# Patent

## Title
Cancer screening method

## Abstract

A method for screening cancer comprises the following steps: (1) providing a test specimen; (2) detecting the methylation state of the CpG sequence in at least one target gene within the genomic DNA of the test specimen, wherein the target genes is consisted of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1; and (3) determining whether there is cancer or cancerous pathological change in the specimen based on the presence or absence of the methylation state in the target gene; wherein method for detecting methylation state is methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC), and pyrosequencing.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a cancer screening method, and in particular, to a cancer screening method using methylated DNA as the biomarker.
2. Description of the Prior Art
Cervical cancer has been one of the main causes of death in females worldwide and in Taiwan. Based on the statistical survey by the World Health Organization (WHO) in 2002, cervical cancer was the second major disease responsible for the death of women worldwide, second to breast cancer. Regular cervical cancer screening is the best way to prevent cervical cancer. Conventional cervical cancer screening includes two approaches: the most commonly used Pap smear, and human papilloma virus testing (HPV testing). Pap smear consists of sampling secreta from cervix uteri, examining under microscope whether there is cancerous pathological change in the exfoliated epithelial cell, so as to detect cervical cancer early. HPV testing, on the other hand, relies on the detection of human papilloma virus (HPV) DNA.
There are, however, many undesired properties of Pap smear. For one, it requires sampling by a physician, and analysis by a medical examiner/pathologist, which is a high cost of manpower that poses difficulty on promoting the test in many developing countries. Also, Pap smear has a high false negative rate which delays diagnosis and proper treatment prior to cancerous pathological change. As for HPV testing, although it is highly sensitive, it tends to create a high false positive rate, which not only leaves patients worry in vain, but also wastes much medical resources in examinations follow up to those false positive patients. Accordingly, one of the important topics in promoting cervical cancer examination relies on increasing the accuracy and convenience of cervical cancer examination method.
Infection with oncogenic human papilloma virus (HPV) is the most significant risk factor in the etiology of cervical cancer. E6/E7 oncoprotein encoded by “high-risk” HPV types has been shown to interact with the tumor-suppressor gene p53/pRB, causing abnormal cell-cycle regulation (zur Hausen 2000). HPV DNA could be detected in virtually all cases of cervical cancers (Walboomers, Jacobs et al. 1999). However, HPV infection is necessary but not sufficient to cause cervical cancer. About 60% of LSIL (low-grade squamous intraepithelial lesion) regress, 30% persists, 5-10% progress to high-grade SIL (HSIL, or High-grade squamous intraepithelial lesion) and only less than 1% becomes cervical cancer (Syrjanen, Vayrynen et al. 1985; Syrjanen 1996). Persistence of HPV infection and viral load may be detrimental accounting the development of HSIL and cancer (Ylitalo, Sorensen et al. 2000). However, the molecular mechanism of cervical carcinogenesis remains illusive.
Other factors, such as environmental and genetic alterations, may also play a decisive role in malignant conversion of cervical keratinocytes (Magnusson, Sparen et al. 1999; Ylitalo, Sorensen et al. 1999). Despite initiation by HPV, genetic changes with resultant genomic instability has long been recognized as an important mechanism for cervical carcinogenesis. Cytogenetic studies have revealed non-random chromosomal changes in cervical cancers (Mitra, Rao et al. 1994; Atkin and Baker 1997; Harris, Lu et al. 2003). Several molecular genetic studies have identified a few frequent loss of heterozygosity (LOH) sites, suggesting the involvement of tumor suppressor genes (TSGs) in the development of cervical cancer. (Mitra, Murty et al. 1994; Mullokandov, Kholodilov et al. 1996; Rader, Kamarasova et al. 1996; Kersemaekers, Hermans et al. 1998; Mitra 1999).
Genomic deletions have long been considered to be an important factor in tumorigenesis. For a long time, we have been accustomed to the idea that the coding potential of the genome lies within the arrangement of the four A, T, G, C bases. The two-hit theory proposed as early as in 1970s indicates concomitant mutations or deletions of some homologous tumor suppressor genes may cause or predispose to cancer development (Knudson, Hethcote et al. 1975; Knudson 2001). However, additional information that affects phenotype can be stored in the modified base 5-methylcytosine. 5-Methylcytosine is found in mammals in the context of the palindromic sequence 5′-CpG-3′. Most CpG dinucleotide pairs are methylated in mammalian cells except some areas called “CpG island.” CpG islands are GC- and CpG-rich areas of approximately 1 kb, usually located in the vicinity of genes and often found near the promoter of widely expressed genes (Bird 1986; Larsen, Gundersen et al. 1992). Cytosine methylation occurs after DNA synthesis, by enzymatic transfer of a methyl group from the methyl donor S-adenosylmethionine to the carbon-5 position of cytosine. The enzymatic reaction is performed by DNA methyltransferases (DNMTs)(Laird 2003). DNMT1 is the main enzyme in mammals, and is responsible for the post-replicative restoration of hemi-methylated sites to full methylation, referred to as maintenance methylation, whereas DNMT3A and DNMT3B are thought to be involved primarily in methylating new sites, a process called de novo methylation (Okano, Bell et al. 1999; Robert, Morin et al. 2003).
Loss of methylation at CpG dinucleotides, i.e., general hypomethylation, was the first epigenetic abnormalities identified in cancer cells (Feinberg and Vogelstein 1983; Cheah, Wallace et al. 1984). However, during the past few years, it has become increasing apparent that site-specific hypermethylation, e.g., some tumor suppressor genes, is associated with loss of function which may provide selective advantages during carcinogenesis (Jones and Baylin 2002; Feinberg and Tycko 2004). Dense methylation of CpG islands at promoter regions can trigger chromatin remodeling through histone modifications with subsequent gene silencing (Geiman and Robertson 2002; Egger, Liang et al. 2004). Therefore, in addition to chromosomal deletions or genetic mutations, epigenetic silencing of tumor suppressor genes by promoter hypermethylation is commonly seen in human cancer (Baylin, Herman et al. 1998; Jones and Laird 1999; Baylin and Herman 2000).
Epidemiologic studies have recently shown the correlation of serum folate level, a major source of methyl group, with the infection and clearance of HPV (Piyathilake, Henao et al. 2004). Genetic polymorphisms of enzymes in the metabolism of methyl cycle were also reported to be associated with the development of cervical intraepithelial lesions (Henao, Piyathilake et al. 2004). As the concept of epigenetics evolves, studies exploring the association between DNA methylation and cervical cancer are also booming. Studies of DNA methylation in cervical cancer are accumulating, which showed the potential of using methylation as markers in cervical screening (Feng, Balasubramanian et al. 2005). With the nature of the interface between genetics and environment, the prevalence of methylation in tumor suppressor genes varies in different genes and different populations. The concept of methylator phenotypes with different disease behaviors was proposed with controversy. The methylator phenotype of cervical cancer and its interaction with HPV genotypes still remains unknown. The extent to which adenocarcinoma can be analogue to squamous cell carcinoma in terms of methylation patterns has never been investigated. What genes are specifically methylated in cervical cancer and how many genes are required to achieve clinical application will remain a blossoming issue in the coming future. The excavation of genes with higher contribution component to cervical carcinogenesis may shed light on the promise of using DNA methylation as a diagnostic marker as well as the development of a novel therapeutic intervention through epigenetic modulation.
