WO2007116417A1

WO2007116417A1 - Novel dna methylation markers useful for the diagnosis of neoplastic diseases

Info

Publication number: WO2007116417A1
Application number: PCT/IT2006/000237
Authority: WO
Inventors: Silvia Sabbioni; Elena Miotto
Original assignee: Silvia Sabbioni; Elena Miotto
Priority date: 2006-04-07
Filing date: 2006-04-07
Publication date: 2007-10-18

Abstract

Novel markers for neoplastic diseases in humans are described, as well as methods, oligonucleotides and kits for detecting thereof, useful for the diagnosis of solid tumours and haemetological neoplastic diseases, based on the analysis of the methylation status of the CpG island of a human gene selected from the group consisting of ALDH 1 A3, ARHGAP20, FLU , FOSL2, HES2, MAPI B, MDM2, NID1 , NID2, PNUTL2, PTGER3 and PTPRM.

Description

Novel DNA methylation markers useful for the diagnosis of neoplastic diseases

The present invention relates to an in vitro method for the diagnosis of neoplastic diseases in humans through the analysis of the methylation state of the DNA of genes which are useful as tumour markers, as well as to nucleic acids and oligonucleotides useful in such method and to kits comprising such oligonucleotides.

DNA methylation, that is to say the bonding of methyl groups (-CH₃) to cytosines which are found in the DNA within the context of the CpG dinucleotide (C = cytosine; G = guanine; p = monophosphate), is one of the commonest and best known epigenetic modifications which DNA may undergo in mammalian cells. Once the methylation pattern is established, the original cell transmits this pattern to all cells deriving therefrom.

DNA methylation is known to have a regulatory function, in particular of gene transcription. It has in fact been observed that the gene promoters often contain regions which are particularly rich in CpG dinucleotides, known as CpG islands, the cytosines of which are generally unmethylated when the gene is transcriptionally active.

It is also known that neoplastic diseases are characterised by the altered functionality of specific genes involved in the normal physiology of the cell, and in the scientific literature several oncosuppressor genes which are activated by the abnormal methylation of their own promoter are described.

These factors gave rise to the idea that the analysis of the methylation status of specific genes involved in the neoplastic transformation process could be a useful tool for the early diagnosis of neoplastic diseases in humans. In fact, both the scientific and the patent literature report several studies into the methylation status of genes involved in the development of human tumours and on the methods which for thereof.

However, a need still remains to identify new markers for neoplastic diseases which, on the one hand, could make it possible to expand the knowledge currently available on the molecular mechanisms underlying carcinogenesis and, on the other hand, could help to provide new methods for the early diagnosis of human tumours. The present invention is based on studies carried out by the inventors which lead to the identification of a group of genes (listed in table 1 ) which were shown to be useful as DNA methylation markers. Specifically, the inventors have found that the methylation status of the CpG islands of the genes listed in table 1 is altered in human tumours and that the analysis of the methylation status of the CpG islands of such genes is a useful marker for the early diagnosis of various types of human neoplastic diseases, comprising solid tumours and haematological neoplastic diseases.

More specifically, the inventors have found that the CpG islands located at the 5' end of the genes listed in table 1 have an higher methylation rate in cells derived from human neoplastic diseases than in normal controls which are unmethylated or have a low methylation rate.

The methylation status of such CpG islands, whose genomic location and extension is specificed in table 1 , thus represent a useful marker for solid tumours and neoplastic haematological diseases in humans.

The CpG islands of the genes listed in table 1 are all located at the 5¹ end of such genes and they cover regions varying from about 2.6 kb to about 8.0 kb, which include the 5' region, the first exon and a portion of the first intron of each gene.

Accordingly, the present invention provides a method for the diagnosis of solid tumours and haematological neoplastic diseases, characterised in that it comprises, in a sample of genomic DNA, the step of analysing the methylation status of the CpG island of a gene selected from the group consisting of the human genes listed in table 1 , the genomic location of the CpG island of each of such genes being as shown in table 1, the methylation od said CpG island being indicative of neoplastic disease.

In an embodiment, the method of the invention comprises the step of analysing the methylation status of the nucleic acid sequence of the ALDH13A gene comprised between the nucleotide positions corresponding to nucleotides 86.701 and 89.700 of the Genbank sequence AC015712, version 10 (SEQ ID NO: 1), the methylation of said sequence being indicative of neoplastic disease.

In another embodiment, the method of the invention comprises the step of analysing the methylation status of the nucleic acid sequence of the ARHGAP20 gene comprised between the nucleotide positions corresponding to nucleotides 42.661 and 45.840 of the Genbank sequence AP003460, version 3 (SEQ ID NO: 2), the methylation of said sequence being indicative of neoplastic disease.

In another embodiment, the method of the invention comprises the step of analysing the methylation status of the nucleic acid sequence of the FLI-1 gene comprised between the nucleotide positions corresponding to nucleotides 40.001 and 47.460 of the Genbank sequence AP001122, version 5 (SEQ ID NO: 3), the methylation of said sequence being indicative of neoplastic disease.

In another embodiment, the method of the invention comprises the step of analysing the methylation status of the nucleic acid sequence of the FOSL2 gene comprised between the nucleotide positions corresponding to nucleotides 52.801 and 57.000 of the Genbank sequence AC104695, version 2 (SEQ ID NO: 4), the methylation of said sequence being indicative of neoplastic disease.

In another embodiment, the method of the invention comprises the step of analysing the methylation status of the nucleic acid sequence of the HES2 gene comprised between the nucleotide positions corresponding to nucleotides 26.641 and 34.670 of the Genbank sequence AL031848, version 11 (SEQ ID NO: 5), the methylation of said sequence being indicative of neoplastic disease.

In another embodiment, the method of the invention comprises the step of analysing the methylation status of the nucleic acid sequence of the MAPI B gene comprised between the nucleotide positions corresponding to nucleotides 38.041 and 42.000 of the Genbank sequence AC093218, version 2 (SEQ ID NO: 6), the methylation of said sequence being indicative of neoplastic disease.

In another embodiment, the method of the invention comprises the step of analysing the methylation status of the nucleic acid sequence of the MDM2 gene comprised between the nucleotide positions corresponding to nucleotides 1.001 and 3.000 of the Genbank sequence AF527840, version 1 (SEQ ID NO: 7), the methylation of said sequence being indicative of neoplastic disease. In another embodiment, the method of the invention comprises the step of analysing the methylation status of the nucleic acid sequence of the NID1 gene comprised between the nucleotide positions corresponding to nucleotides 985.000 and 986.859 of the Genbank sequence NT_004836, version 16 (SEQ ID NO: 8), the methylation of said sequence being indicative of neoplastic disease.

