bims-tumhet Biomed News
on Tumor Heterogeneity
Issue of 2021–06–06
sixteen papers selected by
Sergio Marchini, Humanitas Research



  1. Genome Med. 2021 May 31. 13(1): 96
       BACKGROUND: Cell-free DNA (cfDNA) profiling is increasingly used to guide cancer care, yet mutations are not always identified. The ability to detect somatic mutations in plasma depends on both assay sensitivity and the fraction of circulating DNA in plasma that is tumor-derived (i.e., cfDNA tumor fraction). We hypothesized that cfDNA tumor fraction could inform the interpretation of negative cfDNA results and guide the choice of subsequent assays of greater genomic breadth or depth.
    METHODS: Plasma samples collected from 118 metastatic cancer patients were analyzed with cf-IMPACT, a modified version of the FDA-authorized MSK-IMPACT tumor test that can detect genomic alterations in 410 cancer-associated genes. Shallow whole genome sequencing (sWGS) was also performed in the same samples to estimate cfDNA tumor fraction based on genome-wide copy number alterations using z-score statistics. Plasma samples with no somatic alterations detected by cf-IMPACT were triaged based on sWGS-estimated tumor fraction for analysis with either a less comprehensive but more sensitive assay (MSK-ACCESS) or broader whole exome sequencing (WES).
    RESULTS: cfDNA profiling using cf-IMPACT identified somatic mutations in 55/76 (72%) patients for whom MSK-IMPACT tumor profiling data were available. A significantly higher concordance of mutational profiles and tumor mutational burden (TMB) was observed between plasma and tumor profiling for plasma samples with a high tumor fraction (z-score≥5). In the 42 patients from whom tumor data was not available, cf-IMPACT identified mutations in 16/42 (38%). In total, cf-IMPACT analysis of plasma revealed mutations in 71/118 (60%) patients, with clinically actionable alterations identified in 30 (25%), including therapeutic targets of FDA-approved drugs. Of the 47 samples without alterations detected and low tumor fraction (z-score<5), 29 had sufficient material to be re-analyzed using a less comprehensive but more sensitive assay, MSK-ACCESS, which revealed somatic mutations in 14/29 (48%). Conversely, 5 patients without alterations detected by cf-IMPACT and with high tumor fraction (z-score≥5) were analyzed by WES, which identified mutational signatures and alterations in potential oncogenic drivers not covered by the cf-IMPACT panel. Overall, we identified mutations in 90/118 (76%) patients in the entire cohort using the three complementary plasma profiling approaches.
    CONCLUSIONS: cfDNA tumor fraction can inform the interpretation of negative cfDNA results and guide the selection of subsequent sequencing platforms that are most likely to identify clinically-relevant genomic alterations.
    Keywords:  Cancer; Liquid biopsy; Molecular diagnostic; Noninvasive; Plasma DNA; Sequencing
    DOI:  https://doi.org/10.1186/s13073-021-00898-8
  2. BMC Med Genomics. 2021 May 31. 14(1): 142
       BACKGROUND: Allelic imbalance (AI) in tumors is caused by chromosomal and sub-chromosomal gains and losses.
    RESULTS: We evaluated AI at 109,086 germline exonic SNP loci in four cancer types, and identified a set of SNPs that demonstrate strong tumor allele specificity in AI events. Further analyses demonstrated that these alleles show consistently different frequencies in the cancer population compared to the healthy population and are significantly enriched for predicted protein-damaging variants. Moreover, genes harboring SNPs that demonstrate allele specificity are enriched for cancer-related biological processes and are more likely to be essential in cancer cells.
    CONCLUSIONS: In summary, our study provides a unique and complementary method to identify genes and variants that are relevant to carcinogenesis.
