MIB-1 (Ki-67) immunostaining of breast cancer cells in cytologic smears.

OBJECTIVE: The reliability of immunocytochemical evaluation of proliferation activity was tested using the monoclonal antibody MIB-1 on cytologic specimens. STUDY DESIGN: The study comprised 83 frozen tissue smears (FTSs) and 51 fine needle aspirates (FNAs) from 119 breast cancer patients. MIB-1 labeling indexes (LIs) were compared with various tumor parameters assessed on histologic material. RESULTS: MIB-1 LIs established on cytologic smears were significantly different in ductal and lobular carcinomas (P = .024) and correlated significantly with mitotic activity (P < .0001), histologic grade (P < .0001) and S-phase fraction (P < .0001). Essentially the same results were obtained on FTSs and FNAs. CONCLUSION: Proliferative activity can reliably be evaluated by FNA cytology, and the evaluation of MIB-1 LIs may complement cytologic grading of breast cancer. The evaluation of proliferation activity may, therefore, contribute to the selection of candidates for adjuvant chemotherapy.

Breast cytology. Statistical analysis and cytohistologic correlations.

OBJECTIVE: To analyze the sensitivity, specificity, positive and negative predictive values and the efficacy of fine needle aspiration (FNA) in our material, to investigate the influence of the histologic type and stage of carcinoma on the quality of the aspirates and on the detection rates of mammary carcinoma, and to investigate the rate of inadequate samples and the accuracy of cytologic diagnoses, with an emphasis on the rate of false positive diagnoses in benign mammary lesions. STUDY DESIGN: The results of 1,472 FNAs of the breast obtained over three years were subjected to a retrospective analysis. RESULTS: The cytologic diagnoses were benign in 1,003 cases (68.1%), suspicious in 49 (3.3%) and malignant in 181 (12.3%); 239 (16.2%) of the aspirates were inadequate. In 393 (26.6%) of the cases and in 85% of cytologically malignant smears, the aspirate was compared with histologic examination. The rate of false negative FNAs was 9.0%. The proportion of inadequate cases was clearly related to stage (pT): it was 9.5% in pT1, 5.0% in pT2 and 0% in pT3. Among invasive carcinomas the sensitivity was 89.9%, specificity 99.3% and overall accuracy 88.5%. Among the cases diagnosed cytologically as benign, 182 were compared with biopsies. Of these, 79.9% were true negative, 0.5% (1 case) was false positive, and 15.4% had insufficient cells for evaluation. CONCLUSION: FNA cytology has improved decision making and the selection of patients for biopsy of mammary lesions and has contributed to saving time in the clinical management of breast lumps. In no case did FNA lead to inadequate clinical measures or other disadvantages to patients. Thus, FNA cytology is an indispensible diagnostic tool in the management of breast lesions.

Prognostic importance of DNA-ploidy and S-phase fraction in endometrial cancer.

On fresh frozen tumor tissue from 161 patients with endometrial cancer DNA-ploidy and S-phase fraction were measured in a prospective study to evaluate their prognostic and predictive value. All FIGO stage I or II patients had surgery and were included in an adjuvant trial comparing tamoxifen 30 mg p.o. versus medroxyprogesterone acetate 500 mg p.o. for 2 years versus no therapy. Diploid (DNA index (DI) 5%) in 46 (30%) of the patients. Significant correlations of DNA-ploidy and S-phase fraction were found with classical parameters such as stage, grade, histologic type and estrogen and progesterone receptor status. Patients with FIGO stage I aneuploid tumors showed significantly shorter disease-free interval (DFS) and overall survival (OAS). Recurrences and deaths occurred more often in tumors with raised S-phase fraction. In these early stages clinical outcome was worst if both factors were unfavorable. In multivariate analysis of stage I tumors DNA-ploidy and S-phase fraction were independent of grade, type and estrogen receptor status. Patients whose tumors had elevated S-phase fractions (>5%) gained more benefit from endocrine treatment than patients with low S-phase fractions. Patients with diploid and aneuploid tumors had prolonged DFS and improved OAS, if they had received adjuvant hormonal therapy. In endometrial cancer, DNA-ploidy and S-phase fraction are objective and reliable prognostic and predictive parameters which should be integrated into the clinical management.

