LGR5 expression is associated with prognosis in poorly differentiated gastric adenocarcinoma

We identified 91 PD-AC cases who underwent surgical resection between 2008 and 2018 at six institutes [Shinshu University Hospital (Matsumoto, Japan), Nagano Municipal Hospital (Nagano, Japan), Aizawa Hospital (Matsumoto, Japan), Showa Inan General Hospital (Komagane, Japan), Iida Municipal Hospital (Iida, Japan), and Nagano Matsushiro General Hospital (Nagano, Japan)] and evaluated their clinicopathological features. Of these patients, stage II and III cases were selected. A re-evaluation by two pathologists (T.U. and H.O.) before analysis excluded five cases that did not contain poorly differentiated components, and 41 cases remained as candidates for analysis. This study was approved by the Ethics Committee of Shinshu University, Japan (no. 4088).

Paraffin blocks fixed with 8% formaldehyde containing sufficient tumor for analysis were prepared for hematoxylin and eosin (HE) staining and tissue microarray (TMA) analysis by extracting a core with a diameter of 3 mm from each case. Additionally, the TMA was subjected to immunostaining using antibodies against the following mismatch repair proteins (MMRPs): MLH1 (ES05; mouse monoclonal; dilution 1:50), PMS2 (EP51; rabbit monoclonal; dilution 1:40), MSH2 (FE11; mouse monoclonal; dilution 1:50), or MSH6 (EP49; rabbit monoclonal; dilution 1:50; Agilent Technologies, Santa Clara, CA, USA), as described previously [10]. Representative images of the slides were captured with an Olympus DP74 camera (lens, × 40; Olympus, Tokyo, Japan) using the CellSens Standard software (Olympus, Tokyo, Japan). The images were acquired at a resolution of 96 dpi and Adobe Photoshop was used to enhance the resolution of the images to 300 dpi. As reported in our previous paper, the staining results were scored as positive when a nuclear staining pattern was observed. If at least one of the four antibodies did not show expression, MMR protein deficiency was indicated. In the tumor, the tumor-infiltrating lymphocyte (TIL) score was assessed using a four-tier scale and recorded as follows: none: 0; mild: 1; moderate: 2; and marked: 3 [11]. The TIL score was classified as low grade (scores 0 and 1) and high grade (2 and 3).

The EBER in situ hybridization assay was performed on TMA block sections. Epstein–Barr virus (EBV) was identified using EBER probes (ISH iVIEW Blue detection kit; Ventana Medical Systems Inc., Oro Valley, AZ, USA).

LGR5 mRNA detection was performed using the RNAscope® kit (Advanced Cell Diagnostics, Hayward, CA, USA), as described previously [10]. Additionally, a four-step evaluation method we reported previously was used [10]. Furthermore, LGR5 mRNA expression levels were categorized as low expression (grades 0, 1+, 2+, and 3+) and high expression (4+). We analyzed the relationship between LGR5 expression levels and the clinicopathological data and prognosis in patients with PD-AC, particularly regarding overall survival (OS).

mRNA-seq analysis was performed using the TCGA database. The TCGA data were downloaded from cBioPortal (http://​www.​cbiopor- tal.​org/) in the form of mRNA median values. All clinical and pathological data for the TCGA cohort were downloaded from cBioPortal. The TCGA cohort was classified into high or low LGR5 mRNA expression groups. LGR5 high indicates LGR5 mRNA expression ≥ the median, and LGR5 low indicates LGR5 mRNA expression < the median. Stage II/III patients were included in the first analysis, and then only Stage II/III patients with histological grade 3 were included in the second analysis.

The chi-squared test was applied to assess statistical significance. A P-value < 0.05 was considered to be statistically significant. The OS rates of PD-AC patients were calculated using the Kaplan–Meier method, and differences were compared using the log-rank test. Univariate and multivariate analyses for prognostic factors were performed using the Cox proportional hazard regression model. A P-value < 0.05 was considered to be statistically significant. Statistical analysis was performed using JMP version 13 (SAS Institute Japan, Tokyo, Japan).

In PD-AC patients, 38 of 41 cases showed LGR5 expression. Among them, 17 cases were identified as LGR5 high (Fig. 1a and b). Moreover, LGR5 expression was completely absent in three cases (Fig. 1d and e). LGR5 expression varied from diffuse to scattered.

EBER expression was negative in most cases (Fig. 1c). Although there were few EBV-positive PD-AC cases (Fig. 1f), all exhibited low LGR5 expression. LGR5 expression and clinicopathologic data are shown in Table 1. The number of EBV-positive PD-AC cases tended to be higher in the LGR5-low expression group than in the LGR5-high expression group (P = 0.0764). The frequency of vascular invasion tended to be higher in the LGR5-high expression group than in the LGR5-low expression group (P = 0.0764). No significant difference was found between the LGR5-high expression group and LGR5-low expression group regarding TILs, MSI, histological subtype, or TNM stage.

The prognostic value of LGR5 expression in PD-AC was analyzed by the Kaplan–Meier method and log-rank test (Fig. 2). The median OS for the study patients was 1146 (range; 635.5–1718) days. A significant difference was found in OS between PD-AC cases in the LGR5-high expression group [median OS: 756 (range; 154.5–1306.5) days] and LGR5-low expression group [median OS: 1338 (range; 922.75–2022.75) days] (log-rank test, P = 0.0108).

We evaluated the relationship between clinicopathological factors and LGR5 expression regarding OS using a Cox proportional hazard regression model (Table 2), which revealed that the LGR5-low expression group (HR = 0.29; 95% CI: 0.11–0.74; P = 0.01) had independently better OS for PD-AC.

First, we examined the LGR5 mRNA expression levels in the TCGA cohort of stage II/III patients. There was no significant difference in OS between the LGR5 high and low groups (log-rank test, P = 0.7175). Univariate and multivariate analyses for prognostic factors were performed using the Cox proportional hazard regression model. However, there was no significant difference in the univariate analysis of the TCGA cohort (HR = 0.93; 95% CI: 0.64–1.35; P = 0.72).

We then examined the LGR5 mRNA expression levels in the TCGA cohort when stage II/III patients were limited to those with histological grade 3. There was no significant difference in OS between the LGR5 high and low groups (log-rank test, P = 0.7198). Univariate and multivariate analyses for prognostic factors were performed using the Cox proportional hazard regression model. However, there was no significant difference in the univariate analysis of the TCGA cohort (HR = 1.08; 95% CI: 0.70–1.67; P = 0.72).