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Table S5 shows 16 up-regulated genes and 10 predicted potential transcript factors in the global expression analysis

Table S1 shows clinicopathologic characteristics of 50 CRC patients

Figure S5. Kaplan-Meier analysis demonstrating RFS and OS for all enrolled patients stratified by their status of follow up ctDNA.

Somatic mutations identified in tissue samples and plasma samples of the enrolled patients

Figure S2 indicates that higher PNO1 mRNA levels are associated with poorer overall survival, relapse-free survival and shorter time of first progression of patients with breast or lung cancer. PNO1 knockdown decreased cell viability and cell survival of lung and breast cancer cells.

Table S4 summaries the characteristics of antibodies for analyses of Western-bloting, immunofluorescence, immunohistochemistry and co-immunoprecipitation.

Purpose:Circulating tumor DNA (ctDNA) provides a novel approach for detecting tumor burden and predicting clinical outcomes of hepatocellular carcinoma (HCC). Here, we performed a thorough evaluation of HCC circulating genetic features and further fully integrated them to build a robust strategy for HCC monitoring and prognostic outcome assessment.Experimental Design:We performed target sequencing and low-coverage whole-genome sequencing on plasma samples collected from 34 long-term follow-up patients with HCC to capture tumor somatic SNVs and CNVs, respectively. Clinical information was also obtained to evaluate the prognostic performance of ctDNA comparing with clinically applied protein biomarkers.Results:All plasma samples before surgery showed somatic genetic variations resembling corresponding tumor tissues. During follow-up, SNVs and CNVs dynamically changed correlating to patients' tumor burden. We integrated the comprehensive ctDNA mutation profiles to provide a robust strategy to accurately assess patients' tumor burden with high consistence comparing with imaging results. This strategy could discover tumor occurrence in advance of imaging for an average of 4.6 months, and showed superior performance than serum biomarkers AFP, AFP-L3%, and Des-Gamma-Carboxy Prothrombin (DCP). Furthermore, our strategy could precisely detect minimal residual disease (MRD) in advance and predict patients' prognostic outcomes for both relapse-free survival (P = 0.001) and overall survival (P = 0.001); further combining ctDNA with DCP could increase the sensitivity for MRD detection.Conclusions:We demonstrated that plasma CNV and SNV levels dynamically correlated with patients' tumor burden in HCC. Our strategy of comprehensive mutation profile integration could accurately and better evaluate patients' prognostic risk and detect tumor occurrence in advance than traditional strategies.

Figure S2. The concordance of somatic SNVs detected in preoperative plasma sample and matched tumor in enrolled patients. A. The bar plot demonstrating the R2 of Pearson correlation between the SNVs' mutant allele frequency in preoperative plasma and in corresponding tumor tissues. B. The representative scatter plots showing the correlation of somatic SNVs' mutant allele frequency in preoperative plasma between whole exome sequencing and target sequencing. Each somatic SNV is represented as a single circle.

Clinical characteristics of 34 enrolled HCC patients.

Table S6 indicates that PNO1 mRNA expression is significantly up-regulated in various types of malignancies, including CRC, lung cancer, and breast cancer.

The RNA-binding protein PNO1 is critical for ribosome biogenesis, but its potential role in cancer remains unknown. In this study, online data mining, cDNA, and tissue microarrays indicated that PNO1 expression was higher in colorectal cancer tissue than in noncancerous tissue, and its overexpression was associated with worse patient survival. Gain-of-function and loss-of-function studies demonstrated that PNO1 knockdown suppressed growth of colorectal cancer cells in vitro and in vivo, while PNO1 overexpression promoted colorectal cancer cell proliferation in vitro. In colorectal cancer cells expressing wild-type p53, PNO1 knockdown enhanced expression of p53 and its downstream gene p21, and reduced cell viability; these effects were prevented by p53 knockout and attenuated by the p53 inhibitor PFT-α. Moreover, PNO1 knockdown in HCT116 cells decreased levels of 18S rRNA, of 40S and 60S ribosomal subunits, and of the 80S ribosome. It also reduced global protein synthesis, increasing nuclear stress and inhibiting MDM2-mediated ubiquitination and p53 degradation. Overexpressing EBF1 suppressed PNO1 promoter activity and decreased PNO1 mRNA and protein, inhibiting cell proliferation and inducing cell apoptosis through the p53/p21 pathway. In colorectal cancer tissues, the expression of EBF1 correlated inversely with PNO1. Data mining of online breast and lung cancer databases showed increased PNO1 expression and association with poor patient survival; PNO1 knockdown reduced cell viability of cultured breast and lung cancer cells. Taken together, these findings indicate that PNO1 is overexpressed in colorectal cancer and correlates with poor patient survival, and that PNO1 exerts oncogenic effects, at least, in part, by altering ribosome biogenesis.Significance:This study identifies the ribosome assembly factor PNO1 as a potential oncogene involved in tumor growth and progression of colorectal cancer.

