Optimization and validation of immunocytochemical detection of oestrogen receptors on cytospins prepared from fine needle aspiration (FNA) samples of breast cancer.

OBJECTIVE: To optimize and validate immunocytochemical (ICC) assessment of oestrogen receptors (ERs) on cytospins prepared from fine needle aspiration (FNA) samples. METHODS: Optimal conditions and variability in ICC detection of ERs were established on cytospins prepared from the human breast cancer cell line MCF-7. Protocols that yielded adequate results were further validated on 52 FNA samples of resected breast cancer tumours using analysis of concordance with the ER status, determined by standard immunohistochemistry on corresponding formalin-fixed, paraffin-embedded tissue (FFPET). On 37 diagnostic FNA samples, manual immunostaining with antibody 1D5 was compared with automated immunostaining with antibody 6F11. RESULTS: The highest percentage of ER-positive MCF-7 cells with lowest variability was obtained on methanol-fixed cytospins with or without microwave pre-treatment: 72 ± 5% and 75 ± 7%, respectively. Microwave pre-treatment was mandatory for Papanicolaou-stained cytospins in order to achieve between 63 ± 14% and 67 ± 9% of ER-positive MCF-7 cells. The concordance between ICC assessment of ERs on FNA samples and corresponding FFPET sections was complete for methanol-fixed cytospins (100%, kappa = 1) and adequate for Papanicolaou-stained cytospins (94%, kappa = 0.84) and Papanicolaou-stained smears (92%, kappa = 0.75). Complete agreement in ICC detection of ERs was obtained for manual immunostaining with antibody 1D5 and automated immunostaining with antibody 6F11. CONCLUSIONS: Methanol-fixed cytospins prepared from FNA samples ensure highly reliable ICC assessment of ERs, whereas Papanicolaou-stained cytospins or smears are conditionally suitable because of the small risk of false negative results.

S-phase fraction determined on fine needle aspirates is an independent prognostic factor in breast cancer - a multivariate study of 770 patients.

BACKGROUND: The prognostic significance of DNA ploidy and the S-phase fraction (SPF) have been extensively studied in breast cancer, but their clinical utility remains controversial. The type of tumour material can substantially influence flow cytometric DNA measurements. Material obtained by fine needle aspiration (FNA) biopsy is very suitable for flow cytometric DNA analysis because it contains a low proportion of non-tumour cells and less debris than tissue samples. METHODS: The prognostic significance of DNA ploidy and SPF, determined on FNA samples, was analysed in 770 breast cancer patients, diagnosed between 1992 and 1997. DNA ploidy and SPF were determined at the time of diagnosis as part of the diagnostic work-up. The median follow-up was 90 months. Survival analysis included overall cancer specific survival (OS), disease free survival (DFS) and survival after recurrence (SAR). Other variables included in survival analyses were age, histological grade, histological type, lymph node status and tumour size. Disease free interval and the site of recurrence were also included in SAR analysis. RESULTS: DNA ploidy and SPF correlated with tumour type, size, lymph node involvement and, especially, tumour grade. In a univariate analysis, both aneuploidy and high SPF were associated with shorter OS, DFS and SAR, but only SPF retained its independent prognostic significance in multivariate analyses. Independent prognostic variables for OS were node status, histological grade, SPF and tumour size. Node status, histological grade and SPF were independent predictors of DFS, while the site of recurrence, SPF, histological grade, disease free interval and age were independent predictors of SAR. CONCLUSIONS: DNA ploidy and SPF can be efficiently and routinely determined on FNA samples. High SPF is independently associated with a worse clinical outcome of patients with breast cancer. Although SPF and histological grade share prognostic information to some degree, SPF provides additional, less subjective prognostic information. The prognostic value of SPF determined on FNA samples could be even more relevant in neoadjuvant settings and for patients not amenable for surgical treatment, when histological grade cannot be assessed.

Analysis of routine cytopathologic reports in 1,598 histologically verified benign breast lesions.

