[Diagnosis and grading of cervical intraepithelial neoplasias]. / Diagnose und Graduierung zervikaler intraepithelialer Neoplasien.

Diagnosing and grading of cervical intraepithelial neoplasias (CIN) are part of the routine practice of pathologists. However, discriminating between reactive changes and CIN1 and determining the different degrees of CIN may be challenging. Aim of this study was the evaluation of the proliferation markers Ki-67 and Mcm2 as well as p16 for their potential to aid in the assessment of CIN. 297 samples of normal epithelium, CIN1, CIN2, and CIN3 were assessed for expression of the above mentioned markers using tissue microarrays. There was an increase in the expression of Ki67 and Mcm2 from normal epithelium, CIN1, CIN2 to CIN3 (p<0.001 for both markers). Ki-67 was the most useful marker in differentiating between normal epithelium and CIN1. The number of p16-positive cases was 7% in CIN1, 46% in CIN2 and 86% in CIN3. There were no p16-positive cases in the group with normal epithelium. In order to grade CIN1 vs. CIN2 a combination of Ki-67 and p16 was helpful. Cases with a proliferation rate of <25% assessed with Ki-67 were most likely CIN1 (sensitivity 91.7%, specificity: 54.3%, positive predictive value: 73.3%, negative predictive value 82.6%). P16 was the most helpful marker in distinguishing between CIN2 and CIN 3 as p16 negative cases were more likely to belong into the CIN2 category. In summary, the histopathological assessment of cervical biopsies is based on H&E-stained slides. However, Ki-67 and p16 can be helpful in diagnosing and grading cervical intraepithelial neoplasia.

Association of tumor induced vascularization with clinicopathological parameters in cervical neoplasm.

Angiogenic properties have been shown in preinvasive cervical lesions. Our goal was to determine the angiogenesis in cervical intraepithelial neoplasms (CIN), the relationship between microvessel counts, histopathological parameters and the clinical outcome in invasive cervical carcinoma. Comparison of microvessel counts from normal epithelium with that from CIN and invasive carcinoma showed significant increases in pre-cancerous lesions and invasive cancer (p < 0.0001). Microvessel density, assessed by CD31 immunostaining, was found to be associated with the overall survival in women with clinical stage IB cervical carcinoma (p < 0.03). There was a significant association of microvessel density (p < 0.05) with relapse-free survival in patients with regional lymph node metastasis.

Stereology, flow cytometry, and immunohistochemistry of follicular neoplasms of the thyroid gland.

A retrospective analysis of surgically resected thyroid cold solitary nodules was performed by stereology, DNA flow cytometry, and immunohistochemistry in 15 follicular adenomas and 15 well-differentiated follicular carcinomas to determine if a differential diagnosis of both follicular neoplasms can be done exclusively from cytologic criteria. Between 150 and 200 tumor cell nuclei (TCN) were studied per case for their TCN profile area, perimeter, and density, as well as for stereologic estimates, including the new parameter, volume-weighted mean particle volume (Vv). Flow-cytometric analyses included measurement of the DNA index and the percentage of cells in S phase and G2M phase. The same tumors were examined for the expression of thyroglobulin and the intermediate filaments vimentin and keratin. Follicular adenomas and follicular carcinomas did not show any significant differences in stereologic estimates related to TCN size. Both groups included similar proportions of diploid and aneuploid neoplasms. Aneuploid follicular neoplasms showed a significantly greater area, perimeter, and volume of TCN as compared with diploid tumors, regardless of their histologic diagnosis. Follicular adenomas and follicular carcinomas expressed a similar staining pattern for the tested immunoreagents, with a few cases coexpressing vimentin and keratin. From our results, a differential diagnosis of follicular neoplasms cannot be performed on the basis of cytologic aspirates exclusively. Infiltration of capsula or vessels remains the only safe indicator of malignancy in the absence of metastases. The lack of cytologic differences suggests that some follicular adenomas are preinvasive carcinomas, not yet showing infiltrative growth at the time of resection.

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.

DNA aneuploidy, S-phase fraction, nuclear p53 positivity, and survival in non-small-cell lung carcinoma.

