scholarly journals Spatial and temporal intratumour heterogeneity has potential consequences for single biopsy-based neuroblastoma treatment decisions

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Karin Schmelz ◽  
Joern Toedling ◽  
Matt Huska ◽  
Maja C. Cwikla ◽  
Louisa-Marie Kruetzfeldt ◽  
...  
Keyword(s):  
Risk Stratification ◽  
Copy Number ◽  
Target Genes ◽  
Clonal Evolution ◽  
Gene Mutations ◽  
Therapy Resistance ◽  
Disease Evolution ◽  
Intratumour Heterogeneity ◽  
Spatial And Temporal Heterogeneity ◽  
Transcriptomic Level

AbstractIntratumour heterogeneity is a major cause of treatment failure in cancer. We present in-depth analyses combining transcriptomic and genomic profiling with ultra-deep targeted sequencing of multiregional biopsies in 10 patients with neuroblastoma, a devastating childhood tumour. We observe high spatial and temporal heterogeneity in somatic mutations and somatic copy-number alterations which are reflected on the transcriptomic level. Mutations in some druggable target genes including ALK and FGFR1 are heterogeneous at diagnosis and/or relapse, raising the issue whether current target prioritization and molecular risk stratification procedures in single biopsies are sufficiently reliable for therapy decisions. The genetic heterogeneity in gene mutations and chromosome aberrations observed in deep analyses from patient courses suggest clonal evolution before treatment and under treatment pressure, and support early emergence of metastatic clones and ongoing chromosomal instability during disease evolution. We report continuous clonal evolution on mutational and copy number levels in neuroblastoma, and detail its implications for therapy selection, risk stratification and therapy resistance.

Defining Functional Heterogeneity In Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1365-1365 ◽  
Author(s):  
Stephanie M. Dobson ◽  
Esmé Waanders ◽  
Jessica McLeod ◽  
Ildiko Grandal ◽  
Olga I. Gan ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Survival Rates ◽  
Disease Free Survival ◽  
Therapy Resistance ◽  
High Survival ◽  
Disease Relapse ◽  
Functional Heterogeneity

Abstract Despite high survival rates for children with acute lymphoblastic leukemia (ALL), only 40% of adult patients will achieve long-term disease-free survival, and relapses in both pediatric and adult ALL are often fatal. Most current therapies are directed at molecular markers or dominant pathways present in the bulk of neoplastic cells, yet recent studies have identified many genetically distinct subclones co-existing within a single neoplasm. The functional properties and clinical relevance of these neoplastic subclones remain undefined. Genome wide copy number analysis of matched diagnostic and relapse ALL samples identified that in 50% of patients, the clones present at relapse are not the dominant clones at diagnosis, but have evolved from an ancestral pre-leukemic clone (Mullighan et al., 2008). In order to investigate the functional consequences of clonal evolution in disease progression and therapy resistance, we performed limiting dilution analysis of 3 diagnostic and 14 paired diagnostic/relapse samples from adult and pediatric B-ALL patients of varying cytogenetics, by transplantation into immune-deficient mice (xenografts). In one patient, the leukemia-initiating cell (LIC) frequency was 7.65 fold higher in the relapse sample than at diagnosis, while another patient showed the reverse with a 5.81 fold higher LIC frequency in the diagnostic sample. Two patients showed no significant differences in LIC frequency from diagnosis to relapse. LIC frequency varied from 1 in 14.2 to 1 in 4802 CD19+ blast cells. Interestingly, in 50% of the paired patient samples, transplantation of cells from the relapse sample gave rise to greater leukemic dissemination to the spleen and/or central nervous system of recipient mice in comparison to the diagnostic sample, despite similar levels of engraftment in the bone marrow. This data suggests that although the LIC frequency in B-ALL remains high and relatively static between diagnosis and relapse, relapse cells acquire increased invasive properties. To investigate the clonal composition of 3 diagnostic B-ALL samples, we undertook copy number variation (CNV) analysis of xenografts generated at both limiting and high transplanted cell doses. In all 3 samples, we detected subclones in the xenografts that were distinct from the predominant clone in the primary patient sample. We performed network analysis on these subclones and identified differentially enriched pathways, including differential expression of anti-apoptotic and apoptosis regulation pathways, providing evidence of putative functional differences. These results support the existence of functionally diverse subclones within diagnostic samples as well as functional diversity between the subclones present at diagnosis and relapse. Ongoing in depth genomic analysis of the diagnosis/relapse paired samples will add to our understanding of the functional role of the subclones identified at diagnosis in the establishment of disease relapse. In summary, these experiments will provide further insight into the functional heterogeneity present in B-ALL and the drivers of lymphoid leukemogenesis that lead to therapy failure and disease relapse. Disclosures: Danska: Trillium Therapeutics/Stem Cell Therapeutics: Research Funding.