In another embodiment, the method of the invention comprises the step of analysing the methylation status of the nucleic acid sequence of the NID2 gene comprised between the nucleotide positions corresponding to nucleotides 33.534.100 and 33.536.379 of the Genbank sequence NT_026437, version 11 (SEQ ID NO: 9), the methylation of said sequence being indicative of neoplastic disease.

In another embodiment, the method of the invention comprises the step of analysing the methylation status of the nucleic acid sequence of the PNUTL2 gene comprised between the nucleotide positions corresponding to nucleotides 108.541 and 111.200 of the Genbank sequence AC005666, version 1 (SEQ ID NO: 10), the methylation of said sequence being indicative of neoplastic disease.

In another embodiment, the method of the invention comprises the step of analysing the methylation status of the nucleic acid sequence of the PTGER3 gene comprised between the nucleotide positions corresponding to nucleotides 122.263 and 126.262 of the Genbank sequence AL031429, version 11 (SEQ ID NO: 11), the methylation of said sequence being indicative of neoplastic disease.

In another embodiment, the method of the invention comprises the step of analysing the methylation status of the nucleic acid sequence of the PTPRM gene comprised between the nucleotide positions corresponding to nucleotides 63.001 and 67.500 of the Genbank sequence AP001091 , version 5 (SEQ ID NO: 12), the methylation of said sequence being indicative of neoplastic disease.

Table 1 summarises the correspondence between the CpG islands of each of the above- mentioned genes and the nucleotide positions of the relevent Genbank sequences. Table 1. Novel abnormal ene meth lation markers in human tumours and location of their C G islands

s LO~ «~t- e— K

C

CT U\ 00 tr

CT CD

CT bO

The methylation status of of the CpG island is analysed using techniques which include chemical treatment of the DNA with a reagent capable of converting methylation differences into sequence differences. Preferably, a chemical reagent is used to this end which is capable of converting the unmethylated cytosine bases into uracil or any other base capable of pairing with a base other than guanine.

One preferred reagent for this purpose is bisulfite, preferably sodium bisulfite, in combination with hydroquinone. In this chemical reaction, the unmethylated cytosines of DNA are in fact deaminated to uracil which, in terms of base pairing, corresponds to thymidine. The methylated cytosines, on the contrary, are resistant to the chemical treatment and thus do not undergo any modification. Consequently, the methylcytosines of the original genomic DNA, which could not initially be distinguished from the cytosines because they exhibited the same pairing characteristics, remain the only cytosines in the chemicallly-treated DNA and they may thus be identified using standard molecular biology techniques.

The base sequence of the chemicallly-treated DNA may be analysed for example by direct sequencing. Alternatively, it is possible to use techniques such as Methylation- Specific PCR (MSP), which are based on the methylation-specific amplification of segments of the CpG island of the sample of the chemically-treated DNA. Such methods make use of oligonucleotides which are capable of undergoing selective hybridisation with the modified sequences of the methylated or unmethylated CpG island obtained with the above-described chemical treatment.

The above-descrived chemical treatment of a sample of genomic DNA including the CpG island of one of the genes disclosed in table 1 , results in the formation of two modified strands, each of which is derived from one of the two strands of the treated genomic DNA, which are, however, no longer complementary to one another.

The sequences designated as SEQ ID NO: 13 and SEQ ID NO: 14 are the nucleic acid sequences of the two modified strands which are obtained when all the dinucleotides of the original CpG island of the ALDH1A3 gene are methylated.

The sequences designated as SEQ ID NO: 15 and SEQ ID NO: 16 are the nucleic acid sequences of the two modified strands which are obtained when none of the dinucleotides of the original CpG island of the ALDH 1 A3 gene is methylated.

The sequences designated as SEQ ID NO: 17 and SEQ ID NO: 18 are the nucleic acid sequences of the two modified strands which are obtained when all the dinucleotides of the original CpG island of the ARHGAP20 gene are methylated.

The sequences designated as SEQ ID NO: 19 and SEQ ID NO: 20 are the nucleic acid sequences of the two modified strands which are obtained when none of the dinucleotides of the original CpG island of the ARHGAP20 gene is methylated.

The sequences designated as SEQ ID NO: 21 and SEQ ID NO: 22 are the nucleic acid sequences of the two modified strands which are obtained when all the dinucleotides of the original CpG island of the FLU gene are methylated.

The sequences designated as SEQ ID NO: 23 and SEQ ID NO: 24 are the nucleic acid sequences of the two modified strands which are obtained when none of the dinucleotides of the original CpG island of the FLU gene is methylated.

The sequences designated as SEQ ID NO: 25 and SEQ ID NO: 26 are the nucleic acid sequences of the two modified strands which are obtained when all the dinucleotides of the original CpG island of the FOSL2 gene are methylated.

The sequences designated as SEQ ID NO: 27 and SEQ ID NO: 28 are the nucleic acid sequences of the two modified strands which are obtained when none of the dinucleotides of the original CpG island of the FOSL2 gene is methylated.

The sequences designated as SEQ ID NO: 29 and SEQ ID NO: 30 are the nucleic acid sequences of the two modified strands which are obtained when all the dinucleotides of the original CpG island of the HES2 gene are methylated.

The sequences designated as SEQ ID NO: 31 and SEQ ID NO: 32 are the nucleic acid sequences of the two modified strands which are obtained when none of the dinucleotides of the original CpG island of the HES2 gene is methylated.

The sequences designated as SEQ ID NO: 33 and SEQ ID NO: 34 are the nucleic acid sequences of the two modified strands which are obtained when all the dinucleotides of the original CpG island of the MAPI B gene are methylated.

The sequences designated as SEQ ID NO: 35 and SEQ ID NO: 36 are the nucleic acid sequences of the two modified strands which are obtained when none of the dinucleotides of the original CpG island of the MAPI B gene is methylated.

The sequences designated as SEQ ID NO: 37 and SEQ ID NO: 38 are the nucleic acid sequences of the two modified strands which are obtained when all the dinucleotides of the original CpG island of the MDM2 gene are methylated.

