Hypoxia-driven immunosuppression by Treg and type-2 conventional dendritic cells in HCC.

BACKGROUND AND AIMS: Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However, the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated. APPROACH AND RESULTS: We analyzed the immune landscapes of hypoxia-low and hypoxia-high tumor regions using cytometry by time of light, immunohistochemistry, and transcriptomic analyses. The mechanisms of immunosuppression in immune subsets of interest were further explored using in vitro hypoxia assays. Regulatory T cells (Tregs) and a number of immunosuppressive myeloid subsets, including M2 macrophages and human leukocyte antigen-DR isotype (HLA-DRlo ) type 2 conventional dendritic cell (cDC2), were found to be significantly enriched in hypoxia-high tumor regions. On the other hand, the abundance of active granzyme Bhi PD-1lo CD8+ T cells in hypoxia-low tumor regions implied a relatively active immune landscape compared with hypoxia-high regions. The up-regulation of cancer-associated genes in the tumor tissues and immunosuppressive genes in the tumor-infiltrating leukocytes supported a highly pro-tumorigenic network in hypoxic HCC. Chemokine genes such as CCL20 (C-C motif chemokine ligand 20) and CXCL5 (C-X-C motif chemokine ligand 5) were associated with recruitment of both Tregs and HLA-DRlo cDC2 to hypoxia-high microenvironments. The interaction between Tregs and cDC2 under a hypoxic TME resulted in a loss of antigen-presenting HLA-DR on cDC2. CONCLUSIONS: We uncovered the unique immunosuppressive landscapes and identified key immune subsets enriched in hypoxic HCC. In particular, we identified a potential Treg-mediated immunosuppression through interaction with a cDC2 subset in HCC that could be exploited for immunotherapies.

Uncoupling immune trajectories of response and adverse events from anti-PD-1 immunotherapy in hepatocellular carcinoma.

BACKGROUND & AIMS: While immune checkpoint blockade (ICB) has shown promise in patients with hepatocellular carcinoma (HCC), it is associated with modest response rates and immune-related adverse events (irAEs) are common. In this study, we aimed to decipher immune trajectories and mechanisms of response and/or irAEs in patients with HCC receiving anti-programmed cell death 1 (anti-PD-1) therapy. METHODS: Pre- and on-treatment peripheral blood samples (n = 60) obtained from 32 patients with HCC (Singapore cohort) were analysed by cytometry by time-of-flight and single-cell RNA sequencing, with flow cytometric validation in an independent Korean cohort (n = 29). Mechanistic validation was conducted by bulk RNA sequencing of 20 pre- and on-treatment tumour biopsies and using a murine HCC model treated with different immunotherapeutic combinations. RESULTS: Single-cell analyses identified CXCR3+CD8+ effector memory T (TEM) cells and CD11c+ antigen-presenting cells (APC) as associated with response (p = 0.0004 and 0.0255, respectively), progression-free survival (p = 0.00079 and 0.0015, respectively), and irAEs (p = 0.0034 and 0.0125, respectively) in anti-PD-1-treated patients with HCC. Type-1 conventional dendritic cells were identified as the specific APC associated with response, while 2 immunosuppressive CD14+ myeloid clusters were linked to reduced irAEs. Further analyses of CXCR3+CD8+ TEM cells showed cell-cell interactions specific to response vs. irAEs, from which the anti-PD-1 and anti-TNFR2 combination was harnessed to uncouple these effects, resulting in enhanced response without increased irAEs in a murine HCC model. CONCLUSIONS: This study identifies early predictors of clinical response to anti-PD-1 ICB in patients with HCC and offers mechanistic insights into the immune trajectories of these immune subsets at the interface between response and toxicity. We also propose a new combination immunotherapy for HCC to enhance response without exacerbating irAEs. CLINICAL TRIAL NUMBER: NCT03695952. LAY SUMMARY: Response rates to immune checkpoint blockade (ICB) treatment in hepatocellular carcinoma (HCC) remain modest and adverse events are common. Herein, we identified early predictors of response and gained an in-depth understanding of the immunological mechanisms behind response and adverse events in patients with HCC treated with ICB. We also proposed a new combination immunotherapy for HCC that enhances response without exacerbating adverse events.

Neuroblastoma patient-derived cultures are enriched for a mesenchymal gene signature and reflect individual drug response.