From Favorable Histology to Relapse: The Clonal Evolution of a Wilms Tumor

2019 ◽  
Vol 23 (2) ◽  
pp. 167-171
Author(s):  
Jason Saliba ◽  
Natasha Belsky ◽  
Ami Patel ◽  
Kristen Thomas ◽  
William L Carroll ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Copy Number ◽  
Wilms Tumor ◽  
Clonal Evolution ◽  
Therapy Resistance ◽  
Biological Pathways ◽  
Copy Number Analysis ◽  
Favorable Histology ◽  
Whole Exome ◽  
Genetic Landscape

Favorable histology (FH) Wilms tumor (WT) is one of the most curable of all human cancers, yet a small minority of patients fail treatment. The underlying biological pathways that lead to therapy resistance are unknown. We report a case of initially unresectable, FH WT which revealed limited necrosis and persistent blastemal predominant histology following neoadjuvant chemotherapy. Despite intensification of therapy and whole abdominal radiation, the patient relapsed and succumbed to her disease. In an effort to discover candidate drivers of drug resistance, whole exome sequencing and copy number analysis were performed on samples from all 3 tumor specimens. Sequencing results revealed outgrowth of clones with a dramatically different genetic landscape including dominant mutations that could explain therapy evasion, some of which have not been previously reported in WT. Our results implicate PPM1D, previously shown to be associated with drug resistance in other tumors, as the major driver of treatment failure.

Circulating tumor cell (CTC) genomic signatures of hormone therapy resistance in men with metastatic castration-resistant prostate cancer (mCRPC).

2020 ◽  
Vol 38 (6_suppl) ◽  
pp. 147-147
Author(s):  
Santosh Gupta ◽  
Susan Halabi ◽  
Gabor Kemeny ◽  
Monika Anand ◽  
David M. Nanus ◽  
...  
Keyword(s):  
Clinical Benefit ◽  
Copy Number ◽  
Clonal Evolution ◽  
Circulating Tumor Cell ◽  
Therapy Resistance ◽  
Comparative Genomic ◽  
Copy Number Alterations ◽  
Castration Resistant Prostate Cancer ◽  
Protein Assay ◽  
Poor Outcomes

147 Background: Men with CTC AR-V7 + mCRPC have very poor outcomes when treated with enzalutamide/abiraterone. However, many men lack AR-V7. Here, we determined whether baseline or post-treatment DNA alterations in CTCs from AR-V7 negative mCRPC men could provide clinical utility in predicting outcomes with these hormonal therapies. Methods: We analyzed whole-genome copy number alterations (CNA) using array-comparative genomic hybridization (aCGH) in CTCs from 48 men (45 baseline and 28 progression), and whole-exome sequencing (WES) from 11 mCRPC men treated with abi/enza, longitudinally, and focused exclusively on AR-V7 negative men (N = 40) by the Epic-AR-V7 nuclear protein assay, and comparing those men who benefit from therapy vs. who do not. Results: We observed broad heterogeneity of CNAs between patients; common genomic alterations included gain in KDM6A (44%), FOXA1 (44%), MYCN (32%), and AR (38%), and loss in BRCA1 (30%) and PTEN (25%). Men who had the clinical benefit to abi/enza (n = 23, median PFS 10 mo) were more likely to have CTCs with genomic gains of ATM, HSD17B4, or PTEN, and loss of BRAF, ABL1, or NKX3-1. Likewise, men who did not benefit from abi/enza (n = 14, median PFS 2.6 mo) had CTCs with more copy number alterations than men who had clinical benefit (median 19.5 vs. 14, p = 0.01), and were enriched for gain of BRCA2, APC, KDM5D, SPARC, MYCN, AR, and CYP11B1, and loss of PTEN, CHD1, PHLPP1, and NCOR2. After progression on abi/enza, we observed clonal evolution of CTCs harboring gain of ATM, FOXA1, KDM6A, CYP11B1, MYC, APC, and NCOR2, and loss of NCOR1, ERG, and RUNX2. Several COSMIC-validated non-synonymous pathogenic exome mutations were detected in progressed or non-responding patients’ CTC DNA; TP53 (55 vs 27% at baseline), AKAP9 (36 vs 9%), CDK12, KMT2D, and BRAF (each 36 vs 18%), and BRD4 and SPOP (each 18 vs 0%). Conclusions: We demonstrate that specific CTC genomic profiles associated with TP53, PTEN, WNT, DNA repair, epigenetic, and AR signaling, as well as lineage plasticity pathways are associated with worse clinical outcomes in AR-V7 negative men with mCRPC treated with abi/enza. Further mechanistic and validation studies are warranted. Clinical trial information: NCT02269982.

