Patients and Tissue Samples
For this study, we selected 130 consecutive patients with ICC who underwent surgical treatment at Tianjin Medical University Cancer Institute and Hospital from January 2012 to December 2017. Because of the challenge of determining exact anatomical origin of some tumors, the ICCs with similar features of perihilar carcinoma were excluded. Cases with combined HCC-CCA, composed of typical HCC and typical ICC, were excluded. All patients were reviewed to confirm the diagnosis of ICC and to restage according to the 8th edition 2017 AJCC staging system. After exclusion of 28 patients for prognostic analysis (including 11 cases lost to follow-up, 14 cases received non-R0 resection, 1 cases died of postoperative complications, and 2 cases died of some non-tumor-related causes), 130 and 102 patients were eventually used for comparisons of clinical characteristics and survival analyses respectively.
Formalin-fixed paraffin-embedded (FFPE) samples and hematoxylin-eosin (HE) staining slides of 130 surgical specimens were collected from Pathology Department of Tianjin Medical University Cancer Institute and Hospital. Tissue microarrays (TMAs) composed of 2-mm cores of FFPE tumor tissue were constructed for various staining by selecting a representative tumor areas and a typical peritumoral region from each case.
Clinical Data
Clinical data, including the patients’ gender, age, surgical records, imaging examinations and laboratory tests were carefully reviewed and analyzed. All cases were regularly followed up every 3 months for tumor recurrence by screening of plasma tumor markers and imaging examinations. Disease-free survival (DFS) was measured from the date of surgery to the date of first recurrence or last follow-up, whereas OS was defined as the interval between the date of first diagnosis of ICC and the date of death or last follow-up.
Histopathology
HE staining was performed routinely on a set of TMAs. According to histological features,we subclassified ICCs into two types: large duct and small duct [11, 14, 15].
Type1 (large duct type) is made up of tall columnar tumor cells arranged in a large-sized glandular or tubular pattern and usually shows abundant desmoplastic stroma and low cellularity. Meanwhile, the tumor cells of type1 contain abundant eosinophilic, clear, or mucinous cytoplasm, and their nuclei are usually high grade. Extracellular mucin can be detected in glandular spaces. [Figure 1A]
Type2 (small duct type) has histological features of small-sized tubular or acinar component composed of low columnar to cuboidal tumor cells and usually shows scant stroma and high cellularity. This type can also be arranged in cribrate or solid patterns. The tumor cells of Type2 have a higher nuclear-to-cytoplasmic ratio compared with type1 tumors and usually contain scanty amphophilic or eosinophilic cytoplasm. [Figure 1D]
If there was any doubt in the process of classification, we can refer to whole mount sections for further observation. Ultimately, some cases, not clearly classified, were termed undetermined type.
Mucin production was stained by Alcian Blue Stain Kit (pH 2.5) (G1560; Solarbio Science & Technology Co., Ltd, Beijing, China) on TMAs to distinguish subtypes of ICCs [Figure 1B,E]. Alcian blue staining was scored semiquantitatively by the proportion of glandular lumens with mucin production on a scale from 0 to 2: score 0, <10%; score 1, 10% to 50%; and score 2, >50% or frequent intracytoplasmic mucin [10, 26].
Immunohistochemistry
TMAs sections were dewaxed and rehydrated in xylene and gradient ethanol, respectively. After antigen retrieval and endogenous peroxidase activity blocking, the slides were incubated with primary antibodies (4°C for 14h and 37°C for 1h) and HRP-conjugated secondary antibody (37°C for 1 h) in turn. Then the sections were visualized with 3,3’-diaminobenzidine (ZLI-9017; Zhongshan Goldbridge) for 5 min and counterstained with Hematoxylin. Appropriate internal or external positive controls and negative controls were designed and used for each round.
The primary antibodies used in the present study were as follows: S100P (clone EPR6142; dilution 1:300; Abcam, Cambridge, UK), IDH1/2 mutant (R132/172) (clone MsMab-1; dilution 1:200; EMD Millipore Corp., Billerica, MA, USA), BAP1 (ab199396; dilution 1:200; Abcam), ARID1A (clone EPR13501-73; dilution 1:500; Abcam), PBRM1 (HPA015629; dilution 1:200; Sigma-Aldrich).
The expression of S100P showed nuclear and cytoplasmic staining pattern and was evaluated semiquantitatively according to the percentage of positive tumor cells as follows: score 0, <1%; score 1, 1% to 25% ; score 2, 26% to 50%; score 3, 51% to 75%; and score 4, 76% to 100% [10]. [Figure 1C,F]
IDH1/2 mutant exhibited cytoplasmic and mild nuclear expression, while only nuclear staining was interpreted as immunopositive in the evaluation of BAP1, ARID1A, and PBRM1 expression. [Figure 2-3] A semiquantitative scoring system was used for the above 4 factors in terms of the proportion of positive neoplastic cells: score 0 (negative), < 1%; score 1 (focal or regional positive), 1% to 90%; score 2 (diffuse positive), 91% to 100% [27, 28].
Analysis of IDH1/2 mutation
Genomic DNA of ICCs was extracted from ten 10-um-thick sections micro-dissected from FFPE tissue blocks. Two pairs of primers were designed for hotspot mutations of IDH1/2 [10]. The first pair of primers for IDH1-R132: forward 5’- ACACGACGCTCTTCCGATCTACACATACAAGTTGGAAATTTCTGG-3’ and reverse 5’- GACGTGTGCTCTTCCGATCTAATCACCAAATGGCACCATAC-3’; the second pair of primers for IDH2-R140 and IDH2-R172: forward 5’-ACACGACGCTCTTCCGATCTCAGAGACAAGAGGATGGCTAGG-3’ and reverse 5’-GACGTGTGCTCTTCCGATCTTGTCCTCACAGAGTTCAAGCTG-3’ (Tsingke Biological Technology CO., Ltd, Beijing, China). The target DNA sequences were amplified by polymerase chain reaction using the primers above, and were further sequenced and analyzed for IDH1/2 mutation.
Statistical analysis
Categorical variables were presented as totals and frequencies, and evaluated by chi-square test or Fisher exact test, as appropriate; Continuous variables were described as means with standard deviations or medians with ranges, and compared with t test or Mann-Whitney U test, as appropriate. DFS and OS were calculated by Kaplan-Meier method, and assessed by log-rank test for univariate analysis. Cox proportional hazards model was conducted to adjust for the bias from tumor characteristics (e.g. TNM stage, bilateral involvement) in analyzing prognostic value of gene mutations and other clinical factors. The results were recorded as hazard ratios (HR) and 95% confidence intervals (CIs). A two-tailed P value less than 0.05 was considered significant. SPSS statistical program (IBM SPSS Statistics 24, Chicago, IL, USA) was used for data analysis.