ES2395121T3 - Methods for the diagnosis of cancer based on OBCAM and NTM genes - Google Patents

Abstract

A method of diagnosing cancer in a patient comprising the steps of contacting a sample containing the patient's nucleic acid with (a) a nucleic acid that selectively hybridizes to the OBCAM gene, or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the OBCAM cDNA, or a mutant allele thereof, or its complements; or (b) a nucleic acid that selectively hybridizes to the NTM gene, or a mutant allele thereof, or an acidic acid that selectively hybridizes to the NTM cDNA, or a mutant allele thereof, or its complements; or (c) both (a) and (b).

Description

Methods for the diagnosis of cancer based on OBCAM and NTM genes

0001 The present invention relates to cancer and in particular to ovarian and colorectal cancers.

0002 Cancer is a serious disease and an important cause of death. Although there have been advances in the diagnosis and treatment of certain cancers in recent years, there is still a need for improvements in diagnosis and treatment.

0003 Cancer is a genetic disease and in many cases involves mutations in one or more genes. It is believed that there are about 30-40,000 genes in the human genome but only a handful of these genes have been shown to be involved in cancer. Although it is conjectured that many more genes that have been identified today will be implicated in cancer, progress in this area has been slow despite the availability of molecular analysis techniques. This is due to the varied structure and function of genes that have been identified to date that suggests that cancer genes can take many forms and have many different functions.

0004 Ovarian cancer is the most frequent cause of death from malignant gynecological tumors in the western world, with an incidence of 5,500 new cases each year in England and Wales. It is the fourth most common cause of cancer mortality in American women. The majority of patients with epithelial ovarian cancer present at an advanced stage of the disease. Consequently, the 5-year survival rate is only 30% after surgery and adequate chemotherapy despite the introduction of new drugs such as platinum and taxol (Advanced Ovarian Cancer Trialists Group (1991) BMJ 303, 884-893; Ozols (1995) Semin Oncol 22, 61-66). However, patients who have stage I disease (confined to the ovaries) do better with a 5-year survival rate of 70%. Therefore it is desirable to have techniques to detect cancer before metastasis to have a significant impact on survival.

0005 Epithelial ovarian cancer constitutes 70-80% of ovarian cancer and covers a broad spectrum of lesions, ranging from localized benign tumors and neoplasms of borderline malignant potential to invasive adenocarcinomas. Histologically, common ovarian epithelial cancers are classified into several types, that is, serous, mucinous, endometrioid, clear cell, mixed and undifferentiated epithelial tumors. The heterogeneity of histological subtypes reflects the metaplastic potential of the Mullerian ovarian epithelium surface, which shares a common embryological origin with the peritoneum and the rest of the uro-genital system. Germ cell tumors, sex cords / stroma and sarcomas represent the rest of the ovarian cancers. The histogenesis and biological characteristics of ovarian epithelial cancer are not well understood as are the molecular genetic alterations that may contribute to the development of such tumors or their progression. Epidemiological factors related to ovulation appear to be important, so ovarian epithelial cells undergo several batches of proliferative division and growth to heal the wound on the epithelial surface. This leads to the development of inclusion epithelial cysts and frank malignant tumors can be derived from them (Fathalla (1971) Lancet 2, 163).

0006 An ovarian cancer screening review is given in Bel et al (1998) Health Technology Assessment 2, 1

fifty.

0007 Genetic changes in the tumor are critical for the development of cancer. Many chromosomal regions (chromosomes 3, 5, 6, 8, 11, 13, 17, 18, 22, and X) have been implicated in containing tumor suppressor genes involved in the tumor progression of sporadic ovarian cancer, but only the p53 gene (arm of chromosome 17p) was found to frequently mutate (Shelling et al (1995) Br. J. Cancer 72, 521-527). The BRCA1 gene (arm of chromosome 17q) and the BRCA2 gene (arm of chromosome 13q) isolated in 1994 and 1996 respectively, are mutated in a proportion of patients with familial breast / ovarian cancer (Ford & Easton (1995) Br. J Cancer 72, 805-812). Familial ovarian cancer accounts for only 5-10% of all ovarian tumors. In tumors of patients with sporadic ovarian cancer, only five mutations in the BRCA1 gene and four in the BRCA2 gene have been reported (Takahashi et al (1995) Cancer Res. 55, 2998-3002; Takahashi et al (1996) Cancer Res 56, 27382741) suggesting that they are rare in sporadic ovarian cancer. Mutations in the pairing error repair genes have been reported with a frequency of 10% (Tangi et al (1996) Cancer Res. 56, 25012505; Fujita et al (1995) Int. J. Cancer 64, 361-366 ; Orth et al (1994) Proc. Natl. Acad. Sci. USA 91, 9495-9499). Thus the genes that may be more critical in tumor progression in sporadic ovarian cancer have not yet been fully characterized.

0008 WO 96/05306, WO 96/05307 and WO 96/05308 refer to methods and materials used to isolate and detect a gene (BRCA1) that predisposes to human breast and ovarian cancer, some of whose mutant alleles are alleged to cause susceptibility to cancer, particularly breast and ovarian cancer.

0009 It has been suggested that tumor suppressor activity is encoded on chromosome 11 (Tanaka et al (1991) Nature 349, 340-342; Rimessi et al (1994) Oncogene 9, 3467-3474; Satoh et al (1993) Mol Carcinogenesis 7, 157164; Yoshida et al (1994) Mol. Carcinogenesis 9, 114-121; Gabra et al (1996) Int. J. Oncol. 8, 625-631; Gabra et al (1996) Cancer Res. 56, 950-954; Gabra et al (1995) Br. J. Cancer 72, 367-375; EP 0 727 486; Gabra et al (1998) Proc. AACR 39, Abstract # 4236; and Gabra et al (1998) Br J Cancer 78, Poster P185), but none of these documents identify the candidate gene (s).

0010 Colorectal tumors of the large intestine are a frequent cause of human cancer mortality in the western world with approximately 19,000 deaths in the United Kingdom per year.

0011 Most colon and rectal cancers are adenocarcinomas (Jass and Morson (1987) J. Clin. Pathol. 40,1016-1023; Morson (1974) Proc. R. Soc. Med. 67, 451-457). The literature is still divided on the true origins of colorectal carcinomas and it has been proposed that carcinomas can arise both from within existing benign neoplasms (called adenomas), in what is called the carcinoma adenoma sequence (Muto et al (1975 ) Cancer 30, 2251-2270), or by areas of generalized (de novo) dysplasia without an adenomatous stage. While some colorectal cancers are likely to originate in adenomas, most adenomas do not appear to progress to carcinoma and in fact can even regress (Knoernschild (1963) Surg. Forum XIV 137-138). Although the evidence regarding the environment, diet, age and sex suggests that these are all risk factors for colorectal cancer, the lack of confirmation of the participation of these factors in all cases suggests an underlying genetic basis for tumor formation colorectal Most colorectal cancers are not associated with clear inherited syndromes although there are inherited forms, including Familial Intestinal Polyposis (FPC), Gardner syndrome, colorectal cancer without hereditary polyposis (HNPCC) and Turcot syndrome.

0012 Several oncogenes and tumor suppressor genes have been shown to play a definite role in colorectal tumorigenesis, while in other loci a correlation between LOH and colorectal cancer is not as well defined, especially, it was found that the Barx2 gene is a suppressor of candidate tumor in the 11q24-q25 LOH region involved in ovarian and colorectal cancer (WO 00/77252).

0013 IgLONs (LAMP immunoglobulin, OBCAM and Neurotrimin) are a family of cell adhesion molecules containing immunoglobulin domain (Ig), part of the SuperFamily of proteins containing Ig domain (IgSF). IgLONs consist of LAMP (Membrane Protein Associated with the Limbic System), OBCAM / OPCML (Opioid Binding Cell Adhesion Molecule, previously called OP55A), and Neurotrimin (NTM or HNT in humans, or CEPU-1 in chicks). Recently, it has been shown that the IgLON family includes rat neurotractin (kilon - LON Stems is the chicken homologue). IgLONs are all extracellular proteins, and are not themselves transmembrane proteins or indeed even directly in contact with the cell membrane. Instead, they are attached to the cell membrane through a GPI (glycosylphosphatidylinositol) anchor attached near the Cterminal of the protein, which is then inserted into the cell membrane. It is presumed that signaling to the nucleus after IgLON binding is then carried out through trans-and cis-interaction with, yet undefined, signaling pathways coupled to the G protein (Clarke and Moss (1997) Eur. J Neurosci. 9: 334-41).

0014 The genes that encode OBCAM and NTM are located in the 11q24-q25 region of chromosome 11. The two genes share approximately 80% and 76% identity at the level of nucleotides and amino acids, respectively. In the mouse, both proteins are also encoded by different genes that appear to be clustered at the proximal end of mouse chromosome 9, in a region synthesized to human chromosome 11. The two genes share approximately 80% and 76% identity at the level of nucleotides and amino acids, respectively. In the mouse, both proteins are also encoded by different genes that appear to be confined to the proximal end of chromosome 9 of the mouse, in a region synthesized to chromosome 11q24-q25. It is likely, therefore, that these highly related genes have most likely emerged as a result of an ancestral gene duplication event that occurred at least before the divergence of man and mouse (Struyk AF et al (1995) J Neurosci 15 (3): 2141-56).

0015 The function of IgLON has been described primarily in the context of the guidance of neuronal axon and cell-cell contact in brain development. The members of the IgLON family may be co-expressed in a single cell type, and their relative cell surface levels and resulting heterophilic interactions may be important, in addition to homophilic interactions, in the contextualization of their function. Homodimerization (and also trimerization) in the plane of a membrane is a characteristic of cis-interaction of NTM (Gil et al (1998) J. Neuroscience 18: 9312-9325). It has not been previously suggested that members of the IGLON family are involved in cancer and, in particular, it has not been previously suggested that members of the IgLON family may be tumor suppressor genes.

0016 Nakajima Osamu et al (1997, Neuroreport 8 (14): 3005-3008, XP009010402 ISSN: 0959-4965) (D1) discusses the expression of the opiate-binding cell adhesion molecule (OBCAM) and neurotrimin (NTM) in E. coli and its reactivity with the anti-OBCAM monoclonal antibody.

0017 WO 01/19845 (The Johns Hopkins University School of Medicine) (D2) describes the differentially methylated CACNA1G polypeptide.

0018 EP1077259 (Ono Pharmaceutical Co.) (D4) describes a polypeptide, and a cDNA corresponding thereto, which has 96% that is identified with the sequence shown in Figure 9.

0019 Govitrapong P et al (1993, J. Biol. Chem. 268 (24): 18280-18285, XP000602144 ISSN: 0021-9258) (D6) describes that the transfection of NG108-15 cells with the antisense cDNA of the molecule Opioid-binding cell adhesion alters the interaction of the G-opioid receptor protein.

0020 Surprisingly, it has been found that in addition to the Barx2 tumor suppressor located at 11q24-q25, two additional genes in this region are methylated, mutated and / or eliminated in cancer. It is believed that the OBCAM and NTM genes are implicated in ovarian and colorectal cancer as tumor suppressor genes.

0021 This unexpected observation provides new methods of diagnosis and treatment for cancer, especially for ovarian and colorectal cancers.

0022 A first aspect of the invention provides a method of diagnosing cancer in a patient comprising the steps of

(i)

obtaining a sample containing the patient's nucleic acid; Y

(ii)

the contact of said nucleic acid with

(to)

a nucleic acid that selectively hybridizes to the OBCAM gene, or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the OBCAM cDNA, or a mutant allele thereof, or its complements; or

(b)

a nucleic acid that selectively hybridizes to the NTM gene, or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the NTM cDNA, or a mutant allele thereof, or its complements; or

(C)

both (a) and (b).

0023 A second aspect of the invention provides a method for predicting the relative perspectives of a particular cancer outcome in a patient comprising the steps of

(i)

obtain a sample containing the patient's nucleic acid; Y

(ii)

contact said nucleic acid with

(to)

a nucleic acid that selectively hybridizes to the OBCAM gene, or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the OBCAM cDNA, or a mutant allele thereof, or its complements; or

(b)

a nucleic acid that selectively hybridizes to the NTM gene, or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the NTM cDNA, or a mutant allele thereof, or its complements; or

(C)

both (a) and (b).

0024 The identification of mutations in, or lack of, OBCAM or NTM activity is believed to be particularly useful for prognosis (ie connection with outcome) and in determining whether a patient may be suitable for treatment by gene therapy or agonist therapy / mimetic (see below).

0025 In particular, the lack of OBCAM activity due to loss of heterozygosity is believed to be an early occurrence in, for example, ovarian cancer. The loss of NTM heterozygosity is believed to occur later than the loss of OBCAM in ovarian cancer. Therefore, the identification of a lack of activity or mutation in OBCAM can be particularly informative about the possibility of the onset of cancer, while the analysis of both OBCAM and NTM and the identification of lack of activity or mutation (such as suppression) (eg, by methylation analysis or LOH analysis) in both genes can allow a conclusion as to the degree, or where the method is performed on the same patient at different times, of the disease progression.

0026 As discussed below, the nucleic acid that selectively hybridizes to the OBCAM or NTM gene, or a mutant allele thereof, or its complements, can hybridize with said gene once said gene has been exposed to a modifying treatment, for example bisulfite treatment or methylation sensitive restriction enzymes, as discussed below. Thus, the method of the first or second aspect of the invention may comprise the step of exposing said patient nucleic acid to a modifying treatment, for example bisulfite treatment, before contacting said patient nucleic acid with the test nucleic acid.

0027 Early detection of ovarian cancer is particularly useful since this type of cancer remains asymptomatic until the last stages of tumor development.

0028 Preferably, the patient is a human patient and, generally, the reference to OBCAM and NTM is a reference to human OBCAM and human NTM respectively.

0029 It will be readily appreciated by the expert that the OBCAM and NTM genes or parts thereof can easily be obtained from other suitable libraries of human genes, such as standard cosmid libraries, or artificial yeast chromosome (YAC) or artificial chromosome P1 (PAC). An OBCAM or NTM cDNA can be used as a probe to identify all or parts of the OBCAM or NTM gene respectively.

0030 The OBCAM cDNA sequence is available to the public from the GenBank under Accession Number NM_002545. This sequence is also shown in Figure 7. Additional sequences for OBCAM in rat and cow are available from GenBank under the following Accession Numbers: M88711 (Rattus norvegicus) and X12672 (Bos taurus).

0031 The genomic structure of the gene can be determined by comparing the cDNA sequences, with the sequence of genomic BAC clones from the GenBank database. The following BAC or PAC clones contain the OBCAM gene: AC027631 (although this sequence in GenBank is incorrectly noted, when referring to chromosome 18; the markers contained therein are all chromosome 11) AC027631, AC012234, AP000843 and AP000912. They can be obtained from the relevant sequencing centers as part of the HGS project.

0032 It is believed that the OBCAM gene encompasses the genetic marker D11S4085 on chromosome 11. As described in more detail in Example, the marker D11S4085 has been identified as lost from an allele of chromosome 11 in ovarian or colorectal cancer (in 56 % (24/43) and 32% (8/25) of cases respectively). The D11S4085 marker is known to be located telomeric to the D11S1320 marker. The D11S4085 marker is intronic, in the second intron of OBCAM, approximately 300-600kb telomeric to D11S1320. Therefore, in an alternative embodiment of the first and second aspects of the invention, step (ii) part (a) can be formulated as:

"A nucleic acid that selectively hybridizes to a polynucleotide comprising the microsatellite marker D11S4985, or a mutant allele thereof, or its complements; or".

0033 In this case, the polynucleotide can be any polynucleotide comprising the marker D11S4085. Preferably, the polynucleotide comprises at least a part of the OBCAM gene, more preferably the entire OBCAM gene. Even more preferably, the polynucleotide is chromosome 11, or a portion thereof where the portion is at least 100, 500, 5000, or 50,000 nucleotides in length. Preferably, the nucleotide portion is such that it comprises at least 50, 100, 2500, 5000 or 25000 consecutive nucleotides of chromosome 11 on each side of marker D11S4085.

0034 D11S4085 is flanked by genomic sequence in intron 2 of OBCAM. This is in BAC AC012234. The intron 2 partial nucleotide sequence is shown in Figure 16. However, since Figure 16 only shows intron / exon limits and does not show full length introns, the D11S4085 marker is not shown in this Figure.

0035 Therefore, it will be appreciated that the reference to the "OBCAM gene" below may be a reference to a polynucleotide comprising the marker D11S4085 as defined above.

0036 The NTM cDNA sequence is available to the GenBank public under Accession Number NM_016522. This sequence is also shown in Figure 8. An additional sequence for NTM in rat is available from GenBank under Accession Number NM_017354 (Rattus norvegicus).

0037 The genomic structure of the gene can be determined by comparing the cDNA sequences, for example GenBank cDNA sequences, with the BAC genomic clone sequence of the GenBank database. The following BAC or PAC clones contain the NTM gene: AC012134, AC018368 and AP0000912. They can be obtained from the relevant sequencing centers as part of the HGS project.

0038 Other NTM cDNA sequences may include those of clones 11753149.0.6 and 11753149.0.37 of WO 00/61754 and PRO337 of WO 99/46281.

0039 We have determined additional NTM cDNA sequences shown in Figure 9. These sequences are found, for example, in the superficial epithelium of the human ovary. The predominant form in the superficial epithelium of the human ovary seems to be the "+ 33bp" form. The "+ 69bp" form is another alternative form. Both forms appear to be more abundant in the superficial epithelium of the human ovary than the sequence shown in Figure 8 and in the database entry referred to above. An additional form is the "+ 108bp" form, which contains an additional 108bp, which would be predicted to result in premature termination of protein translation and a resulting truncated NTM protein isoform lacking the GPI anchor binding site at the end. carboxy The truncated protein, therefore, could be predicted to not be anchored to the cell membrane by means of a GPI anchor. This isoform can therefore represent a soluble form of NTM, which can be located extracellularly, and which can potentially interfere with or modulate the normal function of NTM anchored to GPI.

0040 Thus, it is preferred in relation to the first and second aspect of the invention that the nucleic acid that selectively hybridizes to the NTM gene, or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the NTM cDNA, or a mutant allele thereof , or its complements, hybridize to the NTM + 33bp and / or + 69bp and / or + 108bp or cDNA gene

or its supplements (particularly in relation to ovarian cancer).

0041 In one embodiment, it is preferred that said nucleic acid hybridizes to the + 33bp and / or + 69bp and / or + 108bp forms of the NTM gene or cDNA or its complements. In a further embodiment it may be preferred that said nucleic acid also does not hybridize to the "normal" NTM gene to a database or to the cDNA sequence, as exemplified by the cDNA sequence in Figure 8.