Assessment of Clonal Evolution in 42 AML with NPM1 Mutations by Molecular Characterization of Paired Diagnosis and Relapse Samples

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 237-237
Author(s):  
Jan Krönke ◽  
Lars Bullinger ◽  
Frank G. Rücker ◽  
Verena I. Gaidzik ◽  
Michael WM Kühn ◽  
...  
Keyword(s):  
Copy Number ◽  
Conflicts Of Interest ◽  
Clonal Evolution ◽  
Snp Array ◽  
Gene Mutations ◽  
Normal Karyotype ◽  
Genetic Alterations ◽  
Snp Analysis ◽  
Genetic Event ◽  
Npm1 Mutation

Abstract Abstract 237 Mutations in the nucleophosmin 1 (NPM1) gene represent one of the most frequent gene mutations in acute myeloid leukemia (AML), in particular in cytogenetically normal (CN)-AML. NPM1 mutations (NPM1mut) are considered as an early genetic event in the pathogenesis of AML. To address the role of clonal evolution from diagnosis to relapse in NPM1mut AML, we applied high-resolution genome-wide single nucleotide polymorphism (SNP) array analysis using the Affymetrix 6.0 platform to detect copy number alterations (CNAs) and uniparental disomies (UPDs) in paired samples from 42 patients. In addition, we determined NPM1 and FLT3 [internal tandem duplication (ITD) and tyrosine kinase domain (TKD)] mutation status in all samples. Blood or bone marrow samples obtained at complete morphologic remission were available for all patients to exclude germline copy number variations. At diagnosis, 29 cases (69%) had a normal karyotype by cytogenetics and no CNAs and UPDs by SNP analysis. In the 13 remaining cases, we found a total of 10 CNAs in 7 cases (19%), and 6 UPDs in 6 cases (14%): deletions of 9q21 (size range 0.9 to 17 Mb) were detected in 5 cases and were the only recurrent CNA; the only recurrent UPD affected the long arm of chromosome 13 in 4 cases, all resulting in homozygous FLT3-ITD mutations with FLT3-ITD/wildtype ratios >1; heterozygous FLT3-ITD and –TKD mutations were detected in 9 and 7 patients, respectively. At the time of relapse, the number of CNAs increased (34 CNAs in 16 cases, 38%) while the frequency of UPDs remained unchanged (6 UPDs in 6 cases, 14%). Of note, in 6 patients (14%) the NPM1 mutation was no longer detectable at the time of relapse; SNP analysis showed completely distinct CNAs/UPDs in 4 of these patients; 3 of these 4 cases had a small gain at 11q23 corresponding to MLL partial tandem duplications as confirmed by PCR. These findings suggest that these 4 cases were therapy-related AMLs (t-AML) rather than relapsed AML. The median interval from diagnosis to relapse/tAML in these 4 cases was 65 months compared with 9 months for the relapsed cases still having the NPM1 mutation. In the two remaining cases, genetic alterations were neither present at diagnosis nor at relapse. Analysis of other gene mutations (eg, IDH1 and 2, DNMT3A, ASXL1, p53) is currently under way to further elucidate the clonal origin of these cases. Of the 36 NPM1mut positive relapse samples, 15 maintained a “normal karyotype”, and 2 showed the CNAs already present at diagnosis; 19 relapse samples (53%) displayed clonal evolution with acquiring new (n=15) and/or loosing single aberrations (n=4): Acquired recurrent alterations comprised deletions of tumor suppressor genes [ETV6 (n=2), TP53 (n=2), NF1 (n=2), WT1 (n=2)], most of which are uncommon in de novo NPM1mut AML. All 6 UPDs detected in relapse samples affected 13q, of which 3 were already present at diagnosis. One patient with initial heterozygous FLT3-ITD mutation developed a homozygous state by acquiring UPD13q at relapse. Two cases with wild-type FLT3 at diagnosis acquired UPD13q at relapse. Of note, one UPD13q was not present in the corresponding relapse sample anymore. In conclusion, almost half (45%) of NPM1mut AML showed evolution to a more aberrant karyotype at relapse, including acquisition of high-risk genetic changes that may account for the adverse prognosis of relapsed patients. Conversely, other alterations such as UPD13q or del(9q) detected at diagnosis were not always present in relapse samples, implying that relapse had evolved from a more ancestral clone. In addition, our data suggest that in a proportion of cases t-AML rather than relapse had developed. Further analysis, such as gene mutation studies of paired diagnosis/ relapse samples, will provide more detailed information on clonal evolution events in the pathogenesis of NPM1mut AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Single Cell Genetic Profiling of Tumors of Breast Cancer Patients Aged 50 Years and Older Reveals Enormous Intratumor Heterogeneity Independent of Individual Prognosis