Figure S1 shows that the expression levels of endogenous PNO1 in cultured CRC cell lines are relatively higher than that of differentiated Caco2 cells, and PNO1 knockdown reduces cell viability and survival of CRC cells.

The long reads of Nanopore sequencing permit accurate transcript assembly and ease in discovering novel transcripts with potentially important functions in cancers. The wide adoption of Nanopore sequencing for transcript quantification, however, is largely limited by high costs. To address this issue, we developed a bioinformatics software, NovelQuant, that can specifically quantify long‐read‐assembled novel transcripts with short‐read sequencing data. Nanopore Direct RNA Sequencing was carried out on three hepatocellular carcinoma (HCC) patients’ tumor, matched portal vein tumor thrombus, and peritumor to reconstruct the HCC transcriptome. Then, based on the reconstructed transcriptome, NovelQuant was applied on Illumina RNA sequencing data of 59 HCC patients’ tumor and paired peritumor to quantify novel transcripts. Our further analysis revealed 361 novel transcripts dysregulated in HCC and that 101 of them were significantly associated with prognosis. There were 19 novel prognostic transcripts predicted to be long noncoding RNAs (lncRNAs), and some of them had regulatory targets that were reported to be associated with HCC. Additionally, 42 novel prognostic transcripts were predicted to be protein‐coding mRNAs, and many of them could be involved in xenobiotic metabolism. Moreover, the tumor‐suppressive roles of two representative novel prognostic transcripts, CDO1‐novel (lncRNA) and CYP2A6‐novel (protein‐coding mRNA), were further functionally validated during HCC progression. Overall, the current study shows a possibility of combining long‐ and short‐read sequencing to explore functionally important novel transcripts in HCC with accuracy and cost‐efficiency, which expands the pool of molecular biomarkers that could enhance our understanding of the molecular mechanisms of HCC., We used Nanopore RNA sequencing to discover novel transcripts in hepatocellular carcinoma (HCC), and developed NovelQuant to specifically quantify novel transcripts. Many of these novel transcripts were further found to be predictive of prognosis and have functional roles in HCC.

Personalized neoantigen vaccine could induce a robust antitumor immune response in multiple cancers, whose efficacy could be further enhanced by combining with programmed cell death 1 blockade (α-PD-1). However, the corresponding immune response and synergistic mechanisms remain largely unclear. Here, we aimed to develop clinically available combinational therapeutic strategy and further explore its potential antitumor mechanisms in hepatocellular carcinoma (HCC).Neoantigen peptide vaccine (NeoVAC) for murine HCC cell line Hepa1-6 was developed and optimized by neoantigen screening and adjuvant optimization. Then the synergistic efficacy and related molecular mechanisms of NeoVAC combined with α-PD-1 in HCC were evaluated by orthotopic HCC mouse model, single-cell RNA sequencing, tetramer flow cytometry, immunofluorescence, etc. The tumor-killing capacity of CD8NeoVAC, which consisted of seven high immunogenic neoantigen peptides and clinical-grade Poly(I:C), could generate a strong antitumor immune response in HCC mouse models. Significantly, its efficacy could be further improved by combining with α-PD-1, with 80% of durable tumor regression and long-term immune memory in orthotopic HCC models. Moreover, in-depth analysis of the tumor immune microenvironment showed that the percentage of CD8This study showed that NeoVAC plus α-PD-1 could induce a strong antitumor response and long-term tumor-specific immune memory in HCC by increasing CD8

