Contextual analysis complements single-cell analysis in the diagnosis of breast cancer in fine needle aspirates

Image analysis techniques were used to characterize individual nuclei of cells and entire clusters of cells in hematoxylin-and-eosin-stained smears of fine needle aspirates of the breast to determine the ability of these techniques to distinguish benign from malignant cases. Analysis of the individual nuclear features showed significant differences in nuclear area, shape (bending energy), texture and integrated darkness between benign and malignant samples. Analysis of the clusters demonstrated that the benign clusters were fewer in number, more cellular (average gray level) and larger than malignant clusters. A statistical classifier was constructed to test the discriminatory accuracy for benign and malignant cases. Good discrimination was found for both the individual nuclei and the clusters when analyzed separately, although a few cases were misclassified by each type of analysis. When combined, the two classifiers achieved a completely accurate classification. This suggests the complementary nature of high-resolution single-cell analysis and the more global cluster analysis techniques.     

Masquerades of malignancy: a review of 4,241 aspirates from the breast

A total of 4,241 aspirates from the breast were obtained by fine needle aspiration biopsy. Of the 3,809 benign lesions, 61 (1.6%) were interpreted as possibly malignant. In all, excisional biopsy, not mastectomy, was recommended as the next procedure. More than 50% of these misdiagnoses were from aspirates of fibrocystic disease, 15% were from fibroadenomas, and the remainder were from gynecomastia, solitary papillomas, periareolar hyperplasia, pregnancy hyperplasia and granulation tissue. Most errors were based on specimens that were insufficiently cellular or whose poorly preserved cells lacked the majority of the criteria of malignancy.     

Computer-assisted grading of adenocarcinoma in prostatic aspirates

Conventional cytologic grading of fine needle aspirates of prostatic adenocarcinoma has been shown neither to be reproducible nor to correlate well with histologic grading. This study developed a tumor grade classification based on computerized cytomorphometric features and compared the results to conventional grading of companion tissue sections. The image analysis system evaluated architectural features of the aspirates (mainly cell cluster features and interrelationships) as well as nuclear features. Thirty-five prostatic adenocarcinomas (8 well, 19 moderately and 8 poorly differentiated) were evaluated. Discriminant functions based on data collected at medium and high resolution distinguished between aspirates from low-grade (well-differentiated) and high-grade (poorly differentiated) adenocarcinomas with 81% accuracy. Moderately differentiated cancers could not be classified as a distinct group. This study suggests that accurate grading of prostatic adenocarcinoma in fine needle aspirate smears requires the evaluation of medium-resolution features related to specimen cellularity and uniformity or crowding of cell clusters as well as of high-resolution features of nuclear area, perimeter and coarseness of chromatin texture. These findings are compared to those of other schemes for the cytologic grading of prostatic aspirates.     

High-resolution and contextual analysis for the diagnosis of fine needle aspirates of breast.

A study was undertaken to confirm earlier work on a smaller number of patients that had suggested that medium-resolution contextual analysis complements high-resolution individual cell analysis for cytomorphometric classification of fine needle aspirate smears of breast. The objectives of this study were to improve and verify the method. Sixty-one biopsy-confirmed hematoxylin and eosin-stained aspirate smears of breast were restained using the Feulgen technique. Individual nuclei were digitized at a resolution of 0.25 micron. Features describing size, shape, density and texture were extracted from the images. Individual cell analysis correctly classified 84% of cases, contextual analysis correctly classified 70% of cases, and the combined use of both techniques resulted in 87% classification accuracy. However, if fibroadenoma cases are excluded, the combined correct classification rate is 93%. Geometric and densitometric features contributed most to correct classification in individual cell analysis, while the most important contextual feature was the number of clusters per scene. We conclude that the addition of quantitative measures of smear patterns, termed "contextual analysis," improves automated classification schemes.     

Morphometric analysis of fine needle aspirates from breast lesions

In an attempt to discover the morphometric variables with the most diagnostic power in the differentiation of benign from malignant breast disease, 20 unequivocally benign and 20 unequivocally malignant and histologically confirmed breast aspirates were examined on an image analyser. It was found that standard deviation of nuclear area was the most discriminant variable. Then 23 aspirates initially diagnosed as 'suspicious of malignancy' were measured by the same technique, and standard deviation of nuclear area correctly differentiated all but three cases.     

