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Summary Background Therapeutic synergism between radiotherapy and immune checkpoint blockade has been observed in preclinical models of hepatocellular carcinoma. We aimed to study the safety and efficacy of sequential radioembolisation with yttrium-90-resin microspheres (Y90-radioembolisation) followed by nivolumab in patients with advanced hepatocellular carcinoma. Methods Patients with Child-Pugh A cirrhosis and advanced hepatocellular carcinoma not suitable for curative surgery were treated with Y90-radioembolisation followed by intravenous nivolumab 240 mg 21 days after Y90-radioembolisation and every 2 weeks thereafter. The primary endpoint, assessed in the per-protocol population, was the objective response rate, determined by RECIST version 1.1, defined as the proportion of patients with a confirmed complete or partial response observed for lesions both within and outside the Y90-radioembolisation field. This study is registered with ClinicalTrials.gov , NCT03033446 and has been completed. Findings 40 patients were enrolled, of whom 36 received Y90-radioembolisation followed by nivolumab. One (3%) patient had a complete response and ten (28%) had a partial response; the objective response rate was 30·6% (95% CI 16·4–48·1). The most common treatment-related adverse events of any grade were pruritus (18 [50%] of 36 patients) and maculopapular rash (13 [36%]). Two (6%) patients experienced grade 3–4 treatment-related adverse events: one patient had a grade 3 increase in alanine aminotransferase levels, grade 3 bilirubin increase, and grade 4 increase in aspartate aminotransferase levels, while the other had a grade 3 maculopapular rash. Five (14%) patients had a treatment-related serious adverse event (Steven-Johnson syndrome, hepatitis E infection, fever, liver abscesses, and ascites). Interpretation Y90-radioembolisation followed by nivolumab resulted in an encouraging objective response rate in patients with advanced hepatocellular carcinoma, although the activity observed was not as high as the study was powered for. This strategy should be further evaluated in patients with Barcelona Clinic Liver Clinic (BCLC) stage B hepatocellular carcinoma that is ineligible or refractory to transarterial chemoembolisation and patients with BCLC C disease without extrahepatic spread. Funding National Medical Research Council Singapore, Bristol-Myers Squibb, Sirtex.

Introduction: Real-world management of patients with hepatocellular carcinoma (HCC) is crucially challenging in the current rapidly evolving clinical environment which includes the need for respecting patient preferences and autonomy. In this context, regional/national treatment guidelines nuanced to local demographics have increasing importance in guiding disease management. We report here real-world data on clinical outcomes in HCC from a validation of the Consensus Guidelines for HCC at the National Cancer Centre Singapore (NCCS). Method: We evaluated the NCCS guidelines using prospectively collected real-world data, comparing the efficacy of treatment received using overall survival (OS) and progression-free survival (PFS). Treatment outcomes were also independently evaluated against 2 external sets of guidelines, the Barcelona Clinic Liver Cancer (BCLC) and Hong Kong Liver Cancer (HKLC). Results: Overall treatment compliance to the NCCS guidelines was 79.2%. Superior median OS was observed in patients receiving treatment compliant with NCCS guidelines for early (nonestimable vs. 23.5 months p < 0.0001), locally advanced (28.1 vs. 22.2 months p = 0.0216) and locally advanced with macrovascular invasion (10.3 vs. 3.3 months p = 0.0013) but not for metastatic HCC (8.1 vs. 6.8 months p = 0.6300), but PFS was similar. Better clinical outcomes were seen in BCLC C patients who received treatment compliant with NCCS guidelines than in patients with treatment only allowed by BCLC guidelines (median OS 14.2 vs. 7.4 months p = 0.0002; median PFS 6.1 vs. 4.0 months p = 0.0286). Clinical outcomes were, however, similar for patients across all HKLC stages receiving NCCS-recommended treatment regardless of whether their treatment was allowed by HKLC. Conclusion: The high overall compliance rate and satisfactory clinical outcomes of patients managed according to the NCCS guidelines confirm its validity. This validation using real-world data considers patient and treating clinician preferences, thus providing a realistic analysis of the usefulness of the NCCS guidelines when applied in the clinics.

Cancer immunotherapy has revolutionised cancer treatment, with immune checkpoint blockade (ICB) therapy and adoptive cell therapy (ACT) increasingly becoming standard of care across a growing number of cancer indications. While the majority of cancer immunotherapies focus on harnessing the anti-tumour CD8+ cytotoxic T cell response, the potential role of CD4+ ‘helper’ T cells has largely remained in the background. In this review, we give an overview of the multifaceted role of CD4+ T cells in the anti-tumour immune response, with an emphasis on recent evidence that CD4+ T cells play a bigger role than previously thought. We illustrate their direct anti-tumour potency and their role in directing a sustained immune response against tumours. We further highlight the emerging observation that CD4+ T cell responses against tumours tend to be against self-derived epitopes. These recent trends raise vital questions and considerations that will profoundly affect the rational design of immunotherapies to leverage on the full potential of the immune system against cancer.

Background & Aim Under the regulation of various oncogenic pathways, cancer cells undergo adaptive metabolic programming to maintain specific metabolic states that support their uncontrolled proliferation. As it has been difficult to directly and effectively inhibit oncogenic signaling cascades with pharmaceutical compounds, focusing on the downstream metabolic pathways that enable indefinite growth may provide therapeutic opportunities. Thus, we sought to characterize metabolic changes in hepatocellular carcinoma (HCC) development and identify metabolic targets required for tumorigenesis. Methods We compared gene expression profiles of Morris Hepatoma (MH3924a) and DEN (diethylnitrosamine)-induced HCC models to those of liver tissues from normal and rapidly regenerating liver models, and performed gain- and loss-of-function studies of the identified gene targets for their roles in cancer cell proliferation in vitro and in vivo. Results The proline biosynthetic enzyme PYCR1 (pyrroline-5-carboxylate reductase 1) was identified as one of the most upregulated genes in the HCC models. Knockdown of PYCR1 potently reduced cell proliferation of multiple HCC cell lines in vitro and tumor growth in vivo. Conversely, overexpression of PYCR1 enhanced the proliferation of the HCC cell lines. Importantly, PYCR1 expression was not elevated in the regenerating liver, and KD or overexpression of PYCR1 had no effect on proliferation of non-cancerous cells. Besides PYCR1, we found that additional proline biosynthetic enzymes, such as ALDH18A1, were upregulated in HCC models and also regulated HCC cell proliferation. Clinical data demonstrated that PYCR1 expression was increased in HCC, correlated with tumor grade, and was an independent predictor of clinical outcome. Conclusion Enhanced expression of proline biosynthetic enzymes promotes HCC cell proliferation. Inhibition of PYCR1 or ALDH18A1 may be a novel therapeutic strategy to target HCC. Lay summary Even with the recently approved immunotherapies against liver cancer, currently available medications show limited clinical benefits or efficacy in the majority of patients. As such, it remains a top priority to discover new targets for effective liver cancer treatment. Here, we identify a critical role for the proline biosynthetic pathway in liver cancer development, and demonstrate that targeting key proteins in the pathway, namely PYCR1 and ALDH18A1, may be a novel therapeutic strategy for liver cancer.

Background Previous prospective phase II study conducted by our research group at the National Cancer Centre Singapore had shown the efficacy of combined induction chemotherapy followed by cytotoxic T-lymphocyte (CTL) immunotherapy as a first-line treatment for advance nasopharyngeal carcinoma (NPC) – i.e. median survival for patients treated with combined therapy was 29.9 months, compared to 17.7 months for patients who received only standard chemotherapy.1 Using the same data set, we further investigate the correlation between various patient factors (Eastern Cooperative Oncology Group (ECOG) score, gender, age, initial stage of cancer, neutrophil-to-lymphocyte ratio (NLR), initial EBV-DNA titre) on overall survival (OS). This is to further validate our hypothesis that the improved OS is due to an effect of treatment and not due to intrinsic patient factors. Materials and Methods Survival distribution curves were estimated using the Kaplan-Meier method and differences were compared statistically using log-rank test. IBM SPSS statistics software package (v. 22) was used for the purpose of statistical analysis. Overall survival was defined as time from diagnosis to date of event (date of death/date of last follow-up). For analysis of overall survival, data for patients who were alive or who were lost to follow-up were censored at the end of study period. Results It was revealed that lower ECOG score, a scale used to assess the physical condition of patients, correlated with longer OS while other characteristics such as gender, age, initial stage of cancer, NLR, and initial EBV-DNA titre did not correlate with survival outcomes. ECOG0 patients had a median survival of 146.7 weeks, compared to ECOG1 patients, which had a median survival of 86.6 weeks (hazard ratio: 0.35; 95% CI: 0.14-0.84; P = 0.033). Conclusions Even though ECOG performance status is found to be statistically associated with survival outcome of patients with advance stage NPC. This result is unsurprising as the prognostic value of ECOG has been well documented in literature, albeit in other cancer types. Other patient parameters such as gender, age, initial stage of cancer, NLR, and initial EBV titre, did not yield significance and did not prognosticate for survival outcome. This finding supports our hypothesis that the improved survival outcomes observed in advance NPC patients treated with chemotherapy followed by EBV CTL-immunotherapy is due to effects of treatment and not because of intrinsic patient factors. Reference Chia WK, Teo M, Wang WW, Lee B, Ang SF, Tai WM, Chee CL, Ng J, Kan R, Lim WT, Tan SH, Ong WS, Cheung YB, Tan EH, Connolly JE, Gottschalk S, Toh HC. Adoptive T-cell transfer and chemotherapy in the first-line treatment of metastatic and/or locally recurrent nasopharyngeal carcinoma. Mol Ther 2014 Jan;22(1):132–9. Disclosure Information A. Chu: None. S. Han: None. H. Toh: None.

