Growth assay for fixed solid tumors
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- VENTANA MEDICAL SYSTEMS INC
- Filing Date
- 2024-04-03
- Publication Date
- 2026-06-16
Smart Images
Figure 2026519344000001_ABST
Abstract
Claims
1. A method for quantifying the proportion of normal cells and the proportion of tumor cells expressing cell proliferation markers, Obtaining a sample, Regarding the presence of at least one cell proliferation marker, the cells in a first aliquot derived from the sample are stained, To determine the presence of at least one tumor marker, the cells in a second aliquot derived from the sample are stained, Optionally, counterstain the cells in the first and second aliquots for the presence of DNA, To generate a scatter plot of fluorescence paired side scattering for the stained cells in each of the first and second aliquots, Based on the obtained scatter plot, the stained cells are gated to at least one of the cell proliferation marker-positive tumor cell population and the cell proliferation marker-positive normal cell population. To quantify the proportion of normal cells and the proportion of tumor cells in the tumor cell population and the normal cell population, respectively, that are positive for the cell proliferation marker. Methods that include...
2. The method according to claim 1, wherein the at least one tumor marker is an epithelial marker.
3. The method according to claim 2, wherein the epithelial marker is cytokeratin.
4. The method according to claim 3, wherein the cytokeratin is selected from the group consisting of high molecular weight cytokeratin and / or low molecular weight cytokeratin.
5. The method according to claim 3, wherein the cytokeratin is selected from the group consisting of CK8 / 18 or pancytokeratin markers that recognize cytokeratins 1-8, 10, 14-16 and 19.
6. The method according to any one of claims 1 to 5, wherein the at least one cell proliferation biomarker is selected from the group consisting of Ki-67, Ki-S5, Ki-S2, p21, p27, caspase, BAD, CD95, fas-ligand, and parp-protein.
7. The method according to any one of claims 1 to 6, wherein the at least one cell proliferation biomarker is Ki-67.
8. The method according to any one of claims 1 to 7, wherein the gating of the stained cells comprises performing at least two consecutive gatings.
9. The method according to claim 8, wherein the first of the at least two consecutive gatings is performed to identify cells that are positive for the tumor cell marker.
10. The method according to claim 9, wherein the first gating comprises (i) obtaining a scatter plot of fluorescence-paired side scattering for negative control aliquots derived from the sample, (ii) positioning the vertical quadrant gate such that less than a predetermined proportion of the stained cells in the scatter plot of the negative control aliquots are located to the right of the vertical quadrant gate, and (iii) positioning the horizontal quadrant gate in the generated scatter plot for the second aliquots such that less than a predetermined proportion of the stained cells in the generated scatter plot for the second aliquots are located in the lower right corner of the generated scatter plot for the second aliquots.
11. The method according to claim 10, wherein the negative control aliquot is incubated with one or more detection reagents.
12. The method according to claim 8, wherein the second gating includes mapping the first gating to the generated scatter plot corresponding to the first aliquot.
13. The method according to claim 8, further comprising optionally evaluating the DNA content in at least the first aliquot and the second aliquot in order to confirm the at least two consecutive gatings.
14. The method according to any one of claims 1 to 13, further comprising staining cells in a third aliquot derived from the sample in relation to the presence of at least one normal cell marker.
15. The method according to any one of claims 1 to 14, wherein the obtained sample is derived from a heterogeneous input sample that has been mechanically and / or chemically dissociated, and the obtained sample comprises substantially uniformly distributed cells.
16. The method according to claim 15, wherein the heterogeneous input sample is derived from one or more surgical excisions and / or surgical residues.
17. The method according to claim 15, wherein the ratio of cells in any aliquot derived from the sample is substantially the same as the ratio of cells in the heterogeneous input sample.
18. The method according to claim 15, wherein the heterogeneous input sample has a diameter of at least 1 cm at its widest point.
19. The method according to claim 15, wherein the heterogeneous input sample has a diameter of at least 2 cm at its widest point.
20. The method according to claim 15, wherein the heterogeneous input sample has a diameter of at least 5 cm at its widest point.
21. The aforementioned heterogeneous input sample is at least about 10 cm ３ The method according to claim 15, having the volume of
22. The method according to any one of claims 1 to 21, wherein the obtained sample includes dissociated cells.
23. The method according to any one of claims 1 to 22, wherein the obtained sample includes a homogenized fixed tissue sample.
24. The method according to any one of claims 1 to 23, further comprising sequencing genomic material isolated from cells within the aforementioned cell proliferation marker-positive tumor cell population.