Diagnostic schemes for fine needle aspirates of breast masses

A comparison was made of four statistically based schemes for classifying epithelial cells from 243 fine needle aspirates of breast masses as benign or malignant. Two schemes were computer-generated decision trees and two were user generated. Eleven cytologic characteristics described in the literature as being useful in distinguishing benign from malignant breast aspirates were assessed on a scale of 1 to 10, with 1 being closest to that described as benign and 10 to that described as malignant. The original computer-generated dichotomous decision tree gave 6 false negatives and 12 false positives on the data set; another tree generated from the current data improved performance slightly, with 5 false negatives and 10 false positives. Maximum diagnostic overlap occurred at the cut-point of the original dichotomous tree. The insertion of a third node evaluating additional parameters resulted in one false negative and seven false positives. This performance was matched by summing the scores of the eight characteristics that individually were most effective in separating benign from malignant. We conclude that, while statistically designed, computer-generated dichotomous decision trees identify a starting sequence for applying cytologic characteristics to distinguish between benign and malignant breast aspirates, modifications based on human expert knowledge may result in schemes that improve diagnostic performance.     

Carcinoembryonic antigen content in fine needle aspirates of the lung. A diagnostic adjunct to cytology.

Carcinoembryonic Antigen (CEA) was measured in 59 consecutive fine needle aspirates (FNAs) of the lung from 58 patients to determine if the CEA content would enhance the sensitivity of the cytologic diagnosis. Twenty-eight males and 30 females with tumors 1-40 cm in diameter were studied. Final diagnoses were correlated with the clinical history, radiologic studies, tissue (when available) and follow-up. Image-guided FNAs were performed by radiologists using a 22-gauge Chiba needle and 20-mL syringe with one to four passes per specimen. Cytologic examination included rapid assessment in the radiology suite and a final diagnosis in 24 hours. CEA was measured by enzyme immunoassay using monoclonal antibody. Nine benign aspirates and 50 malignant aspirates were diagnosed. The sensitivity of cytology was 86% and specificity, 100%. Using 5 ng/mL as the cutoff, the sensitivity of CEA for malignant aspirates was 50% and specificity, 90%. The combined sensitivity of CEA and cytology was 95%. The mean CEA in malignant aspirates was 131 ng/mL and in benign aspirates, 2.41. The highest mean CEA was seen in adenocarcinoma, 402.6 ng/mL. Lower CEA content was seen in epidermoid carcinoma (58.6 ng/mL), large cell carcinoma (8.09), oat cell carcinoma, metastatic carcinoma of the kidney and breast, thymoma and lymphoma (each less than 1 ng/mL). Elevated CEA alone was diagnostic in two aspirates of bronchioloalveolar carcinoma; carcinoma with an unknown primary source, three; and large cell carcinoma, one. The adjunctive use of CEA in FNAs of the lung enhances the sensitivity of the cytologic diagnosis.     

Immunocytochemical Detection of the Androgen Receptor In Fine Needle Aspirates From Benign and Malignant Human Prostate

A monoclonal antibody to the androgen receptor was applied to fine needle aspirates from patients with benign and malignant prostatic disease. The series includes six patients with benign hyperplasia and 24 patients with prostatic carcinomas. The androgen receptor was detected in most nuclei of both benign and malignant epithelial cells. The intensity of immunostaining varied. No obvious relation was observed between the intensity of the staining in benign versus malignant cells. In addition no clear differences were found in the proportion of androgen receptor positive cells in benign aspirates as compared with aspirates from well differentiated or moderately well differentiated prostatic carcinomas. The relative number of androgen receptor positive cells was highest in smears from poorly differentiated prostatic carcinomas.     

p53 mutations as tumor markers in fine needle aspirates of palpable breast masses

