3.1 Large duct and small duct type of ICCs
3.1.1 Subclassification of ICCs
We classified 130 cases with ICC into 3 subtypes on the basis of histological features, S100P expression and Alcian blue staining in the first round. 12 cases (9.2%) were recognize as typical large duct type, which met three standards:HE, type1; S100P, score 3-4; and Alcian blue, score 1-2. Meanwhile, 73 cases (56.2%) were typical small duct type, which satisfied the following conditions: HE, type2; S100P, score 0-1; and Alcian blue, score 0-1. The remaining 45 cases (34.6%) were intermediate type that exhibited mixtures of two subtypes or indeterminate features. [Supplementary Table S1]
In the second round, according to the above criteria, we set classification efficiency of three factors as follows: HE > S100P > Alcian blue. Then 45 cases of intermediate type were divided into 2 subcategories. [Supplementary Table S2] Finally, we identified 27 (20.8%) and 103 (79.2%) cases as large duct and small duct type ICCs, respectively. [Table 1] The concordant rate and κ value of interpretations between two observer were 96.9% and 0.904, respectively.
3.1.2 Clinical characteristics and prognosis of ICC subtypes
Our study cohort included 71 males (54.6%) and 59 females (45.4%), with a mean age of 57.8±9.7 years (range from 28 to 77). 44 cases (33.8%) underwent lymphadenectomy.
The large duct type was more likely to have higher level of CA19-9 (P=0.002), higher frequencies of lymphadenectomy (P=0.002), nerve invasion (P=0.025), satellite lesions (P=0.009), smaller size of tumor (P=0.021) and more aggressive tumor stage of pT (P=0.041), pM (P=0.019), and TNM classification (P=0.04) than small duct type. [Supplementary Table S3]
The median follow-up period of 102 ICC cases was 25.2 months (range from 4.9 months to 100.0months). Although patients with large duct type had worse prognosis than those with small duct type in univariate analysis (DFS, P=0.063; OS, P=0.031) [Figure S1], the histological subtype was not an independent prognostic factors of ICCs in multivariate analysis (P>0.10).
3.2 IDH1/2 mutations in ICCs
3.2.1 IDH1/2 mutations by DNA sequencing and IHC
DNA sequencing showed 21 patients (16.1%) harbored IDH1/2 mutation, including IDH1-R132C, IDH1-R132G, IDH1-R132H, IDH1-R132L, IDH2-R172K, and IDH2-R172W. But no mutation of IDH2-R140 was detected. Meanwhile, IDH1/2 mutant was detected in 14 cases (10.8%) by MsMab-1, which is specific for IDH1-R132G, IDH1-R132H, and IDH2-R172W according to instructions. [Table 2] IHC analysis verified the specificity of MsMab-1 according to results detected by DNA sequencing, and showed that sensitivity and specificity of MsMab-1 to detect specific types of IDH1/2 mutation were 81.8% (9 of 11) and 95.8% (114 of 119), respectively. Accordingly, MsMab-1 was a relatively effective multi-specific antibody against IDH1/2 mutant in ICCs (κ= 0.691).
3.2.2 Clinical implications of IDH1/2 mutation in small duct type of ICCs.
IDH1/2 mutation was detected in 3.7% (1/27) of cases with large duct type and 19.4% (20/103) of patients with small duct type, respectively. Patients with IDH1/2 mutation had decreased TBIL (total bilirubin) (P=0.039), Fe (ferritin) (P=0.000) and higher histological differentiation (P=0.024) in small duct type. [Table 3]
Univariate and multivariate analyses exhibited that cases with IDH1/2 mutation had significantly improved prognosis than those without IDH1/2mutation in terms of DFS (P=0.006) and OS (P=0.031) in total cohort of ICCs.[Figure 4 A-B, Supplementary Table S4] And more notably, Kaplan-Meier analysis showed IDH1/2 mutation was associated with prolonged DFS (P=0.009)and a trend toward increased OS (P=0.126) in small duct type of ICCs. Further multivariate analysis confirmed IDH1/2 mutation was a favorable independent predictor of DFS (P=0.022, HR=3.452, 95% CI=1.191-10.004), and was not significantly related to OS (P=0.114, HR=2.686, 95%CI=0.789-9.144) in patients with small duct type. [Figure 4C-D, Table 4]
3.3 Expression status of BAP1, ARID1A and PBRM1 in ICCs
3.3.1 Loss of BAP1, ARID1A and PBRM1 expressions
BAP1 expression was retained in 72 cases (55.4%), while 47 cases (36.2%) and 11 cases (8.5%) showed complete and focal loss of BAP1 expression respectively. Complete or regional loss of ARID1A and PBRM1 staining was observed in 28 cases (21.5%) and 45 cases (34.6%), respectively. [Supplementary Table S5] There was obviously positive correlation among their expression loss (P<0.05), especially between BAP1 and PBRM1 (P=0.000). [Supplementary Table S6]
3.3.2 Clinical implications of BAP1, ARID1A and PBRM1 expression loss in subtypes of ICCs.
The loss of BAP1 expression was associated with decreased ALT (alanine aminotransferase), AST (aspartate aminotransferase), TBIL and higher histological differentiation in large type of ICCs, while it was correlated to larger size of tumor in small duct type. [Supplementary Table S7] All clinical factors significantly related to the loss of ARID1A expression, were within small duct type of ICCs, such as decreased ALT, AST, lower incidence of liver cirrhosis and earlier TNM stage. [Supplementary Table S8] PBRM1 loss correlated decreased AST, TBIL, IBIL (indirect bilirubin), and CA19-9 in the large duct type of ICCs. [Supplementary Table S9] In addition, the loss of BAP1, ARID1A and PBRM1 expressions had similar distribution in large duct and small duct type of ICC patients (P>0.05). [Supplementary Table S7-9]
Kaplan-Meier curves showed loss of BAP1, ARID1A and PBRM1 expressions were not significantly associated DFS and OS in patients with ICCs (P>0.05). [Supplementary Table S4] While univariate and multivariate analyses exhibited BAP1 expression loss was correlated to prolonged DFS (P=0.031) and OS (P=0.041) in large duct type of ICCs. [Figure 5] Besides this, none of inactive mutations was statistically associated with DFS or OS in either subtype of ICCs (P>0.05). [Table 4]
3.4 A typical case of ICC patient with IDH1 mutation
A 61-year-old Chinese man was diagnosed with liver cancer and diffuse intrahepatic and abdominal lymph node metastasis by abdominal magnetic resonance (MR) in May 2017, and subsequently confirmed as ICC (stage IV) with IDH mutation (IDH1-R132C) by biopsy and gene sequencing. He received dasatinib (100mg, once a day) combined with chemotherapy (gemcitabine, once every three weeks, twenty-five cycles) since June 2017. Abdominal MR in July 2017 showed stable disease (SD). Abdominal MR in January 2019 revealed progressive disease (PD). Therefore, in the same month, this patient was administered with lenvatinib (12mg, once a day) instead of dasatinib and chemotherapy. Encouragingly, this case still survives with ECOG performance status 1 at the end of this study (September 2019). Figure 6 summarized the patient's serial images of abdominal MR and diameter curves of representative tumors during follow-up period.