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3366
Author(s):  
Anna-Sophie Liegmann ◽  
Kerstin Heselmeyer-Haddad ◽  
Annette Lischka ◽  
Daniela Hirsch ◽  
Wei-Dong Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Patients ◽  
Copy Number ◽  
Genome Instability ◽  
Single Cells ◽  
Gene Mutations ◽  
Intratumor Heterogeneity ◽  
Breast Cancer Patients ◽  
Luminal A ◽  
Pik3ca Mutations

Purpose: Older breast cancer patients are underrepresented in cancer research even though the majority (81.4%) of women dying of breast cancer are 55 years and older. Here we study a common phenomenon observed in breast cancer which is a large inter- and intratumor heterogeneity; this poses a tremendous clinical challenge, for example with respect to treatment stratification. To further elucidate genomic instability and tumor heterogeneity in older patients, we analyzed the genetic aberration profiles of 39 breast cancer patients aged 50 years and older (median 67 years) with either short (median 2.4 years) or long survival (median 19 years). The analysis was based on copy number enumeration of eight breast cancer-associated genes using multiplex interphase fluorescence in situ hybridization (miFISH) of single cells, and by targeted next-generation sequencing of 563 cancer-related genes. Results: We detected enormous inter- and intratumor heterogeneity, yet maintenance of common cancer gene mutations and breast cancer specific chromosomal gains and losses. The gain of COX2 was most common (72%), followed by MYC (69%); losses were most prevalent for CDH1 (74%) and TP53 (69%). The degree of intratumor heterogeneity did not correlate with disease outcome. Comparing the miFISH results of diploid with aneuploid tumor samples significant differences were found: aneuploid tumors showed significantly higher average signal numbers, copy number alterations (CNAs) and instability indices. Mutations in PIKC3A were mostly restricted to luminal A tumors. Furthermore, a significant co-occurrence of CNAs of DBC2/MYC, HER2/DBC2 and HER2/TP53 and mutual exclusivity of CNAs of HER2 and PIK3CA mutations and CNAs of CCND1 and PIK3CA mutations were revealed. Conclusion: Our results provide a comprehensive picture of genome instability profiles with a large variety of inter- and intratumor heterogeneity in breast cancer patients aged 50 years and older. In most cases, the distribution of chromosomal aneuploidies was consistent with previous results; however, striking exceptions, such as tumors driven by exclusive loss of chromosomes, were identified.