OBJECTIVE: Mutations in p53 exons 5-8 are found in 40-50% of breast carcinomas. We performed a retrospective analysis of p53 mutations in fluid-based, archival fine needle aspirates (FNAs) of breast masses to determine their potential diagnostic utility as breast tumor cell markers. STUDY DESIGN: Residual, fluid-based, archival FNAs of 27 breast masses were retrospectively evaluated by polymerase chain reaction (PCR), single-strand conformational polymorphism analysis (SSCP) and sequencing for p53 exons 5-8. Results were compared with the morphologic diagnoses and genotyping of available excisional biopsy tissue. RESULTS: Six of the twenty-seven cases were found to have a clonal mutation in p53; all six mutated cases showed carcinoma on subsequent excisional biopsy. Definitive cytologic diagnosis of cancer had been possible in only four of the six cases. Identical mutations were found in the excised carcinomas in the five cases with available tissue. None of the 14 aspirates with benign cytology had detectable mutations in p53. CONCLUSION: p53 Mutational analysis by PCR/SSCP/sequencing deserves to be critically studied as a diagnostic criterion in patients with indeterminate or suspicious cytology. Validation studies should be performed to test p53 mutations as molecular diagnostic markers in breast cytology specimens.     

Image analysis of low magnification images of fine needle aspirates of the breast produces useful discrimination between benign and malignant cases

Image analysis of low magnification images of fine needle aspirates of the breast produces useful discrimination between benign and malignant cases
Fine needle aspirates of the breast (FNAB) ( n =362; 204 malignant, 158 benign), prepared by cytocentrifuge methods and stained by the Papanicolaou technique, were analysed using a semi‐automated image analysis system at a low magnification which precluded resolution of nuclear detail. The measured parameters were integrated optical density, fractal textural dimension, number of cellular objects (single cells and contiguous groups of cells), distance between cellular objects (mean, s.d., skewness and kurtosis), area of cellular objects (mean, s.d., skewness, kurtosis) and the nearest neighbour statistic. The cases were divided into a 200‐case training set and a 162‐case test set. Analysis was performed by logistic regression and the multi‐layer Perceptron type of artificial neural network. Logistic regression and the neural network produced similar performances with a sensitivity of 82–83%, specificity 85% and a positive predictive value for a malignant result of 85%. A non‐parametric analysis of all the predictor variables showed that all except the mean area of cellular objects and the s.d. of this measurement were significant discriminants ( P <0.05), but most were highly interrelated and this was reflected in the selection of only three predictor variables by forward and backward conditional logistic regression. This study shows that much diagnostic information is present in low power views of FNAB, and that image analysis could form the basis of a semi‐automated decision‐support aid.     

Specific tumor markers in diagnostic cytology. Immunoperoxidase studies of carcinoembryonic antigen, lysozyme and other tissue antigens in effusions, washes and aspirates

The immunoperoxidase technique was used to identify specific tumor markers in exfoliated cells in fine needle aspirates and body fluids. Carcinoembryonic antigen (CEA) and lysozyme staining was evaluated in cytocentrifuge preparations from 42 malignant effusions and aspirates and 16 benign effusions. Reactive mesothelial cells were negative for CEA and lysozyme or showed faint peripheral cytoplasmic staining. Malignant cells from 50% of the adenocarcinomas studied were positive for CEA. All tumors studied were negative for lysozyme. These staining patterns are helpful in the differential diagnosis of reactive mesothelial and adenocarcinoma cells, a frequent diagnostic dilemma. Moreover, demonstration of specific tumor antigens (e.g., prostatic acid phosphatase, calcitonin and immunoglobulin) helped define the origin of metastatic malignancy in selected cases. Estrogen receptor activity was also identified in tumor cells using this technique. Immunoperoxidase was helpful in the evaluation of malignant cytologic specimens from patients with more than one tumor. Interpretation of staining patterns is discussed, with reference to the limitations of the technique. Immunoperoxidase methods maintain cytologic detail, are readily adaptable to diagnostic cytology and increase the specificity of cytologic diagnosis.     