Clinical and pathological features of highly malignant prostatic carcinomas with metastases to the penis.

Since 1885, 73 penile metastases of a primary carcinoma of the prostate have been reported. There is no standardized therapy as various therapeutic methods have produced variable results. The authors present their experiences with 2 recent cases. In some cases, total penectomy can relieve untolerable pain. Immunohistochemistry and flow cytometry for various prognostic factors showed a high malignant potential of carcinomas of the prostate metastasizing to the penis. Screening for various prognostic factors could favor an initial, more radical surgical approach in some cases, thus avoiding later tumor progression.

[Sarcoma of the breast: what is the contribution of immunohistochemistry and flow cytometry?]. / Sarkome der Mamma: Was bringen Immunhistochemie und Flowzytometrie?

Only 0.2-1% of all mammary malignancies are sarcomas of the breast. This study includes 4 cases: 2 osteosarcomas, 1 fibrosarcoma, and 1 malignant undifferentiated stromal sarcoma. The therapy was mastectomy in 3 cases with dissection of axillary lymph nodes and simple mastectomy in one case. One patient demonstrated local recurrence and died. The remaining 3 patients developed neither metastases nor local recurrence. They are still alive after a follow-up period of between 18 months and 17 years. As first-line treatment, wide local excision or simple mastectomy is recommended. Dissection of the axillary lymphatics, adjuvant radiotherapy, or chemotherapy have no established value in the treatment of breast sarcoma. In our 4 cases, flow-cytometric analysis does not always agree with the biological properties of the tumour and the clinical behaviour, in contrast to the results obtained in respect of carcinomas.

Comparative analysis of the expression of tenascin and established prognostic factors in human breast cancer.

Tenascin is an extracellular matrix glycoprotein expressed during morphogenesis in embryonal life. It reappears in the stroma of benign and malignant tumors. The distribution of tenascin in variants of fibrocystic disease and infiltrating breast carcinoma was assessed in cryostat sections by immunofluorescence using a polyclonal antibody. The tenascin immunoreactivity was compared with various prognostic factors. In fibrocystic disease (n = 10), tenascin appeared as periductal and periacinar bands. In infiltrating carcinomas (n = 32) the tenascin expression was markedly increased. Tenascin immunoreactivity was noted around the ducts (78%), extended into the distal stroma (56%), or was distributed in smaller (reticular) septa around and within tumor-cell nests (34%). Nineteen percent of infiltrating carcinomas did not express tenascin. None of the patterns correlated with prognostic factors such as nodal metastasis, tumor necrosis, invasion of blood vessels, or with flow cytometry results, such as ploidy and S-phase fraction. However, a significantly higher reticular and periepithelial tenascin expression was noted in cases with increased stromal inflammatory reaction. These findings indicate that the appearance of tenascin is neither an indicator of malignancy nor predictive of invasiveness or metastasis but that it is related to local inflammatory response.

[Rare breast tumors]. / Seltene Mammatumoren.

The cystosarcoma phylloides, like the sarcoma, represents 0.3 to 0.9% of all tumors of the breast. The clinical features include two different developments, one phase with increase and the other without. After removal of tumors, there are frequent recurrences. The unique therapy is for that reason to remove the lump with a margin in healthy tissue. The sarcoma of the breast represents only 0.2 to 1% of all mammary malignancies. This study reports four cases, including two osteosarcomas, one fibro- and one malignant fibrous sarcoma. The treatment of choice was mastectomy in three cases with dissection of axillary lymph nodes. The remaining patient was treated by simple mastectomy only. One patient demonstrated local recurrence and died. The remaining three patients developed neither metastases nor local recurrence. They are still alive with periods of observation between 18 months and 17 years. As first-line treatment wide local excision or simple mastectomy is recommended. Dissection of the axillary lymphatics, adjuvant radiotherapy or chemotherapy have no proven value in the treatment of breast sarcoma.

