Single-Cell NGS-Based Analysis of Copy Number Alterations Reveals New Insights in Circulating Tumor Cells Persistence in Early-Stage Breast Cancer

Abstract

Circulating tumor cells (CTCs) are a rare population of cells representing a key player in the metastatic cascade. They are recognized as a validated tool for the identification of patients with a higher risk of relapse, including those diagnosed with breast cancer (BC). However, CTCs are characterized by high levels of heterogeneity that also involve copy number alterations (CNAs), structural variations associated with gene dosage changes. In this study, single CTCs were isolated from the peripheral blood of 11 early-stage BC patients at different time points. A label-free enrichment of CTCs was performed using OncoQuick, and single CTCs were isolated using DEPArray. Libraries were prepared from single CTCs and DNA extracted from matched tumor tissues for a whole-genome low-coverage next-generation sequencing (NGS) analysis using the Ion Torrent S5 System. The analysis of the CNA burden highlighted that CTCs had different degrees of aberration based on the time point and subtype. CTCs were found even six months after surgery and shared CNAs with matched tumor tissue. Tumor-associated CNAs that were recurrent in CTCs were patient-specific, and some alterations involved regions associated with BC and survival (i.e., gains at 1q21-23 and 5p15.33). The enrichment analysis emphasized the involvement of aberrations of terms, associated in particular with interferon (IFN) signaling. Collectively, our findings reveal that these aberrations may contribute to understanding the molecular mechanisms involving CTC-related processes and their survival ability in occult niches, supporting the goal of exploiting their application in patients’ surveillance and follow-up.

CNA profiling of single CTCs in locally advanced esophageal cancer patients during therapy highlights unexplored molecular pathways.

“…In this scenario, the analysis of circulating tumor cells (CTCs) represents a key player to understand EC aggressiveness related to tumor spreading. Previously we and others illustrated the presence of CTCs in non-metastatic cancers, proving that tumor spreading starts in cancer early stages [11][12][13] .…”

“…In order to compare and quantitatively assess the genomic imbalance of CTCs relative to a matched diploid genome, we calculated a Jaccard similarity index (JI) de ned as the ratio of shared to all aberrations between single CTCs and leucocytes for each patient. JI values range between 0 and 1, with 0 indicating maximal genomic imbalance and 1 indicating complete overlap with a normal diploid genome 13 .…”

Case Report: Analysis of Circulating Tumor Cells in a Triple Negative Spindle-Cell Metaplastic Breast Cancer Patient

“…CTC enumeration and evaluation have been performed in a delayed period after removal of the primary tumor. Therefore, in the absence of their major source, CTCs are likely to come from secondary homing sites such as bone marrow or other occult niches ( 9 , 20 ).…”

“…Genetic and phenotypic heterogeneity is a hallmark of MpBC and has important reflections for cancer treatment as the presence of multiple clones may hide cells responsible for relapse ( 6 , 7 ). In this context, the characterization of circulating tumor cells (CTCs), a rare population of cells considered as pro-metastasis precursors ( 8 ), may be helpful in the unraveling of tumor heterogeneity ( 9 ).…”

Functional Genomic Analysis of Breast Cancer Metastasis: Implications for Diagnosis and Therapy

“…Hence, the detection and molecular characterization of CTCs in early-stage BC may contribute to the decision-making of clinicians in the selection of patients for strict follow ups for consideration of secondary adjuvant treatments [106]. Rossi and coworkers evaluated the CNA profiles of single CTCs isolated from early-stage BC patients at different time points by exploiting a whole-genome low-coverage next-generation sequencing (NGS) approach [107]. They found that CTCs persisting even months after tumor resection shared several CNAs with matched tumor tissue, suggesting the presence of regions potentially associated with their persistence.…”