    Keywords:  Allelic imbalance; Cancer; Copy number; Exonic variants; Somatic selection
    DOI:  https://doi.org/10.1186/s12920-021-00984-1
  3. Eur J Orthop Surg Traumatol. 2021 May 29.
      Magnetic Resonance Imaging is a fundamental tool in the evaluation of soft tissue sarcoma. Imaging features are relevant for the assessment of treatment strategies, surgical planning and also for patients' prognosis prediction. Among soft tissue sarcoma and also other malignancies, the size of the mass is usually considered the prognostic key element in diagnostic imaging. Moreover, several other features should be obtained from MRI studies with prognostic implications in all type of soft tissue sarcoma: peritumoral enhancement, signs of necrosis, deep location, ill-defined borders/signs of infiltrations. Focusing on soft tissue sarcoma subtypes, some other magnetic resonance imaging features are more specific and related to prognosis. In myxofibrosarcoma the magnetic resonance imaging "tail sign" and a "water-like" appearance on fluid-sensitive sequences, due to rich myxoid matrix content, are both associated with higher risk of local recurrence after surgical excision; nevertheless, the "tail sign" is also related to a higher risk of distant metastases at diagnosis. The "tail sign" is associated with higher risk of local recurrence after surgical excision in undifferentiated pleomorphic sarcoma as well. In patients affected by synovial sarcoma, the "triple sign" identifiable in magnetic resonance imaging (T2w sequences) is associated with decreased disease-free survival and indicates the simultaneous presence of solid cellular elements (intermediate signal intensity), hemorrhage or necrosis (high signal intensity) and fibrotic regions (low signal intensity). In addition, absence of calcifications are associated with reduced disease-free survival in patients affected by synovial sarcoma. Signal heterogeneity is associated with worst prognosis in all type of soft tissue sarcoma, particularly in myxoid liposarcoma. In recent years, several new quantitative tools applied on magnetic resonance imaging have been proved to predict patients' prognosis. Above all the new tools, radiomics seems to be one of the most promising, and, has been proved to have the capability in discriminating low-grade from high-grade soft tissue sarcomas. Therefore, magnetic resonance imaging studies in patients with soft tissue sarcoma should be accurately evaluated and their results should be taken into account for prognostic assessment.
    Keywords:  Local; Magnetic resonance imaging; Neoplasm metastasis; Neoplasm recurrence; Prognosis; Sarcoma; Soft tissue
    DOI:  https://doi.org/10.1007/s00590-021-03003-2
  4. Cancers (Basel). 2021 May 14. pii: 2386. [Epub ahead of print]13(10):
      The lack of a sensitive and specific biomarker and the limits relating to the single primary tumor sampling make it difficult to monitor high-grade serous epithelial ovarian cancer (HGS-EOC) over time and to capture those alterations that are potentially useful in guiding clinical decisions. To overcome these issues, liquid biopsy has emerged as a very promising tool for HGS-EOC. The analysis of circulating tumor DNA appears to be feasible and studies assessing specific pathogenic mutations (i.e., TP53) or copy number alterations have shown a sufficient degree of sensitivity and specificity to be realistically used to monitor the effectiveness of antitumor therapy. Liquid biopsy can also provide potential important information on the mechanisms of sensitivity and resistance, e.g., by the determination of the reversion of BRCA mutations. Perspective studies are needed to test whether the application of liquid biopsy will significantly improve HGS-EOC management and patients' survival.
    Keywords:  circulating tumor DNA; high-grade serous epithelial ovarian cancer; highly sensitive technologies; liquid biopsy
    DOI:  https://doi.org/10.3390/cancers13102386
  5. N Engl J Med. 2021 Jun 03.
    OlympiA Clinical Trial Steering Committee and Investigators
       BACKGROUND: Poly(adenosine diphosphate-ribose) polymerase inhibitors target cancers with defects in homologous recombination repair by synthetic lethality. New therapies are needed to reduce recurrence in patients with BRCA1 or BRCA2 germline mutation-associated early breast cancer.
    METHODS: We conducted a phase 3, double-blind, randomized trial involving patients with human epidermal growth factor receptor 2 (HER2)-negative early breast cancer with BRCA1 or BRCA2 germline pathogenic or likely pathogenic variants and high-risk clinicopathological factors who had received local treatment and neoadjuvant or adjuvant chemotherapy. Patients were randomly assigned (in a 1:1 ratio) to 1 year of oral olaparib or placebo. The primary end point was invasive disease-free survival.
