# Patent

## Title
Method for the analysis of ovarian cancer disorders

## Abstract

The invention relates to a method for the analysis of ovarian cancer disorders, comprising determining the genomic methylation status of one or more CpG dinucleotides in a sequence selected from the group of sequences according to SEQ ID NO. 1 to 10 and/or SEQ ID NO. 50 to SEQ ID NO. 60. Optionally, additionally following steps are performed, the one or more results from the methylation status test is input into a classifier that is obtained from a Diagnostic Multi Variate Model, calculating a likelihood as to whether the sample is from a normal tissue or an ovarian cancer tissue and/or, calculating an associated p-value for the confidence in the prediction.

## Background
FIELD OF THE INVENTION
The present invention is in the field of biology and chemistry, more in particular in the field of molecular biology and human genetics. The invention relates to the field of identifying methylated sites in human DNA, in particular methylated sites in certain defined sequences which when methylated are indicative of ovarian cancer.
BACKGROUND OF THE INVENTION
Ovarian cancer is the fifth leading cause of cancer death in women, the leading cause of death from gynecological malignancy, and the second most commonly diagnosed gynecologic malignancy (The Merck Manual of Diagnosis and Therapy Section 18. Gynecology And Obstetrics Chapter 241. Gynecologic Neoplasms).
It is idiopathic, meaning that the exact cause is usually unknown. The disease is more common in industrialized nations, with the exception of Japan. In the United States, females have a 1.4% to 2.5% (1 out of 40-60 women) lifetime chance of developing ovarian cancer.
More than half of the deaths from ovarian cancer occur in women between 55 and 74 years of age and approximately one quarter of ovarian cancer deaths occur in women between 35 and 54 years of age.
The risk for developing ovarian cancer appears to be affected by several factors.
The link to the use of fertility medication, such as clomiphene citrate, has been controversial. An analysis in 1991 raised the possibility that use of drugs may increase the risk for ovarian cancer. Several cohort studies and case-control studies have been conducted since then without providing conclusive evidence for such a link.
There is good evidence that genetic factors are important. Carriers of certain mutations of the BRCA1 or the BRCA2 gene, more frequent in some populations (e.g. Ashkenazi Jewish women) are at a higher risk of both breast cancer and ovarian cancer, often at an earlier age than the general population. Patients with a personal history of breast cancer or a family history of breast and/or ovarian cancer, especially if at a young age, may have an elevated risk. A strong family history of uterine cancer, colon cancer, or other gastrointestinal cancers may indicate the presence of a syndrome known as hereditary nonpolyposis colorectal cancer (HNPCC, also known as Lynch II syndrome), which confers a higher risk for developing ovarian cancer.
Other factors that have been investigated, such as talc use, asbestos exposure, high dietary fat content, and childhood mumps infection, are controversial and have not been definitively proven.
Ovarian cancer is classified according to the histology of the tumor (ICD-O codes). Histology dictates many aspects of clinical treatment, management, and prognosis.
Ovarian tumors can be classified by their presumed cell of origin. The main categories are, surface epithelial-stromal tumours, sex cord-stromal tumours (ICD-O 8590), germ cell tumours (ICD-O 9060-9090) and secondary or metastatic tumours.
Surface epithelial-stromal tumours are the most common and prototypic ovarian cancers. They are thought to originate from the ovarian surface lining, and include serous cystadenocarcinoma (8441/3), and mucinous cystadenocarcinoma (8470/3). The abdominal cavity is lined with the same cells that make up the ovarian surface lining, and it is possible to have cancer begin there, in which case, it is called primary peritoneal cancer. Treatment, however, is basically the same as treatment for ovarian cancer.
Sex cord-stromal tumors (8590) include lesions that are hormonally active such as the estrogen-producing granulosa cell tumor (8620/3) and the virilizing Sertoli-Leydig cell tumor or arrhenoblastoma.
Germ cell tumors (9060-9090) of the ovary originate from germ cells and tend to occur in young women and girls. These tumors represent approximately 5% of ovarian cancers. They tend to be well encapsulated and many are benign, hence prognosis than for other ovarian tumors.
There are also mixed tumors secondary or metastatic tumors.
Ovarian cancer often is primary, but can also be secondary, i.e. the result of metastasis from primary cancers elsewhere in the body, for example, from breast cancer, or from gastrointestinal cancer, in which case the ovarian cancer is a Krukenberg cancer.
Historically ovarian cancer was called the “silent killer” because symptoms were not thought to develop until the chance of cure was poor. However, recent studies have shown this term is untrue and that the following symptoms are much more likely to occur in women with ovarian cancer than women in the general population. These symptoms include, bloating, pelvic or abdominal pain, difficulty eating or feeling full quickly, urinary symptoms (urgency or frequency).
Early stage diagnosis is associated with an improved prognosis.