Objective Neoantigens derived from tumor-specific genomic alterations have demonstrated great potential for immunotherapeutic interventions in cancers. However, the comprehensive profile of hepatocellular carcinoma (HCC) neoantigens and their complex interplay with immune microenvironment and tumor evolution have not been fully addressed. Methods Here we integrated whole exome sequencing data, transcriptome sequencing data and clinical information of 72 primary HCC patients to characterize the HCC neoantigen profile, and systematically explored its interactions with tumor clonal evolution, driver mutations and immune microenvironments. Results We observed that higher somatic mutation/neoantigen load was associated with better clinical outcomes and HCC patients could be further divided into two subgroups with distinct prognosis based on their neoantigen expression patterns. HCC subgroup with neoantigen expression probability high (NEP-H) showed more aggressive pathologic features including increased incidence of tumor thrombus (P=0.038), higher recurrence rate (P=0.029), more inclined to lack tumor capsule (P=0.026) and with more microsatellite instability sites (P=0.006). In addition, NEP-H subgroup was also characterized by higher chance to be involved in tumor clonal evolution [odds ratio (OR)=46.7, P

Cell survival rate determines engraftment efficiency in adipose-derived mesenchymal stem cell (ADSC)-based regenerative medicine.

Circular RNA (circRNA) can participate in various biological processes, including tumorigenesis, through their microRNA response elements. Alterations in circRNA profiles during hepatocellular carcinoma (HCC) progression and their clinical significance remain unclear. Here, we present extensive analysis of circRNA profiles in tumor and matched peritumor tissues collected from 10 HCC patients, conducted to identify circRNA related to HCC progression. A total of 42 dysregulated circRNA (38 down-regulated and 4 up-regulated) were identified in HCC tumor tissues compared with matched peritumor tissues, revealing the heterogeneity of circRNA profiles in HCC. CircADAMTS13, derived from Exon 13-14 of the ADAMTS13 gene, was significantly downregulated in HCC tumor tissues. Furthermore, clinicopathological analysis revealed that up-regulation of circADAMTS13 was negatively associated with tumor size but positively associated with prognosis. In addition, overexpression of circADAMTS13 could markedly inhibit HCC cell proliferation in vitro. Bioinformatic analysis and luciferase reporter assays further revealed that circADAMTS13 directly interacts with microRNA (miR)-484. Rescue experiments showed that miR-484 mimics can reverse the tumor-suppressing roles of circADAMTS13 in HCC. Therefore, our results demonstrated that circADAMTS13 can serve as a tumor suppressor during HCC progression via the functional pathway of sponging miR-484.

Feng Chen,1,2 Lei He,2,3 Liman Qiu,2,3 Yang Zhou,2,3 Zhenli Li,2,3 Geng Chen,2,3 Fuli Xin,2,3 Xiuqing Dong,2,3 Haipo Xu,2,3 Gaoxiong Wang,4 Jingfeng Liu,2,3 Zhixiong Cai2,3 1College of Life Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, People’s Republic of China; 2The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025, People’s Republic of China; 3Mengchao Med-X Center, Fuzhou University, Fuzhou, 350025, People’s Republic of China; 4Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Fujian Medical University, Quanzhou, 362001, People’s Republic of ChinaCorrespondence: Gaoxiong WangDepartment of Hepatobiliary Surgery, The Second Affiliated Hospital of Fujian Medical University, Zhongshan North Road 34, Quanzhou, 362001, People’s Republic of ChinaTel/Fax +86 591 8370 5927Email wanggaoxiong2013@163.comZhixiong CaiThe United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Xihong Road 312, Fuzhou, Fujian, 350025, People’s Republic of ChinaTel/Fax +86 591 8370 5927Email caizhixiong1985@163.comBackground: Circular RNAs (circRNAs) could interact with miRNAs to regulate gene expression, participating in hepatocellular carcinoma (HCC) initiation and development. This work aimed to determine the potential function and molecular mechanism of circEPB41L2 (hsa_circ_0077837) during HCC progression.Materials and Methods: The expression of circEPB41L2 in HCC tissues and HCC cell lines was quantified using real-time quantitative PCR (qRT-PCR). CCK-8 assays and colony formation assays were utilized to detect the proliferation of HCC cells. Wound healing assay and transwell assay were performed to determine the capability of migration and invasion for HCC cells. Western blot was conducted to determine gene expression on protein levels. The effect of circEPB41L2 on HCC in vivo was investigated via xenograft experiment. Interaction between circEPB41L2 and miR-590-5p was predicted through bioinformatics methods and confirmed via luciferase reporter assay.Results: Extensive analysis of circRNA profiles in tumor and matched para-tumor tissues collected from 61 HCC patients identified that circEPB41L2 was significantly down-regulated in HCC, which was further confirmed in another HCC group by qRT-PCR analysis. The clinicopathological analysis revealed that down-regulation of circEPB41L2 was negatively associated with tumor size, vascular invasion and alpha-fetoprotein, while positively correlated with HCC prognosis. The biological function experiments showed that overexpression of circEPB41L2 could obviously inhibit the proliferation and metastasis of HCC cells in vitro, while knockdown of circEPB41L2 induced opposite results. Moreover, we also found that circEPB41L2 inhibited HCC migration and invasion though EMT signaling pathway. Similarly, overexpression of circEPB41L2 can also significantly inhibit the proliferation of HCC cells in vivo. Bioinformatic analysis and luciferase reporter assay revealed that circEPB41L2 interacts directly with miR-590-5p and the corresponding biological functions were also verified in miRNA rescue experiments.Conclusion: Our results suggest that circEPB41L2 might function as a tumor suppressor during HCC progression by sponging miR-590-5p.Keywords: circular RNA, circEPB41L2, hepatocellular carcinoma, miR-590-5p

