Evaluation of 20 indices in malignant and benign cases
The presence or absence of the following 20 cytological indices was evaluated by 7 reviewers based on the results of previous studies:18,21 (1) necrotic background (Fig. 1A), (2) mucus background (Fig. 1B), (3) hypercellularity (Fig. 1C), (4) two-cell pattern (Fig. 1D), not including contamination of digestive epithelial cells, (5) irregular structure (Fig. 2A), (6) irregular cell polarity (Fig. 2B), (7) overlapping (Fig. 2C), (8) decreased cell adhesion (Fig. 2D), (9) irregular nuclei (Fig. 3A), (10) hyperchromasia (Fig. 3B), (11) nuclear membrane thickening (Fig. 3C), (12) anisonucleosis (Fig. 3D), which is the presence of a mixture of various cell sizes, (13) prominent nucleoli (Fig. 3E), (14) increased mitosis (Fig. 3F), which is indicated by the presence of an increased number of mitotic cells, (15) unclear cell boundary (Fig. 4A), which is indicated by the presence of overlapping cells and unclear cellular boundaries, (16) high nuclear/cytoplasm (N/C) ratio (Fig. 4B), (17) pink intracellular mucus (Fig. 4C), (18) orange-yellow intracellular mucus (Fig. 4D), (19) cannibalism (Fig. 4E), and (20) keratinization (Fig. 4F), which is an important cytologic feature to predict adenosquamous carcinoma. Most of these are typical cytological indices of malignant cells in various organs. The presence of a two-cell pattern, which is the coexistence of malignant and benign cells (Fig. 1D), aids in identifying malignant cells with relatively lower atypia.18 The indices related to mucins are specific to malignant cells of the pancreas based on our previous study.21
Usefulness of 20 indices to distinguish malignant and benign cases
Seven reviewers, including 4 cytotechnologists and 3 medical doctors, evaluated 20 cytological indices using pancreatic specimens obtained by EUS-FNA (malignant, n=111; benign, n=31). Figure 5 shows the percentages of the presence of these 20 indices among the included specimens. This was used to distinguish malignant from benign tumors. Thirteen indices had an incidence rate of more than 40%, when the specimens were evaluated by cytotechnologists (Fig. 5A), medical doctors (Fig. 5B), and all reviewers combined (Fig. 5C). Furthermore, these 13 indices showed statistically significant differences between malignant and benign cases, when evaluated by cytotechnologists (Fig. 5A), medical doctors (Fig. 5B), and all reviewers combined (Fig. 5C), (*P<0.05). Hypercellularity showed both a high presence rate and statistically significant difference between malignant and benign cases, when evaluated by all reviewers and cytotechnologists, but not when evaluated by medical doctors (Fig. 5).
The 13 indices (gray boxes in Table 1) had higher odds ratios for malignancy than benignity. Several odds ratios could not be determined (N.D.) because the agreement was 0 or 100.
The useful indices to distinguish malignant and benign cases should have the following properties: (1) high incidence rate in malignant cases, (2) statistically significant difference (P < 0.05) between malignant and benign cases, and (3) reproducibility in various groups with different backgrounds and skills. Therefore, we determined that the following 13 cytological indices were useful to distinguish malignant and benign cases: irregular structure, hyperchromasia, irregular cell polarity, unclear cell boundary, nuclear membrane thickening, anisonucleosis, overlapping, irregular nuclei, high N/C ratio, decreased cell adhesion, two-cell pattern, prominent nucleoli, and necrotic background.
Cutoff values of the 13 indices indicating tumor malignancy
Each of the 13 indices was given a score of 1 point, based on the evaluation of 4 out of 7 reviewers (Table 2). Malignant cases had higher scores than benign cases (Table 2). To determine the cutoff values of the scores of all 13 malignant indices, we calculated the Youden index.22 A score of 8 points compared with a score of > 9 points showed the highest Youden index (0.950, Table 3). The cutoff value 8/9 showed high sensitivity (98%, Table 3), specificity (97%), accuracy (98%), and high area under the receiver operating characteristic curve value (0.996).
Discrepancy between score and pathological diagnosis
At a cutoff value of 8/9, 3 cases showed a discrepancy between cytological score and pathological diagnosis (Table 2A, B and C and Supplementary Table 1). Cytological specimens of case A lacked malignant features with contamination of many red blood cells (Fig. 6A). The cytological specimens of case B contained only a small number of cells (Fig. 6B). The cytological specimen of case C showed epithelial cells with enlargement of the nucleus, nuclear groove, and yellow mucus (Fig. 6C), and case C was diagnosed as mild atypical epithelium but not malignancy.