The past decade has witnessed the gradual and steady progress of adoptive T cell therapy in treating various types of cancer. In combination with gemcitabine and carboplatin chemotherapy, we previously conducted a clinical trial, NCT00690872, to treat Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) patients with autologous EBV-expanded cytotoxic T lymphocytes (CTLs). While achieving a 2-year overall survival rate of 62.9%, this trial failed to induce an anti-tumor response in a sizable fraction of patients. Thus, the identification of benchmarks capable of evaluating CTL products and predicting clinical immunotherapeutic efficacy remains an urgent need. We conducted T cell receptor (TCR) repertoire sequencing to assess EBV-expanded infusion-ready CTL products. To depict the overall repertoire landscape, we evaluated the individual repertoire diversity by Shannon entropy, and, compared the inter-patient CDR3 similarity to estimate T cells expanded by common antigens. With a recently developed bioinformatics algorithm, termed Motif Analysis, we made a machine-learning prediction of structural regions within the CDR3 of TCRβ that associate with CTL therapy prognosis. We found that long term survivors, defined as patients surviving longer than two years, had a higher CTL repertoire diversity with reduced inter-patient similarity. Furthermore, TCR Motif Analysis identified 11 structural motifs distinguishing long term survivors from short term survivors. Specifically, two motifs with a high area under the curve (AUC) values were identified as potential predictive benchmarks for efficacious CTL production. Together, these results reveal that the presence of diverse TCR sequences containing a common core motif set is associated with a favorable response to CTL immunotherapy against EBV-positive NPC.

Intra-tumor heterogeneity (ITH) is a key challenge in cancer treatment, but previous studies have focused mainly on the genomic alterations without exploring phenotypic (transcriptomic and immune) heterogeneity. Using one of the largest prospective surgical cohorts for hepatocellular carcinoma (HCC) with multi-region sampling, we sequenced whole genomes and paired transcriptomes from 67 HCC patients (331 samples). We found that while genomic ITH was rather constant across stages, phenotypic ITH had a very different trajectory and quickly diversified in stage II patients. Most strikingly, 30% of patients were found to contain more than one transcriptomic subtype within a single tumor. Such phenotypic ITH was found to be much more informative in predicting patient survival than genomic ITH and explains the poor efficacy of single-target systemic therapies in HCC. Taken together, we not only revealed an unprecedentedly dynamic landscape of phenotypic heterogeneity in HCC, but also highlighted the importance of studying phenotypic evolution across cancer types. National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This work is supported in part by the Singapore National Medical Research Council grants (TCR/015-NCC/2016, CIRG18may-0057l, NMRC/CSA-SI/0018/2017, and NMRC/ OFIRG/0064/2017) and the National Research Foundation, Singapore (NRF-NRFF2015-04). W.Z. is supported in part by the National Key R&D Program of China (2018YFC1406902 and 2018YFC0910400), the National Natural Science Foundation of China (31970566), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDPB17). H.Y. is supported by the National Natural Science Foundation of China (32000407).

BackgroundImmune check-point blockade (ICB) is one of the emerging therapeutic options for advanced hepatocellular carcinoma (HCC). However, low response rates amongst patients necessitates the development of robust predictive biomarkers that identify patients who likely benefit from ICB. Previously our group found that immunohistochemical scoring of CD38 in the tumour microenvironment predicts responsiveness to anti-PD-1/anti-PD-L1 immunotherapy in HCC.1 Recently BMS 4-gene inflammatory signature, comprising the 4 genes CD8, PD-L1, LAG-3 and STAT1, has been shown to be associated with better overall response to immunotherapy in various cancer types.2–4 In the present study, we examined the relationship between tissue expression of BMS 4-gene inflammatory signature and the responsiveness of HCC to immunotherapy, and whether BMS 4-gene inflammatory signature increases the predictive power of CD38.MethodsHCC tissue samples from 124 Asian patients that underwent conventional treatment and from 49 Asian patients that underwent ICB were analysed for CD8, PD-L1, LAG-3, STAT1, CD38 and CD68 tissue expression using immunohistochemistry and multiplex immunohistochemistry, followed by survival and statistical analysis.ResultsSurvival analysis of the 124 samples showed that high LAG-3 tissue expression was associated with shorter progression-free survival (PFS). On the other hand, immunohistochemical analyses on the 49 patient samples treated with ICB revealed that high LAG-3 density and high total LAG-3+CD8+ T cell proportion were associated with improved response to ICB (figure 1). However, CD8, PD-L1 and STAT1 levels did not significantly correlate with improved survival. The addition of total LAG-3+ cell proportion to total CD38+ cell proportion significantly increased the predictive value for both PFS (DeltaLRChi2=9.97; P=0.0016; table 1) and overall survival (OS) (DeltaLRChi2=8.84; P=0.0021; table 1), compared with total CD38+ cell proportion alone. Similarly findings were obtained after adding total LAG-3+CD8+ cell proportion to total CD38+ cell proportion (PFS: DeltaLRChi2=7.21; P=0.0072; OS: DeltaLRChi2=8.06; P=0.0045; table 1), compared with total CD38+ cell proportion alone. Lastly, when the total LAG-3+CD8+ cell proportion was added to total CD38+ and CD38+CD68+ cell proportion, the predictive value of the biomarker was significantly increased (PFS: DeltaLRChi2=6.10; P=0.0136; OS: DeltaLRChi2=6.18; P=0.0129; table 1). Ongoing works include further validation of the findings in various cohorts, and correlating with clinical outcome of the patients.Abstract 89 Table 1Log-likelihood of models with added predictive termsAbstract 89 Figure 1HCC patients‘ response to ICB in relation to LAG-3 density. (A) Kaplan-Meier curve showing the association between a high LAG-3 density and improved overall survival after treatment with ICB. (B) Kaplan-Meier curve showing the association between a high total LAG-3+CD8+ T cell proportion and improved overall survival after treatment with ICB. (C) Kaplan-Meier curve showing the association between a high LAG-3 density and improved progression-free survival after treatment with ICB. (D) Kaplan-Meier curve showing the association between a high total LAG-3+CD8+ T cell proportion and improved progression-free survival after treatment with ICB.ConclusionsHigh LAG-3 expression on tissue-infiltrating immune cells predicted greater response to ICB. LAG-3+ and LAG3+CD8+ cell proportion added predictive value to CD38+ cells for predicting survival outcome in immunotherapy-treated HCC. LAG-3 may be used in conjunction with CD38 to predict responsiveness to ICB in HCC.ReferencesNg HHM, Lee RY, Goh S, et al. Immunohistochemical scoring of CD38 in the tumor microenvironment predicts responsiveness to anti-PD-1/PD-L1 immunotherapy in hepatocellular carcinoma. J Immunother Cancer 2020;8.Hodi FS, Wolchok JD, Schadendorf D, et al. Abstract CT037: Genomic analyses and immunotherapy in advanced melanoma. AACR 2019.Lei M, Siemers NO, Pandya D, et al. Analyses of PD-L1 and Inflammatory Gene Expression Association with Efficacy of Nivolumab ± Ipilimumab in Gastric Cancer/Gastroesophageal Junction Cancer. Clinical Cancer Research 2021;27:3926–35.Sangro B, Melero I, Wadhawan S, et al. Association of inflammatory biomarkers with clinical outcomes in nivolumab-treated patients with advanced hepatocellular carcinoma. J Hepatol 2020.Ethics ApprovalThis study was approved by the Centralised Institutional Review Board of SingHealth (CIRB ref: 2009/907/B).ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Proposed short titleY90-Resin Microspheres Radioembolization Followed by Nivolumab in Advanced Hepatocellular CarcinomaBackground : Nivolumab (N) and Y90-resin microspheres radioembolization (Y90-RE) aretherapeutic options in advanced hepatocellular carcinoma (aHCC). Emerging evidence suggests synergy between radiotherapy and immune checkpoint inhibitors. Methods : Eligible Child-Pugh A aHCC patients were treated with Y90-RE followed by N 240mg, 21 days after Y90-RE and every 2 weeks thereafter. Pre- and on-treatment tumor biopsies were obtained. Primary end-point was overall response rate (ORR) (RECIST v1·1), defined as the composite overall response observed for lesions both within and outside Y90-RE field. Secondary end points included progression free survival (PFS), overall survival (OS), and safety. This study is registered with ClinicalTrials.gov, NCT03033446 .Findings : Forty patients were enrolled, 36 received Y90-RE followed by N. At baseline: 61·1% had hepatitis B; 66·7% BCLC stage C; 50·0% had AFP > 400ng/mL; median number of liver lesions was 5 (range 1- >20); median size of largest liver lesion was 78·5 mm (range 14-177mm); 38·9% had prior TACE/RFA/MWA; and 12·5% had prior systemic therapy. ORR was 30·6% (16·4% to 48·1%). ORR was 43·5% in patients with no extra-hepatic spread (EHS) (n=23). 81% of target lesions within Y90-RE field regressed. Median PFS and OS were 5·6 months (95% CI 2·1 to 8·8 months) and 16.9 months (95% CI 8·1 to 27·6 months). Treatment was well tolerated with only 13·8% experiencing grade (G) 3/4 treatment related adverse events (TRAEs). Immune activation predicted response to Y90-RE followed by N. Interpretation : Y90-RE followed by N resulted in an encouraging ORR of 30·6% in aHCC and of 43·5% in subjects with no EHS. 81% of target lesions within Y90-RE field regressed suggesting synergy. Importantly, this combination is safe and tolerable with low G 3/4 TRAEs of 13·8%. Trial Registration: NCT number: NCT03033446 Funding: Funding for this study was acquired from National Medical Research Council Singapore (NMRC/CIRG/1454/2016), Bristol-Myers Squibb (BMS), and SIRTEX Declaration of Interest: David Tai All support for the present manuscript (e.g., funding, provision of study materials, medical writing, article processing charges, etc.): BMS, SIRTEX, NMRC Singapore (CIRG/1470/2017) Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: IPSEN, Eisai, BMS Consulting fees: Novartis, BMS, MSD Choo Su Pin Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: Roche, BMS, Ipsen, Lilly, AZ, Roche. Consulting fees: BMS, Roche, Ipsen, Servier, Eisai, AstraZeneca. Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid: MOH Medishield Life Cancer Drug committee Stock or stock options: BMS David Ng All support for the present manuscript (e.g., funding, provision of study materials, medical writing, article processing charges, etc.): SIRTEX Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: SIRTEX Joycelyn Lee Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: BMS, IPSEN, Bayer Research Funding: Bayer Pierce K.H. Chow Grants or contracts from any entity: Sirtex Medical, Ipsen, IQVIA, New B Innovation, Perspectum, AMiLi, MiRXES, Genentech, Engine Biosciences. Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: Sirtex Medical, Ipsen, Oncosil, Bayer, Roche, New B Innovation, MSD, BTG PLC, Eisai, Abbott, AZ, IQVIA, Genentech, Worrell Guerbet, LEK Consulting, COR2ED. Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid: AVATAMED Stock or stock options: AVATAMED Too Chow Wei Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: SIRTEX Consulting fees: SIRTEX Ethical Approval: All patients provided written informed consent and the institutional review board committee approved the protocol (Singhealth IRB Ref No. 2016/2613). The study was done in accordance with the Declaration of Helsinki and Good Clinical Practice.

