Targeted Next-Generation Sequencing Combined With Circulating-Free DNA Deciphers Spatial Heterogeneity of Resected Multifocal Hepatocellular Carcinoma.
Ontology highlight
ABSTRACT: Background
Hepatocellular carcinoma (HCC) has a high risk of recurrence after surgical resection, particularly among patients with multifocal HCC. Genomic heterogeneity contributes to the early recurrence. Few studies focus on targeted next-generation sequencing (tNGS) to depict mutational footprints of heterogeneous multifocal HCC.Methods
We conducted tNGS with an ultra-deep depth on 31 spatially distinct regions from 11 resected multifocal HCC samples. Matched preoperative peripheral circulating-free DNA (cfDNA) were simultaneously collected. Genomic alterations were identified and compared to depict the heterogeneity of multifocal HCC.Results
Widespread intertumoral heterogeneity of driver mutations was observed in different subfoci of multifocal HCC. The identified somatic mutations were defined as truncal drivers or branchy drivers according to the phylogenetic reconstruction. TP53 and TERT were the most commonly altered truncal drivers in multifocal HCC, while the most frequently mutated branchy driver was TSC2. HCC patients with a higher level of intertumoral heterogeneity, defined by the ratio of truncal drivers less than 50%, had a shorter RFS after surgical resection (HR=0.17, p=0.028). Genome profiling of cfDNA could effectively capture tumor-derived driver mutations, suggesting cfDNA was a non-invasive strategy to gain insights of genomic alterations in patients with resected multifocal HCC.Conclusions
Truncal mutations and the level of genomic heterogeneity could be identified by tNGS panel in patients with resected multifocal HCC. cfDNA could serve as a non-invasive and real-time auxiliary method to decipher the intertumoral heterogeneity and identify oncodrivers of multifocal HCC.
SUBMITTER: Lin J
PROVIDER: S-EPMC8240639 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:BackgroundHepatocellular carcinoma (HCC) often presents with multiple nodules within the liver, with limited effective interventions. The high genetic heterogeneity of HCC might be the major cause of treatment failure. We aimed to characterize genomic heterogeneity, infer clonal evolution, investigate RNA expression pattern and explore tumour immune microenvironment profile of multifocal HCC.Patients and methodsWhole-exome sequencing and RNA sequencing were carried out in 34 tumours and 6 adjacent normal liver tissue samples from 6 multifocal HCC patients. Protein expression of Ki67, AFP, P53, Survivin and CD8 was detected by immunohistochemistry. Fluorescence in situ hybridization was carried out to validate the amplification status of sorafenib-targeted genes.ResultsWe deciphered genomic and transcriptional heterogeneity among tumours in each multifocal HCC patient including mutational profiles, copy number alterations, tumour evolutionary trajectory and tumour immune microenvironment profiles. Of note, sorafenib-targeted alterations were identified in the trunk of phylogenetic tree in only one out of the six patients, which may explain the relative low treatment response rate to sorafenib in clinical practice. Moreover, we demonstrated RNA expression patterns and tumour immune microenvironment profiles of all nodules. We found that RNA expression pattern was associated with Edmondson-Steiner grading. Based on the differential expression of 66 reported immune markers, unsupervised hierarchical clustering analysis of 34 nodules identified immune subsets: one low expression cluster with seven nodules and one high expression cluster with 11 nodules. CD8+ T cells were more enriched in nodules of the high expression cluster.ConclusionsOur study provided a detailed view of genomic and transcriptional heterogeneity, clonal evolution and immune infiltration of multifocal HCC. The heterogeneity of druggable targets and immune landscape might help interpret the clinical responsiveness to targeted drugs and immunotherapy for multifocal HCC patients.
