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Graphical distributions of CD3+ and CD8+ T lymphocyte densities in the TI and IM.

ImageJ plug-in code for image analysis.

Bivariate analysis of TIL pattern and tumor stage, cellular differentiation, and vascular invasion.

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.

Kaplan-Meier analysis of RFS stratified by pattern of CD3+ and CD8+ T lymphocytes.

Example of computer-assisted evaluation of infiltrate densities.

Bivariate analysis of CD3+ and CD8+ cell density and beta-catenin expression.

Flowchart of image analysis process.

Bivariate analysis of tumor stage with vascular invasion and cellular differentiation.

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.

461 Background: Hepatocellular carcinoma (HCC) is the sixth most common cancer and the second leading cause of cancer-related death worldwide. Most HCCs develop in severely damaged liver. Methods: The effect of multiple treatment options on liver function (LF) and disease outcome of patients (pts) with HCC (n = 185) were examined retrospectively. Pt tumor burden (using Barcelona clinic liver cancer [BCLC] classification) and LF (Child Pugh [CP]) were assessed at time of diagnosis and then after treatment. Using Kaplan Meier with log rank and T tests, BCLC and CP scores were correlated with overall survival (OS) following individual treatment regimens. Results: We show that better BCLC and LF scores at time of diagnosis predict a better outcome (median OS; p < 0.05). Pts received one or more of the following: no treatment, experimental treatment, TACE, Y90 radioembolization, radiofrequency ablation, resection, radiation, and sorafenib (SFB). Considering all treatment scenarios, LF improved in 9.5%, did not change in 33%, and worsened in 57% of pts. Sixty percent of untreated pts experienced LF decline, compared with 33, 54, 48 and 50% of pts receiving TACE, SFB, TACE/SFB, and Y90/SFB, respectively (no significant differences [NS]); 72% of pts receiving TACE/Y90/SFB (NS); and 85% of pts receiving other Y90/SFB combinations (p = 0.006). In general, pts who saw no change or an improvement in LF from baseline had longer median OS versus pts who had declining LF (p < 0.002). However, pts receiving TACE/Y90/SFB (n = 29) had similar OS to those receiving TACE/SFB (n = 35), despite the toxicity difference (72% [TACE/Y90/SFB] vs. 48% [TACE/SFB] of pts had declining LF). TACE/SFB or TACE/Y90/SFB led to longer median OS than any other treatment group (p = 0.017). Conclusions: The outcome of pts with HCC depends on disease stage and LF. Most pts experience LF decline during treatment. Despite LF decline in 48% of pts receiving a SFB/TACE, these pts experienced longer median OS than pts on any other treatment. Balancing survival benefit with liver toxicities is critical to the successful treatment of pts with advanced HCC. Agents with good antitumor activity and minimal liver toxicity are desperately needed for these pts.

442 Background: Hepatocellular carcinoma (HCC) is the sixth most common cancer, and the second leading cause of cancer-related death, worldwide. This reflects the challenges facing HCC treatment. Methods: Patients (pts) with HCC receiving TACE treatment (n = 96) were examined retrospectively for clinical outcome and its possible predictors. The number of TACE treatments and the time elapsed between each treatment were assessed and correlated with overall survival (OS) using the log rank test of Kaplan Meier curves. T-stage, level of differentiation, vascular invasion, and Child Pugh score at the time of HCC diagnosis were compared among pts who received different numbers of TACE treatments (Kaplan-Meier survival analysis, ANOVA and student T test). Results: TACE treated pts had a median OS of 46 month (mo) and progression free survival of 12 mo (difference in time between the date of first progression and the date of diagnosis). Pts received 1-2 (n = 52), 3-4 (n = 28), or 5-6 (n = 16) TACE treatments. We found that pts who had only 1-2 TACE treatments had significantly shorter median OS (38 mo) than those who received 3-4 or 5-6 treatments (48 and 83 mo; p < 0.05). Of the 96 pts studied 22, 33, 37, and 3 pts had T1, T2- T3 and T4-stage HCC, respectively. Only 39 pt tumors underwent pathological analysis, and 13 were well-differentiated (WD), while 24 were moderately- or poorly- differentiated (MPD) (p > 0.05). Tumor T-stage and differentiation were correlated with the number of TACE treatments received. Thus, 30% of pts (n = 10) with T2-stage disease compared with 10% (n = 4) with T3-stage disease received 5-6 TACE treatments (p < 0.001). Similarly, 30% of WD cases (n = 4) compared with only 8% of MPD cases (n = 2) received 5-6 TACE treatments (p < 0.001). The duration between the second and third TACE treatments ( < 4 mo, 5-8 mo or > 8 mo) seemed to correlate with outcome (p < 0.05). Conclusions: Pt survival time following TACE treatment is diverse and correlates with the number of TACE treatments. Pts with T3-stage, or MPD HCC tended to receive fewer TACE treatments than those with T2-stage or WD HCC, and have worse outcomes. Other therapies should certainly be considered for pts with T3-stage and/or MPD tumors.

280 Background: The ability to define risk of hepatocellular carcinoma (HCC) recurrence after resection could improve the clinical management of patients. The pathological factors currently indicative of tumor invasiveness, such as vascular invasion, elevated AFP and advanced pTNM stage, are the established risk factors for recurrence. It has been suggested that immune cells that infiltrate a tumor are a prognostic factor in predicting patient outcome. In this study, the prognostic significance of tumor immune infiltration, as defined by the Immunoscore methodology, was assessed in patients with HCC. Methods: The influence of immune infiltration on clinical outcome was evaluated in patients who had undergone resection of HCC. The density of intratumoral immune infiltrates were measured in the center of the tumor (CT) and in the invasive margin (IM) of 45 stage I to IV HCC tissue specimens from a single cohort. The density of total (CD3+) and cytotoxic (CD8+) T lymphocytes in the CT and IM were obtained by immunohistochemistry and quantified using a Nuance FX Multiplex Biomarker Imaging system in tandem with ImageJ image processing software. Immune cell density in the CT and IM was converted to a binary score (0 as Low, 1 as High), with a cutoff threshold determined by the median density of CD3+ and CD8+ cells (273 cells/mm² and 217.5 cells/mm², respectively). The Immunoscore values were correlated with tumor recurrence and recurrence-free survival. Results: High densities of both CD3+ and CD8+ T lymphocytes in the CT and IM, along with a corresponding Immunoscore of 3+ (on a scale from 0 to 4), were significantly correlated with a low rate of recurrence (p-value = 0.0004). High densities of CD3+ lymphocytes alone were correlated with a prolonged recurrence-free survival (p-value = 0.0005). High densities of CD8+ lymphocytes alone were also correlated with a prolonged recurrence-free survival (p-value = 0.0031). Conclusions: The Immunoscore is a useful prognostic marker in patients with HCC who have received primary tumor resection. To better characterize the immune landscape of HCC tumors, the correlation between the Immunoscore and additional immune biomarkers are being evaluated.