Simple Summary In this review, we discuss treatment strategies in biliary tract cancers (gallbladder cancer, intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma). In particular, we will describe advances in cellular therapies and cancer vaccines for biliary tract cancers, followed by our local experience with combining a melanoma-associated antigen (MAGE)-positive cell lysate-based autologous dendritic cell vaccine and anti-angiogenic therapy (bevacizumab) in a case of stage IV gallbladder cancer. Abstract Biliary tract cancer (BTC) is a rare, but aggressive, disease that comprises of gallbladder carcinoma, intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma, with heterogeneous molecular profiles. Advanced disease has limited therapeutic options beyond first-line platinum-based chemotherapy. Immunotherapy has emerged as a viable option for many cancers with a similar unmet need. Therefore, we reviewed current understanding of the tumor immune microenvironment and recent advances in cellular immunotherapy and therapeutic cancer vaccines against BTC. We illustrated the efficacy of dendritic cell vaccination in one patient with advanced, chemorefractory, melanoma-associated antigen (MAGE)-positive gallbladder carcinoma, who was given multiple injections of an allogenic MAGE antigen-positive melanoma cell lysate (MCL)-based autologous dendritic cell vaccine combined with sequential anti-angiogenic therapy. This resulted in good radiological and tumor marker response and an overall survival of 3 years from diagnosis. We postulate the potential synergism of adding anti-angiogenic therapy, such as bevacizumab, to immunotherapy in BTC, as a rational scientific principle to positively modulate the tumor microenvironment to augment antitumor immunity.

The clinical relevance of immune landscape intratumoural heterogeneity (immune-ITH) and its role in tumour evolution remain largely unexplored. Here, we uncover significant spatial and phenotypic immune-ITH from multiple tumour sectors and decipher its relationship with tumour evolution and disease progression in hepatocellular carcinomas (HCC). Immune-ITH is associated with tumour transcriptomic-ITH, mutational burden and distinct immune microenvironments. Tumours with low immune-ITH experience higher immunoselective pressure and escape via loss of heterozygosity in human leukocyte antigens and immunoediting. Instead, the tumours with high immune-ITH evolve to a more immunosuppressive/exhausted microenvironment. This gradient of immune pressure along with immune-ITH represents a hallmark of tumour evolution, which is closely linked to the transcriptome-immune networks contributing to disease progression and immune inactivation. Remarkably, high immune-ITH and its transcriptomic signature are predictive for worse clinical outcome in HCC patients. This in-depth investigation of ITH provides evidence on tumour-immune co-evolution along HCC progression., Intratumoural heterogeneity is a feature of liver cancer. Here, the authors demonstrate that heterogeneity exists at the immune cell level in liver cancer and show that tumours with high intratumoural immune heterogeneity demonstrated an immune suppressive microenvironment, which was associated with tumour evolution and a poor prognosis.

Advanced, late-stage Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) is incurable, and its treatment remains a clinical and therapeutic challenge. Results from a phase II clinical trial in advanced NPC patients employing a combined chemotherapy and EBV-specific T cell (EBVST) immunotherapy regimen showed a response rate of 71.4%. Longitudinal analysis of patient samples showed that an increase in EBV DNA plasma concentrations and the peripheral monocyte-to-lymphocyte ratio negatively correlated with overall survival. These parameters were combined into a multivariate analysis to stratify patients according to risk of death. Immunophenotyping at serial time points showed that low-risk individuals displayed significantly decreased amounts of monocytic myeloid-derived suppressor cells postchemotherapy, which subsequently influenced successful cytotoxic T-lymphocyte (CTL) immunotherapy. Examination of the low-risk group, 2 weeks post-EBVST infusion, showed that individuals with a greater overall survival possessed an increased frequency of CD8 central and effector memory T cells, together with higher levels of plasma interferon (IFN)-γ, and cytotoxic lymphocyte-associated transcripts. These results highlight the importance of the rational selection of chemotherapeutic agents and consideration of their impact on both systemic immune responses and downstream cellular immunotherapy outcomes., Graphical Abstract, Patients with advanced nasopharyngeal carcinoma were treated with a combined chemotherapy and cellular immunotherapy regimen. Those who displayed lower overall survival possessed higher amounts of regulatory leukocytes following chemotherapy. These results demonstrated the importance of the rational selection of chemotherapeutic agents and consideration of their impact on both systemic immune responses and downstream cellular immunotherapy outcomes.

