Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients

Abstract

Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of smallcell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics.cancer diagnostics | personalized therapy A s a genomic disease, cancer involves a series of changes in the genome, starting from primary tumors, via circulating tumor cells (CTCs), to metastases that cause the majority of mortalities (1-3). These genomic alterations include copy number variations (CNVs), single-nucleotide variations (SNVs), and insertions/deletions (INDELs). Regardless of the concentrated efforts in the past decades, the key driving genomic alterations responsible for metastases are still elusive (1).For noninvasive prognosis and diagnosis of cancer, it is desirable to monitor genomic alterations through the circulatory system. Genetic analyses of cell-free DNA fragments in peripheral blood have been reported (4-6) and recently extended to the whole-genome scale (7-9). However, it may be advantageous to analyze CTCs, as they represent intact functional cancer cells circulating in peripheral blood (10). Although previous studies have shown that CTC counting was able to predict progression and overall survival of cancer patients (11,12), genomic analyses of CTCs could provide more pertinent information for personalized therapy (13). However, it is difficult to probe the genomic changes in DNA obtainable from the small number of captured CTCs. To meet this challenge, a single-cell whole-genome amplification (WGA) method, multiple annealing and loopingbased amplification cycles (MALBAC) (14), has been developed to improve the amplification uniformity across the entire genome over previous methods (15,16), allowing precise determination of CNVs and detection of SNVs with a low false-positive rate in a single cell. Here, we present genomic analyses of CTCs from...

Circulating Tumor Cell Analysis in Metastatic Triple-Negative Breast Cancers

“…Direct molecular characterization of CTCs requires further purification beyond current enrichment strategies, which retain a considerable amount of leukocytes (11). For example, CTC enriched samples obtained via modified versions of the CellSearch method have been subjected to molecular profiling (28)(29)(30)(31). In addition, some methods may provide relative advantage in terms of ability to isolate CTCs usable for genomic and expression analysis (11).…”

Circulating Tumor Cells: A Window into Tumor Development and Therapeutic Effectiveness

“…150 The study researchers also found that a small number of individuals with lung adenocarcinoma (N = 4) exhibited nearly identical global CNV patterns (78% of the gain and loss regions were shared). 150 This finding could suggest the possibility that CTC CNV analysis may be a biomarker for lung adenocarcinoma. It is likely that the genetic analysis of CTCs will be an important area in the development of biomarkers for cancer.…”

“…Because CTCs are a heterogeneous population of cells, single-cell genetic analysis will be required, as will searching the profile(s), determining the metastatic potential of the tumor cell, and understanding the copy number variations of specific genes known to be critical in CTCs. In their study of lung cancer, Ni et al 150 illustrated that all CTCs from a given individual, irrespective of cancer subtype, showed reproducible copy number variation patterns similar to the metastatic tumor in the same individual.…”

“…150 Study findings suggest that gains and losses of copy numbers at certain chromosome regions during metastasis are selected for and supply the tumor cells with a survival advantage in the circulatory system. 150 The study researchers also found that a small number of individuals with lung adenocarcinoma (N = 4) exhibited nearly identical global CNV patterns (78% of the gain and loss regions were shared). 150 This finding could suggest the possibility that CTC CNV analysis may be a biomarker for lung adenocarcinoma.…”

Recent Advances in Studying of Copy Number Variation and Gene Expression

“…Single-cell sequencing helped to discover clonal mutations, cryptic cell types, or transcriptional features that would be diluted or averaged out in bulk tissue. [85][86][87] To provide the proof of concept, genome-wide SNPs and CNVs were successfully detected by using a single human cell. 88 Using single-cell sequencing, McConnell et al further showed that mosaic CNV is abundant in human neurons.…”