[DNA cytometry in breast carcinoma. Review of method and value in assessing prognosis]. / DNA-Zytometrie beim Mammakarzinom. Ubersicht zur Methode und zum Stellenwert bei der Prognoseabschätzung.

DNA flow cytometry (FCM) has become a routine method in breast cancer diagnosis for evaluation of ploidy and proliferation kinetics (cell cycle analysis). Image cytometry is less practicable and provides less information than flow cytometry. An optimized technique with a low coefficient of variation is required for optimal results in flow cytometry. The S-phase fraction and the proliferation index (sum of S-phase fraction and G2M fraction) provide prognostic and therapeutically relevant information. A profound knowledge of the technique and its limitations is indispensable for the interpretation of FCM results. It remains to be established whether immunohistological evaluation of cell proliferation has the same prognostic value. Future developments are to be expected from multiparametric analysis and the improvement of mathematical analysis of FCM measurements.

Combination of new biologic parameters as a prognostic index in epithelial ovarian carcinoma.

In a total of 77 patients with epithelial ovarian carcinomas, new biologic parameters [estrogen and progesterone receptor (ER, PR), DNA-ploidy (DP), S-phase fraction (Spf), cycling index (Ki67), Her2b/neu oncoprotein, epidermal growth factor receptor (EGF-R), cathepsin D and P170 glycoprotein] have been simultaneously detected and correlated to the clinical outcome [progression-free (PFI) and overall survival (OAS)] in a preliminary study. Apart from conventional prognosticators (age, stage, grade, residual tumor) and the postoperative serum marker Ca125, DP (P = 0.01), Spf (P = 0.009), Ki67 (P = 0.05) and PR (P = 0.01) could predict a short OAS (log rank test), whereas cathepsin D was of borderline significance only. Prognostic significance could be improved by using combinations of different factors [two markers of differentiation (DP, PR), one marker of proliferation (Spf) and one marker describing local tumor spread (cathepsin D)]. The difference in prognosis between patients with either all or three favorable tumor factors and patients with two to four unfavorable tumor factors reached a similar significance as can be obtained using FIGO stage as a prognostic factor (P = 0.007 for PFI, P = 0.0005 for OAS). These results were similar if early stages (FIGO I and II) were excluded. However, in a Cox regression analysis, including stage and residual tumor, this combination was significantly independent for PFI and OAS and could give additional information. Therefore, the large number of new biologic tumor markers could be restricted to only a few significant prognosticators to predict prognosis in primary epithelial ovarian carcinoma. In the future tumor characterizations may allow more individualized treatment with more aggressive, or even without, cytotoxic therapy after primary surgery.

Heidelberg Cytometry Symposium, 22-24 October 1992.

Lectures and presentations at the 1992 Heidelberg Cytometry Symposium reflected a rapidly growing research field with widespread activities that cover daily routine diagnosis as well as investigations at the molecular level and the diagnosis of genetic alterations. Both flow and image cytometry and their impact on quantitative cytology were backed and combined with new approaches (i.e. magnetic cell sorting) that enable the isolation of rare cells with high purity for cell biological analyses and thus pave the way for new research fields (i.e. arteriosclerosis). Their combination with the multicolor painting of gene sequences (fluorescent in situ hybridization) represents a further improvement of chromosome quantification and thus the analysis of the topology of cell nuclei. This year's meeting will be held from 21 to 23 October. Its topics will comprise chromosome painting, apoptosis, automated cytology, bio-imaging and proliferation. As usual, space is given to the presentation of new techniques and concepts. The deadline for abstracts is 30 June.

Long term tumorigenicity of a single application of indomethacin or Amuno in adolescent and in adult male Sprague Dawley rats.

200 adolescent (Group I) and 200 adult male Sprague Dawley rats (Group II) were divided into 4 subgroups of 50 animals each. Animals were treated on the 29th (Group I) or 98th day of life (Group II) either with acetone or Amuno carrier in acetone or Amuno on acetone (2.5 mg indomethacin/100 g animal weight in acetone) or with the pure substance indomethacin 2.5 mg/100 g of animal weight in acetone, giving a single application on the shaved dorsal skin. Subsequently the animals remained under observation until their deaths, followed by autopsy and histopathologic examination of several organs. The rats treated with Amuno of indomethacin in the adolescent stage showed lower body weights and a shorter total survival time. The adolescent animals treated with Amuno or indomethacin showed a significantly higher rate of interstitial testicular tumors of the Leydig tumor type, adenomas and adenocarcinomas of the small and large intestine as well as hepatocellular tumors. The total number of neoplasias was higher in the animals treated with Amuno or indomethacin compared to those treated with acetone or carrier with acetone. Application of Amuno or indomethacin also resulted in a higher rate of hyperplasias (sole of foot, lymph nodes, prostate). The impairment of the synthesis of prostaglandins caused by indomethacin apparently results in starting complex pathomechanisms which have effects until the late death of the animals. Adolescent animals were affected more frequently by the application of indomethacin as were already adult animals.