This retrospective study was designed to evaluate the accuracy of cytopathologic diagnosis and of correct classification of benign breast diseases. A total of 1,598 FNABs were identified to have met the study criteria; of these, 1,258 (78.7%) cases were cytologically benign, 88 (5.5%) suspicious, 3 (0.18%) false-positive, and in 249 (15.6%) cases an inadequate sample was obtained. A specific diagnosis was made in 847/1,258 (67.3%) cases; the other 411 were diagnosed as benign NOS. Out of 847 specific FNABs diagnoses, 451 were fibroadenomas, 27 phyllodes tumors, 289 fibrocystic diseases, 4 proliferative fibrocystic diseases, 38 papillomas, 22 fat necrosis, 9 mastitis, 1 pseudolymphoma, 2 lipomas, 2 duct ecstasies, and 2 atheromas. In our study group the cytopathologic diagnosis of benign breast diseases excluding unsatisfactory aspirates was correct in 93%. Specific diagnosis was correct on average in 50% of cases, only in FA was its accuracy over 60%; in adequately sampled tumor, the predictive value of FA was 86.2%.

Effect of primary treatment on survival in anaplastic thyroid carcinoma.

AIMS: Anaplastic thyroid carcinoma (ATC) is a fatal disease despite combined treatment consisting of chemotherapy, radiotherapy and surgery. The optimal sequence of treatment modalities is not known. The purpose of our retrospective non-randomized study was to find out whether timing of the treatment modality had any influence on survival, and to find out if primary surgery prolongs survival in comparison to primary chemotherapy and/or radiotherapy. METHODS: From our database of 162 patients with ATC treated at the Institute of Oncology Ljubljana from 1972-98, 79 patients (26 men, 53 women; age: 40-86 years, mean age 65 years) were included in this retrospective study. The 83 patients with distant metastases on admission, with the survival shorter than one month or patients without any treatment were excluded. The 79 patients were classified into (1) primary surgery group (n=26) and (2) primary chemotherapy and/or radiotherapy group (n=53), including the 12 patients in whom surgery was performed after chemotherapy and/or radiotherapy. The survival of both groups was compared by log-rank test and group characteristics by ANOVA and(2 test using SPSS program. RESULTS: In comparison to the primary surgery group, the patients from the primary chemotherapy and/or radiotherapy group were older and had faster growing, and larger tumours, which were not confined to the thyroid, and more frequently had regional metastases. There was no difference in the survival of the two groups (P=0.17). Survival for longer than one year was observed in 25% of patients with primary surgery and in 21% of patients with primary chemotherapy and/or radiotherapy. The best results (50% survival at one year) were obtained in patients in whom the tumour was surgically removed after primary chemotherapy and radiotherapy. CONCLUSION: This study suggests that the timing of the treatment modalities has an impact on survival and that treatment should start with chemotherapy and/or radiotherapy, with surgery to follow if possible.)

Prognostic significance of DNA ploidy determined by high-resolution flow cytometry in breast carcinoma.

OBJECTIVE: To assess the prognostic value of DNA ploidy in breast carcinoma and its relation to other established prognostic factors. STUDY DESIGN: We evaluated DNA ploidy in 303 breast carcinoma patients with a median follow-up of 63 months. Flow cytometry was performed on frozen tumor material, yielding histograms with narrow peaks (median coefficient of variation of 2.08). DNA ploidy pattern was classified as either diploid versus nondiploid, euploid (diploid and tetraploid) versus aneuploid or diploid/near-diploid (DNA index < 1.2) versus other, and correlated with relapse-free (RFS) and cancer-specific survival (CSS) along with tumor size, histologic grade and type, axillary lymph node involvement, menopausal and steroid receptor status, age and type of treatment. RESULTS: Seventy-one percent of tumors were DNA nondiploid (14% tetraploid and 57% aneuploid). There was a strong association between DNA ploidy and histologic grade. Histologic grade, lymph node status, tumor size and DNA ploidy (regardless of the classification used) were all significantly associated with RFS and CSS in multivariate analysis. CONCLUSION: These results suggest that DNA ploidy, at least when determined from frozen tumor tissue, is an independent prognostic factor in breast carcinoma; however, its prognostic power seems to be inferior to that of histologic grade, with which it strongly correlates.

Cytological grading of breast cancer in Giemsa-stained fine needle aspiration smears.