SUMMARY OF THE INVENTION
The invention provides a cancer diagnostic method. The method uses the degree of methylation of a specific gene as the index to diagnose whether there is presence of cancer. The cancer diagnostic method according to the invention is applicable on the detection of cervical cancer. In addition to be the first line screening for cervical cancer, the cancer diagnostic method according to the invention can be used as the second line screening for cervical cancer in combination with or as an assistant to HPV testing in order to achieve a more accurate screening result for cervical cancer. Furthermore, the cancer diagnostic method according to the invention is capable of detecting other cancer types such as ovarian cancer, liver cancer and the like, to facilitate the diagnosis of other abnormal specimens.
In using the cancer diagnostic method according to the invention on the detection of cervical cancer, it exhibits a sensitivity and specificity higher than those of the Pap smear and HPV testing.
Accordingly, the invention provides a method for the diagnosis of cancer, characterized in that it comprises of detecting the methylation state of the target gene in the cell of the test specimen as a screening index to determine the existence of cancer, the method comprising the following steps:
step 1: providing a test specimen;
step 2: detecting the methylation state of the CpG sequence in at least one target gene within the genomic DNA of the test specimen, wherein the target genes is consisted of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1; and
step 3: determining whether there is cancer or cancerous pathological change in the specimen based on the presence or absence of the methylation state in the target gene.
The test specimens may be a cervical smear, ascites, blood, urine, feces, sputum, oral mucosa cell, gastric juice, bile, cervical epithelial cell and the like.
Method for detecting the methylation state of the CpG sequence in the target gene may be a methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC), and pyrosequencing.
The target gene SOX1 has a nucleotide sequence as depicted in SEQ ID No: 1.
The target gene PAX1 has a nucleotide sequence as depicted in SEQ ID No: 2.
The target gene LMX1A has a nucleotide sequence as depicted in SEQ ID No: 3.
The target gene NKX6-1 has a nucleotide sequence as depicted in SEQ ID No: 4.
The target gene WT1 has a nucleotide sequence as depicted in SEQ ID No: 5.
The target gene ONECUT1 has a nucleotide sequence as depicted in SEQ ID No: 6.
The invention provides a method for screening cervical cancer, characterized in that it comprises of detecting the methylation state of the target gene in the cell of the test specimen as a screening index to determine the existence of the cervical cancer, the method comprising the following steps:
step 1: providing a test specimen;
step 2: detecting the methylation state of the CpG sequence in at least one target gene within the genomic DNA of the test specimen, wherein the target genes is consisted of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1; and
step 3: determining whether there is cervical cancer or cancerous pathological change in the specimen based on the presence or absence of the methylation state in the target gene.
The test specimens may be a cervical smear, blood, urine, cervical epithelial cell and the like.
In one embodiment, the test specimen is a cervical smear.
In one embodiment, the test specimen is a cervical cell specimen exhibiting a positive HPV testing.
Method for detecting the methylation state of the CpG sequence in the target gene may be a methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC), and pyrosequencing.
The target gene SOX1 has a nucleotide sequence as depicted in SEQ ID No: 1.
The target gene PAX1 has a nucleotide sequence as depicted in SEQ ID No: 2.
The target gene LMX1A has a nucleotide sequence as depicted in SEQ ID No: 3.
The target gene NKX6-1 has a nucleotide sequence as depicted in SEQ ID No: 4.
The target gene WT1 has a nucleotide sequence as depicted in SEQ ID No: 5.
The target gene ONECUT1 has a nucleotide sequence as depicted in SEQ ID No: 6.
The invention provides further a method for screening ovarian cancer, characterized in that it comprises of detecting the methylation state of the target gene in the cell of the test specimen as a screening index to determine the existence of ovarian cancer, the method comprising following steps:
step 1: providing a test specimen;
step 2: detecting the methylation state of the CpG sequence in at least one target gene within the genomic DNA of the test specimen, wherein the target genes is consisted of SOX1, PAX1, and LMX1A; and
step 3: determining whether there is an ovarian cancer or cancerous pathological change in the specimen based on the presence or absence of the methylation state in the target gene.
The test specimens may be an ascites, blood, urine and the like.
Method for detecting the methylation state of the CpG sequence in the target gene may be a methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC), and pyrosequencing.
The target gene SOX1 has a nucleotide sequence as depicted in SEQ ID No: 1.
The target gene PAX1 has a nucleotide sequence as depicted in SEQ ID No: 2.
The target gene LMX1A has a nucleotide sequence as depicted in SEQ ID No: 3.
The invention provides further a method screening liver cancer, characterized in that it comprises of detecting the methylation state of the target gene in the cell of the test specimen as a screening index to determine the existence of liver cancer, the method comprising following steps:
step 1: providing a test specimen;
step 2: detecting the methylation state of the CpG sequence in at least one target gene within the genomic DNA of the test specimen, wherein the target genes is consisted of SOX1, and NKX6-1; and
step 3: determining whether there is a liver cancer or cancerous pathological change in the specimen based on the presence or absence of the methylation state in the target gene.
The test specimens may be an ascites, blood, urine, feces, gastric juice, bile, and the like.
Method for detecting the methylation state of the CpG sequence in the target gene may be a methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC), and pyrosequencing.
The target gene SOX1 has a nucleotide sequence as depicted in SEQ ID No: 1.
The target gene NKX6-1 has a nucleotide sequence as depicted in SEQ ID No: 4.
These features and advantages of the present invention will be fully understood and appreciated from the following detailed description of the accompanying Drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the analysis of CpG sequences in various target gene used in the cancer screening method according to the invention, wherein CpG sequences in various genes are marked with , positions occupied by synthetic fragments of MSP primers in various MSP genes are marked with , and positions occupied by synthetic fragments of bisulfite-sequenced primer in various genes are marked with .
