Heterogeneity of DNA distribution pattern in colorectal carcinoma. A microspectrophotometric study of fine needle aspirates.

For 50 consecutive patients with colorectal carcinoma, the nuclear DNA content in four separate fine needle aspiration samples taken from the resected tumors was analyzed using single-cell Feulgen cytometry. The DNA distribution patterns were divided into four classes according to their degree of euploidy/aneuploidy. Twenty tumors (40%) displayed a similar DNA pattern in all four samples while 30 tumors (60%) were heterogeneous, with a different DNA pattern within one or more of the four samples. None of the tumors were homogeneously diploid. These results illustrate the importance of multiple biopsy samples in the evaluation of the DNA pattern in colorectal tumors.

Comparative DNA analyses in longstanding ulcerative colitis with aneuploidy.

In seven patients with longstanding ulcerative colitis, multiple mucosal biopsies taken at colonoscopy were assessed for DNA content and histological dysplasia. DNA analyses were performed using flow cytometric analyses in biopsies and microspectrophotometry in imprint slides prepared from the biopsies. Abnormal aneuploid DNA content was detected in five of the patients in eight separate locations in the colon. There was good conformity between the two methods in the detection of aneuploidy. Both methods of DNA analysis are considered to be applicable in mucosal biopsies from patients with ulcerative colitis. In two patients with significant low grade dysplasia in association with macroscopical lesions (DALMs), there was a relationship with aneuploid DNA pattern found by flow cytometric or microspectrophotometric analyses. Indefinite changes, probably dysplastic were found in three other patients and two of those also displayed aneuploidy, although only once in the same biopsy location. In five locations aneuploidy was detected without concomitant dysplasia. DNA-aneuploidy seems therefore to appear earlier than dysplasia and may be an early marker of malignant transformation of the mucosa in ulcerative colitis. Prospective trials are needed before the clinical significance of early findings of aneuploidy can be determined.

Nuclear DNA content and behavior of oxyphil thyroid tumors.

Microspectrophotometric measurement of nuclear DNA content was made on archival smears of fine-needle aspirates from 23 oxyphil thyroid neoplasms. Fourteen tumors were considered benign as judged from the histologic picture as well as follow-up for 7 to 18 years after the operation. Nine tumors were malignant; five of these showed capsular penetration and/or blood vessel invasion as the only signs of malignancy, whereas the remaining four in addition had histologically verified metastases and were the cause of death. The DNA patterns found--diploid, polyploid or aneuploid--appeared to have a limited diagnostic value, since malignancy could not be excluded on this basis. A practically useful finding was, however, that aneuploidy appeared to be associated with a high probability of invasive growth. As regards prognostic information, it was found that euploid patterns occurred in tumors from patients with long survival after surgical treatment, while tumors with aneuploid patterns showed a variable clinical course.

Reversibility of 20-methylcholanthrene-induced bronchial cell atypia in dogs.

The sequence of cellular alterations in the bronchial epithelium of individual dogs was studied by means of combined morphologic and cytochemical techniques during a period of exposure to 20-methylcholanthrene (20-MC) and during a subsequent period without treatment. In agreement with earlier observations, 20-MC was found to induce progressive cellular changes which in four of seven dogs resulted in the occurrence of cells morphologically diagnosed as cancer cells. When the 20-MC treatment was stopped these cellular alterations were found to regress in three of the four dogs. Only in one dog did the advanced cellular atypia persist and cause the death of the animal. The results indicate that cells exhibiting cytomorphologic and cytochemical characteristics of cancer cells may reflect reversible lesions which regress on withdrawal of the carcinogen.

Prognostic significance of nuclear DNA content in mammary adenocarcinomas in humans.

The value of the determination of the DNA content of tumor cells for the assessment of the prognosis of mammary adenocarcinoma was studied in 36 patients, who survived for at least 15 years after the cancer had been found and in another 42 patients who died within a 2-year period of diagnosis. The results show a distinct correlation between the type of the DNA histograms of the carcinoma and the grade of cancer. This correlation was particularly apparent among patients surviving for at least 15 years despite the occurrence of metastases or relapses after primary treatment, i.e., in patients not cured by their first treatment.