0042 In any case, an OBCAM or NTM cDNA can easily be obtained from a human cDNA library using well known techniques and portions of the genomic clones, or portions of the OBCAM or NTM cDNA sequence shown in Figure 7 and 8 or 9 respectively , like a probe. A suitable human cDNA library is a mRNA preparation isolated from a human ovary or a human ovarian tissue or human brain or lymphoblastoid human tissues although different isoforms specific to NTM tissue may exist in non-ovarian tissues. Once an OBCAM or NTM cDNA or gene or fragment thereof has been identified as stated, its nucleotide sequence can easily be determined, for example using Sanger dideoxy sequencing or other methods well known in the art.

0043 It will be appreciated that the OBCAM or NTM gene may exist as a "wild type" gene or may exist as mutant alleles that differ in sequence from the wild type gene, as indicated above. By "mutant alleles" is included not only sequences that lead to changes in function or expression of the OBCAM or NTM polypeptide, but allelic variants (or polymorphisms) that have no or less effect on the function or expression of the OBCAM or NTM polypeptide. Thus, nucleic acids that selectively hybridize in the methods of the invention include those that selectively hybridize the wild-type OBCAM or NTM gene sequence or the wild-type OBCAM or NTM cDNA sequence (or the mRNA sequence) as well as those that selectively hybridize to mutant alleles thereof. Also, it will be readily appreciated that, as described in more detail, the expert can easily identify mutant alleles of the OBCAM or NTM gene and polymorphisms thereof.

0044 An example of a mutant allele of the OBCAM gene is described in Example 5, where a cytosine nucleotide in axon 2 (at position 334 at GenBank entry Number NM_002545 shown in Figure 7; mutation indicated in Figure 18) is present as a guanine. This mutation is seen in the ovarian cancer cell lines PEO1 and PEO4, but it is not seen in fibroblast DNA isolated from the same patient as PEO1 and PEO4. The mutation produces a change in the encoded amino acid sequence; a "wild type" proline residue is replaced by an arginine in the ovarian cancer cell lines (Figure 18).

0045 By "the OBCAM or NTM polypeptide" we include a polypeptide whose sequence comprises or consists of the amino acid sequence given in Figure 7, or 8 or 9, respectively, or whose sequence is encoded by the nucleotide sequence indicated as coding region in Figures 7, or 8, or 9, respectively, and natural variants thereof. Preferably, the OBCAM polypeptide is one whose amino acid sequence comprises the sequence given in Figure 7. Preferably, the NTM polypeptide is one whose amino acid sequence comprises the sequence given in Figure 8 or 9.

0046 By "the OBCAM or NTM polypeptide" we also include any naturally occurring polypeptide comprising a residual portion of 50 consecutive amino acids or their natural variants of the polypeptide sequence given in Figure 7, or Figure 8 or 9, respectively. Preferably, the polypeptide is a human polypeptide.

0047 "Change in expression of the OBCAM or NTM polypeptide" includes any change in the OBCAM or NTM gene that leads to changes in the expression of the OBCAM or NTM polypeptide respectively. For example, changes in the transcription of the OBCAM or NTM gene will lead to changes in the expression of the OBCAM or NTM polypeptide respectively. Similarly, changes in the translation of the OBCAM or NTM mRNA will lead to changes in the expression of the OBCAM or NTM polypeptide respectively.

0048 OBCAM or NTM protein coding sequence mutations can lead to a loss of OBCAM or NTM protein function respectively; Similarly, loss of function may be due to transcriptional silencing of the OBCAM or NTM gene or the presence of dominant negative mutations.

0049 It will be appreciated that the methods of the invention defined above may involve either directly or indirectly comparing the results of the test sample with results of a control sample such as a known (normal) non-cancerous sample or a known cancerous sample .

0050 It will be appreciated that nucleic acids that are useful in the method of the invention can easily be defined as those that selectively hybridize to the OBCAM or NTM cDNA, or a mutant allele thereof or its complements. In addition, the methods of the invention include the use of a nucleic acid that selectively hybridizes to the OBCAM or NTM or cDNA gene, or mutant alleles thereof whatever the source of the gene or cDNA. An example of a mutant OBCAM allele is shown in Figure 18 and described in more detail above. Nucleic acids that selectively hybridize to this mutant can easily be determined using the sequences shown in Figures 7, 16 and 18.

0051 By "selectively hybridizing" it is meant that the nucleic acid has sufficient nucleotide sequence similarity with said DNA or cDNA that can hybridize under moderate or highly stringent conditions. As is well known in the art, the stringency of nucleic acid hybridization depends on factors such as the length of the nucleic acid over which hybridization occurs, degree of identification of hybridization sequences and factors such as temperature, ionic strength and CG or AT content of the sequence. Thus, any nucleic acid that is capable of selectively hybridizing as said is useful in the practice of the invention.

It is preferred that the nucleic acid that selectively hybridizes selectively hybridizes to the human OBCAM or NTM cDNA or gene.

0052 Nucleic acids that can selectively hybridize to said DNA or cDNA (such as human DNA or cDNA) include nucleic acids having> 95% sequence identity, preferably those with 5> 98%, more preferably those with> 99% sequence identity, in at least a portion of the nucleic acid with said DNA or cDNA. As is well known, human genes usually contain introns such that, for example, an mRNA or cDNA derived from a gene within said human DNA would not perfectly coincide along its entire length with said human DNA but could nevertheless being a nucleic acid capable of selectively hybridizing to said human DNA. Thus, the invention specifically includes nucleic acids.

10 that selectively hybridize to an OBCAM or NTM cDNA but may not hybridize to an OBCAM or NTM gene, or vice versa. For example, nucleic acids that extend the intron-exon boundaries of the OBCAM or NTM gene may not be able to selectively hybridize to the OBCAM or NTM cDNA respectively. The intron-exon boundaries for the OBCAM gene are shown in Figure 16; The nucleotide sequence for all introns is incomplete.

0053 Moderately or highly stringent normal hybridization conditions that lead to selective hybridization

15 are known in the art, for example those described in Molecular Cloning, A Laboratory Manual, 2nd edition, Sambrook et al (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA.

0054 An example of a typical hybridization solution when a nucleic acid is immobilized on a nylon membrane and the probe nucleic acid is � 500 bases 0 base pairs is:

6 x SSC (saline sodium citrate)

20 0.5% sodium dodecyl sulfate (SDS)

100 µg / ml denatured, fragmented salmon sperm DNA

0055 Hybridization is carried out at 68 ° C. The nylon membrane, with immobilized nucleic acid, can be washed at 68 ° C in 1 x SSC or, for high stringency, 0.1 x SSC.

0056 20 x SSC can be prepared as follows. 175.3 g of NaCl and 88.2 g of sodium citrate are dissolved

25 in 800 ml of H2O. The pH is adjusted to 7.0 with a few drops of 10 N NaOH solution. The volume is adjusted to 1 liter with H2O. It is dispensed in aliquots. It is sterilized in autoclave.

0057 An example of a typical hybridization solution when a nucleic acid is immobilized on a nylon membrane and the probe is an oligonucleotide of between 15 and 50 bases is:

3.0 M trimethylammonium chloride (TMAC1)

30 0.01 M sodium phosphate (pH 6.8)

1 mm EDTA (pH 7.6)

0.5% SDS

100 µg / ml denatured, fragmented salmon sperm DNA

0.1% skim milk powder

35 0058 The optimum temperature for hybridization is usually chosen as 5 ° C below the Ti for the given chain length. Ti is the irreversible melting temperature of the hybrid formed between the probe and its target sequence. Jacobs et al (1988) Nucl. Acids Res. 16, 4637 discusses determining Tis. The recommended hybridization temperature for 17-mer in 3M TMAC1 is 48-50 ° C; for 19-groupers, it is 55-57 ° C; and for 20-mer, it is 58-66 ° C.

0059 "Nucleic acid that selectively hybridizes" also includes nucleic acids that will amplify the DNA

40 of said region of human DNA by any of the well-known amplification systems such as those described in more detail below, in particular the polymerase chain reaction (PCR). Suitable conditions for PCR amplification include amplification in a suitable 1 x amplification buffer:

10 x amplification buffer is 500 mM KCI; 100mM

Tris.CI (pH 8.3 at room temperature); 15 mM MgCl2;

45 0.1% jelly.

0060 A suitable denaturation agent or method (such as heating to 95 ° C) is used in order to separate the strands of double stranded DNA.

0061 Suitably, the annealing portion of the amplification is between 37 ° C and 60 ° C, preferably 50 ° C.

0062 Although the nucleic acid that is useful in the methods of the invention may be RNA or DNA, DNA is preferred. Although the nucleic acid that is useful in the methods of the invention may be double stranded or single stranded, single stranded nucleic acid is preferred under some circumstances such as in nucleic acid amplification reactions.

0063 The nucleic acid that is useful in the methods of the invention can be very large, such as 100 kb, if it is double stranded. For example, such large nucleic acids are useful as a template for the manufacture of probes for use in FISH analysis (fluorescence in situ hybridization). Normally, labeled probes used in FISH are generally made by translation by notches or random priming from a genomic clone (such as an insert in a suitable PAC clone). Once these probes are made, they are about 50-1000 nucleotides in length. It is more preferably used as a template for notch translation or random primer extension as described above. However, for certain diagnoses, probing or amplification purposes, it is preferred that the nucleic acid be less than 10,000, more preferably less than 1000, more preferably even 10 to 100, and more preferably 15 to 30 pairs of bases (if the nucleic acid is double stranded) or bases (if the nucleic acid is single stranded). As described in more detail below, single-stranded DNA primers, suitable for use in a polymerase chain reaction, are particularly preferred.

0064 The nucleic acid for use in the methods of the invention is a nucleic acid capable of hybridizing to the OBCAM or NTM gene or to the OBCAM or NTM or mRNA cDNA or a mutant thereof. Fragments and variants of this gene, and cDNAs derived from the mRNA encoded by the gene are also preferred nucleic acids for use in the methods of the invention.

0065 Clearly nucleic acids that selectively hybridize to the gene itself or variants thereof are particularly useful. Gene fragments are preferred for use in the method of the invention. The fragments can be made by enzymatic or chemical degradation of a larger fragment, or they can be chemically synthesized. By "gene" not only introns and exons are included, but also regulatory regions related to, and physically close to, introns and exons, particularly exons 5 'to the 5'-most end. By "physically close" is meant within 50 kb, preferably within 10 kb, preferably within 5 kb and even more preferably within 2 kb. It is believed that the basic promoter and regulatory elements of the OBCAM and NTM gene are probably 200-400 base pairs from the transcriptional start site or start of the coding regions. However, tissue-specific or inducible elements may be at 50 kb in any direction of the coding regions (exons) or they may be in introns. Such elements of the OBCAM or NTM genes can be identified or located by DNA hypersensitivity sites (detected in Southern blots) that indicate regulatory protein binding sites.

0066 Alternatively, indicator constructs can be generated using upstream genomic DNA (e.g., upstream of exon 5'-most) and, for example, �-galactosidase as an indicator enzyme. Serial deletions and fingerprint techniques can also be used to identify regulatory regions.

0067 The NTM CpG island overlaps with the ATG translation start site and is about 1.1 kb in length. OBCAM CpG Island is approximately 500bp upstream of the translation start site and is about 900bp in length. When investigating the methylation status of the NTM or OBCAM gene (as discussed below), nucleic acids that selectively hybridize to the CpG island of the selected gene may be particularly useful.

0068 By "fragment" of a gene any portion of the gene of at least 15 nucleotides in length (either single stranded or double stranded) is included but more preferably the fragment is at least 20 nucleotides in length, more preferably at least 50 nucleotides in length and it can be at least 100 nucleotides in length or it can be at least 500 nucleotides in length: Preferably the fragment is not more than 50 kb and, more preferably, not more than 100 kb.

0069 A "cDNA" of a gene specifically includes a cDNA, either partial or full length, or copied from any mRNA splice variant. A nucleic acid is also specifically included in which, compared to the natural gene, nucleotide substitutions (including inversions), insertions and deletions are present either in the gene or a fragment thereof or in a cDNA. Both variants and fragments will be selected according to their intended purposes; For probing, amplification, or diagnostic purposes, shorter fragments are generally required but with a greater degree of sequence identity (e.g., at least 80%, 90%, 95%

or 99%). An example of a variant of the OBCAM gene (and corresponding cDNA) includes the mutant with a cytosine substitution to guanine in nucleotide 334 as numbered in Figure 7 and shown in Figure 18.

0070 It is particularly preferred if the nucleic acid for use in the methods of the invention is a oligonucleotide primer that can be used to amplify a part of the gene or cDNA.

0071 Preferred nucleic acids for use in the invention are those that selectively hybridize to the OBCAM or NTM gene or cDNA and do not hybridize to other genes or cDNAs. Such selectively hybridizing nucleic acids can be readily obtained, for example, by reference to whether or not they hybridize to the OBCAM or NTM cDNA as described in Figures 7 and 8 or 9 respectively.

0072 Preferably, the first and second aspect methods are used to detect the presence or absence of a mutation in any of said genes or cDNA. In other words, if a variant gene or cDNA is present. More preferably, it is determined whether the nucleotide corresponding to nucleotide 334 of the OBCAM as numbered in Figure 7 in the patient's nucleic acid is the same as in Figure 7, or not. Such determination can be made using a polynucleotide according to the invention and as described below.

0073 The methods are suitable for any cancer but it is preferred if the cancer is ovarian cancer, colorectal cancer, or other common adenocarcinomas such as breast, lung, prostate and cervical cancer. Additionally, OBCAM related methods may also be suitable for leukemia and NTM related methods suitable for pancreas and leukemia. The methods are particularly suitable with respect to ovarian or colon cancer; The methods are most suitable in relation to ovarian cancer when the method involves a nucleic acid that selectively hybridizes to the OBCAM or cDNA gene or a polynucleotide comprising the D11S4085 marker, and are most suitable with respect to colorectal cancer when the method involves a nucleic acid that selectively hybridizes to the NTM gene or cDNA. It will be appreciated that the methods of the invention include prognostic methods and methods that aid diagnosis. It will also be appreciated that the methods of the invention are useful for the doctor or surgeon to determine a patient's management or treatment path.

0074 The patient can be any individual in whom a cancer or tumor has been found or is suspected. Particularly preferred patients are those who have a pelvic mass identified by ultrasound and / or who have slightly elevated CA125 levels. CA125 is a serum glycopeptide recently identified as Muc16 and is a tumor marker, not only for ovarian cancer, but mainly used in the clinical management of ovarian tumors.

0075 Although it is believed that any sample containing patient-derived nucleic acid is useful in the methods of the invention, since mutations in the OBCAM or NTM gene may occur in family cancers and not only sporadic cancers, it is, however, preferred. if the nucleic acid is derived from a sample of the tissue in which cancer is suspected or in which the cancer may be or has been found. For example, if the tissue in which cancer is suspected or in which cancer can be or has been found is the ovary, it is preferred if the sample containing nucleic acid is derived from the patient's ovary. Ovarian samples can be obtained by surgical excision, laparoscopy and biopsy, endoscopy and biopsy, and image-guided biopsy. The image can be generated by ultrasound or antibodies labeled with technetium-99 or antibody fragments that bind or selectively locate in the ovary. The well-known monoclonal antibody HMFG1 is an antibody suitable for imaging ovarian cancer. The ascitic fluid / peritoneal cavity, and peritoneal samples, can be obtained by surgery or laparoscopy. Similarly, if the tissue in which the cancer is suspected or in which the cancer can be or has been found is the colon, it is preferred if the sample containing nucleic acid is derived from the patient's colon; and so on. Colon samples can be obtained by colonoscopy.

0076 Other samples in which it may be beneficial to analyze OBCAM or NTM include lymph nodes, blood, serum, and potential or actual sites of metastasis, for example, bone. It is particularly preferred that the sample be blood or lymph nodes, for example for early diagnosis of occult disease, for example, asymptomatic ovarian cancer.

0077 The sample can be derived directly from the patient, for example, by tissue biopsy, or it can be derived from the patient from a site far away from the tissue, for example because the tissue cells have migrated from the tissue to other parts of the body. Alternatively, the sample can be derived indirectly from the patient in the sense that, for example, the tissue or cells thereof can be cultured in vitro, grown in a xenograft model; or the nucleic acid sample may be one that has been replicated (either in vitro or in vivo) of the nucleic acid from the patient's original source. Thus, although the nucleic acid derived from the patient may have been physically within the patient, it may also have been copied from the nucleic acid that was physically within the patient. Tumor tissue can be taken from the primary tumor or from metastasis.

0078 It will be appreciated that a useful method of the invention includes the analysis of mutations in, or the detection of the presence or absence of, the OBCAM or NTM gene in any suitable sample. The sample may suitably be a sample just obtained from the patient, or the sample may be a historical sample, for example a sample stored in a sample library.

Conventionally, the nucleic acid capable of selectively hybridizing to said human DNA and which is used in the methods of the invention also comprises a detectable label.

0080 By "detectable label" any suitable radioactive label such as 32P 33P or 35S is included that can easily be incorporated into a nucleic acid molecule using well known methods; any convenient chemiluminescent or fluorescent label that can easily be incorporated into a nucleic acid is also included. In addition the term "detectable label" also includes a fraction that can be detected by virtue of binding to another fraction (such as biotin that can be detected by streptavidin binding); and a fraction, such as an enzyme, that can be detected by virtue of its ability to convert a colorless compound into a colored compound, or vice versa (for example, alkaline phosphatase can convert colorless onitrophenyl phosphate into colored o-nitrophenol). Conveniently, the nucleic acid probe can occupy a certain position in a fixed assay and it can be determined whether the nucleic acid hybridizes to said region of human DNA by reference to the position of hybridization in the fixed assay. The detectable label may also be a fluorophore-quencher pair as described in Tyagi and Kramer (1996) Nature Biotechnology 14, 303-308.

0081 It will be appreciated that the aforementioned methods can be used for presymptomatic detection of a patient who is in a cancer risk group. Patients at high risk for detection include patients over 50 years of age or patients who carry a gene that results in increased susceptibility (eg, predisposing versions of BRCA1, BRCA2 or p53); patients with a family history of breast / ovarian cancer; patients with affected siblings, nulliparous women; and women who have a long interval between menarche and menopause. Similarly, the methods can be used for the pathological classification of tumors such as ovarian tumors or colon tumors.