Diagnostic significance of 'benign pairs' and signet ring cells in fine needle aspirates (FNAs) of the breast

sturgis c. d., sethi s., cajulis r. s., hidvegi d. f. and yu g. h. (1998) Cytopathology 9, 308–319 Diagnostic significance of ‘benign pairs’ and signet ring cells in fine needle aspirates (FNAs) of the breast Most breast mass lesions are readily characterized by FNA with cytodiagnosis. Occasionally, benign but markedly proliferative lesions are diagnostically difficult to separate from well differentiated malignancies. We present information pertaining to the diagnostic significance of two cytologic findings observed in breast aspiration specimens, namely pairs of stripped bipolar nuclei and signet ring cells (SRC). We have evaluated aspirate smears from 219 cases of histologically proven benign (n= 114) and malignant (n= 105) breast lesions. Both singlets and pairs of bipolar nuclei and SRC were enumerated, and their numbers were correlated to histological diagnosis. Closely associated pairs of stripped bipolar nuclei were found in 68% of benign lesions compared with only 3.8% of carcinomas, establishing their presence as a highly specific indicator of a benign process. Large numbers of such ‘benign pairs’ also favoured the diagnosis of fibroadenoma. SRC were identified in 66% of histologically proven carcinomas (67% of ductal and 70% of lobular). SRC were also present in 10% of histologically benign cases. In the malignant cases, SRC were most frequently noted in a single cell distribution or within small, loosely cohesive tissue fragments. In the benign instances, SRC were most commonly noted within large fragments, and many of these cells were proved by immunohistochemical reactions to be vacuolated myoepithelial cells. We conclude that the presence of bipolar nuclei in closely associated pairs suggests benignity and aids in the subclassification of benign breast masses. In addition, the presence of SRC does not aid in the classification of tumour subtype (ductal vs lobular), and the occurrence of such cells in the proper context should prompt surgical biopsy.     

Diagnostic utility of galectin-3 in aspirates of thyroid follicular lesions

OBJECTIVE: To investigate the expression of galectin-3 in various thyroid follicular lesions, including diffuse hyperplasia, nodular hyperplasia, and benign and malignant follicular neoplasms, to clarify the diagnostic utility of galectin-3 in aspirates of follicular lesions. STUDY DESIGN: A total of 146 follicular lesions diagnosed cytologically, obtained from patients who had undergone thyroidectomy for either benign or malignant nodules, were evaluated using Papanicolaou-stained slides and cell blocks with galectin-3 immunostaining. We primarily categorized the aspirated specimens as benign, indeterminate or suspicious for a follicular neoplasm based on cellularity, architectural arrangement of the follicular cells and presence or absence of colloid. Galectin-3 immunostaining was evaluated according to the proportion and intensity of positively stained cells. Cytologic diagnoses were correlated with the results of galectin-3 and categorized into 3 groups (benign, indeterminate for malignancy, suspicious for malignancy) and compared with the corresponding histologic diagnoses. RESULTS: When the histologic diagnoses were compared with the cytologic diagnoses, the accuracy in the distinction between benign and malignant cases was 79.5% except for 8 cytologically and 3 histologically indeterminate cases. Except for 11 indeterminate cases, there were 18 (12.3%) discrepant cases. False positive cases included 8 of 62 (12.9%) nodular hyperplasias and 1 of 42 (2.4%) follicular adenomas. Of 9 false negative cases, 4 minimally invasive carcinomas and 2 widely invasive carcinomas were included. Three follicular tumors of uncertain malignant potential were cytologically categorized as malignant, and all cases showed positivity for galectin-3. CONCLUSION: Galectin-3 could be used as a useful supplementary marker for cytologic diagnosis, although it was not an absolute marker in determining whether a lesion was benign or malignant.     

Morphologic and immunocytochemical evaluation of 220 fine needle aspirates of malignant lymphoma and lymphoid hyperplasia

A total of 220 fine needle aspiration (FNA) specimens from 212 patients with clinically suspected or previously histologically confirmed lymphoma were evaluated by cytology in conjunction with immunophenotyping analysis of the aspirate; the results were compared with the histologic diagnosis made on previous or current accessions of lymph node or extranodal tissue. Smears of the aspirates were stained with the Diff-Quik and Papanicolaou stains while immunoperoxidase staining using antibodies against kappa and lambda immunoglobulin light chains and Leu-4 was routinely performed on Cytospin preparations. Where indicated, additional marker studies (including T-200, Leu-1, Leu-2a, Leu-3a + 3b, Leu-M1, B1, Leu-12, IgM, CALLA and TdT) were performed. For the non-Hodgkin's lymphomas, specimens were classified by the cytologic characteristics of the neoplastic cells according to the International Working Formulation scheme. The combination of cytologic smears and immunoperoxidase studies resulted in a diagnosis of lymphoma in 173 cases (79%). The remaining aspirates were interpreted as suspicious for lymphoma (7%), benign (10%) or inadequate for diagnosis (4%). Of the 15 suspicious aspirates, 5 proved to be Hodgkin's disease and 2 to be T-cell lymphoma by subsequent biopsy. The cause of failure in the nine inadequate aspirates were necrosis (3 cases), sclerosis (2 cases) and faulty technique (4 cases). In the cases that had concurrent tissue biopsies, no false-positive diagnoses were rendered. These results indicate that FNA used in association with immunocytochemistry is a reliable tool for establishing the diagnosis and classification of the majority of cases of lymphoma. Optimal immunoglobulin light-chain ratios for defining monoclonality in FNA specimens of B-cell lymphomas are proposed.     

