bims-tumhet Biomed News
on Tumor Heterogeneity
Issue of 2022‒03‒27
nineteen papers selected by
Sergio Marchini
Humanitas Research

  1. J Gynecol Oncol. 2022 Mar 08.
      The use of PARP inhibitors (PARPi) in patients with epithelial ovarian cancer is expanding, with the transition from use in recurrent disease to the first-line setting. This is accompanied with an increasing population of patients who develop acquired PARPi resistance. Coupled with those patients with primary PARPi resistance, there is an urgent need to better understand mechanisms of resistance and identify means to overcome this resistance. Combination therapy offers the potential to overcome innate and acquired resistance, by either working synergistically with PARPi or by restoring homologous recombination deficiency, targeting the homologous recombination repair pathway through an alternate strategy. We discuss mechanisms of PARPi resistance and data on novel combinations which may restore PARPi sensitivity.
    Keywords:  Carcinoma, Ovarian Epithelial; Drug Resistance, Neoplasm; Drug Therapy, Combination; Poly(ADP-ribose) Polymerase Inhibitors
  2. Cancers (Basel). 2022 Mar 15. pii: 1511. [Epub ahead of print]14(6):
      The majority of patients with ovarian cancer ultimately develop recurrent chemotherapy-resistant disease. Treatment stratification is mainly based on histological subtype and stage, prior response to platinum-based chemotherapy, and time to recurrent disease. Here, we integrated clinical treatment, treatment response, and survival data with whole-genome sequencing profiles of 132 solid tumor biopsies of metastatic epithelial ovarian cancer to explore genome-informed stratification opportunities. Samples from primary and recurrent disease harbored comparable numbers of single nucleotide variants and structural variants. Mutational signatures represented platinum exposure, homologous recombination deficiency, and aging. Unsupervised hierarchical clustering based on genomic input data identified specific ovarian cancer subgroups, characterized by homologous recombination deficiency, genome stability, and duplications. The clusters exhibited distinct response rates and survival probabilities which could thus potentially be used for genome-informed therapy stratification for more personalized ovarian cancer treatment.
    Keywords:  ovarian cancer; patient stratification; personalized treatment; treatment response; whole-genome sequencing
  3. Diagnostics (Basel). 2022 Mar 19. pii: 748. [Epub ahead of print]12(3):
      Circulating tumor DNA (ctDNA), accurately described by the term liquid profiling (LP), enables real-time assessment of the tumor mutational profile as a minimally invasive test and has therefore rapidly gained traction, particular for the management of cancer patients. By LP, tumor-specific genetic alterations can be determined as part of companion diagnostics to guide selection of appropriate targeted therapeutics. Because LP facilitates longitudinal monitoring of cancer patients, it can be used to detect acquired resistant mechanisms or as a personalized biomarker for earlier detection of disease recurrence, among other applications. However, LP is not yet integrated into routine care to the extent that might be expected. This is due to the lack of harmonization and standardization of preanalytical and analytical workflows, the lack of proper quality controls, limited evidence of its clinical utility, heterogeneous study results, the uncertainty of clinicians regarding the value and appropriate indications for LP and its interpretation, and finally, the lack of reimbursement for most LP tests. In this review, the value proposition of LP for cancer patient management and treatment optimization, the current status of implementation in standard care, and the main challenges that need to be overcome are discussed in detail.
    Keywords:  cancer management; cell-free DNA; circulating tumor DNA; clinical oncology; liquid biopsy; liquid profiling; personalized medicine; standard care
  4. BMC Bioinformatics. 2022 Mar 21. 23(Suppl 3): 98
      BACKGROUND: Although both copy number variations (CNVs) and single nucleotide variations (SNVs) detected by single-cell RNA sequencing (scRNA-seq) are used to study intratumor heterogeneity and detect clonal groups, a software that integrates these two types of data in the same cells is unavailable.RESULTS: We developed Clonal Architecture with Integration of SNV and CNV (CAISC), an R package for scRNA-seq data analysis that clusters single cells into distinct subclones by integrating CNV and SNV genotype matrices using an entropy weighted approach. The performance of CAISC was tested on simulation data and four real datasets, which confirmed its high accuracy in sub-clonal identification and assignment, including subclones which cannot be identified using one type of data alone. Furthermore, integration of SNV and CNV allowed for accurate examination of expression changes between subclones, as demonstrated by the results from trisomy 8 clones of the myelodysplastic syndromes (MDS) dataset.
