Obiettivo
THE DEGREE OF MALIGNANCY OF SPONTANEOUS AND RADIATION-INDUCED RAT MAMMARY TUMOURS WILL BE DETERMINED USING THREE TECHNIQUES, NAMELY FLOW CYTOMETRY, COMPUTER CONTROLLED MORPHOMETRY AND HISTOPATHOLOGY.
The clinical behaviour of rat mammary carcinomas is different from that of human breast cancer ie, they are characterised by a noninvasive or microinvasive growth pattern and a low frequency of metastasis (about 5%). Their volume doubling time is lower and may even be less than that of benign rat mammary tumours.
In this study, deoxyribonucleic acid (DNA) ploidy patterns were determined in spontaneous and radiation and/or estrogen treatment induced rat mammary tumours. The DNA content of the tumour cells was related to DNA content of diploid reference cells and expressed as the DI ie, the ratio of the mode of the relative DNA content of the G 1/0 cells of the sample divided by the mode of the relative DNA content of the diploid G 1/0 reference cells.
According to the convention on nomenclature for DNA cytometry, the reference cells were mixed with the cells of the tumour sample before propidium iodide (PI) lablelling. The reference cells were derived from paraffin blocks of renal tissue of the same rat to assure identical treatment of sample and reference cells before PI staining used for DI estimation. In order to recognise the added reference cells from those of the sample they were prepared and prestained with fluorescein isothiocyanate (FITC) separately. Subsequently, both sample and FITC labelled cells were mixed and stained with PI. The small shift of PI intensity of the reference cells due todouble staining as measured in parallel control experiments using renal nuclei stained with PI only and PI + FITC was corrected for and was never greater than a factor 1.14. Using this method we could clearly recognise the normal reference G 1/0 peak and therefore distinguish DNA hypoploid and DNA hyperploid peaks. This is impossible when normal cells in the tumour are used as a reference. In the latter situation, the first peak is assumed to represent normal G 1/0 cells.
In the study 5 (20%) of 26 benign tumours were DNA hypoploid and 1 (4%) hyperploid. These find ings contrast with data obtain in man where benign mammary tumours are characterised by a DI of about 1. However this may be species related as comparable percentages of DNA aneuploidy have been reported in canine benign mammary tumours (6,9). Of the 39 malignant rat tumours 17 (44$) were aneuploid and 9 (23%) of these were hyperploid. Thus DNA aneuploidy and especially DNA hyperploidy was more frequent in malignant than in benign rat mammary tumours. No differences in this respect were observed between invasive and noninvasive carcinomas.
In the study a lower incidence of aneuploidy was observed (44%) in the rat mammary carcinomas than is normally observed in human mammary carcinomas. Others, however, have also observed a relatively low incidence (44%) of aneuploidy in human mammary carcinomas.
In man DNA aneuploidy is associated with a poor prognosis. However, rats with mammary carcinomas do not die because of generalised metastatic disease but in the majority are euthanised because of the large size of the tumours or ulceration of overlying skin. They, therefore, might have lived for a much longer time. In man prognosis is mostly determined as survival after time of diagnosis and treatment. Rats are left untreated and histological diagnosis is made after necropsy.
In this study DNA hyperploidy was observed more often in malignant than in benign tumours. However, with in th carcinoma category hyperploidy or the degree of DNA hyperploidy was not more frequent in the histologically or clinically more malignant cases as has been reported in man. Histological appearance, quantitative nuclear parameters and the mitotic rate are thought to be good predictors of prognosis in breast cancer. The study found a correlation between histological malignancy, nuclear features and mitotic activity index, however, no differences were observed between invasive and noninvasive malignant tumours.
In conclusion, the study shows that the category of histologically malignant rat mammary tumours differ significantly from benign rat mammary tumours in the relative frequency of DNA hyperploidy, mitotic activity index and some quantitative nuclear characteristics. This lends support to the contention that the histologically malignant rat mammary tumours, although clinically less malignant, are in many aspects comparable to their human counter parts.
Cytological characteristics, such as nuclear area, perimeter and nuclear irregularity, of benign and malignant rat mammary tumours have been assessed by applying morphometrical methods to archival formalin fixed tumour material as used for deoxyribonucleic acid (DNA) flow cytometry and histology. The data on DNA flow cytometry and nuclear characteristics have been evaluated and correlated with histological signs of malignancy such as cellular pleomorphism, mitoticrate, invasiveness and metastatic capacity.