Several other symptoms have been commonly reported by women with ovarian cancer. These symptoms include fatigue, indigestion, back pain, pain with intercourse, constipation and menstrual irregularities. However, these other symptoms are not as useful in identifying ovarian cancer because they are also found in equal frequency in women in the general population who do not have ovarian cancer.
Ovarian cancer at its early stages (I/II) is difficult to diagnose until it spreads and advances to later stages (III/IV). This is due to the fact that most of the common symptoms are non-specific.
Ovarian cancer has a poor prognosis. It is disproportionately deadly because symptoms are vague and non-specific, hence diagnosis is late. More than 60% of patients presenting with this cancer already have stage III or stage IV cancer, when it has already spread beyond the ovaries.
Ovarian cancers that are malignant shed cells into the naturally occurring fluid within the abdominal cavity. These cells can implant on other abdominal (peritoneal) structures included the uterus, urinary bladder, bowel, lining of the bowel wall (omentum) and can even spread to the lungs. These cells can begin forming new tumor growths before cancer is even suspected.
More than 50% of women with ovarian cancer are diagnosed in the advanced stages of the disease because no cost-effective screening test for ovarian cancer exists. The five year survival rate for all stages is only 35% to 38%. If, however, diagnosis is made early in the disease, five-year survival rates can reach 90% to 98%.
Hence, it would be advantageous to have method for the analysis of ovarian cancer disorders as well as a method for detection of ovarian cancer in a subject.
SUMMARY OF THE INVENTION
The present invention teaches a method for the analysis of ovarian cancer disorders, comprising determining the genomic methylation status of one or more CpG dinucleotides in a sequence selected from the group of SEQ ID NO. 1 to 91 and/or determining the genomic methylation status of one or more CpG dinucleotides in particular of sequences according to SEQ ID NO. 1 to 10 and/or SEQ ID NO. 50 to SEQ ID NO. 60.
The regions of interest are designated in table 1A and table 1B (“start” and “end”).
CpG islands are regions where there are a large number of cytosine and guanine adjacent to each other in the backbone of the DNA (i.e. linked by phosphodiester bonds). They are in and near approximately 40% of promoters of mammalian genes (about 70% in human promoters). The “p” in CpG notation refers to the phosphodiester bond between the cytosine and the guanine.
The length of a CpG island is typically 100-3000 base pairs. These regions are characterized by CpG dinucleotide content equal to or greater than what would be statistically expected (≈6%), whereas the rest of the genome has much lower CpG frequency (≈1%), a phenomenon called CG suppression. Unlike CpG sites in the coding region of a gene, in most instances, the CpG sites in the CpG islands of promoters are unmethylated if genes are expressed. This observation led to the speculation that methylation of CpG sites in the promoter of a gene may inhibit the expression of a gene. Methylation is central to imprinting alongside histone modifications. The usual formal definition of a CpG island is a region with at least 200 bp and with a GC percentage that is greater than 50% and with an observed/expected CpG ratio that is greater than 0.6.
Herein, a CpG dinucleotide is a CpG dinucleotide which may be found in methylated and unmethylated status in vivo, in particular in human.
The invention relates to a method, wherein a primary cancer is detected using the methylation pattern of one or more sequences disclosed herein and also, wherein the methylation pattern obtained is used to predict the therapeutic response to a treatment of an ovarian cancer.
Herein, a subject is understood to be all persons, patients, animals, irrespective whether or not they exhibit pathological changes. In the meaning of the invention, any sample collected from cells, tissues, organs, organisms or the like can be a sample of a patient to be diagnosed. In a preferred embodiment the patient according to the invention is a human. In a further preferred embodiment of the invention the patient is a human suspected to have a disease selected from the group of, primary ovarian cancer, secondary ovarian cancer, surface epithelial-stromal tumor, sex cord-stromal tumor, germ cell tumor.
The method is for use in the improved diagnosis, treatment and monitoring of ovarian cell proliferative disorders, for example by enabling the improved identification of and differentiation between subclasses of said disorder and the genetic predisposition to said disorders. The invention presents improvements over the state of the art in that it enables a highly specific classification of ovarian cell proliferative disorders, thereby allowing for improved and informed treatment of patients.
Herein, the sequences claimed also encompass the sequences which are reverse complement to the sequences designated.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the method for determination of differentially methylated regions of the genome. This is outlined in more detail in the Examples.
FIG. 2 shows clustered samples (columns) vs. methylation loci (rows). Methylation signatures can differentiate between tumors (left part of bar on top) and normal tissue (right part of bar on top).
FIG. 3 shows a clustering of ovarian samples based on the methylation features. Unsupervised clustering can differentiate between normal and tumor samples.