Silver-binding nucleoli and nucleolar organizer regions in fine needle aspiration cytology of the breast

Silver staining was used to demonstrate nucleoli and nucleolar organizer regions (NORs) in cytopreparations from breast fine needle aspiration (FNA) biopsies, with the resulting black dots being a combination of silver-binding nucleoli and dispersed NORs. Discrete black dots in 200 nuclei were manually counted in 25 silver-stained biopsies; the cytologic diagnoses were made on routine Papanicolaou-stained cytopreparations from the same aspirates. Although malignant breast lesions showed higher counts than did benign breast disease, an overlap of one malignant and three benign lesions occurred. Therefore, the method cannot be recommended for reliably discriminating between malignant and benign routine breast specimens obtained by FNA; it may have some value in assessing tumor behavior.     

Use of specimen mammography-guided FNA (fine-needle aspirates) for flow cytometric multiple marker analysis and immunophenotyping in breast cancer

A pilot study of a novel translational research method to simultaneously assay multiple molecular markers and DNA in fine-needle aspirates (FNA) of mammographically detected breast lesions is described. Specimen mammography-guided 20-gauge FNAs obtained from 86 lesions and 22 areas of normal tissue were analyzed by multiparameter flow cytometry for DNA content, her2/neu, transforming growth factor alpha (TGF alpha), and the epithelial marker cytokeratin (CK) simultaneously. Epithelial cell her2/neu positivity was detected in 12 of 44 (27%) of invasive ductal carcinomas and 3 of 9 (33%) ductal carcinoma in situ (DCIS), 10 of 30 (33%) benign lesions, and 4 of 22 (18%) normal tissue aspirates. All lesions and normal tissue showed a similar positive rate for TGFalpha ranging from 61 to 76%. The CK(+)TGF alpha(-)her2/neu(+) immunophenotype was more frequently positive in aneuploid tumors (22%) than all other lesions (7%) (P < 0.05). Specimen mammography-guided FNAs provide fresh cells for flow cytometric multiple marker analysis and immunophenotyping of clinically occult breast lesions and normal tissue.     

Asbestos bodies in fine needle aspirates of lung masses. Markers of underlying pathology

Analysis of 52 transthoracic-mass aspirates that contained asbestos bodies (ABs) showed the mass to be due to pathology other than (or superimposed upon) asbestosis in every case. Malignancy accounted for 30 masses, all of which were carcinomas except for one mesothelioma. The remaining 22 lesions were benign, with tuberculosis or lung abscesses accounting for the majority. Fine needle aspiration (FNA) detection of the pathology (benign or malignant) associated with ABs was diminished, probably due to asbestos-induced fibrosis. Other diagnostic methods, including bronchial studies, mediastinoscopy and even exploratory thoracotomy, were required to document 20% of the neoplasms and 50% of the benign lesions. The results of this series support the view that ABs in FNA specimens from localized or dominant parenchymal lung masses are significant markers of underlying pathology, whether or not cellular evidence of that pathology is observed in the aspirated material.     

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.     

Scanning electron microscopic analysis of breast aspirates

Scanning electron microscopic analysis (SEM) of 50 pre-selected breast aspirates was performed after light microscopy and cytomorphological evaluation. SEM analysis of these aspirates revealed the presence of microvilli (mv), microprojections, blebs and irregular contours in malignant cells. Benign cells were regular, globular and smooth. SEM provided additional morphologic data which if used in conjunction with light microscopy can assist in reaching a correct diagnosis.