Cytological Comparison between Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma by Image Analysis Software Using Touch Smear Samples of Surgically Resected Specimens

Abstract

To investigate useful cytological features for differential diagnosis of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), this study cytologically compared HCC to ICC using image analysis software. Touch smear specimens of surgically resected specimens were obtained from a total of 61 nodules of HCC and 16 of ICC. The results indicated that the major/minor axis ratio of ICC is significantly larger than that of HCC (1.67 ± 0.27 vs. 1.32 ± 0.11, p < 0.0001) in Papanicolaou staining. This result means that the nucleus of HCC is close to round and the nucleus of ICC is close to an oval. This significant difference in the major/minor axis ratio between ICC and HCC was consistently observed by the same analyses using clinical samples of cytology (4 cases of HCC and 13 cases of ICC) such a fine-needle aspiration, brushing and ascites (ICC: 1.45 ± 0.13 vs. HCC: 1.18 ± 0.056, p = 0.004). We also confirmed that nuclear position center-positioned nucleus (p < 0.0001) and granular cytoplasm (p < 0.0001) are typical features of HCC tumor cells compared to ICC tumor cells. The research study found a significant difference in the nuclear morphology of HCC (round shape) and ICC (oval shape) in Papanicolaou-stained cytology specimens. This simple and objective finding will be very useful for the differential cytodiagnosis of HCC and ICC.

Keywords: cholangiocarcinoma; cytology; hepatocellular carcinoma; nuclear atypia; touch smear.

Figure 1

Analyzing images of HE-stained tissue section using Tissue Studio. (a) Image of well differentiated HCC (×200). (b) Analyzing image of Figure 1a. The software appropriately recognizes the nuclei of tumor cells and calculate major axis, minor axis, and area of the nucleus. (c) Image of poorly differentiated HCC (×200). The tumor cells are significantly larger than well differentiated HCC. (d) Analyzing image of Figure 1c. The software appropriately recognizes the nuclei of tumor cells although the nuclei are markedly pleomorphic.

Figure 2

(a) The image of Giemsa-stained touch smear cytology of well differentiated HCC (×200, same case of Figure 1a). (b) Analyzing image of Figure 2a. The software Tissue Studio appropriately recognize the nuclei of tumor cells. (c) The image of Papanicolaou-stained touch smear cytology of poorly differentiated HCC (×200, same case of Figure 1c). (d) Analyzing image of Figure 2c. The nuclei were manually selected and then of the major axis, minor axis, and area of the nucleus were calculated by attaching software of EXpath III.

Figure 3

Comparison of major axis, minor axis, major/minor axis ratio and the nucleus area of hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and non-tumorous hepatocytes (NH).

Figure 4

Representative cytological figures of hepatocellular carcinoma (HCC). (a) and intrahepatic cholangiocarcinoma (ICC). (b) In touch smear specimens (Papanicolaou stain, ×400). HCC cells have round and center-positioned nucleus and granular cytoplasm whereas ICC cells have oval and uncenter-positioned nucleus, multiple nucleolus, and foamy cytoplasm.

Figure 5

Comparison of major minor axis ratio in hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) by validation Papanicolaou-stained clinical samples. A significant difference was found in the major/minor axis ratio between HCC and ICC (p = 0.004).