The sequences designated as SEQ ID NO: 39 and SEQ ID NO: 40 are the nucleic acid sequences of the two modified strands which are obtained when none of the dinucleotides of the original CpG island of the MDM2 gene is methylated.

The sequences designated as SEQ ID NO: 41 and SEQ ID NO: 42 are the nucleic acid sequences of the two modified strands which are obtained when all the dinucleotides of the original CpG island of the NID1 gene are methylated.

The sequences designated as SEQ ID NO: 43 and SEQ ID NO: 44 are the nucleic acid sequences of the two modified strands which are obtained when none of the dinucleotides of the original CpG island of the NID1 gene is methylated.

The sequences designated as SEQ ID NO: 45 and SEQ ID NO: 46 are the nucleic acid sequences of the two modified strands which are obtained when all the dinucleotides of the original CpG island of the NID2 gene are methylated.

The sequences designated as SEQ ID NO: 47 and SEQ ID NO: 48 are the nucleic acid sequences of the two modified strands which are obtained when none of the dinucleotides of the original CpG island of the NID2 gene is methylated.

The sequences designated as SEQ ID NO: 49 and SEQ ID NO: 50 are the nucleic acid sequences of the two modified strands which are obtained when all the dinucleotides of the original CpG island of the PNUTL2 gene are methylated. The sequences designated as SEQ ID NO: 51 and SEQ ID NO: 52 are the nucleic acid sequences of the two modified strands which are obtained when none of the dinucleotides of the original CpG island of the PNUTL2 gene is methylated.

The sequences designated as SEQ ID NO: 53 and SEQ ID NO: 54 are the nucleic acid sequences of the two modified strands which are obtained when all the dinucleotides of the original CpG island of the PTGER3 gene are methylated.

The sequences designated as SEQ ID NO: 55 and SEQ ID NO: 56 are the nucleic acid sequences of the two modified strands which are obtained when none of the dinucleotides of the original CpG island of the PTGER3 gene is methylated.

The sequences designated as SEQ ID NO: 57 and SEQ ID NO: 58 are the nucleic acid sequences of the two modified strands which are obtained when all the dinucleotides of the original CpG island of the PTPRM gene are methylated.

The sequences designated as SEQ ID NO: 59 and SEQ ID NO: 60 are the nucleic acid sequences of the two modified strands which are obtained when none of the dinucleotides of the original CpG island of the PTPRM gene is methylated.

Sequences SEQ ID NO: 13 , SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 were determined on the basis of Genbank sequence AC015712, version 10 (nucleotides 86701-89700) of the ALDH 1 A3 gene.

Sequences SEQ ID NO: 17 , SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 were determined on the basis of Genbank sequence AP003460, version 3 (nucleotides 42661- 45840) of the ARHGAP20 gene.

Sequences SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 were determined on the basis of Genbank sequence AP001122, version 5 (nucleotides 40001- 46400) of the FLU gene.

Sequences SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28 were determined on the basis of Genbank sequence AC104695, version 2 (nucleotides 52801- 57000) of the FOSL2 gene. Sequences SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 were determined on the basis of Genbank sequence AL031848, version 11 (nucleotides 26641-34670) of the HES2 gene.

Sequences SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 were determined on the basis of Genbank sequence AC093218, version 2 (nucleotides 38041- 42000) of the MAPI B gene.

Sequences SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 were determined on the basis- of Genbank sequence AF527840, version 1 (nucleotides 1.001- 3.000) of the MDM2 gene.

Sequences SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44 were determined on the basis of Genbank sequence NT_004836, version 16 (nucleotides 985.000 - 986.859) of the NID1 gene.

Sequences SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48 were determined on the basis of Genbank sequence NT_026437, version 11 (nucleotides 33.534.100 - 33.536.379) of the NID2 gene.

Sequences SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52 were determined on the basis of Genbank sequence AC005666, version 1 (nucleotides 108541-111200) of the PNUTL2 gene.

Sequences SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56 were determined on the basis of Genbank sequence AL031429, version 11 (nucleotides 122263-126262) of the PTGER3 gene.

Sequences SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60 were determined on the basis of Genbank sequence AP001091, version 5 (nucleotides 63001- 67500) of the PTPRM gene.

Table 1 summarizes the correspondence between the various genes an the SEQ ID. Oligonucleotides at least 10 nucleotides in length which are complementary or identical to a segment of a target sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, are a useful tool for the analysis of the methylation status of the CpG island of the genes listed in table 1 in a sample of genomic DNA, since the their hybridisation depends on the methylation status of the original CpG island of the sample. Such oligonucleotides fall within the scope of the present invention. Preferably, the oligonucleotides of the invention are complementary or identical to a segment of a target sequence as defined above comprising at least one CpG dinucleotide.

The above-defined oligonucleotides may be used in the diagnostic method of the invention as amplification primers or as hybridisation probes. In the latter case, they preferably have a length of at least 18 nucleotides.

As an example, table 2a reports some specific oligonucleotide primers based on some of the target sequences listed above, which are useful in the diagnostic method of the invention.

The features of the product amplified with the primers of table 2a are reported in table 2b. Table 2a

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CD CD

cT

Table 2b

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O⁾ CD e— *-

cT^"

The diagnostic method of the invention may, for example, be a detection method based on DNA amplification, optionally followed by detection of the amplified DNA fragments by a hybridisation probe.

In such a method, a sample of genomic DNA to be investigated is chemically pretreated with a reagent capable of converting the unmethylated cytosine bases into uracil or any other base capable of pairing with a base other than guanine.

The sample of genomic DNA to be analysed is preferably obtained from a source such as blood, serum, plasma, bronchial washings, expectoration, saliva, intestinal washings, urine, faeces, ejaculate, pads of various origins, needle aspirates, lymph node biopsies, or combinations thereof.

Said chemical pretreatment of the genomic DNA is preferably carried out with sodium bisulfite and hydroquinone.

Following the chemical pretreatment, two modified strands, which are, however, no longer complementary to one another, are obtained from the original double-stranded genomic DNA.

After the chemical pretreatment step, any of the modified strands obtained by pretreatment of the genomic DNA may be subjected to an amplification reaction using as the amplification primers a pair of oligonucleotides at least 10 nucleotides in length, wherein one of said oligonucleotides is complementary to a first segment of a target sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57 and SEQ ID NO: 58 and the other of said oligonucleotides is identical to a second segment of said target sequence, the second segment of the target sequence being downstream from the first segment.