Background The identification of biomarkers predicting a better outcome to adoptive T cell therapy is an emerging, still nascent field. We previously completed a phase 2 clinical trial of autologous adoptive EBV-specific cytotoxic T lymphocyte (CTL) immunotherapy following gemcitabine + carboplatin chemotherapy as first line treatment in 35 advanced incurable stage 4c nasopharyngeal carcinoma (NPC) patients. Here, we aim 1) to evaluate exosome proteins for potential specific predictive biomarkers of benefit to adoptive T cell therapy and 2) to investigate if a specific immunophenotype of our generated EBV targeting CTLs correlates with survival benefit. Methods We isolated exosome from plasma samples by size exclusion chromatography and magnetic-based isolation technology, followed by fluorescence-activated cell sorting (FACS) analysis, Western blot analysis for immune-related checkpoint molecules, or mass spectrometry for exosomal peptide detection. All the generated CTLs were analysed by gene expression microarray as well as a FACS system with the use of T cell-specific 23-antibody panel for comprehensive immunophenotyping. The representative CTL samples were also subject to single-cell (sc) RNA-Seq with DNA barcoded antibodies for comprehensive integrated transcriptomics and immunophenotyping analysis. Results Patients with overall survival longer than 2 years were grouped as long survivors and the remaining patients were grouped as short survivors. Differentially expressed immune-related checkpoint molecules, such as PD1, ICOS, and CD137, were detected in in pre-treatment exosome of long and short survivors. Furthermore, more than 13,000 high confident and unique peptides which belong to 1,500 unique proteins were identified and quantified by mass spectrometry from the purified plasma exosome. Pathway enrichment analysis further showed that plasma exosome of the short survivors had significantly higher innate immune response-activating signal transduction-related peptides detected (p-value = 4.81 x 10-5). The transcriptomic analysis revealed that statistically lower expressions of SELL (CD62L) and LEF1 were found in the EBV CTL of the short survivors, suggesting that the EBV CTL of short survivors were characterized by a lesser central memory T cell phenotype. Conclusions Our data reports that specific pre-treatment plasma exosome proteins, and separately, transcriptomic profile of the generated baseline EBV CTLs prior to infusion into the patients correlate with patient survival, suggesting that they could be used together as biomarkers to predict outcome in the advanced NPC patients treated with this chemo-immunotherapy combination. More in-depth analysis of the immunophenotyping of all the CTLs and the representative CTLs’ scRNA-Seq data will be presented at the meeting. Acknowledgements This work was supported by Singapore government funding (National Medical Research Council (NMRC) – Open Fund - Large Collaborative Grant (OF-LCG) and National Cancer Centre Singapore Block Grant as well as in part by Tessa Therapeutics Ltd.

We demonstrate that in human HCCs with deregulated expression of purine metabolic enzymes, targeting purine metabolism was effective in blocking tumor cell proliferation, leading to tumor shrinkage. Precise regulation of purine metabolic activity was coordinated by the activation status of a PI3K‐E2F1 axis, and potent suppression of purine metabolism via combinatorial targeting of PI3K and the purine synthetic rate‐limiting enzyme IMPDH resulted in enhanced efficacy in a pre‐clinical model. These data provide a strong basis for future precision‐medicine focused clinical trials targeting this tumor‐specific metabolic adaptation as a point of vulnerability in patients with HCC., Tumor‐specific metabolic rewiring, acquired to confer a proliferative and survival advantage over nontransformed cells, represents a renewed focus in cancer therapy development. Hepatocellular carcinoma (HCC), a malignancy that has hitherto been resistant to compounds targeting oncogenic signaling pathways, represents a candidate cancer to investigate the efficacy of selectively antagonizing such adaptive metabolic reprogramming. To this end, we sought to characterize metabolic changes in HCC necessary for tumorigenesis. We analyzed gene expression profiles in three independent large‐scale patient cohorts who had HCC. We identified a commonly deregulated purine metabolic signature in tumors with the extent of purine biosynthetic enzyme up‐regulation correlated with tumor grade and a predictor of clinical outcome. The functional significance of enhanced purine metabolism as a hallmark in human HCC was then validated using a combination of HCC cell lines, patient‐derived xenograft (PDX) organoids, and mouse models. Targeted ablation of purine biosynthesis by knockdown of the rate‐limiting enzyme inosine‐5′‐monophosphate dehydrogenase (IMPDH) or using the drug mycophenolate mofetil (MMF) reduced HCC proliferation in vitro and decreased the tumor burden in vivo. In comparing the sensitivities of PDX tumor organoids to MMF therapy, we found that HCC tumors defined by high levels of IMPDH and guanosine nucleosides were most susceptible to treatment. Mechanistically, a phosphoinositide 3‐kinase (PI3K)–E2F transcription factor 1 (E2F1) axis coordinated purine biosynthetic enzyme expression, deregulation of which altered the activity of mitogen‐activated protein kinase/RAS signaling. Simultaneously abolishing PI3K signaling and IMPDH activity with clinically approved inhibitors resulted in greatest efficacy in reducing tumor growth in a PDX mouse model. Conclusion: Enhanced purine metabolic activity regulated by PI3K pathway‐dependent activation of E2F1 promotes HCC carcinogenesis, suggesting the potential for targeting purine metabolic reprogramming as a precision therapeutic strategy for patients with HCC.

Aim: The new radioimmunotherapeutic regime GAP15R aims to stimulate glucocorticoid-induced TNF-related protein (G) to overcome Treg suppression; add IFN-α (A) to promote inflammatory milieu; block PD1 (P) to disinhibit T effector cytotoxicity; add IL-15 (15) to enhance danger signals & T-cell expansion; and apply radiation (R) at critical time point to sustain localized inflammation. Patients & methods/materials & methods: This was tested in a murine 4T1 metastatic breast carcinoma model given GAP15R with regular monitoring of tumor volume and complications. Results: We had demonstrated long-term complete remission up to 50% of treated mice, which is not associated with major treatment-related complication, in cases with specific tumor burden. Conclusion: GAP15R is efficacious with potential to be applicable to other tumors.

BACKGROUND Pracinostat is a potent histone deacetylase inhibitor with antitumor activity in both solid tumor and acute myeloid leukemia (AML) cell lines. Pracinostat is reported to have modest clinical activity in patients with advanced solid tumors. Given the higher preclinical sensitivity of hematologic malignancies to pracinostat, the authors conducted a phase 1 study to assess the safety, maximum tolerated dose, recommended phase 2 dose, efficacy, pharmacokinetics, and pharmacodynamics of pracinostat in patients with advanced hematological malignancies. METHODS Pracinostat was administered orally 3 times a week for 3 weeks on a 28-day cycle. Patients were assigned to 7 dose levels using a 3 + 3 dose escalation design. RESULTS A total of 44 patients were enrolled, 25 of whom had AML and 14 of whom had myelodysplastic syndrome. The maximum tolerated dose was 120 mg and the recommended phase 2 dose was 60 mg. Two patients with AML achieved a response: 1 complete remission (CR) and 1 complete cytogenetic response. Despite a dose-dependent increase in the plasma concentration of pracinostat, a similar increase in histone acetylation was not observed. As an extension, 10 additional patients with myelodysplastic syndrome were enrolled to assess the safety and efficacy of pracinostat in combination with azacitidine. Six patients achieved a CR and 3 achieved a CR without platelet recovery with no added toxicity. CONCLUSIONS The results of the current study demonstrate that pracinostat is safe, with modest single-agent activity in patients with hematological malignancies. Cancer 2017;123:4851-9. © 2017 American Cancer Society.

// Wei Xia Ang 1, 2, * , Zhendong Li 1, * , Zhixia Chi 1 , Shou-Hui Du 1 , Can Chen 3 , Johan C.K. Tay 1 , Han Chong Toh 4 , John E. Connolly 5 , Xue Hu Xu 6 , Shu Wang 1, 2 1 Department of Biological Sciences, National University of Singapore 117543, Singapore 2 Institute of Bioengineering and Nanotechnology 138669, Singapore 3 Tessa Therapeutics, Pte Ltd., 239351, Singapore 4 Division of Medical Oncology, National Cancer Centre, 169610, Singapore 5 Programme in Translational Immunology, Institute for Molecular and Cell Biology 138648, Singapore 6 Department of Gastrointestinal Surgery, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China * The authors contributed equally to these work Correspondence to: Shu Wang, email: dbsws@nus.edu.sg Keywords: peritoneal carcinomatosis, EpCAM, chimeric antigen receptor, T lymphocytes, adoptive immunotherapy Received: June 16, 2016 Accepted: January 03, 2017 Published: January 10, 2017 ABSTRACT The epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of tumor types, including peritoneal carcinomatosis (PC) from gastrointestinal and gynecological malignancies. To develop a chimeric antigen receptor T (CART) cell therapy approach to treat patients with end-stage PC, we constructed third generation CARs specific to EpCAM using the 4D5MOC-B single chain variable fragment. CART cells were generated with lentiviral transduction and exhibited specific in vitro killing activity against EpCAM-positive human ovarian and colorectal cancer cells. A single intraperitoneal injection of the CART cells eradicated established ovarian xenografts and resulted in significantly prolonged animal survival. Since EpCAM is also expressed on normal epithelium, anti-EpCAM CART cells were generated by mRNA electroporation that display a controlled cytolytic activity with a limited CAR expression duration. Multiple repeated infusions of these RNA CAR-modified T cells delayed disease progression in immunodeficient mice bearing well-established peritoneal ovarian and colorectal xenografts. Thus, our study demonstrates the effectiveness of using anti-EpCAM CAR-expressing T cells for local treatment of PC in mice. The possibility of using this approach for clinical treatment of EpCAM-positive gastrointestinal and gynecological malignancies warrants further validation.

