Working Group: Cytopathology I

Abstracts . 367

213

215

Plasminogen Activators and Plasminogen Activator Inhibitor 1 in A NEW STEREOLOGICAL METHOD ALLOWING AN EASY Dedifferentiated Chondrosarcoma ESTIMATE OF NUMERICAL DENSITIES, VOLUME SHARES, C.Hackel (a.G.), K. Radig, A. Roessner SURFACE STRUCTURES, AND THE TEST OF THE DISTRIBUTION OF Department of Pathology, Otto-von-Guericke University, Magdeburg CELLS OR PARTICLES WITHIN A TIssUE Aims: The high malignant components of dedifferentiated G. Busche, 1. Schlue (a.G.), A. Georgii chondrosarcomas show both an increased proliferative activity and a high Pathologisches Institut der Medizinischen Hochschule Hannover metastatic potential in comparison to the low malignant parts. the plasminogen activator system is well-known to play an important role in Morphometric estimates of tissue volume, surface structure, and the the metastatic spread and growth regulation of different tumor types. In the numerical density of cells (or particles) within a tissue are classical current study, the u-PA, t-P A and PAl-l expressions in 11 dedifferentiated assignments of quantitative pathology. A new method is presented chondrosarcomas have been studied. The main point was the direct allowing the estimate of (a) tissue volume, (b) surface area, (c) the comparison of the findings in the high and low malignant areas. number of cells (or particles) within a tissue, and furthermore, (d) a Methods: We selected the low and high malignant components of 11 statistical test of the distribution of cells (or particles) within a dedifferentiated chondrosarcomas. 17 conventional chondrosarcomas tissue. (grade 1 and 2) served as control cases. The expression ofu-PA, t-PA and The principles of this method are (1) a virtual subdivision of a slide P Al-l was evaluated semiquantitatively by immunohistochemistry into at least 100 squares of equal size by using a morphometric (negative: 1, positive:2-4). machine or a measuring ocular, (2) counting "positive" squares, i.e. Results: In the cartilaginous components of the tumors, the chondrocytes squares with at least 50 % tissue, with the surface structure, or with showed a low to intermediate coexpression ofu-PA (mean intensity 1.9), t- at least one cell of interest, and (3) estimating the tissue volume, the PA (a.i. 1.7) and PAl-l (a.i. 2.3). In locally restricted areas, such as some surface area or the numerical density of cells which are randomly invasive foci and regions of tumor calcification or pseudo enchondral distributed within the tissue. The numerical density of cells is ossification, a higher cellular immunoreactivity for all antigens has been estimated supposing a Binomial distribution of "positive" squares detected. The high malignant cells of the dedifferentiated components, depending on the cell number within the tissue. The statistical test of independent of their morphologic features, displayed a significantly the distribution of cells (or particles) within a tissue allows the increased expression ofu-PA (a.i. 3.7), t-PA (a.i. 3.8) and PAl-l (a.i. 3.6). decision whether the cells are distributed by chance within the In the conventional chondrosarcomas, we found identical results as we did tissue, or whether the cells tend to clustering or to a homogeneous in the cartilaginous areas of the dedifferentiated tumors (u-PA: a.i. 1.9, t- distribution. The advantages of this method are presented by examples of PA: a.i. 1.5, PAl-I: a.i. 2.3). Conclusions: The expression of both the plasminogen activators u-PA and patients with a Ph' -positive CML. The estimated numerical densities t-PA and the plasminogen activator inhibitor 1 strongly increases through of Pseudo-Gaucher cells / rom 2 bone marrow were almost identical the process of dedifferentiation in chondrosarcomas. Subsequently, the when compared with an exact count, but the time of examination enzymes are highly suspicious to play an important role in the invasion and was significantly shortened by the new method (n = 94; r = 0.97; P metastasis andlor growth regulation of these lesions as shown in recent < 0.00001). findings in other tumor types.