    RESULTS: A total of 1836 patients underwent randomization. At a prespecified event-driven interim analysis with a median follow-up of 2.5 years, the 3-year invasive disease-free survival was 85.9% in the olaparib group and 77.1% in the placebo group (difference, 8.8 percentage points; 95% confidence interval [CI], 4.5 to 13.0; hazard ratio for invasive disease or death, 0.58; 99.5% CI, 0.41 to 0.82; P<0.001). The 3-year distant disease-free survival was 87.5% in the olaparib group and 80.4% in the placebo group (difference, 7.1 percentage points; 95% CI, 3.0 to 11.1; hazard ratio for distant disease or death, 0.57; 99.5% CI, 0.39 to 0.83; P<0.001). Olaparib was associated with fewer deaths than placebo (59 and 86, respectively) (hazard ratio, 0.68; 99% CI, 0.44 to 1.05; P = 0.02); however, the between-group difference was not significant at an interim-analysis boundary of a P value of less than 0.01. Safety data were consistent with known side effects of olaparib, with no excess serious adverse events or adverse events of special interest.
    CONCLUSIONS: Among patients with high-risk, HER2-negative early breast cancer and germline BRCA1 or BRCA2 pathogenic or likely pathogenic variants, adjuvant olaparib after completion of local treatment and neoadjuvant or adjuvant chemotherapy was associated with significantly longer survival free of invasive or distant disease than was placebo. Olaparib had limited effects on global patient-reported quality of life. (Funded by the National Cancer Institute and AstraZeneca; OlympiA ClinicalTrials.gov number, NCT02032823.).
    DOI:  https://doi.org/10.1056/NEJMoa2105215
  6. Cancer Chemother Pharmacol. 2021 Jun 02.
       BACKGROUND: Patients with advanced high-grade serous ovarian cancer (HGSOC) are usually treated with paclitaxel and carboplatin; however, predictive markers for this drug combination are unknown.
    METHODS: Tumor samples from 71 consecutive HGSOC patients, who received neoadjuvant chemotherapy with paclitaxel and carboplatin, were subjected to molecular analysis.
    RESULTS: BRCA1/2 germline mutation carriers (n = 22) had longer treatment-free interval (TFI) than non-carriers (n = 49) (9.5 months vs. 3.8 months; P = 0.007). Fifty-one HGSOCs had sufficient quality of tumor DNA for the next-generation sequencing (NGS) analysis by the SeqCap EZ CNV/LOH Backbone Design panel. All 13 tumors obtained from BRCA1/2 germline mutation carriers and 12 sporadic HGSOCs showed a high number of evenly spread chromosomal breaks, which was defined as a BRCAness phenotype; median TFI for this combined group approached 9.5 months. The remaining 26 HGSOCs had similarly high global LOH score (above 20%); however, in contrast to BRCAness tumors, LOH involved large chromosomal segments; these patients had significantly lower TFI (3.7 months; P = 0.006). All patients with CCNE1 amplification (n = 7), TP53 R175H substitution (n = 6), and RB1 mutation (n = 4) had poor response to paclitaxel plus carboplatin.
    CONCLUSION: This study describes a cost-efficient method of detecting the BRCAness phenotype, which is compatible with the laboratory-scale NGS equipment. Some molecular predictors allow the identification of potential non-responders to paclitaxel plus carboplatin, who may need to be considered for other treatment options.
    Keywords:  BRCAness; Neoadjuvant chemotherapy; Ovarian cancer; Targeted sequencing; Treatment response
    DOI:  https://doi.org/10.1007/s00280-021-04301-6
  7. Cancers (Basel). 2021 May 13. pii: 2359. [Epub ahead of print]13(10):
      Soft-tissue sarcomas are rare tumors characterized by pathogenetic, morphological, and clinical intrinsic variability. Median survival of patients with advanced tumors are usually chemo- and radio-resistant, and standard treatments yield low response rates and poor survival results. The identification of defined genomic alterations in sarcoma could represent the premise for targeted treatments. Summarizing, soft-tissue sarcomas can be differentiated into histotypes with reciprocal chromosomal translocations, with defined oncogenic mutations and complex karyotypes. If the latter are improbably approached with targeted treatments, many suggest that innovative therapies interfering with the identified fusion oncoproteins and altered pathways could be potentially resolutive. In most cases, the characteristic genetic signature is discouragingly defined as "undruggable", which poses a challenge for the development of novel pharmacological approaches. In this review, a summary of genomic alterations recognized in most common soft-tissue sarcoma is reported together with current and future therapeutic opportunities.
