Fine needle aspiration biopsy of the breast. Value of nuclear morphometry after different sampling methods

OBJECTIVE: To study the potential of nuclear morphometry in supporting the interpretation of fine needle aspiration biopsy (FNAB) samples of the breast fixed in 50% ethanol and centrifuged on slides. STUDY DESIGN: Computerized morphometry was used to outline the nuclei of breast epithelial cells in breast cancer, fibroadenoma and fibrocystic disease. The diagnoses were histologically confirmed. We applied 2 different sampling methods (measurements done on cell groups and on free cells). RESULTS: The mean nuclear area of cell groups of malignant samples (23) varied from 42 to 125 microns 2, in fibroadenomas from 30 to 50 microns 2 and in fibrocystic disease from 26 to 57 microns 2. The mean nuclear area of free cells varied as follows: cancer, 66-181 microns 2; fibroadenoma, 33-70 microns 2; fibrocystic disease, 35-60 microns 2. Apocrine metaplasia was excluded from comparison on a morphologic basis. CONCLUSION: The study suggests that if the mean nuclear area of cell groups is < 42 microns 2, the lesion is probably benign; if > 57 microns 2, and apocrine metaplasia is excluded, malignancy should be considered. The differential diagnosis between carcinoma and fibroadenoma could be based on free cells: mean area of free cell nuclei < or = 65 microns 2 suggested a benign lesion, and of > or = 71 microns 2 suggested a malignant lesion. Morphometric nuclear size features (exemplified by nuclear area) appeared efficient in distinguishing between malignant and benign lesions when measured from free cells and cell groups.     

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

Cytofluorometric nuclear DNA content analysis of breast tissues after frozen section diagnosis

OBJECTIVE: To establish a suitable method for measurement of nuclear DNA content in breast tissues from frozen storage after frozen section diagnosis. STUDY DESIGN: For fundamental research, rat liver samples preserved in a deep freezer were used. Four protocols were used (1. fixation with 70% ethanol followed by naked nuclei preparation; 2. fixation with 10% neutral buffered formalin followed by naked nuclei preparation; 3. preparation for naked nuclei prior to fixation with 70% ethanol; and 4. preparation for naked nuclei prior to fixation with 70% neutral buffered formalin). For clinical research, 13 separate fresh frozen breast tissue samples were analyzed after frozen section diagnosis. One contained a malignant phyllodes tumor (MPT) consisting of 2 components, benign epithelial cells and malignant stromal cells; 3 were benign tumors containing fibroadenoma; and 9 cases were carcinomas, consisting of 5 scirrhous, 3 papillotubular and 1 mucinous. RESULTS: Protocols 1, 2 and 3 were not suitable methods for our purpose because remaining cytoplasm or cohesive nuclei were observed. In protocol 4 the cytoplasm was completely undetectable, and nuclei were suitably separated for nuclear DNA content measurement. Benign epithelial cell component nuclei presented a diploid pattern, and the malignant stromal cell component nuclei indicated a euploid pattern in MPT. All 3 cases of benign constituents in fibroadenoma showed a diploid pattern, as did the 3 carcinoma cases (1 mucinous, 1 scirrhous and 1 papillary). Four scirrhous and 2 papillary carcinomas showed an aneuploid pattern. CONCLUSION: Our findings show that it is possible to measure nuclear DNA content of human frozen storage tissues after frozen section diagnosis.     

Comparative studies of nuclear DNA content in benign and malignant thyroid lesions