[Fine needle biopsy in diagnosis of cervical lymph node changes]. / Die Feinnadelbiopsie in der Diagnostik zervikaler Lymphknotenveränderungen.

In this study the validity and specificity of fine needle aspiration biopsy in cervical lymph node disease was examined. From 1983 to 1989, 495 outpatients were subjected to fine needle aspiration biopsies. One hundred and seven of the smears were not usable due to insufficient material. Tumour cells were found in 124 of the remaining 388 smears. In 117 of these cases the cytological findings were confirmed by later histology or clinical history. In the 7 remaining cases the cytological diagnosis of malignant lymphoma was disproved by histology. There were 38 (14%) false-negative diagnoses in 264 smears without tumour cells. In most of these cases the patient had undergone prior surgical or radiation therapy or had a malignant lymphoma. In spite of this relatively high number of false-negative results fine needle aspiration biopsy is helpful in the diagnosis of cervical lymph node disease. It must be emphasized that it is a screening method only: in the diagnosis of a tumour the method can only confirm and never disprove the clinical suspicion of malignancy.

[Purification kinetics of human tumors measured by flow cytometry]. / Durchflusszytometrische Untersuchungen zum Proliferationsverhalten menschlicher Tumoren.

We report on the use one-parameter DNA flow cytometry in the evaluation of the proliferation kinetics of malignant tumors. We emphasize the need of a background correction of the histograms in cases where the S-phase portion is used as a prognostic parameter for patients with malignant tumors. Flow cytometric investigations during the last 12 years with many thousand samples of human malignant tumors have shown that the described methodology can be used routinely as "assistant diagnosis" for a distinct group of tumors. These tumors comprise carcinomas of the breast and the ovaries and probably also of the endometrium. Regarding the relevance of the method for oropharyngeal squamous carcinomas, gliomas and meningiomas we have to await longterm results. In contrast, at present the method does not seem to be applicable for colorectal carcinomas and it is apparently not suitable for stomach carcinomas. It is also questionable whether data on proliferation kinetics gained in human tumors transplanted to nude mice can be as a basis in chemotherapeutic treatment of patients from whom the tumor originated.

Detection and chromosomal assignment of SV40-DNA integration in Chinese hamster cell lines by chromosome sorting and dot blot hybridization.

A combination of cytometric (chromosome sorting), molecular (dot blot hybridization using radio-active and/or biotinylated DNA probes) and cytogenetic (G-banding) evaluation is described which allows the rapid identification of single copy and repetitive viral integrates and their assignment to chromosome groups or even individual chromosomes. In the case of Chinese hamster cell line CO 631 it could be demonstrated that SV40 DNA was solely integrated into a submetacentric marker chromosome. Such a cytometric/molecular/cytogenetic "identogram" may prove to be a useful tool in many areas of cell and tumor biology. Furthermore, amounts of chromosomes sufficient for analysis as well as subsequent cloning experiments can be accumulated.

Influence of the elimination of lymphocytes from tumor cell suspensions on the calculation of S-phase fractions by flow cytometry.

Contaminating lymphocytes were eliminated from enzymatically obtained cell suspensions of 260 surgical biopsy specimens by density gradient centrifugation using lymphocyte separation medium (LSM) in an attempt to improve the determination of S-phase fractions by flow cytometry. The elimination of lymphocytes from the cell suspensions was ascertained on cytologic smears prepared from the suspensions before and after LSM centrifugation. Following the elimination of lymphocytes, the calculated S-phase fractions increased significantly in DNA-diploid tumors, but not in DNA-aneuploid ones. The increase of the S-phase fraction was correlated to the numbers of lymphocytes in the tumor cell suspensions before LSM centrifugation. Furthermore, in 12 tumors originally classified as diploid, an aneuploid cell line was detected after LSM centrifugation. These results indicate that samples from diploid tumors containing large numbers of lymphocytes should have the lymphocytes eliminated by methods such as LSM centrifugation in order to obtain reliable results for the calculations of the S-phase fractions.

DNA index and cell cycle analysis of primary breast cancer and synchronous axillary lymph node metastases.