25. The method according to claim 21, wherein the sequencing includes next-generation sequencing.
26. The method according to claim 21, wherein the sequencing includes single-cell sequencing.
27. The method according to any one of claims 1 to 26, wherein the staining of the first aliquot of the sample comprises contacting the first aliquot with a primary antibody specific to the cell proliferation marker in order to form a cell proliferation marker-primary antibody complex.
28. The method according to claim 27, wherein the primary antibody specific to the cell proliferation antibody is an anti-Ki-67 monoclonal antibody.
29. The method according to claim 28, further comprising contacting the first aliquot of the sample with a secondary antibody specific to the primary antibody specific to the cell proliferation marker.
30. The method according to claim 29, wherein the secondary antibody is conjugated with a first fluorescent label.
31. The method according to any one of claims 1 to 30, wherein the staining of the second aliquot of the sample comprises contacting the second aliquot with a primary antibody specific to the tumor marker in order to form a tumor marker-primary antibody complex.
32. The method according to claim 31, wherein the primary antibody specific to the tumor antibody is an anti-cytokeratin monoclonal antibody.
33. The method according to claim 32, further comprising contacting the second aliquot of the sample with a secondary antibody specific to the primary antibody specific to the tumor marker.
34. The method according to claim 33, wherein the secondary antibody, which is specific to the primary antibody that is specific to the tumor marker, is conjugated with a second fluorescent label.
35. A method for evaluating the proportion of normal cells positive for cell proliferation markers and the proportion of tumor cells positive for cell proliferation markers, (i) Obtaining at least two aliquots of a sample, wherein the cells in the first aliquot of the at least two aliquots of the sample are fluorescently stained for the presence of a cell proliferation marker, and the cells in the second aliquot of the at least two aliquots of the sample are fluorescently stained for the presence of a tumor marker. (ii) To generate a first scatter plot of fluorescence-paired side scattering for the fluorescently stained cells in the first aliquot of the sample, (iii) To generate a second scatter plot of fluorescence-paired side scattering for the fluorescently stained cells in the second aliquot of the sample, (iv) To evaluate the proportion of cell proliferation-positive normal cells and the proportion of cell proliferation-positive tumor cells, perform at least two gating operations using at least the first generated scatter plot and the second generated scatter plot. Methods that include...
36. The method according to claim 35, further comprising counterstaining the cells in the first aliquot and the second aliquot with respect to the presence of DNA.
37. The method according to any one of claims 35 and 36, further comprising obtaining a third aliquot of the sample, wherein the cells in the third aliquot of the sample are fluorescently stained for the presence of a normal cell marker.
38. The method according to claim 35, wherein the tumor marker is an epithelial marker.
39. The method according to claim 38, wherein the epithelial marker is cytokeratin.
40. The method according to claim 39, wherein the cytokeratin is selected from the group consisting of CK1, CK2, CK3, CK4, CK5, CK6, CK7, CK8, and CK9.
41. The method according to claim 35, wherein the tumor marker is selected from the group consisting of CK10, CK12, CK13, CK14, CK16, CK17, CK18, CK19, and CK20.
42. The method according to claim 35, wherein the tumor marker is selected from the group consisting of CK8 / 18 or pancytokeratin markers that recognize cytokeratins 1-8, 10, 14-16 and 19.
43. The method according to any one of claims 35 to 42, wherein the cell proliferation marker is selected from the group consisting of Ki-67, Ki-S5, Ki-S2, p21, p27, caspase, BAD, CD95, fas-ligand, and parp-protein.
44. The method according to any one of claims 35 to 42, wherein the cell proliferation marker is Ki-67.
45. The method according to any one of claims 35 to 44, further comprising obtaining a negative control aliquot, wherein the cells in the negative control aliquot are incubated with one or more detection reagents, and cytometry data is generated for the negative control aliquot.
46. The method according to claim 45, further comprising generating a negative control scatter plot of fluorescence paired side scattering for the cells in the negative control aliquot.
47. The method according to any one of claims 35 to 46, wherein the first of the at least two gatings is performed to identify cells that are positive for the tumor cell marker.
48. The method according to any one of claims 35 to 47, wherein the second of the at least two gatings is performed to identify normal cells and tumor cells that are positive for a cell proliferation marker.
49. The method according to any one of claims 35 to 48, wherein the obtained sample is a representative sample derived from a heterogeneous input sample that has been mechanically and / or chemically dissociated, and the representative sample comprises substantially uniformly distributed cells.
50. The method according to claim 49, wherein the heterogeneous input sample is derived from one or more surgical excisions and / or surgical residues.
51. The method according to claim 49, wherein the ratio of cells in any aliquot derived from the representative sample is substantially the same as the ratio of cells in the heterogeneous input sample.
