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

Abstract

BACKGROUND

Tumor-infiltrating lymphocytes (TILs) constitute a prognostic factor in hepatocellular carcinoma (HCC). However, different methods of assessing 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.

AIM

To evaluate the prognostic significance of TILs in H&E-stained slides of HCCs.

METHODS

This was a retrospective study performed in the hospital. HCC patients who underwent liver resection between 2015 and 2017 in Zhongshan Hospital were enrolled in this study. Patients who experienced recurrence or received therapy in addition to antiviral therapy before surgery at this time were excluded. A total of 204 patients were enrolled in the study. The ILs were counted manually in tumor sections stained with H&E under an optical microscope at 400 ×. The ILs were assessed separately in the center of the tumor (TILs^CT), the invasive front (TILs^IF), and peritumor (PILs) areas. Univariate and multivariate survival analyses were performed using a Cox regression model. P < 0.05 was considered statistically significant and all P-values were two-sided.

RESULTS

Among the 204 patients, univariate analysis indicated that macrovascular invasion (MaVI) (P = 0.001), microvascular invasion (MVI) (P = 0.012), multiple tumors (P = 0.008), large tumors (> 10 cm) (P = 0.001), absence of a tumor capsule (P = 0.026), macrotrabecular histological subtype (P = 0.001), low density of TILs^CT (P = 0.039), TILs^IF (P = 0.014), and PILs (P = 0.010) were predictors of progression-free survival (PFS). Cox multivariate analysis indicated that MaVI (P = 0.009), absence of a tumor capsule (P = 0.031), low-density of TILs^IF (P = 0.047) and PILs (P = 0.0495) were independent predictors of PFS. A three-category analysis was carried out by combining TILs^CT, TILs^IF, and PILs, after which HCCs were classified into immune^high [(TILs^CT)^high, (TILs^IF)^high, and PILs^high, 83 cases], immune^mod (tumors other than immune^high and immune^low subtypes, 94 cases), and immune^low [(TILs^CT)^low, (TILs^IF)^low, and PILs^low, 27 cases)] subtypes. The immune^high subtype had a lower rate of MVI (40.96%) than the immune^mod (61.70%, P = 0.017) and immune^low (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.

CONCLUSION

HCC patients with high infiltrating lymphocytes tend to have a lower recurrence rate and less MVI. The evaluation of TILs in H&E-stained specimens could be a prognostic parameter for HCC.

Keywords: Lymphocytes, Tumor infiltration, Hepatocellular carcinoma, Hematoxylin and eosin-stained, Pathology

Core Tip: Successful use of immunotherapy in tumors starve for widely applicable, accessible and reliable immune-oncology biomarkers. Assessing tumor infiltrating lymphocytes (TILs) in hematoxylin and eosin (H&E)-stained tissues showed great clinical validity and utility in many solid tumors. However, barely any research on hepatocellular carcinoma (HCC) has been published. TILs evaluated in H&E-stained HCC tissues showed a great prognostic effect for recurrence in the present study and might be helpful to select patients with the highest likelihood of responding to immunotherapeutic agents. The method has low requirements in terms of technical and economic costs and can be easily applied in routine practice.