Ex vivo evaluation of personalized models can facilitate individualized treatment selection for patients, and advance the discovery of novel therapeutic options. However, for embryonal malignancies, representative primary cultures have been difficult to establish. We developed patient-derived cell cultures (PDCs) from chemo-naïve and post-treatment neuroblastoma tumors in a consistent and efficient manner, and characterized their in vitro growth dynamics, histomorphology, gene expression, and functional chemo-response. From 34 neuroblastoma tumors, 22 engrafted in vitro to generate 31 individual PDC lines, with higher engraftment seen with metastatic tumors. PDCs displayed characteristic immunohistochemical staining patterns of PHOX2B, TH, and GD2 synthase. Concordance of MYCN amplification, 1p and 11q deletion between PDCs and patient tumors was 83.3%, 72.7%, and 80.0% respectively. PDCs displayed a predominantly mesenchymal-type gene expression signature and showed upregulation of pro-angiogenic factors that were similarly enriched in culture medium and paired patient serum samples. When tested with standard-of-care cytotoxics at human Cmax -equivalent concentrations, MYCN-amplified and non-MYCN-amplified PDCs showed a differential response to cyclophosphamide and topotecan, which mirrored the corresponding patients' responses, and correlated with gene signatures of chemosensitivity. In this translational proof-of-concept study, early-phase neuroblastoma PDCs enriched for the mesenchymal cell subpopulation recapitulated the individual molecular and phenotypic profile of patient tumors, and highlighted their potential as a platform for individualized ex vivo drug-response testing.

Microarray profiling shows distinct differences between primary tumors and commonly used preclinical models in hepatocellular carcinoma.

BACKGROUND: Despite advances in therapeutics, outcomes for hepatocellular carcinoma (HCC) remain poor and there is an urgent need for efficacious systemic therapy. Unfortunately, drugs that are successful in preclinical studies often fail in the clinical setting, and we hypothesize that this is due to functional differences between primary tumors and commonly used preclinical models. In this study, we attempt to answer this question by comparing tumor morphology and gene expression profiles between primary tumors, xenografts and HCC cell lines. METHODS: Hep G2 cell lines and tumor cells from patient tumor explants were subcutaneously (ectopically) injected into the flank and orthotopically into liver parenchyma of Mus Musculus SCID mice. The mice were euthanized after two weeks. RNA was extracted from the tumors, and gene expression profiling was performed using the Gene Chip Human Genome U133 Plus 2.0. Principal component analyses (PCA) and construction of dendrograms were conducted using Partek genomics suite. RESULTS: PCA showed that the commonly used HepG2 cell line model and its xenograft counterparts were vastly different from all fresh primary tumors. Expression profiles of primary tumors were also significantly divergent from their counterpart patient-derived xenograft (PDX) models, regardless of the site of implantation. Xenografts from the same primary tumors were more likely to cluster together regardless of site of implantation, although heat maps showed distinct differences in gene expression profiles between orthotopic and ectopic models. CONCLUSIONS: The data presented here challenges the utility of routinely used preclinical models. Models using HepG2 were vastly different from primary tumors and PDXs, suggesting that this is not clinically representative. Surprisingly, site of implantation (orthotopic versus ectopic) resulted in limited impact on gene expression profiles, and in both scenarios xenografts differed significantly from the original primary tumors, challenging the long-held notion that orthotopic PDX model is the gold standard preclinical model for HCC.

Assessment of tumor necrotic fraction by dynamic contrast-enhanced MRI: a preclinical study of human tumor xenografts with histopathologic correlation.

Contrary to the common notion that tumor necrotic regions are non-enhancing after contrast administration, recent evidence has shown that necrotic regions exhibit delayed and slow uptake of gadolinium tracer on dynamic contrast-enhanced MRI (DCE MRI). The purpose of this study is to explore whether the mapping of tumor voxels with delayed and slow enhancement on DCE MRI can be used to derive estimates of tumor necrotic fraction. Patient-derived tumor xenograft lines of seven human cancers were implanted in 26 mice which were subjected to DCE MRI performed using a spoiled gradient recalled sequence. Gadolinium tracer concentration was estimated using the variable flip angle technique. To identify tumor voxels exhibiting delayed and slow uptake of contrast medium, clustering analysis was performed using a k-means clustering algorithm that classified tumor voxels according to their contrast enhancement patterns. Comparison of the percentage of tumor voxels exhibiting delayed and slow enhancement with the tumor necrotic fraction estimated on histology showed a strong correlation (r = 0.962, p < 0.001). The mapping of tumor regions with delayed and slow contrast uptake on DCE MRI correlated strongly with tumor necrotic fraction, and can potentially serve as a non-invasive imaging surrogate for the in vivo assessment of necrotic fraction.

