Ferroptosis in Intrahepatic Cholangiocarcinoma: IDH1105GGT Single Nucleotide Polymorphism Is Associated With Its Activation and Better Prognosis

Abstract

Objectives: Intrahepatic cholangiocarcinoma (ICC) has a dismal prognosis and often demonstrates an anti-apoptotic landscape, which is a key step to chemotherapy resistance. Isocitrate dehydrogenase 1 or 2 (IDH1-2)-mutated ICCs have been described and associated with better prognosis. Ferroptosis is a regulated iron-mediated cell death induced by glutathione peroxidase 4 (GPX4) inhibition, and may be triggered pharmacologically. GPX4 is overexpressed in aggressive cancers, while its expression is inhibited by IDH1 ^R132C mutation in cell lines. We investigated tissue expression of ferroptosis activation markers in ICC and its correlation with clinical-pathological features and IDH1-2 status.

Materials and methods: We enrolled 112 patients who underwent hepatic resection or diagnostic liver biopsy for ICC. Immunostaining for transferrin-receptor 1 and GPX4, and Pearls' stain for iron deposits were performed to evaluate ferroptosis activation. Immunostaining for STAT3 was performed to study pro-inflammatory and anti-apoptotic landscape. Main IDH1-2 mutations were investigated in 90 cases by real-time polymerase chain reaction.

Results: GPX4 overexpression was seen in 79.5% of cases and related to poor histological prognostic factors (grading and perineural and vascular invasion; p < 0.005 for all) and worse prognosis (OS p = 0.03; DFS p = 0.01). STAT3 was expressed in 95.5% of cases, confirming the inflammation-related anti-apoptotic milieu in ICC, and directly related to GPX4 expression (p < 0.0001). A high STAT3 expression correlated to a worse prognosis (OS p = 0.02; DFS p = 0.001). Nearly 12% of cases showed IDH1 ^105GGT single nucleotide polymorphism, which was never described in ICC up to now, and was related to lower tumor grade (p < 0.0001), longer overall survival (p = 0.04), and lower GPX4 levels (p = 0.001).

Conclusion: Our study demonstrates for the first time that in most inflammatory ICCs ferroptosis is not active, and its triggering is related to IDH1-2 status. This supports the possible therapeutic role of ferroptosis-inducer drugs in ICC patients, especially in drug-resistant cases.

Keywords: GPX4; IDH1105GGT single nucleotide polymorphism; STAT3; ferroptosis; intrahepatic cholangiocarcinoma.

FIGURE 1

STAT3 and GPX4 expression in ICC cases. Examples of 1 + and 2 + cytoplasmic positivity for STAT3 and GPX4 [(A) 1 + STAT3, original magnification 20x; (B) 2 + STAT3, original magnification 10x; (C) 1 + GPX4, original magnification 20x; (D) 2 + GPX4, original magnification 10x].

FIGURE 2

TFR1 expression in ICC cases was semi-quantitatively evaluated as negative, 1 + [(A) original magnification 20x], and 2 + [(B) original magnification 20x].

FIGURE 3

Intratumoral iron deposits in ICC were evaluated as absent/present. Negative cases often showed iron deposition in peri-tumoral macrophages, but not in neoplastic cells [(A) original magnification 20x]; in positive cases, iron deposits appear as intracytoplasmic blue granules in tumor cells [(B) original magnification 40x].

FIGURE 4

Kaplan–Meier curves showed reduced overall (A) and disease-free survivals (B) in cases with higher STAT3 expression.

FIGURE 5

Significantly reduced overall (A) and disease-free survivals (B) were observed in cases with higher GPX4 expression. Considering GPX4 1 + and 2 + positive cases together, the association becomes even stronger for both overall (C) and disease-free survivals (D), as shown by the Kaplan–Meier curves.

FIGURE 6

Kaplan–Meier curves showed different overall survivals in cases bearing IDH1^105GGT SNP, IDH1-2 point mutations and wild type IDH1-2.