MSACL 2024 Abstract

INTRODUCTION

The presence of ductal carcinoma in situ (DCIS) increases the likelihood of a positive surgical margin, due to the impalpable nature of the disease, the unpredictable extension of DCIS beyond the edge of a palpable invasive tumour. Rates of DCIS-associated positive margins reported in the literature range between 30-63%, compared to 14-27% for invasive disease. In the UK, data from Hospital Episode Statistics suggests rates of re-operative intervention for close-positive margins following failed breast conserving surgery (BCS) were substantially higher for DCIS (29.5%) versus invasive disease (18%). The recognition of DCIS is fundamental in the development of an accurate intra-operative margin detection tool. DCIS can co-exist with invasive cancer and the in-situ component can extend beyond the invasive component, increasing the likelihood of a positive margin.

For the past decade Desorption Electrospray Ionisation Mass Spectrometry Imaging (DESI-MSI) has extensively been used as an imaging mass spectrometry technique. DESI-MSI allows the determination of small molecule distribution directly from tissue. TOF-based MSI has commonly been demonstrated for untargeted discovery analyses. However, there is an increasing need for methods that enhance specificity for the detection of low-level analytes considering tissue complexity. There is also a need to produce less complex and higher throughput data to strengthen its potential as a routine clinical tool for IVD (in vitro diagnostics). Tandem quadrupole (TQ) MS are renowned for their sensitivity and specificity in targeted applications using MRM modes of acquisition. This technique has been of particular interest in the field of diagnosis, in particular cancer. As a diagnosis tool, DESI-TQ MSI would allow cancer diagnosis to be quicker, more precise, and more accurately compared to routinely used methodologies in histopathology laboratories. Moreover, data acquired via TQ assay is considerably less complex than HRMS imaging, allowing easier translation to digital healthcare.

METHODS

Fresh frozen breast cancer tissue samples containing healthy and cancerous tissue were cryosectioned at 10 µm thickness and mounted onto glass slides. The DESI-MSI analysis was performed using a DESI stage coupled to a TQ mass spectrometer (TQ-XS, Waters) with a flow rate of 2 µl/min 95:5 v/v methanol:water as ionisation solvent. The N2 nebulising gas pressure was set at 10-15 psi and capillary voltage set between 0.55 to 0.8 kV, allowing a focused spray. Different DESI-MSI scan rates were set from 5-10 scans per second, analysis was performed in positive and negative ion modes. The samples were imaged at different pixel sizes from 20-50 μm. Sections of H&E stained samples were digitised with an optical scanner. The optical image and DESI-MSI image of the samples analysed were aligned using an in-house imaging toolbox and the areas of interest were annotated by a Consultant Histopathologist.

RESULTS

The identified candidate markers from LD-REIMS assay and other metabolites and lipids previously identified in the group were set as target molecules to image the DCIS samples. The assay specificity and the identity of isomeric compounds and interferences was studied by targeting 2 or more product ions and comparing their distribution in the tissue. MRM transitions for the energy metabolites were obtained from previous UPLC MS/MS studies and applied to the DESI-TQ, while other metabolite product ions and CE values were optimised from chemical standards via DESI-TQ. We determined that improving specificity and detection limits choosing the product ion with the better S/N ratio is more important than the abundance of that product compared to other fragments. The spatial distribution of the analysed compounds in different tissue regions was determined then compared to H&E stained images. Differences in abundances of biomarkers between cancerous tissue and healthy tissue were identified, limitations and advantages will be discussed.

CONCLUSION

Overall, with DESI-MRM imaging it is possible to accomplish tissue characterization and identification of breast cancer tissue using only a few selected biomarkers. In addition, increased specificity can provide further understanding of the role of molecules in DCIS. Minimal sample preparation (cryosectioning), faster and less complex data processing are the key advantages of DESI-TQ over other MS imaging in cancer diagnostics.