FIG. 2 shows results of methylation-specific PCR (MSP) analysis of various target genes used in the cancer screening method according to the invention, in mixed cervical cancer tissue specimens (a mixture of 30 specimens) as well as in mixed normal cervical smear specimens (a mixture of 10 specimens): the first column, mixed normal cervical smear specimens (a mixture of 10 specimens); the second column mixed cervical cancer tissue specimens (a mixture of 30 specimens); the third column, negative control; the fourth column, positive control; and the fifth column, blank control (water).
FIG. 3 shows results of methylation-specific PCR (MSP) analysis of various target genes used in the cancer screening method according to the invention, in individual cervical cancer tissue specimens as well as in individual normal cervical smear specimens: T1, T2, T3 and T4 represent 4 individual cervical cancer tissue specimens, while N1, N2, N3 and N4 represent 4 normal specimens; entries labeled with U indicate results from the methylation-specific PCR (MSP) conducted with MSP primers (U) that can recognize specifically the non-methylated gene sequences; while entries labeled with M indicate results from the methylation-specific PCR (MSP) conducted with MSP primers (M) that can recognize specifically the methylated gene sequences.
FIG. 4A shows results of methylation-specific PCR (MSP) analysis conducted with various target genes used in the cancer screening method according to the invention in non-5′-aza-2′-deoxycytidine-treated HeLacervical cancer cell line (AZC−, the first and second columns), as well as in 5′-aza-2′-deoxycytidine-treated HeLacervical cancer cell line (AZC+, the third and fourth columns); entries labeled with U indicate results from the methylation-specific PCR (MSP) conducted with MSP primers (U) that can recognize specifically the non-methylated gene sequences; while entries labeled with M indicate results from the methylation-specific PCR (MSP) conducted with MSP primers (M) that can recognize specifically the methylated gene sequences.
FIG. 4B shows results of RT-PCR analysis conducted with various target genes used in the cancer screening method according to the invention in non-5′-aza-2′-deoxycytidine-treated HeLacervical cancer cell line (AZC−, the fifth column), as well as in 5′-aza-2′-deoxycytidine-treated HeLacervical cancer cell line (AZC+, the sixth column).
FIG. 5A shows results of bisulfite sequencing (BS) analysis conducted with various target genes used in the cancer screening method according to the invention in non-5′-aza-2′-deoxycytidine-treated HeLacervical cancer cell line.
FIG. 5B shows results of bisulfite sequencing (BS) analysis conducted with various target genes used in the cancer screening method according to the invention in 5′-aza-2′-deoxycytidine-treated HeLacervical cancer cell line.
FIG. 6A shows results of bisulfite sequencing (BS) analysis conducted with various target genes used in the cancer screening method according to the invention in the cervical squamous cell carcinoma (SCC).
FIG. 6B shows results of bisulfite sequencing (BS) analysis conducted with various target genes used in the cancer screening method according to the invention in the normal specimen.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
EXAMPLE 1
Materials and Methods
1. Tissue Specimens
Cervical tissue specimens were obtained from patients with normal uterine cervixes (n=45) and patients with LSIL (n=45), HSIL (n=58), and invasive squamous cell carcinoma (SCC; n=109) of the uterine cervix. The patients were diagnosed, treated, and tissue banked at the Tri-Service General Hospital, Taipei, Taiwan, since 1993. For diagnostic purposes, cytological, histological, and clinical data for all patients were reviewed by a panel of colposcopists, cytologists, and pathologists. All patients were examined and treated using a standard hospital protocol for cervical neoplasia. Controls were recruited from healthy women who underwent routine Pap screening during the same period. Informed consent was obtained from all patients and control subjects. Exclusion criteria included pregnancy, chronic or acute viral infection, a history of cervical neoplasia, skin or genital warts, an immune-compromised state, the presence of other cancers, and past surgery of the uterine cervix. The study was approved by the Institutional Review Board of the Tri-Service General Hospital.
The tissue specimens also include a series of ovarian tumor samples, which were obtained from the tumor bank of Tri-Service General Hospital, and the ovarian samples include benign ovarian samples (n=36), borderline ovarian tumors (n=6), and malignancy ovarian tumors (n=122).
In addition, the liver samples used in the study includes normal liver samples (n=13), chronic hepatitis (n=15), cirrhosis of the liver (n=40), and hepatocellular carcinoma (HCC, n=54). All the liver samples were from the tumor bank of Tri-Service General Hospital.
2. Preparation of Genomic DNA
Genomic DNA was extracted from specimens using Qiagene DNA Extraction Kits. The concentration of DNA was determined using the PicoGreen fluorescence absorption method, and DNA quality was verified using agarose gel electrophoresis.
3. Differential Methylation Hybridization (DMH) Using CpG Island Microarrays
Differential Methylation Hybridization (DMH) was performed according to Yan et al. Pooled DNA from 30 cancer tissues and 10 normal cervical swabs were used for comparison. DNA was digested using MseI, ligated to linkers, and sequentially digested with methylation-sensitive restriction enzymes (HpaII and BstUI). The digested linker-ligated DNA was used as a template for polymerase chain reaction (PCR) amplification (20 cycles) and coupled to fluorescence dyes (Cy3: pooled normal cervical sample; Cy5: pooled cervical cancer sample) before hybridizing to the CpG island microarray containing 8,640 CpG island tags (University of Toronto). The identity of selected CpG islands (CGIs) was obtained from the CGI database (http://derlab.med.utoronto.ca/CpGIslands/). The microarray data were analyzed using the circular-features mode of GenePix 6.0 software. Spots representing repetitive clones were flagged and unacceptable features were removed by filtering. Loci with ratios >2.0 were accepted as hypermethylated in the pooled cervical cancer sample.
4. Bisulfite Modification, Methylation-Specific PCR (MS-PCR), and Bisulfite Sequencing
A DNA modification kit (Chemicon, Ternecula, Calif.) was used according to the manufacturer's recommendations to convert 1 μg aliquots of genomic DNA with sodium bisulfite to preserve the methylated cytosines. The final precipitate was eluted with 70 μl of prewarmed (55° C.) TE buffer for MS-PCR.
MS-PCR was performed according to Herman et al. (1996). In short, 1 μl of modified DNA was amplified using MS-PCR primers (table 1) that specifically recognized either the unmethylated or the methylated gene sequences present in the bisulfite-converted DNA. Methylation-specific PCR was done in a total volume of 25 μl containing 1 μl of modified template DNA, 1.5 pmol of each primer, 0.2 mmol/L deoxynucleotide triphosphates, and 1 unit of Gold Taq DNA polymerase (Applied Biosystems, Foster City, Calif.). MS-PCR reactions were subjected to an initial incubation at 95° C. for 5 min, followed by 35 cycles of 95° C. for 30 s, and annealing at the appropriate temperature for 30 s and at 72° C. for 30 s. The final extension was done at 72° C. for 5 min.