Currently available data suggest that DNA aneuploidy is associated with aggressive behavior of and unfavorable prognosis in several malignant human tumors as compared with diploid malignancies. However, the diagnostic and prognostic importance of flow cytometric DNA measurements in the case of thyroid neoplasms remains controversial. Therefore, the aim of our study was to evaluate utility of DNA index (DI) and proliferative index (PI) in distinguishing benign from malignant thyroid lesions taking into account the possible influence of intra-tumor heterogeneity and tissue preparation mode on DNA flow-cytometry measurements. A retrospective study was performed on 71 paraffin-embedded specimens from 57 patients with benign and malignant thyroid pathologies: 13 colloid goitres, 12 parenchymatous goitres, 19 adenomas and 13 carcinomas. In 14 of 57 cases two separate specimens taken from different areas of the same lesion were analysed and DNA parameters were compared. Additionally, flow cytometry DNA analysis was parallelly performed on 3 adjacent but differently processed tissue sections (fresh, formalin-fixed and paraffin-embedded) taken from each of 26 surgically excised thyroid lesions. DNA content was also analysed in both fresh and formalin-fixed twin specimens of normal pig thyroid glands (N = 6). We demonstrated that all tumors diagnosed as thyroid carcinomas were associated with abnormal nuclear DNA content although aneuploidy was not found specific to malignant thyroid tumors. Aneuploid samples of benign thyroid lesions exhibited higher proliferative activity, expressed as mean PI values, than diploid ones. In carcinomas the mean PI values were significantly higher than in benign lesions, independently whether they concerned aneuploid or diploid tissues. Considering intra-tumor heterogeneity, the flow cytometric DNA parameters can be assumed as reproducible despite differences in the mode of tissue fixation and preparation for analysis.     

Manual and automated AgNOR count in differentiating reactive mesothelial from metastatic malignant cells in serous effusions

OBJECTIVE: To distinguish reactive mesothelial cells from malignant cells in serous effusions using manual and automated methods of enumeration of argyrophilic nucleolar organizer regions (AgNORs). STUDY DESIGN: In this prospective study, 38 samples of benign (19 cases) and malignant (19 cases) serous effusions were included. AgNOR stain was used in each case along with routine Papanicolaou stain. The smears were examined under an oil immersion objective, and AgNOR dots were counted by direct observation independently by 2 observers. Automated AgNOR counting and morphometry were performed with a Quantimet 600 image cytometer (Leica, Cambridge, England). At least 100 cells were counted in each case. The number of AgNOR dots in individual cells, AgNOR area, nuclear area, AgNOR vs. nuclear area and nuclear perimeter were measured. Data on benign and malignant cells were compared. RESULTS: The AgNOR dots were discrete and smaller in benign effusion cases as compared to coarse and aggregated in malignant effusion cases. In benign reactive effusion cases the mean number of AgNOR dots per nucleus was 2.33 +/- 0.71 and 2.83 +/- 1.15 by the manual and automated method, respectively, whereas that for malignant effusion cases was 7.48 +/- 2.51 and 8.09 +/- 1.69 by the manual and automated method, respectively. Mean total AgNOR areas in benign and malignant groups were 4.77 +/- 2.66 microns 2 and 38.22 +/- 13.71 microns 2, respectively. Mean nuclear area, nuclear perimeter and ratio of AgNORs vs. nuclear area were 48.72 +/- 19.30 microns 2, 24.68 +/- 10.25 microns and .098 in benign effusion cases as compared to 174.25 +/- 82.36 microns 2, 69.03 +/- 27.23 microns and 0.22 in malignant effusion samples. All these values were significantly higher (P < .001, Student's t test) in malignant cells as compared to benign reactive cells. CONCLUSION: AgNOR dot enumeration, AgNOR area and ratio of AgNORs to nuclear area are valuable adjuncts to cytomorphology in differentiating reactive mesothelial cells from malignant cells in serous effusions. Automated AgNOR counting is rapid and less cumbersome.     