The DNA Index (DI) and the percentage of cells in S-phase (S-phase fraction, SPF) were measured by flow cytometry in 80 primary breast carcinomas and in 80 accompanying axillary lymph node metastases. The DI in primary tumors and metastases agreed in 61 cases (76%). Cases with diploid primary tumors revealed more constancy of the DI in comparison to the metastases than the cases with aneuploid primary tumors (91% and 70% respectively). The mean values of the SPF were in close agreement in the primary tumors and in the lymph node metastases (6.1% and 6.0% respectively). Differences between the SPF of the two groups could be detected only by the consideration of case-related data pairs. In 50 cases (62%), the percentage of SPF agreed approximately in primary tumors and in the correspondent metastases. The cases with diploid primary tumors revealed more agreement of the SPF in the primary site and the metastases than did cases with aneuploid primary tumors (78% and 56% respectively). In conclusion, diploid carcinomas and their metastases revealed more constancy of the DI and the percentage of SPF than aneuploid carcinomas. These findings agree well with a better prognosis of diploid mammary carcinomas, as reported in the literature. Comparisons between the DI and the SPF in primary tumors and the corresponding metastases could be a source of valuable information on the biological behaviour and the aggressiveness of mammary carcinomas.

Comparison of S-phase fractions measured by flow cytometry and autoradiography in human transplant tumors.

The 3H-thymidine labeling index (TLI) and the percentage of cells in the S-phase have been determined by autoradiography and by flow cytometry, (FCM), respectively, in six malignant tumors of human origin transplanted on athymic nude mice. The Dean and Jett model and the graphical model were used to determine the percent of S-phase cells by FCM. Cell cycle analysis was performed using 1) no correction for background; 2) an algebraic function for background correction; and 3) an exponential function for background subtraction. Each of these three data sets was evaluated using both the Dean and Jett model and a graphical model for the evaluation of DNA histograms. The S-phase fractions (SPF) were compared to the corresponding labeling index results. SPF without background correction were 1.54 times higher than the TLI. SPF, after correction using the algebraic model, were 1.29-fold higher than the TLI, whereas SPF obtained after background subtraction according to the exponential model were only 1.05-fold higher than the TLI. Student's t-test revealed significant differences between the mean TLI values (16.25 +/- 9.06) and the mean SPF obtained by FCM without background correction (mean 25.0 +/- 9.36, P less than 0.01), but not between the mean TLI values and the mean SPF percentages after algebraic (mean 21.0 +/- 10.29) and exponential background correction (mean 17.11 +/- 11.59), P greater than 0.05 each. There was no difference between the results obtained using the Dean and Jett model and those obtained using the graphic evaluation.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of flow-sorted chromosomes by G-banding and in situ hybridization.

The identification of flow-sorted chromosomes is a very important tool for checking the purity of the fractions obtained. An easy and reproducible method for obtaining G-banded chromosomes with good resolution of bands is described. Also, we are able to show that the percentage of chromosomes which can be clearly distinguished by this procedure depends to a large extent on the duration of mitotic arrest. In particular when sorting chromosomes from human-rodent hybrid cell lines, the possibility of using in situ hybridization in addition to conventional staining techniques to characterize the chromosomes can help overcome the problem of highly condensed chromosomes and chromosomal fragments of unknown origin, which cannot be identified otherwise. Thus, we have developed an in situ hybridization technique, based on biotin-labelled human genomic DNA, which allows a clear distinction between human and rodent chromosomal material to be made.

Correlation of DNA flow cytometric results and other prognostic factors in primary breast cancer.

The percentage of cells in S-phase and DNA-ploidy have been measured in 300 primary mammary carcinomas by means of DNA-flow cytometry (FCM). The data were compared with the age and menopausal status of the patients as well as with the size, regional lymph-node involvement, histologic type, grade and concentration of estrogen (ER) and progesterone (PR) receptors of the tumors. A DNA-diploid distribution of the G0/1-peak was found in 37.6% of the cases. The mean percentage of S-phase fractions was 4.83. DNA-aneuploid tumors had significantly higher amounts of S-phase fractions (6.12%) than DNA-diploid tumors (2.66%). There was also a significant correlation between the DNA measurement data (DNA-ploidy and S-phase fractions) and histologic grade, as well as the content of ER and PR, but not between DNA-ploidy, S-phase fractions, tumor size (T) and evidence of axillary lymph-node metastases. DNA-FCM gives a biological characterization of the tumor in addition to the histopathologic examination. The method can be used as a routine procedure because of the reliability and reproducibility of the results as well as the short time needed for the measurements.

S-phase fractions and DNA-ploidy of oropharyngeal squamous epithelium carcinomas compared with histologic grade, stage, response to chemotherapy and survival.

DNA-ploidy and the percentage of S-phase fractions in 55 primary oropharyngeal squamous epithelium carcinomas were measured by DNA-Flow Cytometry (FCM). The data were compared with the histologic grade, the stage and the response of the tumours to cytostatic chemotherapy. A significant correlation was found between the histologic grade and the mean percentage of S-phase fractions (p less than 0.01). No correlation could be found between the FCM measurement data and the tumour stage. Carcinomas with an amount from 4.0 to 10.4% S-phase fractions responded to chemotherapy by complete remission, and those with 10.0 to 13.3% S-phase fractions by partial remission. The group of non-responders could be subdivided into two subgroups: non-responders with low amounts of S-phase fractions (1.1-3.9%), and non-responders with very high amounts of S-phase fractions (11.6-16.6%). FCM data, histologic and clinical prognostic factors were summed up to a prognostic score. The number of score points showed a significant correlation to the length of survival in months after diagnosis of the tumour (p less than 0.00001). FCM may be used as an additional diagnostic tool for a better biological characterization of the neoplastic tissue, especially as an aid for grading, prediction of the response to chemotherapy and the length of survival.