Cell-based and antibody-based cancer immunotherapies have been widely tested across increasing numbers of cancers with an unprecedented number of successful practice-changing immunotherapy clinical trials, achieving significant survival outcomes and, characteristically, some very long-term survivors. Still, a sizeable proportion of patients, especially with solid tumours, do not benefit from immunotherapy. Here, we summarise key literature on immunotherapy biomarkers and resistance mechanisms and discuss potential strategies to overcome such resistance to improve patient outcomes. The ever-expanding understanding of the tumour-immune interaction and the tumour microenvironment allows a real opportunity to identify predictive biomarkers and tailor immune-based therapies, including designing rational combination drugs to enhance clinical outcomes, and to identify patients most likely to benefit from immunotherapy. Where there has never been a precision chemotherapy clinic in the last 70 years since its inception, even with no shortage of trying, the hope and evolution of a functional precision immunotherapy cancer clinic is a much more likely reality.

CD38 is involved in the adenosine pathway, which represents one of the immunosuppressive mechanisms in cancer. CD38 is broadly expressed across immune cell subsets, including human macrophages differentiated in vitro from monocytes, but expression by tissue-resident macrophages remains to be demonstrated. We analysed tissue samples obtained from 66 patients with hepatocellular carcinoma (HCC) from Singapore and analysed using immunohistochemistry. Tumor-infiltrating leukocytes (TILs) were further examined using DEPArrayTM., and the phenotype of freshly isolated TILs was determined using flow cytometry. It is found that CD38 was frequently co-expressed with the macrophage-specific marker CD68. CD38+CD68+ macrophage density was associated with improved prognosis after surgery, while total CD68+ macrophage density was associated with poor prognosis. DEPArrayTM. analysis revealed the presence of large (>10 µm), irregularly shaped CD45+CD14+ cells that resembled macrophages, with concurrent CD38+ expression. Flow cytometry also revealed that majority of CD14+HLA-DR+ cells expressed CD38. In conclusion, CD38 expression was clearly demonstrated on human macrophages in an in vivo setting. The positive association identified between CD38+ macrophage density and prognosis may have implications for routine diagnostic work.

Background Hepatocellular carcinoma is the second most deadly cancer with late presentation and limited treatment options, highlighting an urgent need to better understand HCC to facilitate the identification of early-stage biomarkers and uncover therapeutic targets for the development of novel therapies for HCC. Methods Deep transcriptome sequencing of tumor and paired non-tumor liver tissues was performed to comprehensively evaluate the profiles of both the host and HBV transcripts in HCC patients. Differential gene expression patterns and the dys-regulated genes associated with clinical outcomes were analyzed. Somatic mutations were identified from the sequencing data and the deleterious mutations were predicted. Lastly, human-HBV chimeric transcripts were identified, and their distribution, potential function and expression association were analyzed. Results Expression profiling identified the significantly upregulated TP73 as a nodal molecule modulating expression of apoptotic genes. Approximately 2.5% of dysregulated genes significantly correlated with HCC clinical characteristics. Of the 110 identified genes, those involved in post-translational modification, cell division and/or transcriptional regulation were upregulated, while those involved in redox reactions were downregulated in tumors of patients with poor prognosis. Mutation signature analysis identified that somatic mutations in HCC tumors were mainly non-synonymous, frequently affecting genes in the micro-environment and cancer pathways. Recurrent mutations occur mainly in ribosomal genes. The most frequently mutated genes were generally associated with a poorer clinical prognosis. Lastly, transcriptome sequencing suggest that HBV replication in the tumors of HCC patients is rare. HBV-human fusion transcripts are a common observation, with favored HBV and host insertion sites being the HBx C-terminus and gene introns (in tumors) and introns/intergenic-regions (in non-tumors), respectively. HBV-fused genes in tumors were mainly involved in RNA binding while those in non-tumors tissues varied widely. These observations suggest that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts may occur during tumorigenesis. Conclusions Transcriptome sequencing of HCC patients reveals key cancer molecules and clinically relevant pathways deregulated/mutated in HCC patients and suggests that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts likely occur during the process of tumorigenesis.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-associated mortality in the world. However, with the associated low five-year survival and high recurrence rates, alternative treatment modalities specifically immunotherapy have been researched. A correlation between CD38+tumour-infiltrating leukocyte (TIL) density and improved prognosis was found in a recent study. However, studies relating to CD38 expression in immune infiltrates within tumours are limited. In the present study, we confirmed the expression of CD38 on macrophages in HCC and determined the relationship between CD38+leukocytes and lymphocytes and patient response to immunotherapy. Using immunohistochemistry, we analysed tissue samples obtained from 20 patients from Singapore with HCC prior to immunotherapy. Tumour infiltrating leukocytes expression within tumour were correlated to the responsiveness of patients to immunotherapy.Expression of CD38 was found within the tumour cells and surrounding immune infiltrates including lymphocytes and macrophages. We then ask whether CD38 expression by the distinct cell populations may acquire theranostic relevance. Patients with higher level of CD38+immune infiltrate subsets had significantly better response to anti-PD-1 immunotherapy, and this is also true for CD38+lymphocytes within the tumour microenvironment. In particular, a cut-off of 13.0% positive out of total leukocytes and 12.4% positive out of total lymphocytes is found to be of strong predictive value of responsiveness to immunotherapy treatment, thus a strong theranostic impact is seen by using CD38 as a biomarker for anti-PD-1 therapy.The establishment of an association between CD38 expression and the response to anti-PD-1 immunotherapy in HCC, could be applied to a larger cohort outside Singapore. These may eventually change the routine testing in clinical practice to identify HCC patients suitable for immunotherapy.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-21556-y.

Objective: To develop and evaluate a screening version of the validated Scored Sodium Questionnaire (SSQ). Design: Development phase-a Scored Sodium Questionnaire-Screening Form (SSQ-SF) and scoring system were developed using data previously collected in 47 chronic kidney disease outpatients participating in the SSQ validation study; evaluation phase-conducted in 49 participants with end-stage kidney disease on hemodialysis and 16 nursing staff. Subjects: A total of 49 outpatients (61% male) aged 63.1 ± 14.8 years attending a hemodialysis kidney clinic at a tertiary referral hospital in Australia. On separate occasions, participants completed the SSQ, the SSQ-SF, and a feasibility questionnaire. Nursing staff from the same clinic completed a feasibility questionnaire. Main Outcome Measures: Time and ease of completion; association and agreement between the SSQ and SSQ-SF scores; assessment of binary classifications for sodium consumption: sensitivity and specificity. Results: Median time to complete the SSQ-SF was 4 minutes versus 10 minutes for the SSQ (P < .01); 93% of participants and nursing staff agreed that the SSQ-SF was easy to complete. There was strong and significant association between total SSQ and SSQ-SF scores (r = 0.777, P ≤ .01), with strong agreement between individual scores and no fixed bias. Compared with the SSQ, the SSQ-SF score cut-point of ≥50 successfully classified 23 of 27 participants as high sodium consumers: sensitivity 85% and specificity 68%. Conclusion: The SSQ-SF is quick and feasible to implement in the clinical setting and may be a useful first step for screening the sodium intakes of kidney disease patients for referral into the nutrition care process.

Hepatocellular carcinoma (HCC) is the 6th most common cancer in the world, but the second most common cause of cancer death. There is no universally accepted consensus practice guidelines for HCC owing to rapid developments in new treatment modalities, the heterogeneous epidemiology and clinical presentation of HCC worldwide. However, a number of regional and national guidelines currently exist which reflect practice relevant to the epidemiology and collective experience of the consensus group. In 2014, clinicians at the multidisciplinary Comprehensive Liver Cancer Clinic (CLCC) at the National Cancer Centre Singapore (NCCS) reviewed the latest published scientific data and existing international and regional practice guidelines, such as those of the National Comprehensive Cancer Network, American Association for the Study of Liver Diseases and the Asian Pacific Association for the Study of the Liver, and modified them to reflect local practice. These would serve as a template by which treatment outcomes can be collated and benchmarked against international data. The NCCS Consensus Guidelines for HCC have been successfully implemented in the CLCC since their publication online on 26th September 2014, and the guidelines allow outcomes of treatment to be compared to international data. These guidelines will be reviewed periodically to incorporate new data.

IntroductionHepatocellular 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.MethodsClinically 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.ResultsIHC 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. CD38himacrophages produce more interferon γ (IFN-γ) and related cytokines, which may explain its predictive value when treated with ICB.ConclusionsA high proportion of CD38+cells, determined by IHC, predicts response to ICB and is associated with superior mPFS and OS in advanced HCC.