Nuclear morphometry and AgNOR quantification: computerized image analysis on ovarian mucinous tumor imprints

OBJECTIVE: To determine the morphometric characteristics of nuclei and silver-stained nucleolar organizer regions (AgNORs) on cytologic imprints and their value in differential cytodiagnosis of benign, atypical proliferative (borderline) and malignant ovarian mucinous tumors. STUDY DESIGN: Forty-six mucinous ovarian tumor imprints (16 benign, 15 borderline, 15 malignant), were analyzed. Nuclear area, outline, "shape factor" and "form factor" were measured on Papanicolaou-stained smears. AgNOR quantification included 7 variables related to the number and area of single, cluster, total and relative AgNOR content per nucleus and the size distribution of AgNORs. RESULTS: Nuclear area and shape factor allowed distinguishing borderline and malignant tumors. The nuclear area in benign tumors was larger than that in borderline tumors; malignant tumors had the highest values. Single and cluster AgNORs were statistically significantly different in borderline tumors compared with malignant tumors, except for the cluster AgNOR area. The total AgNOR area, number and relative area increased from benign through malignant tumors, with statistically significant differences among all groups. By AgNOR size distribution, small AgNORs discriminate malignant from borderline and benign tumors. CONCLUSION: Combining nuclear morphometry and AgNOR analysis on cytologic imprints could be a diagnostically useful method in the assessment of mucinous ovarian tumors.     

Analysis of protein expression in pure cell nuclei populations isolated from human breast cancer tissue by DNA flow cytometric sorting

In this study, cell nuclei from aneuploid breast cancer samples were sorted with respect to DNA content into pure diploid and aneuploid fractions using flow cytometry. The nuclear proteins were then separated by one-dimensional gel electrophoresis (1D-PAGE) and differences in protein expression patterns, between diploid and aneuploid nuclei from the same tumours, were compared. Using a combination of peptide finger printing and peptide identification by MALDI-TOF mass spectrometry, we identified proteins and confirmed that the proteins were of nuclear origins. The results in this study add further information to the knowledge about the breast cancer disease complexity and heterogeneity at molecular level. For some of the tumours studied different nuclei protein patterns were obtained, in the diploid respective aneuploid nuclei populations, whilst other tumours did not show these differences.     

Cytophotomeric DNA-analysis of aspirated cells from prostatic carcinoma.

In the present study material obtained from prostatic lesions by transrectal aspiration biopsy was subjected to a comparative morphologic and cytophotometric DNA analysis. Based on the morphologic pattern, the clinical material was divided into benign lesions (prostatic hyperplasia), suspected prostatic malignancy and highly, moderately and poorly differentiated prostatic carcinoma. The cytochemical analysis, based on quantitative cytophotometric measurements of Feulgen stained nuclei, showed that cell nuclei from benign lesions (prostatic hyperplasia) exhibited the normal diploid amount of DNA. Contrary to this, cell populations from prostatic malignancies, were characterized by various degrees of heteroploidy, i.e. the existence of cells with nuclear DNA quantities increased above the normal diploid level. A general correlation between degree of heteroploidy (frequency of heteroploid cells) and degree of clinical malignancy seemed to exist; high grade malignant prostatic carcinoma (poorly differentiated) exhibited a pronounced degree of heteroploidy with more or less distinct aneuploid stemlines, whereas low-grade prostatic carcinomas (highly differentiated) were more similar to benign cell populations, in showing a large proportion of cells with a diploid DNA quantity suggesting the existence of diploid or near diploid stemlines. Cases morphologically classified as moderately differentiated prostatic carcinoma, which previously have been shown to exhibit individual variations in degree of clinical malignancy, also showed large individual variations in degree of hetyroploidy. Approximately half of these cases had cytochemical DNA characteristics similar to that of highly differentiated prostatic carcinoma in showing a modal DNA value in the diploid region, while the other half showed cytochemical characteristics similar to that of poorly differentiated prostatic carcinoma, i.e. aneuploid DNA distributions. However, no morphologiifferentiated prostatic carcinoma. In conclusion it can be stated that the present investigation suggests the possibility that quantitative cytochemical DNA analysis may be used in combination with, and offer additional information to, morphologic analysis in the malignancy grading of prostatic carcinoma. A future clinical follow-up, now in progress, will hopefully give a more definite answer to that idea.     