Working Group: Cytopathology I

214

216

Extracellular Matrix Analysis in Intervertebral Disc Protrusion K.-R. Greskotter, U. Bios a.G., K. Sauer a.G., T. Aigner a.G., Th. Kirchner Pathologisches Institut, Universitat Erlangen

Targeting aids for fine Needle Aspiration in Mamma-

Aims: The characteristic distribution of various collagen subtypes within the different tissue compartments in intervertebral discs is assumed to be crucial for fulfilling the essential biomechanical function of the collagen network within the disc system. Accordingly differences in the biochemical composition of the collagens could be of great significance for tissue instability with subsequent disc protrusion. In our study we analysed the biochemical composition of surgical disc specimens of patients with disc prolapse to evaluate potential matrix alterations as potential causes of tissue instability. Methods: 40 surgical (prolapsed discs) and 8 normal were analysed immunohistochemically using monospecific antibodies to collagen subtypes I, II, III, and VI. Results: In normal and operated intervertebral discs, type II collagen was restricted to the interterritorial matrix of the inner annulus as well as the nucleus pulposus. Type I and III collagens were in both specimen types found interterritorially in the outer and inner annulus in the characteristic laminated pattem, whereas in the nucleus pulposus pronounced staining was seen in the pericellular and territorial matrix. In all specimens, type VI collagen was in all compartments restricted to the pericellular matrix. A different matrix composition in prolapsed disc specimens was found in areas of tissue repair: The extracellular matrix contained collagen types I, III, and VI and no type II irrespective of the location within the disc tissue. Conclusions: The results of our study show that no significant principle alterations of COllagenous extracellular matrix components are found in degenerated disc tissue. This indicates that qualitative aHerations in the biochemical composition as a causative factor of disc protrusion are rather unlikely and rather focuses attention to aHerations in the fine architecture of the disc matrix and impaired external mechanical loading as a crucial condition for tissue failure in intervertebral disc protrusion. Secondary repair tissue is characterised by a matrix composition typical for normal scar formation and does not originate from a cellular response of disc cells.

Diagnostics M. Bauer, P. Tontsch, G. Birmelin Aims: for cytological diagnostics different targeting aims are in use. Next to the stereotaxical guided FNA the US-guided and the puncture guided by palpation are of most importance. Methods: the methods of localization are identical for cytologic puncture and core biopsy. While in the US-guided FNA the needle is held parallel to the transducer, in the stereotaxic guided FNA a special supplemental device for the mammographic unit is used. Results: Experienced cytodiagnosticians have lowererd the rate of not usable puncture specimens to less than 5 %. For not as experienced examiners a rate of up to 30 % has to be considered. Small palpable foci are localized incorrectly in 113 of the cases. Here, method of first choice is the combination of clinical findings with sonographic contro\. The advantage of stereotaxic punctures is the exact localization, however, due to the system, the clearance for aspiration puncture is limited and therefore the rate of not usable specimens higher. In literature it is determined between 9 and 20% (Bauer, Schulz-Wendtland, Fonschr. Rontgenstr. 156, 286 (1992); Azavedo, Svane, Auer, The Lancet, 1033 (1989».

368 . Abstracts

217

220

Indications and Strategies for a precise application of the Aspiration Cytology in Mammmy Surgery H.Schoendorf