BackgroundCollecting duct carcinoma (CDC) of the kidney is a rare and highly aggressive malignant tumor with the worst prognosis among all renal cancers. Nevertheless, the first-line treatments, including chemotherapy and target therapy, usually show poor response to CDC. Recent studies have suggested that immunotherapy targeting personal tumor-specific neoantigens could be a promising strategy for several solid cancers. However, whether it has therapeutic potential in CDC remains unclear.Case presentationHere, we report a case of an Asian patient who underwent personalized neoantigen-based immunotherapy. The patient was diagnosed with metastatic CDC and suffered extensive tumor progression following sorafenib treatment. Based on the patient’s own somatic mutational profile, a total of 13 neoantigens were identified and corresponding long-peptide vaccine and neoantigen-reactive T cells (NRTs) were prepared. After six cycles of neoantigen-based vaccination and T-cell immunotherapy, the patient was reported with stable disease status in tumor burden and significant alleviation of bone pain. Ex vivo interferon-γ enzyme-linked immunospot assay proved the reactivity to 12 of 13 neoantigens in peripheral blood mononuclear cells collected after immunotherapy, and the preferential reactivity to mutant peptides compared with corresponding wild-type peptides was also observed for 3 of the neoantigens. Surprisingly, biopsy sample collected from CDC sites after 3 months of immunotherapy showed decreased mutant allele frequency corresponding to 92% (12/13) of the neoantigens, indicating the elimination of tumor cells carrying these neoantigens.ConclusionsOur case report demonstrated that the combined therapy of neoantigen peptide vaccination and NRT cell infusion showed certain efficacy in this CDC case, even when the patient carried only a relatively low tumor mutation burden. These results indicated that the personalized neoantigen-based immunotherapy was a promising new strategy for advanced CDC.Trial registration numberChiCTR1800017836.

Tuberculosis is one of the most serious infectious diseases, resulting in death worldwide. Traditional detection methods are not enough to meet the clinical requirements of rapid diagnosis, high specificity, and high sensitivity. Fast, sensitive, and accurate detection of Mycobacterium tuberculosis (MTB) is urgently needed to treat and control tuberculosis disease. Clustered regularly interspaced short palindromic repeats (CRISPR)–associated proteins (Cas12a) exhibit strong nonspecific degradation ability of exogenous single-strand nucleic acids (trans cleavage) after specific recognition of target sequence. We purified Cas12a protein and selected a proper guide RNA based on conserved sequences of MTB from designed guide RNA library. Then, we proposed a novel detection method based on recombinase polymerase amplification and CRISPR/Cas12a nuclease system for specific and sensitive detection of MTB DNA. The assay, based on fluorescence detection, showed 4.48 fmol/L of limit of detection and good linear correlation of concentration with fluorescence value (R2 = 0.9775). It also showed good performance in distinguishing other bacteria. Furthermore, its clinical performance was evaluated by 193 samples and showed sensitivity of 99.29% (139/140) and specificity of 100% (53/53) at 99% CI, compared with culture method. Taken together, the CRISPR/Cas12a system showed good specificity, excellent sensitivity, and excellent accuracy for MTB detection, and it meets requirements of MTB detection in clinical samples and has great potential for clinical translation.