    Keywords:  genome; personalized medicine; precision medicine; sarcoma; translocation
    DOI:  https://doi.org/10.3390/cancers13102359
  8. Int J Mol Sci. 2021 May 24. pii: 5522. [Epub ahead of print]22(11):
      Approximately 23% of metastatic castration-resistant prostate cancers (mCRPC) harbor deleterious aberrations in DNA repair genes. Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) therapy has shown improvements in overall survival in patients with mCRPC who harbor somatic and/or germline alterations of homology recombination repair (HRR) genes. Peripheral blood samples are typically used for the germline mutation analysis test using the DNA extracted from peripheral blood leucocytes. Somatic alterations can be assessed by extracting DNA from a tumor tissue sample or using circulating tumor DNA (ctDNA) extracted from a plasma sample. Each of these genetic tests has its own benefits and limitations. The main advantages compared to the tissue test are that liquid biopsy is a non-invasive and easily repeatable test with the value of better representing tumor heterogeneity than primary biopsy and of capturing changes and/or resistance mutations in the genetic tumor profile during disease progression. Furthermore, ctDNA can inform about mutation status and guide treatment options in patients with mCRPC. Clinical validation and test implementation into routine clinical practice are currently very limited. In this review, we discuss the state of the art of the ctDNA test in prostate cancer compared to blood and tissue testing. We also illustrate the ctDNA testing workflow, the available techniques for ctDNA extraction, sequencing, and analysis, describing advantages and limits of each techniques.
    Keywords:  ATM; BRCA1; BRCA2; PARP inhibitors; circulating tumor DNA; homology recombination repair genes; metastatic castration-resistant prostate cancer; prostate cancer
    DOI:  https://doi.org/10.3390/ijms22115522
  9. JAMA Oncol. 2021 Jun 04.
       Importance: DNA damage repair (DDR) gene mutations represent actionable alterations that can guide precision medicine strategies for advanced prostate cancer. However, acquisition of contemporary tissue samples for molecular testing can be a barrier to deploying precision medicine approaches. We hypothesized that most DDR alterations represent truncal events in prostate cancer and that primary tissue would faithfully reflect mutations found in cell-free circulating tumor DNA (ctDNA) and/or metastatic tissue.
    Objective: To assess concordance in DDR gene alterations between primary prostate cancer and metastases or ctDNA specimens.
    Design, Setting, and Participants: Patients were included if a DDR pathway mutation was detected in metastatic tissue or ctDNA and primary tissue sequencing was available for comparison. Sequencing data from 3 cohorts were analyzed: (1) FoundationOne, (2) University of Washington clinical cases (University of Washington-OncoPlex or Stand Up to Cancer-Prostate Cancer Foundation International Dream Team sequencing pipelines), and (3) University of Washington rapid autopsy series. Only pathogenic somatic mutations were included, and more than 30 days between primary tumor tissue and ctDNA and/or metastatic tissue acquisition was required. Clonal hematopoiesis of indeterminate potential (CHIP) and germline events were adjudicated by an expert molecular pathologist and excluded.
    Main Outcomes and Measures: The DDR gene alterations detected in primary prostate tissue matched with metastatic tissue and/or ctDNA findings.
    Results: A total of 72 men with known DDR alterations were included in the analysis, and primary samples with paired ctDNA and/or metastatic tissue were sequenced. After excluding patients with ctDNA where only CHIP and/or germline events (n = 21) were observed, 51 patients remained and were included in the final analysis. The median (range) time from acquisition of primary tissue to acquisition of ctDNA or tumor tissue was 55 (5-193) months. Concordance in DDR gene mutation status across samples was 84% (95% CI, 71%-92%). Rates of concordance between metastatic-primary and ctDNA-primary pairs were similar when patients with CHIP events were excluded. Multiclonal BRCA2 reversion mutations associated with resistance to PARP inhibitors and platinum chemotherapy were detected in ctDNA from 2 patients.
    Conclusions and Relevance: In this genetic association study of 3 patient cohorts, primary prostate tissue accurately reflected the mutational status of actionable DDR genes in metastatic tissue, consistent with DDR alterations being truncal in most patients. After excluding likely CHIP events, ctDNA profiling accurately captured these DDR mutations while also detecting reversion alterations that may suggest resistance mechanisms.
