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BACKGROUND Fine-needle sampling, although a practical and noninvasive method of tissue acquisition, has rarely been used for HER-2/neu fluorescent in situ hybridization (FISH). To assess HER-2/neu gene amplification in mammary carcinoma, FISH signals on cytology and corresponding tissue biopsies were detected visually and measured by image analysis. The results were correlated with patient and tumor characteristics. METHODS In situ HER-2/neu DNA probe hybridization was performed on 61 cytology specimens and on 47 corresponding frozen sections of breast carcinomas. Tumors were classified by visual evaluation as unamplified, moderately amplified, or highly amplified. Multiparametric image analysis was performed using the Discovery automated image analyzer (Becton Dickinson, Leiden, Netherlands). The integrated fluorescence ratio (IFR) was calculated for each sample as the integrated FISH fluorescence of the tumor cells divided by the integrated FISH fluorescence of internal control cells containing two spots. The percentage positive nuclear area (PPN), calculated as the area of FISH fluorescence divided by the area of nuclear DNA fluorescence, and the PPR, ratio of the PPN of the tumor cells divided by the control cells, were also calculated for each sample. RESULTS Visual analysis yielded 46 unamplified and 15 (24.6%) amplified (seven moderately amplified and eight highly-amplified) tumors. Strong (P < 0.001) correlation between results on cytological and histological materials was obtained. The FISH spots on the cytological preparations were more easily visualized and scored than those on the corresponding tissue sections. Visual HER-2/neu signal scoring was strongly correlated with IFR (P = 0.0001) and PPR (P = 0.0001). Within the tumors classified as highly amplified by visual examination, quantitation of the degree of amplification fluorescence signal was possible using image analysis. CONCLUSIONS Cytologic specimens were a suitable and representative source of materials for detection and quantitation of HER-2/neu gene amplification by FISH and image analysis. Cancer (Cancer Cytopathol) 1999;87:312–8. © 1999 American Cancer Society.