levels of the NDRG2/NDRG4-family gene.
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`15
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`The invention also provides a method of detecting a
`
`predisposition to, or the incidence of, cancer and in
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`particular a gastrointestinal cancer such as colorectal
`
`cancer in a sample comprising detecting an epigenetic change
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`20
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`in at least one gene selected from GATA4, OSMR, NDRG4,
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`GATA5, SFRPl, ADAM23, JPH3, SFRP2, APC, and MGMT, and/or
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`TFPI2, BNIP3, FOXEl, SYNEl, S0Xl7, PHACTR3 and JAM3, wherein
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`detection of the epigenetic change is indicative of a
`
`predisposition to, or the incidence of, cancer and in
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`25 particular a gastrointestinal cancer such as colorectal
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`cancer. These subsets of genes may be particularly useful
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`where faecal test samples are utilised (and plasma in
`
`certain embodiments).
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`30
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`In a related aspect, the invention also provides a method of
`
`detecting a predisposition to, or the incidence of, cancer
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`and in particular a gastrointestinal cancer such as
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`colorectal cancer in a sample and in particular in a blood
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`sample, or derivative thereof comprising detecting an
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`epigenetic change in at least one gene selected from GATA4,
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`OSMR, NDRG4, GATA5, SFRPl, ADAM23, JPH3, SFRP2, APC, MGMT,
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`5
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`TFPI2, BNIP3, FOXEl, SYNEl, S0Xl7, PHACTR3 and JAM3(together
`
`with any suitable subset or panel thereof), wherein
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`detection of the epigenetic change is indicative of a
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`predisposition to, or the incidence of, cancer and in
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`particular a gastrointestinal cancer such as colorectal
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`10
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`cancer.
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`By "NDRG2/NDRG4 subfamily gene" is meant any gene which is
`
`taken from the subfamily to which NDRG4 and NDRG2 belong and
`
`includes according to all aspects of the invention NDRG2 and
`
`15 NDRG4. Note that "NDRGl, NDRG2, NDRG3 and NDRG4" is the
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`standard nomenclature approved by the human genome
`
`organisation for the NDRG family genes, to ensure that each
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`symbol is unique. The listed accession number for these
`
`genes can be found at www.gene.ucl.ac.uk/nomenclature.
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`20
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`NDRG family genes encompass not only the particular
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`sequences found in the publicly available database entries,
`
`but also encompass transcript variants of these sequences.
`
`Variant forms of the encoded proteins may comprise post-
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`25
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`translational modification, may result from spliced
`
`messages, etc.... NDRG4 has transcript variants having the
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`accession numbers NM 020465 and NM 022910. NDRG2 has
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`several transcript variants having the accession numbers,
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`NM_201535, NM_201536, NM_201537, NM_201538, NM_201539,
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`30 NM_201540, NM 2015401 and NM 016250. Variant sequences may
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`have at least 90%, at least 91%, at least 92%, at least 93%,
`
`at least 94%, at least 95%, at least 96%, at least 97%, at
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`least 98%, or at least 99% identity to sequences in the
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`database entries or sequence listing. Computer programs for
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`determining percent identity are available in the art,
`
`including Basic Local Alignment Search Tool (BLASTS)
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`5
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`available from the National Center for Biotechnology
`
`Information.
`
`GATA4 is the gene symbol approved by the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome 8
`
`10
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`(location p23.1-p22) and the gene sequence is listed under
`
`the accession numbers AK097060, NM_002052 and
`
`ENSG00000136574. The gene encodes GATA binding protein 4.
`
`OSMR is the gene symbol approved by the HUGO Gene
`
`15 Nomenclature Committee. The gene is located on chromosome 5
`
`(location p13.2) and the gene sequence is listed under the
`
`accession numbers U60805, NM 003999 and ENSG00000145623. The
`
`gene encodes oncostatin M receptor.
`
`20
`
`NDRG4 is the gene symbol approved by the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome 16
`
`(location q21-q22.3) and the gene sequence is listed under
`
`the accession numbers AB044947 and ENSG00000103034. The gene
`
`encodes NDRG family member 4.
`
`25
`
`30
`
`GATA5 is the gene symbol approved by the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome 20
`
`and the gene sequence is listed under the accession number
`
`ENSG00000130700. The gene encodes GATA binding protein 5.
