Lytic efficiency of immunosuppressive drug-resistant armoured T cells against circulating HBV-related HCC in whole blood.

Recurrence of hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) after liver transplant (LT) is mediated by circulating tumour cells (CTCs) and exacerbated by the immunosuppressants required to prevent graft rejection. To circumvent the effects of immunosuppressants, we developed immunosuppressive drug-resistant armoured HBV-specific T-cell receptor-redirected T cells (IDRA HBV-TCR). However, their ability to eliminate HBV-HCC circulating in the whole blood has never been tested, and whether their lytic efficacy is compatible with the number of adoptively transferred T cells in vivo has never been measured. Hence, we developed a microscopy-based assay to quantify CTCs in whole blood. The assay was then used to quantify the efficacy of IDRA HBV-TCRs to lyse free-floating HBV-HCC cells in the presence of Tacrolimus and Mycophenolate Mofetil (MMF). We demonstrated that a panel of antibodies (AFP, GPC3, Vimentin, pan-Cytokeratin, and CD45) specific for HCC tumour antigens and immune cells can effectively differentiate HCC-CTCs in whole blood. Through dose-titration experiments, we observed that in the presence of immunosuppressive drugs, a minimum of 20 000 IDRA HBV-TCR T cells/ml of whole blood is necessary to lyse ~63.5% of free-floating HBV-HCC cells within 16 hours. In conclusion, IDRA HBV-TCR T cells can lyse free-floating HBV-HCC cells in whole blood in the presence of Tacrolimus and MMF. The quantity of IDRA-HBV TCR T cells required can be achieved by the adoptive transfer of 5 × 106 IDRA-HBV TCR-T cells/kg, supporting the utilisation of IDRA HBV-TCR T cells to eliminate CTCs as prophylaxis against recurrence after LT.

Immunohistochemical scoring of LAG-3 in conjunction with CD8 in the tumor microenvironment predicts response to immunotherapy in hepatocellular carcinoma.

Introduction: Immune checkpoint blockade (ICB) is a systemic therapeutic option for advanced hepatocellular carcinoma (HCC). However, low patient response rates necessitate the development of robust predictive biomarkers that identify individuals who will benefit from ICB. A 4-gene inflammatory signature, comprising CD8, PD-L1, LAG-3, and STAT1, was recently shown to be associated with a better overall response to ICB in various cancer types. Here, we examined whether tissue protein expression of CD8, PD-L1, LAG-3, and STAT1 predicts response to ICB in HCC. Methods: HCC samples from 191 Asian patients, comprising resection specimens from 124 patients (ICB-naïve) and pre-treatment specimens from 67 advanced HCC patients treated with ICB (ICB-treated), were analyzed for CD8, PD-L1, LAG-3, and STAT1 tissue expression using multiplex immunohistochemistry followed by statistical and survival analyses. Results: Immunohistochemical and survival analyses of ICB-naïve samples showed that high LAG-3 expression was associated with shorter median progression-free survival (mPFS) and overall survival (mOS). Analysis of ICB-treated samples revealed that high proportions of LAG-3+ and LAG-3+CD8+ cells pre-treatment were most closely associated with longer mPFS and mOS. Using a log-likelihood model, adding the total LAG-3+ cell proportion to the total CD8+ cell proportion significantly increased the predictive values for mPFS and mOS, compared with the total CD8+ cell proportion alone. Moreover, levels of CD8 and STAT1, but not PD-L1, were significantly correlated with better responses to ICB. After analyzing viral-related and non-viral HCC samples separately, only the LAG3+CD8+ cell proportion was significantly associated with responses to ICB regardless of viral status. Conclusion: Immunohistochemical scoring of pre-treatment levels of LAG-3 and CD8 in the tumor microenvironment may help predict ICB benefits in HCC patients. Furthermore, immunohistochemistry-based techniques offer the advantage of being readily translatable in the clinical setting.

First-Line Systemic Therapies for Advanced Hepatocellular Carcinoma: A Systematic Review and Patient-Level Network Meta-Analysis.