Flow cytometric determination of DNA content in malignant and benign bone tumours

The nuclear DNA content of 38 malignant and 25 benign bone tumours was measured by flow cytometry. The specimens were taken either from biopsies or from surgical specimens. Seventeen of 26 primary malignant bone tumours were aneuploid, 15 had a single aneuploid DNA content, and 2 had a biclonal abnormality. Thirteen of 15 osteosarcomas were aneuploid, but only 2 of 6 chondrosarcomas showed an aneuploid DNA content. Six of 12 metastatic malignant bone tumours were also aneuploid. All 25 benign tumours had a diploid DNA content. Cell cycle analysis showed that the proportion of cells in S- and G2M-phases was higher in the malignant compared to benign tumours, indicating a higher proliferative activity. The increase was statistically significant (p less than 0.05) both in diploid and in aneuploid tumours. Among five tumours studied after chemotherapy, four displayed a marked hyperdiploid abnormality. Aneuploidy and high proliferative activity both were highly associated with malignant bone tumours, suggesting that DNA flow cytometry may be an adjunct in the assessment of malignancy of bone tumours.     

Discrimination between benign and malignant melanocytic skin lesions by multivariate analysis, quantitative S-100 immunohistochemistry, nuclear morphometry and DNA cytometry

OBJECTIVE: To determine whether combined quantitative immunohistochemistry of S-100, nuclear morphometry and DNA image cytometry improves discrimination between benign and malignant melanocytic skin lesions (MSLs). STUDY DESIGN: S-100 protein expression was measured in tissue sections of MSLs using an image cytometry system. Localized areas of high S-100 expression were used to identify regions in sequential, facing sections in which morphometric and cytometric features of nuclei, including DNA ploidy, were also measured. RESULTS: Malignant cases had significantly higher S-100 protein staining intensity, larger nuclei and greater DNA content (P < .05). High staining intensity for S-100 protein weakly correlated with variation in size of the mean nuclear area (P = .04) and DNA content (P = .03). Combining the features of nuclear area and DNA integrated optical density in areas of high-intensity staining for S-100 protein discriminated more accurately between 12 benign and 16 malignant areas than any of the features along (P = .0003). CONCLUSION: Combined multivariate quantitative immunohistochemical, morphometric and DNA cytometric analysis greatly improves discrimination between benign MSLs and malignant melanoma. Larger test sets are required to confirm the promising results of this initial study.     

Flow cytometric analysis of DNA content and keratins by using CK7, CK8, CK18, CK19, and KL1 monoclonal antibodies in benign and malignant human breast tumors.

We have used a double-labelling flow cytometry analysis of keratin (CK) and DNA in breast cancer. Five monoclonal anti-keratin antibodies were tested: KL1 recognizing Mr 55,000-57,000 keratins, and "anti-glandular epithelia," LE41, RGE-53, and LP2K specific for CK n. 7, 8, 18, and 19 of Moll's classification, respectively. Flow cytometric (DNA-CK) analysis was performed on 10 benign and 19 malignant human breast tumors. All the benign tumors were diploid and 63% of the malignant tumors were aneuploid. This technique permits the analysis of DNA in the epithelial fraction alone. In aneuploid tumors, gating the DNA-keratin-positive population allowed accurate DNA analysis without interference due to debris background and non-epithelial cells. Moreover, double-labelling using the CK19 antibody gave a better identification of near-diploid tumors. An enhancement of keratin expression in malignant tumors was observed with CK 19 (P less than 0.001), KL1 (P less than 0.01), CK 8 (P less than 0.05), and CK18 (n.s.) compared to benign tumors. The comparison of keratin expression in aneuploid and diploid malignant tumors revealed reduced CK8, CK18, and CK19 in the former.     

Morphometric studies on nuclei in smears of fine needle aspirates from oxyphilic tumors of the thyroid

Morphometry was applied to smears of fine needle aspirates from 26 oxyphilic thyroid neoplasms. Thirteen tumors were considered benign as judged from histologic findings and clinical follow-up for 2 to 20 years after the operation. Thirteen tumors were considered malignant histologically; four of these had proven metastases. In each case the mean and standard deviation of the projected nuclear area were calculated from 200 nuclei in the smear. Our results indicate that morphometric estimation of mean nuclear size and/or degree of anisokaryosis is of no practical value in distinguishing between benign and malignant thyroid neoplasms of the oxyphilic type.     