Background: Neoantigens derived from tumor specific genomic alterations have demonstrated great potential for immunotherapeutic interventions in cancers. However, the comprehensive profile of HCC neoantigens and their complex interplay with immune microenvironment and tumor evolution have not been fully addressed. Methods: We integrated whole exome sequencing data, transcriptome sequencing data and clinical information of 72 primary HCC patients to characterize the HCC neoantigen profile, and systematically explore its interactions with tumor clonal evolution, driver mutations and immune microenvironments. Findings: Besides the observation that higher somatic neoantigen load indicated a better clinical outcome in HCC, it is important that we discovered two subgroups with distinct neoantigen expression pattern, which exhibited markedly different features including tumor clonal evolution states, microsatellite instability as well as clinical outcomes. Gene set enrichment analysis revealed that upregulation of MYC and its targets could suppress immune responses and elevate neoantigen expression in tumor cells. Furthermore, we discovered an immune escape mechanism that tumors could become more inconspicuous by evolving subclones with less immunogenicity. We also observed that smaller clonal mutation clusters with higher immunogenicity in tumor were more likely to involve in clonal evolution. We also discovered series of neoantigenic hotspot genes, which could serve as potential actionable targets for HCC in future. Interpretation: Our results revealed the landscape of HCC neoantigens and discovered two clinically relevant subgroups with distinct neoantigen expression patterns, suggesting the neoantigen expression should be fully considered in future immunotherapeutic interventions. Funding Statement: This work was supported by the National Science and Technology Major Project of China (grant No.2018ZX10302205), the Science and Technology Development Project of Central government Guiding Local Government (Grant No. 2017L3017, Grant No. 2018L3016, Grant No. 2019L3027), the Scientific Foundation of Fujian Province (Grant No.2018J01145), the Scientific Foundation of Fujian Health and family planning Department (Grant No. 2019-ZQN87), the Joint Funds for the Innovation of Science and Technology of Fujian Province (Grant No. 2018Y9121). Declaration of Interests: All authors declare no competing interests related to this study. Ethics Approval Statement: All human sample collection and the usage of human samples were approved by the Institution Review Board of Mengchao Hepatobiliary Hospital of Fujian Medical University and were conducted according to the principles of the Declaration of Helsinki. Written informed consent was obtained from the enrolled patient.

Background/Aims: Aberrant RNA editing, mediated by adenosine deaminases acting on RNA (ADAR), serves as a post-transcriptional event participating in tumorigenesis and prognosis. However, the RNA editing profiles during HCC progression and their clinical correlations remain unclear. Methods: Multiple tissue samples were collected from an advanced HCC patient. RNA-seq was performed to obtain the RNA editing profiles for each sample. Two RNA editing sites from CDK13 were further validated in 60 HCC patients; and their potential regulatory mechanisms were investigated. Results: In-depth analysis of the RNA-seq data revealed a significant number of editing sites (632-816) in coding regions for each tissue sample, showing branched evolution during tumorigenesis and metastasis. Two editing sites (Q103R and K96R) in CDK13 showed significant over-editing in tumor, and these phenomenon were validated in 60 HCC patients. Furthermore, the clinicopathological analysis revealed that these CDK13 over-editing sites were positively associated with TNM, PVTT and poor prognosis. In addition, the editing level of these sites were significantly correlated with the expression of ADAR1. Loss of function assays further proved that these CDK13 over-editing sites were mediated by ADAR1 in HCC cells. Conclusions: CDK13 RNA over-editing sites mediated by ADAR1 may serve as novel cancer driver events in HCC progression.