Introduction: Sorafenib was historically the standard of care for advanced hepatocellular carcinoma (aHCC) until it was superseded by the combination of atezolizumab and bevacizumab. Thereafter, several novel first-line combination therapies have demonstrated favorable outcomes. The efficacies of these treatments in relation to current and previous standards of care are unknown, necessitating an overarching evaluation. Methods: A systematic literature search was conducted on PubMed, EMBASE, Scopus, and the Cochrane Controlled Register of Trials for phase III randomized controlled trials investigating first-line systemic therapies for aHCC. Kaplan-Meier curves for overall survival (OS) and progression-free survival (PFS) were graphically reconstructed to retrieve individual patient-level data. Derived hazard ratios (HRs) for each study were pooled in a random-effects network meta-analysis (NMA). NMAs were also conducted using study-level HRs for various subgroups, according to viral etiology, Barcelona Clinic Liver Cancer (BCLC) staging, alpha-fetoprotein (AFP) levels, macrovascular invasion, and extrahepatic spread. Treatment strategies were ranked using p scores. Results: Among 4,321 articles identified, 12 trials and 9,589 patients were included for analysis. Only two therapies showed OS benefit over sorafenib: combined anti-programmed-death and anti-VEGF pathway inhibitor monoclonal antibodies (Anti-PD-(L)1/VEGF Ab), including atezolizumab-bevacizumab and sintilimab-bevacizumab biosimilar (HR = 0.63, 95% CI = 0.53-0.76) and tremelimumab-durvalumab (HR = 0.78, 95% CI = 0.66-0.92). Anti-PD-(L)1/VEGF Ab showed OS benefit over all other therapies except tremelimumab-durvalumab. Low heterogeneity (I2 = 0%) and inconsistency (Cochran's Q = 0.52, p = 0.773) was observed. p scores for OS ranked Anti-PD-(L)1/VEGF Ab as the best treatment in all subgroups, except hepatitis B where atezolizumab-cabozantinib ranked highest for both OS and PFS, as well as nonviral HCC and AFP ≥400 µg/L where tremelimumab-durvalumab ranked highest for OS. Conclusion: This NMA supports Anti-PD-(L)1/VEGF Ab as the first-line therapy for aHCC and demonstrates a comparable benefit for tremelimumab-durvalumab which also extends to certain subgroups. Results of the subgroup analysis may guide treatment according to baseline characteristics, while pending further studies.

Immune checkpoint inhibitors and tyrosine kinase inhibitors in patients with advanced hepatocellular carcinoma: Does the sequence matter?

INTRODUCTION: Combination therapy with immune checkpoint inhibitor (ICI) and antivascular endothelial growth factor (anti-VEGF) is currently the first line treatment for advanced hepatocellular carcinoma (aHCC). However, there are many patients who may not be able to receive combination therapy due to underlying comorbidities or resource limitations. For these patients, systemic treatment options include single agent tyrosine kinase inhibitors (TKIs) or ICI monotherapy. However, whether an optimal sequence of systemic therapy exists remains unknown. We aim to explore the impact of sequencing of TKI and ICI therapy in terms of response rates and to examine the safety of their use in sequential order. METHODS: Patients with aHCC treated with both ICI and TKI between December 30, 2013 and June 13, 2018 were retrospectively identified. Patients were classified into two groups: those who received TKI in the first-line (TKI1), followed by ICI (ICI2) and those who received ICI (ICI1) in the first-line followed by TKI (TKI2). The primary objective of the study was to identify differences in objective response rate (ORR) and disease control rate (DCR), as evaluated based on response evaluation criteria in solid tumor v1.1 for TKI1, TKI2, ICI1, and ICI2. Secondary objectives included comparison of progression free survival (PFS) for each line of therapy, overall survival (OS) and adverse events (AEs). RESULTS: Twenty-seven and 23 patients were classified into group 1 and 2, respectively. Objective response rates of TKI1 and TKI2 were 3.8% and 17.6%, respectively (p = .28); DCR to TKI1 versus TKI2 was 23.1% versus 35.3% (p = .49). ORRs of ICI1 and ICI2 were 8.7% and 14.3%, respectively (p = .66); DCR to ICI2 versus ICI1 was 56.5% versus 42.9% (p = .37). Median PFS was not significant between TKI1 and TKI2 (PFS 3.06 versus 1.61 months, p = .097) as well as between ICI2 and ICI1 (PFS 1.84 versus 2.37 month, p = .32). Median OS was also not significantly different between both groups (OS 20.63 versus 13.93 months, p = .20) on univariable and multivariable analysis (OS adjusted hazard ratio [HR] 2.07, 95% CI .83-5.18, p = .118). The proportion of patients who experienced adverse events of any grade was similar in both groups (TKI1 59.3% versus TKI2 52.2%; ICI1 78.3% versus ICI2 70.4%). CONCLUSION: Our study suggests that the sequence of TKI versus ICI therapy in patients with aHCC may not matter, given similar efficacy and toxicity profile when either agent is received in the first or second-line setting. This finding is of value in the real-world setting, where patients may be frail or have comorbidities that render them unable to tolerate combination therapy (ICI and TKI/anti-VEGF). For these patients, sequential exposure to both classes of drugs (ICI and TKI) may be a suitable option.

Impact of Immune-Related Adverse Events on Efficacy of Immune Checkpoint Inhibitors in Patients with Advanced Hepatocellular Carcinoma.