INTRAOPERATIVE HIS TO- AND CYTODIAGNOSIS OF NEOPLASTIC BREAST LESIONS: CORRELATION AND DISCREPANCIES (RESULTS BASED ON 200 CASES) F. MOINFAR, H. DENK Institut fiir Pathologie, Karl-Franzens-Universitiit Graz Aims: The intraoperative examination of frozen sections of mammary lesions is a well established and useful diagnostic tool. Most pathologists do not find it necessary to perform additional cytologic examinations. The aim of our prospective study wasto investigate whether diagnostic preciSIOn can be further improved by a combination of intraoperative cytological and histological examination. Methods: We report the results obtained on the basis of 200 unselected cases. After palpation and inspection of surgical specimens, which were submitted for intraoperative frozen sectIon diagnosis, imprints were performed from the surface of specImens and investigated in additJon to hIstology The im prints were stained wi"th DifT-Quick. The cytologic dIagnoses were made independently of the histologic evaluation. Results: In 3% of the cases cytology clearly revealed the presence of malignant epithelial cells, whereas frozen section histology was negative. However, extensive histological examination of paraffin-embedded material revealed (mostly intraductal) carcinoma. In II additIOnal cases (5.5.% of total cases examined) the cytological examination revealed suspicious or even highly suspicious but not definitively malignant tumor cells. In these cases the frozen section histology was also negative, whereas exammation of paraffin material revealed either carcinoma or atypical ductal or lobular hyperplasia. In 4% of the cases the cytological examination revealed suspicious epithelial cells, whereas after examination of frozen section and paraffin material neither carcinoma nor atypIcal hyperplaSIa could be detected. In 0.5% of the cases the frozen section histology was false positive (as related to paraffin histology). Conclusions: The following conclusions can be drawn: (i) sensitivity of cytology is 96% (ii) sensitivity of frozen section histology is 80% (iii) specIficity of cytology is 90% (iv) specificity of frozen section histology is 97.5%. Since additional intraoperative application of cytology of breast lesions clearly reduces the num ber of false negative diagnoses we conclude that intraoperative cytology of breast lesions is a valuable supplement to frozen section histology. We, therefore, strongly recommend the combination of cytology and frozen section histology.

Ketteler Krankenhaus Offenbach

Aspiration Cytology of the breast today has become a reliable method in diagnosis of breast diseases by its high sensitivity and specifity . This method benefits of rapid diagnosis, minimal disruption of the tumor bed, low costs, great economic value and few complications. In identifYing malignant lesions important indications are the diagnosis before definitive therapy (mastectomy, breast conserving therapy), diagnosis of recurrences and palpable metastases, definitive diagnosis before chemotherapy, and by the stereotactic-cytology diagnosis of occult lesions. In the field ofbenigne lesions Aspiration Cytology has become a useful method in the diagnosis of pseudotumors and cysts, so avoiding unnecessary biopsies. The diagnosis ofpalpabel solid tumors is of high accuracy. The application and ease of procuring cell samples from tumors for cell image analysis, for flow cytometry and ploidy studies place this method in the forefront of biologic research and clinical medicine.

218 ACCURACY, CLASSIFICATION OF FINDINGS AND DIAGNOSTIC LIMITS OF FINE NEEDLE ASPIRATION BIOPSY (FNA) IN BREAST

TUMORS

G. W. LOCHER Pathologie-Institut, Bnmnhofweg 45, CH-3000 Bern 14, Switzerland 'This review discusses indications ofbreast FNA, main cytological criterias of malignancy, problems of cytological typing and grading of mammary carcinomas as well as possibilities and limits of cytological typing in benign lesions. A cytological "grayish zone" between lUlequivocal morphological malignancy and benignancy is described in detail; the handling of its difficulties is a central aspect in evaluating the relative importance of the FNA cytology within the various diagnostic methods in breast diseases. With the alertness of the public, and using more sophisticated imaging technics, the morphological spectrum of breast cancers lUldergoing FNA has changed. For example, the rate ofFNAs from smaJ\ carcinomas (PTl), frequently of the low-grade tubulo-Iobular type, is today higher than 10 years ago. In these tumors, a cytodiagnostic sensitivity of only about 70-80% has to be accepted if false-positive diagnoses are to be avoided. Other issues are rates and management of the so-called "false diagnoses", the furmulation of the cytopathological reports, and the quality assurance. Our experiences are compared with published results from other institutions.

(X

the

lnbbtly needle biq>Sy (te) is a dlag1)Stic alternative to fine needle asplratial biWSY (f1Wl) for the exanlnation of turors of thE! famle breast. Ibth nBthxfs eM be awl ied for the sme irdlcations as \leB, gnraBy lnvovif'Y;l the lOll diag-05tic wecificy of clinical ard iJragit-1,/Iretoods. The ally contrairdication for te is tlEmlm1agic diathesi s. CaTplications (l1EIMtanas, infl tmTBtlons) of both N3 an:! FW6 FWI8 are extrerely rare. Between 1.1.94 ard 31.9.95 120 Nl \lere carr~a:I out with the follOllif'Y;l results: 69 I of the cases \En'! carcil'lln3S, 25 Illete mastq;lathies, 3 I \lere inflannrtlms ard In 15 ,; of the cases II carcirana was suspecta:I. In 1,5 ,; tre rraterial