Significance of AgNOR measurement in thyroid lesions

OBJECTIVE: To evaluate the discriminating potential of AgNOR area measurement and count in thyroid tumors using static cytometry equipment. STUDY DESIGN: Slides were analyzed by a computerized system for image analysis, CAS 200 (Becton & Dickinson, U.S.A.), using the Cell Measurement computer program (CAS 200, Becton & Dickinson). The argyrophilic reaction (NORs) was evaluated with a 400-fold amplification directly from the computer monitor. RESULTS: Thirty-three cases were analyzed for AgNOR staining. The cases studied included 3 goiters, 10 follicular adenomas, 6 Hürthle adenomas, 4 follicular carcinomas, 7 papillary carcinomas, and 3 Hürthle carcinomas. A total of 6,600 nuclei were evaluated. For statistical purposes, lesions were classified as benign and malignant, and both the number and the area of counted NORs showed very similar values. The NORs median among 19 benign tumors was 1.484 (SD +/- 0.265) and of 14 malignant tumors was 1.436 (SD +/- 0.414); the NORs areas were 2.6584 (SD +/- 1.0653) and 2.3643 (SD +/- 0.6320), respectively. CONCLUSION: Our results showed that AgNOR evaluation was not a significant parameter to discriminate between malignant and benign thyroid lesions.     

Cytophotometric and electron microscopic study of chronic human skin lymphomas. I. A study of skin manifestations

Electron microscopic and cytophotometric studies of low-grade malignant cutaneous lymphomas and non-malignant skin diseases revealed substantial differences between these groups of diseases. In the latter case, the DNA content per nucleus is diploid but in the former an atypical distribution of DNA content per nucleus (more than 5% aneuploid and polyploid nuclei) is observed in addition to a significant excess of the mean DNA content per nucleus above the diploid standard level. In lymphomas electron microscopy reveals clusters of atypical lymphoid cells with irregularly shaped nuclei, nuclear pockets, nuclear extrusions, network of cytoplasmic microfilaments. These features never occur with skin diseases. The data obtained can be helpful in the diagnostics of the low-grade malignant cutaneous lymphomas.     

Flow cytometric study of DNA distribution in cytopunctures of benign and malignant breast lesions

One hundred seventy-eight cytopunctures of mammary lesions were obtained for cytologic diagnosis and flow cytometric (FCM) analysis of the nuclear DNA content. All lesions were excised and evaluated histologically; 106 were carcinomas and 72 were benign lesions. The benign lesions showed a diploid DNA content, with one exception. Among the 106 carcinomas, 35 (33%) were diploid, 14 (13%) were tetraploid and 57 (54%) were aneuploid. For 79 carcinomas, the relationship between ploidy and (1) "T" and "N" of TNM staging, (2) the histologic grading of Scarff, Bloom and Richardson, (3) axillary nodal involvement, (4) the presence of estrogen and progesterone receptors, (5) age and (6) menopausal status was investigated. The percentage of aneuploidy was significantly higher (P less than .05) in grade III tumors as compared to grade I tumors. There was no significant relationship between aneuploidy and the other factors. However, a trend was observed between the lack of steroid receptors and a high probability of the tumor being aneuploid. FCM DNA analysis was also carried out on breast carcinomas obtained at surgery in 40 patients for whom FCM DNA analysis had previously been performed on breast cytopuncture specimens. The FCM DNA analyses were found to be best performed on the samples obtained by cytopuncture, which may increase the yield of tumor cells.     