0082 Conventionally, in the methods of the first, second and third aspects of the invention the nucleic acid that is capable of said selective hybridization (whether labeled with a detectable label or not) is contacted with a nucleic acid derived from the patient under hybridization conditions. . Suitable hybridization conditions include those described above.

0083 It is preferred that if the sample containing the patient-derived nucleic acid is not a substantially pure sample of the tissue or cell type in question that the sample be enriched in said tissue or cells. For example, enrichment of ovarian cells in a sample such as a blood sample can be achieved using, for example, cell sorting methods, such as fluorescence activated cell sorting (FACS) using a selective ovarian cell antibody. , or at least one antibody that is selective for an epithelial cell. For example. Cam 5.2 Antitokeratin 7/8, from Becton Dickinson, 2350 Qume Drive, San Jose, California, USA, may be useful.

0084 In a preferred embodiment, the invention provides a diagnostic blood test for early ovarian disease or other types of tumors. The blood test allows to analyze small numbers of circulating tumor cells with respect to OBCAM and / or NTM, for example with respect to the state of methylation of one or both of these genes.

0085 The source of said sample also includes biopsy material as discussed above and tumor samples, including also fixed specimens mounted on paraffin, as well as fresh or frozen tissue. The patient's nucleic acid sample can be processed before contact with the nucleic acid that selectively hybridizes to OBCAM or NTM. For example, the patient's nucleic acid sample can be treated by selective amplification, reverse transcription, immobilization (such as sequence specific immobilization), or incorporation of a detectable label.

0086 It is particularly preferred if the methods of the invention include determining methylation of, mutations in,

or the detection of the presence or absence of the OBCAM or NTM gene.

0087 The methods of the first or second aspects of the invention may involve sequencing DNA in one

or more of the relevant positions within the relevant region, including direct sequencing; direct sequencing of exons amplified by PCR; differential hybridization of an oligonucleotide probe designed to hybridize in the relevant positions within the relevant region (conventionally this use immobilizes oligonucleotide probes in so-called "chip" systems that are well known in the art); denaturing gel electrophoresis after digestion with an appropriate restriction enzyme, preferably after amplification of relevant regions of DNA; S1 nuclease sequence analysis; non-denaturing gel electrophoresis, preferably after amplification of the relevant DNA regions; conventional RFLP assays (fragment length restriction polymorphism); heteroduplex analysis; selective amplification of DNA using oligonucleotides; fluorescent in situ hybridization (FISH) of interface chromosomes; ARMS-PCR (Mutation Refractory System Amplification PCR) for specific mutations; cleavage at mismatch sites in hybridized nucleic acids (chemical or enzymatic cleavage being); single-chain conformational SSCP polymorphism or DGGE (discontinuous or denaturing gradient gel electrophoresis); analysis to detect mismatch in DNA amplified by normal / annealed mutant PCR; and protein truncation assay (translation and transcription of exons - if a mutation introduces a stop codon it will result in a truncated protein product). Other methods may be employed such as the detection of changes in the secondary structure of the single stranded DNA resulting from the changes in the primary sequence, for example, using the cleavage enzyme I. This system is commercially available from GibcoBRL, Life Technologies, 3 Fountain Drive, Inchinnan Business Park, Paisley PA4 9RF, Scotland.

0088 It will be appreciated that the methods of the invention can also be carried out in "DNA chips". Such "chips" are described in US 5,445,934 (Affymetrix; probe arrays), WO 96/31622 (Oxford, probe array plus ligase or polymerase extension), and WO 95/22058 (Affymax; fluorescently marked targets bind to oligomer substrate, and location in array detected).

0089 Detailed methods of mutation detection are described in "Llaboratory protocols for mutation detection" 1996, ed. Landegren, Oxford University Press on behalf of HUGO (Human Genome Organization).

0090 It is preferred if RFLP is used for the detection of large deletions or insertions (500bp). Southern blots can be used for this method of the invention.

0091 PCR amplification of smaller regions (maximum 300bp) to detect small changes greater than 3-4 bp insertions or deletions may be preferred. The amplified sequence can be analyzed on a sequencing gel, and small changes can be visualized (minino size 3-4 bp). Suitable primers are designed as described herein.

0092 In addition, using either Southern blot or PCR analysis, variant restriction enzyme sites can be detected. For example, to analyze variant sites in the enzymatic digestion of genomic DNA restriction, gel electrophoresis, Southern Blot, and specific hybridization probe (for example any suitable fragment derived from cDNA or OBCAM or NTM gene).

0093 For example, to analyze variant sites that use DNA amplification by PCR, restriction enzyme digestion, gel detection by ethidium bromide, silver staining or incorporation of radionuclotide or fluorescent primer in the PCR.

0094 Other suitable methods include the development of allele specific oligonucleotides (ASOs) for specific mutational events. Similar methods are used on RNA and cDNA for suitable tissue, such as ovarian or colon tissue.

0095 While it is useful for detecting mutations in any part of the OBCAM and / or NTM gene, it is preferred if the mutations are detected in the exons of the gene and it is more preferred if the mutations are ones that change the sense of coding. The detection of these mutations is a preferred aspect of the invention. An example of a cytosine to guanine mutation in exon 2 (nucleotide 334 as numbered in GenBank Entry No. NM_002545) of the OBCAM gene is described in Example 5.

0096 The methods of the invention also include checking for loss of heterozygosity (LOH; shows a lost copy). LOH may be a sufficient marker for diagnosis; seeking mutation / loss of the second allele may not be necessary. LOH of the gene can be detected using polymorphisms in the coding sequence, and introns, of the gene. LOH in a tumor cell, from any source, compared to blood is useful as a diagnostic tool, eg, it can show that the tumor has progressed and requires more rigorous treatment.

0097 Particularly preferred nucleic acids for use in the aforementioned methods of the invention are those selected from the group consisting of primers suitable for amplifying nucleic acid.

0098 Conveniently, the primers are selected from the group consisting of primers that hybridize to the nucleotide sequences shown in any of the Figures showing the OBCAM or NTM gene or cDNA sequences. It is particularly preferred if the primers hybridize to the introns of the OBCAM or NTM gene or if the primers are ones that will prime the synthesis of the DNA from the OBCAM or NTM gene or cDNA but not from other genes or cDNAs. The intron-exon boundaries of the OBCAM gene are shown in Figure 16.

0099 Primers that are suitable for use in a polymerase chain reaction (PCR; Saiki et al (1988) Science 239, 487-491) are preferred. Preferred PCR primers may have the following properties:

It is well known that the sequence at the 5 ’end of the oligonucleotide does not need to match the target sequence to be amplified.

0100 It is usual that PCR primers do not contain any complementary structure to each other greater than 2 bases, especially at their 3 ’ends, since this characteristic can promote the formation of an artifactual product called“ primer dimer ”. When the 3 'ends of the two primers hybridize, they form a "barley template" complex, and the extension of the primer results in a short duplex product called "primer dimer."

0101 Internal secondary structure should be avoided in primers. For symmetric PCR, 40-60% of G + C content is often recommended for both primers without long segments of any base. Classic melting temperature calculations used in conjunction with DNA probe hybridization studies often predict that a given primer must anneal at a specific temperature or that the 72 ° C extension temperature will dissociate the primer / template hybrid early. . In practice, hybrids are more effective in the PCR process than is generally predicted by simple Tm calculations.

0102 The optimum annealing temperatures can be determined empirically and can be higher than the expected Taq DNA polymerase has activity in the region 37-55 ° C, so that the extension of the primer will take place in the annealing step and the hybridization will stabilize. The concentrations of the primers are the same in conventional (symmetric) PCR and, normally, within the range 0.1 - to 1-µM.

0103 Any of the nucleic acid amplification protocols can be used in the method of the invention including the polymerase chain reaction, QB replicase and the ligase chain reaction. Also, NASBA (nucleic acid sequence based amplification) also called 3SR can be used, as described in Compton (1991) Nature 350, 91-92 and AIDS (1993), Vol 7 (Suppl 2), S108 or SDA ( strand shift amplification) can be used as described in Walker et al (1992) Nucl. Acids Res. 20, 1691-1696. The polymerase chain reaction is particularly preferred due to its simplicity.

0104 When a pair of suitable nucleic acids of the invention are used in a PCR, it is convenient to detect the product by gel electrophoresis and ethidium bromide staining. As an alternative to detect the DNA product, amplification using agarose gel electrophoresis and staining with DNA ethidium bromide, it is convenient to use a labeled oligonucleotide capable of hybridizing to the amplified DNA as a probe. When the amplification is by a PCR the oligonucleotide probe hybridizes to the interceptor sequence as defined by the two primers. The oligonucleotide probe is preferably between 10 and 50 nucleotides in length, more preferably between 15 and 30 nucleotides in length. The probe can be labeled with a radionuclide such as 32P, 33P and 35S using standard techniques, or it can be labeled with a fluorescent dye. When the oligonucleotide probe is labeled with fluorescence, the amplified DNA product can be detected in solution (see for example Balaguer et al (1991) "Quantification of DNA sequences obtained by polymerase chain reaction by adsorbent bioluminescence" Anal. Biochem. 195, 105-110 and Dilesare et al (1993) "A sensibility detection system based on electrochemiluminescence for the quantification of the automated PCR product" Bio Tecniques 15, 152-157.

0105 PCR products can also be detected using a probe that can have a fluorophoreser or pairing pair or can be attached to a solid support or can have a biotin label or can be detected using a combination of a capture probe and a detection probe.

0106 Fluorophore-quencher pairs are particularly suitable for quantitative measurements of PCR reactions (eg, RT-PCR). Fluorescent polarization can also be used using a suitable probe to detect PCR products.

0107 Oligonucleotide primers can be synthesized using methods well known in the art, for example using solid phase phosphoramidite chemistry.

0108 The present invention provides the use of a nucleic acid that selectively hybridizes to the OBCAM or NTM gene or a polynucleotide comprising the microsatellite D11S4085 (as described above), or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the cDNA OBCAM or NTM or a mutant allele thereof, or its supplements in a method of diagnosing cancer or predicting cancer or determining susceptibility to cancer,

or in the manufacture of a reagent to carry out these methods.

0109 Also, the present invention provides a method for determining the presence or absence of, or mutation in, said OBCAM and / or NTM gene. Preferably, the method uses a suitable sample from a patient. An example of a suitable mutation includes the cytosine to guanine mutation in nucleotide 334 in the OBCAM, as described in Example 5 and shown in Figure 18.

0110 The methods of the invention include the detection of mutations in the OBCAM and / or NTM gene.

0111 The methods of the invention can make use of a difference in restriction enzyme cleavage sites caused by mutation. A non-denaturing gel can be used to detect different lengths of fragments that result from digestion with an appropriate restriction enzyme.

0112 An "appropriate restriction enzyme" is one that will recognize and cut the wild type sequence and not the mutated sequence or vice versa. The sequence that is recognized and cut by the restriction enzyme (or not, as the case may be) may be present as a consequence of the mutation or may be introduced into the normal allele.

or mutant using unpaired oligonucleotides in the PCR reaction. It is convenient if the enzyme cuts DNA infrequently, in other words if it recognizes a sequence that happens only rarely.

0113 In another method, a pair of PCR primers that fit (e.g. hybridize to) with either the wild type genotype or the mutant genotype but not both are used. If the amplified DNA is produced then it will indicate the wild type or mutant genotype (and therefore phenotype). However, this method is based in part on a negative result.

(e.d. the absence of amplified DNA) that could be due to a technical failure. Therefore it may be less reliable and / or require additional control experiments. In a preferred embodiment, one of the pair of primers selectively hybridizes to a portion of the OBCAM gene that includes nucleotide 334 as numbered in Figure 7, or the corresponding portion of the OBCAM cDNA.

0114 A preferable method employs similar PCR primers but, as well as hybridizing only one of the wild-type or mutant sequences, they introduce a restriction site that is not there in any of the wild-type or mutant sequences.

0115 It will be appreciated that the nucleic acid that selectively hybridizes as said can selectively hybridize to the OBCAM and NTM because these two genes are within the relatively close proximity of each other on chromosome 11. As shown in Figure 1, the two genes are probably adjacent to each other.

0116 Nucleic acids that selectively hybridize to the OBCAM and / or NTM gene or to the OBCAM and / or NTM cDNA are useful for a number of purposes.

0117 They can be used in Southern hybridization to genomic DNA and in the RNase protection method to detect point mutations already discussed above. The probes can be used to detect PCR amplification products. They can also be used to detect mismatches with the OBCAM or NTM or mRNA gene in a sample using other techniques. The mismatches can be detected using either enzymes (e.g., nuclease S1 or resolvase), chemicals (e.g., hydroxylamine or osmium tetroxide and piperidine), or changes in electrophoretic mobility of mismatched hybrids compared to fully matched hybrids. These techniques are known in the art. Generally, the probes are complementary to the coding sequences of the OBCAM and / or NTM gene although probes to certain introns are also contemplated. A nucleic acid probe battery can be used to compose a kit to detect loss of or mutation in the wild-type OBCAM and / or NTM gene. The kit allows hybridization to the OBCAM and / or NTM gene in its entirety. The probes can overlap each other or be contiguous. In a preferred embodiment, the probe detects a portion of the OBCAM gene (for example, after amplification as described above), which includes nucleotide 334 as numbered in Figure 7, or the corresponding nucleotide in the OBCAM cDNA.

0118 If a ribosonde is used to detect mismatches with mRNA, it is complementary to the mRNA of the human OBCAM or NTM gene. The ribosonde is therefore an antisense probe because it does not code for the protein encoded by the OBCAM or NTM gene because it is of opposite polarity to the sense strand. The ribosonde will generally be labeled, for example, radioactively labeled which can be achieved by any means known in the art. If the ribosonde is used to detect errors of pairing with the DNA it can be of any polarity, sense or antisense. Similarly, DNA probes can also be used to detect pairing errors.

0119 Nucleic acid probes may also be complementary to mutant alleles of the OBCAM or NTM genes. These are useful for detecting similar mutations in other patients based on hybridization rather than pairing errors. As mentioned above, probes of the OBCAM and NTM gene can also be used in Southern hybridizations to genomic DNA to detect gross chromosomal changes such as deletions and insertions.

0120 According to the diagnostic and prognostic method of the present invention, loss of, or modification of, the function of the wild-type gene can be detected. The loss may be due to any event of insertion, deletion, or point mutation. If only a single allele is mutated, an early neoplastic state may be indicated. However, if both alleles are mutated then a malignant state is indicated or a greater probability of malignancy is indicated. The search for such mutations therefore provides diagnostic and prognostic information. An allele of the OBCAM or NTM gene that is not deleted (e.g., on the sister chromosome of a chromosome that carries a gene deletion) can be analyzed for other mutations, such as insertions, small deletions, and point mutations. We believe that the detection of a mutation in a single copy (allele) of the gene is useful. For example, the mutation of the OBCAM gene in cytosine 334 as numbered in GenBank Accession Number NM_002545 is seen as an early event in the development of ovarian cancer. Loss of the second allele may be necessary for carcinogenesis. If the second copy was routinely lost by a rude mechanism, this could be a useful event to detect. Some gene mutations may have a dominant negative effect on the remaining allele. Mutations that lead to non-functional gene products can also lead to a malignant state or a greater probability of malignancy. Mutational events (such as point mutations, deletions, insertions and the like) can occur in regulatory regions, such as in the promoter of the gene, leading to the loss or decrease of mRNA expression. Point mutations can also suppress adequate RNA processing, leading to the loss of or alteration in the expression of the OBCAM or NTM gene product or to the fact that the OBCAM or NTM polypeptide is nonfunctional or has an altered expression. It is preferred if the amount of OBCAM or NTM mRNA is a test sample is quantified and compared to that present in a control sample. It is also preferred if the splicing patterns or structure of the OBCAM or NTM mRNA in a test sample are determined and compared with those present in a control sample. However, detection of OBCAM or NTM expression is less preferred.

0121 The amount of OBCAM or NTM mRNA is suitably determined per unit mass of sample tissue

or by the number of units of sample cells and this is compared to the unit mass of known normal tissue or by number of units of normal cells. RNA can be quantified using, for example, Northern blot or quantitative RT-PCR.

0122 The genes have two alleles each, and it will be appreciated that alterations of both alleles may have a greater effect on cellular behavior than the alteration of one. At least one mutant allele is expected to have mutations that result in an altered coding sequence. Modifications of the second allele, other than the coding sequence, may include total or partial deletion of the gene, and loss or mutation of regulatory regions.

0123 As mentioned earlier, it may be useful to determine the loss or inactivation of an OBCAM or NTM allele, since the loss or inactivation of OBCAM may be an event earlier than the loss or inactivation in NTM, and determine the presence or Absence of loss or inactivation in either can be informative as to the stage of cancer progression. For example, a patient in which a deletion (or other losses or inactivations) is identified in the OBCAM, but not in the NTM, may have an earlier stage of cancer than a patient in which a loss or inactivation is identified in the OBCAM and NTM. For example, it is believed that a subsequent NTM LOH occurs almost exclusively in cases with OBCAM LOH, and that NTM LOH almost never occurs alone. There may be a correlation with a higher stage, or degree, or adverse survival. For colorectal cancer, the rates of OBCAM and LOH neutrimin are considered to be approximately 32% and 50%, respectively.

0124 Since the progression of a cancer or a precancerous condition can be indicated by the detection of loss or inactivation in NTM, in addition to the loss or inactivation in OBCAM, then it may be useful to carry out the method of the first or second aspect of the invention more than once in the same patient.

0125 Therefore, the present invention also provides a method for determining the progression of a cancerous disease, for example the progression of a tumor, in a patient comprising the steps of

(i)

obtain a sample of the patient's nucleic acid in which the patient's OBCAM gene has been lost or inactivated;

(ii)

contacting said nucleic acid with a nucleic acid that selectively hybridizes to the NTM gene or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the NTM cDNA, or a mutant allele thereof, or its complements.

0126 Preferably, the sample is a sample of the tissue in which the cancer is or is suspected, and more preferably the sample is from the ovary or colon.

0127 Such information regarding the progression of, or change in, any cancer or precancerous condition may be useful for the physician in the control of the efficacy of a treatment, or in the diagnosis of the exact condition or in the prognosis or prediction of lss relative perspectives of a particular outcome in a patient.

0128 The invention also includes the following methods: in vitro transcription and translation of the OBCAM and / or NTM gene to identify truncated gene products, or altered properties such as substrate binding; immunohistochemistry of tissue sections to identify cells in which protein expression is reduced / lost, or its distribution is altered in the cells or on their surfaces; and the use of RT-PCR using random primers, before the detection of mutations in the region as described above. It is preferred if the altered distribution of the OBCAM or NTM polypeptide is analyzed.

