2.2. Image Analysis

We evaluated 20 representative nuclei per each sample of the test set (n = 27) in IND areas using Image J (National Institutes of Health, Bethesda, United States). The average areas of the nuclei were measured to evaluate mean nuclear areas for nuclear enlargement, standard deviation of nuclear areas for pleomorphism, brightness ratio of tumor to lymphocytes for hyperchromasia, and Feret diameters for nuclear irregularity. The area ratios of structural atypia to IND were calculated to quantify structural atypia. The structural atypia area (SAA) was defined as areas with ≥1 of the following: variable glandular size, irregular glandular arrangement, glandular branching/budding, and glandular cribriform patterns (Figure 1) [16]. The area of IND was defined as an area with any well-known structural anomalies (LOSM, margination, glandular cribriform patterns, glandular branching/budding, glandular arrangement, and glandular crowding) or nuclear atypia (loss of nuclear polarity (LONP), nuclear pseudostratification in more than half, nuclear pleomorphism, nuclear hyperchromasia, and prominent nucleoli) [16]. These measurements were discussed among three pathologists (K Yim, JH Shin, and J Yoo). Receiver operating characteristic (ROC) curves were plotted to determine the cut-off values in the SAA to IND that could predict neoplasm, above high-grade dysplasia (HGD), or carcinoma. The sum of the sensitivity and specificity was obtained, and cut-off values were determined using the maximum sum.

Representative images of structural atypia area (red-lined inner area) and area with total indefinite for dysplasia (black-lined inner area) In the red-lined inner area, one or more of the following are seen: variable glandular size, irregular glandular arrangement, glandular branching/budding, and glandular cribriform. In the black-lined inner area, one or more of structural or nuclear atypia are visible. (a) Structural atypia was present in 6.18% of the area, with a final diagnosis of nonneoplasm (hematoxylin and eosin; ×40 magnification). (b) The area ratio of the structural atypia was 61.1%, with a final diagnosis of carcinoma (hematoxylin and eosin; ×40 magnification). (c) A representative image of an area of nuclear atypia without structural atypia. Loss of surface maturation (arrow), nuclear enlargements, and hyperchromasia are visible (hematoxylin and eosin; ×100 magnification). (d) A representative image of an area of structural atypia. Glandular variation in size, irregular arrangement, and branching/budding (arrow) are visible (hematoxylin and eosin; ×200 magnification).