Implications of nuclear diameter in small cell lung carcinoma

The nuclear diameter of 5,117 malignant cells from 42 small cell lung carcinoma (SCLC) patients was assessed either on pretreatment tissue sections (35 cases) or cytologic smears (7 cases) by ocular micrometry. The SCLCs were subtyped as 30 oat cell carcinomas and 12 intermediate cell carcinomas according to the World Health Organization classification, based on the predominant histology of the tumor. The median number of nuclei measured from each patient was 110. All patients were treated identically by sequential hemibody and local irradiation combined with chemotherapy and had a median follow-up time of 310 days. The mean nuclear diameter (+/- standard error) obtained from tissue sections was 8.2 +/- 0.03 microns (median = 8.0), including 7.3 +/- 0.03 microns (median = 7.0) for oat cell cases and 9.5 +/- 0.06 microns (median = 9.0) for intermediate cell cases (P less than .001). In 28.6% of these patients, the nuclear diameter overlapped in the range of 8 microns to 9 microns between both subtypes. Comparisons between the nuclear diameter of primary and metastatic SCLC cells revealed no statistically significant differences. The nuclear diameter of malignant cells correlated with the mitotic index and stage of disease, but did not correlate with the other nuclear morphologic variables or with survival. The only identified prognostic factor was the stage of disease; these results indicate that the nuclear diameter of malignant cells should not be considered a prognosticator or a guide for therapy in SCLC patients.     

Nucleolar organizer regions in aspirates of malignant lymphomas and benign disorders of the lymph nodes.

Silver staining of nucleolar organizer regions (AgNOR) was used to differentiate malignant lymphoma and chronic lymphadenitis. Aspiration smear samples from lymph nodes of 120 cases, including 43 non-Hodgkin's lymphoma, 3 Hodgkin's disease, 56 chronic lymphadenitis, 7 tuberculosis, 6 reactive hyperplasia and 5 samples from other diseases (epidermoid cyst, branchial cyst, mixed tumor, lymphoepithelioma and nodulous disease), were investigated. The number of AgNORs in 200 cells in each sample was counted, and the mean +/- SD in each disease was calculated: non-Hodgkin's lymphoma, 6.58 +/- 2.37; Hodgkin's disease, 4.22 +/- 0.5; chronic lymphadenitis, 1.16 +/- 0.1; tuberculosis, 1.13 +/- 0.14; reactive hyperplasia, 1.48 +/- 0.25; other diseases, 1.47 +/- 0.31. The results indicate that the AgNOR count in malignant lymphoma differed highly significantly from that in benign disease (P less than .001). The size of AgNORs in malignant lymphoma and chronic lymphadenitis was measured, and the maximum diameter and area of lymphocyte and lymphoma cell were: lymphocyte, 0.93 +/- 0.12 microns, 0.61 +/- 0.13 microns 2; lymphoma cell, 0.83 +/- 0.22 microns, 0.50 +/- 0.25 microns 2. The AgNOR sizes in malignant lymphoma were significantly smaller than in chronic lymphadenitis (P less than .001).     

Prognostic value of DNA flow cytometry in sympathoadrenal paragangliomas.

OBJECTIVE: To determine whether ploidy patterns are related to prognosis in sympathoadrenal paragangliomas (SAP) using flow cytometry. STUDY DESIGN: DNA flow cytometric analysis of formalin-fixed, paraffin-embedded tumor samples from 36 patients with SAP was performed. Eight cases fulfilled at least one of the following malignancy criteria: (1) extensive invasion of adjacent structures (5 cases), (2) local recurrence (3 cases), or (3) metastases (4 cases). RESULTS: Of the 36 tumors, 22 (61%) showed nondiploid patterns (12 aneuploid, 10 tetraploid). All diploid tumors were benign, while all malignant cases showed nondiploid patterns (P = .0131). The differences between diploid and aneuploid tumors and between diploid and tetraploid tumors, with regard to the malignancy of the disease, were statistically significant (P = .03311 and .01976, respectively). Only one malignant tumor had a DNA index < 1.75 (P = .00259). CONCLUSION: Anomalous DNA ploidy patterns are frequent in SAP, without necessarily implying malignancy. However, diploid DNA content may be a marker of a good prognosis. The likelihood of malignancy is greater in the tetraploid and peritetraploid range.