[Cytology and cytometry of breast cancer]. / Zytologie und Zytometrie des Mammakarzinoms.

The cells of mammary carcinoma are obtained by means of fine-needle aspiration (FNA) and assessed by light microscopy. The cytologic diagnosis is supplemented by a steadily increasing series of various cytologic methods such as cytometry (morphometry and flow cytometry) as well as immunocytochemistry. This article deals with the basic principles of a correct FNA technique, its sources of error, and the pitfalls of cytologic differential diagnosis. The additive methods and some of their applications are presented briefly.

Flow cytometry of breast cancer.

Flow cytometry (FCM) has become a generally accepted analytical method of worldwide application. Considerably achievements in flow cytometry hardware, staining techniques, especially immunoflow cytometry, together with progress in software programmes have significantly contributed to persistent expansion of the method. Some of the inbuilt rapidity of FCM has been lost because of additional preparatory steps and incubations. Mammary carcinoma is one of those malignant tumours which have been most extensively investigated by FCM. Evidence has been repeatedly produced to the existence of correlations among the most important FCM data, prognostic factors, and follow-up findings. Further improvement of the method and search for the best possible clinical application actually are the main challenges to FCM of breast cancer, today.

[Extent of psammoma carcinomas of the ovary--a clinical, DNA flow cytophotometric and morphometric image analysis study]. / Zur Dignität von Psammomkarzinomen des Ovars--eine klinische, DNA-durchflusszytophotometrische und morphometrisch-bildanalytische Studie.

There is no agreement in literature on the biological behavior of psammoma carcinomas of the ovary. The majority of authors consider psammoma bodies to be the result of tumour regression, associating the occurrence of psammoma bodies with longer survival. On the other hand, several studies reveal a poor prognosis for psammoma carcinomas, similar to that of other epithelial malignant tumours. In our study, the psammoma body content in 174 serous carcinomas stage III/IV was morphometrically quantified by image analysis and the results correlated to survival time and progression time. In 20 carcinomas the psammoma body content was extremely high. In such cases, DNA flow cytometry revealed these tumours to be slowly-growing. The DNA index was 1.0 (DNA-diploid) and the number of S phases was low (max. 5.9%). The five year estimate survival was 50%, as opposed to 10% for other tumours. If no methods are available for cell kinetic analysis and for objectification and quantification of psammoma body content in serous carcinomas it is sufficient to make a semiquantitative assessment of the psammoma body content to differentiate tumours with longer survival from carcinomas of poor prognosis.

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.

[Prognostic significance of cell kinetic parameters of endometrial cancer]. / Prognostische Bedeutung zellkinetischer Parameter beim Endometriumkarzinom.

In 120 patients suffering from carcinoma of the endometrium of a wide variety of histological types, conventional clinical and morphological criteria of prognosis were compared with the DNA index (DI) and the S-phase fraction. The cellular kinetics parameters were obtained via flow cytophotometry. Adenocarcinomas (n = 101) were more frequently DNA diploid and had a lower S-phase share (less than 5%) than adenoacanthomas, clear-cell carcinomas and malignant Müller's mixed tumors. There are close correlations between the conventional prognostic factors such as FIGO stage, relative tumor invasion depth, grading and oestrogen or progesteron receptor content and the DNA content or S-phase fraction. Carcinomas with a DI less than or equal to 1.1 (n = 83) and the S-phase fraction less than 5% (n = 77), have a significantly longer survival time and recurrence-free interval than DNA-aneuploid tumors with a high S-phase fraction. The studies underline the need to use flow cytophotometry in the prognostic assessment of the carcinoma of the endometrium. The method is superior to histological grading, since the results obtained can be objectively assessed.

DNA flow cytometry, clinical and morphological parameters as prognostic factors for advanced malignant and borderline ovarian tumors.

