Trans-arterial chemoembolization as a loco-regional inducer of immunogenic cell death in hepatocellular carcinoma: implications for immunotherapy

Abstract

Background: Modulation of adaptive immunity may underscore the efficacy of trans-arterial chemoembolization (TACE). We evaluated the influence of TACE on T-cell function by phenotypic lymphocyte characterization in samples of patients undergoing surgery with (T+) or without (T-) prior-TACE treatment.

Methods: We profiled intratumoral (IT), peritumoral (PT) and non-tumoral (NT) background tissue to evaluate regulatory CD4+/FOXP3+ (T-reg) and immune-exhausted CD8+/PD-1+ T-cells across T+ (n=58) and T- (n=61). We performed targeted transcriptomics and T-cell receptor sequencing in a restricted subset of samples (n=24) evaluated in relationship with the expression of actionable drivers of anti-cancer immunity including PD-L1, indoleamine 2,3 dehydrogenase (IDO-1), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), Lag-3, Tim-3 and CD163.

Results: We analyzed 119 patients resected (n=25, 21%) or transplanted (n=94, 79%) for Child-Pugh A (n=65, 55%) and Barcelona Clinic Liver Cancer stage A (n=92, 77%) hepatocellular carcinoma. T+ samples displayed lower IT CD4+/FOXP3+ (p=0.006), CD8+ (p=0.002) and CD8+/PD-1+ and NT CD8+/PD-1+ (p<0.001) compared with T-. Lower IT (p=0.005) and NT CD4+/FOXP3+ (p=0.03) predicted for improved recurrence-free survival. In a subset of samples (n=24), transcriptomic analysis revealed upregulation of a pro-inflammatory response in T+. T+ samples were enriched for IRF2 expression (p=0.01), an interferon-regulated transcription factor implicated in cancer immune-evasion. T-cell clonality and expression of PD-L1, IDO-1, CTLA-4, Lag-3, Tim-3 and CD163 was similar in T+ versus T-.

Conclusions: TACE is associated with lower IT density of immune-exhausted effector cytotoxic and T-regs, with significant upregulation of pro-inflammatory pathways. This highlights the pleiotropic effects of TACE in modulating the tumor microenvironment and strengthens the rationale for developing immunotherapy alongside TACE.

Keywords: immunotherapy; liver neoplasms.

Figure 1

Representative sections illustrating architectural features of an HCC sample resected after TACE on standard H&E stain (Panel A). Red arrows highlight deposition of embolic material to the immediate vicinity of a viable HCC tumor deposit, marked by a blue dashed line (magnification 200×). Panel B highlights T-cell phenotypic characteristics by multiplex immunohistochemistry for CD4 (brown chromogen), FOXP3 (green chromogen), CD8+ (red chromogen), PD-1 (blue chromogen) and CD8+/PD-1 co-expression (purple chromogen). TACE pre-treatment (Panel B) is associated with less intense T-cell infiltrate compared with a TACE-untreated case (Panel C). Panels D–G illustrate representative sections of PD-L1 (Panel D), IDO-1 (Panel E), Tim-3/CD163 (Panel F) and Lag-3/CTLA-4 (Panel G) intratumoral staining corresponding to the H&E section shown in Panel B. CTLA-4, cytotoxic T-lymphocyte associated protein 4; HCC, hepatocellular carcinoma; IDO-1, indoleamine 2,3 dehydrogenase; PD-1, programmed-cell death 1; TACE, trans-arterial chemoembolization.

Figure 2

Histograms illustrating the relationship between CD4+, CD8+, CD4+/FOXP3+ and CD8+/PD-1+tumor infiltrating lymphocytes and prior-treatment with TACE (T+/T−) in patients with HCC, assayed across intratumoral (IT), peritumoral (PT) and non-tumoral (NT) background liver areas. TACE pre-treatment (T+) was associated with lower T-cell density values for IT CD8+, CD4+/FOXP3+, CD8+/PD-1+ cells and NT CD4+/FOXP3+ cells. **p<0.01, ****p<0.001. HCC, hepatocellular carcinoma; PD-1, programmed-cell death 1; TACE, trans-arterial chemoembolization.

Figure 3

Kaplan-Meier curves illustrating the relationship between characteristics of the T-cell infiltrate found differentially regulated in association with TACE and recurrence-free survival of patients with HCC. IT and CD4+/FOXP3+ and NT CD4+/FOXP3+ but not IT CD8+/PD-1+ T-cell density were significantly associated associated with RFS. HCC, hepatocellular carcinoma; IT, intratumoral; NT, non-tumoral; PD-1, programmed-cell death 1; RFS, recurrence-free survival; TACE, trans-arterial chemoembolization.

Figure 4

Targeted transcriptomic analysis of tumorous HCC tissue (n=24) using Nanostring PanCancer Immune profiling illustrates the differential regulation of 11 gene expression signatures on the basis of TACE pre-treatment using directed gene set analysis (GSA) and confirms significant upregulation of IRS2 in TACE-pre-treated samples (Panel B). Panel C: histograms illustrating the distribution of productive clonality, productive entropy and number of most frequently identified clonotypes as assayed by deep sequencing of the TCR-beta chain. HCC, hepatocellular carcinoma; TACE, trans-arterial chemoembolization; TCR, T-cell receptor.