A potential cytological nuclear grading based on a semi-quantitative evaluation of three basic nuclear features, size of cell nuclei, anisonucleosis and the proportion of nucleoli-containing-nuclei, was tested on 74 Giemsa-stained fine needle aspiration of breast smears for its reliability in establishing the malignant potential of breast cancer. The prognostic impact of DNA-ploidy and S-phase fraction was also assessed. A good correlation between the three basic nuclear features, DNA-ploidy, S-phase fraction, cytological nuclear grade and histological grade, was shown. Using the cytological nuclear grade proposed, correct classification of cases between low histological grade (HG I) and high histological grade (HG II + HG III) was achieved in 79.73%. A statistically significant difference in 5-year survival rate was also observed between low malignancy grade and high malignancy grade breast cancer patients, regardless of the grading method used. DNA-ploidy and S-phase fraction were not statistically significant in establishing the malignant potential of breast cancer.

DNA ploidy of human granulosa cells from natural and stimulated in vitro fertilization cycles.

OBJECTIVE: To analyze and compare the DNA ploidy of granulosa cells from natural and gonadotropin-stimulated follicles obtained during IVF. DESIGN: Retrospective analysis of laboratory data. SETTING: University medical center. PATIENT(S): Seventy-three aspirates of dominant follicles from natural IVF cycles and 113 aspirates from gonadotropin-stimulated cycles were analyzed. INTERVENTION(S): Cytospins were prepared and stained by the Feulgen-thionine method. MAIN OUTCOME MEASURE(S): Image DNA analysis was performed on an automated high-resolution image cytometer. DNA content and the number of nuclei with DNA content >5c were measured. RESULT(S): All samples from natural and gonadotropin-stimulated follicles were found to be diploid. Single cells with DNA content >5c were found in follicular fluid samples of four women with natural IVF cycles and in samples of nine women with gonadotropin-stimulated cycles. CONCLUSION(S): DNA ploidy of granulosa cells from natural follicles has not been studied before. In natural samples, granulosa cells were only diploid, without euploid polyploidization. We were unable to confirm DNA aneuploidy of granulosa cells in gonadotropin-stimulated follicles of women undergoing IVF.

Papillary thyroid carcinoma with exuberant nodular fasciitis-like stroma in a fine needle aspirate. A case report.

BACKGROUND: Papillary thyroid carcinoma (PTC) with exuberant nodular fasciitis-like stroma (PTC-ES) is a new morphologic variant of conventional PTC. It is characterized by extensive reactive stromal proliferation, which may occupy 60-80% of the tumor. CASE: A 42-year-old female developed a tender, left-sided thyroid mass. The fine needle aspiration biopsy specimen contained, besides diagnostic epithelial features of PTC, many cohesive tissue fragments of cellular stroma. CONCLUSION: A correct cytopathologic diagnosis of PTC-ES can be established if both epithelial and stromal components are present in needle aspirates.

The same luteinized granulosa-luteal cells from normal and superovulated human chorionic gonadotropin (hCG)-stimulated cycles contain hCG and insulin-like growth factor binding protein-1.

OBJECTIVE: To determine whether hCG and IGFBP-1 appear in the same or different cells and in what sequence. DESIGN: Retrospective analysis of laboratory data. SETTING: University medical center. PATIENT(S): Twenty-five women undergoing IVF-ET with natural cycles and 25 women having stimulated IVF-ET. INTERVENTION(S): Cells were obtained from dominant follicles in women with natural cycles and from the follicles from hMG- and hCG-stimulated cycles. MAIN OUTCOME MEASURE(S): Detection and localization of hCG and IGFBP-1 in granulosa-luteal cells using double immunocytochemical staining. Measurement of hCG and IGFBP-1 in follicular fluid and serum. RESULT(S): Three types of hCG staining were found: on the cell surface, on the cell surface and in the cytoplasm, and in the cytoplasm alone. IGFBP-1 stained diffusely in the cytoplasm and was found only in those cells that were luteinized and contained hCG. IGFBP-1 and hCG were colocalized in the same cells. There was a positive correlation between follicular fluid hCG and IGFBP-1 levels, but only in natural IVF-ET cycles. CONCLUSION(S): HCG-driven luteinization is required for IGFBP-1 synthesis to take place in granulosa cells.