DETAILED DESCRIPTION OF EMBODIMENTS
The inventors have astonishingly found that a small selection of DNA sequences may be used to analyze ovarian cancer disorders. This is done by determining genomic methylation status of one or more CpG dinucleotides in either sequence disclosed herein or its reverse complement. About 900 sequences were identified in total that are suited for such an analysis. It turns out that 91 sequences are particularly suited.
Based on just 10 sequences, such as the first ten from table 1A or B (Pvalue 0.0001), it is possible to arrive at a classification accuracy for of 94%. The sequences may be found in genes as can be seen in table 1A below.













TABLE 1A





SEQ ID





Gene


NO.
ID
Chromosome
Start
End
P-val
Promoter







 1
ID88611
chr19
 5631787
 5631904
0.0000315
AY313896


 2
ID175860
chr9
 5440496
 5442012
0.0000634
BC069381


 3
ID83251
chr18
42804624
42805591
0.0000758
TCEB3C


 4
ID123662
chr22
46348054
46348410
0.0000952
FLJ46257


 5
ID90252
chr19
12706515
12706562
0.000100964
ASNA1


 6
ID88853
chr19
 6410747
 6411538
0.000109899
CRB3


 7
ID106715
chr2
1.53E+08
1.53E+08
0.000132458
FMNL2


 8
ID76937
chr17
45397876
45398117
0.000136
DLX4


 9
ID22548
chr10
1.01E+08
1.01E+08
0.000148441
NKX2-3


10
ID45743
chr13
35818685
35818932
0.000157858
SPG20


11
ID106499
chr2
1.39E+08
1.39E+08
0.000180479
LOC339745


12
ID131616
chr3
1.63E+08
1.63E+08
0.000188374
BC071875


13
ID33153
chr11
77528404
77528466
0.00021029
ALG8


14
ID69601
chr16
88084505
88084566
0.000224742
ANKRD11


15
ID55562
chr15
39412526
39412630
0.000231649
OIP5


16
ID82852
chr18
31178284
31178321
0.00026884
AF542097


17
ID188098
chrX
1.14E+08
1.14E+08
0.000276279
BC028688


18
ID125695
chr3
28365531
28365798
0.000277255
AZI2


19
ID69407
chr16
87450852
87451117
0.000303853
BC011369


20
ID147776
chr5
1.75E+08
1.75E+08
0.000354
DRD1


21
ID129197
chr3
1.12E+08
1.12E+08
0.000378924
BC067808


22
ID39382
chr12
54509420
54509575
0.000415538
AK057179


23
ID138427
chr4
1.21E+08
1.21E+08
0.00047605
MAD2L1


24
ID5570
chr1
31752564
31752750
0.000526242
HCRTR1


25
ID120807
chr22
23313706
23314029
0.000548859
LOC388886


26
ID175953
chr9
 6747544
 6747604
0.000597361
AB018323


27
ID163464
chr7
89868741
89869864
0.000623
PFTK1


28
ID119641
chr22
17268168
17268417
0.000639985
BC047039


29
ID43355
chr12
1.24E+08
1.24E+08
0.000681142
BRI3BP


30
ID148329
chr5
1.77E+08
1.77E+08
0.000707
NY-REN-7


31
ID178503
chr9
88845701
88845932
0.000731649
AK129921


32
ID21652
chr10
88717549
88718107
0.000744071
C10orf116


33
ID179700
chr9
1.07E+08
1.07E+08
0.000767134
RAD23B


34
ID77161
chr17
46299407
46299451
0.000793328
TOB1


35
ID40416
chr12
74712124
74712190
0.000802
PHLDA1


36
ID149652
chr6
 6572127
 6575902
0.000808
FLJ33708


37
ID56526
chr15
54812868
54813104
0.000829585
SUHW4


38
ID18295
chr10
14960697
14960796
0.000868353
SUV39H2


39
ID68291
chr16
82398697
82399030
0.000934789
HSBP1


40
ID51334
chr14
72673142
72673174
0.000939
PSEN1


41
ID128265
chr3
62836031
62836284
0.000954756
CADPS


42
ID100401
chr2
25387018
25387063
0.000974638
DNMT3A


43
ID184276
chrX
 550487
 550772
0.00100771
SHOX


44
ID2370
chr1
 7778659
 7778715
0.001043041
PER3


45
ID34541
chr11
1.18E+08
1.18E+08
0.001059334
MIZF


46
ID78653
chr17
68699787
68700038
0.001078955
COG1


47
ID55183
chr15
35180110
35180409
0.001083766
MEIS2


48
ID160402
chr7
27993506
27993623
0.00112
JAZF1


49
ID121081
chr22
27793491
27793540
0.001117003
BC063787









The sequences may also be found in intergenic regions as can be seen in table 1B below.