0129 An example of a mutation that may be useful to detect is the proline to arginine mutation in the OBCAM polypeptide described in Example 5. The mutated residue is located within the first immunoglobulin domain, and may introduce a conformation change. in the OBCAM polypeptide. This mutation is believed to be an indicator of cancer, particularly an early indicator of ovarian cancer. Such a mutation in the OBCAM polypeptide can be detected using an antibody that is able to distinguish between the wild type (e.g. without proline to arginine mutation) and the mutant. Such antibodies are described in more detail below.

0130 The methods of the inventions also include the detection or inactivation of the OBCAM or NTM gene by investigating its state of DNA methylation. DNA methylation of the OBCAM or NTM gene can be evaluated using standard techniques such as those described in Herman et al (1996) Proc. Natl Acad. Sci. USA 93, 9821-9826. Aberrant methylation of the OBCAM or NTM gene may be associated with its inactivation.

0131 As will be known by the person skilled in the art, the cytosine regions located 5 ’to guanines, called CpG islands, are present in the regulatory regions of many genes. These cytosines are generally not methylated under normal circumstances. However, these cytosines can become methylated within certain genes in association with cancer. These methylated genes can be repressed as a consequence of this by either biallelic methylation or in combination with a second inactivation mechanism (for example LOH or mutation). Specific Methylation PCR (MS-PCR) involves deamination of unmethylated cytosines in genomic DNA to uracil by modification using sodium bisulfite. Methylated cytosines are not deaminated by sodium bisulfite. The primers are designed so that mismatches are created, depending on the methylation status of the genomic DNA under investigation. Typical experiments involve two PCR reactions using the same template. One experiment uses a primer that counts modified methylated DNA and the other designed to anneal modified unmethylated DNA, producing specific bands for methylated and non-methylated DNA, respectively, by MS-PCR sequencing of MS-PCR products further confirms the scope of CpG island methylation for the genes tested. Examples of suitable primers are shown in Figure 12.

0132 The following references refer to MS-PCR: Maekawa et al (2001) Clin Chem Lab Med Feb; 39 (2): 121-8; Maekawa M et al (1999) Biochem Biophys Res Commun 262 (3): 671-6.

0133 The Examples show that there is a correlation between the methylation status of the OBCAM or NTM gene and its level of expression: the down-regulation of the expression of OBCAM or NTM correlates with the methylation of OBCAM or NTM. Therefore, the invention includes methods for determining the level of expression of OBCAM or NTM by evaluating the level or degree of methylation of the OBCAM or NTM gene, and for using this information in diagnosing or predicting the relative perspectives of a particular outcome of a Cancer in a patient.

0134 A further aspect of the invention provides a method for diagnosing cancer in a patient comprising the steps of

(i)

obtain a sample containing the patient's OBCAM and / or NTM gene;

(ii)

determine the degree of methylation of the OBCAM and / or NTM gene;

(iii) compare the level of methylation of the OBCAM and / or NTM gene of the patient sample with the level of methylation in a control sample; Y

(iv) If the patient sample has a higher degree of methylation of the OBCAM and / or NTM gene compared to the control sample, this is indicative of cancer.

0135 Determining the degree of methylation includes determining the presence or absence of methylation, for example the presence or absence of methylation in a particular residue or region. Thus, for example, methylation in the primer region can be detected by the presence or absence of a product using MS-PCR as described above, while product sequencing can indicate whether or how many of the potential methylation sites in the Interposition region (amplified) are methylated (and therefore mutated as a consequence of the deaminating agent or treatment, for example bisulfite treatment).

0136 If the patient sample shows methylation of the OBCAM and / or NTM gene while the control sample shows no or insignificant methylation of the OBCAM and / or NTM gene this is indicative of cancer. It is considered that methylation can occur in an essentially "all or nothing" way.

0137 A further aspect of the invention provides a method for predicting the relative perspective of a particular outcome of a cancer patient comprising the steps of

(i)

obtain a sample containing the patient's OBCAM and / or NTM gene;

(ii)

determine the degree of methylation of the OBCAM and / or NTM gene;

(iii) compare the level of methylation of the OBCAM and / or NTM gene of the patient sample with the level of methylation in a control sample; Y

(iv) if the patient sample has a higher degree of methylation of the OBCAM and / or NTM gene compared to the control sample, this is indicative of a lower probability of a successful outcome.

0138 By “control sample” we include the meaning of a non-tumor sample or a sample that may be tumor but that is in an earlier stage of development than suspected in the patient sample.

0139 Preferably, the control sample is a non-tumor sample. In the case of measuring the progression of a tumor, it may be preferable if the control sample is a tumor sample from an earlier stage of development, usually of the same patient.

0140 A progression in the disease or tumor can be identified by determining the degree of methylation in the NTM alone, or it can be identified by determining the degree of methylation in NTM and OBCAM. Preferably, the degree of methylation is determined in NTM and OBCAM. By "progression" we include an increase in the likelihood of the tumor becoming malignant.

0141 Unlike colorectal cancer that has a clear path of progression, ovarian tumors do not have a premalignant / malignant indicator that is detectable. Therefore, the progression of ovarian disease refers to an increasingly aggressive tumor with, for example, decreased prognosis or increased local spread.

0142 LOH seems to be temporarily separated with OBCAM LOH rather than with NTM LOH. Methylation can also be temporarily defined for OBCAM and NTM but it is more common for methylation, if it occurs, to occur in both genes at the same time, as described in Example 1.

0143 The identification of any methylation in NTM, or of additional methylation in NTM where previously only OBCAM was methylated, may indicate a progression in the disease or tumor from the stage in which no methylation was identified in NTM.

0144 Therefore, a further aspect of the invention provides a method for determining the progression of a cancerous disease, for example the progression of a tumor, in a patient comprising the steps of

(i)

obtain a sample of the patient containing the NTM gene in which the patient's OBCAM gene has been methylated;

(ii)

determine the degree of methylation of the NTM gene;

(iii) compare the level of methylation of the NTM gene of the patient sample with the level of methylation in a control sample; and

(iv) if the level of NTM methylation of the patient sample is increased compared to the control sample, this is indicative of a progression in the disease or tumor.

0145 The tumor can be benign or malignant. As described above in relation to the first and second aspect of the invention, it is preferable if the tumor is an ovarian or colorectal tumor, and it is also preferable if the sample is a sample of the tissue in which cancer is suspected or the cancer is found. tumor.

0146 Methods for determining methylation differences between nucleic acids are well known in the art and include (a) the use of a methylation-sensitive single nucleotide primer extension (Ms-SNuPE);

(b) digestion of genomic DNA with methylation sensitive restriction enzymes by Southern analysis; and (c) PCR-based methylation assays using genomic DNA digestion with methylation sensitive restriction enzymes before PCR amplification. The above methods can be carried out after digestion of bisulfite-converted DNA. Bisulfite treatment causes unmethylated cytosine in the nucleic acid sample to be converted to uracil, but does not cause deamination of the methylated cytosine (to thymine).

0147 Aspects of the invention can be carried out using a system (or it could also be called a kit of parts) to detect the presence or absence of, or mutation in, the relevant region of human DNA, the system comprising a nucleic acid capable of selectively hybridize to the relevant region of human DNA and a nucleoside triphosphate or deoxynucleoside triphosphate or derivative thereof. Preferred nucleic acids capable of selectively hybridizing to the relevant region of human DNA are the same as those preferred above.

The "relevant region of human DNA" includes the OBCAM or NTM gene or the OBCAM or NTM cDNA. Preferably, the relevant region of human DNA is the OBCAM or NTM gene as defined herein.

0148 The kit of parts may comprise a nucleic acid that selectively hybridizes to the OBCAM or NTM gene or a mutant allele thereof, and means for detecting a mutation in the OBCAM or NTM gene where said mutation is a mutation in OBCAM or NTM found In a cancer cell.

0149 “Mutation” includes insertions, substitutions and deletions. An example of a mutation in which the nucleic acid is capable of selectively hybridizing is the cytosine to guanine mutation of OBCAM in nucleotide 334 as numbered in Figure 7.

0150 By "nucleoside triphosphate or deoxynucleoside triphosphate or derivative thereof" any natural nucleoside triphosphate or deoxynucleoside triphosphate such as ATP, GTP, CTP, and UTP, dATP, dGTP, dCTP, TTP as well as unnatural derivatives such such as those that include a phosphorothioate bond (for example aS derivatives).

0151 Conveniently, the nucleoside triphosphate or deoxynucleoside triphosphate is radiolabelled or derived therefrom, for example with 32P, 33P or 35S, or is fluorescently labeled or labeled with a chemiliminiscent compound or with digoxigenin.

0152 Conveniently the deoxynucleotides are in a concentration suitable for dilution for use in a PCR.

0153 The kit of parts may include a nucleic acid capable of selectively hybridizing to said relevant region of human DNA and means for detecting the presence or absence of, or a mutation in, said region. Means for detecting the presence or absence of, or mutation in, said region include, for example, a diagnostic restriction enzyme or a mutant-specific nucleic acid probe or the like.

0154 Aspects of the invention can be carried out using a kit of parts comprising:

(to)

at least two nucleic acids that selectively hybridize to the OBCAM gene or a mutant allele thereof, or at least two nucleic acids that selectively hybridize to the OBCAM cDNA or a mutant allele thereof; or

(b)

at least two nucleic acids that selectively hybridize to the methylated OBCAM gene treated by bisulfite or a mutant allele thereof; or

(C)

at least two nucleic acids that selectively hybridize to the unmethylated OBCAM gene treated by bisulfite or a mutant allele thereof; or

(d)

both (a) and (b), or (a) and (c), or (b) and (c), or (a), (b) and (c) and a source of bisulfite.

0155 Aspects of the invention can also be carried out using a kit of parts comprising:

(to)

at least two nucleic acids that selectively hybridize to the NTM gene or a mutant allele thereof, or at least two nucleic acids that selectively hybridize to the NTM cDNA or a mutant allele thereof; or

(b)

at least two nucleic acids that selectively hybridize to the methylated NTM gene treated by bisulfite or a mutant allele thereof; or

(C)

at least two nucleic acids that selectively hybridize to the unmethylated NTM gene treated by bisulfite or a mutant allele thereof; or

(d)

both (a) and (b), or (a) and (c), or (b) and (c), or (a), (b) and (c) and a source of bisulfite.

0156 The kit may further comprise means for carrying out an amplification reaction, as described below (for example a PCR reaction) and / or for sequencing an amplification product, which may indicate the presence or absence of methylated CpG in the amplification product body, for example PCR.

0157 Conveniently, these kits comprising a bisulfite source further comprise methylated control DNA. Such control DNA is known to be methylated in at least one cytosine, and allows a positive comparison with test DNA. Any methylated human DNA can be used, for example DNA that has been artificially methylated using enzymatic methods. Blood-derived DNA can be useful since it represents a renewable source. Methylated human DNA is commercially available from Intergen (Purchase, New York, USA / Oxford, United Kingdom): CpGenome Universal Methylated DNA Cat No. S7821.

0158 Also conveniently, these kits further comprise a DNA polymerase. DNA polymerases are useful for amplifying bisulfite modified DNA before sequence analysis. The use of bisulfite in DNA modification, and subsequent DNA amplification in the investigation of the state of DNA methylation is described above in the Example.

0159 Aspects of the invention can be carried out using a system for the detection of the presence or absence of, or mutation in, the relevant region of DNA, the system comprising a nucleic acid that hybridizes selectively to the relevant region of human DNA and an enzyme which modifies nucleic acid. Preferred nucleic acids capable of selectively hybridizing to the relevant region of human DNA are the same as those preferred above.

0160 "Mutation" includes insertions, substitutions (including transitions) and deletions.

0161 "Enzyme that modifies nucleic acid" includes any enzyme capable of modifying an RNA or DNA molecule.

0162 Preferred enzymes are selected from the group consisting of DNA polymerases, DNA ligases, polynucleotide kinases or restriction endonucleases. A particularly preferred enzyme is a thermostable DNA polymerase such as Taq DNA polymerase. Nucleases such as Cleavase I that recognize secondary structure, for example mismatches, may also be useful.

0163 The detection of mutations in the gene will be useful in determining the appropriate treatment for a patient, eg, therapy of the OBCAM and / or NTM gene (see below). The detection of mutations in the gene can be useful to identify a subset of patients whose tumors have this common characteristic, and can be analyzed as a group for the prognosis or response to various therapies. An example of a mutation in OBCAM is described in Example 5 and is shown in Figure 18.

0164 Mutations in the gene may be related to the response or resistance to certain treatments, this can be investigated using cell lines with known sensitivity to various therapies, or by clinical correlation studies.

0165 It is possible that the genes were used as part of a panel of markers and assays, whose combined results would direct the therapy. Detection of mutations in one or both of the genes may be useful for monitoring the spread and burden of the disease.

0166 Gene analysis can be useful for differential diagnosis in the case where mutations in the gene are common in one tumor, but not in another. For example, secondary tumors of gastrointestinal origin are frequently found in the ovaries and are difficult to distinguish from tumors of real ovarian origin.

0167 A further aspect of the invention provides a method for the diagnosis of cancer in a patient comprising the steps of

(i)

obtain a sample containing protein derived from the patient; and

(ii)

decide:

(to)

the amount or activity or sequence of the OBCAM polypeptide; or

(b)

the amount, or activity or sequence of, the NTM polypeptide; or

(C)

both (a) and (b).

0168 A further aspect of the invention provides a method for predicting the relative perspectives of a particular cancer outcome in a patient comprising the steps of

(i)

obtain a sample containing protein derived from the patient; and

(ii)

decide:

(to)

the amount or activity or sequence of the OBCAM polypeptide; or

(b)

the amount, or activity or sequence of the NTM polypeptide; or

(C)

both (a) and (b).

0169 The methods of the invention also include the measurement and detection of the OBCAM and / or NTM polypeptide or mutants thereof in test sample and their comparison in a control sample. It may also be useful to detect altered polypeptide activity. It will be appreciated that measurements taken with respect to the OBCAM and / or NTM polypeptide (or mutants thereof) in the test sample can be compared to equivalent measurements in control samples that can be derived from known non-cancerous (normal) cells or derived from known cancer cells.

0170 The sample containing protein derived from the patient is conveniently a sample of the tissue in which the cancer is suspected or in which the cancer can be or has been found. These methods can be used for any cancer, but are particularly suitable in relation to ovarian cancer, colorectal cancer, and other common adenocarcinomas such as breast, lung or upper gastrointestinal cancer; The methods are especially suitable in relation to ovarian cancer or colorectal cancer. The methods for obtaining suitable samples are described in relation to previous methods.

0171 The methods of the invention involving detection of the OBCAM and / or NTM polypeptide are particularly useful in relation to historical samples such as those containing sections of paraffin embedded tumor samples.

0172 The amount of the OBCAM or NTM polypeptide can be determined in any suitable way.

0173 The OBCAM polypeptide sequence is given in the GenBank data library under Accession No. NM_002545 (see Figure 7). The NTM polypeptide sequence is given in the GenBank data library under Access Nos Nos NM_016522 (see Figures 8 and 9). NTM polypeptide sequences may also include those encoded by clones 11753149.0.6 and 11753149.0.37 of WO 00/61754 and PRO337 of WO 99/46281.

0174 By determining the sequence of the polypeptide, we include, for example, determining the presence of differences such as insertions, deletions, substitutions, etc., between the wild-type sequence as shown in Figures 7 and the sequence of the polypeptide present in the patient sample. It may be useful to determine if the polypeptide is a variant, such as the mutant described in Example 5 in the case of OBCAM, since these variants can be informative. For example, determine that a sample of a patient contains an OBCAM variant in which residue 95 (as numbered in the immature polypeptide shown in Figure 7 and in the reference sequence of the corresponding protein under GenBank Accession No. NP_002536) is an arginine instead of proline may be indicative of ovarian cancer. Since it is believed that this variant is detectable from an early stage of ovarian cancer, its detection allows a rapid diagnosis, prognosis and a determination of the relative perspectives of a particular result in the patient, all of which allows a more adequate treatment to select, thus improving the chances of a favorable outcome for the patient.

0175 It is preferred that the amount of, or sequence of the OBCAM or NTM polypeptide is determined using a molecule that selectively binds to the OBCAM or NTM polypeptide or that selectively binds to a mutant form of the OBCAM or NTM polypeptide. As the expert will know, OBCAM and NTM are extracellular and secreted cell adhesion molecules and therefore mature wild-type molecules are generally not intracellular. Suitably, the molecule that selectively binds to OBCAM or NTM or that selectively binds to an OBCAM or NTM mutant is an antibody. The antibody can also bind to a natural variant or fragment of the OBCAM or NTM polypeptide.

0176 Antibodies to OBCAM or NTM can be made by methods well known in the art.

0177 It is preferable that the antibodies used are selective for OBCAM or NTM. By "selective for OBCAM or NTM" we mean that they bind OBCAM or NTM but do not substantially bind to other polypeptides. Preferably the antibody selectively binds only to the OBCAM or NTM polypeptide, and more preferably, the antibody binds only to one of OBCAM and NTM, and not both.

0178 Antibodies that can selectively bind to a mutant form of OBCAM or NTM can be made, for example, using peptides that span the changed or otherwise modified region of amino acid of OBCAM or NTM, or using fusion proteins that express a portion of the polypeptide OBCAM or NTM that includes the amino acid region changed or otherwise modified.

0179 An example of an OBCAM variant (ie, a mutant OBCAM polypeptide sequence, against which it may be useful to make an antibody, which is capable of selective binding, is described in Example 5, and the amino acid sequence immediately surrounding the residue The mutated amino acid is shown in Figure 18. The mutated amino acid corresponds to residue 95 of the immature polypeptide (ie, with the peptide signal still attached), as numbered in Figure 7. Residue 95 is believed to be in the first domain of OBCAM immunoglobulin.

0180 In any case, based on the genetic code, it is possible to easily deduce the change in the amino acid sequence.

0181 Antibodies can be monoclonal or polyclonal. Suitable monoclonal antibodies can be prepared by known techniques, for example those described in "Monoclonal Antibodies: A Manual of Techniques", H Zola (CRC Press, 1988) and in "Hybridoma Monoclonal Antibodies: Techniques and Applications", JGR Hurrel (CRC Press , 1982).

