Evaluating tumor-infiltrating lymphocytes in hepatocellular carcinoma using hematoxylin and eosin-stained tumor sections

ARTICLE HIGHLIGHTS

Research background

As successful use of immune checkpoint inhibitors and other forms of immunotherapy has become a clinical reality, the need for widely applicable, accessible and reliable biomarkers is clear. Different methods of assessing tumor infiltrating lymphocytes (TILs) have various pre-analytical, analytical, and post-analytical challenges. The evaluation of TILs in hematoxylin and eosin (H&E) stained tumor sections proposed by the International Immuno-Oncology Biomarker Working Group was demonstrated to be a reproducible, affordable and easily applied method in many tumors. However, this method has barely been conducted in hepatocellular carcinoma (HCC). The exploration of TILs in H&E sections of HCC could provide a detailed information for the selection of patients who receive the immunotherapy and evaluation of the prognostic effect of immunotherapy.

Research motivation

There have been few suggestions to evaluate HCC by examining TILs in H&E sections. The key problem is to build a method suitable for the tissue specificity of HCC. Once a consensus of the method is established, it will be helpful to manifest the inflammatory condition of the tumor and help to select patients that will experience the greatest benefit of immunotherapy as well as to gain deep insight into immunotherapy.

Research objectives

The main objective of this study was to explore whether evaluating TILs in H&E-stained sections has a prognostic effect in HCC. Based on this study, evaluating TILs in H&E-stained sections could be a prognostic method for HCC. Increasing multicenter research to validate and improve this method should be implemented in the future.

Research methods

H&E staining was performed on a high-throughput fast automatic platform (Dako coverstainer, United States) according to standard protocols. Programmed cell death-ligand 1 (PD-L1) (SP142) rabbit monoclonal primary antibody (Ventana Medical Systems Inc, Tucson, AZ, United States) was optimized for a fully automated immunohistochemical (IHC) assay on the BenchMark ULTRA (Ventana Medical Systems Inc) staining platform using the OptiView DAB IHC Detection Kit and OptiView Amplification Kit (Ventana Medical Systems Inc). The method to record TILs was described as follows: (1) The number of ILs on full sections was scanned at low magnification and evaluated manually at higher magnification (400 ×) under an optical microscope; (2) ILs were assessed in the areas of the tumor center (TILs^CT), the invasive front (TILs^IF) and on the portal areas of the peritumor 1 cm away from the border (PILs). The “invasive front” (IF) is defined as the region centered on the border separating the host tissue from the malignant nests by 1 mm. Areas with crush artifacts, necrosis, and previous biopsy sites were excluded; and (3) All mononuclear cells, including lymphocytes and plasma cells, were counted. Kaplan-Meier univariate and multivariate survival analyses were performed using a Cox regression model. A nonpaired t-test was conducted to compare the clinicopathological parameters of the immune subtypes.

Research results

Based on this research, low density of TILs^CT (P = 0.039), TILs^IF (P = 0.014), and PILs (P = 0.010) were independent predictors of progression-free survival (PFS). The immune^high subtype [(TILs^CT)^high, (TILs^IF)^high, and PILs^high, 83 cases] had a lower rate of microvascular invasion (MVI) (40.96%) than the immune^mod (tumors other than immune^high and immune^low subtypes, 94 cases) (61.70%, P = 0.017) and immune^low [(TILs^CT)^low, (TILs^IF)^low, and PILs^low, 27 cases] (66.67%, P = 0.020) subtypes. The recurrence rates of the immune^high, immune^mod and immune^low subtypes were 10.8%, 25.5% and 33.3%, respectively.

Research conclusions

This study proposed that the density of TILs in HCC tissues can predict the recurrence of the patient. The method of evaluating TILs in H&E-stained specimens may also be meaningful in HCC.

Research perspectives

Increasing multicenter research to validate and improve this method should be implemented in the future.