According to one embodiment, at least one of said first and second segments of the target sequence comprises at least one CpG dinucleotide. In this case, the amplification reaction is methylation-specific, since the oligonucleotide primer pair used is designated to specifically amplify a modified strand derived from the sequence of the original methylated CpG island. In this embodiment, detection of the amplified DNA fragments, if present, may be performed by any method known per se, for example by electrophoresis on agarose gel and staining with a DNA intercalating agent, such as for example ethidium bromide.

According to another embodiment, neither the first nor the second segment of the target sequence comprises a CpG dinucleotide. In this case, the amplification reaction is not methylation-specific. Consequently, the detection of the amplified fragments, if present, will be performed by hybridisation with an oligonucleotide probe at (east 18 nucleotides in length, which is complementary to a third segment of the target sequence, said third segment being located between the said first and second segments and comprising at least one CpG dinucleotide. The third segment of the target sequence may optionally be partially overlapped to the first or the second segment.

In both embodiments, the amplification reaction is preferably a polymerase chain reaction (PCR). Still more preferred is a two-step amplification reaction comprising a primary PCR followed by a semi-nested or nested secondary PCR.

Since the modified genomic DNA representing the target of the amplification reaction is initially single-stranded, in the first PCR cycle only one of the two primers will initially hybridise with the target strand. The first hybridised primer will be extended by a polymerase using the target strand as template. Then, the second primer will hybridise with the newly synthesised chain and will be extended by the polymerase using the newly synthesised chain as the template. In this manner, the initial single-stranded target DNA will be converted into double-stranded DNA. In the subsequent cycles of the PCR reaction, in contrast, the two amplification primers will each simultaneously hybridise with one strand of the double-stranded target DNA.

Table 2a shows some specific examples of oligonucleotides suitable as primers or probes in the diagnostic method of the invention.

The oligonucleotides ALDH1A3_MF (SEQ ID NO: 61) and ALDH1A3_MR (SEQ ID NO: 62) were designed to specifically hybridise in SEQ ID NO: 13. When used as primers in a PCR amplification, the oligonucleotide pair ALDH1A3_MF and ALDH1A3_MR amplifies a 227 bp sequence comprised between nucleotide 1238 and nucleotide 1465 of SEQ ID NO: 13.

The oligonucleotides ARHGAP20_MF (SEQ ID NO: 63) and ARHGAP20_MR (SEQ ID NO: 64) were designed to specifically hybridise in SEQ ID NO: 18. When used as primers in a PCR amplification, the oligonucleotide pair ARHGAP20_MF and ARHGAP20_MR amplifies a 153 bp sequence comprised between nucleotide 1777 and nucleotide 1930 of SEQ ID NO: 18.

The oligonucleotides FLI1_MF (SEQ ID NO: 65) and FLI1_MR (SEQ ID NO: 66) were designed to specifically hybridise in SEQ ID NO: 21. When used as primers in a PCR amplification, the oligonucleotide pair FLI1_MF and FL11_MR amplifies a 199 bp sequence comprised between nucleotide 5744 and nucleotide 5943 of SEQ ID NO: 21.

The oligonucleotides FOSL2_MF (SEQ ID NO: 67) and FOSL2_MR (SEQ ID NO: 68) were designed to specifically hybridise in SEQ ID NO: 25. When used as primers in a PCR amplification, the oligonucleotide pair FOSL2_MF and FOSL2_MR amplifies a 264 bp sequence comprised between nucleotide 2496 and nucleotide 2760 of SEQ ID NO: 25.

The oligonucleotides HES2_MF (SEQ ID NO: 69) and HES2_MR (SEQ ID NO: 70) were designed to specifically hybridise in SEQ ID NO: 29. When used as primers in a PCR amplification, the oligonucleotide pair HES2_MF and HES2__MR amplifies a 205 bp sequence comprised between nucleotide 6072 and nucleotide 6277 of SEQ ID NO: 29

The oligonucleotides MAP1B_MF (SEQ ID NO: 71) and MAP1B_MR (SEQ ID NO: 72) were designed to specifically hybridise in SEQ ID NO: 33. When used as primers in a PCR amplification, the oligonucleotide pair MAP1B_MF and MAP1B_MR amplifies a 216 bp sequence comprised between nucleotide 1635 and nucleotide 1851 of SEQ ID NO: 33.

The oligonucleotides MDM2_MF (SEQ ID NO: 73) and MDM2_MR (SEQ ID NO: 74) were designed to specifically hybridise in SEQ ID NO: 37. When used as primers in a PCR amplification, the oligonucleotide pair MDM2_MF and MDM2_MR amplifies a 328 bp sequence comprised between nucleotide 1175 and nucleotide 1503 of SEQ ID NO: 37. The oligonucleotides NID1_MF (SEQ ID NO: 75) and NID1_MR (SEQ ID NO: 76) were designed to specifically hybridise in SEQ ID NO: 41. When used as primers in a PCR amplification, the oligonucleotide pair NID1_MF and NID1_MR amplifies a 144 bp sequence comprised between nucleotide 1319 and nucleotide 1462 of SEQ ID NO: 41.

The oligonucleotides NID2_MF (SEQ ID NO: 77) and NID2_MR (SEQ ID NO: 78) were designed to specifically hybridise in SEQ ID NO: 45. When used as primers in a PCR amplification, the oligonucleotide pair NID2_MF and NID2_MR amplifies a 141 bp sequence comprised between nucleotide 1310 and nucleotide 1438 of SEQ ID NO: 45

The oligonucleotides PNUTL2_MF (SEQ ID NO: 79) and PNUTL2_MR (SEQ ID NO: 80) were designed to specifically hybridise in SEQ ID NO: 49. When used as primers in a PCR amplification, the oligonucleotide pair PNUTL2_MF and PNUTL2_MR amplifies a 216 bp sequence comprised between nucleotide 826 and nucleotide 1042 of SEQ ID NO: 49.

The oligonucleotides PTGER3_MF (SEQ ID NO: 81) and PTGER3_MR (SEQ ID NO: 82) were designed to specifically hybridise in SEQ ID NO: 54. When used as primers in a PCR amplification, the oligonucleotide pair PTGER3_MF and PTGER3_MR amplifies a 191 bp sequence comprised between nucleotide 2089 and nucleotide 2280 of SEQ ID NO: 54.

