Frequent loss of RUNX3 gene expression in remnant stomach cancer and adjacent mucosa with special reference to topography

Abstract

Our previous studies suggest that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer. This study was conducted to determine whether alteration of RUNX3 gene expression could be detected in the normal-looking gastric remnant mucosa, and to ascertain any difference in the potential of gastric carcinogenesis between the anastomotic site and other areas in the remnant stomach after distal gastrectomy for peptic ulcer (RB group) or gastric cancer (RM group), by analysing RUNX3 expression with special reference to topography. A total of 89 patients underwent distal gastrectomy for gastric cancer from the intact stomach (GCI group) and 58 patients underwent resection of the remnant stomach for gastric cancer (RB group: 34 cases, RM group: 24 cases). We detected RUNX3 and gene promoter methylation by in situ hybridisation, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and methylation-specific PCR. The interval between the initial surgery and surgery for remnant gastric cancer (interval time) was 10.4 years in the RM group, and 27.5 years in the RB group. Cancers in the RB group were significantly more predominant in the anastomosis area (P<0.05). Within the tumour, downregulation of RUNX3 expression ranged from 74.7 to 85.7% in the three groups. The rate of downregulation of RUNX3 of adjacent mucosa was 39.2% (11 in 28 cases) in RB and 47.6% (10 in 21 cases) in RM, which are significantly higher than that of the GCI group (19.5%, 17 in 87 cases). In noncancerous mucosa of the remnant stomach in the RB group, RUNX3 expression decreased more near the anastomosis area. In the RM group, however, there were no significant differences in RUNX3 expression by sampling location. Based on RUNX3 downregulation and clinical features, residual stomach mucosa of the RM group would have a higher potential of gastric carcinogenesis compared to the RB or GCI group. Gastric stump mucosa of the RB group has higher potential especially than other areas of residual stomach mucosa. Measurement of RUNX3 expression and detection of RUNX3 methylation in remnant gastric mucosa may estimate the forward risk of carcinogenesis in the remnant stomach.

Figure 1

In situ hybridisation of RUNX3 mRNA in a remnant gastric cancer specimen. Haematoxylin and eosin staining (A) and in situ hybridisation using an antisense probe (B). Cancer tissue faces the lumen (upper side). Scale bars are equal to 1 mm. Enlargement of boxed regions marked (C–H) in (A) and (B). (C, D) show cancer cells (Ca.); (E and F) show normal mucosa; and (G and H) show intestinal metaplasia (IM). Scale bars are 100 μm.

Figure 2

RUNX3 expression in noncancerous remnant gastric mucosa (RB group, Billroth-II reconstruction). (A, B) are gastric cystica polyposa in the anastomosis area, (C, D) are distal, and (E, F) are proximal. Haematoxylin and eosin staining (A, C, E) and in situ hybridisation using antisense probe (B, D, F). Mucosa faces the lumen (upper side). Scale bars are 300 μm.

Figure 3

Downregulation rate of RUNX3 expression in mucosa and tumour in GCI, RB, and RM groups. T; tumour. N; noncancerous adjacent mucosa.

Figure 4

Quantitative RT–PCR of noncancerous mucosa in RB and RM groups. Resected remnant stomach; RM group, Billroth-I reconstruction (A). RB group, Billroth-II reconstruction (B). Gastric mucosa samples from remnant stomach are 2–3 cm lengths from distal to proximal (a–d). (C) Quantitative analysis of tumour and noncancerous mucosa (a–d) in RM (black bars) and RB groups (white bars). Control; normal gastric mucosa (fundic glands). Mean±s.d. values.

Figure 5

(A) Methylation-specific PCR of remnant gastric cancer and adjacent mucosa. (M) Methylated-sequence-specific PCR. (U) Unmethylated-sequence-specific PCR. Methylated PCR product present in T and N of RM22 and RB32, and T in RB29, unmethylated PCR products in T and N of RB29, T in RM22, and N in RB32. Lanes: T, tumour; N, noncancerous mucosa; P, positive control; DW, distilled water; and SM, size marker. (B) Methylation status of the C residues between −218 and −69 relative to the translation initiation site of the RUNX3 exon 1 region. The nucleotide sequences of the products of MSP of the DNA samples from remnant gastric cancer that do not express RUNX3 (Exp−) (RB30T, RM23T) and noncancerous mucosa that express RUNX3 (Exp+) (RB30N, RM23N) together with wt RUNX3 sequence are at the top. The red C indicates that it was resistant to bisulphite treatment due to methylation. The blue T indicates that it was converted from C by bisulphite treatment, that is, not methylated.