Hydrolysis profiles of formalin fixed paraffin-embedded tumors based on IOD (integrated optical density) and nuclear texture feature measurements.

The aim of the study was to determine optimal hydrolysis time for the Feulgen DNA staining of archival formalin fixed paraffin-embedded surgical samples, prepared as single cell suspensions for image cytometric measurements. The nuclear texture features along with the IOD (integrated optical density) of the tumor nuclei were analysed by an automated high resolution image cytometer as a function of duration of hydrolysis treatment (in 5 N HCl at room temperature). Tissue blocks of breast carcinoma, ovarian serous carcinoma, ovarian serous tumor of borderline malignancy and leiomyosarcoma were included in the study. IOD hydrolysis profiles showed plateau between 30 and 60 min in the breast carcinoma and leiomyosarcoma, and between 40 and 60 min in the ovarian serous carcinoma and ovarian serous tumor of borderline malignancy. Most of the nuclear texture features remained stable after 20 min of hydrolysis treatment. Our results indicate that the optimal hydrolysis time for IOD and for nuclear texture feature measurements, was between 40 and 60 min in the cell preparations from tissue blocks of three epithelial and one soft tissue tumor.

Medullary carcinoma of the thyroid glad: diagnostic cytopathological characteristics.

Sixty fine needle aspiration biopsy samples of the medullary carcinoma of the thyroid, 49 primary and 11 recurrent/metastatic, were reexamined in order to determine diagnostic features and value of auxiliary techniques. In the smears, tumor cells were present either as single cells or in loose cohesive groups; in about one third of cases, three-dimensional groups predominated. Cells were of different shapes round-to-oval, polyhedral and spindle. The aspirates contained one or all three cell types. Large mononucleated cells were present in 47/60 cases and, in addition, in 34 of these multinucleated cells with nuclei arrayed in semicircular rows were present. Plasmacytoid and dendritic cells, observed in 58/60 cases, appear to be an important diagnostic feature. Red cytoplasmic granules and amyloid deposits could serve as an additional diagnostic clue. Among the auxiliary techniques, the demonstration of calcitonin and CEA immunoreactive cells proved to be the most helpful.

The effect of primary fixation with standard postfixation and the duration of hydrolysis on nuclear features in image cytometry.

The effect of three primary fixation procedures, used in the preparation of routine cytological samples: air-drying, Delaunay, and Saccomanno fixation, with postfixation in modified Böhm-Sprenger fixative, on nuclear features as a function of hydrolysis time is reported. Three different cell types: lymphatic cells (tonsil), epithelial cells (buccal mucosa) and mesenchymal cells (uterine myometrium) were used for the study. Our findings show, that generally not all features have the same plateau times as the IOD (integrated optical density), and that many features show different values depending on cell type and fixation method. It is therefore recommended that for any primary fixative used in routine clinical work and for each cell type, the hydrolysis curve for all nuclear features to be used in sample analysis should be established.

Detection of compact low-chromatin areas in cell nuclei images.

When analysing some DNA stained human cell nuclei using a light microscope or an quantitative image cytometer, compact low-chromatin areas (CLCA) can be observed. We are still not certain about the meaning and source of this phenomena. To enable the detection of CLCA by an image cytometer, a special image processing algorithm has to be developed and new nuclear cell features have to be designed. The presented image processing algorithm automatically detects CLCA in nuclei cell images taken from different tissues. The algorithm is composed of many basic image processing operations. The sequence of operations is determined by a priory knowledge about the properties of CLCA as a set of heuristic roles. The calculated CLCA features are CLCA area, perimeter, average intensity, compactnes and edge sharpness. The matching of the automatic CLCA detection and manual detection (performed by a biologist) was tested using 1400 cell nuclei. The results show a 83.4% match for the nuclei without CLCA and a 93.8% match for the cells with CLCA.

Prognostic value of DNA ploidy in breast cancer stage I-II.

