Protein profiling of fine-needle aspirates reveals subtype-associated immune signatures and involvement of chemokines in breast cancer.

Bo Franzén,Giuseppe Masucci,Lena Kanter,Jonas Kierkegaard,Torbjörn Ramqvist,Gert Auer,Thomas Hatschek,Rolf Lewensohn,Ulf Landegren,Andrey Alexeyenko,Masood Kamali‐Moghaddam

doi:10.1002/1878-0261.12410

Abstract

There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and for follow‐up of personalized cancer therapy, including immunotherapy. Fine‐needle aspiration (FNA) biopsy provides ready access to relevant tissue samples; however, the minute amounts of sample require sensitive multiplex molecular analysis to be of clinical biomarker utility. We have applied proximity extension assays (PEA) to analyze 167 proteins in FNA samples from patients with breast cancer (BC; n = 25) and benign lesions (n = 32). We demonstrate that the FNA BC samples could be divided into two main clusters, characterized by differences in expression levels of the estrogen receptor (ER) and the proliferation marker Ki67. This clustering corresponded to some extent to established BC subtypes. Our analysis also revealed several proteins whose expression levels differed between BC and benign lesions (e.g., CA9, GZMB, IL‐6, VEGFA, CXCL11, PDL1, and PCD1), as well as several chemokines correlating with ER and Ki67 status (e.g., CCL4, CCL8, CCL20, CXCL8, CXCL9, and CXCL17). Finally, we also identified three signatures that could predict Ki67 status, ER status, and tumor grade, respectively, based on a small subset of proteins, which was dominated by chemokines. To our knowledge, expression profiles of CCL13 in benign lesions and BC have not previously been described but were shown herein to correlate with proliferation (P = 0.00095), suggesting a role in advanced BC. Given the broad functional range of the proteins analyzed, immune‐related proteins were overrepresented among the observed alterations. Our pilot study supports the emerging role of chemokines in BC progression. Due to the minimally traumatic sampling and clinically important molecular information for therapeutic decisions, this methodology is promising for future immunoscoring and monitoring of treatment efficacy in BC.

Highlights

One of the main current challenges for breast cancer (BC) therapy is to device methods that allow repeated and minimally invasive sampling with the capacity to identify informative molecular signatures
We have previously demonstrated that ‘leftover’ fineneedle aspiration (FNA) sample material from BC can be analyzed by proximity extension assay (PEA) with high sensitivity, and with results that correlated with routine assessments
38 samples from patients with benign breast lesions and samples (≥ 8 mm in size) from 25 BC patients were analyzed for protein expression

Summary

Introduction

One of the main current challenges for breast cancer (BC) therapy is to device methods that allow repeated and minimally invasive sampling with the capacity to identify informative molecular signatures. This may provide support for the choice of neoadjuvant and/or immune therapy, and a possibility to monitor the disease characteristics during the course of the disease. The minimally traumatic FNA is often used for diagnosis of small nonpalpable, deeply located, or otherwise hard-to-reach lesions, both for primary tumors and for lymph node and other metastases These samples have been subjected to cytological analysis with staining for a small number of proteins. Modern molecular technologies allow more comprehensive analyses of the state of the sampled tissues

Methods

Discussion

Conclusion