Cytochemical changes in the nucleus during tumour development.

The general background to tumour analytic work using quantitative optical cytochemical methods is first presented. An instrument complex, constructed especially for multiparameter work in cytopathological material has been developed. Nuclear changes have been followed in cell populations during their development through different grades of atypia to cancer and conspicuous cytochemical changes were observed. In a comprehensive series of clinically verified mammary carcinomas, a large percentage of cases was found in which the DNA values were within the normal range, while the others showed pronounced aneuploidy. A clear correlation was found between DNA profile-type and patient survival, the latter of which reflects the degree of malignancy in the individual case. The shift from resting state (G0) to growth activated G1-stage is initiated by a large increase of the nuclear proteins. Mammary tumours of a high malignancy grade, as judged by their DNA profile type, showed an especially great accumulation of nuclear protein and thus a high degree of activation. DNA-profile measurements, preferably combined with determinations of nuclear proteins can thus be used for judging malignancy grades in mammary tumours, which is also of considerable clinical interest. An as yet limited observational material also indicates similar situations in some other types of tumour.

Determination of the fraction of G0 cells in cytologic samples by means of simultaneous DNA and nuclear protein analyses.

The ratio of protein to amount of DNA in the nucleus was cytophotometrically determined by means of a combined Feulgen-naphthol yellow S staining procedure. Quiescent normal lymphocytes, bronchial epithelial cells and mammary epithelial cells all showed ratios that were significantly lower than in the proliferating counterparts of the different cell types. Hence, combined determinations of DNA and nuclear protein make it possible to determine the fraction of growth-arrested cells (G0 cells) in cytologic preparations.

Cytochemical identification of quiescent and growth-activated tumor cells.

Cytochemical determinations of the amount of protein in the cell nucleus together with the amount of DNA give a background for judging the growth activity of the individual cell. A series of human breast carcinomas of different malignancy grades was studied, and it was shown that judged by their types of DNA profiles, the tumors of low malignancy grades were characterized by growth-arrested cells while the majority of cells in highly malignant tumors were intensely growth activated. This indicates that determinations of proteins in the nucleus together with DNA determinations can be used to better judge malignancy grades in individual breast carcinomas, which is of considerable clinical importance.

Cytochemical classification of nonpalpable breast carcinoma.

A stereotaxic needle biopsy technique makes it possible to obtain diagnostic cytologic material from nonpalpable breast lesions down to sizes of 2 to 3 mm in diameter. Considering the high correlation between the type of DNA distribution pattern and patient survival demonstrated in palpable breast carcinomas, cytophotometric DNA measurements were performed on smear preparations from nonpalpable tumors. In a series of 30 consecutive nonpalpable breast carcinomas, the same types of DNA profiles as shown in palpable tumors were found. This indicates that malignancy grading of breast carcinomas by cytochemical means can be performed on stereotaxic needle biopsy material and that an improved prognostic evaluation may be expected by the use of the methods described. This should result in a better therapeutic approach to patients with nonpalpable breast tumors.

Reversibility of bronchial cell atypia.

Various degrees of cellular atypia were induced in the bronchial epithelium of dogs by means of repeated submucous 20-methylcholanthrene injections. Thereafter, the 20-methylcholanthrene treatment was stopped, and the outcome of the bronchial cell atypias in individual dogs was studied using cytomorphological and cytochemical methods. The results suggest that the various degrees of 20-methylcholanthrene-induced cellular atypias, including those cytologically interpreted as malignant, may reflect reversible cellular alterations which disappear after removal of the carcinogen. Similar observations were made in a group of cigarette smokers who, after malignant-appearing cells were observed in the sputum material, stopped smoking or significantly reduced their cigarette consumption.

Relationship between nuclear DNA distribution and estrogen receptors in human mammary carcinomas.

