Detection and quantitation by fluorescence in situ hybridization (FISH) and image analysis of HER-2/neu gene amplification in breast cancer fine-needle samples

BACKGROUND. Fine-needle sampling, although a practical and noninvasive meth od of tissue acquisition, has rarely been used for HER-2/neu fluorescent in situ hybridization (FISH). To assess HER-2/neu gene amplification in mamma ry carcinoma, FISH signals on cytology and corresponding tissue biopsies we re detected visually and measured by image analysis. The results were corre lated with patient and tumor characteristics. METHODS. In situ HER-2/neu DNA probe hybridization was performed on 61 cyto logy specimens and on 47 corresponding frozen sections of breast carcinomas . Tumors were classified by visual evaluation as unamplified, moderately am plified, or highly amplified. Multiparametric image analysis was performed using the Discovery automated image analyzer (Becton Dickinson, Leiden, Net herlands). The integrated fluorescence ratio (IFR) was calculated for each sample as the integrated FISH fluorescence of the tumor cells divided by th e integrated FISH fluorescence of internal control cells containing two spo ts. The percentage positive nuclear area (PPN), calculated as the area of F ISH fluorescence divided by the area of nuclear DNA fluorescence, and the P PR, 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 (s even 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 eas ily visualized and scored than those on the corresponding tissue sections. Visual HER-2/neu signal scoring was strongly correlated with IFR (P = 0.000 1) and PPR (P = 0.0001). Within the tumors classified as highly amplified b y visual examination, quantitation of the degree of amplification fluoresce nce 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. (C) 1999 American Cancer Society.