`
`SFRPl is the gene symbol approved by the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome 8
`
`Geneoscopy Exhibit 1046, Page 15
`
`
`
`WO 2008/084219
`
`PCT/GB2008/000056
`
`- 15 -
`
`(location pll.21) and the gene sequence is listed under the
`
`accession numbers AF017987, NM_003012 and ENSG00000104332.
`
`The gene encodes secreted frizzled-related protein 1.
`
`5 ADAM23 is the gene symbol approved by the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome 2
`
`(location q33) and the gene sequence is listed under the
`
`accession numbers AB009672 and ENSG00000114948. The gene
`
`encodes ADAM metallopeptidase domain 23.
`
`10
`
`JPH3 is the gene symbol approved by the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome 16
`
`(location q24.3) and the gene sequence is listed under the
`
`accession numbers AB042636 and ENSG00000154118. The gene
`
`15
`
`encodes junctophilin 3.
`
`SFRP2 is the gene symbol approved by the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome 4
`
`(location q31.3) and the gene sequence is listed under the
`
`20
`
`accession numbers AF017986 and ENSG00000145423. The gene
`
`encodes secreted frizzled-related protein 2.
`
`APC is the gene symbol approved by the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome 5
`
`25
`
`(location q21-q22) and the gene sequence is listed under the
`
`accession numbers M74088 and ENSG00000134982. The gene
`
`encodes adenomatosis polyposis coli.
`
`The MGMT gene encodes 06-methylguanine-DNA methyltransferase
`
`30
`
`(MGMT), which is a cellular DNA repair protein that rapidly
`
`reverses alkylation (e.g. methylation) at the 06 position of
`
`guanine, thereby neutralizing the cytotoxic effects of
`
`Geneoscopy Exhibit 1046, Page 16
`
`
`
`WO 2008/084219
`
`PCT/GB2008/000056
`
`- 16 -
`
`alkylating agents such as temozolomide (TMZ) and carmustine
`
`(1-3). MGMT is the gene symbol approved by the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome
`
`10 (location 10q26) and the gene sequence is listed under
`
`5
`
`the accession numbers M29971, NM 002412 and ENSG00000170430.
`
`BNIP3
`
`is
`
`the gene
`
`symbol
`
`approved by
`
`the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome 10
`
`(location 10q26.3) and the gene sequence is listed under the
`
`10
`
`accession numbers Ul5174
`
`and ENSG00000176171. The gene
`
`encodes the BCL2/adenovirus ElB 19kDa interacting protein 3.
`
`FOXEl
`
`is
`
`the gene
`
`symbol
`
`approved by
`
`the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome 9
`
`15
`
`( location 9q22) and the gene sequence is listed under the
`
`accession numbers U89995
`
`and ENSG00000178919. The gene
`
`encodes the forkhead box El (thyroid transcription factor 2)
`
`JAM3
`
`is
`
`the gene
`
`symbol
`
`approved by
`
`the HUGO Gene
`
`20 Nomenclature Committee. The gene is located on chromosome 11
`
`(location llq25) and the gene sequence is listed under the
`
`accession numbers AF356518, NM 032801 and ENSG00000166086.
`
`The gene encodes the junctional adhesion molecule 3.
`
`25
`
`PHACTR3
`
`is
`
`the gene
`
`symbol approved by
`
`the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome 20
`
`(location 20ql3. 32) and the gene sequence is listed under
`
`30
`
`the
`
`accession
`
`numbers
`
`AJ311122,
`
`NM 080672
`
`and
`
`ENSG00000087495. The gene encodes the phosphatase and actin
`
`regulator 3.
`
`Geneoscopy Exhibit 1046, Page 17
`
`
`
`WO 2008/084219
`
`PCT/GB2008/000056
`
`- 17 -
`
`TFPI2
`
`is
`
`the gene
`
`symbol
`
`approved by
`
`the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome 7
`
`(location 7q22) and the gene sequence is listed under· the
`
`5
`
`accession numbers L27624
`
`and ENSG00000105825. The gene
`
`encodes the tissue factor pathway inhibitor 2.
`
`SOX17
`
`is
`
`the gene
`
`symbol
`
`approved by
`
`the HUGO Gene
`
`Nomenclature Committee. The gene is located on chromosome 8
`
`10
`
`(location Bqll.23) and the gene sequence is listed under the
`
`accession numbers AB073988 and ENSG00000164736. The gene
`
`encodes the SRY (sex determining region Y)-box 17.
