A Multi-Center Clinical Study to Harvest and Characterize Circulating Tumor Cells from Patients with Metastatic Breast Cancer Using the Parsortix® PC1 System

Evan N Cohen, Gitanjali Jayachandran, Richard G Moore, Massimo Cristofanilli, Julie E Lang, Joseph D Khoury, Michael F Press, Kyu Kwang Kim, Negar Khazan, Qiang Zhang, Youbin Zhang, Pushpinder Kaur, Roberta Guzman, Michael C Miller, James M Reuben, Naoto T Ueno, Evan N Cohen, Gitanjali Jayachandran, Richard G Moore, Massimo Cristofanilli, Julie E Lang, Joseph D Khoury, Michael F Press, Kyu Kwang Kim, Negar Khazan, Qiang Zhang, Youbin Zhang, Pushpinder Kaur, Roberta Guzman, Michael C Miller, James M Reuben, Naoto T Ueno

Abstract

Circulating tumor cells (CTCs) captured from the blood of cancer patients may serve as a surrogate source of tumor material that can be obtained via a venipuncture (also known as a liquid biopsy) and used to better understand tumor characteristics. However, the only FDA-cleared CTC assay has been limited to the enumeration of surface marker-defined cells and not further characterization of the CTCs. In this study, we tested the ability of a semi-automated device capable of capturing and harvesting CTCs from peripheral blood based on cell size and deformability, agnostic of cell-surface markers (the Parsortix® PC1 System), to yield CTCs for evaluation by downstream techniques commonly available in clinical laboratories. The data generated from this study were used to support a De Novo request (DEN200062) for the classification of this device, which the FDA recently granted. As part of a multicenter clinical trial, peripheral blood samples from 216 patients with metastatic breast cancer (MBC) and 205 healthy volunteers were subjected to CTC enrichment. A board-certified pathologist enumerated the CTCs from each participant by cytologic evaluation of Wright-Giemsa-stained slides. As proof of principle, cells harvested from a concurrent parallel sample provided by each participant were evaluated using one of three additional evaluation techniques: molecular profiling by qRT-PCR, RNA sequencing, or cytogenetic analysis of HER2 amplification by FISH. The study demonstrated that the Parsortix® PC1 System can effectively capture and harvest CTCs from the peripheral blood of MBC patients and that the harvested cells can be evaluated using orthogonal methodologies such as gene expression and/or Fluorescence In Situ Hybridization (FISH).

Keywords: biomarkers; biopsy; blood; breast neoplasms/pathology; circulating; circulating tumor cells; circulating/pathology; liquid biopsy; neoplasms/diagnosis; neoplastic cells; tumor.

Conflict of interest statement

The authors ENC, GJ, KKK, NK, QZ, YZ, PK and RG declare no conflict of interest. RGM reports consulting fees from Fujirebio Diagnostics; MC reports AstraZeneca (Consulting Fees (e.g., advisory boards), Contracted Research) Celcuity (Consulting Fees (e.g., advisory boards)) Eli Lilly (Consulting Fees (e.g., advisory boards), Contracted Research, Fees for Non-CMEServices Received Directly from Commercial Interest or their Agents (e.g., speakers’ bureaus)) Ellipses (Consulting Fees (e.g., advisory boards)) Foundation Medicine (Fees for Non-CME Services Received Directly from Commercial Interest or their Agents (e.g., speakers’ bureaus)) Guardant (Fees for Non-CME Services Received Directly from Commercial Interest or their Agents (e.g., speakers’ bureaus)) Menarini (Consulting Fees (e.g., advisory boards)) Olaris (Consulting Fees (e.g., advisory boards)) Pfizer (Contracted Research, Fees for Non-CME Services Received Directly from Commercial Interest or their Agents (e.g., speakers’ bureaus)) Semonix (Consulting Fees (e.g., advisory boards)); JEL reports grant from ANGLE (Contracted Research); JDK reports Angle, plc (Consulting Fees (e.g., advisory boards), Contracted Research); MFP reports AstraZeneca (Consulting Fees (e.g., advisory boards)) Biocartis SA (Consulting Fees (e.g., advisory boards)) CEPHEID (Consulting Fees (e.g., advisory boards)) Eli Lilly & Company (Consulting Fees (e.g., advisory boards)) Lilly USA, LLC (Consulting Fees (e.g., advisory boards)) Merck & Co. (Consulting Fees (e.g., advisory boards)) Puma Biotechnology (Consulting Fees (e.g., advisory boards)) TORL BIOTHERAPEUTICS LLC (Ownership Interest (stocks, stock options, patent or other intellectual property or other ownership interest excluding diversified mutual funds)) Zymeworks Inc. (Consulting Fees (e.g., advisory boards)); MCM reports ANGLE plc (Full-time employee, Ownership Interest (stocks, stock options, patent or other intellectual property or other ownership interest excluding diversified mutual funds)); JMR reports Angle plc (Consulting Fees (e.g., advisory boards), Contracted Research); NTU reports Angle plc (Contracted Research) AnHeart Therapeutics Inc. (Consulting Fees (e.g., advisory boards)) AstraZeneca Pharmaceutical (Consulting Fees (e.g., advisory boards)) Carisma Therapeutics, Inc. (Consulting Fees (e.g., advisory boards), Contracted Research) CARNA Biosciences, Inc. (Consulting Fees (e.g., advisory boards)) ChemDiv, Inc. (Consulting Fees (e.g., advisory boards), Contracted Research) Chugai Pharmaceutical (Consulting Fees (e.g., advisory boards)) Daiichi Sankyo, Inc. (Consulting Fees (e.g., advisory boards), Contracted Research) DualityBio (Consulting Fees (e.g., advisory boards), Contracted Research) DynaMed (Consulting Fees (e.g., advisory boards)) Eisai Medical Research Inc (Consulting Fees (e.g., advisory boards), Contracted Research) Epic Science (Contracted Research) Gilead Sciences, Inc. (Consulting Fees (e.g., advisory boards), Contracted Research) Kechow Pharma (Consulting Fees (e.g., advisory boards)) Kirilys Therapeutics, Inc. (Consulting Fees (e.g., advisory boards)) Kyowa Hakko Kirin Co., Ltd. (Speaker) LARVOL (Consulting Fees (e.g., advisory boards)) Merck Co. (Contracted Research) OBI Pharma Inc. (Contracted Research) OncoCyte Co. (Consulting Fees (e.g., advisory boards)) Oncolys BioPharma Inc. (Contracted Research) Ourotech, Inc., DBA Pear Bio (Royalty) Peptilogics, Inc. (Consulting Fees (e.g., advisory boards)) Pfizer Inc. (Consulting Fees (e.g., advisory boards), Contracted Research) Phoenix Molecular Designs (Consulting Fees (e.g., advisory boards)) Preferred Medicine, Inc. (Consulting Fees (e.g., advisory boards), Contracted Research) Rakuten Medical, Inc. (Consulting Fees (e.g., advisory boards)) Sumitomo Dainippon Pharma, Inc. (Contracted Research) Sysmex Co. Ltd. (Consulting Fees (e.g., advisory boards)) Takeda Pharmaceuticals, Ltd. (Consulting Fees (e.g., advisory boards)) Unitech Medical, Inc. (Consulting Fees (e.g., advisory boards)). MCM, a full-time employee of ANGLE North America, Inc., a subsidiary of ANGLE plc, and was involved in the design of the study, the analysis and interpretation of the data, the decision to publish the results, and in the writing of the manuscript. The information and results reported in the manuscript, as well as their interpretations, are based on generally accepted scientific principals and methods and as such were not inappropriately influenced by the commercial interest of the funder (ANGLE Europe Limited).