    DOI:  https://doi.org/10.1001/jamaoncol.2021.2350
  10. Mol Oncol. 2021 May 31.
      Despite impressive and durable responses, non-small cell lung cancer (NSCLC) patients treated with anaplastic lymphoma kinase inhibitors (ALK-Is) ultimately progress due to development of resistance. Here, we have evaluated the clinical utility of circulating tumor DNA (ctDNA) profiling by next-generation sequencing (NGS) upon disease progression. We collected26 plasma and two cerebrospinal fluid samples from 24 advanced ALK-positive NSCLC patients at disease progression to an ALK-I. These samples were analyzed by NGS and digital PCR. A tool to retrieve variants at the ALK locus was developed (VALK tool).We identified at least one resistance mutation in the ALK locus in ten (38.5%) plasma samples; the G1269A and G1202R mutations were the most prevalent among patients progressing to first- and second-generation ALK-Is, respectively. Overall, 61 somatic mutations were detected in 14 genes: TP53, ALK, PIK3CA, SMAD4, MAP2K1 (MEK1), FGFR2, FGFR3, BRAF, EGFR, IDH2, MYC, MET, CCND3 and CCND1. Specifically, a deletion in exon 19 in EGFR, a non-V600 BRAF mutation (G466V) and the F129L mutation in MAP2K1 were identified in four patients who showed no objective survival benefit from ALK-Is. Potential ALK-I-resistance mutations were also found in PIK3CA and IDH2. Finally, a c-MYC gain, along with a loss of CCND1 and FGFR3, were detected in a patient progressing on a first-line treatment with crizotinib. We conclude that NGS analysis of liquid biopsies upon disease progression identified different putative ALK-I-resistance mutations in most cases, and could be a valuable approach for therapy decision making.
    Keywords:  ALK-TKI; EML4-ALK; Liquid Biopsy; NGS; NSCLC
    DOI:  https://doi.org/10.1002/1878-0261.13033
  11. Brief Bioinform. 2021 May 31. pii: bbab201. [Epub ahead of print]
      Genetic and epigenetic contributions to various diseases and biological processes have been well-recognized. However, simultaneous identification of single-nucleotide variants (SNVs) and DNA methylation levels from traditional bisulfite sequencing data is still challenging. Here, we develop double strand bisulfite sequencing (DSBS) for genome-wide accurate identification of SNVs and DNA methylation simultaneously at a single-base resolution by using one dataset. Locking Watson and Crick strand together by hairpin adapter followed by bisulfite treatment and massive parallel sequencing, DSBS simultaneously sequences the bisulfite-converted Watson and Crick strand in one paired-end read, eliminating the strand bias of bisulfite sequencing data. Mutual correction of read1 and read2 can estimate the amplification and sequencing errors, and enables our developed computational pipeline, DSBS Analyzer (https://github.com/tianguolangzi/DSBS), to accurately identify SNV and DNA methylation. Additionally, using DSBS, we provide a genome-wide hemimethylation landscape in the human cells, and reveal that the density of DNA hemimethylation sites in promoter region and CpG island is lower than that in other genomic regions. The cost-effective new approach, which decodes DNA methylome and genomic variants simultaneously, will facilitate more comprehensive studies on numerous diseases and biological processes driven by both genetic and epigenetic variations.
    Keywords:  CpG context; cytosine modification; epigenomic alteration; genomic mutation; population genomics
    DOI:  https://doi.org/10.1093/bib/bbab201
  12. Int J Mol Sci. 2021 May 26. pii: 5683. [Epub ahead of print]22(11):
      Despite recent progress, gastric cancer remains one of the most common cancers and has a high mortality rate worldwide. Aberrant DNA methylation pattern and deregulation of noncoding RNA expression appear in the early stages of gastric cancer. Numerous investigations have confirmed their significant role in gastric cancer tumorigenesis and their high potential as diagnostic and prognostic biomarkers. Currently, it is clear that these epigenetic regulators do not work alone but interact with each other, generating a complex network. The aim of our review was to summarize the current knowledge of this interaction in gastric cancer and estimate its clinical potential for the diagnosis, prognosis, and treatment of the disease.
