Discovery of Novel and Clinically Relevant Markers in Formalin-Fixed Paraffin-Embedded Esophageal Cancer Specimen

Abstract

Due to the ineffectiveness of chemoradiation and targeted therapy in esophageal anticancer care and the subsequent low survival rates, we constructed a high throughput method to discover and investigate new markers with prognostic, diagnostic, and therapeutic clinical utility. This was accomplished by developing a quick, inexpensive, and dependable platform to simultaneously quantify thousands of proteins which subsequently revealed novel markers involved in the pathogenesis of esophageal adenocarcinoma (EAC) via discovery mass spectrometry paired with conservative biostatistics. Our method uncovered a perfect storm of tumor suppressors being downregulated, proliferation markers ramped up, and chemoresistance markers overexpressed-many of which could serve as new therapy targets for EAC. The 12 markers discovered by this method are novel regarding their involvement in the pathogenesis of EAC. The molecular oncology arena now has a dozen new proteomic targets suitable for validation and elucidation of their clinical utility via gene knockdown in cellular and animal models. This new method can be replicated and applied to other cancers or disease states for research and development and discovery-based investigations. Our findings, which serve as a proof of concept, will hopefully motivate research groups to further expound on the molecular processes involved in the aggressiveness of EAC and other solid tumor diseases, ultimately leading to improved patient management strategies.

Keywords: SWATH analysis; chemoresistance markers; formalin-fixed paraffin-embedded tissue; mass spectrometry; molecular oncology; proliferation markers; proteomics.

Figure 1

Discovery LT-SWATH-MS Workflow. H&E stained sections of our specimen were marked by a board-certified pathologist and used as a guide for microdissections of serial sections stained with hematoxylin. We aimed to attain 12–15 mm² of pure tumor, Barrett’s esophagus (precancerous tissue) or normal squamous esophageal epithelium in each sample. Pre- and post-microdissection images were taken to ensure that tissue retrieval occurred within the specified margins. The microdissected formalin-fixed paraffin-embedded tissue was placed in Eppendorf tubes and covered in roughly 40 µL of Liquid Tissue^® buffer and through a series of heating and digestion stages turned our fixed tissue into a mass spec-friendly lysate. The resultant digest was quantified for protein concentration via nanodrop, cleaned via MCX, and analyzed by TripleTOF mass spectrometry. SWATH analysis and our outlined series of biostatistics (ANOVA, Benjamini–Hochberg procedure, post hoc T-tests, Tukey–Kramer range test), and simple bioinformatics yielded a list of a dozen markers, never before described as being involved in EAC pathogenesis.

Figure 2

Upregulated proliferation markers. These six markers are associated with more advanced progression in various cancers and were found to be upregulated or overexpressed in our cohort’s esophageal adenocarcinoma tumor tissue compared to normal squamous esophageal epithelium, possibly contributing to enhanced invasiveness and shorter overall survival. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.1.

Figure 3

Downregulated tumor suppressors. These six markers are associated with inducing programmed cell death in various cancers and were found to be downregulated in our cohort’s esophageal adenocarcinoma tumor tissue compared to normal squamous esophageal epithelium, possibly contributing to enhanced invasiveness and shorter overall survival. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.1.

Figure 4

Novel marker heat maps—proliferation up, tumor suppression down. Heat maps of the two groups of novel markers (cellular proliferation and tumor suppression) found to be upregulated or downregulated during esophageal pathogenesis (Normal → Barrett’s → Adenocarcinoma). This provides for an individualized bird’s eye view of the expression trends of our newly discovered 12 markers in 50 patient samples, showing consistent trends with very few outliers. Here, we see how the mass spectrometry platform can yield statistically significant biomarkers with consistent and reliable expression trends. Notably, Barrett’s tissue shows expression levels similar to adenocarcinoma, meaning that these protumoral expression trends manifest in the precancerous Barrett’s tissue and carries over throughout carcinogenesis.