    CONCLUSIONS: CAISC is a powerful tool for integration of CNV and SNV data from scRNA-seq to identify clonal clusters with better accuracy than obtained from a single type of data. CAISC allows users to interactively examine clonal assignments.
    Keywords:  Copy number variation; Entropy-based weighted integration; Single nucleotide variation; Single-cell RNA sequencing
  5. Cancers (Basel). 2022 Mar 08. pii: 1384. [Epub ahead of print]14(6):
      Human solid malignancies harbour a heterogeneous set of cells with distinct genotypes and phenotypes. This heterogeneity is installed at multiple levels. A biological diversity is commonly observed between tumours from different patients (inter-tumour heterogeneity) and cannot be fully captured by the current consensus molecular classifications for specific cancers. To extend the complexity in cancer, there are substantial differences from cell to cell within an individual tumour (intra-tumour heterogeneity, ITH) and the features of cancer cells evolve in space and time. Currently, treatment-decision making usually relies on the molecular characteristics of a limited tumour tissue sample at the time of diagnosis or disease progression but does not take into account the complexity of the bulk tumours and their constant evolution over time. In this review, we explore the extent of tumour heterogeneity with an emphasis on ITH and report the mechanisms that promote and sustain this diversity in cancers. We summarise the clinical strikes of ITH in the management of patients with cancer. Finally, we discuss the current material and technological approaches that are relevant to adequately appreciate ITH.
    Keywords:  circulating tumour DNA; liquid biopsy; multi-region sampling; next-generation sequencing; single-cell approaches; treatment resistance; tumour heterogeneity
  6. Front Oncol. 2022 ;12 798680
      Background: High-grade serous ovarian cancer (HGSOC) is the predominant and deadliest form of ovarian cancer. Some of its histological subtypes can be distinguished by frequent occurrence of cancer-associated myofibroblasts (CAFs) and desmoplastic stroma reaction (DSR). In this study, we want to explore the relationship between therapy outcome and the activity of CAF-associated signaling pathways in a homogeneous HGSOC patient collective. Furthermore, we want to validate these findings in a general Epithelial ovarian cancer (EOC) cohort.Methods: The investigation cohort consists of 24 HGSOC patients. All of them were treated with platinum-based components and clinical follow-up was available. The validation cohort was comprised of 303 patients. Sequencing data (whole transcriptome) and clinical data were extracted from The Cancer Genome Atlas (TCGA). RNA of HGSOC patients was isolated using a Maxwell RSC instrument and the appropriate RNA isolation kit. For digital expression analysis a custom-designed gene panel was employed. All genes were linked to various DSR- and CAF- associated pathways. Expression analysis was performed on the NanoString nCounter platform. Finally, data were explored using the R programming environment (v. 4.0.3).
    Result: In total, 15 CAF-associated genes were associated with patients' survival. More specifically, 6 genes (MMP13, CGA, EPHA3, PSMD9, PITX2, PHLPP1) were linked to poor therapy outcome. Though a variety of different pathways appeared to be associated with therapy failure, many were related to CAF paracrine signaling, including MAPK, Ras and TGF-β pathways. Similar results were obtained from the validation cohort.
    Discussion: In this study, we could successfully link CAF-associated pathways, as shown by increased Ras, MAPK and PI3K-Akt signaling to therapy failure (chemotherapy) in HGSOC and EOCs in general. As platinum-based chemotherapy has been the state-of-the-art therapy to treat HGSOC for decades, it is necessary to unveil the reasons behind resistance developments and poor outcome. In this work, CAF-associated signaling is shown to compromise therapy response. In the validation cohort, CAF-associated signaling is also associated with therapy failure in general EOC, possibly hinting towards a conserved mechanism. Therefore, it may be helpful to stratify HGSOC patients for CAF activity and consider alternative treatment options.