TABLE 1B





SEQ







ID

Chromo-





NO.
ID
some
Start
End
P-val




















50
ID89944
chr19
10843569
10843613
0.0000227


51
ID102184
chr2
63152348
63153687
0.0000231


52
ID28331
chr11
27698553
27698834
0.0000338


53
ID144851
chr5
114908035
114908080
0.0000553


54
ID128185
chr3
58546910
58547629
0.00008


55
ID93003
chr19
40483018
40483248
0.000110513


56
ID136801
chr4
68239844
68239927
0.000144546


57
ID146275
chr5
140146252
140146717
0.000215464


58
ID131177
chr3
148621317
148621647
0.000216975


59
ID12952
chr1
158307786
158308067
0.000242093


60
ID39999
chr12
63439190
63439288
0.000286113


61
ID116585
chr20
61967316
61967544
0.000287984


62
ID73971
chr17
26742913
26742971
0.000311873


63
ID125133
chr3
13654044
13654318
0.000372628


64
ID99092
chr2
1654591
1654895
0.000372925


65
ID69936
chr16
88767910
88769082
0.000397396


66
ID78601
chr17
67623230
67623629
0.00041658


67
ID148836
chr5
179854129
179854384
0.000420579


68
ID21285
chr10
79714238
79714714
0.000425735


69
ID158039
chr7
922643
922835
0.000459


70
ID32408
chr11
70345916
70347923
0.000464267


71
ID76532
chr17
43973948
43974107
0.00051907


72
ID178855
chr9
93408533
93408596
0.0005196


73
ID81125
chr17
78514384
78516444
0.000559665


74
ID100286
chr2
24625709
24625843
0.000582927


75
ID155118
chr6
119711684
119711950
0.000636


76
ID89463
chr19
8668749
8668987
0.000646711


77
ID9880
chr1
94718230
94718935
0.000655989


78
ID177108
chr9
37016858
37016916
0.000661917


79
ID68281
chr16
81219051
81219377
0.000665387


80
ID178263
chr9
83765733
83765839
0.000669707


81
ID34175
chr11
113165828
113166488
0.00067498


82
ID147847
chr5
175420376
175420628
0.000688163


83
ID47981
chr13
111756373
111756614
0.000693087


84
ID146308
chr5
140181734
140181814
0.000694524


85
ID17523
chr10
1273925
1274241
0.00074504


86
ID166673
chr7
149355326
149355615
0.000762


87
ID91016
chr19
15399966
15400044
0.000779943


88
ID101572
chr2
45143519
45143913
0.000805715


89
ID39294
chr12
52897679
52898035
0.000854819


90
ID50743
chr14
61349222
61349293
0.00087795


91
ID157888
chr7
750241
750295
0.000912









The genes that form the basis of the present invention are preferably to be used to form a “gene panel”, i.e. a collection comprising the particular genetic sequences of the present invention and/or their respective informative methylation sites. The formation of gene panels allows for a quick and specific analysis of specific aspects of ovarian cancer. The gene panel(s) as described and employed in this invention can be used with surprisingly high efficiency for the diagnosis, treatment and monitoring of and the analysis also of a predisposition to ovarian cell proliferative disorders in particular however to the detection of ovarian tumor.
In addition, the use of multiple CpG sites from a diverse array of genes allows for a relatively high degree of sensitivity and specificity in comparison to single gene diagnostic and detection tools.
The invention relates to a method for the analysis of ovarian cancer disorders, comprising determining the genomic methylation status of one or more CpG dinucleotides in a sequence selected from the group of sequences according to SEQ ID NO. 1 to SEQ ID NO. 10 and/or SEQ ID NO. 50 to SEQ ID NO. 60.
In one embodiment it is preferred that the methylation status of one or more of the sequences according to SEQ ID NO. 1 to 91 is determined, wherein the sequence has a p-value which is smaller than 0.0001 as designated in table 1A or 1B.
The methylation status of CpG islands is indicative of ovarian cancer. Preferably, however the methylation status is determined for each CpG and the differential methylation pattern is determined, because not all CpG islands necessarily need to be methylated.
In one embodiment of the method according to the invention the analysis is detection of ovarian cancer in a subject and wherein the following steps are performed, (a) providing a sample from a subject to be analyzed, (b) determining the methylation status of one or more CpG dinucleotides in a sequence selected from the group of sequences according to SEQ ID NO. 1 to SEQ ID NO. 10 and/or SEQ ID NO. 50 to SEQ ID NO. 60.
Optionally, additionally the following steps are performed, (a) the one or more results from the methylation status test is input into a classifier that is obtained from a Diagnostic Multi Variate Model, (b) the likelihood is calculated as to whether the sample is from a normal tissue or an ovarian cancer tissue and/or, (c) an associated p-value for the confidence in the prediction is calculated.
For example, we use a support vector machine classifier for “learning” the important features of a tumor or normal sample based on a pre-defined set of tissues from patients. The algorithm now outputs a classifier (an equation in which the variables are the methylation ratios from the set of features used). Methylation ratios from a new patient sample are then put into this classifier. The result can be 1 or 0. The distance from the marginal plane is used to provide the p-value.
It is preferred that the methylation status is determined for at least four of the sequences according to SEQ ID NO. 1 to 10 and/or SEQ ID NO. 50 to SEQ ID NO. 60.
It is preferred that additionally the methylation status is determined for one or more of the sequences according to SEQ ID NO. 11 to 49 and/or 61 to 91.
In one embodiment the methylation status is determined for at least ten sequences, twenty sequences, thirty sequences forty sequences or more than forty sequences of the sequences according to SEQ ID. NO. 1 to SEQ ID NO. 91. It is particularly preferred that the methylation status is determined for all of the sequences according to SEQ ID NO. 1 to SEQ ID NO. 91.
In one embodiment the methylation status is determined for the sequences according to SEQ ID. NO. 1 to SEQ ID NO. 10 and SEQ ID NO. 50 to SEQ ID NO. 60. In principle the invention also relates to determining the methylation status of only one of the sequences according to SEQ ID NO. 1 to SEQ ID NO. 91.