INTRODUCTION: Development of immune-related adverse events (irAEs) has been associated with enhanced efficacy with the use of immune checkpoint inhibitors (ICIs). It remains unknown whether such an association exists in advanced hepatocellular carcinoma (aHCC). This study aims to evaluate the association between irAEs and ICI efficacy in patients with aHCC. METHODS: We performed a retrospective cohort study on patients with aHCC who received at least one dose of an ICI between May 2015 and November 2019 at the National Cancer Centre Singapore. The primary study objectives were to compare the overall survival (OS) and progression-free survival (PFS) between patients with and without irAEs. Complementary multivariable landmark analyses were performed at the 6-week and 12-week landmarks. Data cutoff was December 31, 2020. RESULTS: One hundred and sixty-eight patients were included. Median age was 69 years, 85.7% were male, 57.7% had hepatitis B infection, 60.7% had ECOG 0, and 78.0% had Child-Pugh A liver cirrhosis. 82.7% received ICI monotherapy, while 17.3% received ICI in combination. Development and severity of irAE were correlated with survival. The median PFS for grade ≥3 irAE versus grades 1-2 irAE versus no irAE was 8.5 versus 3.6 versus 1.3 mths (p < 0.001). The median OS for grade ≥3 irAE versus grades 1-2 irAE versus no irAE was 26.9 versus 14.0 versus 4.6 mths (p < 0.001). Patients with ≥2 irAEs had a significantly longer OS on multivariable analysis (adjusted hazard ratio [aHR]0.35, p < 0.001). The presence of grade ≥3 irAEs was associated with a significantly longer OS on the multivariable analysis at the 6-week landmark (aHR0.34, p = 0.030) and 12-week landmark (aHR0.28, p = 0.011). The use of systemic corticosteroids in patients with irAE was associated with a trend toward a longer OS (20.7 vs. 14.3 mths, p = 0.064). CONCLUSION: Our study suggests that the presence of all-grade irAEs may be a potential prognostic biomarker in patients with aHCC treated with ICI. Patients with more severe irAEs and multisystem involvement have better prognosis. The prompt use of systemic corticosteroids to treat patients with irAEs is key to ensure the best long-term outcomes for these patients.

Resected pancreatic adenocarcinoma: An Asian institution's experience.

BACKGROUND: Pancreatic adenocarcinoma (PDAC) is highly lethal. Surgery offers the only chance of cure, but 5-year overall survival (OS) after surgical resection and adjuvant therapy remains dismal. Adjuvant trials were mostly conducted in the West enrolling fit patients. Applicability to a general population, especially Asia has not been described adequately. AIM: We aimed to evaluate the clinical outcomes, prognostic factors of survival, pattern, and timing of recurrence after curative resection in an Asian institution. METHODS AND RESULTS: The clinicopathologic and survival outcomes of 165 PDAC patients who underwent curative resection between 1998 and 2013 were reviewed retrospectively. Median age at surgery was 62.0 years. 55.2% were male, and 73.3% had tumors involving the head of pancreas. The median OS of the entire cohort was 19.7 months. Median OS of patients who received adjuvant chemotherapy was 23.8 months. Negative predictors of survival include lymph node ratio (LNR) of >0.3 (HR = 3.36, P = .001), tumor site involving the body or tail of pancreas (HR = 1.59, P = .046), presence of perineural invasion (PNI) (HR = 2.36, P = .018) and poorly differentiated/undifferentiated tumor grade (HR = 1.86, P = .058). The median time to recurrence was 8.87 months, with 66.1% and 81.2% of patients developing recurrence at 12 months and 24 months respectively. The most common site of recurrence was the liver. CONCLUSION: The survival of Asian patients with resected PDAC who received adjuvant chemotherapy is comparable to reported randomized trials. Clinical characteristics seem similar to Western patients. Hence, geographical locations may not be a necessary stratification factor in RCTs. Conversely, lymph node ratio and status of PNI ought to be incorporated.

Real-world efficacy and safety of immune checkpoint inhibitors in advanced hepatocellular carcinoma: Experience of a tertiary Asian Center.