TABLE 1







The sequences of MS-PCR primers










Gene
Primer
Sequence















SOX1
M
Forward (F′)
5′ CGTTTTTTTTTTTTCGTTATTGGC 3′
(SEQ ID No: 7)





Reverse (R′)
5′ CCTACGCTCGATCCTCAACG 3′
(SEQ ID No: 8)



U
Forward (F′)
5′ TGTTTTTTTTTTTTTGTTATTGGTG 3′
(SEQ ID No: 9)




Reverse (R′)
5′ CCTACACTCAATCCTCAACAAC 3′
(SEQ ID No: 10)


 


LMX1A
M
Forward (F′)
5′ TTTAGAAGCGGGCGGGAC 3′
(SEQ ID No: 11)




Reverse (R′)
5′ CCGAATCCAAACACGCG 3′
(SEQ ID No: 12)



U
Forward (F′)
5′ GAGTTTAGAAGTGGGTGGGATG 3′
(SEQ ID No: 13)




Reverse (R′)
5′ CAACCAAATCCAAACACACAAAAC 3′
(SEQ ID No: 14)


 


ONECUT1
M
Forward (F′)
5′ TTGTAGCGGCGGTTTTAGGTC 3′
(SEQ ID No: 15)




Reverse (R′)
5′ GCCAAACCCTTAACGTCCCG 3′
(SEQ ID No: 16)



U
Forward (F′)
5′ GATTGTAGTGGTGGTTTTAGGTTG 3′
(SEQ ID No: 17)




Reverse (R′)
5′ CACCAAACCCTTAACATCCCAATAC 3′
(SEQ ID No: 18)


 


PAX1
M
Forward (F′)
5′ TATTTTGGGTTTGGGGTCGC 3′
(SEQ ID No: 19)




Reverse (R′)
5′ CCCGAAAACCGAAAACCG 3′
(SEQ ID No: 20)



U
Forward (F′)
5′ GTTTATTTTGGGTTTGGGGTTGTG 3′
(SEQ ID No: 21)




Reverse (R′)
5′ CACCCAAAAACCAAAAACCAC 3′
(SEQ ID No: 22)


 


NKX6.1
M
Forward (F′)
5′ CGTGGTCGTGGGATGTTAGC 3′
(SEQ ID No: 23)




Reverse (R′)
5′ ACAAACAACGAAAAATACGCG 3′
(SEQ ID No: 24)



U
Forward (F′)
5′ GTGTGGTTGTGGGATGTTAGTG 3′
(SEQ ID No: 25)




Reverse (R′)
5′ CAACAAACAACAAAAAATACACAAC 3′
(SEQ ID No: 26)


 


WT1
M
Forward (F′)
5′ TGTTGAGTGAATGGAGCGGTC 3′
(SEQ ID No: 27)




Reverse (R′)
5′ CGAAAAACCCCCGAATATAAACG 3′
(SEQ ID No: 28)



U
Forward (F′)
5′ GTTGTTGAGTGAATGGAGTGGTTG 3′
(SEQ ID No: 29)




Reverse (R′)
5′ AATTACAAAAAACCCCCAAATATAAACAC 3′
(SEQ ID No: 30)





M: The primers can specifically recognize the methylated gene sequences present in the bisulfite-converted DNA.


U: The primers can specifically recognize the unmethylated gene sequences present in the bisulfite-converted DNA.






Normal DNA from human peripheral blood was modified with sodium bisulfite and used as a control for the unmethylated promoter sequence. Normal human DNA was treated with SssI methyltransferase (New England Biolabs, Beverly, Mass.) to generate a positive control for methylated alleles. Amplification products were visualized on 2.5% agarose gel containing ethidium bromide and illuminated under UV light. All MS-PCR data were derived from at least two independent modifications of DNA. The absence of signal in duplicate experiments was scored as negative methylation. Bisulfite-treated genomic DNA was amplified using primers (table 2) and amplified PCR product was purified and cloned into pCR4-TOPO vectors (Invitrogen, Carlsbad, Calif.). Bisulfite sequencing was performed on at least five individual clones using the 377 automatic sequencer (Applied Biosystems, Foster City, Calif.).








TABLE 2







The sequences of Bisulfite sequencing primers










Gene
Primer
Sequence















Sox1

Forward (F′)
5′ GTTGTTTTYGGGTTTTTTTTTGGTTG 3′
(SEQ ID No: 31)





Reverse (R′)
5′ ATTTCTCCTAATACACAAACCACTTACC 3′
(SEQ ID No: 32)


 


LMX1A

Forward (F′)
5′ TAGTTATTGGGAGAGAGTTYGTTTATTAG 3′
(SEQ ID No: 33)




Reverse (R′)
5′ CTACCCCAAATCRAAAAAAAACAC 3′
(SEQ ID No: 34)


 


ONECUT1

Forward (F′)
5′ GAGTTTATTTAAGTAAGGGAGG 3′
(SEQ ID No: 35)




Reverse (R′)
5′ CAACTTAAACCATAACTCTATTACTATTAC 3′
(SEQ ID No: 36)


 


PAX1
BS1
Forward (F′)
5′ GTGTTTTGGGAGGGGGTAGTAG 3′
(SEQ ID No: 37)




Reverse (R′)
5′ CCCTCCCRAACCCTACCTATC 3′
(SEQ ID No: 38)



BS2
Forward (F′)
5′ GATAGAAGGAGGGGGTAGAGTT 3′
(SEQ ID No: 39)