0182 By "the amount of OBCAM polypeptide" is meant the amount of OBCAM polypeptide per unit of tissue sample mass or by the number of sample cell units, usually compared to the amount of OBCAM polypeptide per unit of tissue mass. normal known or by number of normal cell units. The amount can be determined using any suitable protein quantification method. In particular, it is preferable that antibodies are used and that the amount of OBCAM is determined using methods that include quantitative Western Blot, enzyme-linked immunosorbent assays (ELISA) or quantitative immunohistochemistry. Similarly, by "the amount of NTM polypeptide" is meant the amount of NTM polypeptide per unit mass of the sample etc, as described below in relation to OBCAM.

0183 Neoplastic lesion condition can also be detected based on the alteration of the OBCAM polypeptide

or wild type NTM. Such alterations can be determined by sequence analysis according to conventional techniques. More preferably, antibodies (polyclonal or monoclonal) are used to detect differences in, or absence of, OBCAM or NTM polypeptide or peptides derived therefrom. Antibodies can be prepared as described herein.

0184 Other techniques for creating and purifying antibodies are well known in the art and any such technique can be chosen.

0185 In a preferred embodiment of the invention, antibodies can be used to immunoprecipitate OBCAM or NTM solution proteins as well as react with the OBCAM or NTM protein in Western or polyacrylamide gel immunoblots. In another preferred embodiment, antibodies can be used to detect OBCAM or NTM proteins in paraffin or frozen tissue sections, using immunocytochemical techniques.

0186 Preferred embodiments in relation to methods for detecting OBCAM or NTM or its mutations include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal antibodies and / or polyclonal (exemplary sandwich assays are described by David et al in US Patent Nos. 4,376,110 and 4,486,530, the secondary antibody that recognizes the first, is labeled with an enzyme, a fluorescent tag, a radioisotope) and image analysis assisted by computer of sections stained by IHC.

0187 The activity of OBCAM or NTM can be determined by measuring the activity of the OBCAM or NTM polypeptide per unit mass of the tissue sample or by number of units of sample cells and usually comparing this activity with the activity of the OBCAM or NTM polypeptide per unit. of known normal tissue mass or by the number of normal cell units. The amount can be determined using any suitable OBCAM or NTM activity assay. Preferably, the assay is selective for the activity of OBCAM or NTM polypeptide.

0188 The activity of the NTM and OBCAM genes include tumor suppressor activity, neuronal axon targeting, promotion of cell aggregation and a still poorly defined cell signaling function.

0189 Aspects of the invention can be carried out with an antibody that reacts with an OBCAM polypeptide

or mutant NTM or fragment thereof, wherein said OBCAM or NTM mutant is a mutant found in a cancer cell. Preferably, the antibody does not react with the wild type OBCAM or NTM polypeptide. Such antibodies are useful in diagnostic assays and methods of the invention and can be made, for example, using peptides whose sequence is derived from the mutant OBCAM or NTM polypeptide as immunogens. An example of a mutant OBCAM is described in Example 5 and is shown in Figure 18.

0190 The invention can also be carried out using a nucleic acid that selectively hybridizes to a nucleic acid encoding a mutant OBCAM or NTM polypeptide, whose mutant is one found in a cancer cell. Such nucleic acids are useful in diagnostic assays and methods of the invention.

0191 It will be appreciated that in relation to certain methods based on diagnostic nucleic acid, susceptibility determination and prediction of relative outcome perspectives, the methods involve determining whether the status of the OBCAM or NTM nucleic acid (either DNA or mRNA) is altered in a sample that is tested in comparison to a sample of an equivalent tissue or other source that is known to be normal or disease free.

0192 Peptides based on mutant sequences may be useful in stimulating an immune response.

0193 A further aspect of the invention provides a nucleic acid that selectively hybridizes to the OBCAM gene.

or NTM or a nucleic acid that selectively hybridizes to the OBCAM or NTM cDNA for use in treating cancer in a patient.

0194 A further aspect of the invention provides a nucleic acid encoding the OBCAM and / or NTM polypeptide or a fusion thereof for use in the treatment of cancer in a patient.

0195 The invention also includes all or part of the OBCAM and / or NTM gene or cDNA for use in treating cancer in a patient. Preferably, the cancer to be treated is an ovarian cancer or colorectal cancer.

0196 Suitably, the nucleic acid encodes the OBCAM and / or NTM polypeptide or a fusion thereof. Preferably, the OBCAM and / or NTM polypeptide is a wild-type polypeptide or a variant polypeptide that has substantially wild-type activities. It is less preferred if the OBCAM and / or NTM polypeptide is a polypeptide with mutations found in cancer cells such as ovarian cancer cells; however, such polypeptides may be useful in eliciting an anti-cancer cellular immune response. Therefore, according to the present invention, a method is also provided for supplying wild-type OBCAM and / or NTM function to a cell carrying mutant OBCAM and / or NTM alleles. The provision of such a function should suppress the neoplastic growth of recipient cells. The gene or a part of the wild-type OBCAM and / or NTM gene can be introduced into the cell in a vector so that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location. If a gene fragment is introduced and expressed in a cell that carries a mutant OBCAM and / or NTM allele, the gene fragment should encode a part of the OBCAM and / or NTM protein that is required for non-neoplastic cell growth. . More preferred is the situation where the wild-type OBCAM and / or NTM gene or a part thereof is introduced into the mutant cell such that it is recombined with the endogenous mutant OBCAM and / or NTM gene present in the cell. Such recombination requires a double recombination event that results in the correction of the genetic mutation of OBCAM and / or NTM. Vectors for the introduction of genes for both recombination and extrachromosomal maintenance are known in the art, and any suitable vector can be used. Methods for the introduction of DNA into cells such as electroporation, coprecipitation with calcium phosphate and viral transduction are known in the art, and the choice of method is within the competence of the person skilled in the art. Cells transformed with the wild-type OBCAM and / or NTM gene can be used as model systems to study cancer remission and drug treatments that promote such remission.

0197 As generally discussed above, the OBCAM and / or NTM gene or fragment, if applicable, can be used in gene therapy methods in order to increase the amount of expression products of such genes in cancer cells. Such gene therapy is particularly suitable for use in both cancer and precancerous cells, in which the level of OBCAM and / or NTM polypeptide is absent or decreased or otherwise changed compared to normal cells. It may also be useful to increase the expression level of an OBCAM and / or NTM gene even in those tumor cells in which the mutant gene is expressed at a "normal" level, but the gene product is not fully functional or has a function. altered

0198 Gene therapy can be carried out according to generally accepted methods, for example, as described by Friedman, 1991. A patient's tumor cells can first be analyzed by diagnostic methods described herein, to determine the production of the polypeptide. OBCAM and / or NTM and its physical form (ie what mutations it contains) in tumor cells. A virus or plasmid vector is prepared (see more details below), which contains a copy of the OBCAM and / or NTM gene linked to the expression control elements and capable of replicating within the tumor cells. Suitable vectors are known, such as those described in US Patent 5,252,479 and published PCT Application WO 93/07282. The vector is then injected into the patient, either locally at the tumor site or systemically (in order to reach any tumor cell that may have metastasized to other sites). If the transfected gene is not permanently incorporated into the genome of each of the specific tumor cells, the treatment may have to be repeated periodically.

0199 Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and non-viral transfer methods. A number of viruses have been used as gene transfer vectors, including papovavirus, e.g., SV40 (Madzak et al, 1992), adenovirus (Berkner, 1992; Berkner et al, 1988; Gorziglia and Kapikian, 1992; Quantin et al , 1992; Rosenfeld et al, 1992; Wilkinson et al, 1992; Stratford-Perricaudet et al, 1990), vaccinia virus (Moss, 1992), adeno-associated virus (Muzyczka, 1992; Ohi et al, 1990), herpesvirus including HSV and EBV (Margolskee, 1992; Johnson et al, 1992; Fink et al, 1992; Breakfield and Geller, 1987; Freese et al, 1990), and avian influenza retroviruses (Brandyopadhyay and Temin, 1984; Petropoulos et al., 1992), murine (Miller, 1992; Miller et al, 1985; Sorge et al, 1984; Mann and Baltimore, 1985; Miller et al, 1988), and of human origin (Shimada et al, 1991; Helseth et al, 1990 ; Page et al, 1990; Buchschacher and Panganiban, 1992). To date, most human gene therapy protocols have been based on deactivated mouse retroviruses.

0200 It may be preferred, particularly in relation to OBCAM, that the gene therapy vector is not a vaccinia virus vector.

0201 Methods for transferring non-viral genes known in the art include chemical techniques such as coprecipitation with calcium phosphate (Graham and van der Eb, 1973; Pellicer et al, 1980); mechanical techniques, for example microinjection (Anderson et al, 1980; Gordon et al, 1980; Brinster et al, 1981; Constantini and Lacy, 1981); fusion-mediated membrane transfer via liposomes (Felgner et al, 1987; Wang and Huang, 1989; Kaneda et al, 1989; Stewart et al, 1992; Nabel et al, 1990; Lim et al, 1992); and direct DNA absorption and receptor-mediated DNA transfer (Wolff et al, 1990; Wu et al, 1991; Zenke et al 1990; Wu et al, 1989b; Wolff et al, 1991; Wagner et al, 1990; Wagner et al, 1991; Cotten et al, 1990; Curiel et al, 1991a; Curiel et al, 1991b). Virus-mediated gene transfers can be combined with direct gene transfer in vivo using liposome delivery, allowing one to direct viral vectors to tumor cells and not in surrounding cells that do not divide. Alternatively, the retroviral vector producer cell line can be injected into tumors (Culver et al, 1992). Injection of producing cells will then provide a continuous source of vector particles. This technique has been approved for use in humans with inoperable brain tumors.

0202 Other suitable systems include the adenovirus-retroviral hybrid system described by Feng et al (1997) Nature Biotechnology 15, 866-870, or viral systems with focused ligands such as suitable single chain Fv fragments.

0203 In an approach that combines the biological and physical methods of gene transfer, plasmid DNA of any size is combined with a polylysine-conjugated antibody specific for the adenovirus hexon protein, and the resulting complex is ligated to an adenovirus vector. The trimolecular complex is then used to infect cells. The adenovirus vector allows efficient binding, internalization and degradation of the endosome before the coupled DNA is damaged.

0204 It has been shown that liposome / DNA complexes are capable of mediating direct gene transfer in vivo. While in standard preparations of liposomes the process of gene transfer is non-specific, localized absorption and expression in tumor deposits have been reported, for example, after in situ administration (Nabe1 (1992) Hum. Gene Ther 3, 399-410).

0205 Gene transfer techniques that direct DNA directly to ovarian tissue, eg, epithelial cells of the ovaries, are preferred. Receptor-mediated gene transfer, for example, is accomplished by conjugating DNA (usually in the form of a covalently closed superspiral plasmid) to a protein ligand through polylysine. The ligands are chosen based on the presence of the corresponding ligand receptors on the cell surface of the cell / target tissue type. These DNA ligand conjugates can be injected directly into the blood if desired and are directed to the target tissue where binding and internalization of the DNA-protein complex receptor occurs. To overcome the problem of intracellular destruction of DNA, coinfection with adenovirus can be included to disrupt endosome function.

0206 In the event that gene replacement therapy using wild-type OBCAM and / or NTM functionality is used, it may be useful to control the treatment by detecting the presence of OBCAM and / or NTM mRNA or polypeptide, or OBCAM and / or functional NTM, at various sites in the body, including the target tumor, metastatic sites, blood serum, and body secretions / excretions, for example urine.

0207 Aspects of the invention can be carried out using a gene therapy vector that is capable of expressing the OBCAM and / or NTM polypeptide or a functional or variant fragment or fusion thereof in a mammalian cell. Normally, the functional fragment or variant or portion or fusion of the OBCAM or NTM polypeptide has tumor suppressor activities of the wild-type OBCAM or NTM respectively.

0208 OBCAM tumor suppressor activity is shown in Figure 14, and is described in more detail in Example3.

0209 Preferably, the vector is one that can be replicated in a human cell. Preferably, the vector is one that has been described above in more detail in connection with the aspects of gene therapy of the invention.

0210 A further aspect of the invention provides an effective amount of the OBCAM and / or NTM polypeptide or a fusion thereof for use in the treatment of cancer in a patient.

0211 Peptides that have OBCAM or NTM activity can be delivered to cells that carry mutant or missing OBCAM or NTM alleles. The sequence of the OBCAM or NTM protein is described in Figures 7 and 8 or 9 respectively. The protein can be produced by expression of the cDNA sequence in the bacterium, for example, using known expression vectors. Alternatively, the OBCAM or NTM polypeptide can be extracted from mammalian cells that produce OBCAM or that produce NTM. In addition, synthetic chemistry techniques can be used to synthesize the OBCAM or NTM protein. Any such technique can provide the preparation of the present invention comprising the OBCAM or NTM protein. The preparation is substantially free of other human proteins. This is most easily achieved by synthesis in a microorganism or in vitro.

0212 The OBCAM or NTM cDNA or gene can be expressed by any suitable method. Generally, the DNA is inserted into an expression vector, such as a plasmid, in the correct orientation and the correct reading frame for the expression. If necessary, the DNA can be linked to the appropriate transcription and translation regulatory control nucleotide sequences recognized by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host by standard techniques. Generally, not all guests will be transformed by the vector. Therefore, it will be necessary to select transformed host cells. A selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control element, that codes for a selectable characteristic in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such a selectable characteristic may be in another vector, which is used to co-transform the desired host cell.

0213 Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings described herein to allow expression of the polypeptide, which may Then be recovered.

0214 Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.

0215 Vectors include a prokaryotic replicon, such as CoIE1 ori, for propagation in a prokaryotic, even if the vector is to be used for expression in other types of cells, not prokaryotes. Vectors may also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of genes in a bacterial host cell, such as E. coli, transformed therewith.

0216 A promoter is an expression control element formed by a DNA sequence that allows the binding of RNA polymerase and that transcription occurs. Promoter sequences compatible with example bacterial hosts are normally provided in plasmid vectors containing restriction sites suitable for insertion of a DNA segment of the present invention.

0217 Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway, NJ, USA.

0218 A typical vector plasmid of mammalian cells is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive the expression of cloned genes, the highest level of expression being found in cells that produce T antigen, such as COS-1 cells.

0219 An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid inducible promoter of the long terminal repeat of the mouse mammary tumor virus to drive the expression of the cloned gene.

0220 Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integration Plasmids (Yips) and incorporate selectable yeast markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromer (YCps) plasmids.

0221 A number of methods have been developed to operatively link DNA to vectors through complementary cohesive terms. For example, complementary homopolymer zones can be added to the DNA segment to be inserted into the vector DNA. The vector and the DNA segment are then joined by hydrogen bonds between complementary homopolymer tails to form recombinant DNA molecules.

0222 Synthetic linkers containing one or more restriction sites provide an alternative method of linking the DNA segment to vectors. The DNA segment, generated by restriction endonuclease digestion as described above, is treated with bacteriophage T4 DNA polymerase I or E. coli DNA polymerase, enzymes that remove 3'-single stranded leaving terms with their exonucleolytic activities. 3'-5 ', and filled in 3' ends embedded with its polymerization activities.

0223 The combination of these activities therefore generates blunt-end DNA segments. The blunt end segments are then incubated with a large molar excess of connecting molecules in the presence of an enzyme that is capable of catalyzing the ligation of blunt end DNA molecules, such as bacteriophage T4 ligase DNA. Thus, the products of the reaction are segments of DNA that carry sequences of linker polymers at their ends. These segments of DNA are then cleaved with the appropriate restriction enzyme and linked to an expression vector that has been cleaved with an enzyme that produces ends compatible with those of the DNA segment.

0224 Active OBCAM and / or NTM molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some active molecules can be captured by cells, actively

or by diffusion. The extracellular application of the OBCAM and / or NTM gene product may be sufficient to affect tumor growth. The supply of molecules with OBCAM and / or NTM activity should lead to the partial reversal of the neoplastic state. Modified polypeptides with substantially similar function are also used for peptide therapy.

0225 Treatment aspects of the invention can be carried out using a pharmaceutical composition comprising a gene therapy vector that includes a nucleic acid encoding the OBCAM and / or NTM polypeptide or a functional variant or portion or fusion thereof and a pharmaceutically carrier. acceptable; a pharmaceutical composition comprising a gene therapy vector including a nucleic acid that selectively hybridizes to the OBCAM and / or NTM gene, or a mutant allele thereof, or an OBCAM and / or NTM cDNA, or a mutant allele thereof, and a pharmaceutically acceptable carrier; a pharmaceutical composition comprising the OBCAM and / or NTM polypeptide or fusion thereof, and a pharmaceutically acceptable carrier.

0226 Suitable gene therapy vectors have been described before. Suitable OBCAM and NTM polypeptides have been described before. As indicated, it is preferable, particularly in relation to OBCAM, that the gene therapy vector is not a vaccinia virus vector.

0227 By "pharmaceutically acceptable" it is included that the formulation is sterile and pyrogen free. Pharmaceutically acceptable carriers are well known in the pharmacy art.

0228 The present invention will now be described in more detail with reference to the following, non-limiting, Examples and Figures.

0229 Figure 1.

Map contiguous sequences BAC (bacterial artificial chromosome) of the 11q25 region that contains the NTM and OBCAM genes, organized according to physical position. The relative position of relevant microsatellite markers is shown. It is not to scale.

0230 Figure 2.

Relationship of discrete LOH regions on chromosome 11q24-q25 showing the Barx2 region (region 2) and the OBCAM / NTM region (region 5).

0231 Figure 3.

LOH values for microsatellite markers in relation to Figure 2 from centromere to telomere for ovarian and colorectal cancer.

0232 Figure 4.

This figure shows how the markers have been reordered and documents the number of cases with LOH as a percentage of the number of informative cases for each marker. 66% (43/65) of ovarian tumors and 69% (27/39) of colorectal tumors had LOH that included at least 1 locus within the 11q24-q25 region. 8 tumors in each group had LOH in all informative loci.

0233 Figure 5.

Examples of LOH D11S4085. 2 cases with retention of both alleles in the blood and complete loss of an allele in ovarian cancer tissue.

0234 This example of the LOH profile in D11S4085 shows heterozygosity retention in the flanking microsatellite markers D11S4085 (centromeric) and D11S969 (telomeric). The markers were amplified by PCR (with a primer for each label labeled with a fluorescent dye) of Normal (N) and Tumor (T) DNA from two ovarian cancer patients. The PCR products (fluorescently labeled) were separated by size on an AB1310 Genetic Analyzer, detected by a laser and the data analyzed with the GeneScan ABI software. The peaks represent the allele pattern for each of the markers in the Normal and Tumor DNA for the two patients, Patient 1 and Patient 2. The comparison of the D11S4085 allele pattern between N and T for each patient shows two alleles (heterozygosity ) present in Normal DNA, while only a single allele is present in Tumor DNA, indicating that loss of heterozygosity has occurred. The loss of allele has been complete in both cases, indicating a lack of heterogeneity in the Tumor sample. This suggests that the loss of D11S4085 is an early event in ovarian carcinogenesis. In contrast, heterozygous allele patterns for the two flanking markers do not change between Normal and Tumor for each patient, which represents heterozygosity retention.