scholarly journals Copy Number Alteration Profile Provides Additional Prognostic Value for Acute Lymphoblastic Leukemia Patients Treated on BFM Protocols

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3289
Author(s):  
Μirella Αmpatzidou ◽  
Lina Florentin ◽  
Vassilios Papadakis ◽  
Georgios Paterakis ◽  
Marianna Tzanoudaki ◽  
...  
Keyword(s):  
Risk Stratification ◽  
Copy Number ◽  
Copy Number Alteration ◽  
Lymphoblastic Leukemia ◽  
Risk Index ◽  
Outcome Predictor ◽  
Distinct Subgroup ◽  
Genomic Risk ◽  
Good Risk

We present our data of a novel proposed CNA-profile risk-index, applied on a Greek ALLIC-BFM-treated cohort, aiming at further refining genomic risk-stratification. Eighty-five of 227 consecutively treated ALL patients were analyzed for the copy-number-status of eight genes (IKZF1/CDKN2A/2B/PAR1/BTG1/EBF1/PAX5/ETV6/RB1). Using the MLPA-assay, patients were stratified as: (1) Good-risk(GR)-CNA-profile (n = 51), with no deletion of IKZF1/CDKN2A/B/PAR1/BTG1/EBF1/PAX5/ETV6/RB1 or isolated deletions of ETV6/PAX5/BTG1 or ETV6 deletions with a single additional deletion of BTG1/PAX5/CDKN2A/B. (2) Poor-risk(PR)-CNA-profile (n = 34), with any deletion of ΙΚΖF1/PAR1/EBF1/RB1 or any other CΝΑ. With a median follow-up time of 49.9 months, EFS for GR-CNA-profile and PR-CNA-profile patients was 96.0% vs. 57.6% (p < 0.001). For IR-group and HR-group patients, EFS for the GR-CNA/PR-CNA subgroups was 100.0% vs. 60.0% (p < 0.001) and 88.2% vs. 55.6% (p = 0.047), respectively. Among FC-MRDd15 + patients (MRDd15 ≥ 10−4), EFS rates were 95.3% vs. 51.7% for GR-CNA/PR-CNA subjects (p < 0.001). Similarly, among FC-MRDd33 + patients (MRDd33 ≥ 10−4), EFS was 92.9% vs. 27.3% (p < 0.001) and for patients FC-MRDd33 − (MRDd33 < 10−4), EFS was 97.2% vs. 72.7% (p = 0.004), for GR-CNA/PR-CNA patients, respectively. In a multivariate analysis, the CNA-profile was the most important outcome predictor. In conclusion, the CNA-profile can establish a new genomic risk-index, identifying a distinct subgroup with increased relapse risk among the IR-group, as well as a subgroup of patients with superior prognosis among HR-patients. The CNA-profile is feasible in BFM-based protocols, further refining MRD-based risk-stratification.

scholarly journals Unravelling the Sequential Interplay of Mutational Mechanisms during Clonal Evolution in Relapsed Pediatric Acute Lymphoblastic Leukemia

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 214
Author(s):  
Željko Antić ◽  
Stefan H. Lelieveld ◽  
Cédric G. van der Ham ◽  
Edwin Sonneveld ◽  
Peter M. Hoogerbrugge ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Leukemic Cells ◽  
Proliferative Potential ◽  
Disease Evolution ◽  
Pediatric Acute Lymphoblastic Leukemia ◽  
First Relapse ◽  
Mutational Processes

Pediatric acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and is characterized by clonal heterogeneity. Genomic mutations can increase proliferative potential of leukemic cells and cause treatment resistance. However, mechanisms driving mutagenesis and clonal diversification in ALL are not fully understood. In this proof of principle study, we performed whole genome sequencing of two cases with multiple relapses in order to investigate whether groups of mutations separated in time show distinct mutational signatures. Based on mutation allele frequencies at diagnosis and subsequent relapses, we clustered mutations into groups and performed cluster-specific mutational profile analysis and de novo signature extraction. In patient 1, who experienced two relapses, the analysis unraveled a continuous interplay of aberrant activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) activity. The associated signatures SBS2 and SBS13 were present already at diagnosis, and although emerging mutations were lost in later relapses, the process remained active throughout disease evolution. Patient 2 had three relapses. We identified episodic mutational processes at diagnosis and first relapse leading to mutations resembling ultraviolet light-driven DNA damage, and thiopurine-associated damage at first relapse. In conclusion, our data shows that investigation of mutational processes in clusters separated in time may aid in understanding the mutational mechanisms and discovery of underlying causes.