Reverse (R′)
5′ TACTACCCCCTCCCAAAACAC 3′
(SEQ ID No: 40)


 


NKX6.1
BS1
Forward (F′)
5′ GGTATTTTTGGTTTAGTTGGTAGTT 3′
(SEQ ID No: 41)




Reverse (R′)
5′ AATACCCTCCATTACCCCCACC 3′
(SEQ ID No: 42)



BS2
Forward (F′)
5′ GGTGGGGGTAATGGAGGGTATT 3′
(SEQ ID No: 43)




Reverse (R′)
5′ CCTAAATTATAAATACCCAAAAAC 3′
(SEQ ID No: 44)


 


WT1

Forward (F′)
5′ GTGTTGGGTTGAAGAGGAGGGTGT 3′
(SEQ ID No: 45)




Reverse (R′)
5′ ATCCTACAACAAAAAAAAATCCAAAATC 3′
(SEQ ID No: 46)









5. Re-Expression of Methylated Genes by 5′-aza-2′-Deoxycytidine Treatment in Cancer Cell Lines

The methylation status of candidate genes was tested in HeLa cervical cancer cell line using MS-PCR. Re-expression of methylated genes in cervical cancer cell lines after treatment with 10 μM of 5′-aza-2′-deoxycytidine (AZC) (Sigma Chemical Co.) for four days was assessed by RT-PCR. Total RNA was extracted using a Qiagen RNeasy kit (Qiagen, Valencia, Calif.). An additional DNase I digestion procedure (Qiagen) was included in the isolation of RNA to remove DNA contamination. One microgram of total RNA from each sample was subjected to cDNA synthesis using Superscript II reverse transcriptase and random hexamer (Invitrogen). The cDNA that was generated was used for PCR amplification with the reagents in the PCR master mix reagents kit (Applied Biosystems) as recommended by the manufacturer. The reactions were carried out in a thermal cycler (GeneAmp 2400 PE, Applied Biosystems). The primers and conditions for the PCR are listed in Table 3.








TABLE 3







The sequences of MSP primers for RT-PCR










Gene
Primer
Sequence














SOX1
Forward (F′)
5′ AGACCTAGATGCCAACAATTGG 3′
(SEQ ID No: 47)




Reverse (R′)
5′ GCACCACTACGACTTAGTCCG 3′
(SEQ ID No: 48)


 


LMX1A
Forward (F′)
5′ GCTGCTTCTGCTGCTGTGTCT 3′
(SEQ ID No: 49)



Reverse (R′)
5′ ACGTTTGGGGCGCTTATGGTC 3′
(SEQ ID No: 50)


 


ONECUT1
Forward (F′)
5′ CAAACCCTGGAGCAAACTCAA 3′
(SEQ ID No: 51)



Reverse (R′)
5′ TGTGTTGCCTCTATCCTTCCC 3′
(SEQ ID No: 52)


 


PAX1
Forward (F′)
5′ CCTACGCTGCCCTACAACCACATC 3′
(SEQ ID No: 53)



Reverse (R′)
5′ TCACGCCGGCCCAGTCTTCCATCT 3′
(SEQ ID No: 54)


 


NKX6.1
Forward (F′)
5′ CACACGAGACCCACTTTTTCC 3′
(SEQ ID No: 55)



Reverse (R′)
5′ CCCAACGAATAGGCCAAACG 3′
(SEQ ID No: 56)


 


WT1
Forward (F′)
5′ GCTGTCCCACTTACAGATGCA 3′
(SEQ ID No: 57)



Reverse (R′)
5′ TCAAAGCGCCAGCTGGAGTTT 3′
(SEQ ID No: 58)









6. HPV Detection

The presence of HPV DNA in SCC was detected by L1 consensus PCR followed by a reverse line blot (Gravitt, et al., 1998; Lai, et al., 2005). DNA sequencing was used to verify novel HPV types that exceeded the detection spectrum of the hybridization procedure.
7. Statistical Analysis
Data analysis was carried out using statistical package SAS version 9.1. Associations between the methylation of genes and clinical parameters, including HPV status, were analyzed using a X2 test and Fisher's exact test, wherever appropriate. Odds ratios (ORs) and 95% confidence intervals (95% CI) were calculated and adjusted for age and HPV infection using a logistic regression model. The alpha level of statistical significance was set at p=0.05. The sensitivity and specificity using HPV and methylation markers for the diagnosis of cervical lesions were calculated. The 95% CI was estimated using the BINOMIAL option in the EXACT statement.
EXAMPLE 2
Identification of Methylated Genes in Invasive Squamous Cell Carcinoma of the Cervix
Differential methylation hybridization (DMH) was carried out by means of CpG island microarrays to screen out the highly methylated gene in cervical squamous cell carcinoma (SCC). The result from CpG island microarrays revealed that there were 216 points exhibited differential methylation between cervical cancer tissue specimens and normal cervical smears, of which, after taking off those having overlapped sequences, 26 gene promoter domain CpG islands (promoter CGIs).
Sequencing and analysis were carried out on these gene promoter and 6 genes were selected. These genes included: SOX1 (SEQ ID No: 1), PAX1 (SEQ ID No: 2), LMX1A (SEQ ID No: 3), NKX6-1 (SEQ ID No: 4), WT1 (SEQ ID No: 5) and ONECUT1 (SEQ ID No: 6). Their detailed information were shown in Table 4. All of these 6 genes are important transcription factors in the development course, of which, SOX1, PAX1, LMX1A, NKX6-1, and WT1 were vital for the development of brain, roof plate, extremities, pancreatic island and urogenital organ, respectively, while ONECUT1 is important for the performance of hepatic and pancreatic genes. However, little correlation between these genes and cancer has been disclosed so far.