There are numerous methods for determining the methylation status of a DNA molecule. It is preferred that the methylation status is determined by means of one or more of the methods selected form the group of, bisulfite sequencing, pyrosequencing, methylation-sensitive single-strand conformation analysis (MS-SSCA), high resolution melting analysis (HRM), methylation-sensitive single nucleotide primer extension (MS-SnuPE), base-specific cleavage/MALDI-TOF, methylation-specific PCR (MSP), microarray-based methods, msp I cleavage. An overview of the further known methods of detecting 5-methylcytosine may be gathered from the following review article: Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998, 26, 2255. Further methods are disclosed in US 2006/0292564A1.
In a preferred embodiment the methylation status is determined by mspI cleavage, ligation of adaptors, McrBC digestion, PCR amplification, labeling and subsequent hybridization.
In a preferred embodiment the methylation status is determined as follows.
It is preferred that the sample to be analyzed is from a tissue type selected from the group of tissues such as, a tissue biopsy from the tissue to be analyzed, vaginal tissue, tongue, pancreas, liver, spleen, ovary, muscle, joint tissue, neural tissue, gastrointestinal tissue, tumor tissue, body fluids, blood, serum, saliva, and urine.
In a preferred embodiment a primary cancer is detected.
In one embodiment of the method according to the invention the methylation pattern obtained is used to predict the therapeutic response to the treatment of an ovarian cancer.
The invention relates to probes such as oligonucleotides which are in the region of up CpG sites. The oligomers according to the present invention are normally used in so called “sets” which contain at least one oligonucleotide for each of the CpG dinucleotides within SEQ ID NO. 1 through SEQ ID NO. 91 or at least for 10, preferred, 20, more preferred 30 most preferred more than 50 of said sequences. The invention also relates to the reverse complement of the oligonucleotides which are in the region of the CpG sites.
The probes to be used for such analysis are defined based on one or more of the following criteria: (1) Probe sequence occurs only once in the human genome; (2) Probe density of C/G nucleotides is between 30% and 70%; (3) Melting characteristics of hybridization and other criteria are according to Mei R et al, Proc Natl Acad Sci USA. 2003 Sep. 30; 100(20):11237-42.
In a very preferred embodiment the mention relates to a set of oligonucleotides, which are specific for the sequences according to SEQ ID NO. 1 to 10 and/or SEQ ID NO: 50 to 60, or SEQ ID NO. 50 to 60. The oligonucleotide according to the invention may be specific for the sequence as it occurs in vivo or it may be specific for a sequence which has been bisulfite treated. Such a probe is between 10 and 80 nucleotides long, more preferred between 15 and 40 nucleotides long.
In the case of the sets of oligonucleotides according to the present invention, it is preferred that at least one oligonucleotide is bound to a solid phase. It is further preferred that all the oligonucleotides of one set are bound to a solid phase.
The present invention further relates to a set of at least 10 probes (oligonucleotides and/or PNA-oligomers) used for detecting the cytosine methylation state of genomic DNA, by analysis of said sequence or treated versions of said sequence (according to SEQ ID NO. 1 through SEQ ID NO. 91 and sequences complementary thereto).
These probes enable improved detection, diagnosis, treatment and monitoring of ovarian cell proliferative disorders.
The set of oligonucleotides may also be used for detecting single nucleotide polymorphisms (SNPs) by analysis of said sequence or treated versions of said sequence according to one of SEQ ID NO. 1 through SEQ ID NO. 91.
According to the present invention, it is preferred that an arrangement of different oligonucleotides and/or PNA-oligomers (a so-called “array”) made available by the present invention is present in a manner that it is likewise bound to a solid phase.
This array of different oligonucleotide- and/or PNA-oligomer sequences can be characterised in that it is arranged on the solid phase in the form of a rectangular or hexagonal lattice. The solid phase surface is preferably composed of silicon, glass, polystyrene, aluminium, steel, iron, copper, nickel, silver, or gold. However, nitrocellulose as well as plastics, such as nylon which can exist in the form of pellets or also as resin matrices, are suitable alternatives.
Therefore, a further subject matter of the present invention is a method for manufacturing an array fixed to a carrier material for the improved detection, diagnosis, treatment and monitoring of ovarian cell proliferative disorders and/or detection of the predisposition to ovarian cell proliferative disorders. In said method at least one oligonucleotide according to the present invention is coupled to a solid phase. Methods for manufacturing such arrays are known, for example, from U.S. Pat. No. 5,744,305 by means of solid-phase chemistry and photolabile protecting groups. A further subject matter of the present invention relates to a DNA chip for the improved detection, diagnosis, treatment and monitoring of ovarian cell proliferative disorders. Furthermore, the DNA chip enables detection of the predisposition to ovarian cell proliferative disorders.
The DNA chip contains at least one nucleic acid and/or oligonucleotide according to the present invention. DNA-chips are known, for example, in U.S. Pat. No. 5,837,832.
The invention relates to a composition or array comprising nucleic acids with sequences which are identical to at least 10 of the sequences according to SEQ ID NO. 1 to 91, wherein the composition or array comprises no more than 100 different nucleic acid molecules.