    Keywords:  cancer-associated fibroblasts; chemoresistance; epithelial ovarian cancer; high-grade serous ovarian cancer; tumor microenvironment
  7. Cancers (Basel). 2022 Mar 10. pii: 1420. [Epub ahead of print]14(6):
      PolyADP-ribose polymerase (PARP) inhibitors (PARPis) represent the first clinically approved drugs able to provoke "synthetic lethality" in patients with homologous recombination-deficient (HRD) tumors. Four PARPis have just received approval for the treatment of several types of cancer. Besides, another three additional PARPis underlying the same mechanism of action are currently under investigation. Despite the success of these targeted agents, the increasing use of PARPis in clinical practice for the treatment of different tumors raised the issue of PARPis resistance, and the consequent disease relapse and dismal prognosis for patients. Several mechanisms of resistance have been investigated, and ongoing studies are currently focusing on strategies to address this challenge and overcome PARPis resistance. This review aims to analyze the mechanisms underlying PARPis resistance known today and discuss potential therapeutic strategies to overcome these processes of resistance in the future.
    Keywords:  BRCA; DNA damage repair; PARP inhibitor resistance; homologous recombination; ovarian cancer; polyADP-ribose polymerase (PARP) inhibitor; replication fork
  8. Biochim Biophys Acta Rev Cancer. 2022 Mar 17. pii: S0304-419X(22)00047-6. [Epub ahead of print] 188722
      DNA methylation is an epigenetic mechanism regulating gene expression. Changes in DNA methylation were suggested to be useful biomarkers for diagnosis, and for the determination of prognosis and treatment response. Here, we provide an overview of methylation-based biomarkers in colorectal cancer. First, we start with the two methylation-based diagnostic biomarkers already approved for colorectal cancer, SEPT9 and the combination of NDRG4 and BMP3. Then, we provide a list-based overview of new biomarker candidates depending on the sample source including plasma, stool, urine, and surgically removed tumor tissues. The most often identified markers like SDC2, VIM, APC, MGMT, SFRP1, SFRP2, and NDRG4 have distinct functions previously linked to tumor progression. Although numerous studies have identified tumor-specific methylation changes, most of these alterations were observed in a single study only. The lack of validation in independent samples means low reproducibility and is a major limitation. The genome-wide determination of methylation status (methylome) can provide data to solve these issues. In the third section of the review, methylome studies focusing on different aspects related to CRC, including precancerous lesions, CRC-specific changes, molecular subtypes, aging, and chemotherapy response are summarized. Notably, techniques simultaneously analyzing a large set of regions can also uncover epigenetic regulation of genes which have not yet been associated with tumorigenesis previously. A remaining constraint of studies published to date is the low patient number utilized in these preventing the identification of clinically valuable biomarker candidates. Either future large-scale studies or the integration of already available methylome-level data will be necessary to uncover biomarkers sufficiently robust for clinical application.
    Keywords:  Biomarker discovery; Diagnosis; Epigenetics; HumanMethylation450K; HumanMethylationEPIC; Methlyome; Plasma; Stool
  9. Lab Invest. 2022 Mar 22.
      Morphologic and immunohistochemical analysis of preoperative core needle biopsies (CNB) is important in the management of patients with soft tissue and bone tumors (STBTs). Most SBTB subtypes have more or less extensive DNA copy number aberrations (CNA), potentially providing useful diagnostic information. To evaluate the technical feasibility of single nucleotide polymorphism (SNP) array analysis and the diagnostic usefulness of the copy number profiles, we studied CNBs from 171 patients with suspected STBTs. SNP array analysis could be performed on 168 (98%) of the samples. The CNA profile was compatible with the CNB diagnosis in 87% of the cases. Discrepant cases were dominated by false-negative results due to nonrepresentative material or contamination with normal cells. 70 genomic profiles were indicative of specific histopathologic tumor entities and in agreement with the corresponding CNB diagnoses in 83%. In 96 of the cases with aberrant CNA profiles, the SNP profiles were of sufficient quality for segmentation, allowing clustering analysis on the basis of the Jaccard similarity index. The analysis of these segment files showed three major CNA clusters, based on the complexity levels and the predominance of gains versus losses. For 43 of these CNB samples, we had SNP array data also from their corresponding surgical samples. In 33 of these pairs, the two corresponding samples clustered next to each other, with Jaccard scores ranging from 0.61 to 0.99 (median 0.96). Also, for those tumor pairs that did not cluster together, the Jaccard scores were relatively high (median 0.9). 10 cases showed discrepant results, mainly due to varying degrees of normal cell contamination or technical issues. Thus, the copy number profile seen in a CNB is typically highly representative of the major cell population in the tumor.
