Method for the cytological analysis of cervical cells
View Patent ↗The invention provides for a diagnostic test to monitor cancer-specific genetic abnormalities to diagnose cervical cell disorders and predict which patients might progress to cancer. Genetic abnormalities are detected by identification in chromosomal copy number of chromosome 3 and chromosome 5 using FISH analysis of probes targeted to 3q and/or 5p.
1. A method for detecting chromosomal abnormalities in a plurality of cells in a cervical sample, said method comprising:
a) hybridizing a first nucleic acid sequence to a target nucleic acid sequence on chromosome 3q;
b) hybridizing a second nucleic acid sequence to a target nucleic acid sequence on a control chromosome;
c) detecting a hybridization signal of the first and the second nucleic acid sequence, wherein the hybridization signal of said first and the second nucleic acid sequences is indicative of chromosomal copy number for chromosome 3q and the control chromosome;
d) scoring the chromosomal copy number for chromosome 3q; and e) reporting whether the sample contains chromosomal abnormalities, wherein a lack of chromosomal abnormalities in the sample exists when a gain of 3q copy number is in less than 0.9% of the cells.
2. The method of claim 1 , wherein the scoring of the chromosomal copy number for chromosome 3q is performed by counting cells having 3q chromosomal abnormalities.
3. The method of claim 1 , wherein the scoring of the
chromosomal copy number for chromosome 3q is performed by counting 3q hybridization signals.
4. The method of claim 1 , wherein the target nucleic acid sequence on chromosome 3q is at locus 3q26 or 3q27.
5. The method of claim 1 , wherein the target nucleic acid sequence on chromosome 3q is at locus 3q26.1, 3q26.2, 3q26.31, 3q26.32, or 3q26.33.
6. The method of claim 1 , wherein the target nucleic acid sequence on chromosome 3q is the TERC gene.
7. The method of claim 1 , wherein the target nucleic acid sequence on the control chromosome is CEN3.
8. The method of claim 1 , wherein the method further comprises hybridizing a third nucleic acid sequence to a target nucleic acid sequence on chromosome 5p.
9. The method of claim 8 , wherein the target nucleic acid sequence on chromosome 5p is TERT, TRIP13, or Cri du Chat locus at 5p15.2.
10. The method of 8 , wherein the target nucleic acid sequence on chromosome 5p is at locus 5p15, 5p15.3 or 5p15.2.
11. The method of claim 8 , wherein a lack of chromosomal abnormalities in the sample exists when either (a) a gain of 3q copy number is in less than 0.9% of the cells, or (b) a gain of 5p copy number is in less than 0.7% of the analyzed cells.
12. The method of claim 8 , wherein a lack of chromosomal abnormalities in the sample exists when (a) a gain of 3q copy number is in less than 0.9% of the cells, (b) a gain of 5p copy number is in less than 0.7% of the analyzed cells, or (c) a gain in both 3q and 5p copy number is in less than 1.0% of the analyzed cells.
13. The method according to claim 1 , wherein the method further comprises hybridizing at least one additional nucleic acid sequence to a target nucleic acid sequence on chromosome 1q, 20q, 12q, 19q, 11q, 6q, 17p, 7, 8q, 9q, 16q, 2q, 9p, 10q, or 18p.
14. The method according to claim 1 , wherein the method further comprises hybridizing at least one additional nucleic acid sequence to a target nucleic acid sequence on chromosome 1q21-31, 20q12, 12q13-24, 19q13, 11q21, 7q1-22, 8q24, 9q33-34, 16q23, 2q32, 9p22, 10q21-24, or 18p11.
15. The method of claim 1 , wherein the hybridization signal is detected by FISH, CISH, PCR, ELISA, CGH, Array CGH or flow cytometry.
16. The method of claim 1 , wherein the cervical sample is derived from a cervical biopsy, a punch biopsy, a pap smear, a thin layer cytological specimen, a thin layer suspension, fine needle aspiration, loop electrosurgical excision procedure (LEEP), hysterectomy, CONE biopsy, or endocervical curettage (ECC).
17. The method of claim 1 , wherein at least 800 cells are examined.
18. The method of claim 17 , wherein at least 1000 cells are examined.
19. The method of claim 18 , wherein 1000 cells are examined.
20. The method of claim 1 , wherein the target nucleic acid sequence on a control chromosome is CEN7.
21. The method of claim 20 , wherein CEN7 is amplified.
22. The method of claim 12 , wherein the target nucleic acid sequence on chromosome 3q is TERC and the target nucleic acid sequence on chromosome 5p is at locus 5p15.
23. The method according to claim 22 , wherein the method further comprises hybridizing a fourth nucleic acid sequence to a target nucleic acid sequence on chromosome 20q.
24. The method according to claim 23 , wherein the target nucleic acid sequence on the control chromosome is CEN7.
25. The method of claim 24 , wherein CEN7 is amplified.
26. The method of claim 22 , wherein the target nucleic acid sequence on chromosome 5p is SEMA5A.
27. The method according to claim 26 , wherein the method further comprises hybridizing a fourth nucleic acid sequence to a target nucleic acid sequence on chromosome 20q.
28. The method according to claim 27 , wherein the target nucleic acid sequence on the control chromosome is CEN7.
29. The method of claim 28 , wherein CEN7 is amplified.
30. The method of claim 28 , wherein the hybridization signals are detected using differently colored fluorescently labeled probes.