The tumor biological significance of RNF43 and LRP1B in gastric cancer is complex and context-dependent

Abstract

Gastric cancer (GC) is the fifth most common cancer in the world with a poor prognosis. Both RNF43 and LRP1B function as tumor suppressors in the Wnt signaling pathway and have been described to be frequently mutated in GC. In this study of a large and well characterized cohort of 446 GCs we explored the significance of expression of RNF43 and LRP1B and their correlations with clinicopathological patient characteristics. Immunostaining of whole mount tissue sections was documented with the histoscore. Dichotomized at the median, we separated the cohort into a low/negative and a high/positive group of RNF43 and LRP1B expression, respectively. Apart from the entire cohort, we also examined the intestinal and diffuse type GCs separately. Regarding the entire cohort, the expression of RNF43 and LRP1B correlated significantly with the Lauren phenotype and with each other. Interestingly, differences were noted regarding RNF43 between the intestinal and diffuse type GCs. Survival analysis of the intestinal type GCs showed that RNF43 low/negative GCs tended to have a better outcome compared with RNF43 high/positive GCs [24.5 months overall survival (OS) and 25.0 months tumor-specific survival (TSS) vs. 14.1 months OS and 17.9 months TSS, respectively]. To the contrary, diffuse type GCs with RNF43 low/negative had a worse outcome compared with RNF43 high/positive GCs (12.9 months OS and 18.2 months TSS vs. 17.1 months OS and 21.5 months TSS, respectively). On multivariate analysis, RNF43 low/negative versus high/positive was an independent prognosticator of survival in diffuse type GC (hazard ratio 2.393 for OS and 2.398 for TSS). These data support the contention that the expression and biological effect of RNF43 and LRP1B in GC is context-dependent.

Figure 1

Wnt signaling pathway and its regulatory mechanisms. Wnt ligand binds to FZD-LRP5/6 complex. DVL inhibits the destruction complex (DC) so that β-catenin is not degraded but accumulates in the cytoplasm, translocates into the nucleus and activates transcription factors. RNF43 and LRP1B act as negative regulators of the Wnt signaling pathway (a). Mutations in RNF43 or LRP1B or their loss lead to increased Wnt signaling (b).

Figure 2

Reference slides for RNF43 (a–c) and LRP1B (d–g) immunostaining in gastric cancer. Staining intensities of RNF43 ranged from 0 (a; negative) over 1+ (b; weak staining) to 2+ (c; moderate staining). Images of LRP1B display H-score of 0 (e, negative), 1+ (d, weak staining), 2+ (f; moderate staining) and 3+ (g, strong staining). Anti-RNF43-immunostaining and anti-LRP1B-immunostaining, hematoxylin counterstain. Original magnifications ×400.

Figure 3

Waterfall-plots illustrating the distribution of RNF43 (a) and LRP1B (b) immunostaining among 446 patients. The waterfall plots show case-by-case the results of immunohistochemistry (RNF43-IHC and LRP1B-IHC; linear data presentation). The different colours illustrate the different staining intensities: RNF43-IHC 0 (blue), RNF43-IHC 1+ (light green) and RNF43-IHC 2+ (medium green) as well as LRP1B-IHC 0 (blue), LRP1B-IHC 1+ (light green), LRP1B-IHC 2+ (medium green) and LRP1B-IHC 3+ (dark green). The sum total of all staining intensities found in a single case always added to a total H-score of 100% according to the following formulas: P (RNF43-IHC 0) + P (RNF43-IHC 1+) + P (RNF43-IHC 2+) = 100% and P (LRP1B-IHC 0) + P (LRP1B-IHC 1 +) + P (LRP1B-IHC 2+) + P (LRP1B-IHC 3+) = 100%.

Figure 4

RNF43 and survival depending on Lauren-type. There was no significant correlation with overall or tumor specific survival for either the intestinal (a, b) or the diffuse gastric cancer (c, d). However, an interesting trend is that in the intestinal cohort, patients with low RNF43 expression lived longer than those with high RNF43 expression (a, b). In the diffuse type gastric cancer, patients with high RNF43 expression tended to live slightly longer (c, d).

Figure 5

Images of RNF43 (a–c) and LRP1B (d–f) immunostaining in gastric cancer for different types of mutations. The wild type shows strong protein expression of RNF43 (a) and LRP1B (d). The frameshift mutation shows an attenuated protein expression of RNF43 (b) and LRP1B (e) and the missense mutations shows an almost complete loss of protein expression of RNF43 (c) and LRP1B (f). Anti-RNF43-immunostaining and anti-LRP1B-immunostaining, hematoxylin counterstain. Original magnifications ×400.