TABLE 4







Characteristics of methylated genes in cervical cancer that were


identified using a CpG island microarray













Chromosomal




Gene
UniGene
location
Full name
Known molecular function





SOX1
NM_005986
13q34
Sex
DNA binding





determining
Transcription factor activity





region Y-box 1


PAX1
NM_006192
20p11.2
Paired box
DNA binding





gene 1


LMX1A
NM_177398
1q22-q23
LIM
Transcription factor activity





homeobox
Zinc ion binding





transcription





factor 1 alpha


NKX6-1
NM_006168
4q21.2-q22
NK6
Transcription factor activity





transcription





factor related





locus 1


ONECUT1
NM_004498
15q21.1-q21.2
One cut
Transcription factor activity





domain family
Transcriptional activator





member 1
activity


WT1
NM_024426
11p13
Wilm's tumor 1
Transcription factor activity






Zinc ion binding









CpG sequence analysis was carried out over about 500 bp nucleotides before and after each gene transcription initiation point (+1). As shown in FIG. 1, various genes containing CpG sequence are marked with . MSP primer (as shown in Table 1) and bisulfite sequencing (BS) primer (as shown in Table 2) were designed with respect to each gene. Positions occupied by fragments synthesized during methylation-specific PCR (MSP) and bisulfite sequencing (BS) over various genes are shown also in FIG. 1.
Then, methylation-specific PCR (MSP) analysis were carried out on mixed cervical cancer tissue specimens (a mixture of 30 specimens) as well as on mixed normal cervical smear specimens (a mixture of 10 specimens) in order to confirm whether the methylation phenomena of these 6 genes were different in different tissue specimens. As indicated from results shown in FIG. 2, these 6 genes exhibited methylation in mixed cervical cancer tissue specimens (as shown at column 2 in FIG. 2), while no methylation was occurred in mixed normal cervical smear specimens (as shown at column 1 in FIG. 2). Further testing was carried out with individual cervical cancer tissue specimen. Methylation-specific PCR (MSP) was performed on 4 cervical cancer tissue specimens (T1, T2, T3, T4) and 4 normal specimens (N1, N2, N3, N4), respectively, with MSP primer (U) that could recognize specifically non-methylated gene sequence as well as with MSP primer (M) that could recognize specifically methylated gene sequence. Results shown in FIG. 3 revealed that all of these 6 genes exhibited methylation in individual cervical cancer tissue specimen (as shown at columns 1, 3, 5, and 7 in FIG. 3), while no methylation could be detected in normal specimens with these same genes (as shown at columns 9, 11, 13, and 15 in FIG. 3). Based on the above-described results, these 6 genes were used as the methylation indicator genes for screening cervical cancer.
EXAMPLE 3
Association of DNA Methylation and Gene Expression in HeLa Cervical Cancer Cell Line
In order to confirm whether the expression of cervical cancer methylation indicator gene is regulated through DNA methylation, HeLa cervical cancer cell line was treated with 10 μM of DNA methyltransferase inhibitor, 5′-aza-2′-deoxycytidine (AZC) (Sigma Chemical Co.), for 4 days, following by checking the demethylation by the 6 gene promoters described above by means of methylation-specific PCR (MSP) carried out with MSP primer (U) that could recognize specifically non-methylated gene sequence, as well as with MSP primer (M) that could recognize specifically methylated gene sequence, respectively. Results as shown in FIG. 4A indicated that among non-5′-aza-2′-deoxycytidine (AZC)-treated HeLa cervical cancer cell lines (AZC−), 6 gene promoters exhibited methylated conditions (as shown at column 1 in FIG. 4A), and no non-methylated gene was detected (as shown at column 2 in FIG. 4A). On the other hand, after treated with 5′-aza-2′-deoxycytidine for 4 days, non-methylated target gene could be detected in HeLa cervical cancer cell lines (AZC+) (as shown at column 4 in FIG. 4A), indicating that through treated with methyltransferase inhibitor, 5′-aza-2′-deoxycytidine (AZC), the above-described 6 target genes had been demethylated partially.
Next, expressions of these 6 genes in HeLa cervical cancer cell line were analyzed through RT-PCR. Results shown in FIG. 4B indicated that in cell lines treated with 5′-aza-2′-deoxycytidine (AZC), mRNA of these 6 target genes could be detected (as shown at column 6 in FIG. 4B), while in those cell lines that had not been treated with 5′-aza-2′-deoxycytidine (AZC), no mRNA of any one target gene could be detected (as shown at column 5 in FIG. 4B). It is evident from these results that gene expression of these 6 target genes in cervical cancer cell could be modified actually through DNA methylation. As gene had been methylated, its expression could be inhibited, whereas after demethylated, the target gene could be re-expressed.
Furthermore, bisulfite sequencing (BS) was used to analyze whether the target gene in HeLa cervical cancer cell line exhibited hypermethylation condition. Results shown in FIG. 5 indicated that the number of hypermethylated target gene specimens in cell lines that had not been treated with 5′-aza-2′-deoxycytidine(AZC) (FIG. 5A) is higher than that in cell lines that had been treated with 5′-aza-2′-deoxycytidine (AZC) (FIG. 5B). Furthermore, cervical squamous cell carcinoma (SCC) and normal specimens were analyzed by means of bisulfite sequencing (BS) analysis. Results shown in FIG. 6 indicated that the number of hypermethylated target gene specimens in cervical squamous cell carcinoma (SCC) specimens (FIG. 6A) is considerably higher than that in normal specimens (FIG. 6B).
EXAMPLE 4
Methylation Analysis of Genes in Clinical Cervical Samples
The mean ages of patients with normal cervix and with LSIL, HSIL and SCC were 51.0±11.3, 39.7±9.6, 46.4±14.4 and 53.3±10.9 years, respectively (p<0.05). As shown in Table 5, the positive rate of high risk HPV DNA is 21.4%, 47.7%, 59.3% and 88.9% in normal, LSIL, HSIL and SCC, respectively (p<0.05). Patients with HPV infection showed significantly higher risk of the full spectrum of cervical lesions (OR: 3.1, 95% CI: 1.1-8.3; OR: 5.2, 95% CI: 2.1-13.0; OR: 29.9, 95% CI: 11.5-77.7 for LSIL, HSIL and SCC, respectively). All six genes (SOX1, PAX1, LMX1A, NKX6-1, WT1, and ONECUT1) showed frequent methylation in SCC (81.5%, 94.4%, 89.9%, 80.4%, 77.8%, and 20.4%, respectively), which was significantly greater than the methylation frequencies of their normal counterparts (2.2%, 0%, 6.7%, 11.9%, 11.1% and 0%, respectively; p≦0.001).
The methylation frequency of NKX6-1 was 53.3% in LSIL, 55.1% in HSIL, and 80.4% in SCC. Patients with methylations of NKX6-1 showed higher risks of SCC (OR: 29.8, 95% CI: 10.4-85.2). The methylation frequency of PAX1 was 2.3% in LSIL, 42.1% in HSIL, and 94.4% in SCC. Patients with methylations of PAX1 showed higher risks of HSIL and SCC (OR: >999.9, 95% CI:<0.1→999.9; OR:>999.9, 95% CI:<0.1→999.9, respectively).
The methylation rates of SOX1, LMX1A, and ONECUT1 were low in precancerous lesions, but increased substantially between HSIL and SCC (9.3% vs. 81.5%, 16% vs. 89.9%, and 7.4% vs. 20.4%, respectively). Patients with methylations of SOX1, LMX1A and ONECUT1 showed higher risks of SCC (OR: 200.2, 95% CI: 25.8-999.9; OR: 124.5, 95% CI: 33.0-470.1; OR: 7.3, 95% CI: 2.0-25.9, respectively). WT1 exhibited a severity-dependent increase in methylation frequency (11.1% in normal, 20.0% in LSIL, 42.1% in HSIL, and 77.8% in SCC). Patients with methylations of WT1 showed higher risks of HSIL and SCC (OR: 6.7, 95% CI: 2.2-19.8; OR: 27.9, 95% CI: 9.8-78.9, respectively).
EXAMPLE 5
Diagnostic Performance of DNA Methylation Markers
The sensitivities and specificities of HPV and DNA methylations were determined to assess their usefulness as biomarkers for diagnosis of high-grade cervical lesions and invasive cervical cancer. As shown in Table 6, the sensitivity and specificity for the diagnosis of SCC using HPV testing were 83.1% and 85.5%, respectively (95% CI: 77.6-88.5 and 79.6-91.4, respectively). SOX1, PAX1, LMX1A, NKX6-1, and WT1 methylations had high sensitivities (77.8%-94.4%) and specificities (88.1%-100%) for diagnosis of SCC.
When combined parallel testing (CPT) was applied for HPV and each methylation marker, which means that either one being positive was counted as positive, the sensitivities and specificities were in the ranges of 97.2%-98.2% and 66.7%-79.5%, respectively. When combined sequential testing (CST) was applied for HPV and each methylation marker, which means testing for HPV first with methylation detection following for HPV (+) patients, the sensitivities were in the ranges of 69.4%-85.0%. All the specificities were 100%.
When HSIL and SCC were present, the sensitivity and specificity for the diagnosis of HSIL/SCC using HPV testing were 75.0% (95% CI 70.2-79.8) and 85.5% (95% CI 79.6-91.4), respectively. The sensitivities and specificities of SOX1, PAX1, LMX1A, NKX6-1 and WT1 methylations were in the ranges of 57.4%-76.2% and 88.1%-100%, respectively. Using CPT for HPV and each methylation marker, the sensitivities could be improved to 85.8%-94.9%. Using CST for HPV and each methylation marker, all the specificities were 100%. When CPT was done using HPV and the methylations of SOX1, PAX1 and LMX1A, the sensitivities could be 100% for SCC and 93.4% for HSIL/SCC. PAX1 conferred the best performance when used alone with sensitivities of 94.4% (95% CI 90.0-98.8) and 76.2% (95% CI 69.7-82.7) for SCC and HSIL/SCC, respectively. The specificities were both 100%.







