Intratumoral CD3 and CD8 T-cell Densities Associated with Relapse-Free Survival in HCC

Abstract

Immune cells that infiltrate a tumor may be a prognostic factor for patients who have had surgically resected hepatocellular carcinoma (HCC). The density of intratumoral total (CD3(+)) and cytotoxic (CD8(+)) T lymphocytes was measured in the tumor interior and in the invasive margin of 65 stage I to IV HCC tissue specimens from a single cohort. Immune cell density in the interior and margin was converted to a binary score (0, low; 1, high), which was correlated with tumor recurrence and relapse-free survival (RFS). In addition, the expression of programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) was correlated with the density of CD3(+) and CD8(+) cells and clinical outcome. High densities of both CD3(+) and CD8(+) T cells in both the interior and margin, along with corresponding Immunoscores, were significantly associated with a low rate of recurrence (P = 0.007) and a prolonged RFS (P = 0.002). In multivariate logistic regression models adjusted for vascular invasion and cellular differentiation, both CD3(+) and CD8(+) cell densities predicted recurrence, with odds ratios of 5.8 [95% confidence interval (CI), 1.6-21.8] for CD3(+) and 3.9 (95% CI, 1.1-14.1) for CD8(+) Positive PD-L1 staining was correlated with high CD3 and CD8 density (P = 0.024 and 0.005, respectively) and predicted a lower rate of recurrence (P = 0.034), as well as prolonged RFS (P = 0.029). Immunoscore and PD-L1 expression, therefore, are useful prognostic markers in patients with HCC who have undergone primary tumor resection. Cancer Immunol Res; 4(5); 419-30. ©2016 AACR.

Figure 1

Representative images of CD3⁺ TILs in HCC from different patient samples. A, B, and E are focused on the interior of the tumors, whereas C, D, and F are focused on the invasive margin. The pattern of TILs in each region fell into 3 categories: dense nodular clusters of lymphocytes as in A; diffuse, evenly distributed lymphocytes as in B; or rare, or scattered, lymphocytes as in E. Some tumors had clusters of TILs from the IM to the TI, as in C and D, marked by red arrows; whereas others had no obvious TILs as in F.

Figure 2

Immunohistochemical (IHC) staining of CD3 (A and E), CD8 (B and G), PD-L1 (C and I), negative controls (E and H), PD-1 (K), or β-catenin (L) in a lesion from a HCC surgical sample (A, B, C, and D) or in a HCC nodule, shown at 20× magnification (E–L).

Figure 3

Kaplan Meier curves comparing RFS in patients with high and low intratumoral CD3 cell density in the TI (A) and IM (B), as well as high and low intratumoral CD8 cell density in the TI (C) and IM (D). Patients were stratified in high and low groups using median immune cell density as the threshold. Kaplan-Meier curves comparing RFS in patients with high and low total intratumoral CD3 density (CD3TI + CD3IM) and total CD8 density (CD8TI + CD8IM), using median cut-off points as the threshold (E and F, respectively). Kaplan-Meier curves comparing RFS in patients with different Immunoscores (G). Kaplan-Meier curves comparing RFS in patients with and without PD-L1 expression (H).

Figure 4

Kaplan Meier curves comparing OS in patients with high and low intratumoral CD3 cell density in the TI (A) and IM (B), as well as high and low intratumoral CD8 cell density in the TI (C) and IM (D). Patients were stratified in high and low groups using median immune cell density as the threshold. Kaplan-Meier curves comparing OS in patients with high and low total intratumoral CD3 density (CD3TI + CD3IM) and total CD8 density (CD8TI + CD8IM), using median cut-off points as the threshold (E and F, respectively). Kaplan-Meier curves comparing OS in patients with different Immunoscores (G). Kaplan-Meier curves comparing OS in patients with and without PD-L1 expression (H).