The oligonucleotides PTPRM_MF (SEQ ID NO: 83) and PTPRM_MR (SEQ ID NO: 84) were designed to specifically hybridise in SEQ ID NO: 57. When used as primers in a PCR amplification, the oligonucleotide pair PTPRM-MF and PTPRM_MR amplifies a 245 bp sequence comprised between nucleotide 2061 and nucleotide 2306 of SEQ ID NO: 57.

A kit for performing the diagnostic method of the invention also falls within the scope of the present invention, which kit may comprise, for example, a pair of amplification primers as defined above optionally in combination with a reagent capable of converting the unmethylated cytosine bases into uracil or any other base capable of pairing with a base other than guanine, for example sodium bisulfite in combination with hydroquinone. The kit may further comprise DNA detection means, such as for example a methylation- specific hybridisation probe as defined above, and/or DNA amplification means, for example a polymerase enzyme. As an alternative to the probe, the kit may comprise a DNA detection reagent such as a DNA intercalating agent, for example ethidium bromide.

The kit of the invention may also comprise a second pair of amplification primers at least 10 nucleotides in length, one of said oligonucleotides being complementary to a first segment of a target sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 60 and the other oligonucleotide being identical to a second segment of said target sequence, the second segment being located downstream from the first segment. Preferably, at least one of said first and second segments of the target sequence comprises at least one TpG dinucleotide. The second pair of primers is used as a control that the DNA actually has been modified following treatment with bisulfite, so making it possible to validate a negative result of amplification with primers designed on the basis of the modified nucleotide sequences derived from the methylated CpG island.

Experimental section (examples^

The methylation status of the CpG island of the genes liested in table 1 was analysed in tumor and normal human samples, using the primers and reaction conditions indicated in table 2. The results obtained are summarized below in table 3.

Table 3

Tessuto ALDH1A3 ARHGAP20 FLH FOSL2 HES2 MDM2 NID1

Carcinoma 21% (6/28) 61% (17/28) 82% (45/50) nd 0% (0/28) nd 69% (33/48)

Colon Mucosa 0% (0/10) 0% (0/10) 21% . (5/24) 70% (7/10) 0% (0/10) 40% (4/10) 33% (8/24)

Carcinoma 37% (7/19) 58% (11/19) 86% (13/16) nd 21% (3/19) nd 90% (18/20)

Stomach Mucosa 0% (0/10) 0% (0/10) 31% » (4/13) 40% (4/10) 0% (0/10) 30% (3/10) 38% (5/13)

Carcinoma 35% (7/20) 14% (4/28) 25% (6/24) 69% (18/26) 14% (4/28) 50% (13/26) 29% (8/28)

Liver Mucosa 0% (0/9) 0% (0/9) 22% (2/9) 22% (2/9) 0% (0/9) 0% (0/9) 0% (0/9)

Breast Carcinoma 21% (6/28) 25% (7/28) 69% (35/51) 68% (19/28) 4% (1/28) nd 62% (30/48)

C= Tissue NID1 NID2 PNUTL2 PTGER3 PTPRM MAPiB

CX

Carcinoma 69% (33/48) 29% (14/48) 0% (0/28) 61% (17/28) 75% (21/28) 43% (12/28)

Colon Mucosa 33% (8/24) 0% (0/24) 0% (0/10) I D% (0/10) 0% (0/10) 0% (0/10) r C-t- Carcinoma 90% (18/20) 95% (19/20) 26% i (5/19) 68% (13/19) 74% (14/19) 26% (5/19) O) Stomach

CΛ Mucosa 38% (5/13) 0% (0/13) 0% (0/10) i 0% (0/10) 0% (0/10) 0% (0/10) vo

Carcinoma 29% (8/28) 25% (7/28) 0% (0/24) 36% (10/28) 17% (4/23) 29% (7/23)

Liver

O Mucosa 0% (0/9) 0% (0/9) 0% , (0/9) 11% (1/9) 0% (0/9) 0% (0/9)

Breast Carcinoma 62% (30/48) 40% (19/47) 7% (2/28) 11% (3/28) 50% (14/28) 4% (1/28)

to

ON

From the analysis carried out on normal and tumour samples, the methylation percentages of each gene in the various tissues analysed may be calculated. The analysed tissues derive from colon, stomach, liver and breast. These results indicate that the genes listed in table 1 are often methylated in at least one of the tumour tissues analyzed. These tumours are to be considered as an example indicative of the high methylation frequency of such genes in neoplastic diseases, without limitation to the examples described in table 3.

The nucleic acid sequences referred to in the present description are illustrated in the annexed sequence listing, in which:

