Blood-derived extracellular proteins as potential biomarkers for the diagnosis of early ER+ breast cancer and detection of lymph node involvement

Extracellular vesicles (EVs) are membrane surrounded structures released by different cell types that are involved in cellular communication and are emerging as potential therapeutic and diagnostic targets in cancer¹ as in the case of early pancreatic cancer². Structurally, EVs are contained within a phospholipid bilayer, whose composition is very similar to the cell membrane. EVs carry proteins, nucleic acids (DNA, mRNA, and miRNA), and lipids and are classified into three types: exosomes, ectosomes (microvesicles, MVs) and apoptotic bodies.

Tumor-derived EVs are also critical components for preparing the tumor microenvironment because they enable tumor cells to escape from the immunological surveillance³ and help in the setting of a pre-metastatic niche for the engraftment of detached cancer cells⁴. Both exosomes and MVs have been extensively studied and attributed various important physiological roles in cancer^5,6. For instance, EVs have been found to play an important role in every phase of cancer development from cancer initiation, invasion and metastasis⁷. For these reasons, EVs are potential therapeutic and diagnostic targets in cancer and EV-derived biomarkers maybe useful for predicting future metastatic development and identify metastasis sites⁸.

ER+ (estrogen receptor positive) breast cancer (BC) represents 60–80% of all BC cases^9,10. Here we describe our preliminary findings exploring the role of tumour derived EVs biomarkers that could ultimately be used as part of a test kit for the detection of early ER+ BC and lymph node involvement.

Methods

Results and discussion

Clinical data on the EVs isolated from BC patient’s plasma samples and cell lines can be found in Table 1. The method used for EV isolation also precipitates lipoproteins and immunocomplexes (IC) which are known possible contaminants¹⁴. However, samples submitted for mass spectrometry analysis showed none of the recognised contaminants of high speed centrifugation. In the two patients with early BC (Table 2a), we detected HCG1745306 isoform CRA-a, a protein from the family of alpha type haemoglobins and for the patient with lymph node involvement, we detected histone H1.2 (Table 2 a–b). HCG1745306 isoform CRA-a was only present in the two patients with early BC with Mascot scores of 3208.8 and 3966.5, respectively and absent in all controls and other patient samples.

Histone H1.2 was also detected in samples from the two patients with bone metastases, a parental primary BC sample and metastatic SKBr-3, MDA468, MDA231 cell lines. However, histone H1.2 was absent from the plasma sample of a patient with multiple metastases, from the non-metastatic MCF7 cell line (a non significant mascot score) and from MCF10A cells EVs (Table 2b). These observation suggests that histone H1.2 might represent a potential marker for LN involvement and metastatic potential. Recent studies suggest histone H1.2 phosphorylation may be useful as a clinical biomarker of breast and other cancers because of its ability to recognize proliferative cell populations. Both MCF7 (expressing an allelic variant A142T) and MDA231, have a greater number of histone H1.2 phosphorylations when compared to MCF10A cell line¹⁵. Curiously, phosphorylation of histone H1.2 at S173 increases during the M phase relative to the S phase, suggesting that this event is cell cycle-dependent and may serve as a marker for proliferation of cancer cells during BC invasion^16,17. Also, histone H1.2 is a novel component of the nucleolar organizer regions during mitosis¹⁸ and H1.2 depletion was observed in a human BC cell line caused cell cycle G1-phase arrest¹⁹. Indeed, a higher mitotic index (≥ 7) in primary tumors is significantly associated with LN involvement²⁰ and higher mitotic indices accurately predict axillary LN involvement at operation²¹.

Our study does suffer from a number of recognised limitations. Firstly, the isolation method used is insufficient to purify EVs from other contaminants. Indeed, the EV literature is quite confusing due, in part, to the over-interpretation of observations from samples prepared using inadequate isolation methods. Secondly, it is not intuitive that EVs would be likely to contain a histone (normally confined to the nucleus) or haemoglobin, therefore our current data does not support the idea that the biomarkers we found are EV-associated and may just represent blood-derived extracellular proteins. Although we only have data for a small number of patients, a strength of our study is that samples were drawn from those with confirmed non-metastatic and metastatic disease at different sites and so are likely to be representative patients.

Nonetheless, our initial observations suggest the possibility that HCG1745306 isoform CRA-a could be used as a potential biomarker for the detection of early ER+ BC. Moreover, histone H1.2 might represent a marker for LN involvement. Further work in a larger cohort of patients clearly needed to refute or confirm these initial findings.

Conclusion

Our early results suggest that blood- derived extracellular biomarkers could represent a means of identifying both patients with early ER+ BC and those in which the disease has spread to the sentinel nodes. Confirmation of these findings in a larger patient cohort might lead to the development of an antibody-antigen based test kit which could be used to provide an instant diagnosis of BC for patients visiting their primary care physician with a suspicious lump, thereby avoid the need for mammography and invasive biopsy.

Data availability

Dataset 1: The mass spectrometry analysis results from all patient samples 10.5256/f1000research.14129.d196597²⁴