| S-EPMC6594462 | biostudies-literature
Project description:BACKGROUND:Because imaging has a high sensitivity to diagnose hepatocellular carcinoma (HCC) and tissue biopsies carry risks such as bleeding, the latter are often not performed in HCC. Blood-derived circulating tumor DNA (ctDNA) analysis can identify somatic alterations, but its utility has not been characterized in HCC. MATERIALS AND METHODS:We evaluated 14 patients with advanced HCC (digital ctDNA sequencing [68 genes]). Mutant relative to wild-type allele fraction was calculated. RESULTS:All patients (100%) had somatic alterations (median?= 3 alterations/patient [range, 1-8]); median mutant allele fraction, 0.29% (range, 0.1%-37.77%). Mutations were identified in several genes: TP53 (57% of patients), CTNNB1 (29%), PTEN (7%), CDKN2A (7%), ARID1A (7%), and MET (7%); amplifications, in CDK6 (14%), EGFR (14%), MYC (14%), BRAF (7%), RAF1 (7%), FGFR1 (7%), CCNE1 (7%), PIK3CA (7%), and ERBB2/HER2 (7%). Eleven patients (79%) had ?1 theoretically actionable alteration. No two patients had identical genomic portfolios, suggesting the need for customized treatment. A patient with a CDKN2A-inactivating and a CTNNB1-activating mutation received matched treatment: palbociclib (CDK4/6 inhibitor) and celecoxib (COX-2/Wnt inhibitor); des-gamma-carboxy prothrombin level decreased by 84% at 2 months (1,410 to 242 ng/mL [normal: ?7.4 ng/mL]; alpha fetoprotein [AFP] low at baseline). A patient with a PTEN-inactivating and a MET-activating mutation (an effect suggested by in silico molecular dynamic simulations) received sirolimus (mechanistic target of rapamycin inhibitor) and cabozantinib (MET inhibitor); AFP declined by 63% (8,320 to 3,045 ng/mL [normal: 0-15 ng/mL]). CONCLUSION:ctDNA derived from noninvasive blood tests can provide exploitable genomic profiles in patients with HCC. IMPLICATIONS FOR PRACTICE:This study reports that blood-derived circulating tumor DNA can provide therapeutically exploitable genomic profiles in hepatocellular cancer, a malignancy that is known to be difficult to biopsy.
| S-EPMC5947459 | biostudies-literature
Project description:Cancer is a disease driven by evolutionary selection on somatic genetic and epigenetic alterations. Here, we propose Canopy, a method for inferring the evolutionary phylogeny of a tumor using both somatic copy number alterations and single-nucleotide alterations from one or more samples derived from a single patient. Canopy is applied to bulk sequencing datasets of both longitudinal and spatial experimental designs and to a transplantable metastasis model derived from human cancer cell line MDA-MB-231. Canopy successfully identifies cell populations and infers phylogenies that are in concordance with existing knowledge and ground truth. Through simulations, we explore the effects of key parameters on deconvolution accuracy and compare against existing methods. Canopy is an open-source R package available at https://cran.r-project.org/web/packages/Canopy/.
| S-EPMC5027458 | biostudies-literature
Project description:Multifocal tumors developed either as independent tumors or as intrahepatic metastases, are very common in primary liver cancer. However, their molecular pathogenesis remains elusive. Herein, a patient with synchronous two hepatocellular carcinoma (HCC, designated as HCC-A and HCC-B) and one intrahepatic cholangiocarcinoma (ICC), as well as two postoperative recurrent tumors, was enrolled. Multiregional whole-exome sequencing was applied to these tumors to delineate the clonality and heterogeneity. The three primary tumors showed almost no overlaps in mutations and copy number variations. Within each tumor, multiregional sequencing data showed varied intratumoral heterogeneity (21.6% in HCC-A, 20.4% in HCC-B, 53.2% in ICC). The mutational profile of two recurrent tumors showed obvious similarity with HCC-A (86.7% and 86.6% respectively), rather than others, indicating that they originated from HCC-A. The evolutionary history of the two recurrent tumors indicated that intrahepatic micro-metastasis could be an early event during HCC progression. Notably, FAT4 was the only gene mutated in two primary HCCs and the recurrences. Mutation prevalence screen and functional experiments showed that FAT4, harboring somatic coding mutations in 26.7% of HCC, could potently inhibit growth and invasion of HCC cells. In HCC patients, both FAT4 expression and FAT4 mutational status significantly correlated with patient prognosis. Together, our findings suggest that spatial and temporal dissection of genomic alterations during the progression of multifocal liver cancer may help to elucidate the basis for its dismal prognosis. FAT4 acts as a putative tumor suppressor that is frequently inactivated in human HCC.
| S-EPMC4823077 | biostudies-literature
Project description:Recently, oncology has moved to a new clinical practice, more personalized, called Predictive Oncology (PO).
PO comes from our knowledge about tumor heterogeneity that implies that each disease, thus each patient, is unique. PO’s goal is to identify and administrate the right treatment to the right patient.
For this, PO requires to go through 3 majors steps:
1. A good characterization of the tumor to identify candidates,
2. A well-established panel of drugs targeting the identified candidates,
3. A relevant model to functionally test these candidates.
The first point could easily be addressed with recent technologies that now allow the Next Generation Sequencing (NGS) and/or the simultaneous analysis of transcriptomic profiles from thousands of patients. The last two points have not been efficiently achieved so far, which prevents PO to be really efficient.
Indeed, even if NGS allows the identification of potential targets, the presence of a molecular candidate does not necessary means obligatory functional response.