SEQ ID NO. 1 is the CpG Island (isola CpG) of ALDH1A3, corresponding to the 3.000 nucleotide (nt) segment comprised between nt 86701-89700 of the Genbank sequence AC015712, versione 10; SEQ ID NO. 2 is the CpG island of ARHGAP20, corresponding to the 3.180 nt segment comprised between nt 45840 and 42661 of the Genbank sequence AP003460, version 3; SEQ ID NO: 3 is the CpG Island of FLU corresponding to the 7.460 nt segment comprised between nt 40001-47460 of the Genbank sequence AP001122, version 5; SEQ ID NO: 4 is the CpG Island of FOSL2 corresponding to the 4.200 nt segment comprised between nt 52801-57000 of the Genbank sequence AC104695, version 2; SEQ ID NO: 5 is the CpG Island of HES2 corresponding to the 8.030 nt segment comprised between nt 26641- 34670 of the Genbank sequence AL031848, version 11; SEQ ID NO: 6 is the CpG Island of MAPI B corresponding to the 3.960 nt segment comprised between nt 38041-42000 of the Genbank sequence AC093218 versione 2; SEQ ID NO: 7 is the CpG Island of MDM2 corresponding to the 2.000 nt segment comprised between nt 1001-3000 of the Genbank sequence AF527840, version 1; SEQ ID NO. 8 is the CpG island of the NID1 gene, corresponding to the 1.860 nt segment comprised between nt 985.000-986.859 of the Genbank sequence NT_004836, version 16; SEQ ID NO. 9 is the CpG island of the NID2 gene, corresponding to the 2.280 nt segment comprised between nt 33534100- 33536379 of Genbank sequence NT_026437, version 11; SEQ ID NO. 10 is the CpG island of the PNUTL2 gene, corresponding to the 2.660 nt segment comprised between nt 111200-108541 of Genbank AC005666, version 1; SEQ ID NO. 11 is the CpG island of the PTGER3 gene, corresponding to the 4.000 nt segment comprised between nt 122.263 and 126.262 of Genbank AL031429, version 11; SEQ ID NO. 12 is the CpG island of the PTPRM gene, corresponding to the 4.500 nt segment comprised between nt 63001 and 67500 of Genbank AP001091 , version 5; SEQ ID NO. 13 is the sequence derived from the methylated sequence of the ALDH1A3 gene (top strand), derived from SEQ ID NO. 1; SEQ ID NO. 14 is the sequence derived from the methylated sequence of the ALDH 1 A3 gene (bottom strand), derived from SEQ ID NO. 1; SEQ ID NO. 15 is the sequence derived from the unmethylated sequence of the ALDH1A3 gene (top strand), derived from SEQ ID NO. 1; SEQ ID NO. 16 is the sequence derived from the unmethylated sequence of the ALDH 1 A3 gene (bottom strand), derived from SEQ ID NO. 1; SEQ ID NO. 17 is the sequence derived from the methylated sequence of the ARHGAP20 gene (top strand), derived from SEQ ID NO. 2; SEQ ID NO. 18 is the sequence derived from the methylated sequence of the ARHGAP20 gene (bottom strand), derived from SEQ ID NO. 2; SEQ ID NO. 19 is the sequence derived from the unmethylated sequence of the ARHGAP20 gene (top strand), derived from SEQ ID NO. 2; SEQ ID NO. 20 is the sequence derived from the unmethylated sequence of the ARHGAP20 gene (bottom strand), derived from SEQ ID NO. 2; SEQ ID NO. 21 is the sequence derived from the methylated sequence of the FLU gene (top strand), derived from SEQ ID NO. 3; SEQ ID NO. 22 is the sequence derived from the methylated sequence of the FLU gene (bottom strand), derived from SEQ ID NO. 3; SEQ ID NO. 23 is the sequence derived from the unmethylated sequence of the FLU gene (top strand), derived from SEQ ID NO. 3; SEQ ID NO. 24 is the sequence derived from the unmethylated sequence of the FLU gene (bottom strand), derived from SEQ ID NO. 3; SEQ ID NO: 25 is the sequence derived from the methylated sequence of the FOSL2 gene (top strand), derived from SEQ ID NO. 4; SEQ ID NO: 26 is the sequence derived from the methylated sequence of the FOSL2 gene (bottom strand), derived from SEQ ID NO. 4; SEQ ID NO: 27 is the sequence derived from the unmethylated sequence of the FOSL2 gene (top strand), derived from SEQ ID NO. 4; SEQ ID NO: 28 is the sequence derived from the unmethylated sequence of the FOSL2 gene (bottom strand), derived from SEQ ID NO. 4; SEQ ID NO: 29 is the sequence derived from the methylated sequence of the HES2 gene (top strand), derived from SEQ ID NO.5; SEQ ID NO: 30 is the sequence derived from the methylated sequence of the HES2 gene (bottom strand), derived from SEQ ID NO.5; SEQ ID NO: 31 is the sequence derived from the unmethylated sequence of the HES2 gene (top strand), derived from SEQ ID NO.5; SEQ ID NO: 32 is the sequence derived from the unmethylated sequence of the HES2 gene (bottom strand), derived from SEQ ID NO.5; SEQ ID NO: 33 is the sequence derived from the methylated sequence of the MAPI B gene (top strand), derived from SEQ ID NO.6; SEQ ID NO: 34 is the sequence derived from the methylated sequence of the MAPI B gene (bottom strand), derived from SEQ ID NO.6; SEQ ID NO: 35 is the sequence derived from the unmethylated sequence of the MAPI B gene (top strand), derived from SEQ ID NO.6; SEQ ID NO: 36 is the sequence derived from the unmethylated sequence of the MAPI B gene (bottom strand), derived from SEQ ID NO.6; SEQ ID NO: 37 is the sequence derived from the methylated sequence of the MDM2 gene (top strand), derived from SEQ ID NOJ; SEQ ID NO: 38 is the sequence derived from the methylated sequence of the MDM2 gene (bottom strand), derived from SEQ ID

N0.7; SEQ ID NO: 39 is the sequence derived from the unmethylated sequence of the MDM2 gene (top strand), derived from SEQ ID NOJ; SEQ ID NO: 40 is the sequence derived from the unmethylated sequence of the MDM2 gene (bottom strand), derived from SEQ ID NOJ;

SEQ ID NO: 41 is the sequence derived from the methylated sequence of the NID1 gene (top strand), derived from SEQ ID N0.8; SEQ ID NO: 42 is the sequence derived from the methylated sequence of the NID1 gene (bottom strand), derived from SEQ ID N0.8; SEQ ID

NO: 43 is the sequence derived from the unmethylated sequence of the NID1 gene (top strand), derived from SEQ ID N0.8; SEQ ID NO: 44 is the sequence derived from the unmethylated sequence of the NID1 (bottom strand), derived from SEQ ID N0.8; SEQ ID

NO: 45 is the sequence derived from the methylated sequence of the NID2 gene (top strand), derived from SEQ ID N0.9; SEQ ID NO: 46 is the sequence derived from the methylated sequence of the NID2 gene (bottom strand), derived from SEQ ID N0.9; SEQ ID NO: 47 is the sequence derived from the unmethylated sequence of the NID2 gene (top strand), derived from SEQ ID N0.9; SEQ ID NO: 48 is the sequence derived from the unmethylated sequence of the NID2 gene (bottom strand), derived from SEQ ID N0.9; SEQ ID NO: 49 is the sequence derived from the methylated sequence of the PNUTL2 gene (top strand), derived from SEQ ID NO.10; SEQ ID NO: 50 is the sequence derived from the methylated sequence of the PNUTL2 gene (bottom strand), derived from SEQ ID NO.10; SEQ ID NO: 51 is the sequence derived from the unmethylated sequence of the PNUTL2 gene (top strand), derived from SEQ ID NO.10; SEQ ID NO: 52 is the sequence derived from the unmethylated sequence of the PNUTL2 gene (bottom strand), derived from SEQ ID NO.10; SEQ ID NO: 53 is the sequence derived from the methylated sequence of the PTGER3 gene (top strand), derived from SEQ ID NO.11; SEQ ID NO: 54 is the sequence derived from the methylated sequence of the PTGER3 gene (bottom strand), derived from SEQ ID NO.11; SEQ ID NO: 55 is the sequence derived from the unmethylated sequence of the PTGER3 gene (top strand), derived from SEQ ID NO.11; SEQ ID NO: 56 is the sequence derived from the unmethylated sequence of the PTGER3 gene (bottom strand), derived from SEQ ID NO.11; SEQ ID NO: 57 is the sequence derived from the methylated sequence of the PTPRM gene (top strand), derived from SEQ ID NO.12; SEQ ID NO: 58 is the sequence derived from the methylated sequence of the PTPRM gene (bottom strand), derived from SEQ ID NO.12; SEQ ID NO: 59 is the sequence derived from the unmethylated sequence of the PTPRM gene (top strand), derived from SEQ ID NO.12; SEQ ID NO: 60 is the sequence derived from the unmethylated sequence of the PTPRM gene (bottom strand), derived from SEQ ID NO.12; SEQ ID NO: 61 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on SEQ ID NO: 006/000237

