Method for the quantification, qualitative genetic characterization and gene expression characterization of predetermined cells

Abstract

In the present invention, a method for the qualitative genetic characterization and/or gene expression characterization of predetermined cells in a fluid sample containing such cells is provided. The inventive method for the qualitative genetic and/or gene expression characterization of predetermined cells in a fluid sample containing such cells, comprises: a) selectively extracting at least a part of the predetermined cells from the sample forming a cell suspension cs.sub.0; and b) repeating the extraction step a) n times with the same sample of step a), with n1, forming at least one cell suspension cs.sub.n; c) determining a gene expression profile gep.sub.n and/or a first copy number count cnc.sub.0 of at least one DNA and/or RNA with at least a part of the cell suspension cs.sub.0; d) determining at least one further gene expression profile gepn and/or a further copy number count cnc.sub.n of at least one DNA and/or RNA with at least a part of at least one further cell suspension csn; e) calculating the predetermined cells' gene expression profile gep(P) of at least one predetermined DNA and/or RNA by subtracting gepn from gep, and/or the predetermined cells' copy number count cnc(P) of at least one predetermined DNA and/or RNA by subtracting cncn from cnc.sub.0; and f) evaluating the qualitative genetic and/or gene expression characteristics of the predetermined cells from gep(P) and/or cnc(P).

Claims

1. Method for qualitative genetic and/or gene expression characterization of predetermined cells in a fluid sample containing such cells, comprising: a) selectively extracting at least a part of the predetermined cells from the sample thereby obtaining an extracted cell suspension cs.sub.0, and a sample remainder comprising cells that remain after selective extraction; and b) selectively extracting at least a further part of the predetermined cells from the remainder sample thereby obtaining at least one cell suspension cs.sub.n, where n represents a number of times selective extraction is repeated and n is 1; c) determining a gene expression profile gep.sub.0 and/or a first copy number count cnc.sub.0 of at least one DNA and/or RNA with at least a part of the cell suspension cs.sub.0; d) determining at least one further gene expression profile gep.sub.n and/or a further copy number count cnc.sub.n of at least one DNA and/or RNA with at least a part of the at least one cell suspension cs.sub.n; e) calculating the predetermined cells' gene expression profile gep(P) of at least one predetermined DNA and/or RNA by subtracting gep.sub.n from gep.sub.0 and/or the predetermined cells' copy number count cnc(P) of at least one predetermined DNA and/or RNA by subtracting cnc.sub.n from cnc.sub.0; and f) evaluating the qualitative genetic and/or gene expression characteristics of the predetermined cells from gep(P) and/or cnc(P) by applying a specific molecular marker and detecting a marker signal.

2. Method according to claim 1, wherein the at least one RNA is selected from the group consisting of mRNA, ncRNA, rRNA, tRNA, snRNA, snoRNA, miRNA, dsRNA and viral RNA and/or the at least one DNA is selected from the group consisting of cellular DNA, viral DNA and bacterial DNA.

3. Method according to claim 1, wherein the gene expression profile and/or the first copy number count of at least one DNA and/or RNA is determined by RT-PCR, qRT-PCR and/or by microchip technology.

4. Method according to claim 1, wherein the qualitative genetic and/or gene expression characterization comprises or consists of a characterization of genes involved in metabolism.

5. Method according to claim 1, wherein n is 1 and 10.

6. Method according to claim 1, wherein selective extraction is performed using identical extraction reagents in step a) and in step b).

7. Method according to claim 1, wherein step a) is effected by contacting the sample at least one time with a solid phase that preferentially binds the predetermined cells, subsequently removing unbound cells from the solid phase, and subsequently removing bound cells from the solid phase.

8. Method according to claim 7, wherein the solid phase is selected from the group consisting of polymers, plastics, ceramics, glasses, metals, sepharose, agarose and latex.

9. Method according to claim 7, wherein the solid phase comprises antibodies and/or antibody derivatives.

10. Method according to claim 1, wherein the fluid sample comprises at least one selected from the group consisting of peripheral blood, bone marrow, urine, ascites and sputum obtained from a patient.