  10. Cancers (Basel). 2022 Mar 10. pii: 1424. [Epub ahead of print]14(6):
      A large proportion of tumours is characterised by numerical or structural chromosomal instability (CIN), defined as an increased rate of gaining or losing whole chromosomes (W-CIN) or of accumulating structural aberrations (S-CIN). Both W-CIN and S-CIN are associated with tumourigenesis, cancer progression, treatment resistance and clinical outcome. Although W-CIN and S-CIN can co-occur, they are initiated by different molecular events. By analysing tumour genomic data from 33 cancer types, we show that the majority of tumours with high levels of W-CIN underwent whole genome doubling, whereas S-CIN levels are strongly associated with homologous recombination deficiency. Both CIN phenotypes are prognostic in several cancer types. Most drugs are less efficient in high-CIN cell lines, but we also report compounds and drugs which should be investigated as targets for W-CIN or S-CIN. By analysing associations between CIN and bio-molecular entities with pathway and gene expression levels, we complement gene signatures of CIN and report that the drug resistance gene CKS1B is strongly associated with S-CIN. Finally, we propose a potential copy number-dependent mechanism to activate the PI3K pathway in high-S-CIN tumours.
    Keywords:  PI3K oncogenic activation; integrative analysis; structural chromosomal instability; whole chromosomal instability; whole genome doubling
  11. Int J Mol Sci. 2022 Mar 17. pii: 3238. [Epub ahead of print]23(6):
      Deficiency in DNA damage response (DDR) genes leads to impaired DNA repair functions that will induce genomic instability and facilitate cancer development. However, alterations of DDR genes can serve as biomarkers for the selection of suitable patients to receive specific therapeutics, such as immune checkpoint blockade (ICB) therapy. In addition, certain altered DDR genes can be ideal therapeutic targets through adapting the mechanism of synthetic lethality. Recent studies indicate that targeting DDR can improve cancer immunotherapy by modulating the immune response mediated by cGAS-STING-interferon signaling. Investigations of the interplay of DDR-targeting and ICB therapies provide more effective treatment options for cancer patients. This review introduces the mechanisms of DDR and discusses their crucial roles in cancer therapy based on the concepts of synthetic lethality and ICB. The contemporary clinical trials of DDR-targeting and ICB therapies in breast, colorectal, and pancreatic cancers are included.
    Keywords:  DNA damage response; PARP; cGAS-STING; cancer immunotherapy; clinical trial; immune checkpoint; synthetic lethality
  12. Mol Cancer. 2022 Mar 21. 21(1): 81
      BACKGROUND: The promise of precision cancer medicine presently centers around the genomic sequence of a patient's tumor being translated into timely, actionable information to inform clinical care. The analysis of cell-free DNA from liquid biopsy, which contains circulating tumor DNA (ctDNA) in patients with cancer, has proven to be amenable to various settings in oncology. However, open questions surrounding the clinical validity and utility of plasma-based analyses have hindered widespread clinical adoption.MAIN BODY: Owing to the rapid evolution of the field, studies supporting the use of ctDNA as a biomarker throughout a patient's journey with cancer have accumulated in the last few years, warranting a review of the latest status for clinicians who may employ ctDNA in their precision oncology programs. In this work, we take a step back from the intricate coverage of detection approaches described extensively elsewhere and cover basic concepts around the practical implementation of next generation sequencing (NGS)-guided liquid biopsy. We compare relevant targeted and untargeted approaches to plasma DNA analysis, describe the latest evidence for clinical validity and utility, and highlight the value of genome-wide ctDNA analysis, particularly as it relates to early detection strategies and discovery applications harnessing the non-coding genome.
    CONCLUSIONS: The maturation of liquid biopsy for clinical application will require interdisciplinary efforts to address current challenges. However, patients and clinicians alike may greatly benefit in the future from its incorporation into routine oncology care.