The present invention relates to a composition or array comprising at least 5 sequences with a cumulative p-value of under 0.001, preferred under 0.0001.
Moreover, a subject matter of the present invention is a kit which may be composed, for example, of a bisulfitecontaining reagent, a set of primer oligonucleotides containing at least two oligonucleotides whose sequences in each case correspond to or are complementary to an at least 15 base long segment of the base sequences specified in SEQ ID NO. 1 to SEQ ID NO. 91. It is preferred that the primers are for SEQ ID NO. 1 through 10 and/or SEQ ID NO. 50 through SEQ ID NO. 60.
EXAMPLES
Samples
Patient samples were obtained from Norwegian Radium Hospital, Oslo, Norway and patient consent obtained as per legal requirements.
CpG Islands
Annotated CpG islands were obtained from the UCSC genome browser. These islands were predicted using the published Gardiner-Garden definition (Gardiner-Garden, M. and M. Frommer (1987). “CpG islands in vertebrate genomes.” J Mol Biol 196(2): 261-82) involving the following criteria: length >=200 bp, % GC>=50%, observed/expected CpG >=0.6. There are  ̃26219 CpG islands in the range of 200 bp to 2000 bp in the genome. These islands are well covered by Msp I restriction fragmentation.
Arrays were manufactured by Nimblegen Systems Inc using the 390K format to the following specifications. The CpG island annotation from human genome build 33 (hg17) was used to design a 50 mer tiling array. The 50 mers were shifted on either side of the island sequence coordinates to evenly distribute the island. The 390K format has 367,658 available features which would not fit all islands with a 50 mer tiling. Therefore we made a cutoff on the islands to be represented based on size, with only CpG islands of size 200b-2000b being assayed. Control probes were designed to represent background signal. Sample preparation: representations, has been described previously (Lucito, R., J. Healy, et al. (2003). “Representational oligonucleotide microarray analysis: a high-resolution method to detect genome copy number variation.” Genome Res 13(10): 2291-305.), with the following changes. The primary restriction endonuclease used is MspI. After the digestion the following linkers were ligated (MspI24mer, and MSPI12mer). The 12 mer is not phosphorylated and does not ligate. After ligation the material is cleaned by phenol chloroform, precipitated, centrifuged, and re-suspended. The material is divided in two, half being digested by the endonuclease McrBC and the other half being mock digested. As few as four 2500 tubes were used for each sample pair for amplification of the representation each with a 100 ul volume reaction. The cycle conditions were 95° C. for 1 min, 72° C. for 3 min, for 15 cycles, followed by a 10-min extension at 72° C. The contents of the tubes for each pair were pooled when completed. Representations were cleaned by phenol:chloroform extraction, precipitated, resuspended, and the concentration determined. DNA was labeled as described with minor changes (Lucito, R., J. Healy, et al. (2003). “Representational oligonucleotide microarray analysis: a high-resolution method to detect genome copy number variation.” Genome Res 13(10): 2291-305.). Briefly, 2 ug of DNA template was placed (dissolved in TE at pH 8) in a 0.2 mL PCR tube. 5 μl of random nonomers (Sigma Genosys) were added brought up to 25 μL with dH2O, and mixed. The tubes were placed in Tetrad at 100° C. for 5 min, then on ice for 5 min. To this 5 μl of NEB Buffer2, 5 μL of dNTPs (0.6 nm dCTP, 1.2 nm dATP, dTTP, dGTP), 5 μl of label (Cy3-dCTP or Cy5-dCTP) from GE Healthcare, 2 μl of NEB Klenow fragment, and 2 μl dH2O was added. Procedures for hybridization and washing were followed as reported previously (Lucito, R., J. Healy, et al. (2003). “Representational oligonucleotide microarray analysis: a high-resolution method to detect genome copy number variation.” Genome Res 13(10): 2291-305) with the exception that oven temperature for hybridization was increased to 50° C. Arrays were scanned with an Axon GenePix 4000B scanner set at a pixel size of 5 μm. GenePix Pro 4.0 software was used to quantify the intensity for the arrays. Array data were imported into S-PLUS for further analysis.
Data Analysis
Microarray images were scanned on GenePix 4000B scanner and data extracted using Nimblescan software (Nimblegen Systems Inc). For each probe, the geometric mean of the ratios (GeoMeanRatio) of McrBc and control treated samples were calculated for each experiment and its associated dye swap. The GeoMeanRatios of all the samples in a dataset were then normalized using quantile normalization method (Bolstad, B. M., R. A. Irizarry, et al. (2003). “A comparison of normalization methods for high density oligonucleotide array data based on variance and bias” Bioinformatics 19(2): 185-93). The normalized ratios for each experiment were then collapsed to get one value for all probes in every MspI fragment using a median polish model. The collapsed data was then used for further analysis.
Analysis of variance was used to identify the most significant islands. In order to determine the most consistently occurring changes in methylation between tumor and normal samples, we used a t-test approach. Using a p-value cutoff of 0.001 after correction for multiple testing (False Discovery Rate, Benjamini and Hotchberg (Benjamini 1995)), we obtained a list of 916 MspI fragments that show differential methylation
Supervised learning: We used a supervised machine learning classifier to identify the number of features required to differentiate tumor samples from normal. A publicly available support vector machine (SVM) library (LibSVM Ver 2.8) was used to obtain classification accuracy using a leave one out method (Lin, C.-C. C. a. C.-J. (2001). LIBSVM: a library for support vector machines). The methylation features for classification were first selected using t-test among the training data alone. The SVM was then trained on the top 10, 50 and 100 features using the radial basis function (RBF) kernel.