4590 Background: Nivolumab (N) and Y90-radioembolization (RE) are both therapeutic options in advanced hepatocellular carcinoma (aHCC). Increasing evidence suggests that radiotherapy synergizes with immune checkpoint inhibitors to augment anti-tumour effects. Methods: Eligible Child-Pugh A aHCC patients (pts) were treated with Y90-RE followed by N 240mg, 21 days after Y90-RE and every 2 weeks thereafter. Pre- and on-treatment tumor biopsies together with circulating biomarkers were obtained. Primary end-point was overall response rate (ORR) (per RECIST v 1.1). Overall response was defined as the composite overall response observed for the lesions within Y90-RE field and outside Y90-RE field. Key secondary end points included disease control rate (DCR), progression free survival (PFS), overall survival (OS), and safety. 36 evaluable pts were needed to assess whether the addition of N improved the ORR of Y90-RE from 21% to 41% as determined by Simon two-stage optimal design with 80% power and one sided significance level of 0.05. Results: Forty pts were enrolled of which 36 were evaluable. At baseline: 63.9% were HepB in aetiology; 63.9% BCLC stage C; 47.2% had AFP > 400ng/mL; number of liver lesions – median 5 (range 1- 20); size of largest liver lesion – median 80mm (range 14-177mm); 27.8% had prior TACE; and 13.9% had prior systemic therapy. ORR was 31% (95% CI 16.4 - 48.1%). Eight out of 11 responders had not progressed at study cut-off. DCR was 58.3%. 81% of target lesions within Y90-RE field regressed. With a median follow up of 16.4 months, median PFS and OS were 4.6 months (95% CI 2.3m - 8.4m) and 15.1 months (95% CI 7.8m - NE) respectively. Six- and 12-month PFS rates were 44.2% (95% CI 27.3% - 59.9%) and 26.1% (95% CI 11.2% - 43.8%) respectively. Overall, N+ Y90-RE was well tolerated and safe; only 11% had grade 3/4 treatment related adverse events (AEs). Responders demonstrated significant alterations of LIF, MIG and Eotaxin3 levels in the pre-treatment cytokine analyses. Conclusions: Combination N+Y90-RE resulted in an encouraging ORR of 31% (95% CI 16.4 - 48.1%) in aHCC. 81% of target lesions within Y90-RE field regressed suggesting synergy in combining Y90-RE with nivolumab. This combination is safe and tolerable with low G3/4 treatment related AEs of 11%. Further biomarker analyses will be presented at the meeting. Clinical trial information: NCT03033446 .

Background and aimsChronic inflammation induced by chronic hepatitis B virus (HBV) infection increases the risk of hepatocellular carcinoma (HCC). However, little is known about the immune landscape of HBV-related HCC and its influence on the design of effective cancer immunotherapeutics.MethodsWe interrogated the immune microenvironments of HBV-related HCC and non-viral-related HCC using immunohistochemistry and cytometry by time-of-flight (CyTOF). On identifying unique immune subsets enriched in HBV-related HCC, we further interrogated their phenotypes and functions using next-generation sequencing (NGS) and in vitro T-cell proliferation assays.ResultsIn-depth interrogation of the immune landscapes showed that regulatory T cells (TREG) and CD8+ resident memory T cells (TRM) were enriched in HBV-related HCC, whereas Tim-3+CD8+ T cells and CD244+ natural killer cells were enriched in non-viral-related HCC. NGS of isolated TREG and TRM from HBV-related HCC and non-viral-related HCC identified distinct functional signatures associated with T-cell receptor signalling, T-cell costimulation, antigen presentation and programmed cell death protein 1 (PD-1) signalling. TREG and TRM from HBV-related HCC expressed more PD-1 and were functionally more suppressive and exhausted than those from non-virus-related HCC. Furthermore, immunosuppression by PD-1+ TREG could be reversed with anti-PD-1 blockade. Using multiplexed tissue immunofluorescence, we further demonstrated that TREG and TRM contributed to overall patient survival: TREG were associated with a poor prognosis and TRM were associated with a good prognosis in HCC.ConclusionWe have shown that the HBV-related HCC microenvironment is more immunosuppressive and exhausted than the non-viral-related HCC microenvironment. Such in-depth understanding has important implications in disease management and appropriate application of immunotherapeutics.

Prognostic role of surgical resection of the primary tumor and baseline CEA among patients with synchronous stage IV colorectal cancer (CRC) remains an area of debate. The objective of this study was to determine the prognostic value of baseline CEA and surgical resection of the primary among patients with synchronous stage IV CRC in the era of modern chemotherapy and biologic therapy. The Surveillance, Epidemiology and End Results Registry was searched to identify patients with synchronous stage IV CRC diagnosed between 2004 and 2009. Colorectal-cancer-specific survival (CCS) was estimated using the Kaplan-Meier product limit method. Cox models were fitted to assess the multivariable relationship of various patient and tumor characteristics and CCS. Three hundred thirty-three thousand, three hundred ninety nine patients were identified in the SEER registry. Median CCS among patients with their primary tumor removed was 21 M vs. 7 M (primary intact) respectively (p

Purpose This open-label phase III trial evaluated efficacy and tolerability of linifanib versus sorafenib in patients with advanced hepatocellular carcinoma (HCC) without prior systemic therapy. Patients and Methods Patients were randomly assigned in a 1:1 ratio to linifanib 17.5 mg once daily or sorafenib 400 mg twice daily. Patients were stratified by region (Outside Asia, Japan, and rest of Asia), Eastern Cooperative Oncology Group performance score (ECOG PS; 0 or 1), vascular invasion or extrahepatic spread (yes or no), and hepatitis B virus (HBV) infection (yes or no). The primary end point of the study was overall survival (OS). Secondary end points were time to progression (TTP) and objective response rate (ORR) per RECIST v1.1. Results We randomly assigned 1,035 patients (median age, 60 years; Asian, 66.6%; ECOG PS 0, 65.2%; HBV, 49.1%; vascular invasion or extrahepatic spread, 70.1%). Median OS was 9.1 months on the linifanib arm (95% CI, 8.1 to 10.2) and 9.8 months on the sorafenib arm (95% CI, 8.3 to 11.0; hazard ratio [HR], 1.046; 95% CI, 0.896 to 1.221). For prespecified stratification subgroups, OS HRs ranged from 0.793 to 1.119 and the 95% CI contained 1.0. Median TTP was 5.4 months on the linifanib arm (95% CI, 4.2 to 5.6) and 4.0 months on the sorafenib arm (95% CI, 2.8 to 4.2; HR, 0.759; 95% CI, 0.643 to 0.895; P = .001). Best response rate was 13.0% on the linifanib arm versus 6.9% on the sorafenib arm. Grade 3/4 adverse events (AEs); serious AEs; and AEs leading to discontinuation, dose interruption, and reduction were more frequent with linifanib (all P < .001). Conclusion Linifanib and sorafenib had similar OS in advanced HCC. Predefined superiority and noninferiority OS boundaries were not met for linifanib and the study failed to meet the primary end point. TTP and ORR favored linifanib; safety results favored sorafenib.

Cancer immunotherapy has finally joined the pillars of cancer treatment—surgery, radiation, chemotherapy, hormonal therapy, and targeted therapy—in improving cancer patient lives. In the last 5 years, the development of immune checkpoint inhibitor and T cell therapy, particularly chimeric antigen receptor (CAR) T-cell therapy, has been remarkable for the speed, scale, and number of drug approvals. Still, these treatments may also bring unusual adverse effects and clinical outcomes including unprecedented long-term survival. Interrogating the tumor microenvironment and identifying better biomarkers hold the key to improving cancer immunotherapies. CAR T-cell therapy has dramatic effect on leukemias and lymphomas with significant cure rates, but has yet to show comparable effect on solid tumors. Cutting-edge technology will improve both processing and clinical effect of such therapies. Asia has the largest, most rapidly aging population and the largest number of cancer patients in the world. Research and development and clinical trial conduct of cancer immunotherapy in Asia remain nascent, but should be a crucial priority.

The majority of patients with hepatocellular carcinoma (HCC) are inoperable and results with conventional chemotherapy are dismal. Many end up with no treatment options and resort to alternative medicine. The authors report the use of Coriolus versicolor (CV) in advanced HCC patients with poor liver function or who were unfit to receive standard therapy.Fifteen eligible cases were randomized 2:1 to either CV or placebo. The primary endpoint was the median time to progression (TTP) between both arms. Secondary endpoints include evaluating response rates, toxicity, quality of life (QOL), progression-free survival (PFS), and overall survival (OS). Further correlative studies were performed looking at the effect of CV on the immune system.The median treatment duration was 1.5 cycles and 3 cycles on the placebo and CV arm, respectively. Median TTP was 2.5 (1.4-5.3) months compared to 4.2 (0.4-4.2) months in the CV and placebo arm, respectively, hazard ratio (HR) 0.70 (0.16-3.05 p = 0.634). Median PFS was 2.5 (1.4-5.3) months in the CV and 1.1 (0.4-4.2) months in the placebo arm, HR 0.42 (0.13-1.34, p = 0.144). Median OS was 6.5 (3.3-24.1) and 2.2 (0.8-23.3) months, respectively, HR 0.35 (0.10-1.25, p = 0.105). Social and emotional functioning scores were higher in the CV group compared to placebo group on treatment. CV subjects had less appetite loss and pain symptoms compared to placebo subjects during treatment.There was no difference in TTP with use of CV compared to placebo. CV subjects generally had better QOL on treatment compared to placebo subjects. The utility of this supplement in patients whose primary treatment goal is palliation should be further explored.