Inactivation of the p53 gene plays a key role in tumour biology, probably through a disturbed cell cycle control and an increased genetic instability in p53-inactivated tumours. To learn more about the relationship between p53 alterations, proliferation and genetic instability (DNA aneuploidy) in lung cancer patients, specimens of 220 surgically resected lung carcinomas with clinical follow-up information were examined by immunohistochemistry (p53; CM1) and flow cytometry. Nuclear p53 positivity--found in 49.5% of the tumours--was associated with both high S-phase fraction (SPF) and DNA ploidy aberrations. SPF was higher in p53-positive tumours (15.9 +/- 10.2) than in p53-negative tumours (10.3 +/- 8.7; P = 0.03). The rate of p53 positivity was higher in 101 DNA-aneuploid and DNA-multiploid tumours (55%) than in 27 diploid and peridiploid carcinomas (33%; P = 0.0512). These results are consistent with an in vivo role of p53 inactivation for increased proliferative activity and development of genomic instability in lung cancer. There was no association between SPF and prognosis. Although prognosis was worse in DNA-aneuploid and multiploid tumours than in diploid, peridiploid and tetraploid carcinomas (P = 0.029), DNA ploidy was not an independent predictor of poor prognosis in multivariate analysis. These data show that DNA-flow cytometry has little prognostic value for patients with resected non-small-cell lung carcinoma.

Immunocytochemistry in diagnostic cytology.

As the decision for immunocytochemistry is usually made on the basis of findings in Papanicolaou-stained smears and uncovering of the smears takes time, the immunocytochemical results are often reported with some delay. But they are of clinical interest only if reported within a short time. Therefore, immunocytochemistry on cytologic preparations must be carefully organized. The decision for immunocytochemistry must be made before the mounting medium has completely hardened to keep the time of uncovering short. The method of immunocytochemistry should fulfill the following prerequisites: 1. Cell sampling and fixation should be easy to handle for the clinician who sends the specimen to the laboratory. 2. Unspecific background staining, especially in cytologic preparations rich in blood and protein, should not occur. 3. The immunostaining method should be applicable to all kinds of cytologic material, fixed and stained smears included. 4. The nuclear structure of tumor cells should not be destroyed by the immunocytochemical procedure so that tumor cells after incubation are clearly distinguishable from normal cells showing a similar reaction as the tumor cells. There has hitherto been no such all-round method fulfilling all these prerequisites since the properties of the antigenic epitopes of the cells and of the antibodies recognizing them are too heterogeneous. Therefore several methods have to be considered and a variety of technical aspects such as fixation, storage of cytologic material, properties of tinctorial stains, of antibodies and of the antigenic epitopes must be studied to find out the two or three standard methods which meet the requirements in most cases. We recommend the ABC method for Papanicolaou-stained smears and the APAAP method for demonstration of lymphocyte markers. The indication of immunocytochemistry in diagnostic cytology is restricted by the limited number of specimens. Therefore, the following rules have to be observed: 1. The conventional light-microscopic examination must have priority over the immunocytochemical examination. 2. The cytologic specimens assigned for immunocytochemical examination must have been adequately fixed and stored. 3. As the number of smears is limited, the immunocytochemical examinations must be carefully planned and restricted to the absolutely necessary incubations. If possible, an informative smear has to be spared for documentation and future training of cytologists and cytotechnicians. 4. Immunocytochemical examinations in cytology are only justified if the diagnostic problem can be clearly defined. 5. The panel of antibodies should be selected carefully so that the results may give an answer to alternative questions. At least two antibodies should be applied.(ABSTRACT TRUNCATED AT 400 WORDS)

A fluorescent study of ligands for guanidinobenzoatase, a protease associated with tumour cells.