For N samples, t-tests were performed for (N−1) samples to identify fragments with significant differences in methylation ratios. For the ovarian dataset this was performed 18 times for all 18 ovarian samples, so that each sample is left out once during the t-test calculations. The methylation ratios of top 10 fragment features from (N−1) samples were then used for training the SVM and the ratios from one untrained sample was used for testing. Based on just 10 features, we can arrive at a classification accuracy of 94%. Interestingly the two tumor samples that were classified as normal in this analysis were also the closest to normal in both gene expression and ROMA analysis.
Detection of Methylated Sites
In a preferred embodiment, the method comprises the following steps: In the first step of the method the genomic DNA sample must be isolated from sources such as cell lines, tissue or blood samples. Extraction may be by means that are standard to one skilled in the art these include the use of detergent lysates, sonification and vortexing with glass beads. Once the nucleic acids have been extracted the genomic double stranded DNA is used in the analysis.
In a preferred embodiment the DNA may be cleaved prior to the next step of the method, this may by any means standard in the state of the art, in particular, but not limited to, with restriction endonucleases.
In the second step of the method, the genomic DNA sample is treated in such a manner that cytosine bases which are unmethylated at the 5′-position are converted to uracil, thymine, or another base which is dissimilar to cytosine in terms of hybridisation behaviour. This will be understood as ‘pretreatment’ hereinafter.
The above described treatment of genomic DNA is preferably carried out with bisulfite (sulfite, disulfite) and subsequent alkaline hydrolysis which results in a conversion of non-methylated cytosine nucleobases to uracil or to another base which is dissimilar to cytosine in terms of base vairine behaviour. If bisulfite solution is used for the reaction, then an addition takes place at the non-methylated cytosine bases. Moreover, a denaturating reagent or solvent as well as a radical interceptor must be present. A subsequent alkaline hydrolysis then gives rise to the conversion of non-methylated cytosine nucleobases to uracil. The converted DNA is then used for the detection of methylated cytosines.
Fragments are amplified. Because of statistical and practical considerations, preferably more than ten different fragments having a length of 100-2000 base pairs are amplified. The amplification of several DNA segments can be carried out simultaneously in one and the same reaction vessel. Usually, the amplification is carried out by means of a polymerase chain reaction (PCR). The design of such primers is obvious to one skilled in the art. These should include at least two oligonucleotides whose sequences are each reverse complementary or identical to an at least 15 base-pair long segment of the base sequences specified in the appendix (SEQ ID NO. 1 through SEQ ID NO. 91). Said primer oligonucleotides are preferably characterised in that they do not contain any CpG dinucleotides. In a particularly preferred embodiment of the method, the sequence of said primer oligonucleotides are designed so as to selectively anneal to and amplify, only the ovarian cell specific DNA of interest, thereby minimising the amplification of background or non relevant DNA. In the context of the present invention, background DNA is taken to mean genomic DNA which does not have a relevant tissue specific methylation pattern, in this case, the relevant tissue being ovarian cells, both healthy and diseased.
According to the present invention, it is preferred that at least one primer oligonucleotide is bound to a solid phase during amplification. The different oligonucleotide and/or PNA-oligomer sequences can be arranged on a plane solid phase in the form of a rectangular or hexagonal lattice, the solid phase surface preferably being composed of silicon, glass, polystyrene, aluminium, steel, iron, copper, nickel, silver, or gold, it being possible for other materials such as nitrocellulose or plastics to be used as well. The fragments obtained by means of the amplification may carry a directly or indirectly detectable label. Preferred are labels in the form of fluorescence labels, radionuclides, or detachable molecule fragments having a typical mass which can be detected in a mass spectrometer, it being preferred that the fragments that are produced have a single positive or negative net charge for better detectability in the mass spectrometer. The detection may be carried out and visualized by means of matrix assisted laser desorptiodionisation mass spectrometry (MALDI) or using electron Spray mass spectrometry (ESI).
In the next step the nucleic acid amplicons are analyzed in order to determine the methylation status of the genomic DNA prior to treatment.
The post treatment analysis of the nucleic acids may be carried out using alternative methods. Several methods for the methylation status specific analysis of the treated nucleic acids are known, other alternative methods will be obvious to one skilled in the art.
Using several methods known in the art the analysis may be carried out during the amplification step of the method. In one such embodiment, the methylation status of preselected CpG positions within the nucleic acids comprising SEQ ID NO. 1 through SEQ ID NO. 91 may be detected by use of methylation specific primer oligonucleotides. This technique has been described in U.S. Pat. No. 6,265,171.