52. The aforementioned heterogeneous input sample is at least about 10 cm ３ The method according to claim 49, having the volume of
53. The method according to any one of claims 35 to 48, wherein the obtained sample includes dissociated cells.
54. The method according to any one of claims 35 to 48, wherein the obtained sample includes a homogenized fixed tissue sample.
55. The method according to any one of claims 35 to 54, further comprising separating the cell proliferation marker-positive cells into a cell proliferation marker-positive normal cell population and a cell proliferation marker-positive tumor cell population.
56. The method according to claim 56, further comprising sequencing genomic material isolated from cells within the aforementioned cell proliferation marker-positive tumor cell population.
57. The method according to claim 57, wherein the sequencing includes next-generation sequencing.
58. The method according to claim 57, wherein the sequencing includes single-cell sequencing.
59. A method for evaluating the proportion of normal cells positive for cell proliferation markers and the proportion of tumor cells positive for cell proliferation markers, a. Obtaining a residual surgical tumor sample from a human subject having a diameter of at least 1 cm at its widest point, wherein the residual surgical tumor material is fixed but not paraffin-embedded, and the residual surgical tumor material is not deparaffinized. b. To provide a representative sample, the obtained residual surgical tumor material is mechanically blended such that any subpopulation of cells that were originally spatially isolated within the residual surgical tumor material is homogeneously distributed throughout the representative sample, and any aliquot removed from the representative sample contains one or more subclone populations in the proportion that was present in the obtained residual surgical tumor material. c. Obtaining at least two aliquots of the representative sample, wherein the cells in the first aliquot of the representative sample are fluorescently stained for the presence of a cell proliferation marker, and the cells in the second aliquot of the representative sample are fluorescently stained for the presence of a tumor marker. d. To generate a first scatter plot of fluorescence-paired side scattering for the fluorescently stained cells in the first aliquot of the representative sample, e. To generate a second scatter plot of fluorescence-paired side scattering for the fluorescently stained cells in the second aliquot of the representative sample, f. To evaluate the proportion of cell proliferation-positive normal cells and the proportion of cell proliferation-positive tumor cells, perform at least two consecutive gating operations using at least the first generated scatter plot and the second generated scatter plot. Methods that include...
60. A method for quantifying the proportion of normal cells and the proportion of tumor cells expressing cell proliferation markers, a. Obtaining a residual surgical tumor sample from a human subject having a diameter of at least 1 cm at its widest point, wherein the residual surgical tumor material is fixed but not paraffin-embedded, and the residual surgical tumor material is not deparaffinized. b. To provide a representative sample, the obtained residual surgical tumor material is mechanically blended such that any subpopulation of cells that were originally spatially isolated within the residual surgical tumor material is homogeneously distributed throughout the representative sample, and any aliquot removed from the representative sample contains one or more subclone populations in the proportion that was present in the obtained residual surgical tumor material. c. In relation to the presence of at least one cell proliferation marker, stain the cells in the first aliquot derived from the representative sample, d. To confirm the presence of at least one tumor marker, stain the cells in a second aliquot derived from the representative sample, e. Optionally, counterstain the cells in the first and second aliquots for the presence of DNA, f. To generate a scatter plot of fluorescence paired side scattering for the stained cells in each of the first and second aliquots, g. Based on the obtained scatter plot, the stained cells are gated to at least one of the cell proliferation marker-positive tumor cell population and the cell proliferation marker-positive normal cell population. h. To quantify the proportion of normal cells and the proportion of tumor cells in the cell proliferation marker-positive tumor cell population and the cell proliferation marker-positive normal cell population, respectively. Methods that include...
61. A method for quantifying the proportion of normal cells and the proportion of tumor cells expressing cell proliferation markers, Obtaining a sample, Regarding the presence of at least one cell proliferation marker, the cells in a first aliquot derived from the sample are stained, To determine the presence of at least one tumor marker, the cells in a second aliquot derived from the sample are stained, Optionally, counterstain the cells in the first and second aliquots for the presence of DNA, To generate a scatter plot of fluorescence paired side scattering for the stained cells in each of the first and second aliquots, Based on the scatter plot obtained, the stained cells are gated to at least two populations, To quantify the proportion of normal cells and the proportion of tumor cells in the at least two populations. Methods that include...
62. The method according to claim 61, wherein the cells in the first aliquot and the second aliquot are counterstained in relation to the presence of DNA.
63. The method according to claim 62, wherein the obtained sample is derived from residual surgical tumor material.
64. The method according to claim 63, wherein the residual surgical tumor material is fixed but not paraffin-embedded, or does not originate from a paraffin-embedded sample.