Background: Myocardial infarction is one of the leading causes of morbidity and mortality worldwide, triggering irreversible myocardial cell damage and heart failure. The role of low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) as coreceptors of the Wnt/β-catenin pathway in the adult heart remain unknown. Insulin-like growth factor binding protein 4 and dickkopf-related protein 1 (Dkk1) are 2 secreted LRP5/6 binding proteins that play a crucial role in heart development through preventing Wnt/β-catenin pathway activation. However, their roles in the adult heart remain unexplored. Methods: To understand the role of LRP5/6 and β-catenin in the adult heart, we constructed conditional cardiomyocyte-specific LRP5/6 and β-catenin knockout mice and induced surgical myocardial infarction. We also directly injected recombinant proteins of insulin-like growth factor binding protein 4 and Dkk1 into the heart immediately following myocardial infarction to further examine the mechanisms through which these proteins regulate LRP5/6 and β-catenin. Results: Deletion of LRP5/6 promoted cardiac ischemic insults. Conversely, deficiency of β-catenin, a downstream target of LRP5/6, was beneficial in ischemic injury. It is interesting to note that although both insulin-like growth factor binding protein 4 and Dkk1 are secreted Wnt/β-catenin pathway inhibitors, insulin-like growth factor binding protein 4 protected the ischemic heart by inhibiting β-catenin, whereas Dkk1 enhanced the injury response mainly through inducing LRP5/6 endocytosis and degradation. Conclusions: Our findings reveal previously unidentified dual roles of LRP5/6 involved in the cardiomyocyte response to ischemic injury. These findings suggest new therapeutic strategies in ischemic heart disease by fine-tuning LRP5/6 and β-catenin signaling within the Wnt/β-catenin pathway.

As key players in HCC antitumor response, the functions of tumor infiltrated CD8+ T cells are significantly affected by surrounding microenvironment. A detailed profiling of their genomic and transcriptional changes could provide valuable insights for both future immunotherapy development and prognosis evaluation. We performed whole exome and transcriptome sequencing on tumor infiltrated CD8+ T cells and CD8+ T cells isolated from other tissue origins (peritumor tissues and corresponding PBMCs) in eight treatment-naive HCC patients. The results demonstrated that transcriptional changes, rather than genomic alterations were the main contributors to the functional alterations of CD8+ T cells in the process of tumor progression. The origins of CD8+ T cells defined their transcriptional landscape, while the tumor infiltrated CD8+ T cells shared more similarity with peritumor-derived CD8+ T cells compared with those CD8+ T cells in blood. In addition, tumor infiltrated CD8+ T cells also showed larger transcriptional heterogeneity among individuals, which was modulated by clinical features such as HBV levels, preoperative anti-viral treatment and the degree of T cell infiltration. We also identified multiple inter-connected pathways involved in the activation and exhaustion of tumor infiltrated CD8+ T cells, among which IL-12 mediated pathway could dynamically reflect the functional status of CD8+ TILs and activation of this pathway indicated a better prognosis. Our results presented an overview picture of CD8+ TILs' genomic and transcriptional landscape and features, as well as how the functional status of CD8+ TILs correlated with patients' clinical course.

Purpose: Circulating tumor DNA (ctDNA) provides a novel approach for detecting tumor burden and predicting clinical outcomes of hepatocellular carcinoma (HCC). Here, we performed a thorough evaluation of HCC circulating genetic features and further fully integrated them to build a robust strategy for HCC monitoring and prognostic outcome assessment. Experimental Design: We performed target sequencing and low-coverage whole-genome sequencing on plasma samples collected from 34 long-term follow-up patients with HCC to capture tumor somatic SNVs and CNVs, respectively. Clinical information was also obtained to evaluate the prognostic performance of ctDNA comparing with clinically applied protein biomarkers. Results: All plasma samples before surgery showed somatic genetic variations resembling corresponding tumor tissues. During follow-up, SNVs and CNVs dynamically changed correlating to patients' tumor burden. We integrated the comprehensive ctDNA mutation profiles to provide a robust strategy to accurately assess patients' tumor burden with high consistence comparing with imaging results. This strategy could discover tumor occurrence in advance of imaging for an average of 4.6 months, and showed superior performance than serum biomarkers AFP, AFP-L3%, and Des-Gamma-Carboxy Prothrombin (DCP). Furthermore, our strategy could precisely detect minimal residual disease (MRD) in advance and predict patients' prognostic outcomes for both relapse-free survival (P = 0.001) and overall survival (P = 0.001); further combining ctDNA with DCP could increase the sensitivity for MRD detection. Conclusions: We demonstrated that plasma CNV and SNV levels dynamically correlated with patients' tumor burden in HCC. Our strategy of comprehensive mutation profile integration could accurately and better evaluate patients' prognostic risk and detect tumor occurrence in advance than traditional strategies.

