METHOD FOR DETECTING IGF1R/Chr 15 in CIRCULATING TUMOR CELLS USING FISH
The present invention describes methods and probe composition for an automated FISH assay of a blood sample containing circulating tumor cells expressing the IGF-1R gene. The assay provides genetic analysis of suspect circulating tumor cells that have been identified after immunomagnetic selection and fluorescent labeling. Using unique, repeat-free probes to the IGF-1R locus and a chromosome 15 reference probe, cell lines expressing an aberrant number of IGF-1R and Chr 15 signals were detected, including one cell line with a low level of IGF-1R amplification. The ability to directly examine the genetic profile of IGF-1R on circulating tumor cells may provide an automated means for assessing disease and patient response to therapy.
1 . A method for assessing IGF-1R gene aberrations in circulating tumor cells from a patient sample comprising:
a. obtaining a blood sample from a patient, said sample comprising a mixed cell population suspected of containing circulating tumor cells;
b. isolating a subpopulation of epithelial cells by immunomagnetic enrichment;
c. identifying suspect circulating tumor cells; and
d. hybridizing said suspect circulating tumor cells with an IGF-1R repeat-free probe configuration capable of detecting chromosomal aberrations.
2 . The method of claim 1 wherein said immunomagnetic enrichment comprises:
a. mixing said sample with colloidal immunomagnetic particles coupled to a ligand which binds specifically to suspect circulating tumor cells, to the substantial exclusion of other populations; and
b. subjecting the sample-immunomagnetic particle mixture to a high gradient magnetic field to produce a separated cell fraction enriched in immunomagnetic particle-bound tumor cells.
3 . The method of claim 1 wherein said phenotypic profile is determined from a method selected from a group consisting of multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis, manual cell analysis, automated cell analysis and combinations thereof.
4 . The method of claim 3 wherein said phenotypic profile is determined from automated immunofluorescent cell analysis.
5 . The method of claim 1 wherein said probe configuration consists of clone sequences surrounding the IGF-1R locus and an alpha satellite probe lacking cross-hybridization.
6 . The method of claim 4 wherein said clone sequence is selected from bacterial artificial chromosome clones.
7 . The method of claim 5 wherein said alpha satellite probe is selected from a group consisting of SE-17, SE-15, and combinations thereof.
8 . The method of claim 5 wherein said alpha satellite probe is specific for the q1 region of chromosome 15.
9 . The method of claim 1 wherein said hybridizing suspect target cell provides diagnostic, prognostic, or therapeutic information of said patient.
10 . A kit for determining IGF-1R gene aberrations in circulating tumor cells from a patient sample comprising:
a. a polynucleotide probe sequence surrounding the IGF-1R locus wherein said probe is depleted of repetitive sequences;
b. an alpha satellite probe lacking cross-hybridization; and
c. labeling moieties linked to said (a) and (b).
11 . The kit of claim 10 wherein said polynucleotide probe sequence is depleted of said repetitive sequences by digestion with duplex-specific nuclease.
12 . The kit of claim 31 wherein said labeling moiety is a fluorophore linked by a platinum-based coordinative bond.