The DNA content of paraffin-embedded tumor tissue has been measured by flow cytometry in 169 patients with operable breast cancer Stage I-II. The medium follow-up period was 123 months. Aneuploid primary tumors were found in 49% of patients. Tumor ploidy significantly correlated with histological type of tumor (p < 0.05), whereas no clear correlation between DNA ploidy and tumor size, histological grade and lymph node involvement was found. After 10-year follow-up, recurrence-free survival (RFS) of patients with diploid tumors was slightly better than the survival of those with aneuploid tumors, but the difference was not statistically significant (p = 0.39). In a Cox multivariate analysis only the axillary lymph node involvement and tumor size proved to be independent prognostic factors for recurrence, whereas DNA ploidy lost its prognostic value already in the univariate analysis. Therefore, we can conclude that the information on DNA ploidy, obtained from archival material, does not contribute significantly to better discrimination between good-risk and poor-risk operable breast cancer patients.

Cytomorphology of Langerhans cell histiocytosis.

OBJECTIVE: To determine the diagnostic accuracy in recognizing Langerhans cell histiocytosis (LCH) in cytologic smears obtained between 1977 and 1994 and to analyze the composition of these smears and thus identify various cytomorphologic patterns. STUDY DESIGN: The study group consisted of 37 cytologic smears (34 patients) taken from 30 bone lesions, 5 lymph nodes, 1 parotid gland and 1 conjunctival lesion. We noted the presence of various components in each smear and estimated their abundance. Immunocytochemical staining for S-100 protein was performed in seven cases. RESULTS: LCH was recognized in 22 cases, suspected in 5 and unrecognized in 1; one case was suspected of malignancy. Six smears were nondiagnostic. Three reactive lesions were misinterpreted as LCH. Smears contained numerous Langerhans cells (LCs), eosinophils, neutrophils, lymphocytes, macrophages and multinucleated giant cells. Some capillary fragments were usually present. The morphology of smears varied somewhat in the percentage of individual constituents present as well as in the appearance of the LCs. Immunocytochemical staining for S-100 protein was positive in six cases and equivocal in one. CONCLUSION: The cytomorphologic pattern of LCHs in fine needle aspiration biopsy smears is usually characteristic, and a correct diagnosis is possible, especially with the aid of immunocytochemistry. One must be aware, however, of different morphologic patterns of LCHs and of unusual appearances of LCs, which may lead to diagnostic errors.

Anaplastic thyroid carcinoma in fine needle aspirates.

OBJECTIVE: To analyze the diagnostic problems with fine needle aspiration biopsy in anaplastic thyroid carcinoma (ATC) and to describe the cytomorphologic characteristics in 113 cases. STUDY DESIGN: A retrospective analysis of 113 fine needle aspirates and 67 surgical specimens from 113 patients with ATC admitted to the Institute of Oncology, Ljubljana, in 1972-1992. RESULTS: In a series of 113 fine needle aspirates of ATC, 3 (2.7%) were inadequate, 3 (2.7%) suboptimal and 107 (94.7%) diagnostic of malignancy. On reexamination, 96/107 (89.7%) were diagnosed as ATC, 6 (5.6%) as differentiated thyroid carcinoma, and 5 (4.6%) as a malignant tumor not otherwise specified. As to the predominant cell population, fine needle aspirates showed three different cell patterns: (1) pleomorphic cell (43 cases), (2) round cell (33 cases), and (3) spindle cell pattern (7 cases). In the present retrospective analysis we identified three main reasons for inadequate or nonrepresentative fine needle aspiration biopsy sampling: (1) tumor regressive changes (necrosis, hemorrhage, leukocytic infiltration), (2) extensive tumor fibrosis, and (3) distinct differentiated and anaplastic patterns in the same tumor. CONCLUSIONS: The major diagnostic problem with fine needle aspiration biopsy (FNAB) of ATC is related to sample quality. Cytomorphologic features of ATC are highly specific and easy to recognize. Due to the simple technique and high diagnostic accuracy, FNAB is the method of choice in patients with ATC.

Marginal vacuoles in fine-needle aspirates of follicular thyroid carcinoma.