TABLE 5







Clinical relevance of DNA methylations in the full spectrum of cervical neoplasias














Clinical status/









genes
SOX1
PAX1
LMX1A
NKX6-1
WT1
ONECUT1
HPV





Normal
 1/45
 0/41
 3/45
 5/42
 5/45
 0/45
 9/42


(n = 45)
(2.2%)
  (0%)
(6.7%)
(11.9%)
(11.1%)
  (0%)
(21.4%)


LSIL
 2/45
 1/44
 6/45
24/45
 9/45
 3/45
21/44


(n = 45)
(4.4%)
(2.3%)
(13.3%) 
(53.3%)
(20.0%)
(6.7%)
(47.7%)


OR
 2.0
—
2.2
8.5
2.0
—
3.3


(95% CI)
(0.2-23.4)
—
(0.5-9.2) 
(2.8-25.5)
(0.6-6.5)
—
(1.3-8.6)


OR*
 3.1
—
2.2
9.6
2.7
—
3.1


(95% CI)
(0.3-36.7)
—
(0.5-9.7) 
(3.1-30.4)
(0.8-9.3)
—
(1.1-8.3)


HSIL
 5/54
24/57
 8/50
27/49
24/57
 4/54
32/54


(n = 58)
(9.3%)
(42.1%) 
(16.0%) 
(55.1%)
(42.1%)
(7.4%)
(59.3%)


OR
 4.5
>999.9
2.7
9.1
5.8
2.3
5.3


(95% CI)
(0.5-39.9)
(<0.1->999.9)
 (0.6-10.7)
(3.1-27.0)
 (2.0-16.9)
(0.5-10.8)
 (2.1-13.3)


OR*
 5.1
>999.9
2.7
9.6
6.7
2.3
5.2


(95% CI)
(0.6-45.9)
(<0.1->999.9)
 (0.7-10.9)
(3.2-29.1)
 (2.2-19.8)
(0.5-10.8)
 (2.1-13.0)


SCC
 88/108
101/107
 98/109
 86/107
 84/108
 22/108
 96/108


(n = 109)
(81.5%) 
(94.4%) 
(89.9%)
(80.4%)
(77.8%)
(20.4%) 
(88.9%)


OR
193.5
>999.9
124.7 
30.3 
28.0 
7.4
29.3 


(95% CI)
(25.2-1000) 
(<0.1->999.9)
(33.1-469.9)
(10.6-86.5) 
(10.0-78.8)
(2.1-25.7)
(11.3-75.8)


OR*
200.2
>999.9
124.5 
29.8 
27.9 
7.3
29.9 


(95% CI)
(25.8-999.9)
(<0.1->999.9)
(33.0-470.1)
(10.4-85.2) 
 (9.8-78.9)
(2.0-25.9)
(11.5-77.7)


Probability
p < 0.0001
p < 0.0001
p < 0.0001
p < 0.0001
p < 0.0001
p = 0.001
p < 0.0001





*adjusted for age and HPV infection













TABLE 6







The sensitivities and specificities of HPV testing and DNA methylations


for high-grade cervical lesions and invasive cervical cancer.










