OFR-2 The SPIT study: creating a saliva based epigenetic biomarker panel to diagnose oesophgeal cancer

Field: Medicine (Health Sciences)

Introduction

The SPIT study aims to investigate the methylation changes in saliva that result from oesophagael cancer with the ultimate aim of developing a diagnostic biomarker.

Methods

We recruited volunteers with adenocarcinoma, intramucosal carcinoma, Barrett’s Oesophagus and High-Grade Dysplasia. We also recruited two distinct classes of control: healthy volunteers and people with no positive diagnosis of abnormalities after an endoscopy. Study participants completed an extensive questionnaire on relevant lifestyle factors and provided a saliva sample. We had the capacity to analyse 192 saliva samples from our wider cohort using two plates of a standard human methylation array technology. We performed stringent DNA quality-control on our samples and we selected case-control matched samples. A series of quality-control measures were calculated for the array data. This necessitated the removal of 81 samples, leaving 111 samples for differential methylation analysis. Stringent batch-effect removal was applied by calculating the residuals of a batch-fitted linear model on each locus. Differential methylation analysis was performed on the residuals using standard Bioconductor packages (limma). In order to facilitate generalisation of the results, this process was repeated in a manner similar to hold-out cross validation, where a sub-cohort generates a model and the remaining left-out samples are used to test it. This was repeated over a thousand times. The loci that were the most often selected were used as a candidate classifier.

Results

The 50 probes most frequently selected in repeated sub-sampling showed a marked pattern of separation between controls and adenocarcinoma, but individual probes also showed strong potential to function as a commercially viable biomarker to distinguish between cases and controls. Two probes in particular, which were both strongly implicated in oesophagael cancer in the literature, showed individual diagnostic success accuracies of 87.6% and 86.2% (calculated from AUC-ROC values). A candidate biomarker panel using just 4 probes was able to classify held-out samples with an accuracy of 94%, and a panel of 6 probes was able to achieve classification rates of 94%. Furthermore, this panel of 6 probes appeared to be achieve optimal classification success, as the addition of further probes did not produce any tangible improvement of classification unless a more substantial set of probes were used (24 probes at 95% accuracy). The biological plausibility of individual probes was further encouraging. It included probes that were strongly overexpressed in oesophagael cancer, and probes associated with genes implicated in oesophagael cancer survival. An additional probe was, interestingly, associated with a critical tumor-suppressive region with known importance in oesophageal squamous cell carcinoma.

Conclusions

We have evidence that a commercially-viable candidate biomarker panel could be implemented to offer a first-pass screening method for oesophagael cancer. This would enable healthcare providers to implement intelligent prioritisation of resources at a time when it is an urgent priority.