Marginal vacuoles (MV) found in Giemsa-stained fine-needle aspirates of the thyroid gland have been observed in toxic and also in non-toxic goiters. The aim of our retrospective study was to disclose the incidence and diagnostic significance of MV in 46 smears from 43 patients with primary and/or metastatic follicular thyroid carcinoma (FTC). Typical MV, MV-like structures, or both were found in 14 of 36 specimens (39%) of the primary tumor and in four of seven specimens (57%) obtained from metastases of FTC. No association between the appearance of MV/MV-like structures and degree of tumor differentiation was demonstrated. On the other hand, MV or/and MV-like structures were more frequently (69%) documented in hyperthyroid patients (P < 0.05). Accordingly, our study demonstrated relatively frequent appearance of MV or MV-like structures in FTC independent of tumor differentiation. Their appearance, however, seems to be associated with hyperthyroidism.

Hepatoblastoma in fine needle aspirates.

OBJECTIVE: To analyze cytomorphologic characteristics of hepatoblastoma (HB) and evaluate the feasibility of recognizing its histologic subtypes in smears. STUDY DESIGN: Fine needle aspirates from 14 primary and 1 metastatic HB were reexamined. The diagnosis of HB was confirmed by tissue examination (10 cases) and by clinical and laboratory findings alone (5 cases). RESULTS: In 12 samples, neoplastic cells resembled immature hepatocytes but were smaller and had a higher nuclear/cytoplasmic ratio. In nine of these smears the cells were rather uniform, while the other three presented with moderate pleomorphism. The cells were arranged in three-dimensional clusters, loose sheets, cords, rosettelike structures and occasional pseudopapillae and were dispersed. CONCLUSION: With knowledge of the cellular features and architectural patterns of HB, a reliable diagnosis could be obtained in 12/15 cases without the use of special techniques. In the remaining three aspirates the tumor cell population partly or entirely differed from normal hepatocytes, requiring ancillary techniques for proper diagnosis. On reexamination of the 10 cases with tissue diagnoses, 4/6 mixed HBs could be correctly subtyped, whereas the distinction between embryonal and fetal cells in four cases of epithelial HB seemed questionable.

3D presentation of the nuclear cell features in quantitative cytometry.

The use of an image cytometer in analysis of smears and needle aspirates provides valuable information to a cytologist. It allows to combine the overall impression, formed by visually inspecting the cells, with measured and numerically expressed nuclear cell features. Both types of information can be used efficiently only if presented to the expert in an appropriate way. Cell images (as they are seen with a microscope) are easily analysed by the experts. However, measured nuclear features can not be presented as a list of numerical values. Instead, an user interface should be developed, providing graphical presentation of the nuclear features. It should show as much information as possible and provide a comprehensive link between nuclear features and cell images. The user interface described in this paper shows nuclear features in three dimensions. It is based on a perspective projection of the three dimensional feature space onto a two dimensional surface. It allows the user to dynamically change the perspective, i.e., to look at the virtual three dimensional structure from different viewpoints. Each nucleus is represented by a single object in the three dimensional space. When an object in the three dimensional feature space is selected, the image (or the visual appearance) of the corresponding cell is shown. When a nucleus image is selected, its position in the feature space is highlighted. This provides an interconnection between nuclear cell features and cell images allowing simultaneous analysis of both types of information.

Effects of vinblastine on cell membrane fluidity and the growth of SA-1 tumor in mice.

Cell membranes can be targets of some anti-cancer drugs. Therefore, the purpose of this study was to determine whether vinblastine (VLB) can also affect the tumor cell membrane. On the in vivo SA-1 tumor model, alteration of cell membrane fluidity (measured by electron paramagnetic resonance, EPR), cytotoxicity and morphological changes of the SA-1 tumor cells after VLB treatment were studied. The cytotoxic effect of VLB was biphasic, with an initial fast increase in cytotoxicity followed by a plateau. The surviving cells had increased membrane fluidity and were morphologically changed. The dose-response curve of VLB on membrane fluidity was also biphasic with an initial fast increase in membrane fluidity followed by a plateau. Since dose-response curves of VLB cytotoxicity and its effect on membrane fluidity were similar, there was a high correlation between both effects. The effect of VLB on membrane fluidity was the most pronounced at 24 h and 48 h after treatment. The results of this study indicate that VLB affects cell membrane by increasing the membrane fluidity of SA-1 tumor cells in vivo in a dose-and time-dependent manner. Therefore, this finding may be beneficially implemented also in priming cells for other cytotoxic drugs and for appropriate timing of drug sequence in combined schedules.