Patients with malignant ovarian (n = 111) and borderline (n = 8) tumors (FIGO stage III/IV) underwent surgery and chemotherapy and were analyzed clinically (age, residual tumor after surgery) and morphologically (type, grade, psammoma body content), and by means of flow cytometry (DNA ploidy, S-phase fraction). Follow-up was 12-72 months for investigation of survival. Patients under 60 years of age (n = 18) with malignant tumors showed longer survival than patients over 60 (n = 93) (P = 0.078). Residual tumor was relevant for prognosis in malignant tumors only if macroscopically there was no residual disease (n = 13). There were no significant differences between residual tumors less than or equal to 2 cm (n = 61) and greater than 2 cm (n = 37). WHO typing was of little importance for survival analysis. Compared to borderline tumors (n = 8), serous (n = 65), endometrioid (n = 13), nonclassifiable (n = 12), mucinous carcinomas (n = 8), and nonepithelial tumors (n = 12) had a poor prognosis. Psammoma bodies were found in 25 patients with serous carcinomas, 7 of them had a high content. The prognosis for these 7 patients was much better than that for patients with a moderate or low psammoma body content (P = 0.006). Twenty-three epithelial tumors were graded G1, 28 were G2, and 47 were G3. However, grading was considered only as a prognostic factor in serous carcinomas (n = 65) (P = 0.028). A total of 199 DNA histograms from 119 patients were analyzed by flow cytometry (FCM). There were no correlations between tumor type and DNA ploidy or S-phase fraction. Seven of eight borderline tumor and all serous carcinomas with a high content of psammoma bodies were diploid combined with a low (less than or equal to 4%) S-phase fraction. DNA ploidy and S-phase fraction were excellent prognosticators. Of 99 epithelial malignant tumors, 35 were diploid and 64 were aneuploid. An S-phase fraction less than or equal to 4% was found in 39 patients, 4.1-10% in 73 patients, and greater than 10% in 23 patients. Diploid tumors and tumors with a low S-phase fraction showed the best survival (P = 0.007, resp. 0.0001). Our study emphasizes the importance of an accurate histology, including information on psammoma body content, and the importance of DNA flow cytometry. The advantage of FCM is that the results are simple, reproducible, and objective.

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.

[Flow cytophotometry parameters, hormone receptors and axillary lymph node status as prognostic factors in primary breast cancer]. / Flow-zytophotometrische Parameter, Hormonrezeptoren und axillärer Lymphknotenstatus als Prognosefaktoren beim primären Mammakarzinom.

The prognostic significance of DNA flow cytometric examinations of 247 patients (mean age 57 years; 27-84) with operable breast cancer was analyzed. The findings were compared with biochemically determined steroid-hormone receptors and lymph node status. Forty-two percent of the tumors were diploid and 58% aneuploid; 32% of them had a high S-phase portion (greater than 5%; definition by the crit-level method). S-phase fractions were lower in diploid than in aneuploid tumors (2.5 +/- 2.2% versus 5.5 +/- 3.4%). Both with diploid tumors as well as tumors with a low S-phase fraction (less than 5%), recurrence-free survival was better than with aneuploid tumors (p = 0.02) and tumors with a high S-phase fraction (p = 0.004). A direct proportional relationship was found between the S-phase fraction and the mean recurrence-free interval. As regards overall survival, no significant differences have been detected so far, either for diploid and aneuploid tumors or tumors with low and high S-phase fractions. Using the Cox regression model, the prognostic significance of ploidy status and S-phase, as new factors, independent of age, lymph node and steroid-hormone receptor status, is shown. DNA flow cytometry is suitable for routine clinical use and should be used in particular as an independent prognostic factor for planning and stratification in adjuvant therapeutic studies.

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.

Psammomabody content and DNA-flow cytometric results as prognostic factors in advanced ovarian carcinoma.