11. Method according to claim 1, wherein the predetermined cells are selected from the group consisting of tumor stem cells and tumor cells in epithelial-mesenchymal transition.

12. Method for designing a cancer therapy, comprising: i) performing the method according to claim 1, wherein the predetermined cells are circulating tumor cells (CTC); and ii) tailoring of the cancer therapy based on the evaluation.

13. Method according to claim 1, wherein n is 1 and 6.

14. Method according to claim 1, wherein n is 1 or 2.

15. Method according to claim 1, wherein removing unbound cells from the solid phase is by washing the solid phase with at least one buffer and subsequently removing bound cells from the solid phase is by washing the solid phase with at least one further buffer.

16. Method according to claim 7, wherein the solid phase is magnetic beads.

17. Method according to claim 9, wherein the antibodies and/or antibody derivatives are immobilized on a surface of the solid phase.

18. Method according to claim 1, wherein the predetermined cells are circulating tumor cells (CTC).

Description

(1) With reference to the following figures and examples, the subject according to the invention is intended to be explained in more detail without restricting said subject to the special embodiments shown here.

(2) FIG. 1 shows marker signals after the first extraction and second extraction of a sample comprising 0, 10 or 100 IGROV1 tumor cells as well as the calculated gene expression profile of the IGROV1 tumor cells.

(3) FIG. 1 (A) shows is the tumor marker signal (=copy number counts=cnc) of Pi3K, Akt and Twist determined with three different cell suspensions cs.sub.0 which were obtained by a first extraction of IGROV1 cells from three blood samples of healthy donors. The first sample did not contain any IGROV1 cells (0), the second sample was spiked with 10 IGROV1 cells (10) and the third sample was spiked with 100 IGROV1 cells (100). (B) The tumor marker signal (=cnc) of Pi3K, Akt and Twist determined for three different cell suspensions cs.sub.1 which were obtained by a second extraction of IGROV1 cells from the same blood samples as for the first extraction. (C) The tumor marker signals of Pi3K, Akt and Twist which belong only to IGROV1 cells (=cnc(P)) were calculated by subtracting each gep.sub.1 from each gep.sub.0 for the 0, 10 and 100 sample, respectively.

EXAMPLE 1: Qualitative Gene Expression Characterization of CTC in a Sample Containing CTC and Leukocytes

(4) The AdnaTests use an immunobead based technique to enrich circulating tumor cells (CTC) from the blood of cancer patients followed by a molecular determination characterization of such cells using tumor-associated marker gene expression profiles. However, even if the enrichment is quite effective the samples contain finally about 1000 leukocytes or other nucleated cells as a cross contamination.

(5) According to one embodiment of the invention, all CTC plus about 1000 contaminating leukocytes are analyzed in a first enrichment step. Using the same blood sample after this first enrichment step and again extract it with the same immunobeads, the same amount and composition of contaminating leukocytes is captured, but no tumor cells any more. So the second enrichment is the perfect blank sample and can be subtracted from the first sample to mathematically get access to an expression profile that can only be dedicated to the CTC, if there are any.

EXAMPLE 2: Quantitative Determination of IGROV1 Cells in a Blood Sample

(6) In a further example of the invention, the predetermined cells were IGROV1 tumor cells and the fluid sample was a 5 ml blood sample of a healthy person. Firstly, three separate samples of 5 ml blood were spiked with 0, 10 or 100 IGROV1 tumor cells. Then, at least a part of the IGROV1 tumor cells were selectively extracted from the sample forming three different cell suspensions cs.sub.0. Secondly, the extraction step was repeated for each of the three samples one further time forming three further cell suspensions cs.sub.1. Thirdly, the three copy number counts of the first extraction (cnc.sub.0 of 0, 10 and 100) and the three copy number counts of the second extraction (cnc.sub.1 of 0, 10 and 100) of the cellular markers Pi3K, Akt and Twist were determined by qRT-PCR (see FIGS. 1A and 1B). Finally, the copy number count cnc(P) of Pi3K, Akt and Twist was calculated by mathematically subtracting each cnc.sub.1 from its respective cnc.sub.0 (see FIG. 1C).