    Keywords:  Cell-free DNA; Circulating tumor DNA; Liquid biopsy; Next-generation sequencing; Open chromatin; Precision oncology; Whole-genome sequencing
  13. Mol Cancer Ther. 2022 Mar 21. pii: molcanther.MCT-21-0689-E.2021. [Epub ahead of print]
      This paper investigates mechanisms of resistance to the VEGF receptor inhibitor cediranib in high-grade serous ovarian cancer, HGSOC, and defines rational combination therapies. We used three different syngeneic orthotopic mouse HGSOC models that replicated the human tumor microenvironment, TME. After 4-5 weeks treatment of established tumors, cediranib had anti-tumor activity with increased tumor T cell infiltrates and alterations in myeloid cells. However, continued cediranib treatment did not change overall survival or the immune microenvironment in two of the three models. Moreover, treated mice developed additional peritoneal metastases not seen in controls. Cediranib-resistant tumors had intrinsically high levels of IL-6 and JAK/STAT signaling and treatment increased endothelial STAT3 activation. Combination of cediranib with a murine anti-IL-6 antibody was superior to monotherapy, increasing mouse survival, reducing blood vessel density and pSTAT3, with increased T cell infiltrates in both models. In a third HGSOC model, that had lower inherent IL-6 JAK/STAT3 signaling in the TME but high PD1 signaling, long-term cediranib treatment significantly increased overall survival. When the mice eventually relapsed, pSTAT3 was still reduced in the tumors but there were high levels of immune cell PD1 and PDL1. Combining cediranib with an anti-PD1 antibody was superior to monotherapy in this model, increasing T cells and decreasing blood vessel densities. Bioinformatics analysis of two human HGSOC transcriptional datasets revealed distinct clusters of tumors with IL-6 and PD-1 pathway expression patterns that replicated the mouse tumors. Combination of anti-IL-6 or anti-PD1 in these patients may increase activity of VEGFR inhibitors and prolong disease-free survival.
  14. Clin Chem. 2022 Mar 22. pii: hvac020. [Epub ahead of print]
      BACKGROUND: No circulating biomarker is available for endometrial carcinoma (EC). We aimed to identify DNA positions universally hypermethylated in EC, and to develop a digital droplet PCR (ddPCR) assay for detection of hypermethylated circulating tumor DNA (meth-ctDNA) in plasma from patients with EC.METHODS: DNA positions hypermethylated in EC, and without unspecific hypermethylation in tissue/cell types releasing circulating cell-free DNA in plasma, were identified in silico from TCGA/Gene Expression Omnibus (GEO) data. A methylation-specific ddPCR (meth-ddPCR) assay following bisulfite conversion of DNA extracted from plasma was optimized for detection of meth-ctDNA according to dMIQE guidelines. Performances were validated on a retrospective cohort (n = 78 tumors, n = 30 tumor-adjacent tissues), a prospective pilot cohort (n = 33 stage I-IV patients), and 55 patients/donors without cancer.
    RESULTS: Hypermethylation of zinc finger and SCAN domain containing 12 (ZSCAN12) and/or oxytocin (OXT) classified EC samples from multiple noncancer samples with high diagnostic specificity/sensitivity [>97%; area under the curve (AUC) = 0.99; TCGA/GEO tissues/blood samples]. These results were confirmed in the independent retrospective cohort (AUC = 0.99). Meth-ddPCR showed a high analytical specificity (limit of blank = 2) and sensitivity (absolute lower threshold of detection = 50 pgmethDNA/mLplasma). In the pilot cohort, meth-ctDNA was detected in pretreatment plasma samples from 9/11 and 5/20 patients with advanced and non-advanced EC, respectively. 2 of 9 patients had ctDNA detected after macroscopic complete surgery and experienced progression within 6 months. No healthy donors had any copy of hypermethylated DNA detected in plasma.
    CONCLUSIONS: Meth-ddPCR of ZSCAN12/OXT allows a highly specific and sensitive detection of ctDNA in plasma from patients with EC and appears promising for personalized approaches for these patients.
