Gastric Cancer Pre-Stage Detection and Early Diagnosis of Gastritis Using Serum Protein Signatures

Abstract

Background: A gastric cancer (GC) diagnosis relies on histopathology. Endoscopy rates are increasing. Helicobacter pylori infection is a major GC risk factor. In an effort to elucidate abundant blood biomarkers, and potentially reduce the number of diagnostic surgical interventions, we investigated sera and biopsies from a cohort of 219 H. pylori positive and negative patients diagnosed with GC, gastritis, and ulcers. This allowed the comparative investigation of the different gastroduodenal diseases, and the exclusion of protein changes resulting from bacterial infection or inflammation of the gastric mucosa when searching for GC-dependent proteins.

Methods: High-definition mass spectrometry-based expression analysis of tryptically digested proteins was performed, followed by multivariate statistical and network analyses for the different disease groups, with respect to H. pylori infection status. Significantly regulated proteins differing more than two-fold between groups were shortlisted, and their role in gastritis and GC discussed.

Results: We present data of comparative protein analyses of biopsies and sera from patients suffering from mild to advanced gastritis, ulcers, and early to advanced GC, in conjunction with a wealth of metadata, clinical information, histopathological evaluation, and H. pylori infection status. We used samples from pre-malignant stages to extract prospective serum markers for early-stage GC, and present a 29-protein marker panel containing, amongst others, integrin β-6 and glutathione peroxidase. Furthermore, ten serum markers specific for advanced GC, independent of H. pylori infection, are provided. They include CRP, protein S100A9, and kallistatin. The majority of these proteins were previously discussed in the context of cancer or GC. In addition, we detected hypoalbuminemia and increased fibrinogen serum levels in gastritis.

Conclusion: Two protein panels were suggested for the development of multiplex tests for GC serum diagnostics. For most of the elements contained in these panels, individual commercial tests are available. Thus, we envision the design of multi-protein assays, incorporating several to all of the panel members, in order to gain a level of specificity that cannot be achieved by testing a single protein alone. As their development and validation will take time, gastritis diagnosis based on the fibrinogen to albumin serum ratio may be a quick way forward. Its determination at the primary/secondary care level for early diagnosis could significantly reduce the number of referrals to endoscopy. Preventive measures are in high demand. The protein marker panels presented in this work will contribute to improved GC diagnostics, once they have been transferred from a research result to a practical tool.

Keywords: Helicobacter pylori; gastric cancer; gastritis; proteomics; ulcer.

Figure 1

Experimental design and workflow for the elucidation of a GC serum protein marker profile. (A) Biopsies (133, 68 N site, 65 D site) and sera (219) were collected. More than half of the probands were H. pylori positive (59%). Proteins were isolated, trypsinized, and subjected to data-independent MS. Proteins were assigned, and the differences between disease groups were determined considering gender, infection status, and biopsy site, followed by functional classification and network analysis. (B–E) HPp patients with different gastroduodenal clinical manifestations. (B) NGM (no inflammation in antrum), (C) severe antral gastritis, (D) polyp (1st stage GC), (E) GC (differentiated adenocarcinoma).

Figure 2

Comparative biopsy analysis. (A) PCA of combined MoG and MaG (blue) and PanG (purple) shortlisted proteins, compared to NGM (green) and GC (orange), demonstrate the separation of gastritis protein profiles. (B) Venn diagrams showing the number of shortlisted proteins common among the different biopsy groups. Proteins with (B) higher and (C) lower concentration than in NGM.

Figure 2

Comparative biopsy analysis. (A) PCA of combined MoG and MaG (blue) and PanG (purple) shortlisted proteins, compared to NGM (green) and GC (orange), demonstrate the separation of gastritis protein profiles. (B) Venn diagrams showing the number of shortlisted proteins common among the different biopsy groups. Proteins with (B) higher and (C) lower concentration than in NGM.

Figure 3

Comparison of GC vs. NGM shortlisted proteins. (A) Heatmap (Spearman rank correlation). (B) PCA. (C,D) Zero-order networks of (C) up-, and (D) downregulated proteins. For details, see Supplementary Figures S6 and S7.

Figure 3

Comparison of GC vs. NGM shortlisted proteins. (A) Heatmap (Spearman rank correlation). (B) PCA. (C,D) Zero-order networks of (C) up-, and (D) downregulated proteins. For details, see Supplementary Figures S6 and S7.

Figure 3

Comparison of GC vs. NGM shortlisted proteins. (A) Heatmap (Spearman rank correlation). (B) PCA. (C,D) Zero-order networks of (C) up-, and (D) downregulated proteins. For details, see Supplementary Figures S6 and S7.

Figure 4

Heatmaps of serum sample results (Spearman rank). A clear distinction in the protein profiles between gastritis samples (B) and all other groups (A) is detected. Most interestingly, a part of the MiG cases (called MiGa below) cluster with GC samples. (C) Zoom-in. (D) Heatmap of MiG and GC samples clustered by rows and columns. Brackets indicate MiG samples; all other samples are from GC patients. Regular MiG (MiGn) is set apart from GC/MiGa.

Figure 5

FAR identifies gastritis cases. (A) Average MS intensities for serum albumin and fibrinogen detected per patient group. (B) FAR-MS significantly distinguished gastritis samples from NGM, GC, U, and MiGa, as a result of hypoalbuminemia and increased serum fibrinogen. (C) Current procedure for patients with gastroduodenal symptoms: If the efforts of the primary care physician are not successful, a gastroenterologist is consulted. Persistent cases are referred to endoscopy, which clarifies the clinical diagnosis by histopathological examination of gastric biopsies. We suggest that a FAR lab test, in addition to the determination of the H. pylori infection status, can identify the gastritis cases without the need for endoscopy and, thus, reduce the number of medical procedures by about half (as calculated based on the present patient cohort and data).