Figures

Figure 1
Figure 1
Study enrollment, eligibility, and evaluation. HV, healthy volunteer; MBC, metastatic breast cancer; Cyto, cytology evaluation; qPCR, quantitative polymerase chain reaction; FISH, fluorescence in situ hybridization.
Figure 2
Figure 2
(a) Flow diagram for cytopathology evaluation in 207 eligible MBC patients. (b) Representative images of cells classified as CTCs (red arrows) from MBC patients that were harvested by the Parsortix PC1 system and deposited onto cytology slides by cytocentrifugation (images not to same scale) and Wright-Giemsa stained. (c) CTC numbers from the review of evaluable Wright-Giemsa-stained cytology slides. CTC, circulating tumor cell; HV, healthy volunteer; MBC, metastatic breast cancer. * p < 0.05, *** p < 0.001.
Figure 3
Figure 3
(a) Flow diagram for qPCR evaluation in eligible HV and MBC patients. (b) CTC count correlates with total CTC-related gene expression (40-Sum of KRT19, EPCAM, ERBB2, TWIST, and SNAI2 Ct values). Heat map and scatter plots of CTC gene expression show correlation of gene expression with the number of CTCs observed (c) Sum CTC related genes expression = 0.3701 ∗ CTC count + 6.771; R square = 0.3405, slope significantly different from zero (p < 0.0001). HV, healthy volunteer; MBC, metastatic breast cancer; qPCR, quantitative polymerase chain reaction. NS = p ≥0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 4
Figure 4
(a) Sum of CTC-related gene expression (40-Sum of EPCAM, KRT19, ERBB2, SNAI2, and TWIST Ct values) in HV, newly diagnosed MBC, and MBC with recurrence or progression. (b) ERBB2 expression in CTC-enriched samples by tissue HER2 status. (c) SNAI2 expression in CTC-enriched samples by tissue HER2 status. CTC, circulating tumor cell; HV, healthy volunteer; MBC, metastatic breast cancer.
Figure 5
Figure 5
(a) Flow diagram for RNA-seq evaluation in eligible HV and MBC patients. (b) Sum of TPM values for all genes differentially expressed (p < 0.001) between Parsortix PC1 harvests obtained from HV comparators and MBC patients. (c) Genes from the KEGG Cancer Pathway were differentially expressed between the HV and MBC harvests (p < 0.05) (d). Net TPM score of these 20 genes for HV and MBC samples. CTC, circulating tumor cell; HV, healthy volunteer; MBC, metastatic breast cancer.
Figure 6
Figure 6
(a) Flow diagram for HER2 FISH evaluation in eligible MBC patients. (b) Example images of HER2 FISH-stained CTCs from MBC patients that were harvested by the Parsortix PC1 system and deposited onto cytology slides (images not to scale). CTC, circulating tumor cell; HV, healthy volunteer; MBC, metastatic breast cancer; FISH, fluorescence in situ hybridization.

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