scholarly journals The Role of microRNAs in Cholangiocarcinoma

2021 ◽  
Vol 22 (14) ◽  
pp. 7627
Author(s):  
Tingting Shi ◽  
Asahiro Morishita ◽  
Hideki Kobara ◽  
Tsutomu Masaki
Keyword(s):  
Cell Cycle Progression ◽  
Target Genes ◽  
Epithelial Mesenchymal Transition ◽  
Survival Rates ◽  
Therapy Resistance ◽  
Resistance Mechanisms ◽  
Mesenchymal Transition ◽  
Diagnosis And Prognosis ◽  
Related Risk

Cholangiocarcinoma (CCA), an aggressive malignancy, is typically diagnosed at an advanced stage. It is associated with dismal 5-year postoperative survival rates, generating an urgent need for prognostic and diagnostic biomarkers. MicroRNAs (miRNAs) are a class of non-coding RNAs that are associated with cancer regulation, including modulation of cell cycle progression, apoptosis, metastasis, angiogenesis, autophagy, therapy resistance, and epithelial–mesenchymal transition. Several miRNAs have been found to be dysregulated in CCA and are associated with CCA-related risk factors. Accumulating studies have indicated that the expression of altered miRNAs could act as oncogenic or suppressor miRNAs in the development and progression of CCA and contribute to clinical diagnosis and prognosis prediction as potential biomarkers. Furthermore, miRNAs and their target genes also contribute to targeted therapy development and aid in the determination of drug resistance mechanisms. This review aims to summarize the roles of miRNAs in the pathogenesis of CCA, their potential use as biomarkers of diagnosis and prognosis, and their utilization as novel therapeutic targets in CCA.

scholarly journals A Modified Method to Isolate Circulating Tumor Cells and Identify by a Panel of Gene Mutations in Lung Cancer

2021 ◽  
Vol 20 ◽  
pp. 153303382199527
Author(s):  
Helin Wang ◽  
Jieqing Wu ◽  
Qi Zhang ◽  
Jianqing Hao ◽  
Ying Wang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Detection Rate ◽  
Target Genes ◽  
White Blood Cells ◽  
Gene Mutations ◽  
Copy Number Variations ◽  
Immunomagnetic Beads ◽  
Membrane Rupture

The CellSearch system is the only FDA approved and successful used detection technology for circulating tumor cells(CTCs). However, the process for identification of CTCs by CellSearch appear to damage the cells, which may adversely affects subsequent molecular biology assays. We aimed to explore and establish a membrane-preserving method for immunofluorescence identification of CTCs that keeping the isolated cells intact. 98 patients with lung cancer were enrolled, and the efficacy of clinical detection of CTCs was examined. Based on the CellSearch principle, we optimized an anti-EpCAM antibody and improved cell membrane rupture. A 5 ml peripheral blood sample was used to enrich CTCs with EpCAM immunomagnetic beads. Fluorescence signals were amplified with secondary antibodies against anti-EpCAM antibody attached on immunomagnetic beads. After identifying CTCs, single CTCs were isolated by micromanipulation. To confirm CTCs, genomic DNA was extracted and amplified at the single cell level to sequence 72 target genes of lung cancer and analyze the mutation copy number variations (CNVs) and gene mutations. A goat anti-mouse polyclonal antibody conjugated with Dylight 488 was selected to stain tumor cells. We identified CTCs based on EpCAM+ and CD45+ cells to exclude white blood cells. In the 98 lung cancer patients, the detection rate of CTCs (≥1 CTC) per 5 ml blood was 87.76%, the number of detections was 1–36, and the median was 2. By sequencing 72 lung cancer-associated genes, we found a high level of CNVs and gene mutations characteristic of tumor cells. We established a new CTCs staining scheme that significantly improves the detection rate and allows further analysis of CTCs characteristics at the genetic level.

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