was

TUMOR CELL DETECTION IN INTRAOPERATIVE PLEURAL LAVAGE IN LUNG CANCER R. SCHAFFER, J. BUHR * (a.G.), K.H. BERGHAUSER (a.G.) Funktionsbereich Zytopathologie am Institut fiir Pathologie und *Klinik fiir Allgemeinund Thoraxchirurgie der Justus-Liebig-Universitiit GieJ3en Aims: According to the TNM classification, a malignant pleural effusion equals a T4 spread of lung cancer. The criterion is the cytologic demonstration of tumor cells. Methods: We present the results of a prospective study in which pleural lavage was done with 300 ml physiological saline solution after opening the chest to discover whether tumor cell dissemination into the leural cavity occured in the early stages of lung cancer = lavage I). A second lavage after resection of lung cancer ( = lavage II) showed whether surgical manipulation of the lung parenchyma led to a tumor cell contamination of the pleural cavity. Results: 391 patients (306 patients with the first manifestation of lung cancer and 85 patients with nonneoplastic lung disease) underwent pleural lavage. Tumor cells were found in lavage I in 85 patients (27.8%), in 78 of them also in lavage II. Among the previously classified as stage I (pTI NO, pT2 NO) lung cancer positive tumour cell detection was in 38,5% possible. All controls were negative, i.e., there were no false positive fmdings. The cumulative five-year survival rate of curative resected non-small cell lung cancer in stage I (n =78) was 22,1 % if lavage was ~sitive (n =30), and 64,3% if lavage was negative (n =48) (p < 0,05). Conclusions: Therefore, detection of tumor cells in pleural lavage before resection proves that tumor cells have spread into the pleural cavity. The positive result in pleural lavage seems to be a prognostic predictor for patients with lung cancer. An adjuvant therapy should be perfonned for those patients with a positive intraoperative pleural lavage in lung cancer.

f

219 Elcp!riences with the hi~ speBi needle biq,sy tedTli
220 a

insufficient.

For carclranas trere was ~lete aJlCOI"dance betI«ie1 Nl ai-d final histology for tI'e histol~ical classificatial , the receptor status

en:I the proJiferatiCJ1 Irdex. False-lXlSitive results were not cttalna:l Ilhereas false-regatiYe results occurred In 11,7 %of the cases~ This COJld be I'9iC8l to belOll 10 ,; by tecmical If1llI"ClV8I91t ex the fII!thod. In SUTmary, te Is CI'I acceptable alternative tD FWI8 In ~ive dlag-ostic _inatlal of breest turors. It ~ be S~lor ItIere experterce In FNAB IIIXI cytological eltIJTIinatlCJ1 is laddll;!.

Abstracts· 369

Working Group: Cytopathology II

221

223

Mm·1 (Kl·67) IMMUNOSTAINING OF BREAST CANCER CELLS IN CYTOLOGIC SMEARS P. DALQUEN*. B. BASCHIERA (a.G.)*. R. CHAFf.ARD (a.G.)*. H. DIETERICH (a.G.)**. O.E. FEICHTER*. K. KRAMER (a.O.)**. J. TORHORST (a.G.)*. *Institute for Pathology. University of Basel. **Frauenklinik Rheinfelden