0235 Figure 6.

Representative sample of 13 ovarian cancer cases showing methylation status for OBCAM and neurotrimin, and concordance and discordance rates for both between individuals and across the sample.

M = methylated U = not methylated

C = concordant

D = jarring

0236 Figure 7.

The nucleotide sequence of the human OBCAM cDNA with the encoded amino acid sequence. The sequence corresponds to the GenBank database with entry number NM_002545.

0237 Figure 8.

The nucleotide sequence of human Neurotrimin cDNA (NTM) with the encoded amino acid sequence. The sequence corresponds to the GenBank database with entry number NM_016522.

0238 Figure 9.

NTM isoform sequences. The predominant form in the superficial epithelium of the normal human ovary is the + 33bp form (about 69%). The + 69bp form is another alternative form that is a minor isoform (about 19%), compared to the wild-type sequence of the database that forms about 4%. An additional minor isoform contains an additional 108bp, which would be predicted to result in premature termination of protein translation and a resulting truncated NTM isoform protein.

0239 The + 33bp form contains a 33bp inserted nucleotide sequence, which is derived from an alternative single exon within the NTM gene. This exon is one of two exons that contributes to 69bp insertion (see below). The nucleotide sequence with protein translation (below the nucleotide sequence) of the isoform + 33 bp of the superficial epithelium of the human neurotrimin ovary is shown. The additional 33bp of the nucleotide sequence and 11 resulting amino acids are shown in bold underlined in the context of the wild-type human NTM sequence (GenBank NM_015622). The stop codon is denoted by *. The framed insertion results in the inclusion of an additional 11 amino acids near the C-terminus of the NTM protein: EVKTTALTPWK.

0240 The + 69bp form contains an inserted 69bp nucleotide sequence, which is derived from the junction of 2 alternative exons within the NTM gene. The nucleotide sequence with protein translation (under the nucleotide sequence) of the isoform + 69bp of the ovary surface epithelium of human NTM is shown. The additional 69bp of the nucleotide sequence and 23 resulting amino acids framed are shown in bold underlined in the context of the wild-type human NTM sequence (GenBank NM_015622). The stop codon is denoted by *. The framed insertion results in the inclusion of 23 additional amino acids near the C-terminus of the NTM protein: ELNEPTSSTLLQEVKTTALTPWK.

0241 The + 108bp form contains an inserted nucleotide sequence of 108bp, which is derived from the splicing of 3 alternative exons within the NTM gene. The nucleotide sequence with protein translation (under the nucleotide sequence) of the isoform + 108bp of the human NTM ovary surface epithelium is shown. Additional 108bp of the nucleotide sequence would be predicted to result in premature termination of protein translation and a resulting truncated NTM protein isoform lacking the GPI anchor binding site in the carboxy terminus. The truncated protein, therefore, could be predicted not to be anchored to the cell membrane through a GPI anchor. This isoform can therefore represent a soluble form of NTM, which can be located extracellularly, and which can potentially interfere with or modulate the normal function of NTM anchored to GPI. Additional 108bp of the nucleotide sequence and protein translation are shown in bold underlined in the context of the wild-type human NTM sequence (GenBank NM_015622). Stop codons are denoted by *.

0242 Figure 10. IgLONs are highly expressed in normal ovary.

The full-length coding sequence OBCAM and NTM RT-PCR multi-tissue cDNA panel (BD Clontech) with the addition of a normal ovary sample prepared in ICRF Medical Oncology Unit, Edinburgh, UK. Strong expression of both genes is observed in normal brain and ovarian samples prepared internally (ie not the Clontech cDNA panel).

Primer Sequences:

OBCAM:

OPCML F1: 5’-AGTTGTGGCTGTCGAGAATG-3 ’20’ number nucs 34-53

OPCML R1: 5'-TCAGAGGACCTAGGATTTCT-3 '20'number nucs 1110-1091 OBCAM Nucleotide Numbering of NM_002545 NTM: NTM F2: 5'-AGTTGTGGCTGTCGAGAATG-3' nucs 248-267 NTM R1: 5'-AGAGGTTG 1600-1581 NTM nucleotide numbering of NM_016522 0244 Figure 11. Re-expression of IgLON MDAMB23.1 and T47D cancer cell lines were cultured in the presence (+) or absence (-) of azacitidine

for 4 days plus TSA (Aza / TSA) for the fourth day. OBCAM and NTM RT-PCR shows re-expression of OBCAM in the MDAMB23.1 cell line, and NTM re-expression in the MDAAMB23.1 and T47D cell lines. Negative controls of reverse transcriptase are included. NTM RT-PCR shows re-expression of multiple neurotrimin isoforms.

0245 The primers used were: OBCAM: OPCML F4: 5'-TACCATAGATGACCGGGTAA-3 'nucs: 221-240 OPCML R6: 5'-TTCCGCACATCGGGCGCAGC-3' nucs: 694-675 NBC OTMAM nucleotide number: N_00254545: '-ACATGACTATGGGAACTACA-3' nucs 1125-1144 NTM R2: 5'-GGAAGTGGCACTCACATCAA-3 'nucs 1315-1296 NTM nucleotide numbering of NM_016522 0246 Figure 12. Representative primers useful in detecting methylation of OBCAM and NTM genes. 0247 Figure 13. Demethylation and re-expression of OBCAM in SKNV3.3 cells after exposure to 5’-aza-2 ’

deoxcytidine (AZA). CON are untreated control SKNV3.3 cells. Mix is a PCR control reaction that

It contains all the components except the template DNA. 0248 Upper panel: OBCAM RT-PCR of the 1st cDNA chain prepared from SKNV3.3 cells after 4 culture days in the presence (AZA) or absence (NOC) of 20µM 5’-aza 2’-deoxcytidine. Mix refers to a reaction Control PCR that contains all reaction components except the DNA template. OBCAM PCR Products They were then transferred to a nylon membrane and hybridized with an OBCAM probe. The OBCAM expression It is clearly evident after exposure to AZA but is absent in control cells. Bottom panel: Actin RT-PCR of the 1st cDNA chain of similar cell lines as a control for the integrity of samples. Actin expression is similar in treated and untreated SKVN3.3 cells.

0249 Figure 14. OBCAM transfection in SKVN3.3 cells suppresses subcutaneous tumor growth in mice

naked 0250 Graph of mean tumor volume (cm3) of tumors in nude mice after injection OBCAM transfected sense subcutaneous and SKVN3.3 control cells. Tumor volumes were measured. weekly for 4 weeks. The difference in tumor growth s.c. It is statistically significant.

0251 Figure 15. Transfection of OBCAM into SKVN cells increases cell aggregation.

0252 Graph of the number of individual leftover cells in the OBCAM cultures transfected sense, OBCAM transfected anti-sense, and SKNV3.3 progenitor cells measured with a hemocytometer at timed intervals. The expression of OBCAM results in a reduced number of individual cells that remain in the culture, which match an improved observed rate of cell aggregation.

0253 Figure 16. Predicted Exon Structure of the Human OBCAM Gene.

0254 The exonic sequence is highlighted in bold and the intron sequence flanking the exons is in normal text. Nucleotide numbers refer to those corresponding to the GenBank database sequences as follows. Exon 1, refers to AC027631.4; Exon 2, refers to AC012234.6; Exons 3-7, refers to AP000843.3. The nucleotide sequence for Exon 1 is incomplete in the area that covers the exon / intron 1 boundary due to the lack of the Human Genome Project Sequence available in the GenBank with Access AC027631.4. The sense and anti-sense PCR primers for SSCPE are highlighted with single and double underlines, respectively. Exon sizes provided are given.

0255 Figure 17. PEO4 contains a somatic mutation for OBCAM in Exon 2.

0256 Trace sequence files of F1 / R1 SSCPE PCR products of OBCAM exon 2 DNA of PEO4 and PEO4 fibroblasts obtained using OBCAM EX2 F1 as sequence primer. PEO4 fibroblasts are homozygous for a nucleotide C in the marked position (*); PEO4 is heterozygous in this position and has two alleles C and G.

0257 Figure 18. PEO4 contains a somatic mutation in the wrong direction for OBCAM.

0258 The ExPasy translation of the two identified alleles of the nucleotide sequence of the F1 / R1 SSCPE PCR products from OBCAM EX2 from PEO4, predicts a missense mutation from proline (P) to arginine (R). Somatic nucleotide change: cag at nucleotide position 75365 (AP000843.3) / 334 (NM_002545.2) results in an amino acid substitution: arginine (R) for proline (P) at position 95 (immature protein numbering) , within the first immunoglobulin domain of OBCAM.

0259 PEO4 fibroblasts are homozygous for the wild-type proline allele, while PEO4 (and PEO1 and PEO1CDDP) are heterozygous for the wild-type and mutated sequence, both containing the wild-type proline and somatic arginine missense mutation. Wild type refers to the reference sequence as found in the GenBank sequences.

0260 The wild type OBCAM nucleotide sequence and the sequence containing a somatic mutation are each shown under their respective protein translations (single letter code of amino acids). Nucleotides and affected amino acids are shown in bold. Wild type refers to the reference sequence as found in the GenBank database (NM_002545.2 and AP 000843.3).

0261 Figure 19. OBCAM CpG island bisulfite sequencing.

0262 The nucleotide sequence of the predicted CpG island of OBCAM, corresponding to nucleotides 53134-54032 (GenBank Accession No. AC027631.4) is shown. The locations of the PCR primers designed to especially amplify a 529bp product of Methylated (M) or Nonmethylated (U) DNA modified by sodium bisulfite are shown in italics, and their sequences detailed below. The amplified PCR product is in bold, with the surrounding sequence in normal text. CpGs methylable nucleotides are underlined.

Primers:

0263 specific methylated DNA modified by sodium bisulfite:

OBCAM F1M: 5’-AGGCGTTTAGTGGAGGGGTACGGGC-3 ’

OBCAM R3M: 5’-TCCCGATACCGCCTCGAAACGAACG-3 ’

Specific non-methylated DNA modified by sodium bisulfite:

OBCAM-F1U: 5’-AGGTGTTTAGTGGAGGGGTATGGGT-3 ’

OBCAM R3U: 5’-TCCCAATACCACCTCAAAACAAACA-3 ’

0264 Figure 20. OBCAM CpG Island is methylated in ovarian tumors and not methylated in normal ovaries.

0265 Figure 21. The expression of OBCAM in SKNV3.3 almost completely suppresses tumorigenicity in nude mice.

0266 The SKNV3.3 progenitor cell line and two examples of cell lines that re-express SKNV3.3 transfected with OBCAM 1 were injected intraperitoneally (i.p.) in nude mice: 3 injections per cell line with one mouse injection. After 65 days, the mice were sacrificed and the peritoneal cavity tumors removed and photographed. The total amount of tumor present in the cavity i.p. It is markedly decreased in mice injected with SKNV3.3 cells transfected with OBCAM compared to SKNV3.3 progenitor cells. This Figure shows the total tumor removed from three mice injected with SKNV3.3 and d five mice injected with SKNV3.3 cells transfected with OBCAM: three from an OBCAM-transfected cell line and two from the second OBCAM-transfected cell line. No tumor was detectable in the third mouse injected with the second SKNV3.3 OBCAM-transfected cell line. Intraperitoneal propagation of tumors expressing OBCAM after transfection of OBCAM is also considerably reduced compared to progenitor SKNV3.3 cells.

Example 1: The role of OBCAM and NTM in the progression of ovarian epithelial cancer (EOC)

LOH 11q24-q25 DNA analysis of Blood / Ovarian Tumor DNA

0267 Fluorescently labeled polymorphic microsatellite markers selected from the 11q24-q25 chromosome region were amplified by PCR from DNAs extracted from whole ovarian tumors and also from blood or normal ovarian tissue as a normal coupled control. The markers used, in order of centromere at 11qter, were: 11cen-D11S910-D11S1320-D11S874-D11S4085-D11S969-11qter. PCR amplified products were separated on an ABI 310 Genetic Analyzer using GeneScan software (PE Biosystems). LOH is defined as an imbalance of 30% or greater difference between alleles in the tumor compared to normal tissues. The most prominent feature of LOH observed in D11S4085 is the integrity of LOH. This is unusual when one considers that the ovarian tumor DNA was extracted from the entire tumor rather than from microdissected tissue. Tumor tissue could normally be considered to contain a proportion of normal contaminating cells, eg, stromal cells. However, since the loss is so complete in this case, it can be inferred from the lack of normal contaminating tissue that the loss of the D11S4085 allele is an extremely early occurrence in the process of ovarian carcinogenesis.

Physical mapping of the OBCAM / NTM region

0268 OBCAM and Neurotrimin are the 11q24-q25 genes associated with LOH: After having identified the LOH regions, we wanted to identify below the genes of that region interrupted by LOH in ovarian cancer. In order to identify the BAC clones of the 11q24-q25 region containing the markers used in the LOH study, their corresponding nucleotide sequences were used for a BLAST search in the GenBank HTGS database at NCBI. Of the markers used, all except D11S969 identified BAC clones in the homology search of the HTGS database. The nucleotide sequences of the corresponding BAC clones were analyzed using the Nucleotide Identify X (NIX) algorithm at the UK Human Genome Mapping Project Resource Center (HGMP-RC). NIX allows several bioinformatics programs on a nucleotide sequence to be carried out simultaneously, such as BLAST searches against multiple databases, identifying nucleotide and protein translation homologies, carrying out exon predictions, island predictions CpG etc. NIX has allowed us to draw a contig map of the region incorporating overlapping BAC clones, identifying the genes in the region and their positions in relation to the markers in the study (Figure 1).

0269 NIX has identified that the two highly related genes OBCAM and Neurotrimin are the only genes present in the LOH region. The major LOH marker, D11S4085, is contained within OBCAM, while Neumtrimin encompasses two of the markers: D11S1320 and D11S874. OBCAM and NTM are highly related, sharing 80% and 76% identity of nucleotides and proteins, respectively. In the mouse, their respective counterparts are located close to each other in a region of chromosome 9 with the human chromosome 11q24-q25. It is likely, therefore, that the two genes have arisen as a result of a gene duplication event before the divergence of man and mouse. In a man, the two genes are arranged in a 3'-3 'orientation, with convergent transcriptional addresses (Figure 1).

OBCAM and NTM are expressed in the Superficial Ovarian Superficial Epithelium (OSE) but not in the Ovarian Cancer Cell Lines

0270 RNA was extracted from normal human cultured OSE (HOSE), preoperatively naked main HOSE, and from the entire normal normal ovary. RT-PCR showed that OBCAM and NTM are expressed in the preoperatively naked main epithelium, while only the expression of NTM and not OBCAM is detectable in cultured HOSE. The expression of both was detectable of the entire ovary, although OSE only comprises a minor component of the total organ.

0271 In contrast, the expression of one gene or another was not substantially evident in a panel of RNAs isolated from ovarian, breast, lung, colon and pancreatic cancer cell lines. By Northern blot, the expression was completely undetectable in all cell lines with two exceptions: CaOv3 (ovary) and WX330 (small cell lung cancer), in which NTM expression was easily detectable. Interestingly, the size of NTM transcript in CaOv3 is smaller than expected for full-length NTM. The RT-PCR analysis indicated that the transcript is smaller than expected due to a 5 'truncated mRNA, the exact extent of which is undetermined, and possibly arises as a 5' mutation result coupled with the use of an intronic alternative promoter .

CpG Islands OBCAM and NTM are Methylated in Cell Lines and Correlates with Lack of Expression

0272 The methylation status of the CpG islands of OBCAM and NTM was evaluated by MS PCR with primer pairs to detect methylated and unmethylated alleles in a range of ovarian, breast, lung, colon and of prostate. The results are presented in Tables 1 and 2. We tested the same range of cell lines for the level of OBCAM and NTM expression by RT-PCR and the results were compared with the methylation status determined in the MS PCR assay. A correlation was found between methylation of the respective CpG islands and the lack of detectable expression; Conversely, the lack of methylation correlates with gene expression. An exception to this correlation is the OAW28 ovarian cancer cell line, which despite no apparent methylation, shows no expression of any of the genes. This may be attributable to methylation not detectable by this particular test: it is either outside the region being amplified or alternatively among MS PCR primers since MSP only detects the presence / absence of methylation at the primer binding site in yes.

Table 1: NTM methylation status in cancer cell lines as determined by MS-PCR analysis.

FM = fully methylated Table 2: OBCAM methylation status in cancer cell lines by MS-PCR analysis. FM = fully methylated HM = semi-methylated FM / HM = fully or semi-methylated U = not methylated = not determined PE01 FM / HM PEO1 CDDP FM PE016 FM OVCAR 3 FM OVCAR 4 FM OSCAR5 FM OAW 42 FM A2780 FM PEA1 -PEA2 FM PE04 FM PE06 FM PE014 -OAW 28 U 59 MU CaOV3 U MCF7 FM ZR75.1 FM / HM T47D HM HT-29 FM HRT-18 FM HCT-15 FM SW48 FM DU145 FM

HM = semi-methylated

U = not methylated

PE016

FM

PEA1

FM

PEA2

FM

OVCAR 4

FM

OVCAR 5

FM

OAW 42

FM

A2780

FM

SKOV3

FM

OVCAR 3

HM

PE01

HM

PEO1 CDDP

HM

PE04

HM

PE06

HM

PE014

HM

PE023

HM

OAW28

OR

59 m

OR

CaOV3

OR

MDA.MB.2

FM

31

ZR75.1

FM

MCF7

FM

T47D

HM

LoVo

FM

HT-29

FM

HRT-18

FM

HCT-15

FM

SW48

FM

DU145

FM

PC-3

FM

LNCaP

HM

PANC1

FM

HELA

FM

K562

HM

FATO

 OR

WX330

OR

HL60

OR

PC-3 FM / HM

LNCaP HM

HELA FM

K562 HM

PANC1 HM

FATO U

WX330 U

HL60 U

OBCAM and NTM are Methylated in Ovarian Major Tumors

0273 We performed MS PCR to detect methylated and non-methylated alleles for OBCAM and NTM in 13 corresponding ovarian pairs of tumor / normal (or blood) DNAs. A representative set of MSP assays in 13 blood / tumor pairs including control samples of methylated DNA (Intergen) and unmethylated DNA (HL60 cell line) are shown in Figure 3, and a summary of the results is presented in the Figure 6. We note that NTM frequently accompanies OBCAM methylation and both can be considered concordant. This is an agreement with the lack of survival association with LOH in OBCAM (D11S4085, which indicates that the inactivation of OBCAM is an early event in this process).