The natural course of early HCC is unknown, and its progression to intermediate and advanced HCC can be diverse. Some early stage HCC patients enjoy prolonged disease-free survival, whereas others suffer aggressive relapse to stage IV metastatic cancer within a year. Comparative proteomics of HCC tumor tissues was carried out using 2D-DIGE and MALDI-TOF/TOF MS to identify proteins that can distinguish these two groups of stage I HCC patients. Twelve out of 148 differentially regulated protein spots were found to differ by approximately 2-fold for the relapse versus nonrelapse patient tissues. Four proteins, namely, heat shock 70 kDa protein 1, argininosuccinate synthase, isoform 2 of UTP-glucose-1-phosphate uridylyltransferase, and transketolase, were shown to have the potential to differentiate metastatic relapse (MR) from nonrelapse (NR) HCC patients after validation by western blotting and immunohistochemical assays. Subsequent TMA analysis revealed a three marker panel of HSP70, ASS1, and UGP2 to be statistically significant in stratifying the two groups of HCC patients. This combination panel achieved high levels of sensitivity and specificity, which has potential for clinical use in identifying HCC tumors prone to MR. This stratification will allow development of clinical management, including close follow-up and possibly treatment options, in the near future.

Cellular immunotherapy, harnessing the patient’s own and donor immune cells, has emerged as a form of highly personalized treatment in cancer. Adoptive transfer of genetically engineered T cells modified to express chimeric antigen receptor (CAR) has demonstrated remarkable anti-tumor response and long-term remissions in selected hematologic malignancies. However, cancer relapse is still a challenge in some of these patients, and CAR T cell therapy is still investigational and of limited efficacy against solid tumors. On the other hand, tumor infiltrating lymphocyte (TIL) therapy, genetically modified T cell receptor (GM-TCR) therapy and viral-specific T cell therapy are emerging treatment options in solid organ cancers. There is a great interest and increasing effort towards studying biomarkers that can predict for improved outcomes of patients undergoing cellular therapy, still a nascent, emerging field. Identification of such biomarkers may allow enrichment strategies to improve clinical trial design aimed at accelerating clinical development and approvals of such therapies. Given the current high financial costs of cellular therapy, biomarkers can potentially better identify patients that derive maximum benefit as well as potentially reducing pressure on reimbursement-based healthcare systems. Here, we review the current understanding and body of literature on biomarkers for cellular therapy, with a specific focus on T cell therapies. Biomarkers predictive for cellular therapy-based adverse events have been reviewed elsewhere and is not discussed herein.

Summary Tumors display profound changes in cellular metabolism, yet how these changes aid the development and growth of tumors is not fully understood. Here we use a multi-omic approach to examine liver carcinogenesis and regeneration, and find that progressive loss of branched-chain amino acid (BCAA) catabolism promotes tumor development and growth. In human hepatocellular carcinomas and animal models of liver cancer, suppression of BCAA catabolic enzyme expression led to BCAA accumulation in tumors, though this was not observed in regenerating liver tissues. The degree of enzyme suppression strongly correlated with tumor aggressiveness, and was an independent predictor of clinical outcome. Moreover, modulating BCAA accumulation regulated cancer cell proliferation in vitro, and tumor burden and overall survival in vivo. Dietary BCAA intake in humans also correlated with cancer mortality risk. In summary, loss of BCAA catabolism in tumors confers functional advantages, which could be exploited by therapeutic interventions in certain cancers.

The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC, we performed a near-saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers and a very large number (2,860) of candidate later stage drivers that were enriched for genes that are mutated, deregulated or functioning in signaling pathways important for human HCC, with a striking 1,199 genes being linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC.

Currently, only one drug, sorafenib, is FDA approved for the treatment of advanced hepatocellular carcinoma (HCC), achieving modest objective response rates while still conferring an overall survival benefit. Unlike other solid tumors, no oncogenic addiction loops have been validated as clinically actionable targets in HCC. Outcomes of HCC could potentially be improved if critical molecular subclasses with distinct therapeutic vulnerabilities can be identified, biomarkers that predict recurrence or progression early can be determined and key epigenetic, genetic or microenvironment drivers that determine best response to a specific targeting treatment can be uncovered. Our group and others have examined the molecular heterogeneity of hepatocellular carcinoma. We have developed a panel of patient derived xenograft models to enable focused pre-clinical drug development of rationally designed therapies in specific molecular subgroups. We observed unique patterns, including synergies, of drug activity across our molecularly diverse HCC xenografts, pointing to specific therapeutic vulnerabilities for individual tumors. These efforts inform clinical trial designs and catalyze therapeutic development. It also argues for efficient strategic allocation of patients into appropriate enriched clinical trials. Here, we will discuss some of the recent important therapeutic studies in advanced HCC and also some of the potential strategies to optimize clinical therapeutic development moving forward.

Chronic hepatitis B virus (HBV) infection is epidemiologically associated with hepatocellular carcinoma (HCC), but its role in HCC remains poorly understood due to technological limitations. In this study, we systematically characterize HBV in HCC patients. HBV sequences were enriched from 48 HCC patients using an oligo-bead-based strategy, pooled together and sequenced using the FLX-Genome-Sequencer. In the tumors, preferential integration of HBV into promoters of genes (P < 0.001) and significant enrichment of integration into chromosome 10 (P < 0.01) were observed. Integration into chromosome 10 was significantly associated with poorly differentiated tumors (P < 0.05). Notably, in the tumors, recurrent integration into the promoter of the human telomerase reverse transcriptase (TERT) gene was found to correlate with increased TERT expression. The preferred region within the HBV genome involved in integration and viral structural alteration is at the 3'-end of hepatitis B virus X protein (HBx), where viral replication/transcription initiates. Upon integration, the 3'-end of the HBx is often deleted. HBx-human chimeric transcripts, the most common type of chimeric transcripts, can be expressed as chimeric proteins. Sequence variation resulting in non-conservative amino acid substitutions are commonly observed in HBV genome. This study highlights HBV as highly mutable in HCC patients with preferential regions within the host and virus genome for HBV integration/structural alterations.

BACKGROUND and AIMS: Recent observational studies showed that post-operative aspirin use reduces cancer relapse and death in the earliest stages of colorectal cancer. We sought to evaluate the cost-effectiveness of aspirin as an adjuvant therapy in Stage I and II colorectal cancer patients aged 65 years and older. METHODS: Two five-state Markov models were constructed separately for Stage I and II colorectal cancer using TreeAge Pro 2014. Two hypothetical cohorts of 10,000 individuals at a starting age of 65 years and with colorectal cancer in remission were put through the models separately. Cost-effectiveness of aspirin was evaluated against no treatment (Stage I and II) and capecitabine (Stage II) over a 20-year period from the United States societal perspective. Extensive one-way sensitivity analyses and multivariable Probabilistic Sensitivity Analyses (PSA) were performed. RESULTS: In the base case analyses, aspirin was cheaper and more effective compared to other comparators in both stages. Sensitivity analyses showed that no treatment and capecitabine (Stage II only) can be cost-effective alternatives if the utility of taking aspirin is below 0.909, aspirin's annual fatal adverse event probability exceeds 0.57%, aspirin's relative risk of disease progression is 0.997 or more, or when capecitabine's relative risk of disease progression is less than 0.228. Probabilistic Sensitivity Analyses (PSA) further showed that aspirin could be cost-effective 50% to 80% of the time when the willingness-to-pay threshold was varied from USD 20,000 to USD 100,000. CONCLUSION: Even with a modest treatment benefit, aspirin is likely to be cost-effective in Stage I and II colorectal cancer, thus suggesting a potential unique role in secondary prevention in this group of patients.

Intravesical instillation of Bacillus Calmette-Guérin is currently used as adjuvant therapy for superficial, non-muscle invasive bladder cancer (NMIBC). However, nearly 40% of patients with NMIBC will fail Bacillus Calmette-Guérin therapy. In an attempt to investigate the feasibility of using insect baculovirus-based vectors for bladder cancer therapy, we observed that intravesical instillation of baculoviruses without transgene up-regulated a set of Th1-type of cytokines and increased the survival rate of mice bearing established orthotopic bladder tumors. When baculoviral vectors were used to co-deliver the mouse CD40 ligand and IL-15 genes through intravesical instillation, the immunogene therapy triggered significantly increased bladder infiltrations of inflammatory monocytes, CD4+, CD8+ and γδ T lymphocytes. All treated animals survived beyond 12 months whereas control animals died around 2 months after tumor inoculation. We conclude that direct intravesical instillation of baculoviral gene transfer vectors holds the potential to be a novel therapeutic modality for NMIBC.

