Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1996-06-04
1998-03-10
Marschel, Ardin H.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
435 6, 435 691, 435810, 436501, 436 63, 514 2, 514 44, 935 77, 935 78, C12Q 168, G01N 3353
Patent
active
057260095
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
1. Technical Field
The present invention relates to a method of using an in vitro culture system to measure the cell proliferation and cell viability of human tissues, particularly tumor tissues, and thereby to measure the efficacy of anti-neoplastic drugs upon the proliferation and viability of the cultured cells.
2. Background
Cancer is a disease involving inappropriate cell division. A realistic model is greatly needed to understand the biology of altered proliferation in cancer as compared to normal tissue and to use information on proliferation capacity as a basis of cancer prognosis and treatment.
Measurements of proliferation capacity of tumors currently are obtained by thymidine-labeling index (TLI), by flow cytometric measurements of cells presumed to be in S phase, or by measuring a nuclear antigen, Ki-67, found in at least certain proliferating cell types. Meyer et al., Breast Cancer Rest. Treat., 4: 79-88 (1984); McDivitt et al., Cancer, 57: 269-76 (1986); and McGurrin et al., Cancer, 59: 1744-50 (1987). Whichever method is used, the results obtained show that the higher the S-phase fraction is, the poorer the prognosis. Clinical studies utilizing the TLI procedure have been successful in identifying and determining therapy of a subgroup of lymph node-negative women with breast cancer having a 48% relapse rate. Meyer et al., Cancer, 51: 1879-86 (1983). There is therefore great potential value for cancer prognosis, therapy, and biology in determining the proliferative capacity of tumors.
However, As important as the measurement of the TLI seems to be, current methods of measuring the TLI are impractical and are not physiological. For breast tumors, assays must be conducted within approximately 2 hours of surgery, precluding a central laboratory from carrying out the measurement. Generally, the TLI is measured under very high atmospheric pressure in a salt solution to allow penetration of .sup.3 H-thymidine into the tissue. Under these conditions the tumor loses viability after a few hours and in many cases it must be assumed that cells capable of cycling are not measured since the time of measurement is so much less than the generation time of the asynchronous cells within the tumor. With regard to other human tumor types, there is very little information regarding measurement of proliferation capacity of surgical specimens.
While flow cytometry provides a more rapid method of measuring cell cycle kinetics and cells can also be assessed for aneuploidy, it presents the following technical problems: (i) Dissociation, either mechanically or enzymatically, into a single-cell suspension is required, resulting in loss of ability to observe tissue architecture and the potential selective loss of one or more specific populations of cells. Full evaluation of all the heterogeneous cell types of an individual tumor, including their spatial organization, is of obvious importance in the development of prognostic tests. (ii) Flow cytometry does not unambiguously distinguish between S-phase diploid cells and aneuploid resting or nonviable cells. This becomes an important issue, as studies have demonstrated that tumor cell subpopulations that are enriched in aneuploid cells are largely nonviable by dye-exclusion analysis. Frankfurt et al., Cytometry, 5: 71-80 (1984); Slocum et al., Cancer Res., 41: 1428-34 (1981); and Ljung et al., Proc. Am. Assoc. Cancer Res., 28: 34 (1987). (iii) In addition, the S-phase factions of diploid tumors are likely to be underestimated by flow cytometry due to contamination with non-proliferating, nonneoplastic cells. The invasive capacity of diploid cells in vitro from primary breast carcinomas has been clearly demonstrated. Smith et al., Proc. Natl. Acad. Sci. USA, 82: 1805-9 (1985).
The nuclear antigen Ki-67 seems to be present in proliferating breast relevance to other tissue types is not yet known.
Perhaps most importantly, these techniques measure cells in S phase at a single point in time (flow cytometry, Ki-67) or after a very short labeling time (TLI). Thus, th
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Connors Kenneth M.
Monosov Anna
Anticancer Inc.
Marschel Ardin H.
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