Heterogeneity of PIK3CA mutational status at the single cell level in circulating tumor cells from metastatic breast cancer patients

Abstract

Circulating Tumor Cells (CTCs) represent a "liquid biopsy of the tumor" which might allow real-time monitoring of cancer biology and therapies in individual patients. CTCs are extremely rare in the blood stream and their analysis is technically challenging. The CellSearch(®) system provides the enumeration of CTCs with prognostic significance in patients with metastatic breast cancer (mBC), but it does not allow their molecular characterization, which might be useful to identify therapeutically relevant targets for individualized treatment. Combining the CellSearch(®) and DEPArray™ technologies allows the recovery of single CTCs as a pure sample for molecular analysis. The purpose of the study was to investigate the heterogeneity of PIK3CA mutational status within single CTCs isolated from individual mBC patients. CTCs were enriched and enumerated by CellSearch(®) in blood samples collected from 39 mBC patients. In 20 out of 39 patients enriched samples with ≥5 CTCs were sorted using DEParray™ to isolate single CTCs or pools of CTCs to be submitted to Whole Genome Amplification (WGA) before sequencing analysis. In 18 out of 20 patients, it was possible to perform PIK3CA sequencing on exons 9 and 20. Twelve subjects were wild type (wt) for the PIK3CA gene. PIK3CA status could also be assessed in pools of CTCs in seven of these patients, with consistent wt status found. Six patients (33%) had a PIK3CA mutation identified. In 2 of the six patients, molecular heterogeneity was detected when mutational analysis was performed on more than one single CTC, including one patient with loss of heterozygosity on both single and pooled CTCs, and one patient with three different PIK3CA variants on single CTCs but PIK3CA wt status on pooled CTC samples. In six out of the 18 cases PIK3CA status was also evaluable on a primary tumor sample. In one of the six cases a discordance in PIK3CA status between the primary (wild-type) and the matched CTC (exon 20 mutation) was observed. This study demonstrates the feasibility of a non-invasive approach based on the liquid biopsy in mBC patients. Moreover, our data suggest the importance of characterizing CTCs at the single cell level in order to investigate the molecular heterogeneity within cells from the same patient.

The Landscape of Circulating Tumor Cell Research in the Context of Epithelial-Mesenchymal Transition

“…The relative rarity of CTCs in patients, especially at early disease stages, should also bring our attention to the representativeness of molecular characteristics of the few captured cells. Single-cell genomics [180][181][182][183] and transcriptomics [41,138,180,182,184] revealed significant interpatient molecular heterogeneity among CTCs.…”

Clinical Applications of Circulating Tumor Cells and Circulating Tumor DNA as Liquid Biopsy

“…Schneck and colleagues analyzed CTCs using the SNaPshot method and found PIK3CA mutations in enriched CTC pools of 15.9% of patients with metastatic breast cancer ( 80 ). Other authors isolated single CTCs and reported strong heterogeneity in the PIK3CA mutational status among CTCs from individual patients (81)(82)(83). These single CTC analyses resulted in higher detection rates of PIK3CA mutations; however, more information about the occurrence of these mutations in the context of therapeutic interventions is urgently needed.…”

Liquid Biopsies for Monitoring Temporal Genomic Heterogeneity in Breast and Colon Cancers

“…They reported that patients with a baseline CTC count ≥5/7.5 mL of blood had worse progression-free (HR 1.92; p < 0.0001) and overall survival (HR 2.78; p < 0.0001) than patients with a CTC count of <5/7.5 mL. More recently, Pestrin et al [92] reported the feasibility of using CTCs to test the heterogeneity of metastatic breast cancer by evaluating the mutational status of the PIK3CA exons 9 and 20 in both the single and pooled CTCs from 18 patients. Two patients were heterogeneous for the presence of PIK3CA mutations when sequencing analysis was performed on >1 single CTC and 1 patient showed a negative correlation between the PIK3CA status in the primary tumour (wild-type) and the matched CTCs (exon 20 mutation).…”