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 of reporting chromosomal abnormalities in a cervical sample, wherein said method comprises:
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 centromere;
c) detecting a hybridization signal of the first and the second nucleic acid sequence, wherein the hybridization signal is indicative of chromosomal copy number for 3q and the centromere;
d) scoring the chromosomal copy number for 3q and the centromere in a cell, wherein the cell is determined to be:
a. abnormal if the chromosomal copy number for 3q or the centromere is greater than 2; or
b. normal if chromosomal copy number of 3q is 2 and the centromere is 2; and
e) reporting the status of the sample, wherein the sample is determined to be:
(1) positive for chromosomal abnormalities if 1.0% or more of the analyzed cells are scored as abnormal; or
(2) negative for chromosomal abnormalities if less than 1.0% of the analyzed cells are scored as normal.
2. The method of claim 1 , wherein the target nucleic acid sequence on chromosome 3q is at locus 3q26 or 3q27.
3. 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.
4. The method of claim 1 , wherein the target nucleic acid sequence on chromosome 3q is selected from EVI1, MDS1, TERC, ARPM1, MYNN, LRRC34, LRRIQ4, LRRC31, SAMD7, LOC100128164, SEC62, GPR160, PHC3, or PRCKI.
5. The method of claim 4 , wherein the target nucleic acid sequence on chromosome 3q is the TERC gene.
6. The method of claim 1 , wherein the target nucleic acid sequence on the centromere is CEP3.
7. The method of claim 1 , wherein the cell is determined to be abnormal if the chromosomal copy number for both 3q and the centromere is greater than 2.
8. The method of claim 7 , wherein the target nucleic acid sequence on chromosome 3q is at locus 3q26 or 3q27.
9. The method of claim 7 , wherein the target nucleic acid sequence on chromosome 3q is at locus 3q26.1, 3q26.2, 3q26.31, 3q26.32, or 3q26.33.
10. The method of claim 7 , wherein the target nucleic acid sequence on chromosome 3q is selected from EVI1, MDS1, TERC, ARPM1, MYNN, LRRC34, LRRIQ4, LRRC31, SAMD7, LOC100128164, SEC62, GPR160, PHC3, or PRCKI.
11. The method of claim 7 , wherein the target nucleic acid sequence on chromosome 3q is the TERC gene.
12. The method of claim 7 , wherein the target nucleic acid sequence on the centromere is CEP3.
13. The method of claim 7 , wherein the method further comprises:
(a) hybridizing a third nucleic acid sequence to a target nucleic acid sequence on chromosome 5p at locus 5p15, 5p15.3 or 5p15.2;
(b) detecting the hybridization signal of the first, the second and the third nucleic acid sequence, wherein the hybridization signal is indicative of chromosomal copy number for 3q, the centromere, and 5p; and
(c) scoring the chromosomal copy number for 3q, the centromere, and 5p in a cell, wherein the cell is determined to be:
1. abnormal if the chromosomal copy number for 3q or the centromere or 5p is greater than 2; or
2. normal if chromosomal copy number of 3q is 2, the centromere is 2, and 5p is 2.
14. The method of claim 13 , wherein the target nucleic acid sequence on chromosome 5p is selected from ADCY2, C5orf49, FASTKD3, MTRR, SEMA5A, SNORD123, TAS2R1, LOC285692, TRIP13, TERT, NKD2, SLC12A7, SLC6A18, SLC6A19, CLPTM1L, SLC6A3, or CTNND2.
15. The method of claim 1 , wherein the method further comprises:
(a) hybridizing a third nucleic acid sequence to a target nucleic acid sequence on chromosome 5p at locus 5p15, 5p15.3 or 5p15.2;
(b) detecting the hybridization signal of the first, the second and the third nucleic acid sequence, wherein the hybridization signal is indicative of chromosomal copy number for 3q, the centromere, and 5p; and
(c) scoring the chromosomal copy number for 3q, the centromere, and 5p in a cell, wherein the cell is determined to be:
1. abnormal if the chromosomal copy number for 3q or the centromere or 5p is greater than 2; or
2. normal if chromosomal copy number of 3q is 2, the centromere is 2, and 5p is 2.
16. The method of claim 15 , wherein the target nucleic acid sequence on chromosome 5p is TERT, TRIP13, or Cri du Chat locus at 5p15.2.
17. The method of claim 1 , wherein the hybridization signal is detectable by FISH, CISH, PCR, ELISA, CGH, Array CGH or flow cytometry.
18. 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, a fine needle aspiration, a loop electrosurgical excision procedure (LEEP), a hysterectomy, a CONE biopsy, or an endocervical curettage (ECC).
19. The method of claim 1 , wherein at least 50 cells are examined.
20. The method of claim 19 , wherein at least 800 cells are examined.
21. The method of claim 1 , wherein the target nucleic acid sequence on a centromere is CEN7.
22. The method of claim 21 , wherein CEN7 is amplified.
23. The method of claim 21 , wherein the target nucleic acid sequence on chromosome 3q is selected from EVI1, MDS1, TERC, ARPM1, MYNN, LRRC34, LRRIQ4, LRRC31, SAMD7, LOC100128164, SEC62, GPR160, PHC3, or PRCKI.
24. The method of claim 23 , wherein the method further comprises:
(a) hybridizing a third nucleic acid sequence to a target nucleic acid sequence on chromosome 5p at locus 5p15, 5p15.3 or 5p15.2;
(b) detecting the hybridization signal of the first, the second and the third nucleic acid sequence, wherein the hybridization signal is indicative of chromosomal copy number for 3q, the centromere, and 5p; and
(c) scoring the chromosomal copy number for 3q, the centromere, and 5p in a cell, wherein the cell is determined to be:
1. abnormal if the chromosomal copy number for 3q or the centromere or 5p is greater than 2; or
2. normal if chromosomal copy number of 3q is 2, the centromere is 2, and 5p is 2.
25. The method of claim 24 , wherein the target nucleic acid sequence on chromosome 5p is TERT, TRIP13, or at the Cri du Chat locus at 5p15.2.
26. The method of claim 24 , wherein the target nucleic acid sequence on chromosome 5p is selected from ADCY2, C5orf49, FASTKD3, MTRR, SEMA5A, SNORD123, TAS2R1, LOC285692, TRIP13, TERT, NKD2, SLC12A7, SLC6A18, SLC6A19, CLPTM1L, SLC6A3, or CTNND2.
27. The method according to claim 24 , wherein the method further comprises:
(a) hybridizing a fourth nucleic acid sequence to a target nucleic acid sequence on chromosome 20q;
(b) detecting the hybridization signal of the first, the second, the third and the fourth nucleic acid sequence, wherein the hybridization signal is indicative of chromosomal copy number for 3q, the centromere, 5p, and 20q; and
(c) scoring the chromosomal copy number for 3q, the centromere, 5p, and 20q in a cell, wherein the cell is determined to be:
1. abnormal if the chromosomal copy number for 3q or the centromere or 5p or 20q is greater than 2; or
2. normal if chromosomal copy number of 3q is 2, the centromere is 2, 5p is 2, and 20q is 2.
28. The method of claim 27 , wherein CENT is amplified.
29. The method of claim 27 , wherein the hybridization signals are detected using a mixture of differently colored fluorescently labeled probes.
30. The method of claim 27 , wherein at least 50 cells are examined.