The number of drugs approved by the Food and Drug Administration remains limited and most frequent targets in solid tumors (for ex. RAS, P53, MYC, RB1 ...) still do not have specific drugs approved in clinic.
Finally, available pre-clinical models still present many major inconvenient:
* Chimiogrammes on 2D cultures are not sufficiently relevant to be really predictive of the in vivo situation;
* Patient derived xenograft (PDX) are not adapted for clinical use because not all tumors graft and the time to develop a PDX is too long (several months), thus incompatible with the history of the disease (especially for most severe patients). Furthermore the host (NOD-SCID mouse) is immuno-depressed, preventing to objectively test antibodies-mediated drugs.
Recently, the 3D cell culture technology has proven its superiority to predict drug response over classical 2D chimiogrammes. It consists in growing "mini-tissues", or organoid-derived from tumor/healthy tissues, thanks to the amplification of stem cells contained within the sample. The generated organoids are personalized and biologically relevant (organoids are expend form the patient’s stem cells which self-organized according to the architecture of the tissue they are originating from), they are genetically stable, their growth is compatible with patient’s disease history (organoids grow in few weeks), easy and convenient to achieve, even from small biological material quantities (0.5< x < 1cm3), and they can be amplified, frozen and thawed on demand. Moreover, organoids can be made more complex with the addition of other cell types (fibroblasts, immune cells ...). None of the actual available pre-clinical model regroups all these characteristics.
The constitution of a "next generation" biobank of liver samples (Metastases to the liver and Hepato Cellular Adenocarcinoma) will be very useful in the context of predictive oncology.
For this, a biopsy needs to be dissociated and grown in Matrigel, in presence of a well-defined list of growth factors. Once the culture is established, organoids can be frozen then defrost on demand.
Our main objective is to evaluate the feasibility for building a biobank of liver-derived organoids, from liver metastases of colorectal cancers, hepatocellular adenoma and adenocarcinoma (waste tissues).
Applications related to organoids derived from tumors are quasi indefinite, from drug screening assays, tests for novel therapies or original drug combinations, to patients’ stratifications or fundamental research.
In our case, we are interested in building this a biobank in the prospect of using it to build the "next generation of model for predictive oncology" to study liver-related cancers and related drugs testing. Briefly, we want to implement these organoids with cells from the microenvironment in order to makes the global model more pertinent for drug testing.
If successful, the generation of such biobank, including both tumor-derived organoids and healthy counterpart, could be really helpful for the scientific and medical community.
| 2731334 | ecrin-mdr-crc
Project description:Hepatocellular carcinoma (HCC) has limited treatment options. Molecular analysis of its mutational landscape may enable the identification of novel therapies. However, biopsy is not routinely performed in HCC. The utility of analyzing cell-free circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) is not established. We performed 32 ctDNA NGS analyses on 26 patients; 10 of these patients had tissue NGS (236 to 626 genes). ctDNA was evaluated using an assay that detects single nucleotide variants, amplifications, fusions, and specific insertion/deletion alterations in 54 to 70 genes. The ctDNA demonstrated that 23 of 26 patients (88.5%) had ?1 characterized alteration, and all these individuals had ?1 potentially actionable alteration. The most frequently mutated gene was TP53 (16 of 26 patients, 61.5%). There were 47 unique characterized molecular alterations among 18 total gene alterations [variants of unknown significance (VUS) excluded)]. ctDNA and tissue NGS frequently showed different profiles, perhaps due to length of time between tissue and blood samples [median = 370 days (range, 29 to 876 days)]. Serial ctDNA evaluation in an illustrative patient treated with capecitabine demonstrated emergence of a new TP53 alteration after progression. In conclusion, ctDNA profiling is feasible in advanced HCC, and serial assessment using ctDNA NGS can reveal genomic changes with time. NGS of ctDNA provides a minimally invasive alternative for identifying potentially actionable gene alterations and potential molecular targeted therapies. Dynamic changes in molecular portfolio associated with therapeutic pressure in difficult-to-biopsy patients can be observed. Mol Cancer Ther; 17(5); 1114-22. ©2018 AACR.
| S-EPMC5932233 | biostudies-literature
Project description:Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular carcinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co-expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4 + anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addition of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive T cell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 regimens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance T cell infiltration and the intratumoral performance of T lymphocytes.