Tongue carcinoma infrequently harbor common actionable genetic alterations.

BACKGROUND: Oral tongue squamous cell carcinomas (TSCC) are a unique subset of head and neck cancers with a distinct demographic profile, where up to half of the cases are never smokers. A small proportion of patients with OSCC are known to respond to EGFR TKI. We used a high-sensitivity mass spectrometry-based mutation profiling platform to determine the EGFR mutation status, as well as other actionable alterations in a series of Asian TSCC. METHODS: 66 TSCC patients treated between 1998-2009 with complete clinico-pathologic data were included in this study. Somatic mutation profiling was performed using Sequenom LungCarta v1.0, and correlated with clinical parameters. RESULTS: Mutations were identified in 20/66(30.3%) of samples and involved TP53, STK11, MET, PIK3CA, BRAF and NRF2. No activating EGFR mutations or KRAS mutations were discovered in our series, where just over a third were never smokers. The most common mutations were in p53 (10.6%; n = 7) and MET (10.6%, n = 11) followed by STK11 (9.1%, n = 6) and PIK3CA (4.5%, n = 3). BRAF and NRF2 mutations, which are novel in TSCC, were demonstrated in one sample each. There was no significant correlation between overall mutation status and smoking history (p = 0.967) or age (p = 0.360). Positive MET alteration was associated with poorer loco-regional recurrence free survival (LRFS) of 11 months [vs 90 months in MET-negative group (p = 0.008)]. None of the other mutations were significantly correlated with LRFS or overall survival. Four of these tumors were propagated as immortalized cell lines and demonstrated the same mutations as the original tumor. CONCLUSIONS: Using the Sequenom multiplexed LungCarta panel, we identified mutations in 6 genes, TP53, STK11, MET, PIK3CA, BRAF and NRF2, with the notable absence of EGFR and HER2 mutations in our series of Asian OSCC. Primary cell line models recapitulated the mutation profiles of the original primary tumours and provide an invaluable resource for experimental cancer therapeutics.

Exosomal mRNA Cargo are biomarkers of tumor and immune cell populations in pediatric osteosarcoma.

Osteosarcoma is the commonest malignant bone tumor of children and adolescents and is characterized by a high risk of recurrence despite multimodal therapy, especially in metastatic disease. This suggests the presence of clinically undetected cancer cells that persist, leading to cancer recurrence. We sought to evaluate the utility of peripheral blood exosomes as a more sensitive yet minimally invasive blood test that could aid in evaluating treatment response and surveillance for potential disease recurrence. We extracted exosomes from the blood of pediatric osteosarcoma patients at diagnosis (n=7) and after neoadjuvant chemotherapy (n=5 subset), as well as from age-matched cancer-free controls (n=3). We also obtained matched tumor biopsy samples (n=7) from the cases. Exosome isolation was verified by CD9 immunoblot and characterized on electron microscopy. Profiles of 780 cancer-related transcripts were analysed in mRNA from exosomes of osteosarcoma patients at diagnosis and control patients, matched post-chemotherapy samples, and matched primary tumor samples. Peripheral blood exosomes of osteosarcoma patients at diagnosis were significantly smaller than those of controls and overexpressed extracellular matrix protein gene THBS1 and B cell markers MS4A1 and TCL1A. Immunohistochemical staining of corresponding tumor samples verified the expression of THBS1 on tumor cells and osteoid matrix, and its persistence in a treatment-refractory patient, as well as the B cell origin of the latter. These hold potential as liquid biopsy biomarkers of disease burden and host immune response in osteosarcoma. Our findings suggest that exosomes may provide novel and clinically-important insights into the pathophysiology of cancers such as osteosarcoma.

