Detecting PD-L1 Expression on CTCs in Patients With Metastatic Breast Cancer: A New Liquid Biomarker?

Catherine Alix-Panabières, PhD

Catherine Alix-Panabières, PhD

Immune checkpoint therapy currently enjoys a high profile in several areas of oncology. In a paper published inMolecular Oncology, senior author Catherine Alix-Panabières, PhD, director of the Laboratory of Rare Human Circulating Cells, University of Montpellier Medical Centre, Montpellier, France, and her colleagues, demonstrate the expression of programmed cell death ligand 1 (PD-L1) on circulating tumor cells (CTCs) in patients with hormone receptor positive (HR+), human epidermal growth factor negative (HER2-) metastatic breast cancer.¹

“It’s a unique site in Europe where we put key technologies together to isolate, detect, [and] characterize single CTCs,” explained Alix-Panabières, who established the Laboratory of Rare Human Circulating Cells at the university and has studied CTCs for 15 years.²

In the introduction to its paper, the team describes how one of the challenges of immune checkpoint therapy is identifying patients likely to respond to treatment. A suitable liquid biopsy to detect a marker would save potentially unresponsive patients from exposure to the toxicity profile, identify the development of resistance, and thus provide potential cost savings.¹

“A crucial question today is to identify precisely which patients will benefit from these new drugs … [and] CTCs can provide a real-time liquid biopsy of the tumor,” said Alix-Panabières.”²

CTCs isolated from blood are derived from the primary tumor, as well as from metastases, and provide the opportunity to assess the tumor burden in real time.³Work published by Jilaveanu et al revealed how PD-L1 expression at metastatic sites may exceed that in the primary tumor, and therefore, a core biopsy of the primary tumor may not reflect the PD-L1 expression profile of the metastatic sites.⁴Efforts to define the role of PD-L1 as a biomarker are underway in clinical trials in non-small cell lung cancer (NSCLC), and results of some of these trials were recently reported.⁵

Setting up the assay to detect PD-L1+ CTCs

As a first step, the team needed to devise an assay capable of identifying CTCs expressing PD-L1. Central to the assay was a monoclonal antibody to PD-L1 (IgG1 Clone #130021, catalogue number MAB1561, R & D Systems) and its sensitivity, specificity, and dynamic range were established by western blot, flow cytometry, and immunocytochemical analysis in a range of experiments using several cell lines (eg, SKBR3, MCF-7, and MDA-MB-231). The researchers found that the antibody could detect very low levels of PD-L1 with linear response characteristics. Using the CellSearch^®system, they were able to devise a 3-level PD-L1 immunoscore that rated the intensity of PD-L1 expression: no PD-L1 expression, score 0; low PD-L1 expression, score 1; high PD-L1 expression, score 2.¹

These experiments were carried out with cell cultures, so to ensure the assay would work on cells in human blood, they added some of the cultured cells (200 of SKBR3, which express PD-L1) to 7.5 mL blood samples from healthy volunteers. They found that by using the CellSearch^®system with the CellSearch^®CXC Kit (Janssen) they were able to detect 79.5% of the breast cancer cells.¹

Detection of PD-L1 expressing CTCs from patients with metastatic breast cancer

Blood samples were taken at the University Center of Montpelier and the Montpelier Cancer Institute from patients with HR+ and HER2- metastatic breast cancer, prior to receiving treatment.¹

They selected 16 patients (15 female, 1 male) and performed the assay on blood samples. The 16 patients had a CTC count ≥1. Thirteen of the 16 patients had a CTC count ≥5 (mean, 312.5; median, 27; range, 7-3146). A score of 1 or 2 was given to 11 of 16 (68%) patients with CTCs expressing PD-L1, and the fraction of CTCs expressing PD-L1 ranged from 0.2% to 100%. Describing the results, the authors cautioned, “This information may also depend on the number of detected CTCs. If only patients with ≥10 CTCs are considered, the fraction of PD-L1+ CTCs varied from 0.2% to 50%.”¹

The analysis also revealed interesting heterogeneity. The intensity of staining varied between patients and within patient samples. In addition, there was evidence of patients with mixed PD-L1 phenotypes (immunoscores 1 and 2, 6 patients) and with homogenous phenotypes (1 patient). As a control, performing the assay on blood samples from healthy controls revealed no PD-L1+ CTCs.¹

Future steps

“I believe we have demonstrated that CTC characterization for PD-L1 expression is feasible using a CTC platform. This paves the way for the utilization of a CTC/PD-L1 assay in future clinical trials to explore whether it can stratify patients according to response, and potentially predict the efficacy of immune checkpoint blockade,” said Alix-Panabières.⁶

References:

1. Mazel M, Jacot W, Pantel K, et al. Frequent expression of PD-L1 on

circulating breast cancer cells [published online ahead of print June 9, 2015].Mol Oncol. doi:10.1016/j.molonc.2015.05.009.

2. Genomeweb. Study Detects PD-L1 Expression on Breast Cancer Patient CTCs, Points to Potential New CellSearch Use.https://www.genomeweb.com/molecular-diagnostics/study-detects-pd-l1-expression-breast-cancer-patient-ctcs-points-potential-new. Accessed August 7, 2015.

3. Alix-Panabières C, Pantel K. Challenges in circulating tumour cell research.Nat Rev Cancer. 2014;14:623-631.

4. Jilaveanu LB, Shuch B, Zito CR, et al. PD-L1 expression in clear cell renal cell carcinoma: an analysis of nephrectomy and sites of metastases.J Cancer. 2014;5:166-172.

5. Spira AI, Park K, Mazières J, et al. Efficacy, safety and predictive biomarker results from a randomized phase II study comparing MPDL3280A vs docetaxel in 2L/3L NSCLC (POPLAR).J Clin Oncol. 2015;(suppl): Abstract 8010.

6. PRNewswire. CELLSEARCH® System chosen as platform for new research on PD-L1 and Circulating Tumor Cells.http://www.prnewswire.com/news-releases/cellsearch-system-chosen-as-platform-for-new-research-on-pd-l1-and-circulating-tumor-cells-300110058.html. Accessed August 7, 2015.