MALDI imaging identifies prognostic seven-protein signature of novel tissue markers in intestinal-type gastric cancer

Abstract

Proteomics-based approaches allow us to investigate the biology of cancer beyond genomic initiatives. We used histology-based matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry to identify proteins that predict disease outcome in gastric cancer after surgical resection. A total of 181 intestinal-type primary resected gastric cancer tissues from two independent patient cohorts were analyzed. Protein profiles of the discovery cohort (n = 63) were directly obtained from tumor tissue sections by MALDI imaging. A seven-protein signature was associated with an unfavorable overall survival independent of major clinical covariates. The prognostic significance of three individual proteins identified (CRIP1, HNP-1, and S100-A6) was validated immunohistochemically on tissue microarrays of an independent validation cohort (n = 118). Whereas HNP-1 and S100-A6 were found to further subdivide early-stage (Union Internationale Contre le Cancer [UICC]-I) and late-stage (UICC II and III) cancer patients into different prognostic groups, CRIP1, a protein previously unknown in gastric cancer, was confirmed as a novel and independent prognostic factor for all patients in the validation cohort. The protein pattern described here serves as a new independent indicator of patient survival complementing the previously known clinical parameters in terms of prognostic relevance. These results show that this tissue-based proteomic approach may provide clinically relevant information that might be beneficial in improving risk stratification for gastric cancer patients.

Figure 1

MALDI imaging reveals cell type-specific profiles, as shown in this comparison of gastric carcinoma (red) and normal gastric mucosa (green) from an individual patient's tissue. In this study, one example of differentially expressed masses (m/z 8406), exclusively present in cancer cells (right inset, red visualization), was found to correlate significantly with the patients' overall survival.

Figure 2

M/z 3445 and 10098, as measured by MALDI imaging and identified as HNP-1 and S100-A6 correlate with the survival of patients (A, B). Kaplan-Meier analyses in the immunohistochemical validation confirmed their prognostic value although this effect was only observed in patients with UICC stage I cancer (C, n = 29) for HNP-1 and in those with UICC stages II and III for S100-A6 (D, n = 68).

Figure 3

CRIP1, a previously unknown protein in gastric cancer, was found by MALDI imaging as a novel prognostic factor in the discovery cohort (A, C). Immunohistochemical validation confirmed this by showing a strong relationship between the high expression of CRIP1 (E) and poor survival (B) and vice versa (D, B), as calculated by Kaplan-Meier analysis (n = 114).

Figure 4

The prognostic power of a combined pattern was investigated by clustering all patients according to the seven protein signals (A, C). The main two branches of the tree were found to represent a good (blue) and a poor prognosis group (red) (B). Moreover, this pattern predicts patient outcome independently of major clinicopathological parameters (Table 2).