GATA4 downregulation enhances CCL20-mediated immunosuppression in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a deadly cancer with a high global mortality rate, and the downregulation of GATA binding protein 4 (GATA4) has been implicated in HCC progression. In this study, we investigated the role of GATA4 in shaping the immune landscape of HCC. METHODS: HCC tumor samples were classified into "low" or "normal/high" based on GATA4 RNA expression relative to adjacent non-tumor liver tissues. The immune landscapes of GATA4-low and GATA4-normal/high tumors were analyzed using cytometry by time-of-flight, bulk/spatial transcriptomic analyses and validated by multiplex immunofluorescence. RESULTS: GATA4-low tumors displayed enrichment in exhausted programmed cell death protein 1+ T cells, immunosuppressive regulatory T cells, myeloid-derived suppressor cells, and macrophages, highlighting the impact of GATA4 downregulation on immunosuppression. Spatial and bulk transcriptomic analyses revealed a negative correlation between GATA4 and C-C Motif Chemokine Ligand 20 (CCL20) expression in HCC. Overexpressing GATA4 confirmed CCL20 as a downstream target, contributing to an immunosuppressive tumor microenvironment, as evidenced by increased regulatory T cells and myeloid-derived suppressor cells in CCL20-high tumors. Lastly, the reduced expression of GATA4 and higher expression of CCL20 were associated with poorer overall survival in patients with HCC, implicating their roles in tumor progression. CONCLUSIONS: Our study reveals that GATA4 downregulation contributes to an immunosuppressive microenvironment, driven by CCL20-mediated enrichment of regulatory T cells and myeloid-derived suppressor cells in HCC. These findings underscore the critical role of GATA4 reduction in promoting immunosuppression and HCC progression.

Transcriptomic convergence despite genomic divergence drive field cancerization in synchronous squamous tumors.

Introduction: Field cancerization is suggested to arise from imbalanced differentiation in individual basal progenitor cells leading to clonal expansion of mutant cells that eventually replace the epithelium, although without evidence. Methods: We performed deep sequencing analyses to characterize the genomic and transcriptomic landscapes of field change in two patients with synchronous aerodigestive tract tumors. Results: Our data support the emergence of numerous genetic alterations in cancer-associated genes but refutes the hypothesis that founder mutation(s) underpin this phenomenon. Mutational signature analysis identified defective homologous recombination as a common underlying mutational process unique to synchronous tumors. Discussion: Our analyses suggest a common etiologic factor defined by mutational signatures and/or transcriptomic convergence, which could provide a therapeutic opportunity.

CD38 marks the exhausted CD8+ tissue-resident memory T cells in hepatocellular carcinoma.

Introduction: Despite recent advances in immunotherapy for hepatocellular carcinoma (HCC), the overall modest response rate underscores the need for a better understanding of the tumor microenvironment (TME) of HCC. We have previously shown that CD38 is widely expressed on tumor-infiltrating leukocytes (TILs), predominantly on CD3+ T cells and monocytes. However, its specific role in the HCC TME remains unclear. Methods: In this current study, we used cytometry time-of-flight (CyTOF), bulk RNA sequencing on sorted T cells, and single-cell RNA (scRNA) sequencing to interrogate expression of CD38 and its correlation with T cell exhaustion in HCC samples. We also employed multiplex immunohistochemistry (mIHC) for validating our findings. Results: From CyTOF analysis, we compared the immune composition of CD38-expressing leukocytes in TILs, non-tumor tissue-infiltrating leukocytes (NIL), and peripheral blood mononuclear cells (PBMC). We identified CD8+ T cells as the dominant CD38-expressing TILs and found that CD38 expression was significantly higher in CD8+ TRM in TILs than in NILs. Furthermore, through transcriptomic analysis on sorted CD8+ TRM from HCC tumors, we observed a higher expression of CD38 along with T cell exhaustion genes, including PDCD1 and CTLA4, compared to the circulating memory CD8 T cells from PBMC. This was validated by scRNA sequencing that revealed co-expression of CD38 with PDCD1, CTLA4, and ITGAE (CD103) in T cells from HCC tumors. The protein co-expression of CD38 and PD-1 on CD8+ T cells was further demonstrated by mIHC on HCC FFPE tissues, marking CD38 as a T cell co-exhaustion marker in HCC. Lastly, the higher proportions of CD38+PD-1+ CD8+ T cells and CD38+PD-1+ TRM were significantly associated with the higher histopathological grades of HCC, indicating its role in the aggressiveness of the disease. Conclusion: Taken together, the concurrent expression of CD38 with exhaustion markers on CD8+ TRM underpins its role as a key marker of T cell exhaustion and a potential therapeutic target for restoring cytotoxic T cell function in HCC.