    Keywords:  DNA methylation; digital droplet PCR; endometrial carcinoma; molecular characterization; personalized medicine
  15. Cancers (Basel). 2022 Mar 16. pii: 1533. [Epub ahead of print]14(6):
      Epigenetic therapies describe drug molecules such as DNA methyltransferase, histone methyltransferase and histone acetylase/deacetylase inhibitors, which target epigenetic mechanisms such as DNA methylation and histone modifications. Many DNA damage response (DDR) genes are epigenetically regulated in cancer leading to transcriptional silencing and the loss of DNA repair capacity. Epigenetic marks at DDR genes, such as DNA methylation at gene promoters, have the potential to be used as stratification biomarkers, identifying which patients may benefit from particular chemotherapy treatments. For genes such as MGMT and BRCA1, promoter DNA methylation is associated with chemosensitivity to alkylating agents and platinum coordination complexes, respectively, and they have use as biomarkers directing patient treatment options. In contrast to epigenetic change leading to chemosensitivity, DNA methylation of DDR genes involved in engaging cell death responses, such as MLH1, are associated with chemoresistance. This contrasting functional effect of epigenetic modification on chemosensitivity raises challenges in using DNA-demethylating agents, and other epigenetic approaches, to sensitise tumours to DNA-damaging chemotherapies and molecularly targeted agents. Demethylation of MGMT/BRCA1 could lead to drug resistance whereas demethylation of MLH1 could sensitise cells to chemotherapy. Patient selection based on a solid understanding of the disease pathway will be one means to tackle these challenges. The role of epigenetic modification of DDR genes during tumour development, such as causing a mutator phenotype, has different selective pressures and outcomes compared to epigenetic adaptation during treatment. The prevention of epigenetic adaptation during the acquisition of drug resistance will be a potential strategy to improve the treatment of patients using epigenetic therapies.
    Keywords:  DNA methylation; DNA repair; cancer; epigenetics
  16. Semin Cancer Biol. 2022 Mar 21. pii: S1044-579X(22)00068-2. [Epub ahead of print]
      The development of most solid cancers, including pancreatic, breast, lung, liver, and ovarian cancer, involves a desmoplastic reaction: a process of major remodelling of the extracellular matrix (ECM) affecting the ECM composition, mechanics, and microarchitecture. These properties of the ECM influence key cancer cell functions, including treatment resistance. Furthermore, emerging data show that various chemotherapeutic treatments lead to alterations in ECM features and ECM-cell communication. Here, we summarise the current knowledge around the effects of chemotherapy on both the ECM remodelling and ECM-cell signalling and discuss the implications of these alterations on distinct mechanisms of chemoresistance. Additionally, we provide an overview of current therapeutic strategies and ongoing clinical trials utilising anti-cancer drugs to target the ECM-cell communication and explore the future challenges of these strategies.
    Keywords:  ECM; cancer; chemoresistance; chemotherapy; plasticity
  17. Med (N Y). 2021 Sep 10. 2(9): 1004-1010
      Tumor evolution drives tumor progression, therapeutic resistance, and metastasis. Therefore, new predictive medicine strategies that adapt with a tumor are needed to improve patient outcomes. The techniques used in weather prediction are mathematically proven to enable prediction of evolving systems, and thus provide a framework for a new predictive medicine paradigm for cancer.
  18. J Mol Cell Biol. 2022 Mar 23. pii: mjac019. [Epub ahead of print]
      Cytosolic DNA is prevalent in cells constituting the tumor microenvironment (TME) and can activate the cGAS-STING innate immune pathway. The initiation, transmission, and execution of cGAS-STING pathway can take place among different cell types within the TME and thus cGAS-STING may play opposing roles in driving tumor progression in addition to its tumor cell-intrinsic role. Herein, we review recent advances in the cGAS-STING field with a focus on its crosstalk with other signaling pathways in the TME. Future efforts to depict a more detailed picture of the roles of cGAS-STING signaling in the TME will help design better cancer treatment regime by targeting cGAS-STING more precisely.
    Keywords:  DNA sensing; immunity; the tumor microenvironment
  19. Trends Biotechnol. 2022 Mar 17. pii: S0167-7799(22)00035-X. [Epub ahead of print]
      Multimodal analysis of circulating tumour cells (CTCs) has the potential to provide remarkable insight for cancer development and metastasis. CTCs and CTC clusters investigation using single-cell analysis, enables researchers to gain crucial information on metastatic mechanisms and the genomic alterations responsible for drug resistance, empowering treatment, and management of cancer. Despite a plethora of CTC isolation technologies, careful attention to the strengths and weaknesses of each method should be considered in order to isolate these rare cells. Here, we provide an overview of cutting-edge technologies used for single-cell isolation and analysis of CTCs. Additionally, we highlight the biological features, clinical application, and the therapeutic potential of CTCs and CTC clusters using single-cell analysis platforms for cancer management.
    Keywords:  CTC isolation; circulating tumour cell clusters; circulating tumour cells; single-cell analysis; targeted therapy