## Claims

1. A method for analyzing methylation status in a human subject, comprising:
providing a sample from the subject;
determining a genomic methylation status of CpG dinucteotides in DNA from the subject, the DNA including sequences consisting of SEQ ID NO. 1 to SEQ ID NO. 10;
wherein the methylation status is determined by means of at least one of the methods selected from the group of:
bisulfite sequencing;
pyrosequencing;
methylation-sensitive single-strand conformation analysis (MS-SSCA);
high resolution melting analysis (HRM);
methylation-sensitive single nucleotide primer extension (MS-SnuPE);
base-specific cleavage/MALDI-TOF;
methylation-specific PCR (MSP);
microarray-based methods; and
msp I cleavage.

2. The method according to claim 1, wherein the methylation status is further determined in DNA sequences consisting of SEQ ID NO. 50 to SEQ ID NO. 60.

3. The method according to claim 1, wherein the methylation status is further determined in DNA sequences consisting of at least one of SEQ ID NO. 11 to 49 and SEQ ID NO. 61 to 91.

4. The method according to claim 1, wherein the sample to be analyzed is from a tissue type selected from the group of tissues:
a tissue biopsy from the tissue to be analyzed, vaginal tissue, tongue, pancreas, liver, spleen, ovary, muscle, joint tissue, neural tissue, gastrointestinal tissue, tumor tissue, body fluids, blood, serum, saliva and urine.