IFNγ-IL-17+ CD8 T cells contribute to immunosuppression and tumor progression in human hepatocellular carcinoma.

IL-17-producing CD8 (Tc17) T cells have been shown to play an important role in infection and chronic inflammation, however their implications in hepatocellular carcinoma (HCC) remain elusive. In this study, we performed cytometry by time-of-flight (CyTOF) and revealed the distinctive immunological phenotypes of two IFNγ+ and IFNγ- Tc17 subsets that were preferentially enriched in human HCC. Single-cell RNA-sequencing analysis further revealed regulatory circuits governing the different phenotypes of these Tc17 subsets. In particular, we discovered that IFNγ- Tc17 subset demonstrated pro-tumoral characteristics and expressed higher levels of CCL20. This corresponded to increased tumor infiltration of T regulatory cells (Treg) validated by immunohistochemistry in another independent HCC cohort, demonstrating the immunosuppressive functions of IFNγ- Tc17 subset. Most importantly, higher intra-tumoral proportions of IFNγ- Tc17 were associated with poorer prognosis in patients with HCC and this was further validated in The Cancer Genome Atlas (TCGA) HCC cohort. Taken together, this compendium of transcriptomic and proteomic data of Tc17 subsets sheds light on the immunosuppressive phenotypes of IFNγ- Tc17 and its implications in HCC progression.

Pro-metastatic and mesenchymal gene expression signatures characterize circulating tumor cells of neuroblastoma patients with bone marrow metastases and relapse.

Existing marker-based methods of minimal residual disease (MRD) determination in neuroblastoma do not effectively enrich for the circulating disease cell population. Given the relative size differential of neuroblastoma tumor cells over normal hematogenous cells, we hypothesized that cell size-based separation could enrich circulating tumor cells (CTCs) from blood samples and disseminated tumor cells (DTCs) from bone marrow aspirates (BMA) of neuroblastoma patients, and that their gene expression profiles could vary dynamically with various disease states over the course of treatment. Using a spiral microfluidic chip, peripheral blood of 17 neuroblastoma patients at 3 serial treatment timepoints (diagnosis, n=17; post-chemotherapy, n=11; and relapse, n=3), and bone marrow samples at diagnosis were enriched for large intact circulating cells. Profiling the resulting enriched samples with immunohistochemistry and mRNA expression of 1490 cancer-related genes via NanoString, 13 of 17 samples contained CTCs displaying cytologic atypia, TH and PHOX2B expression and/or upregulation of cancer-associated genes. Gene signatures reflecting pro-metastatic processes and the neuroblastoma mesenchymal super-enhancer state were consistently upregulated in 7 of 13 samples, 6 of which also had metastatic high-risk disease. Expression of 8 genes associated with PI3K and GCPR signaling were significantly upregulated in CTCs of patients with bone marrow metastases versus patients without. Correspondingly, in patients with marrow metastases, differentially-expressed gene signatures reflected upregulation of immune regulation in bone marrow DTCs versus paired CTCs samples. In patients who later developed disease relapse, 5 genes involved in immune cell regulation, JAK/STAT signaling and the neuroblastoma mesenchymal super-enhancer state (OLFML2B, STAT1, ARHGDIB, STAB1, TLR2) were upregulated in serial CTC samples over their disease course, despite urinary catecholamines and bone marrow aspirates not indicating the disease recurrences. In summary, using a label-free cell size-based separation method, we enriched and characterized intact circulating cells in peripheral blood indicative of neuroblastoma CTCs, as well as their DTC counterparts in the bone marrow. Expression profiles of pro-metastatic genes in CTCs correlated with the presence of bone marrow metastases at diagnosis, while longitudinal profiling identified persistently elevated expression of genes in CTCs that may serve as novel predictive markers of hematogenous MRD in neuroblastoma patients that subsequently relapse.

Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis.