23

13; SEQ ID NO: 62 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on the inverted complement of SEQ ID NO: 13; SEQ ID NO: 63 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on SEQ ID NO: 18; SEQ ID NO: 64 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on the inverted complement of SEQ ID NO: 18; SEQ ID NO: 65 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on SEQ ID NO:21; SEQ ID NO: 66 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on the inverted complement of SEQ ID NO:21; SEQ ID NO: 67 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on SEQ ID NO:25; SEQ ID NO: 68 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on the inverted complement of SEQ ID NO:25; SEQ ID NO: 69 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on SEQ ID NO:29; SEQ ID NO: 70 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on the inverted complement of SEQ ID NO:29; SEQ ID NO: 71 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on SEQ ID NO:33; SEQ ID NO: 72 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on the inverted complement of SEQ ID NO:33; SEQ ID NO: 73 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on SEQ ID NO:37; SEQ ID NO: 74 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on the inverted complement of SEQ ID NO:37; SEQ ID NO: 75 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on SEQ ID NO:41; SEQ ID NO: 76 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on the inverted complement of SEQ ID NO:41; SEQ ID NO: 77 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on SEQ ID NO:45; SEQ ID NO: 78 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on the inverted complement of SEQ ID NO:45; SEQ ID NO: 79 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on SEQ ID NO:49; SEQ ID NO: 80 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on the inverted complement of SEQ ID NO:49; SEQ ID NO: 81 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on SEQ ID NO:54; SEQ ID NO: 82 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on the inverted complement of SEQ ID NO:54; SEQ ID NO: 83 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on SEQ ID NO:57; SEQ ID NO: 84 is the oligonucleotide sequence for Methylation-Specific PCR (MSP) designed on the inverted complement of SEQ ID NO:57.

Claims

1. A nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60.

2. An oligonucleotide at least 10 nucleotides in length, which is complementary to or identical to a segment of a target sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60.

3. The oligonucleotide according to claim 2, at least 18 nucleotides in length.

4. The oligonucleotide according to claim 2 or claim 3, wherein said target sequence is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58, and wherein said segment of the target sequence comprises at least one CpG dinucleotide.

substitute sheet Rule 26

5. The oligonucleotide according to claim 2 or claim 3, wherein said target sequence is selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 60 and wherein said segment of the target sequence comprises at least one TpG dinucleotide.

6. The oligonucleotide according to claim 1 , selected from the group consisting of SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84.

7. A method for the diagnosis of neoplastic disease comprising the step of analysing, in a sample of genomic DNA, the methylation status of the nucleic acid sequence of the CpG island of a human gene selected from the group of genes listed in table 1 , the methylation of said sequence being indicative of neoplastic disease.

8. The method according to claim 7, comprising the step of analysing, in a sample of genomic DNA, the methylation status of the nucleotide sequence of the ALDH1A3 gene comprised between the nucleotide positions corresponding to nucleotides 86701 and 89700 of Genbank sequence AC015712 version 10, the methylation of said sequence being indicative of neoplastic disease.

9. The method according to claim 7, comprising the step of analysing, in a sample of genomic DNA₁ the methylation status of the nucleotide sequence of the ARHGAP20 gene comprised between the nucleotide positions corresponding to nucleotides 45840 and 42661 of Genbank sequence AP003460 version 3, the methylation of said sequence being indicative of neoplastic disease.

10. The method according to claim 7, comprising the step of analysing, in a sample of genomic DNA, the methylation status of the nucleotide sequence of the FLU gene comprised between the nucleotide positions corresponding to nucleotides 47460 and 40001 of

substitute sheet Rule 26 Genbank sequence AP001122 version 5, the methylation of said sequence being indicative of neoplastic disease.

11. The method according to claim 7, comprising the step of analysing, in a sample of genomic DNA, the methylation status of the nucleotide sequence of the FOSL2 gene comprised between the nucleotide positions corresponding to nucleotides 52801 and 57000 of Genbank sequence AC104695 version 2, the methylation of said sequence being indicative of neoplastic disease.

12. The method according to claim 7, comprising the step of analysing, in a sample of genomic DNA, the methylation status of the nucleotide sequence of the HES2 gene comprised between the nucleotide positions corresponding to nucleotides 26641 and 34670 of Genbank sequence AL031848 version 11, the methylation of said sequence being indicative of neoplastic disease.

13. The method according to claim 7, comprising the step of analysing, in a sample of genomic DNA, the methylation status of the nucleotide sequence of the MAPI B gene comprised between the nucleotide positions corresponding to nucleotides 38041 and 42000 of Genbank sequence AC093218 version 2, the methylation of said sequence being indicative of neoplastic disease.

14. The method according to claim 7, comprising the step of analysing, in a sample of genomic DNA, the methylation status of the nucleotide sequence of the MDM2 gene comprised between the nucleotide positions corresponding to nucleotides 1001 and 3000 of Genbank sequence AF527840 version 1, the methylation of said sequence being indicative of neoplastic disease.

15. The method according to claim 7, comprising the step of analysing, in a sample of genomic DNA, the methylation status of the nucleotide sequence of the NID1 gene comprised between the nucleotide positions corresponding to nucleotides 985000 and 986859 of Genbank sequence NT_004836 version 16, the methylation of said sequence being indicative of neoplastic disease.

