Enhanced CLU canalicular staining pattern was observed in majority of HCCs but not in nonneoplastic liver tissue
In normal and cirrhotic liver tissues, CLU immunostaining highlighted intercellular canaliculi with a delicate, fine granular and “railroad track”-like pericanalicular pattern (Fig. 1A). However, this “benign” pattern had changed when there was a malignant transformation. In HCCs, a much enhanced and exaggerated canalicular staining pattern (Fig. 1B) was observed in 89 of 128 (70%) HCCs. This included cases with pseudoglandular/pseudoacinar formation that exhibited an intraluminal staining pattern (Fig. 1C). Of the 54 surgically resected HCCs where whole tissue sections were used for the study, 42 (77.8%) showed enhanced canalicular CLU staining pattern. Among them, 10 (18.5%) were positive in >50% of tumor cells (diffuse), 20 (37%) in 26-50% of tumor cells (patchy), and 12 (22.2%) in 10-25% of tumor cells (focal). There was no significant difference in aberrant CLU expression between well and moderately differentiated HCCs, seen in 10 of 14 (71.4%) and 32 of 40 (80%) cases, respectively. None of the 55 normal liver tissue samples on TMA and none of 17 non-cirrhotic background liver tissues on whole tissue sections showed this enhanced staining pattern. In 37 cases with a cirrhotic background on whole tissue sections, 20 (54%) showed focal enhanced canalicular pattern of CLU in RNs, but positive cells in all these cases were <10% (thus considered negative), usually <3%, involving only one or a few canaliculi (Fig. 1D). The difference between HCC and RN was statistically significant (p<0.001) (Table 1). The sensitivity and specificity for enhanced canalicular staining pattern of CLU in HCCs were 0.70 and 1.00, respectively. Other expression patterns of CLU observed in HCCs included cytoplasmic (without canalicular staining), paranuclear dot-like, and membranous staining, seen in 6, 4 and 3 cases, respectively.
Enhanced CLU canalicular staining pattern was not observed in non-hepatocellular tumors
Three CLU staining patterns were observed in non-hepatocellular tumors: cytoplasmic with (Fig. 2A) or without (Fig. 2B) membranous staining, luminal/apical staining (Fig. 2C), and paranuclear dot-like staining (Fig. 2D). Cytoplasmic pattern was most common, which was seen in almost all tumor types. Among them, pancreatic neuroendocrine tumor showed strong and diffuse cytoplasmic and membranous expression in 12 of 14 (86%) cases. A small fraction (4/48, 8.3%) of pancreatic ductal adenocarcinoma also showed strong cytoplasmic expression. Cytoplasmic and membranous staining was seen in cholangiocarcinoma (10/13, 76.9%), and PEComa (7/14, 50%). Weak cytoplasmic staining with or without paranuclear dot pattern was seen in lung adenocarcinoma (30/97, 30.9%), breast ductal adenocarcinoma (75/86, 87.2%), clear cell renal cell carcinoma (67/78, 85.9%), clear cell carcinoma of the uterus and ovary (22/28, 78.6%), adrenocortical tumor (22/30, 73.3%) and germ cell tumors (127/184, 69.0%). Luminal/apical staining pattern was commonly seen in tumors that had glandular or papillary structures, such as esophageal adenocarcinoma (15/48, 31.3%), papillary thyroid carcinoma (39/48, 81.3%), prostatic adenocarcinoma (18/96, 18.8%), endometrioid carcinoma of the uterus (41/93, 44.1%), serous carcinoma of the ovary (10/40, 25.0%). Colorectal carcinoma (6/86, 7.0%) and mesothelioma (4/31, 12.9%) showed the least CLU positivity. None of the non-hepatocellular tumors showed enhanced canalicular CLU staining pattern.
CD10 and pCEA also showed an enhanced canalicular pattern in HCC but with a much lower frequency
Positive canalicular staining for CD10 and pCEA was observed in 50 (53%) and 68 (72%) of 94 HCCs examined for these immunomarkers (Fig. 3A and 3B). Though more sensitive for the canalicular pattern than CD10, pCEA also showed cytoplasmic staining and positive staining in inflammatory cells, giving rise to a higher degree of background staining. The majority of CD10 and pCEA positive cases showed equivalent staining intensity between HCC and surrounding benign liver tissue (Fig. 3C and 3D). Enhanced canalicular pattern was observed in only 24 (26%) and 22 (23%) HCCs for CD10 and pCEA, respectively (Table 1). The sensitivity and specificity for enhanced canalicular staining pattern in HCC were 0.26 and 1.000 for CD10, and 0.23 and 1.000 for pCEA, respectively. Of the 54 surgically resected HCCs where whole tissue sections were used for the study, 4 (7.4%) were negative for all 3 markers. Eight cases (14.8%) were positive for CLU, but negative for both CD10 and pCEA. Eleven cases (20.4%) were positive for only one marker, either CLU, CD10 or pCEA.