Peritoneal carcinomatosis (PC) present a ubiquitous clinical conundrum in all intra-abdominal malignancies. Via functional and transcriptomic experiments of ascites-treated PC cells, we identify STAT3 as a key signaling pathway. Integrative analysis of publicly available databases and correlation with clinical cohorts (n = 7,359) reveal putative clinically significant activating ligands of STAT3 signaling. We further validate a 3-biomarker prognostic panel in ascites independent of clinical covariates in a prospective study (n = 149). Via single-cell sequencing experiments, we uncover that PAI-1, a key component of the prognostic biomarker panel, is largely secreted by fibroblasts and mesothelial cells. Molecular stratification of ascites using PAI-1 levels and STAT3 activation in ascites-treated cells highlight a therapeutic opportunity based on a phenomenon of paracrine addiction. These results are recapitulated in patient-derived ascites-dependent xenografts. Here, we demonstrate therapeutic proof of concept of direct ligand inhibition of a prognostic target within an enclosed biological space.

Trajectory of immune evasion and cancer progression in hepatocellular carcinoma.

Immune evasion is key to cancer initiation and later at metastasis, but its dynamics at intermediate stages, where potential therapeutic interventions could be applied, is undefined. Here we show, using multi-dimensional analyses of resected tumours, their adjacent non-tumour tissues and peripheral blood, that extensive immune remodelling takes place in patients with stage I to III hepatocellular carcinoma (HCC). We demonstrate the depletion of anti-tumoural immune subsets and accumulation of immunosuppressive or exhausted subsets along with reduced tumour infiltration of CD8 T cells peaking at stage II tumours. Corresponding transcriptomic modification occur in the genes related to antigen presentation, immune responses, and chemotaxis. The progressive immune evasion is validated in a murine model of HCC. Our results show evidence of ongoing tumour-immune co-evolution during HCC progression and offer insights into potential interventions to reverse, prevent or limit the progression of the disease.

Clinicopathological-Associated Regulatory Network of Deregulated circRNAs in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide. Here, we present a novel strategy to identify key circRNA signatures of clinically relevant co-expressed circRNA-mRNA networks in pertinent cancer-pathways that modulate prognosis of HCC patients, by integrating clinic-pathological features, circRNA and mRNA expression profiles. Through further integration with miRNA expression profiles, clinically relevant competing-endogenous-RNA (ceRNA) networks of circRNA-miRNA-mRNAs were constructed. At least five clinically relevant nodal-circRNAs, co-expressed with numerous genes, were identified from the circRNA-mRNA networks. These nodal circRNAs upregulated proliferation (except circRaly) and transformation in cells. The most upregulated nodal-circRNA, circGPC3, associated with higher-grade tumors and co-expressed with 33 genes, competes with 11 mRNAs for two shared miRNAs. circGPC3 was experimentally demonstrated to upregulate cell-cycle and migration/invasion in both transformed and non-transformed liver cell-lines. circGPC3 was further shown to act as a sponge of miR-378a-3p to regulate APSM (Abnormal spindle-like microcephaly associated) expression and modulate cell transformation. This study identifies 5 key nodal master circRNAs in a clinically relevant circRNA-centric network that are significantly associated with poorer prognosis of HCC patients and promotes tumorigenesis in cell-lines. The identification and characterization of these key circRNAs in clinically relevant circRNA-mRNA and ceRNA networks may facilitate the design of novel strategies targeting these important regulators for better HCC prognosis.

Intratumoral CD39+CD8+ T Cells Predict Response to Programmed Cell Death Protein-1 or Programmed Death Ligand-1 Blockade in Patients With NSCLC.

INTRODUCTION: Programmed cell death protein-1 (PD-1) and programmed death-ligand 1 (PD-L1) blockade is currently widely used in the treatment of metastatic NSCLC. Despite available biomarker stratification, clinical responses vary. Thus, the search for novel biomarkers with improved response prediction is ongoing. Previously, using mass cytometry or cytometry by time-of-flight (CyTOF), our group demonstrated that CD39+CD8+ immune cells represent tumor antigen-specific, cytotoxic T cells in treatment-naive NSCLC. We hypothesized that accurate quantitation of this T cell subset would predict immunotherapy outcome. METHODS: To translate this to a clinical setting, the present study compared CyTOF data with a range of clinically relevant methods, including conventional immunohistochemistry (IHC), multiplex IHC or immunofluorescence (mIHC), and gene expression assay by NanoString. RESULTS: Quantification using mIHC but not conventional IHC or NanoString correlated with the CyTOF results. The specificity and sensitivity of mIHC were then evaluated in a separate retrospective NSCLC cohort. CD39+CD8+ T cell proportion, as determined by mIHC, successfully stratified responders and nonresponders to PD-1 or PD-L1 inhibitors (objective response rate of 63.6%, compared with 0% for the negative group). This predictive capability was independent from other confounding factors, such as total CD8+ T cell proportion, CD39+ lymphocyte proportion, PD-L1 positivity, EGFR mutation status, and other clinicopathologic parameters. CONCLUSIONS: Our results suggest that the mIHC platform is a clinically relevant method to evaluate CD39+CD8+ T cell proportion and that this marker can serve as a potential biomarker that predicts response to PD-1 or PD-L1 blockade in patients with NSCLC. Further validation in additional NSCLC cohorts is warranted.