The cumulative effects of hepatic injury due to hepatitis B virus (HBV) infections and aflatoxin-B1 (AFB1) exposure are the major risk factors of HCC. Understanding early metabolic changes involving these risk factors in an animal model closely resembling human hepatocellular carcinoma (HCC) is critical for biomarker discovery and disease therapeutics. We have used the hepatitis B surface antigen (HBsAg) transgenic mouse model that mimics HBV carriers with and without AFB1 treatment. We investigated early metabolic changes from preneoplastic state to HCC by non-invasive longitudinal imaging in three HCC groups of mice: HBsAg + AFB1(Gp-I), AFB1 alone (Gp-II), HBsAg alone (Gp-III) and a control group (wild-type untreated; Gp-IV). For the first time, we have identified acylcarnitine signals in vivo in the liver prior to the histological manifestation of the tumors in all three groups. Acylcarnitine concentration increased with increase in tumor growth in all HCC mouse models, indicating elevated metabolic activity and increased cell turnover. This was confirmed in a pilot study using human serum from HCC patients, which revealed a higher concentration of acylcarnitine compared with normal subjects. Translational clinical studies can be designed to detect acylcarnitine in patients with high risk factors for HCC.

Hepatocellular Carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. However, the role of epigenetic changes such as aberrant DNA methylation in hepatocarcinogenesis remains largely unclear. In this study, we examined the methylation profiles of 59 HCC patients. Using consensus hierarchical clustering with feature selection, we identified three tumor subgroups based on their methylation profiles and correlated these subgroups with clinicopathological parameters. Interestingly, one tumor subgroup is different from the other 2 subgroups and the methylation profile of this subgroup is the most distinctly different from the non-tumorous liver tissues. Significantly, this subgroup of patients was found to be associated with poor overall as well as disease-free survival. To further understand the pathways modulated by the deregulation of methylation in HCC patients, we integrated data from both the methylation as well as the gene expression profiles of these 59 HCC patients. In these patients, while 4416 CpG sites were differentially methylated between the tumors compared to the adjacent non-tumorous tissues, only 536 of these CpG sites were associated with differences in the expression of their associated genes. Pathway analysis revealed that forty-four percent of the most significant upstream regulators of these 536 genes were involved in inflammation-related NFκB pathway. These data suggest that inflammation via the NFκB pathway play an important role in modulating gene expression of HCC patients through methylation. Overall, our analysis provides an understanding on aberrant methylation profile in HCC patients.

BACKGROUND: The efficacy and safety of linifanib (ABT-869), a selective inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinases, were assessed in this phase 2, single-arm, open-label, multicenter trial. METHODS: Eligible patients had unresectable or metastatic hepatocellular carcinoma and had received ≤ 1 prior systemic therapy. Patients received oral linifanib at a fasting dose of 0.25 mg/kg,. The primary endpoint was the progression-free rate at 16 weeks. Tumor response was assessed every 8 weeks. Secondary endpoints included the time to disease progression, overall survival, and objective response rate. Safety was also assessed. RESULTS: Of the 44 patients enrolled, the majority were Asian (89%), had received no prior systemic therapy (82%), had Child-Pugh class A hepatic function (86%), and had hepatitis B virus infection (61%). The estimated progression-free rate at 16 weeks was 31.8% (34.2% for patients with Child-Pugh class A hepatic function), the estimated objective response rate was 9.1% (10.5% for patients with Child-Pugh class A hepatic function), the median time to disease progression was 3.7 months (3.7 months for patients with Child-Pugh class A hepatic function), and the median overall survival was 9.7 months (10.4 months for patients with Child-Pugh class A hepatic function). The most common linifanib-related adverse events were diarrhea (55%) and fatigue (52%). The most common linifanib-related grade 3/4 adverse events were hypertension (25%) and fatigue (14%). Serum levels of biomarkers cancer antigen (CA) 125, cytokeratin fragment (CYFRA)21.1, and protein induced by vitamin K absence or antagonist II (PIVKA) demonstrated potential as prognostic indicators of patient response or outcome. CONCLUSIONS: Single-agent linifanib was found to be clinically active in patients with advanced hepatocellular carcinoma, with an acceptable safety profile. Cancer 2013. © 2012 American Cancer Society.

Background: Individuals with metastatic Epstein–Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) continue to have poor outcomes. To evaluate the ability of a dendritic cell (DC) vaccine to target subdominant EBV antigens LMP1 and LMP2 expressed by NPC cells, we vaccinated patients using autologous DCs transduced with an adenovirus encoding a truncated LMP1 (ΔLMP1) and full-length LMP2 (Ad-ΔLMP1-LMP2). Materials and methods: Sixteen subjects with metastatic NPC received Ad-ΔLMP1-LMP2 DC vaccines i.d. biweekly for up to five doses. Toxicity, immune responses and clinical responses were determined. Results: Most patients had extensive disease, with a median of three visceral sites of involvement (range 1–7). No significant toxicity was observed. Ad-ΔLMP1-LMP2 DCs induced delayed type hypersensitivity responses in 9 out of 12 patients, but although these DCs activated LMP1/2-specific T cells in vitro, no such increase in the frequency of peripheral LMP1/2-specific T cells was detected. Three patients had clinical responses including one with partial response (for 7½ months) and two with stable disease (for 6½ and 7½ months). Conclusions: Ad-ΔLMP1-LMP2 transduced DCs can be successfully generated and safely administered to patients with advanced NPC. Since efficacy was limited, future studies should focus on DC vaccines with greater potency administered to subjects with less tumor burden.

Objective Gastric cancer is a major gastrointestinal malignancy for which targeted therapies are emerging as treatment options. This study sought to identify the most prevalent molecular targets in gastric cancer and to elucidate systematic patterns of exclusivity and co-occurrence among these targets, through comprehensive genomic analysis of a large panel of gastric cancers. Design Using high-resolution single nucleotide polymorphism arrays, copy number alterations were profiled in a panel of 233 gastric cancers (193 primary tumours, 40 cell lines) and 98 primary matched gastric non-malignant samples. For selected alterations, their impact on gene expression and clinical outcome were evaluated. Results 22 recurrent focal alterations (13 amplifications and nine deletions) were identified. These included both known targets ( FGFR2 , ERBB2 ) and also novel genes in gastric cancer ( KLF5 , GATA6 ). Receptor tyrosine kinase (RTK)/RAS alterations were found to be frequent in gastric cancer. This study also demonstrates, for the first time, that these alterations occur in a mutually exclusive fashion, with KRAS gene amplifications highlighting a clinically relevant but previously underappreciated gastric cancer subgroup. FGFR2 -amplified gastric cancers were also shown to be sensitive to dovitinib, an orally bioavailable FGFR/VEGFR targeting agent, potentially representing a subtype-specific therapy for FGFR2 -amplified gastric cancers. Conclusion The study demonstrates the existence of five distinct gastric cancer patient subgroups, defined by the signature genomic alterations FGFR2 (9% of tumours), KRAS (9%), EGFR (8%), ERBB2 (7%) and MET (4%). Collectively, these subgroups suggest that at least 37% of gastric cancer patients may be potentially treatable by RTK/RAS directed therapies.

Nasopharyngeal carcinoma (NPC) is associated with the Epstein-Barr virus (EBV) and characterized by peritumoral immune infiltrate. Advanced NPC has high lethality. Immunotherapy directed against EBV antigen targets has been previously explored in clinical trials, and is likely to be validated as an important target in NPC as randomized data emerges in the future. Cancer vaccines and adoptive T cell therapy have been explored in the clinic, with the latter showing the greatest success. Recent advances in gene sequencing technology now allow personalized tumor epitope mapping, whilst the advent of immune checkpoint inhibitors targeting the PD-1/PD-L1 axis offers the opportunity to activate adaptive T cell response in vivo. Anti-PD1 antibodies have shown promising activity in early phase clinical trials, and randomized studies against chemotherapy are underway. As immunotherapy is incorporated into standard treatment paradigms, issues of optimal combinations with targeting agents, immune adjuvants, and sequence with chemotherapy and radiation therapy will need to be addressed. Effective strategies to increase tumor antigenicity, improve immunological memory and reduce immune escape, will need to be developed to improve treatment outcomes. Here we present a brief history of the evolution of immunotherapy in NPC, and highlight key concepts relevant to its further development in the clinic.