Materials and methods

Ovarian Cancer Matching Paired Blood Samples (normal) / Tumor

0274 DNA from 65 paired samples of blood (or normal ovarian tissue embedded in paraffin) and ovarian tumor embedded in paraffin was extracted using the QIAamp DNA minikit according to the manufacturer's protocol (QIAGEN).

Heterozygosity Loss Analysis

0275 PCR products were amplified from 6 fluorescently labeled polymorphic microsatellite markers of the 11q24-q25 region from the normal / tumor paired ovarian DNA panel: Cen-D11S910-2D11S1320-D11S874-D11S4085-D11S969-11qter PCR products were separated and analyzed in an ABI 310 Genetic Analyzer using Genescan software (PE Biosystems).

Bioinformatic Analysis of the 11q24-q25 Region of the Human Chromosome:

0276 BAC clones containing the six polymorphic 11q24-q25 markers used to detect LOH were identified from the High Performance Genomic Sequences (HTGS) database in the GenBank by BLAST search with marker sequences. The sequences of identified BAC clones were then analyzed using the Nucleotide Identify X (NIX) algorithm (Resource Center of the Human Genome Mapping Project, Hinxton, UK). In this way, a contig BAC map of the region detailing positions of known genes in relation to microsatellite markers was mounted.

Reverse PCR Transcriptase (RT-PCR)

0277 Total RNA extraction from cell lines was carried out using TRI Reagent (Sigma, Dorset, United Kingdom). The first cDNA chain was prepared from 1 µg DNaseI-treated using a first strand DNA synthesis kit (Roche, United Kingdom), and 2 µl aliquots then used as template in 25 µl of PCR reactions. Alternatively, for smaller cell numbers, total RNA treated with DnaseI was prepared using Absolutely RNA Miniprep spin (Stratagene) columns and the first DNA strand was prepared as described.

Tissue Expression

0278 Northern Blots of Multiple Tissue (Human I and Human II) and cDNA panels of Multiple Tissue (Human I and Human II) were purchased from BD Clontech, Basingstoke, United Kingdom. MTNs were hybridized with full length OBCAM and NTM cDNA probes amplified by PCR, using ExpressHyb buffer (BD Clontech) as recommended. The blots were re-hybridized with a probe of c -ntr0l of -actin (BD Clontech). MTC panels were analyzed by PCR with pairs of OBCAM and NTM primers designed to amplify full-length cDNA products.

Specific Methylation PCR (MS PCR)

0279 genomic DNAs isolated from the paired normal / tumor ovarian pairs (above) and cell line panels (ovarian, breast, lung, colon and pancreatic cancer) were modified by bisulfite treatment using the CpG Modification Kit (Intergen) according to the recommended protocol. Methylated control DNA was purchased from Intergen. Bisulfite-modified DNA was amplified by PCR with primer pairs that specifically recognize methylated and non-methylated alleles, respectively, of the CpG islands of human NTM and OBCAM. The primer pairs and amplification conditions were as follows.

0280 In order to determine the extent of CpG island methylation in the CpG islands of OBCAM and NTM, bisulfite treated DNA amplified by methylation-specific PCR was subcloned into the cloning vector pGEM-T Easy TA (Promega). Six subclones corresponding to each PCR product were sequenced using Big Dye chemistry (PE BioSystems) following standard methods.

Azacitidine and TSA Re-expression

0281 In re-expression demethylation experiments, 5x106 cells (MDAMB23.1 for OBCAM; MDAMB23.1 and T47D for neurotrimin) were seeded and allowed to adhere for 24 hours. The cells were incubated in the presence of 10 µM azacitidine (Sigma) and 0.3 µM TSA was added for the final 24 hours of the 4 days. The cells were harvested, the isolated RNA, the first synthesized cDNA chain and the RT-PCR reactions carried out with OBCAM, NTM and actin PCR primers.

OBCAM transfection in SKOV-3

0282 The total coding sequence of human OBCAM plus the Kozak consensus sequence and the 3'LTTR overlay were amplified by PCR from RNA of the normal human ovary surface epithelium and subcloned into the cloning vector TA pGEM-T Easy (Promega ). The pair of PCR primers used for amplification were:

OPCML F1: 5’-AGTTGTGGCTGTCGAGAATG-3 ’

nucs 34-53

OPCML R1: 5’-TCAGAGGACCTAGGATTTCT-3 ’

Nucs 1110-1091

Nucleotide numbering of GenBank with Access NM_002545 mRNA (Reference Sequence NM_002454.2). The OBCAM insert was then cleaved with NotI and re-subcloned into the mammalian expression vector Zeo ADNpc3 digested with NotI.1 (Invitrogen). The sequence of the insert was then verified before use in transfection. Plasmid DNAs corresponding to OBCAM sense and antisense constructs and the parental vector were prepared by standard methods (QIAGEN) and digested with Pvul. 1µg and 2µg of constructs linearized by Pwl and the vector were transfected into the SKOV-3 cell line resistant to the clonal neomycin SKNV3.3 in the presence of lipofectin.

Selection on Zeomycin (Invitrogen)

0283 40 hours after transfection, the cells were divided 1: 6 and cultured in the presence of the Zeomycin antibiotic selection. Individual colonies were then selected for analysis 3 weeks after the imposition of antibiotic selection.

0284 Transfections indicate that OBCAM anti-sense transfectants and control vectors grow more virulently than OBCAM sense transfectants suggesting that there is a functionally suppressive effect on growth.

Discussion

0285 We have performed a more refined LOH analysis in EOC of the 11q24-q25 region in 65 normal samples / matched tumors and identified a high rate of 56% of LOH in the OBCAM gene in the D11S4085 marker. In addition, 40% LOH was also detected within the homologous gene, neurotrimin (NTM or NTM), in markers D11S1320 and D11S874. The analysis of clinical-pathological parameters does not show any association of LOH with the adverse survival of the patient, indicating that the loss of these genes is an early event in ovarian carcinogenesis. It also proves that the survival gene is not detected in our previous LHO studies. Specific DNA methylation PCR isolated from 43 pairs of paired normal / ovarian tumors and 6 tumor-only DNAs has identified 76% CpG island methylation rate for both OBCAM and NTM, with 86% agreement between genes. Bisulfite sequencing confirmed the presence of extensive methylation in the CpG islands of both genes. Of 12 EOC lines, OBCAM was fully methylated in 75% of the cell lines, 0% were partially methylated and 25% were not methylated. For HNT / NTM, in 13 EOC lines, 54% were fully methylated, 23% partially methylated, 23% unmethylated. Re-expression of both genes was achieved by treatment of azacitidine with Trichostatin A (TSA), providing conclusive evidence that CpG island methylation is the mechanism that underlies the lack of expression of these genes. It is evident from the MS-PCR studies that CpG OBCAM island methylation meets that of NTM, and may be a prerequisite for NTM methylation to occur. This combined with the LOH data suggests that of the two genes, OBCAM is the most important in the early events of the disease. Consequently, we have transfected OBCAM into a clonal derivative of the SKOV-3 ovarian cancer cell line, under the control of the CMV promoter, and shows the following effects. Three observations suggest NTM / OBCAM function as suppressors. First of all there is evidence that some SKOV3 sense clones transfected with NTM demonstrate tumor suppressiveness compared to antisense clones, but the clonal heterogeneity of SKOV3 makes these data difficult to interpret. On the other hand, there was some evidence that morphologically there was contact inhibition associated with the SKOV3 sense clones transfected with NTM. Finally, we have noticed that the clonal derivative of SKOV3 (SKNV3.3) transfected with OBCAM antisense and control vector demonstrates faster growth compared to OBCAM sense transfected in the same clone line SKOV3. There is an apparent 50% reduction in growth rates of transfected OBCAM SKOV3 clones compared to OBCAM SKOV3 transfected clonal cell lines. Correlating the LOH and methylation studies, we provide evidence of the existence of two inactivation coincidences according to the classic Knudsen mechanism of two inactivation coincidences of the tumor suppressor gene (Knudsen AG, 1971 Proc Natl Acad Sci 68 (4) p820-823) . He also highlighted tumor samples in which only one mechanism of inactivation (either LOTH or methylation) was presented, allowing us to objectify these samples for the detection of mutations in the OBCAM gene.

0286 This is the first description of the involvement of the IgLON family, and in particular of OBCAM and Neurotrimin, in the development of any form of cancer, or indeed, in any form of human disease.

Example 2: SKNV3.3 is methylated for OBCAM and does not express OBCAM

0287 The SKNV3.3 cell line is a neomycin-resistant clonal derivative of the SKOV3 ovarian cancer cell line. We have shown by quantitative RT-PCR that does not express OBCAM and by MS-PCR that the CpG island of OBCAM is methylated. On the other hand, demethylation after in vitro exposure of SKVN3.3 to 5’-aza-2’-deoxycytidine results in re-expression of OBCAM. He was therefore selected for the functional studies of OBCAM.

0288 The CpG island of OBCAM is fully methylated as determined in the MS-PCR assay. Consequently, the OBCAM expression in SKNV3.3 is repressed by this epigenetic mechanism. In order to prove that CpG island methylation is the mechanism of repression of OBCAM expression, SKNV3.3 cells were exposed to 5-aza-2'-deoxicitidine and tested for re-expression of OBCAM by RT-PCR, Southern blot and hybridization with an OBCAM specific probe.

0289 1x105 SKNV3.3 cells (Passage 6) were seeded in a 25cm3 tissue culture flask in 10ml of medium. The medium was replaced after 24 hours and 5’-aza 2’-deoxycytidine (Sigma A3656) was added to give a final concentration of 20µM. A duplicated cell flask did not receive azacitidine (control) exposure. After 4 days the cells of both flasks were harvested and total RNA treated with DNasel was prepared using the Absolutely RNA Miniprep kit (Stratagene). The first cDNA chain was synthesized using the cDNA First Chain Synthesis Kit (Roche) and 2 µl aliquots of cDNA were used per RT-PCR reaction.

0290 RT-PCR actin was carried out to confirm the integrity of the first cDNA chain from control SKVN3.3 cells and treated with azacitidine. Equal aliquots of each Actin PCR reaction separated on agarose gel confirmed the integrity of both samples and the equal concentration of cDNA per sample.

OBCAM RT-PCR reaction:

OPCML F4 / R6 RT-PCR (product 474bp) performed in a 25µl reaction.

PCR primers used are (nucleotide numbering corresponds to GenBank Accession Number NM_002545)

OPCML F4: 5’-TACCATAGATGACCGGGTAA-3 ’

nucs: 221-240

OPCML R6: 5’-TTCCGCACATCGGGCGCAGC-3 ’

nucs: 694-675

0292 The products of the OBCAM PCR reactions were separated in size on a 2% agarose gel, Southern blot overnight on MSI nylon membrane, and the DNA then UV crosslinked to the membrane.

0293 The OBCAM Exon2 F2 / OPCML R6 PCR product, purified through a QIAquick PCR purification column (QIAGEN), was labeled with a32P-dCTP PCR. The PCR primers were the following (nucleotide numbering corresponding to GenBank Accession Number NM_002545):

OBCAM Exon 2 F2: 5’-ATAGACCCTCGTGTGATCAT

3 ’nucs 300-319

OPCML R6: 5’-TTCCGCACATCGGGCGCAGC-3 ’

nucs: 694-675

0294 After overnight hybridization, the blot was washed to remove the unbound probe, and the blot was exposed to X-ray film for 1 hour at -70 ° C and then developed.

0295 Re-expression of OBCAM is clearly evident in SKNV3.3 cells exposed to 5'-aza 2'-20'M deoxycytidine for 4 days. In contrast, no expression of OBCAM is detectable in control SKNV3.3 cells after 4 days without the 5’-aza 2’-deoxycytidine treatment (Fig. 13). The actin PCR product was amplified at the same intensity of both control and treated cells, confirming the integrity of the isolated RNA and synthesized first strand cDNA.

Example 3: Functional Studies of OBCAM in SKVN3.3 (clonal derivative of SKOV3)

OBCAM transfection in SKNV3.3

0296 The complete coding sequence of human OBCAM plus the Kozak consensus sequence and the 3'UTR overlay was amplified with PCR from RNA of normal human ovarian surface epithelium and subcloned into the cloning vector TA p-GEM-T Easy . The pair of PCR primers used to amplify nucleotides 34-1110 (Reference Sequence NM_002454.2) was:

OPCML F1: 5’-AGTTGTGGCTGTCGAGAATG-3 ’

nucs 34-53

OPCML R1: 5’-TCAGAGGACCTAGGATTTCT-3 ’

nucs 1110-1091

amplifying a 1077bp PCR product. The OBCAM insert was then excised with Notl and resubcloned into the mammalian expression vector ADNpc3.1 Zeo (zeomycin-resistant) digested with NotI (Invitrogen) in both sense and antisense orientations. The insert sequence was then verified before use in transfection. Plasmid DNAs corresponding to the OBCAM sense and antisense constructs and the parental vector were prepared by standard methods (QIAGEN) and digested with Pvul.

0297 1µg and 2µg of linearized constructs with Pvul and the vector were transfected into the SKOV-3 clonal derived cell line labeled with neomycin, SKNV3.3.

0298 Cell lines were maintained in RPMI 1640 with 10% Fetal Calf Serum heat inactivated (FCS) and penicillin (100 units / ml), streptomycin (100 µg / ml) and G418 and Zeocin (Invitrogen) as appropriate.

0299 2x105 SKNV3.3 cells were seeded by 60 mm plate in 4 ml of medium. 24 hours later when they were 50% confluent, the cells were transfected separately with 2µg of linearized constructs: OBCAM sense or antisense constructs ADNpc3.1 zeo, or ADNpc3.1 zeo vector that does not contain insert, using LIPOFECTIN reagent (Life Technologies GIRBO BRL ) according to the manufacturers protocols. Forty-eight hours after transfection, each cell plate was divided one in six and the transfected cells were then selected with 250 µg / ml zeocin. At three weeks the colonies were collected in 24-well plates and established clonal cell lines.

OBCAM suppresses growth in vitro:

0300 Transfection of OBCAM into SKNV3.3 cells results in suppressed growth compared to SKNV3.3 control cells in vitro (figure not shown).

OBCAM Suppresses Growth and Propagation of Tumor in vivo

0301 SKNV3.3 control phase control cells and transfected OBCAM cells were harvested and 5 x 106 cells were injected either intraperitoneally (i.p.) or subcutaneously (s.c.) on the flanks of nude mice, with 6 s.c. injections. and 3 i.p. by cell line. Measurements of tumor size s.c. They were taken every week for 4 weeks. The mice that received the cells via injection i.p. They were sacrificed 65 days after the injection and the tumors removed from the peritoneal cavity, weighed and photographed.

0302 Transfection of OBCAM in SKNV3.3 results in markedly suppressed growth of the subcutaneous tumor (Fig. 14) by comparing SKNV3.3 cells transfected with the OBCAM construct of 'sense' expression (sense transfectants) and SKNV3.3 control cells ( Controls)

0303 Transfection of OBCAM in SKNV3.3 almost completely eliminated tumor growth and intraperitoneal propagation, compared to tumor growth and i.p. observed with the parental SKNV3.3 cells (Figure 21; see Example 8).

OBCAM Transfection Improves Cell Aggregation:

0304 The control phase SKNV3.3 control cells and transfected by OBCAM were trypsinized and resuspended in medium containing 10% FCS. 1 x 106 cells were resuspended in 1ml of medium and passed through a 21 gauge needle to ensure the creation of a suspension of individual cells. The cell suspensions were incubated in 5% CO2 at 37 ° C. At defined times, aliquots were removed with a wide-hole pipette, and the individual cells were counted with a hemocytometer.

0305 OBCAM transfection in SKNV3.3 (construct expressing meaning) results in an improved cell aggregation rate compared to that observed for SKNV3.3 control cells (Fig. 15). Transfection of the OBCAM construct that expresses antisense results in a reduced rate of cell aggregation compared to parental SKNV3.3.

Example 4: Exon Structure of the Human OBCAM Gene

0306 The exon structure of the human OBCAM gene was determined in a bioinformatic analysis. The human OBCAM mRNA reference sequence NM_002545.2 was compared with the Human Genome Project sequence available in the GenBank HTGS database using a BLAST2 homology search. The comparison identified that OBCAM consists of at least 7 exons. The exon structure derived with the location of intron-exon boundaries is shown in Fig. 16. The exon sequence is highlighted in yellow and the sequence of introns flanking the exons is in normal text. Nucleotide numbers refer to those corresponding to the GenBank database (access numbers for which they are given). The nucleotide sequence for Exon 1 is incomplete in the area that covers the exon / intron 1 boundary due to the lack of Sequence available from the Human Genome Project in the GenBank with Access AC027631.4.

Example 5: Detection of OBCAM Mutation by Single Chain Conformation Polymorphism Electrophoresis (SSCPE)

0307 After the prediction of the bioinformatic analysis of the structure of the human OBCAM gene, primers were designed to amplify the 7 identified exons of the OBCAM gene. Exon 2 was analyzed with two sets of overlapping primers due to the limitation of PCR product size useful for SSCPE analysis. DNA samples used in the SSCPE analysis of the ovarian tumor were extracted and mated to ovarian tumors of normal tissue file embedded in paraffin, and ovarian cancer cell lines.

0308 The exon-specific OBCAM primers used were as follows. The sizes of the PCR product are given in parentheses.

0309 In Fig. 16, the locations of sense and antisense primers for SSCPE are highlighted in single and double underlines, respectively. Intronic and exonic sequences are not highlighted and bold, respectively.