    Keywords:  DNA methylation; biomarker; epigenetics; gastric cancer; noncoding RNAs
    DOI:  https://doi.org/10.3390/ijms22115683
  13. Front Cardiovasc Med. 2021 ;8 631650
      Background: To explore the association of DNA methylation and gene expression in the pathology of obesity. Methods: (1) Genomic DNA methylation and mRNA expression profile of visceral adipose tissue (VAT) were performed in a comprehensive database of gene expression in obese and normal subjects. (2) Functional enrichment analysis and construction of differential methylation gene regulatory networks were performed. (3) Validation of the two different methylation sites and corresponding gene expression was done in a separate microarray dataset. (4) Correlation analysis was performed on DNA methylation and mRNA expression data. Results: A total of 77 differentially expressed mRNAs matched with differentially methylated genes. Analysis revealed two different methylation sites corresponding to two unique genes-s100a8-cg09174555 and s100a9-cg03165378. Through the verification test of two interesting different expression positions [differentially methylated positions (DMPs)] and their corresponding gene expression, we found that methylation in these genes was negatively correlated to gene expression in the obesity group. Higher S100A8 and S100A9 expressions in obese subjects were validated in a separate microarray dataset. Conclusion: This study confirmed the relationship between DNA methylation and gene expression and emphasized the important role of S100A8 and S100A9 in the pathogenesis of obesity.
    Keywords:  DNA methylation; correlation analyses; function enrichment; mRNA expression; obesity
    DOI:  https://doi.org/10.3389/fcvm.2021.631650
  14. Curr Pharm Des. 2021 Jun 03.
       BACKGROUND: Epigenetic modifications have recently attracted much attention in the study of the biological mechanisms of acute myelocytic leukemia (AML) for therapy and prognosis. However, studies on DNA methylation changes during AML treatment are limited.
    OBJECTIVE: The comprehensive DNA methylation-transcriptome profiles association analysis in this study aimed to establish whole-genome DNA methylation profiles and explore DNA methylation-related genes and their potential functions before and after treatment. And more appropriate biomarkers are expected to be identified for therapy strategies in AML.
    METHOD: Illumina 450K and RNA-Seq data were obtained from The Cancer Genome Atlas. We performed comprehensive DNA methylation-transcriptome profiles association analysis, pathway analysis, correlation analysis, and survival analyses. The StarBase database was utilized to predict interactions between lncRNAs, miRNAs and target mRNAs.
    RESULTS: In total, 1592 distinct CpG sites and 2419 different expression transcripts were identified between pre-treatment and post-treatment AML. The significantly enriched functions of methylated genes were stem cell differentiation, cell population maintenance, and cell development. The expression of UGT3A2, MOG, and VSTM1 was correlated with DNA methylation levels (r2>0.5). Lastly, we identified 4 lncRNAs, 9 miRNAs and 142 mRNAs to construct a lncRNA-miRNA-mRNA ceRNA network.
    CONCLUSION: Our results revealed that DNA methylation was altered before and after treatment. Alterations in DNA methylation affected target gene expression and participated in the key biological processes of AML. Therefore, ceRNA networks may provide further insight into the study of favorable therapeutic markers in AML.
    Keywords:  DNA methylation; acute myelocytic leukemia; ceRNAs; epigenetics; lncRNAs
    DOI:  https://doi.org/10.2174/1381612827666210603165253
  15. Cancers (Basel). 2021 May 10. pii: 2274. [Epub ahead of print]13(9):
      Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related death worldwide. Diagnostic, prognostic, and predictive biomarkers are urgently needed in order to improve patient survival. Indeed, the most widely used biomarkers, such as alpha-fetoprotein (AFP), have limited accuracy as both diagnostic and prognostic tests. Liver biopsy provides an insight on the biology of the tumor, but it is an invasive procedure, not routinely used, and not representative of the whole neoplasia due to the demonstrated intra-tumoral heterogeneity. In recent years, liquid biopsy, defined as the molecular analysis of cancer by-products, released by the tumor in the bloodstream, emerged as an appealing source of new biomarkers. Several studies focused on evaluating extracellular vesicles, circulating tumor cells, cell-free DNA and non-coding RNA as novel reliable biomarkers. In this review, we aimed to provide a comprehensive overview on the most relevant available evidence on novel circulating biomarkers for early diagnosis, prognostic stratification, and therapeutic monitoring. Liquid biopsy seems to be a very promising instrument and, in the near future, some of these new non-invasive tools will probably change the clinical management of HCC patients.
    Keywords:  biomarkers; circulating nucleic acids; circulating tumor cells; diagnosis; extracellular vesicles; hepatocellular carcinoma; liquid biopsy; prognosis
    DOI:  https://doi.org/10.3390/cancers13092274