`
`SYNEl
`
`is
`
`the gene
`
`symbol
`
`approved by
`
`the HUGO Gene
`
`15 Nomenclature Committee. The gene is located on chromosome 6
`
`(location 6q25) and the gene sequence is listed under the
`
`accession numbers AB018339 and ENSG00000131018. The gene
`
`encodes the spectrin repeat containing, nuclear envelope 1.
`
`20 Of course, as appropriate, the skilled person would
`
`appreciate that functionally relevant variants of each of
`
`the gene sequences may also be detected according to the
`
`methods of the invention. For example, the methylation
`
`status of a number of splice variants may be determined
`
`25
`
`according to the methods of the invention. Variant
`
`sequences preferably have at least 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 sequence identity with the nucleotide sequences
`
`30
`
`in the database entries. Computer programs for determining
`
`percentage nucleotide sequence identity are available in the
`
`art, including the Basic Local Alignment Search Tool (BLAST)
`
`Geneoscopy Exhibit 1046, Page 18
`
`
`
`WO 2008/084219
`
`PCT/GB2008/000056
`
`- 18 -
`
`available from the National Center for Biotechnology
`
`Information.
`
`The methods of the invention are generally ex vivo or in
`
`5
`
`vitro methods carried out on a test sample, in particular on
`
`an isolated test sample. The methods can be used to
`
`diagnose any suitable type of cancer. The cancer comprises,
`
`consists essentially of or consists of a neoplasia of the
`
`gastrointestinal tract such as gastrointestinal cancer in
`
`10
`
`one embodiment.
`
`In specific embodiments, the methods of the
`
`invention are applied to colorectal cancer, gastric cancer
`
`and/or oesophageal cancer.
`
`In more specific embodiments,
`
`the methods are used to diagnose colorectal cancer, and more
`
`particularly to diagnose hereditary nonpolyposis colon
`
`15
`
`cancer and/or sporadic colorectal cancer. Alternatively, the
`
`methods are aimed at diagnosis of gastric cancer.
`
`Preferably, the methods are used to diagnose colorectal
`
`cancer and/or gastric cancer. The methods may be used to
`
`detect carcinoma or adenoma, in particular advanced adenoma.
`
`20
`
`The methods may be employed in the diagnosis of both diffuse
`
`type and intestinal type carcinomas of the stomach,
`
`particularly when the methylation status of NDRG4 is
`
`determined.
`
`In one embodiment the methods may also include
`
`the step of obtaining the sample.
`
`25
`
`In one specific embodiment, the methods are used to diagnose
`
`oesophageal adenocarcinoma.
`
`In particular, the methylation
`
`status of the NDRG4 gene (promoter) has been shown for the
`
`first time herein to be linked with high sensitivity and
`
`30
`
`specificity to the incidence of this particular cancer type.
`
`Oesophageal adenocarcinoma may be distinguished from
`
`oesophageal squamous cell carcinomas on this basis.
`
`Geneoscopy Exhibit 1046, Page 19
`
`
`
`WO 2008/084219
`
`PCT/GB2008/000056
`
`- 19 -
`
`The "test sample" can be any tissue sample or body fluid.
`
`Preferably, the test sample is obtained from a human
`
`subject.
`
`In specific embodiments, the sample is taken from
`
`5
`
`the gastrointestinal tract. The sample may be a colorectal
`
`tissue sample or a colon, rectal, oesophageal, stomach or
`
`appendix tissue sample or a faecal or blood based sample
`
`from a subject. For faecal samples the methods are
`
`preferably used with respect to detecting gastrointestinal
`
`10
`
`cancers such as colorectal cancer as discussed herein, but
`
`may also be useful in identifying potentially dangerous
`
`adenomas. Different markers and panels of markers may be
`
`most useful with a specific sample type, such as a tissue,
`
`blood based or faecal sample as discussed herein in detail.
`
`15
`
`Thus, for example, in one embodiment, the methods of the
`
`invention involve detecting an epigenetic change, and in
`
`particular determining the methylation status, of (at least)
`
`the NDRG4 gene in a faecal test sample, wherein detection of
`
`20
`
`the epigenetic change, in particular (hyper)methylation of
`
`the NDRG4 gene (promoter) is indicative of gastrointestinal
`
`neoplasias/cancer, in particular colorectal cancer, such as
`
`adenomas and carcinomas, gastric cancer and other
`
`adenocarcinomas of the gastrointestinal tract (such as
`
`25
`
`oesophageal adenocarcinoma) and/or diffuse type and
`
`intestinal type carcinomas of the stomach.
`
`The subject may be suspected of being tumorigenic. More
`
`specifically the subject ma
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