Highly deregulated lncRNA LOC is associated with overall worse prognosis in Hepatocellular Carcinoma patients.

Although numerous long non-coding RNAs (lncRNAs) were reported to be deregulated in Hepatocellular Carcinoma (HCC), experimentally characterized, and/or associated with patient's clinical characteristics, there is, thus far, minimal concerted research strategy to identify deregulated lncRNAs that modulate prognosis of HCC patients. Here, we present a novel strategy where we identify lncRNAs, which are not only de-regulated in HCC patients, but are also associated with pertinent clinical characteristics, potentially contributing to the prognosis of HCC patients. LOC101926913 (LOC) was further characterized because it is the most highly differentially expressed amongst those that are associated with the most number of clinical features (tumor-stage, vascular and tumor invasion and poorer overall survival). Experimental gain- and loss-of-function manipulation of LOC in liver cell-lines highlight LOC as a potential onco-lncRNA promoting cell proliferation, anchorage independent growth and invasion. LOC expression in cells up-regulated genes involved in GTPase-activities and downregulated genes associated with cellular detoxification, oxygen- and drug-transport. Hence, LOC may represent a novel therapeutic target, modulating prognosis of HCC patients through up-regulating GTPase-activities and down-regulating detoxification, oxygen- and drug-transport. This strategy may thus be useful for the identification of clinically relevant lncRNAs as potential biomarkers/targets that modulate prognosis in other cancers as well.

Individualized Molecular Profiling for Allocation to Clinical Trials Singapore Study-An Asian Tertiary Cancer Center Experience.

PURPOSE: Precision oncology has transformed the management of advanced cancers through implementation of advanced molecular profiling technologies to identify increasingly defined subsets of patients and match them to appropriate therapy. We report outcomes of a prospective molecular profiling study in a high-volume Asian tertiary cancer center. PATIENTS AND METHODS: Patients with advanced cancer were enrolled onto a prospective protocol for genomic profiling, the Individualized Molecular Profiling for Allocation to Clinical Trials Singapore study, at the National Cancer Center Singapore. Primary objective was to identify molecular biomarkers in patient's tumors for allocation to clinical trials. The study commenced in February 2012 and is ongoing, with the results of all patients who underwent multiplex next-generation sequencing (NGS) testing until December 2018 presented here. The results were discussed at a molecular tumor board where recommendations for allocation to biomarker-directed trials or targeted therapies were made. RESULTS: One thousand fifteen patients were enrolled with a median age of 58 years (range 20-83 years). Most common tumor types were lung adenocarcinoma (26%), colorectal cancer (15%), and breast cancer (12%). A total of 1,064 NGS assays were performed, on fresh tumor tissue for 369 (35%) and archival tumor tissue for 687 (65%) assays. TP53 (39%) alterations were most common, followed by EGFR (21%), KRAS (14%), and PIK3CA (10%). Of 405 NGS assays with potentially actionable alterations, 111 (27%) were allocated to a clinical trial after molecular tumor board and 20 (4.9%) were enrolled on a molecularly matched clinical trial. Gene fusions were detected in 23 of 311 (7%) patients tested, including rare fusions in new tumor types and known fusions in rare tumors. CONCLUSION: Individualized Molecular Profiling for Allocation to Clinical Trials Singapore demonstrates the feasibility of a prospective broad molecular profiling program in an Asian tertiary cancer center, with the ability to develop and adapt to a dynamic landscape of precision oncology.

Radioembolisation with Y90-resin microspheres followed by nivolumab for advanced hepatocellular carcinoma (CA 209-678): a single arm, single centre, phase 2 trial.

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.

Intratumoural immune heterogeneity as a hallmark of tumour evolution and progression in hepatocellular carcinoma.

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.