As key players in HCC antitumor response, the functions of tumor infiltrated CD8(+) T cells are significantly affected by surrounding microenvironment. A detailed profiling of their genomic and transcriptional changes could provide valuable insights for both future immunotherapy development and prognosis evaluation. We performed whole exome and transcriptome sequencing on tumor infiltrated CD8(+) T cells and CD8(+) T cells isolated from other tissue origins (peritumor tissues and corresponding PBMCs) in eight treatment-naive HCC patients. The results demonstrated that transcriptional changes, rather than genomic alterations were the main contributors to the functional alterations of CD8(+) T cells in the process of tumor progression. The origins of CD8(+) T cells defined their transcriptional landscape, while the tumor infiltrated CD8(+) T cells shared more similarity with peritumor-derived CD8(+) T cells compared with those CD8(+) T cells in blood. In addition, tumor infiltrated CD8(+) T cells also showed larger transcriptional heterogeneity among individuals, which was modulated by clinical features such as HBV levels, preoperative anti-viral treatment and the degree of T cell infiltration. We also identified multiple inter-connected pathways involved in the activation and exhaustion of tumor infiltrated CD8(+) T cells, among which IL-12 mediated pathway could dynamically reflect the functional status of CD8(+) TILs and activation of this pathway indicated a better prognosis. Our results presented an overview picture of CD8(+) TILs’ genomic and transcriptional landscape and features, as well as how the functional status of CD8(+) TILs correlated with patients’ clinical course.

As a key player in HCC antitumor response, the functions of tumor infiltrated CD8 T cells are significantly affected by surrounding microenvironment. A detailed profiling of their genomic and transcriptional changes could provide valuable insights for both future immunotherapy development and prognosis evaluation. We performed whole exome and transcriptome sequencing on tumor infiltrated CD8 T cells and CD8 T cells isolated from other tissue origins (peritumor and corresponding PBMCs) in eight treatment-naive HCC patients. The results demonstrated that transcriptional changes, rather than genomic alterations were the main contributors to the functional alterations of CD8 T cells in the process of carcinogenesis. The origins of CD8 T cells defined their transcriptional landscape, while tumor infiltrated CD8 T cells shared more similarity with peritumor-derived CD8 T cells compared with those CD8 T cells in blood. In addition, tumor infiltrated CD8 T cells also showed larger transcriptional heterogeneity among individuals, which was modulated by clinical features such as HBV levels, preoperative anti-viral treatment and the degree of T cell infiltration. We also identified multiple inter-connected pathways involved in the activation and exhaustion of tumor infiltrated CD8 T cells, among which IL-12 mediated pathway could dynamically reflect the functional status of CD8 TILs and activation of this pathway indicated a better prognosis. Our results presented an overview picture of CD8 TILs' genomic and transcriptional landscape and features, as well as how the functional status of CD8 TILs correlated with patients' clinical course. Funding: This work was supported by the Science and Technology major project of Fuzhou (Grant No. RongKe 16 in 2016); the Scientific Foundation of Fujian Health and family planning Department (Grant No. 2018-1-92); the Scientific Foundation of Fuzhou City (Grant No. 2017-S-131-3); the Startup Fund for scientific research, Fujian Medical University (Grant No.2016QH085); the Scientific Foundation of Fuzhou Health Department (Grant No. 2017-S-wq38); the Science and Technology development project of central government guiding local government (Grant No.2017L3017). Declaration of Interests: All authors declare no competitive interests related to this study. Ethical Approval Statement: A written informed consent was obtained from each participant, and all of the study protocols were approved by the Institution Review Board of Mengchao Hepatobiliary Hospital of Fujian Medical University.