Exon 1 (188bp)

OBCAM EX1 F3: 5’-GACCAGGACTGTGCGGCTGC-3 ’

nucs 54514-54533 AC027631.4

OPCML R3: 5’-CGTCACGTTGTCCATAGCTT-3 ’

nucs: 188-169 NM_002545.2

Exon 2/1 (175bp): (GenBank nucleotide numbering AC012234.6)

OBCAM EX2 F1: 5’-CACCACTCCCTGCCTCACTG-3 ’

nucs 75226-75245

OBCAM EX2 R1: 5’-CATCCACATTTTGGATCATG-3 ’

nucs 75400-75381

Exon 2/2 (180bp): (GenBank nucleotide numbering AC012234.6)

OBCAM EX2 F2: 5’-ATAGACCCTCGTGTGATCAT-3 nucs 75331-75350 OBCAM EX2 R2: 5’-TGGCAACCCCAGATCCAGCT-3 ’ nucs 75510-75491 Exon 3 (179bp): (GenBank AP000843.3 nucleotide numbering) OBCAM EX3 F1: 5’-CAGGTATTTCTTCTATCCTG-3 ’ nucs 37032-37051 OBCAM EX3 R1: 5’-GTCCTCCAGGTCAGCACCTT-3 ’ nucs 37210-37191 Exon 4 (214bp): (GenBank nucleotide numbering AP000843.3) OBCAM EX4 F1: 5’-TGGTTACACAGTTTCCTGAT-3 ’ nucs 2881-2900 OBCAM EX4 R1: 5’-AGAACCCCCTGGCTGCAGGT-3 ’ nucs 3094-3075 Exon 5 (195bp): (GenBank AP000843.3 nucleotide numbering) OBCAM EX5 F1: 5’-GTGCGTGCATGCCTGTGCAT-3 ’ nucs 3466-3485 OBCAM EX5 R1: 5’-CAGAACTGTCCAGGTGTCAT-3 ’ nucs 3660-3641 Exon 6 (198bp): (GenBank AP000843.3 nucleotide numbering) OBCAM EX6 F1: 5’-TAGCAATGTCTTCCCTCTTG-3 ’ nucs 4028-4047 OBCAM EX6 R1: 5’-GCATCCAGGCTTCCAGCACT3 ’ nucs 4225-4206 Exon 7 (176bp): (GenBank AP000843.3 nucleotide numbering) OBCAM EX7 F1: 5’-TCCTTGGGTGTATGCTAATG-3 ’ nucs 19945-19964 OBCAM EX7 R1: 5’-GCGTTGCTCAGAGGACCTAG-3 ’ nucs 20120-20101 0310 SSCPE has been carried out for each OBCAM exon in normal / tumor DNAs, paired with cancer

ovary, tumor DNAs and cell lines. Maintaining the high rate of LOH and methylation of CpG islands observed

for OBCAM, the expected frequency of somatic mutation is low. A somatic mutation of wrong sense that

It has been detected by SSCPE and sequencing is described below.

Somatic OBCAM Mutation in the PEO Ovarian Cancer Cell Line Series

0311 SSCPE of PCR products OBCAM Exon2 F1 / R1 identified a ‘dsplantation in the DNA of a

series of cell lines derived from a patient with ovarian cancer during the course of his illness. PEO1

represents a platinum sensitive ovarian cancer cell line derived from the patient early in the course

Of his sickness. The PEO4 platinum-resistant cell line was derived from the same patient at relapse after

of chemotherapy with cisplatin. The PEO1CDDP cell line was derived from PEO1 by in vitro exposure to cisplatin and represents an in vitro model of platinum resistance. Fibroblast DNA, which represents DNA

normal, it was isolated from the patient at the same time that the PEO4 cell line was established (PEO4 Fibroblasts).

0312 The amplified Exon2 F1 / R1 PCR products of PEO1, PEO1CDDP, PEO4 and PEO4 Fibroblasts were sequenced with the same primers used in the original PCR. The sequence tracking files (Fig. 17) clearly indicate a heterozygous peak corresponding to the presence of a nucleotide C and a nucleotide G at position 334 (GenBank Accession Number NM_002545, shown in Figure 7) in the products PCR of PEO1, PEO1CDDP, and PEO4. Position 334 (NM_002545) is homozygous for a C in the PEO4 Fibroblast PCR product (marked by *). The wild type sequence (nucleotide sequence reference NM_002545) contains a C residue in this position, altering a CCA codon to CGA. Translation (using ExPasy) of the nucleotide sequence encompassing this position predicts that the corresponding wild-type amino acid proline (P) is altered to an arginine (R), corresponding to amino acid residue 95 of the immature OBCAM protein sequence ( Fig. 18). It is believed that this residue is located in the first immunoglobulin domain of OBCAM (Figure 7 and GenBank Accession Number NP_002536).

0313 PEO4 fibroblasts are homozygous wild type (proline) while PEO1, PEO1CDDP, and PEO4 are heterozygous wild-type / missense mutants (proline / arginine). The substitution of an arginine residue for a proline in this position can lead to an altered structural confirmation of OBCAM, and therefore an altered OBCAM function.

0314 As all cell lines derived during the temporary course of this patient's disease contain this missense mutation, we can assume that the mutation was an early event in the course of his illness. Since PEO4 Fibroblasts, corresponding to normal DNA, are wild-type, this alteration is a somatic event.

0315 This is the first somatic mutation identified for OBCAM in cancer, including ovarian cancer.

Example 6: OBCAM is not methylated in the normal human ovary

0316 We extracted DNA and RNA from 5 samples of normal human ovary. OBCAM MS-PCR of these samples shows that the CpG island of OBCAM is not methylated. The 600bp product amplified by MS-PCR contains 58 CpGs. Sequencing along the MS-PCR product of OBCAM from these normal ovaries showed no evidence of methylated CpGs in the product.

0317 In contrast, sequencing of the MS-PCR product from the ovarian cancer cell lines and major tumors that are methylated in the MS-PCR assay show wide methylation throughout the region of the amplified CpG island.

Example 7: CpG Island OBCAM is methylated in ovarian tumors and not methylated in normal ovary.

0318 The DNAs of two examples of ovarian tumors and two examples of normal ovaries were chemically modified by bisulfite treatment. Specific methylation PCR was then carried out with primers designed to discriminate bispulite modified DNA from CBC Island of OBCAM Methylated (M) and Unmethylated (U). A specific methylated or non-methylated 529bp PCR product was specifically amplified from DNAs from the normal ovarian or ovarian tumor, respectively, using the primers detailed above (CpG Island Sequencing OBCAM). The amplified PCR product corresponds to nucleotides 25-553 of Fig. 19 (Bispulite Sequencing Island CpG OBCAM). The PCR products were then subcloned into pGEM-T Easy and individual subclones, representing individual alleles, were then sequenced and the presence or absence of methylated C nucleotides in CpGs was noted. Fig. 20 depicts the extent of methylated CpG Cs present in examples of normal ovarian and ovarian tumors. Nucleotide numbering at this location of CPG Cs as shown in Fig. 19, and the CpG number is the sequential numbering of CpGs located within 526bp of the sequenced OBCAM CpG island. The sequencing results of six alleles are shown for each of the two ovarian tumor examples and two alleles for each of the normal ovarian examples. The filled black square represents a methylated CpG, the empty square represents an unmethylated CpG, and the square containing vertical lines represents cases where the methylation status of the CpG was not determined. Fig. 20 shows that the CpG island is widely methylated in ovarian tumors and not methylated in normal ovaries.

Claims (49)

1. A method of diagnosing cancer in a patient comprising the steps of contacting a sample containing the patient's nucleic acid with

(to)

a nucleic acid that selectively hybridizes to the OBCAM gene, or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the OBCAM cDNA, or a mutant allele thereof, or its complements; or

(b)

a nucleic acid that selectively hybridizes to the NTM gene, or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the NTM cDNA, or a mutant allele thereof, or its complements; or

(C)

both (a) and (b).

2. A method of predicting the relative perspectives of a particular cancer outcome in a patient comprising the steps of contacting a sample containing the patient's nucleic acid with

(to)

a nucleic acid that selectively hybridizes to the OBCAM gene, or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the OBCAM cDNA, or a mutant allele thereof, or its complements; or

(b)

a nucleic acid that selectively hybridizes to the NTM gene, or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the NTM cDNA, or a mutant allele thereof, or its complements; or

(C)

both (a) and (b).

3.

A method for determining the progression of a cancerous disease in a patient comprising the steps of contacting a sample containing the patient's nucleic acid where the patient's OBCAM gene has been lost or inactivated with a nucleic acid that selectively hybridizes to the NTM gene or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the NTM cDNA, or a mutant allele thereof, or its complements.

Four.

A method according to any one of Claims 1 to 3 in which it is determined whether the nucleotide corresponding to nucleotide 334 as numbered in Figure 7 in the patient's nucleic acid is the same as in Figure 7 or not.

5.

A method according to Claim 4 in which the determination involves a nucleic acid according to Claim 40.

6.

A method of diagnosing cancer in a patient comprising the steps of determining the degree of methylation of the OBCAM or NTM gene in a sample containing the patient's OBCAM or NTM gene; and compare the level of methylation of the OBCAM or NTM gene of the patient sample with the level of methylation in a control sample; in which if the patient sample has a higher degree of methylation of the OBCAM or NTM gene compared to the control sample this is indicative of cancer.

7.

A method of predicting the relative perspective of a particular outcome of a cancer patient comprising the steps of

(i)

Determine the degree of methylation of the OBCAM or NTM gene in a sample containing the patient's OBCAM or NTM gene:

(ii)

compare the level of methylation of the OBCAM or NTM gene of the patient sample with the level of methylation in a control sample; Y

(iii) if the patient sample has a higher degree of methylation of the OBCAM or NTM gene compared to the control sample, this is indicative of a lower probability of a successful outcome.

8.

A method for determining the progression of a cancerous disease in a patient comprising the steps of

(i)

determine the degree of methylation of the NTM gene in a patient sample containing the patient's NTM gene;

(ii)

compare the level of methylation of the NTM gene of the patient sample with the level of methylation in a control sample;

and if the level of NTM methylation is increased compared to the control sample this is indicative of a progression in the disease.

9.

A method according to any one of Claims 6 to 8 in which the methylation of the CpG island of OBCAM or NTM is analyzed.

10.

A method according to any of the preceding claims wherein the cancer or tumor is an ovarian cancer or tumor or a colon cancer or tumor.

eleven.

A method according to any one of Claims 1-2 wherein the nucleic acid is contacted with a nucleic acid according to option (a) and the cancer is ovarian cancer.

12.

A method according to any one of Claims 1-2 wherein the nucleic acid is contacted with a nucleic acid according to option (b) and the cancer is colorectal cancer.

13.

A method according to any of the preceding claims wherein the sample is a sample of the tissue in which cancer is suspected or in which the cancer can be or has been found.

14.

A method according to claims 1 to 11 wherein the sample is an ovarian sample and the cancer is ovarian cancer.

fifteen.

A method according to any one of Claims 1 to 9, 12 or 13 wherein the sample is a colon sample and the cancer is colorectal cancer.

16.

A method according to any of the preceding claims wherein the nucleic acid that selectively hybridizes to the DNA of said OBCAM or NTM gene or said OBCAM or NTM cDNA sequence, or a mutant allele thereof, or its complements, further comprises a detectable label.

17.

A method according to any of the preceding claims wherein the nucleic acid that selectively hybridizes as said is single stranded.

18.

A method according to any of the preceding claims wherein the nucleic acid that selectively hybridizes as said has less than 10,000 base pairs when the nucleic acid is double stranded or base when the nucleic acid is single stranded.

19.

A method according to any of the preceding claims wherein the nucleic acid that selectively hybridizes as said has less than 1000 base pairs when the nucleic acid is double stranded or base when the nucleic acid is single stranded.

twenty.

A method according to any of the preceding claims wherein the nucleic acid that hybridizes as said has 10 to 100 base pairs when the nucleic acid is double stranded or base when the nucleic acid is single stranded.

twenty-one.

A method according to any of the preceding claims wherein the nucleic acid that hybridizes as said has 15 to 30 base pairs when the nucleic acid is double stranded or base when the nucleic acid is single stranded.

22

A method according to any one of Claims 1 or 2 wherein the nucleic acid that hybridizes as said comprises a portion of OBCAM cDNA.

2. 3.

A method according to any one of Claims 1 or 2 wherein the nucleic acid that hybridizes as said comprises a portion of NTM cDNA.

24.

A method according to Claim 22 or 23 wherein the portion is a single chain portion.

25.

A method according to claim 24 wherein said portion is capable of amplifying a portion of the OBCAM gene

or the NTM gene or the OBCAM cDNA or mRNA or the NTM cDNA or mRNA in a nucleic acid amplification reaction. 26. A method of diagnosing cancer in a patient that comprises the steps of

(i)

determine in a sample containing patient derived protein

(to)

the amount or activity of the OBCAM polypeptide; or

(b)

the amount or activity of the NTM polypeptide; or

(C)

both (a) and (b); Y

(ii)

compare:

(to)

the amount or activity of the OBCAM polypeptide of the patient sample with the respective amount or activity of the OBCAM polypeptide of a control sample; or

(b)

the amount or activity of the NTM polypeptide of the patient sample with the respective amount or activity of the NTM polypeptide of a control sample; or

(c) both (a) and (b); in which if the patient sample has a lower activity or amount of the OBCAM and / or NTM polypeptide compared to the control sample, this is indicative of cancer. 27. A method of predicting the relative perspectives of a particular cancer outcome in a patient that comprises the steps of

(i)

determine in a sample containing patient derived protein

(to)

the amount or activity of the OBCAM polypeptide; or

(b)

the amount or activity of the NTM polypeptide; or

(C)

both (a) and (b); Y

(ii)

compare:

(to)

the amount or activity of the OBCAM polypeptide of the patient sample with the respective amount or activity of the OBCAM polypeptide of a control sample; or

(b)

the amount or activity of the NTM polypeptide of the patient sample with the respective amount or activity of the NTM polypeptide of a control sample; or

(C)

both (a) and (b); where

If the patient sample has a lower activity or amount of the OBCAM and / or NTM polypeptide compared to the control sample, this is indicative of a less favorable result. 28. A method of diagnosing cancer in a patient who understands the steps of

(i)

determine in a sample containing patient derived protein

(to)

the sequence of the OBCAM polypeptide; or

(b)

the sequence of the NTM polypeptide; or

(C)

both (a) and (b); Y

(ii)

determine the presence of differences between

(to)

 said OBCAM polypeptide sequence and the wild type OBCAM polypeptide sequence; or

(b)

said sequence of the NTM polypeptide and the sequence of the wild-type NTM polypeptide; or

(C)

both (a) and (b); where

If the sequence of the OBCAM and / or NTM polypeptide in the patient sample is different from the sequence of the wild-type OBCAM or NTM polypeptide, respectively, this is indicative of cancer. 29. A method of predicting the relative perspectives of a particular cancer outcome in a patient that comprises the steps of

(i)

determine in a sample containing patient derived protein

(to)

the sequence of the OBCAM polypeptide; or

(b)

the sequence of the NTM polypeptide; or

(C)

both (a) and (b); Y

(ii)

determine the presence of differences between

(to)

said OBCAM polypeptide sequence and the wild type OBCAM polypeptide sequence; or

(b)

said sequence of the NTM polypeptide and the sequence of the wild-type NTM polypeptide; or

(C)

both (a) and (b); where

if the sequence of the OBCAM and / or NTM polypeptide in the patient sample is different from the sequence of the wild-type OBCAM or NTM polypeptide respectively, this is indicative of a less favorable result.

30

A method according to any one of Claims 26 to 29 wherein the cancer is ovarian cancer or colorectal cancer.

31.

A method according to any one of Claims 26 to 30 in which the sample is a sample of the tissue in which cancer is suspected or in which the cancer can be or has been found.

32

A method according to any one of Claims 26 to 30 wherein the sample is an ovarian sample and the cancer is ovarian cancer.

33.

A method according to any one of Claims 26 to 30 in which the sample is a colon sample and the cancer is colorectal cancer.

3. 4.

A method according to any one of Claims 26, 27 and 30 to 33 wherein the amount of the OBCAM polypeptide is determined using a molecule that selectively binds to the OBCAM polypeptide.

35

A method according to any one of Claims 26, 27 and 30 to 33 wherein the amount of the NTM polypeptide is determined using a molecule that selectively binds to the NTM polypeptide.

36.

A method according to Claim 34 or 35 wherein the molecule that selectively binds to the OBCAM or NTM polypeptide is an anti-OBCAM or anti-NTM antibody.

37.

A method according to Claim 36 wherein the OBCAM antibody reacts with a mutant OBCAM polypeptide, wherein said mutant OBCAM is a mutant found in a cancer cell, for example with an arginine in residue 95 as numbered in Figure 7 instead. of a proline, and / or wherein said mutant OBCAM comprises an inserted amino acid sequence shown in Figure 9, and

wherein said antibody does not react with the wild type OBCAM polypeptide.

38.

A method according to any one of Claims 34 to 37 in which the molecule that selectively binds to OBCAM or NTM comprises a detectable label.

39.

A method according to any one of Claims 26, 27 and 30 to 33 wherein the amount of the OBCAM or NTM polypeptide is determined by assay or detection of the activity of the OBCAM or NTM polypeptide.

40

The use of a nucleic acid that selectively hybridizes to:

(to)

the OBCAM gene, or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the OBCAM cDNA, or a mutant allele thereof, or its complements; or

(b)

the NTM gene, or a mutant allele thereof, or a nucleic acid that selectively hybridizes to the NTM cDNA, or a mutant allele thereof, or its complements; or

(C)

both (a) and (b)

in the manufacture of a reagent to diagnose cancer. 41. Use of a molecule that selectively binds to:

(to)

OBCAM polypeptide; or

(b)

NTM polypeptide

in the manufacture of a reagent for the diagnosis of cancer.

42

Use of a nucleic acid according to Claim 40 in a method for diagnosing cancer.

43

Use of a molecule that selectively binds to:

(to)

OBCAM polypeptide; or

(b)

NTM polypeptide

in a method of cancer diagnosis. 44. A use according to any one of claims 41 or 43 wherein the molecule selectively binds to an OBCAM polypeptide wherein the proline in residue 95 as numbered in Figure 7 is an arginine. 45. Use of a nucleic acid that selectively hybridizes to the OBCAM or NTM gene or a nucleic acid that selectively hybridizes to the OBCAM or NTM cDNA in the manufacture of a medicament for treating cancer.

46.

A nucleic acid that selectively hybridizes to the OBCAM or NTM gene or a nucleic acid that selectively hybridizes to the OBCAM or NTM cDNA for use in the treatment of cancer.

47

Use of the OBCAM or NTM polypeptide or a fusion thereof, or a nucleic acid molecule encoding said OBCAM or NTM polypeptide or fusion thereof, in the manufacture of a medicament for treating cancer.

10 48. An OBCAM or NTM polypeptide or a fusion thereof, or a nucleic acid molecule encoding said OBCAM or NTM polypeptide or fusion thereof, for use in treating cancer.

49.

A use according to Claim 40 wherein the gene or nucleic acid is a mutant allele of OBCAM with a guanine in nucleotide 334 as numbered in Figure 7 instead of a cytosine.

fifty.

A method according to any one of Claims 1 to 13 or 16 to 25 in which the sample is blood.