16. The method according to claim 7, comprising the step of analysing, in a sample of genomic DNA, the methylation status of the nucleotide sequence of the NID2 gene

substitute sheet Rule 26 comprised between the nucleotide positions corresponding to nucleotides 33534100 and 33536379 of Genbank sequence NT_026437 version 11, the methylation of said sequence being indicative of neoplastic disease.

17. The method according to claim 7, comprising the step of analysing, in a sample of genomic DNA, the methylation status of the nucleotide sequence of the PNUTL2 gene comprised between the nucleotide positions corresponding to nucleotides 111200 and 108541 of Genbank sequence AC005666 version 1 , the methylation of said sequence being indicative of neoplastic disease.

18. The method according to claim 7, comprising the step of analysing, in a sample of genomic DNA, the methylation status of the nucleotide sequence of the PTGER3 gene comprised between the nucleotide positions corresponding to nucleotides 122263 and 126262 of Genbank sequence AL031429 version 11, the methylation of said sequence being indicative of neoplastic disease.

19. The method according to claim 7, comprising the step of analysing, in a sample of genomic DNA, the methylation status of the nucleotide sequence of the PTPRM gene comprised between the nucleotide positions corresponding to nucleotides 63001 and 67500 of Genbank sequence AP001091 versione 5, the methylation of said sequence being indicative of neoplastic disease.

20. The method according to claim 7, comprising the steps of:

(i) chemically pretreating a genomic DNA sample with a reagent capable of converting the unmethylated cytosine bases into uracil or any other base capable of pairing with a base other than guanine;

(ii) subjecting a strand of the pretreated genomic DNA to amplification using as amplification primers at a pair of oligonucleotides each at least 10 nucleotides in length, one of said oligonucleotides being complementary to a first segment of a target sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58 and the other of said oligonucleotides being identical to a second segment of said target sequence, the second segment being located

substitute sheet Rule 26 downwstream from the first segment; at least one of said first and second segments of the target sequence comprising at least one CpG dinucleotide; and

(iii) detecting the presence of amplified fragments of the pretreated genomic DNA, the presence of amplified fragments being indicative of neoplastic disease.

21. The method according to claim 7, comprising the steps of: ^'

(i) chemically pretreating a sample of genomic DNA with a reagent capable of converting the unmethylated cytosine bases into uracil or any other base capable of pairing with a base other than guanine;

(ii) subjecting a strand of the pretreated genomic DNA to amplification using as amplification primers at least one pair of oligonucleotides each at least 10 nucleotides in length, one of said oligonucleotides being complementary to a first segment of a target sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58 and the other of said oligonucleotides being identical to a second segment of said target sequence, the second segment being downstream from the first segment; and

(iii) detecting the presence of amplified fragments of the pretreated genomic DNA by hybridisation with an oligonucleotide probe at least 18 nucleotides in length which is complementary to a third segment of said target sequence, said third segment being located between said first and said second segment and comprising at least one CpG dinucleotide, the presence of amplified fragments being indicative of neoplastic disease.

22. The method according to claim 21 , wherein said probe is labeled with a detectable marker.

23. The method according to any of claims 20 to 22, wherein said amplification is a polymerase chain reaction (PCR).

24. The method according to claim 23, wherein said amplification is a primary PCR followed by a secondary semi-nested or nested PCR.

25. The method according to any of claims 20 to 24, wherein said reagent is bisulfite,

substitute sheet Rule 26 preferably sodium bisulfite, in combination with hydroquinone.

26. The method according to any of claims 7 to 25, wherein said neoplastic disease is a solid tumour or an haematological neoplastic disease.

27. The method according to claim 26, wherein said neoplastic disease is selected from the group consisting of primary tumours of the central nervous system, cerebral metastases, tumours of the eye, tumours of the pituitary gland, tumours of the thyroid gland, tumours of the adrenal cortex, tumours of the diffuse endocrine system, tumours of the head and neck, tumours of the lungs, malignant mesothelioma, thymomas and thymic tumours, tumours of the heart and major vessels, primary thoracic germ cell tumours, metastatic tumours of the thorax, oesophageal tumours, liver tumours, gastric tumours, tumours of the gallbladder and bile ducts, tumours of the exocrine pancreas, tumours of Vater's ampulla, tumours of the small intestine, tumours of the appendix and peritoneum, tumours of the colon and the rectum, tumours of the anus, renal tumours, tumours of the pelvis and the ureter, tumours of the bladder, tumours of the prostate gland, tumours of the penis and the urethra, testicular tumour, tumours of the vulva and the vagina, tumours of the cervix, endometrial cancer, Fallopian tube tumours, ovarian tumour, gynaecological sarcomas, mammary tumours, malignant melanoma, tumours of the skin, tumours of the bone, soft tissue sarcomas, Wilms¹ tumour, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, myelodysplastic syndrome, acute myeloid leukaemia of the adult and the child, chronic myeloid leukaemia, acute lymphocytic leukaemia of the adult, acute lymphoblastic leukaemia of the child, chronic lymphocytic leukaemia, hairy cell leukaemia, mast cell leukaemia, Hodgkin's disease, non-Hodgkin's lymphomas, fungoid mycosis and Sezary syndrome, plasma cell tumours, chronic myeloproliferative disorders.

28. The method according to claim 26, wherein said neoplastic disease is selected from the group consisting of gastric carcinoma, colorectal carcinoma and mammary carcinoma.

29. A kit for the diagnosis of neoplastic disease, comprising a pair of amplification primers as defined in claim 20 or 21.

30. The kit according to claim 29, additionally comprising a hybridisation probe as defined in claim 21.

substitute sheet Rule 26

31. The kit according to claim 29 or 30, additionally comprising a reagent capable of converting the unmethylated cytosine bases into uracil or any other base capable of pairing with a base other than guanine.

32. The kit according to any of claims 29 to 31, additionally comprising a second pair of oligonucleotide amplification primers at least 10 nucleotides in length, one of said oligonucleotides being complementary to a first segment of a target sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 60 and the other of said oligonucleotides being identical to a second segment of said target sequence, the second segment being downstream from the first segment.

33. The kit according to claim 32, wherein at least one of said first and second segments of the target sequence comprises at least one TpG dinucleotide.

34. The kit according to any of claims 29 to 33, additionally comprising DNA amplification means and/or DNA detection reagents.

substitute sheet Role 26