INTRODUCTION: Immune checkpoint inhibitor (ICI) use in advanced hepatocellular carcinoma (HCC) is increasing. Real-world data on efficacy and safety, however, are lacking. METHODS: We conducted a retrospective review of all patients with advanced HCC seen at our center who received at least one dose of an ICI between May 2015 and June 2018. Data cutoff was December 31, 2018. Responses were evaluated using Response Evaluation Criteria in Solid Tumors version 1.1 criteria. RESULTS: Of 114 patients, 88.6% were male. Median age was 66 years, 96.5% had an Eastern Cooperative Oncology Group of 0-1. 62.3% received monotherapy ICI. 18.4% of patients had Child-Pugh (CP) B disease on initiation of ICI, and 69.3% had an ALBI grade of 2. 54.4% were known to have chronic hepatitis B (HBV) or were previously infected, and 11.4% had hepatitis C. Baseline HBV viral load (VL) ranged from undetectable to 8 210 000 IU/mL. 35.1% received prior systemic treatment. 28.9% received prior sorafenib. Over a median follow-up duration of 13.8 months (10.4-15.8), ORR was 18.4%, and DCR was 50.9%. Median progression-free survival was 2.7 months (1.3-4.0), and median overall survival (OS) was 13.9 months (6.9-16.2). Thirty-one patients (27.2%) received further systemic therapy after ICI discontinuation. On multivariable analyses, lower albumin level, higher bilirubin level, diuretic-refractory ascites, and HBV-associated HCC were associated with poorer OS. 69.3% of patients experienced adverse events (AE) of any grade, 14.9% of these being grade 3-4. No grade 5 AE were observed. Use of antiviral therapy was associated with a lower risk of grade 3 or above hepatic AEs (P = 0.048), whereas high baseline HBV VL was not associated with an increased risk of reactivation or hepatic AE. DISCUSSION: We have demonstrated that the real-world performance of ICIs in advanced HCC appears comparable to that observed in clinical trials for HCC patients with CP A cirrhosis. While prognosis of patients with advanced HCC and CP B cirrhosis remains poor even with ICI, usage of ICI is likely to be safe. Patients with HBV with a baseline HBV VL ≥100 IU/mL may receive ICI safely, especially if they are on antiviral treatment.

Immunohistochemical scoring of CD38 in the tumor microenvironment predicts responsiveness to anti-PD-1/PD-L1 immunotherapy in hepatocellular carcinoma.

INTRODUCTION: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-associated mortality globally. Immune-checkpoint blockade (ICB) is one of the systemic therapy options for HCC. However, response rates remain low, necessitating robust predictive biomarkers. In the present study, we examined the expression of CD38, a molecule involved in the immunosuppressive adenosinergic pathway, on immune cells present in the tumor microenvironment. We then investigated the association between CD38 and ICB treatment outcomes in advanced HCC. METHODS: Clinically annotated samples from 49 patients with advanced HCC treated with ICB were analyzed for CD38 expression using immunohistochemistry (IHC), multiplex immunohistochemistry/immunofluorescence (mIHC/IF) and multiplex cytokine analysis. RESULTS: IHC and mIHC/IF analyses revealed that higher intratumoral CD38+ cell proportion was strongly associated with improved response to ICB. The overall response rates to ICB was significantly higher among patients with high proportion of total CD38+cells compared with patients with low proportion (43.5% vs 3.9%, p=0.019). Higher responses seen among patients with a high intratumoral CD38+cell proportion translated to a longer median progression-free survival (mPFS, 8.21 months vs 1.64 months, p=0.0065) and median overall survival (mOS, 19.06 months vs 9.59 months, p=0.0295). Patients with high CD38+CD68+macrophage density had a better mOS of 34.43 months compared with 9.66 months in patients with low CD38+CD68+ macrophage density. CD38hi macrophages produce more interferon γ (IFN-γ) and related cytokines, which may explain its predictive value when treated with ICB. CONCLUSIONS: A high proportion of CD38+ cells, determined by IHC, predicts response to ICB and is associated with superior mPFS and OS in advanced HCC.

Human epidermal growth factor receptor 2 positive rates in invasive lobular breast carcinoma: The Singapore experience.

BACKGROUND: Invasive lobular carcinomas (ILC) form 5%-10% of breast cancer and rarely show overexpression of human epidermal growth factor receptor 2 (HER2). AIM: To describe the prevalence and prognostic factors of HER2 positive (HER2+) ILC in an Asian population. METHODS: A retrospective review of patients with ILC seen between January 1985 and March 2018 at various SingHealth medical institutions was conducted. Demographic and clinical data were collected from medical records. We examined clinicopathological characteristics and survival in relation to HER2 status. RESULTS: A total of 864 patients were included. Prevalence of HER2 positivity was 10.1% (87 patients). Compared with HER2 negative (HER2-) ILC, HER2+ ILC was associated with a higher proportion of estrogen receptor negative (24.4% vs 5.9%, P < 0.001), progesterone receptor negative (PR-) (40.2% vs 24%, P = 0.002) and grade 3 tumours (Grade 3, 29.0% vs 10.2%, P < 0.001). Overall survival rate was poorer in patients with HER2+ compared to HER2- ILC (56.7% vs 72.9% alive at 10 years; hazard ratio 1.87, 95% confidence interval: 1.21-2.90, P = 0.004). Based on multivariate analysis, negative prognostic factors for overall survival included HER2 positivity, PR negativity, older age, Indian ethnicity and higher tumour stage. CONCLUSION: Prevalence of HER2+ ILC was 10.1%. HER2+ ILC was more likely to have poorer prognostic features such as estrogen receptor negative, PR- and higher tumour grade, and have a poorer survival.