Influence of systematic errors on the evaluation of the S phase portions from DNA distributions of solid tumors as shown for 328 breast carcinomas.

The portion of cells in S phase has proved to be a valuable prognostic indicator of early relapse and life expectancy, particularly in breast carcinoma. Comparisons of published data on samples of primary breast carcinoma biopsies showed that the values obtained by analyses of flow cytometric DNA distributions were generally higher than those of determinations based on the tritiated thymidine (3H-ThdR) labeling index (LI). Flow cytometric DNA analyses of 328 biopsy samples of primary breast carcinomas revealed that these differences could be explained by varying contributions of debris background. Since this influence is inversely proportional to the cell counts in each channel, it may cause considerable errors, particularly in the S phase channels, which normally contain the lowest counts of the DNA distributions. Two different mathematical rationales were tested in order to separate DNA distributions from the debris superimposition. No appreciable differences were found with respect to the essential results. After appropriate subtraction of the background levels, the previously reported discrepancies between cytometrically determined S phase portions and 3H-ThdR LI values disappeared, and good agreement was achieved for the comparable tumor samples of the present study. In conclusion, debris background subtractions should generally precede the DNA histogram analyses, particularly of solid tumors, in order to obtain reliable S phase values.

Benzoyl peroxide promotes the formation of melanotic tumors in the skin of 7,12-dimethylbenz[a]anthracene-initiated Syrian golden hamsters.

A two-stage carcinogenesis experiment was performed in Syrian golden hamsters using a single intragastric initiation with 10 mg/kg body weight of 7,12-dimethylbenz[a]anthracene (DMBA) and repetitive topical promotion on the back skin with two different doses (80 and 160 mg, respectively) of benzoyl peroxide in 1 ml acetone. Benzoyl peroxide was administered three times per week over a period of 16 months. The treatment with benzoyl peroxide alone leads to both a generalized hyperpigmentation and skin scaling without formation of any tumors. DMBA initiation alone induces a moderate number of melanotic foci and a small number of palpable melanotic tumors. Both lesions are located in the dermis. Papillomas develop in the epithelia of the tongue, esophagus and especially of the forestomach but not, however, on the back skin. The combined treatment with DMBA and both doses of benzoyl peroxide drastically increases the incidence of dermal melanotic foci and at late stages also that of melanotic tumors. Therefore, in addition to its known ability to promote papilloma and carcinoma formation in the back skin of mice, benzoyl peroxide is also able to promote the formation of melanotic tumors in the dermis of hamsters.

Does in vitro colony formation and chemosensitivity relate to DNA ploidy and S-phase fractions?

In vitro colony formation and chemosensitivity were analyzed in 65 human solid tumors and compared to proliferation parameters simultaneously obtained by DNA flow cytometry of the same tumor specimens. Colony growth in the human tumor colony assay was enhanced in aneuploid tumors (39/65) in comparison to diploid tumors (26/65, P less than 0.05). In addition, there was a relationship between % S-phase and colony growth. The existence of polyploid sublines (23/65) improved in vitro growth even in tumors with a diploid main G0/1-peak or with a low % S-phase. Metastases exhibited a higher proportion of aneuploidy and showed slightly better growth in vitro than primary tumors. Sensitivity testing in 34 of the 65 tumors showed no convincing relation between DNA parameters and the inhibition of colony formation by five standard anticancer agents with different mechanisms of action. This indicates additional factors other than the proliferative activity of the tumor to be responsible for drug sensitivity or resistance.

Inhibition of the prenatal dimethylbenz[a]anthracene-induced tumour initiation in mice by prior administration of 7,8-benzoflavone.

7,8-Benzoflavone (BF) was applied orally via stomach tube in doses of 50, 100, 200 and 400 mg/kg body weight to pregnant NMRI mice on the 18th day of gestation. BF application was followed 1 h later by oral administration of 60 mg/kg body weight dimethylbenz[a]anthracene (DMBA). As a rule this dose of DMBA does not lead to prenatal secondary effects and is not carcinogenic to either the mother animals or the F1 generation. Subsequent promotion of the F1 generation with the tumour promoter 12-O-tetradecanoylphorbol-13-acetate over a period of 12 weeks resulted in a high yield of skin papillomas and lung adenomas when no BF had been applied. With prior application of BF, the tumour yield could be significantly reduced.