Static and flow cytometry for tumor DNA analysis

• The analysis of cellular DNA content in human tumors has shown to be of prognostic importance. The techniques used involve measurements in slide preparations by absorption cytophotometry and static cytofluorometry as well as the analysis of cell suspensions by flow cytometry. The aim of this work has been to improve these techniques by the development of computer aided systems in order to facilitate the use in a larger scale for clinical purposes. Another aim has been to study the prognostic significance of DNA analysis in breast cancer.Software for determination of DNA content and nuclear area by scanning absorption cytophotometry was developed. The system, HISTOSCAN, is insensitive to light scattering and may therefore be used in tissue sections. Thus, measurements in morphologically well-defined areas may be performed, but the method is less suitable for extensive use due to the slow procedure of the mechanical scanning.More rapid analyses are achieved by the system developed for static cytofluorometry in cytocentrifuged specimens. Cells to be measured are not positioned in the ordinary way, but are just passed through the excitation light beam as the specimen is visually scanned. DNA content and nuclear size are estimated simultaneously from the fluorescence recorded. For estimation of proliferative activity a sufficient number of cells may be analysed within a reasonable time.Similar results were obtained by static cytofluorometry and flow cytometry in a series of primary breast cancers. A close correlation was found for DNA index and, if 200 cells or more were measured, the same was true for S-phase fraction.Tumors from 472 women with primary breast cancer were analysed by flow cytometry. DNA ploidy showed significant association with disease recurrence and mortality but did not show a prognostic value in addition to that of traditional factors. The prognostic significance of S-phase fraction was independent of nodal status, tumor size and estrogen receptor content concerning early relapse and mortality. The survival of 116 women with recurrent breast cancer was correlated with both DNA ploidy and S-phase fraction in a multivariate analysis including nodal status, tumor size, ER content and site of recurrence.

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