The relationship between DNA distribution pattern and estrogen receptor (ER) content was investigated in primary mammary carcinomas from 56 female patients. The data showed that mammary carcinomas in which the DNA amounts of the tumor cells do not deviate conspicuously from diploid or tetraploid DNA values of normal mammary epithelium are characterized by high levels of ER. On the other hand, mammary carcinomas containing a sizable number of cells with DNA amounts in the normal DNA synthesis region or with high degrees of aneuploidy are distinguished by low or even unmeasurable ER levels. These results strongly indicate a correlation between nuclear DNA distribution patterns and ER levels in primary breast carcinomas.

PIP: An investigation was conducted to determine if a relationship exists between the estrogen receptor (ER) content and the type of nuclear DNA distribution patterns in human mammary carcinomas. Primary breast carcinomas from 56 female patients (52 postmenopausal and 4 premenopausal women) were examined. All cases were treated by mastectomy or local excision, and none of the patients had received any treatment for their disease before cell sampling for DNA and ER analysis was performed. The DNA histograms of these tumors could be grouped into 4 distinctly different classes or types: type 1 is characterized by a single modal DNA value in the normal diploid or near-diploid region; histograms of type 2 have a distinct single modal value in the normal tetraploid or near-tetraploid region or pronounced peaks in the 2c region as well as in the 4c region; type 3 populations differ from type 2 populations by showing a sizable number of cells in the normal DNA synthesis region; and the type 4 histograms are characterized by pronounced irregular aneuploidy, with individual DNA values ranging from levels near 2c up to values beyond 6c or even 8c. The data showed that mammary carcinomas in which the DNA amounts of the tumor cells do not deviate conspicuously from diploid or tetraploid DNA values of normal mammary epithelium were characterized by high levels of ER. Mammary carcinomas which contained a sizable number of cells with DNA amounts in the normal DNA synthesis region or with high degrees of aneuploidy were distinguished by low or even unmeasurable ER levels. The findings indicate a correlation between nuclear DNA distribution patterns and ER levels in primary breast carcinoma.

DNA content and survival in mammary carcinoma.

In a retrospective study of archival fine needle aspiration biopsy material from 112 patients with primary mammary carcinoma, the DNA distribution patterns of the cancer cell populations were determined. A distinct correlation was found between the occurrence of certain types of DNA distribution histograms and the survival time of the patients. Thus, the data indicate that DNA determinations can give prognostic information, in the individual case, over and above that furnished by clinical staging and morphologic criteria.

Quantitative tumor cytochemistry--G.H.A. Clowes Memorial Lecture.

Quantitative optical cytochemistry permits the determination of many different parameters in whole cells or parts of cells. Total amounts of DNA, RNA, and protein and/or the amounts of these substances in nucleus or nucleolus are examples. Techniques in this field have contributed considerably to the development of our present conceptions of normal cell growth and regulation. Normal cell study has, however, predominated so that tumor work has not yet been very extensive, even though quite eqrly on, in the 1940's, a conspicuous cytochemical peculiarity was found in cancers, namely, a pronounced variability between cells with regard to several measurable parameters. The result has been different kinds of technical difficulties, which are especially great in work on tumor material. However, as years passed, a fair amount of information was accumulated from studies of different parameters in tumors. Only a few years ago this experience clearly indicated that, provided very considerable improvements were made in technique, several cytochemical methods could be used as efficient supplements to morphological cytodiagnostic work, especially in such important clinical fields as preneoplasia identification, malignancy grading, and judgment of the state of growth of for instance, hormone-stimulated populations and also the distinction, so difficult in morphology, between "atypical hyperplasia" and "truly malignant" states. In an effort to eliminate the foremost of these technical difficulties, during the last few years we have developed a comprehensive system for large-scale uni- and multiparameter measurements aimed specifically at the types of specimens encountered in cytopathological work. The system is based on cytophotometry in ultraviolet and visible light, interferometry, and fluorometry and also encompasses special arrangements to simplify the identification and recovery of individual cells for different types of study. Instruments for facilitation of the comparison of cytochemical results with morphological findings are also included.