138 advanced ovarian serous carcinoma--all FIGO stages III/IV--were investigated by prognostic morphological factors like tumour grading and rate of psammomabody content--subdivided in to serous carcinoma with a high, a moderate and a low rate of psammomabodies and a group of serous carcinoma without psammomabodies. Additionally 91 of these tumours were examined by DNA-flow-cytometry--respectively DNA-ploidy and s-phase-fraction. All prognostic factors were correlated to the overall survival time. The psammomabody content factor is only important if histologically a high or moderate rate of psammomabodies is found in the tumour tissue. Tumours with a low rate of psammomabodies do not differ from serous tumours without psammomabodies. DNA-flow-cytometry is a qualified method to demonstrate a slow tumour growth tendency. The majority of carcinomas with a high rate of psammomabodies is DNA-diploid and has a low -phase-fraction. The overall survival rate for these tumours is much better. Tumour grading--a more subjective method--is also of high value, but there is a certain difficulty in reproducing the results in individual cases. By the combination of the two morphological methods--semiquantitative assessment of psammomabodies and DNA-flow-cytometry--we were able to discover a small group of patients with advanced ovarian cancers who have a favourable prognosis.

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.

[Clinical course of ovarian cancer in relation to morphologic prognostic factors and cell kinetic parameters]. / Klinischer Verlauf des Ovarialkarzinoms in Abhängigkeit von morphologischen Prognosefaktoren und zellkinetischen parametern.

Apart from the stage of the tumour and the method of surgical approach, factors of special importance in prognosis are the histological type of tumour and the histological and cytological grading of malignant ovarian tumours. To supplement the prognostic factors, DNA flow cytometry has recently been introduced to determine parameters of cell kinetics such as ploidy (the status of the chromosome set in the karyotype) and the number of DNA-synthesized cells. In the present study 81 patients with a malignant tumour of the ovary--most of them classified according to FIGO stage III/IV--were followed up for 12 to 56 months (FIGO = Fédération Internationale de Gynécologie et d'Obstétrique). The survival times are correlated with clinical, morphological and cell kinetics data. The FIGO stage is the most important prognostic factor. None of the patients in stage I/II (FIGO) died during the study period, whereas of 63 women in stage III/IV (FIGO) 41 have already died. The histological tumour type is not so significant for prognosis. The survival curves show that of all the examined prognostic factors in stage III/IV (FIGO) the S-phase proportion (i.e. the proportion of DNA-synthesized cells) is the most important one. Statistically significant differences are also seen in ploidy and in grading. However, in histological grading of tumours the differences are noticeable only in grading G 1 and in grading G 3. G 2 carcinomas--which, by the way, account for one-third of all carcinomas-are a kind of collecting "pool" of tumours whose prognosis cannot be assessed.(ABSTRACT TRUNCATED AT 250 WORDS)

[Morphologic criteria of prognosis and clinical course in ovarian tumors of the borderline type]. / Morphologische Prognosekriterien und klinischer Verlauf bei Ovarialtumoren vom "Borderline-Typ".

Borderline tumors of the ovary are morphologically characterized by histologic and cellular criteria and by lack of evidence of invasion. During an observation period of 12 years at Heidelberg University Gynecological Clinic, 49 patients with borderline tumors underwent surgery and the clinical course was followed up. In 15 cases the tumor was at an advanced stage (FIGO III/IV); chemotherapy was instituted after surgery in 12 of these women. None of the patients with early-stage tumors (FIGO I/II) were lost due to the tumor during the period of observation; the death rate among patients with stage III/IV tumors (FIGO) was 40%. However, the death of three of these women was also due to their advanced age and high internal risks. As in the case of ovarian carcinomas, the survival rates improve if the postoperative residual tumor mass is smaller than 2 cm. Subsequent histologic work-up of archived, formalin-fixed tumor tissue and metastases thereof showed that two forms of the metastasizing borderline tumor exist: one with a favorable prognosis, a "pure" form with borderline changes in the ovary and metastases, and one with a less favorable prognosis, with borderline changes in the ovary and destructive-invasive portions of tumor in the metastazation areas. Impulse cytophotometric studies (ICP) showed that all primary tumors had the same degree of ploidy i.e., diploid. However, detection of an aneuploid distribution pattern in a metastasis correlated with subsequent histological confirmation of tumor invasion and rapid tumor death. The S-phase fraction was low, at 2.02% +/- 1.3.(ABSTRACT TRUNCATED AT 250 WORDS)