Abstract 2301: Copy number profiling of circulating CD45+/cytokeratin+ cells in a patient with metastatic breast cancer
Authors: Higa Nikki , Limb Audrey , Rivera Andrés , Kolatkar Anand , Sakura Sara , Joseph Julie , Kildee Margaret , Mullican Lynne , Shriver Craig D. , Hicks James , Kuhn Peter 
Published: June 2023

Background: In carcinomas, circulating tumor cells (CTCs) are classically defined as nucleated cells with expression of epithelial markers (e.g. cytokeratins (CK)) and lacking expression of the pan-leukocyte marker, CD45. While circulating cells expressing both epithelial and leukocyte markers (double-positive cells, DPs) have also been reported in cancer patients, they are typically excluded from further characterization per this classical definition. The leading hypothesis for the presence of DPs is heterotypical cell fusion between tumor cells and macrophages. However, there is insufficient evidence to exclude alternative biological origins or to demonstrate that patient DPs indeed contain tumor-derived genomes. Here, we examined the clonality and copy number status of DPs together with CTCs from a metastatic breast cancer (MBC) case.

Methods: Two blood samples from an ER+/HER2- MBC patient collected prior to and after three weeks of endocrine-based therapy were analyzed. Cells were plated onto glass slides and stained with immunofluorescence antibodies to identify CTCs (DAPI+, CK+, CD45-) and DPs (DAPI+, CK+, CD45+) using a published, enrichment-free detection workflow. Individual cells were then isolated via micro-manipulation and subjected to low-pass whole-genome sequencing for single-cell copy number profiling.

Results: Over 3000 CK+ cells were detected per slide analyzed (&gt;6920/mL blood) and approximately 80% were CD45+ (DPs). Copy number profiling of 40 CK+ cells and 3 white blood cell (WBC) controls revealed clonal alterations that were shared across CTCs and DPs, but not WBCs. These included gains and losses on chromosome (chr) 1, loss on chr 4q, a “firestorm”-like signature on chr 8, chr 16p gain, and chr 16q loss. Alteration amplitudes were also consistent between CTCs and DPs, with no indication of amplitude dampening in DPs. Loss of chr X was observed in a subclone of CK+ cells, including 8/18 CTCs and 4/22 DPs. Copy number gain of the PTPRC gene, which encodes for the CD45 protein, was observed across 38/40 CK+ cells and was not associated with CD45 expression. No other CNAs were unique to CTCs or DPs.

Conclusions: This is the first direct comparison of DP and CTC copy number profiles from the same patient. We show that DPs share clonal CNAs with CTCs, confirming their tumor lineage. Importantly, DP CNA amplitudes did not exhibit dampening, which would be expected if non-tumor DNA was present, and DP profiles did not contain additional CNAs, which often result when hybrid cells undergo division or ploidy reduction. Ongoing work is focused on proteomic characterization to examine expression of additional leukocyte markers. However, these preliminary results were not consistent with the hypothesis of DPs as tumor-immune cell hybrids and highlights the need for further studies to understand their biological and clinical significance.

Citation Format: Nikki Higa, Audrey Limb, Andrés Rivera, Anand Kolatkar, Sara Sakura, Julie Joseph, Margaret Kildee, Lynne Mullican, Craig D. Shriver, James Hicks, Peter Kuhn. Copy number profiling of circulating CD45+/cytokeratin+ cells in a patient with metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2301.