SCC
HSIL/SCC



















Sensitivity

Specificity

Sensitivity

Specificity


Biomarker
Test
OR
(95% CI)
OR
(95% CI)
OR
(95% CI)
OR
(95% CI)



















HPV
alone
83.1
(77.6-88.5)
85.5
(79.6-91.4)
75.0
(70.2-79.8)
85.5
(79.6-91.4)


SOX1
alone
81.5
(74.2-88.8)
97.6
 (93.0-100.0)
57.4
(49.8-65.0)
97.6
 (93.0-100.0)



CPT
98.1
 (95.6-100.0)
76.2
(63.3-89.1)
85.8
(80.4-91.2)
76.2
(63.3-89.1)



CST
72.2
(63.8-80.7)
100.0
(100.0-100.0)
50.6
(42.9-58.3)
100.0
(100.0-100.0)


PAX1
alone
94.4
(90.0-98.8)
100.0
(100.0-100.0)
76.2
(69.7-82.7)
100.0
(100.0-100.0)



CPT
98.1
 (95.6-100.0)
79.5
(66.8-92.2)
89.6
(85.0-94.3)
79.5
(66.8-92.2)



CST
85.0
(78.3-91.8)
100.0
(100.0-100.0)
65.2
(58.0-72.5)
100.0
(100.0-100.0)


LMX1A
alone
89.9
(84.3-95.6)
92.9
 (85.1-100.0)
66.7
(59.3-74.0)
92.9
 (85.1-100.0)



CPT
98.2
 (95.7-100.0)
71.4
(57.8-85.1)
89.3
(84.5-94.1)
71.4
(57.8-85.1)



CST
80.7
(73.3-88.1)
100.0
(100.0-100.0)
58.5
(50.8-66.2)
100.0
(100.0-100.0)


NKX6-1
alone
80.4
(72.9-87.9)
90.0
(80.7-99.3)
72.4
(65.4-79.5)
90.0
(80.7-99.3)



CPT
98.1
 (95.6-100.0)
70.0
(55.8-84.2)
94.9
(91.4-98.3)
70.0
(55.8-84.2)



CST
71.0
(62.4-79.6)
100.0
(100.0-100.0)
59.0
(51.3-66.7)
100.0
(100.0-100.0)


WT1
alone
77.8
(69.9-85.6)
88.1
(78.3-97.9)
65.5
(58.2-72.7)
88.1
(78.3-97.9)



CPT
97.2
 (94.1-100.0)
66.7
(52.4-80.9)
90.3
(85.8-94.8)
66.7
(52.4-80.9)



CST
69.4
(60.8-78.1)
100.0
(100.0-100.0)
53.9
(46.3-61.5)
100.0
(100.0-100.0)


HPV
CPT
100.0
(100.0-100.0)
69.2
(54.8-83.7)
93.4
(89.4-97.3)
69.2
(54.8-83.7)


+SOX1


+PAX1


+LMX1A









EXAMPLE 6
Methylation Analysis of Genes in Ovarian Samples
MS-PCR was performed to analyze the methylation status of the target genes in ovarian samples. As shown in Table 7, the promoters of SOX1, PAX1, and LMX1A were methylated neither in benign ovarian samples nor in borderline ovarian tumors. However, the methylation frequency of these 3 genes, SOX1, PAX1, and LMX1A, was significantly greater in malignancy ovarian tumors. The methylation frequency of SOX1, PAX1, and LMX1A was 55.7%, 49.2%, and 32.8%, respectively.







TABLE 7







Clinical relevance of DNA methylations in ovarian samples










Clinical status/genes
SOX1
PAX1
LMX1A





Benign (n = 36)
0/36 (0.0)
0/36 (0.0)
0/36 (0.0)


Borderline (n = 6)
 0/6 (0.0)
0/36 (0.0)
0/36 (0.0)


Malignancy (n = 122)
68/54 (55.7)
60/122 (49.2) 
40/122 (32.8) 


Probability
p < 0.0001
p < 0.0001
p < 0.0001









EXAMPLE 7
Methylation Analysis of Genes in Liver Samples
MS-PCR was performed to analyze the methylation status of the target genes in liver samples. As shown in Table 8, the methylation frequency of SOX1 was significantly greater in abnormal liver samples than in normal liver samples, and the frequency was 7.7%, 33.3%, 27.5%, and 53.7% in normal liver samples, chronic hepatitis, cirrhosis of the liver, and hepatocellular carcinoma (HCC) respectively. Moreover, the methylation frequency of NKX6-1 was significantly greater in hepatocellular carcinoma (HCC) (57%) than in normal liver samples (10%).







TABLE 8







Clinical relevance of DNA methylations in liver samples











Clinical status/genes
SOX1
NKX6-1







Normal liver (n = 13)
1/13 (7.7)
 1/10 (10)



Chronic Hepatitis (n = 15)
 5/15 (33.3)
—



Cirrhosis (n = 40)
11/40 (27.5)
—



HCC (n = 54)
29/54 (53.7)
12/21 (57)



Probability
P = 0.005
P < 0.05










Many changes and modifications in the above described embodiment of the invention can, of course, be carried out without departing from the scope thereof. Accordingly, to promote the progress in science and the useful arts, the invention is disclosed and is intended to be limited only by the scope of the appended claims.

## Claims

1. A method of screening for cervical cancer in a human patient comprising:
(i) obtaining a biological sample comprising cervical cells from the patient,
(ii) determining a degree of methylation of a CpG sequence of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 in the sample, and
(iii) comparing a level of methylation of the CpG sequence of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1 in the sample with the level of methylation in normal cervical cells; wherein a higher degree of methylation in the sample compared to normal cervical cells indicates an increased likelihood of cervical cancer.

2. A method of screening for cervical cancer as recited in claim 1, wherein a target gene SOX1 has a nucleotide sequence as depicted in SEQ ID No: 1.

3. A method of screening for cervical cancer as recited in claim 1, wherein a target gene PAX1 has a nucleotide sequence as depicted in SEQ ID No: 2.

4. A method of screening for cervical cancer as recited in claim 1, wherein a target gene LMX1A has a nucleotide sequence as depicted in SEQ ID No: 3.

5. A method of screening for cervical cancer as recited in claim 1, wherein a target gene NKX6-1 has a nucleotide sequence as depicted in SEQ ID No: 4.

6. A method of screening for cervical cancer as recited in claim 1, wherein a target gene WT1 has a nucleotide sequence as depicted in SEQ ID No: 5.

7. A method of screening for cervical cancer as recited in claim 1, wherein a target gene ONECUT1 has a nucleotide sequence as depicted in SEQ ID No: 6.