Detection and characterisation of p53 aggregates in liquid biopsy with ultrasensitive immunoassays

Background p53 is a pivotal tumour-suppressor that regulates various critical cellular processes. However, p53 can form amyloid aggregates that lose tumour-suppressing functions and can promote tumour growth.

Although p53 aggregates have been observed in cancer cells and tissue, their detection in liquid biopsies has been limited, potentially due to their low concentrations. Detecting p53 aggregates in liquid biopsy may present a promising approach for early cancer detection.

Understanding the roles of p53 aggregates in cancer can lead to advances in cancer biology and novel treatment strategies.

Aims Our previous study established an ultrasensitive immunoassay and revealed significantly higher p53 aggregate concentrations in the plasma of glioblastoma (GB) patients compared to non-cancer controls, demonstrating a diagnostic accuracy of 90.9%.

We further showed that p53 aggregate concentrations may indicate GB recurrence, reflect treatment response, and indicate other primary cancers with brain metastasis. In this study, we aim to fully release the clinical potential of p53 aggregates and elucidate their biological roles.

Clinically, plasma samples from small cohorts of patients with various cancer types will be tested using the established immunoassay.

Additional analysis of p53 aggregate size and assays detecting anti-p53 autoantibodies will be performed to further enhance diagnostic accuracy.

These assays will then be applied to a large number of patient samples to validate p53 aggregates as an important biomarker.

Biologically, p53 aggregate concentrations will be correlated with patients’ clinical and genetic profiles to understand the key factors influencing p53 aggregation. Samples from animal models will also be examined with the assays to understand p53 aggregate production.

Methods Our immunoassay is based on single-molecule array (SiMoA) technology, a commercialised platform capable of detecting proteins at sub-picomolar levels.

SiMoA utilises antibody-conjugated single paramagnetic beads to specifically capture target proteins, which are then bound to a detector antibody and an enzyme.

The beads are sealed into separate microwells, allowing enzyme-carrying beads to catalyse the hydrolysis of a fluorogenic substrate in the microwell, enabling protein concentration quantification from the ratio of fluorescent microwells.

We have identified a particular antibody pair for specifically detecting p53 aggregates with the largest discrimination between the patients and controls.

Use of the research This pioneering study is expected to validate p53 aggregate as a valuable biomarker for early cancer detection, paving the way for population-wide cancer screening programmes.

Elucidating the mechanisms of p53 aggregate production may also inspire innovative diagnostic and therapeutic strategies to benefit more patients.