A RECENTLY DEVELOPED METHOD PERMITS THE ANALYSIS USING FLOW CYTOMETRY OF CELL SUSPENSIONS DERIVED FROM TISSUE WHICH HAS BEEN FIXED IN FORMALIN AND EMBEDDED IN PARAFFIN WAX. THIS CREATES THE POSSIBILITY OF USING MATERIAL FROM AN ARCHIVE OF RAT MAMMARY TUMOURS CONSISTING OF MORE THAN 5,000 SPONTANEOUS AND RADIATION INDUCED TUMOURS, SOME OF WHICH ARE BENIGN, SOME NON-INVASIVE MALIGNANT AND SOME INVASIVE MALIGNANT WITH AND WITHOUT METASTASES.THE DNA CONTENT OF CELLS CAN BE MEASURED USING FLOW CYTOMETRY SO THAT THE PERCENTAGE OF CELLS IN THE S-PHASE OF THE CELL CYCLE AND THE DEGREE OF ANEUPLOIDY OF THE DIFFERENT TYPES OF TUMOUR CAN BE DETERMINED. THESE TWO PARAMETERS PROVIDE INFORMATION ABOUT THE SPEED OF GROWTH AND AGGRESSIVE NATURE OF THE TUMOUR TYPES. IN ORDER TO DO THIS THICK SLICES OF THE EMBEDDED TUMOUR ARE FREED OF THE PARAFFIN WAX AND REHYDRATED. THE SLICES ARE THEN INCUBATED AT 37 DEGREES CELSIUS IN AN ACID SOLUTION CONTAINING A PROTEIN DEGRADING ENZYME SO THAT THE NUCLEI OF THE CELLS ARE RELEASED. AFTER FILTRATION AND CENTRIFUGATION THE NUCLEI CAN BE RESUSPENDED IN A STAINING SOLUTION. THE STAINING MEDIUM IS A DNA FLUORESCENT DYE THAT BINDS TO THE A-T BASE PAIRS RESULTING IN AN INCREASE IN THE FLUORESCENCE WITH A FACTOR OF 20 TO 25. AFTER FILTRATION OF THE STAINING SUSPENSION THE DNA CONTENT OF THE NUCLEI WILL BE MEASURED USING A SINGLE LASER FLOW CYTOMETER. LYMPHOCYTES ISOLATED FROM RAT SPLEENS TREATED IN THE SAME WAY WILL BE USED AS REFERENCE CELLS IN ORDER TO DEFINE THE NORMAL DIPLOID DNA PATTERN. THE DATA OBTAINED WILL BE CORRELATED WITH HISTOPATHOLOGICAL INFORMATION SUCH AS CELLULAR PLEOMORPHISM, MITOTIC INDEX, INVASIVE GROWTH, METASTASES ETC., AND WITH MORPHOMETRIC PARAMETERS. IN ORDER TO DETERMINE THESE PARAMETERS, USE WILL BE MADE OF A MOP-VIDEOPLAN. ALL THE DATA OBTAINED WILL BE ANALYSED IN ORDER TO ARRIVE AT A DEFINITION OF THE DEGREE OF MALIGNANCY OF THE RAT MAMMARY TUMOURS. THIS IS IMPORTANT BECAUSE THE RAT IS USED AS A MODEL FOR THE ESTIMATION OF RADIATION RISKS IN MAN.
THE RESEARCH WILL PROCEED AS FOLLOWS. AS THE FLOW CYTOMETER TECHNIQUE USING THE PRE-FIXED TISSUES HAS ONLY RECENTLY BEEN DEVELOPED, EMPHASIS WILL BE PLACED ON THE INTRODUCTION OF THIS TECHNIQUE IN THE INSTITUTE IN THE FIRST YEAR. ATTENTION WILL ALSO BE GIVEN TO GETTING CELL SUSPENSIONS FROM THE EMBEDDED TISSUE AND TO ACHIEVING THE OPTIMAL ENZYME AND DYE CONCENTRATIONS FOR THE RESEARCH MATERIAL. DURING THE DEVELOPMENT OF THE TECHNIQUE, REPRESENTATIVE TYPES OF TUMOUR WILL BE SELECTED FROM THE TUMOUR BANK. THE CHOSEN TUMOURS WILL BE EXAMINED MICROSCOPICALLY BEFORE BEING INVESTIGATED WITH THE FLOW CYTOMETER AND MORPHOMETRICALLY DURING THE SECOND YEAR OF THE RESEARCH CONTRACT.
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2280 HV RIJSWIJK
Paesi Bassi