We have employed ethanol-fixed wax embedded sections of human breast tumours and smears of rat leukaemia cells to provide test systems with recognisable tumour cells amongst normal cells. We have used 9-aminoacridine to locate cells possesing guanidinobenzoatase, an enzyme which degrades fibronectin and which binds 9-aminoacridine to its active centre. The binding of 9-aminoacridine to tumour cells allows these cells to be located by fluorescent microscopy. Pre-treatment of these sections with BZAR, a known inhibitor of guanidinobenzoatase inhibited the binding of 9-aminoacridine to the tumour cells. These techniques defined the tumour cells in the sections; we then demonstrated by fluorescent microscopy that both Texas red-agmatine and BZAR also bound to the guanidinobenzoatase of these tumour cells. These fluorescent probes have been used as model compounds to illustrate the ability of both N-substituted agmatines and N-substituted arginines to deliver desired molecules to an enzyme on the surface of tumour cells. Replacement of these fluorescent moieties by cytotoxic moieties attached to the same ligands could lead to selective drug delivery to tumour cells.

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.

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.

A simple "expert system" for morphometric evaluation of cells in pleural effusions.

Quantitation in diagnostic pathology serves to improve both diagnostic reliability and the prognosis of various malignant tumors. Unfortunately, flexible, user-friendly, reasonably priced and compatible morphometric systems are hardly available on the market. A modular Apple II PC-based system was therefore developed in-house. It records stereologic, planimetric and digital image analysis data and calculates secondary parameters. Detailed statistical analyses can be performed, after data transfer, on larger computers. A typical application of diagnostic morphometry is to determine whether, and with which probability a patient with an unclear histopathologic finding can be assigned to one or more groups of patients with known diseases and prognoses. To this end morphometric data of the patient under investigation are compared with other patient data stored in an expert system. This paper describes the concept of an expert system carried out on the Apple II PC system mentioned above and outlines the evaluation procedures. Invariant moments were used to describe nucleus textures. Results obtained from the cytological analysis of pleural effusions demonstrate that with this approach it is possible to differentiate between normal mesothelium, mesothelioma and metastases of adenocarcinomas.

Breast cancer in previously burned skin: a postburn skin adnexal malignancy?

An unstable postburn scar at the submammarian fold 6.5 months after a burn injury turned out to be breast cancer. The unexpected histological findings and a possible correlation between the burn and the malignancy are discussed. This care report emphasizes the importance of histopathologic examination and the chance of unusual findings in a postburn ulcer.

Clinical experience with a new endometrial cell sampling kit (Isaacs curity endometrium cell sampler) in the early detection of endometrial cancer.

The Isaacs Curity Endometrium Cell Sampler (ICECS) is a new device for the detection of precancerous lesions of the endometrium and asymptomatic endometrial carcinoma. Our clinical experience with the ICECS in a group of 136 women 45 years of age and older with an increased risk for endometrial cancer showed that it was easily used, caused minimal discomfort to the patients and gave good results. The cytologic smears were acceptable for screening in 130 of the 136 cases (95.6%). Normal premenopausal or postmenopausal endometrial cells were found in 100 cases. Hyperplastic changes were found in 18 cases. Carcinoma were diagnosed in 12 cases; all were proven by histologic examination of tissue obtained by curettage. Our results indicate that the new device is valuable for mass cytologic screening for endometrial cancer.

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.

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.

Fluorescence in situ hybridization for detecting erbB-2 amplification in breast tumor fine needle aspiration biopsies.

OBJECTIVE: To evaluate the feasibility of erbB-2 amplification analysis of fine needle aspiration (FNA) biopsies. STUDY DESIGN: FNA smears and dissociated nuclei from 58 breast cancer samples were examined by dual-labeling fluorescence in situ hybridization (FISH) with probes for centromere 17 and the erbB-2 gene. The results were compared with the outcome of erbB-2 immunohistochemistry. RESULTS: Tumors were categorized according to the erbB-2/centromere 17 signal ratio. There were 23 tumors with high-level amplification, four cases with a low-level erbB-2 gain and 27 tumors with normal erbB-2 content. Four tumors showed an erbB-2 deletion, all in patients < or = 42 years of age. ErbB-2 amplification was strongly associated with positive erbB-2 immunostaining (P < .0001). Comparison of FISH analysis on dissociated cells and on FNA biopsies showed high correspondence (P < .0001). CONCLUSION: FISH allows reliable detection of erbB-2 gene amplification on FNA biopsies.

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.

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.

[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.