Single-molecule localisation microscopy: a potential clinical diagnostic tool for breast cancer patients

Session

New and Emerging Concepts in Microscopy

Authors

Lauren Toms (2), Hervé Barjat (2), Tiffany Allen (2), Michael Eyres (2), Denis Alferez (1), Robert Clarke (1), Isabel Peset (3)

Affiliations

1. Breast Biology Group, Division of Cancer Sciences, School of Medical Sciences, University of Manchester
2. Medicines Discovery Catapult
3. Spanish National Cancer Research Centre, CNIO

Keywords

Super resolution, STORM, Breast Cancer, HER2, Clinical, FFPE tissue

Abstract text

Single molecule localisation microscopy (SMLM) enables a 10-fold improvement in resolution compared to standard fluorescence imaging techniques. This powerful tool has been applied across a range of cell sample types to gain a better insight into molecular mechanisms, protein interactions and subcellular structures. This super resolution imaging technique can be used to investigate the cellular nano-environment including subtle changes in structures, protein levels and distributions, that may contribute to disease phenotype. To date, only a few studies have applied SMLM in both healthy and disease tissues.

The aims of this study were to (1) establish a method of SMLM of formalin-fixed paraffin embedded (FFPE) tissue, (2) develop an analysis method to investigate protein clustering and cellular distribution and determine if SMLM could be used to improve patient stratification of tumours, and (3) optimise a SMLM dual staining protocol to improve prediction of treatment response.

The data presented here gives an example of the use of SMLM for the study of HER2 expression in breast cancer. HER2 is overexpressed in about 20% of breast cancers and is often associated with the most aggressive form of the disease. On diagnosis, HER2 expression status is determined to assign the most appropriate treatment course. Current diagnosis relies on immunohistochemistry (IHC) to grade the level of HER2 present in a tumour biopsy sample. A patient tumour with grade HER2 3+, or ‘HER2 positive’, will be given anti-HER2 treatment, such as trastuzumab, whereas a grade of 0 or 1+ is defined as ‘HER2 negative’ and will not be treated with anti-HER2. If a tumour is graded as HER2 2+, or ‘HER2 equivocal’, further tests, such as in situ hybridisation, are required to determine the correct route of treatment. The current diagnostic IHC test uses antibodies that bind to the intracellular region of HER2. However, the most prescribed treatment for HER2 positive tumours is trastuzumab, which binds to the extracellular domain. This can create discrepancies between diagnosis and the expected treatment response due to alterations, restricted access, or absence of the extracellular binding site on HER2.

The SMLM method was first developed using patient derived FFPE xenographs from breast cancer patients. SMLM, in agreement with previous studies, showed that HER2 expression correlated with HER2 diagnostic status, and that the protein was predominantly found in the cell membrane of HER2 2+ and 3+ tumour samples, with much less present in HER2 0 and 1+ samples. The measured HER2 cluster areas increased with the IHC clinical grade. Interestingly, HER2 clusters were smaller when labelled with the extracellular binding antibody, trastuzumab, compared to the intracellular antibody. This suggests that binding of trastuzumab to the extracellular domain may be restricted. Following this, SMLM was carried out on 8 FFPE breast cancer patient samples of varying HER2 grades.

In conclusion, SMLM has unprecedented resolution that can be applied to clinical samples and potentially used in a clinical diagnostic setting. This may reduce the diagnostic tests required, time for diagnosis and lead to the most appropriate treatment course. Larger investigations are required, including monitoring patient’s response to treatment and correlating SMLM to protein and RNA levels.

References

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