INTERPHASE CYTOGENETICS AS A NEW DIAGNOSTIC TOOL IN CYTOPATHOLOGY

AIMS: Recent studies have shown that "neoadjuvant" chemotherapy downstagi~g large primary breast cancers prior to surgery may allow more often breast conservation. Since proliferation index correlates with the biological behaviour of breast cancer and since it has been demonstrated that tumor responsiveness to cytotoxic drugs may depend on proliferation, the assessment of proliferating capacity of a carcinoma may contribute to the selection of high risk patients who could be candidates for adjuvant chemotherapy. The purpose of this study was to test the reliability of immunocytochemical evaluation of the proliferation capacity on cytologic specimens. METHOD: The monoclonal antibody MIB·l was employed to 83 smears prepared from frozen tissue (FTS) and to 51 fine-needle aspirates (FNA) from 119 breast cancer patients. MlB-l staining indexes were compared with various tumor parameters assessed on histologic material. RESULTS: MIB-l positive cell fraction established on cytologic smears was significantly different in ductal and lobular carcinomas (p=O.024) and was significantly correlated with mitotic activity (p
Interphase or molecular cytogenetics allows the detection of karyotype changes in non-dividing cells. The various applications of this technique are based on recent developments in non-radioactive in situ hybridization. The possibility to examine intact interphase cells enables one to assess cytomorphology together with genetic characteristics. Thus, gains and losses as well as structural changes of chromosomes or genes can be detected at a single-cell basis.

M. WERNER and A. GEORGII Pathologisches Institut der Medizinischen Hochschule Hannover

Enzymatic visualization of hybridized probes, e.g. by peroxidase or alkaline phosphatase. is a stable reaction, and morphological details can be evaluated by conventional light microscopy. The use of fluorescing dyes, e.g. fluorescein or rhodamin, is necessary to detect specific chromosomal translocations. Comparative genomic hybridization can demonstrate non-ballanced changes of the genome but requires homogenized cells. Molecular cytogenetics is well established for the diagnosis and detection of residual disease in chronic and acute leukemias. In these disorders, cells affected by clinically significant structural aberrations such as t(9;22), t(15;17) or inv(16) can be quantified by using triple color fluorescence. This technique also promises to be suitable in the classification of non-Hodgkin's lymphomas or the differential diagnosis of solid tumors, especially those of soft tissue origin.

222

224

PROGNOSTIc!! SIGNrFICAlfCB OF CHROMATIN FEATURES IN BREAST CARCINOMAS

Detection of human papilloma virus In cytologic specimens by hybrid

K.D. KUNZE, V. DIMMER (a.G.), G. HAROSKE (a.G.), W. MEYER (a.G.), F. THEISSIG (a.G.) Institute of pathology, Technical University Dresden

capture-technlque

Aims: The prognostic significance of chromatin features was studied on 187 invasive ductal breast carcinomas (mean follow up 63 months) and compared with other clinicopathological factors (lymph node status, histopathological grading, tumor size). Methods: The studies were performed with two different image cytometry workstations. Image cytometric features included 54 parameters of nuclear size, shape and chromatin structure. Multivariate, discriminant analysis and the Cox regression proportional model were used for statistical evaluation. Results: Combining clinicopathological and cytometric features a correct prediction of prognosis could be made in 83 t. By statistical evaluation only three out of 70 features were found to contribute significantly to the discrimination of subgroups: the number of coarse chromatin particles, lymph node involvement and tumor size. For prediction of a 5 or 7 year survival the significance of the chromatin particle number is equivalent to the significance of the nodal stage. Independent measurements with two different image cytometric systems indicated a high reproducibility of chromatin features. Conclusion: 'The image cytometric stUdies on breast carcinomas substantiate the prognostic significance and reproducibility of chromatin features.

Postfach 10 03 46

HEINE M, FLENKER H

27503 Brememaven

The hybrid capture method is an easy to uSe technique for the evaluation

of DNA sequences in HPV infections. The nonradioactive test kit is designed to detect 14 different types of HPV divided into two groups: the "low risk" group ( HPV 6/11/42143/44 ) and the "high risk" group ( HPV 16/18131/33135/45151/52156 ) for the genesis of cervical cancer. The

sensibility of this RNA-DNA technique is higher than in Southern blOtting. An outline of the method: after denaturation of DNA in infected cells of the cervical smear, a specific RNA probe is added thus forming RNADNA hybrids. These are detected by an immunological reaction with monoclonal antibodies. The test is recommended for follow-up after a cytologic diagnosis of HPV associated mild or moderate dysplasia , ( group III 0 in the MUNICH classification ). Practical value of the test, corresponding cytologic diagnoses as well as different therapeutiC consequences will be discussed.