| S-EPMC10140615 | biostudies-literature
Project description:Background: Many patients with advanced non-small cell lung cancer manifested with metastasis, and molecular heterogeneity may exhibit between primary and metastatic tumors. We sought to investigate the clinical detection strategy of primary and metastatic tumors in Chinese patients with NSCLC.Methods: Here, 77 paired tumors of Chinese patients with lung adenocarcinoma were analyzed, and 1836 mutation in hotspot regions of 22 genes were identified by next-generation sequencing. The expression of ALK in these paired tumors was also detected by immunohistochemistry.Results: The results showed that the concordance rate in multiple pulmonary nodules, primary-LN metastasis pairs and primary-distant metastasis pairs was 67.7%, 94.1% and 86.7%, respectively. In multiple pulmonary nodules, the concordance rate was 100% when the pathologic diagnosis was intrapulmonary metastasis, whereas the concordance rate was 23.1% when the pathologic diagnosis was multiple primary tumors. TP53 and CTNNB1 mutations were detected as the recurrent alterations in LN metastasis. Moreover, the concordance of ALK status was observed in these pairs.Conclusions: Our data suggested that hotspot mutations and ALK status in the primary-metastasis pairs had a high concordance in lung adenocarcinoma. Clinical detection of one lesion may be enough to identify the key alterations except that patients are diagnosed with multiple primary tumors or have disease progression after benefiting from target therapy.
| S-EPMC7012153 | biostudies-literature
Project description:Next-generation sequencing (NGS) of surgically resected solid tumor samples has become integral to personalized medicine approaches for cancer treatment and monitoring. Liquid biopsies, or the enrichment and characterization of circulating tumor cells (CTCs) from blood, can provide noninvasive detection of evolving tumor mutations to improve cancer patient care. However, the application of solid tumor NGS approaches to circulating tumor samples has been hampered by the low-input DNA available from rare CTCs. Moreover, whole genome amplification (WGA) approaches used to generate sufficient input DNA are often incompatible with blood collection tube preservatives used to facilitate clinical sample batching.To address this, we have developed a novel approach combining tumor cell isolation from preserved blood with Repli-G WGA and Illumina TruSeq Amplicon Cancer Panel-based NGS. We purified cell pools ranging from 10 to 1000 cells from three different cell lines, and quantitatively demonstrate comparable quality of DNA extracted from preserved versus unpreserved samples.Preservation and WGA were compatible with the generation of high-quality libraries. Known point mutations and gene amplification were detected for libraries that had been prepared from amplified DNA from preserved blood.These spiking experiments provide proof of concept of a clinically applicable workflow for real-time monitoring of patient tumor using noninvasive liquid biopsies.
| S-EPMC4947859 | biostudies-other
Project description:BackgroundThe efficacy of adjuvant targeted therapy for operable lung cancer is still under debate. Comprehensive genetic profiling is needed for detecting co-mutations in resected epidermal growth factor receptor (EGFR)-mutated lung adenocarcinoma (ADC), which may interfere the efficacy of adjuvant tyrosine kinase inhibitor (TKI) treatment.Materials and methodsMutation profiling of 416 cancer-relevant genes was conducted for 139 resected stage I-IIIa lung ADCs with EGFR mutations using targeted next-generation sequencing. Co-mutation profiles were systematically analyzed.ResultsRare EGFR alterations other than exon 19 deletion and L858R, such as L861Q (∼3%) and G719A (∼2%), were identified at low frequencies. Approximately 10% of patients had mutations in EGFR exon 20 that could confer resistance to first-generation TKIs. Ninety-one percent of patients harbored at least one co-mutation in addition to the major EGFR mutation. TP53 was the top mutated gene and was found more frequently mutated at later stage. Markedly, NF1 mutations were found only in stage II-III ADCs. Conversely, RB1 mutations were more frequent in stage I ADCs, whereas APC mutations were observed exclusively in this group. Thirty-four percent of patients with EGFR TKI-sensitizing mutations had genetic alterations involving EGFR downstream effectors or bypass pathways that could affect the response to EGFR TKIs, such as PIK3CA, BRCA1, and NOTCH1.ConclusionOperable lung ADCs with EGFR TKI-sensitizing mutations are associated with a high proportion of co-mutations. Mutation profiling of these resected tumors could facilitate in determining the applicability and efficacy of adjuvant EGFR TKI therapeutic strategy.Implications for practiceThe efficacy of adjuvant epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy for lung cancer harboring EGFR mutation after surgical resection is still under debate. Next-generation sequencing of 416 cancer-relevant genes in 139 resected lung cancers revealed the co-mutational landscape with background EGFR mutation. Notably, the study identified potential EGFR TKI-resistant mutations in 34.71% of patients with a drug-sensitizing EGFR mutation and who were naive in terms of targeted therapy. A comprehensive mutation profiling of these resected tumors could facilitate in determining the applicability and efficacy of adjuvant EGFR TKI therapeutic strategy for these patients.
| S-EPMC6795151 | biostudies-literature