Method for early diagnosis of carcinomas of the anogenital tract

1. A method for detecting anogenital cancer or preliminary stages thereof in a sample, comprising determining the methylation status of ASTN1 (astrotactin 1) and ZNF671 (zinc finger protein, transcription factor), wherein ASTN1 and/or ZNF671 are methylated in a positive sample, and wherein primer pairs are used which comprise the following sequences:

(a)

CGTAAGCGTTGTTAGCGTAGC (ASTN1_F)

(SEQ ID No.: 1)

and

CGCGAAATCGAAACGAAAACG (ASTN1_R);

(SEQ ID No.: 2)

(b)

CGGAGGACGTAGTATTTATTCGC (ZNF671_F)

(SEQ ID No.: 11)

and

CTACGTCCCCGATCGAAACG (ZNF671_R).

(SEQ ID No.: 12)

2. The method according to claim 1 , wherein at least one probe is used which comprises one of the following sequences:

(a)

GTAATTCGTTTGTTTCGTAAGTTGTTCG (ASTN1);

(SEQ ID No.: 13)

(b)

CGTGGGCGCGGACAGTTGTCGGGAGCG (ZNF671).

(SEQ ID No.: 18)

3. The method according to claim 1 , additionally comprising the detection of HPV.

4. The method according to claim 3 , wherein the detection of HPV is via PCR.

5. The method according to claim 4 , wherein the HPV is detected using a primer pair comprising the following sequences:

(a)

GGTTATAATAATGGTATTTGTTGGG (HPV_F);

(SEQ ID No.: 19)

and

(b)

TAAAAAATAAACTATAAATCATATTCC (HPV_R).

(SEQ ID No.: 20)

6. A kit for detecting anogenital cancer or preliminary stages thereof in a sample, comprising: gene specific primer pairs that determine methylation and/or hypermethylation of ASTN1 (astrotactin 1) and ZNF671 (zinc finger protein, transcription factor).

7. The kit according to claim 6 , wherein primer pairs are used which comprise the following sequences:

(a)

CGTAAGCGTTGTTAGCGTAGC

(SEQ. ID No.: 1)

(ASTN1_F)

and

CGCGAAATCGAAACGAAAACG

(SEQ. ID No.: 2)

(ASTN1_R);

(b)

CGGAGGACGTAGTATTTATTCGC

(SEQ. ID No.: 11)

(ZNF671_F)

and

CTACGTCCCCGATCGAAACG

(SEQ. ID No.: 12)

(ZNF671_R).

8. The kit according to claim 6 , wherein at least one probe is used which comprises one of the following sequences:

(a)

GTAATTCGTTTGTTTCGTAAGTTGTTCG

(SEQ. ID No.: 13)

(ASTN1);

(b)

CGTGGGCGCGGACAGTTGTCGGGAGCG

(SEQ. ID No.: 18)

(ZNF671).

9. The kit according to claim 6 , additionally containing a primer pair for the detection of HPV.

10. The kit according to claim 9 , wherein the HPV is detected using a primer pair comprising the following sequences:

(a)

GGTTATAATAATGGTATTTGTTGGG

(SEQ. ID No.: 19)

(HPV_F);

and

(b)

TAAAAAATAAACTATAAATCATATTCC

(SEQ. ID No.: 20)

(HPV_R).

11. The kit according to claim 6 , containing in separate containers additionally at least one of the following constituents:

(a) reagents for isolating DNA;

(b) enzyme for DNA amplification;

(c) sodium bisulfate;

(d) one or more buffers.

12. The method of claim 1 wherein the anogenital cancer is cancer of the cervix uteri.

13. The method of claim 1 wherein the sample is a cervical smear.

14. The method of claim 1 where the methylation status of the ASTN1 and ZNF671 are determined at the promoter regions.

15. The method of claim 1 wherein the methylation status of the genes is determined by means of methylation-specific PCR (MSP).

16. The method of claim 15 wherein the MSP is a quantitative MSP (QMSP).

17. The method of claim 16 wherein the quantitative MSP is based on the MethyLight technique.

18. The method according to claim 1 wherein the method is a multiplex method.

19. The kit according to claim 7 wherein at least one probe is used which comprises one of the following sequences:

(a)

GTAATTCGTTTGTTTCGTAAGTTGTTCG

(SEQ. ID No.: 13)

(ASTN1);

(b)

CGTGGGCGCGGACAGTTGTCGGGAGCG

(SEQ. ID No.: 18)

(ZNF671).

20. The kit according to claim 19 , additionally containing a primer pair for the detection of HPV and wherein the HPV is detected using a primer pair comprising the following sequences:

(c)

GGTTATAATAATGGTATTTGTTGGG

(SEQ. ID No.: 19)

(HPV_F);

and

(d)

TAAAAAATAAACTATAAATCATATTCC

(SEQ. ID No.: 20)

(HPV_R).