MUC5AC/β-catenin expression and KRAS gene alteration in laterally spreading colorectal tumors

Abstract

Aim: To clarify differences in mucin phenotype, proliferative activity and oncogenetic alteration among subtypes of colorectal laterally spreading tumor (LST).

Methods: LSTs, defined as superficial elevated lesions greater than 10 mm in diameter with a low vertical axis, were macroscopically classified into two subtypes: (1) a granular type (Gr-LST) composed of superficially spreading aggregates of nodules forming a flat-based lesion with a granulonodular and uneven surface; and (2) a non-granular type (NGr-LST) with a flat smooth surface and an absence of granulonodular formation. A total of 69 LSTs, comprising 36 Gr-LSTs and 33 NGr-LSTs, were immunohistochemically stained with MUC2, MUC5AC, MUC6, CD10 (markers of gastrointestinal cell lineage), p53, β-catenin and Ki-67 antibodies, and examined for alteration in exon 1 of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and exon 15 of v-raf murine sarcoma viral oncogene homologue B1 (BRAF) by polymerase chain reaction followed by direct sequencing.

Results: Histologically, 15 Gr-LST samples were adenomas with low-grade dysplasia (LGD), 12 were high-grade dysplasia (HGD) and 9 were adenocarcinomas invading the submucosa (INV), while 12 NGr-LSTs demonstrated LGD, 14 HGD and 7 INV. In the proximal colon, MUC5AC expression was significantly higher in the Gr-type than the NGr-type. MUC6 was expressed only in NGr-LST. MUC2 or CD10 did not differ. P53 expression demonstrated a significant stepwise increment in progression through LGD-HGD-INV with both types of LST. Nuclear β-catenin expression was significantly higher in the NGr-type. Ki-67 expression was significantly higher in the Gr-type in the lower one third zone of the tumor. In proximal, but not distal colon tumors, the incidence of KRAS provided mutation was significantly higher in the Gr-type harboring a specific mutational pattern (G12V). BRAF mutations (V600E) were detected only in two Gr-LSTs.

Conclusion: The two subtypes of LST, especially in the proximal colon, have differing phenotypes of gastrointestinal cell lineage, proliferation and activation of Wnt/β-catenin or RAS/RAF/extracellular signal-regulated kinase signaling.

Keywords: Adenoma-carcinoma sequence; Colon; Direct sequencing; Immunohistochemistry; Laterally spreading tumor; Mucin core protein; v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; β-catenin.

Figure 1

Macroscopic appearance (chromoscopic image) of laterally spreading tumors. (A) Granular type (Gr-LST) and (B) flat- or non-granular type (NGr-LST). Whole mount view of cut sections of LSTs stained with hematoxylin and eosin: (C) A Gr-LST showing tubulovillous structures with nodular surfaces and (D) an NGr-LST consisting of small tubular glands with flat surfaces. LSTs: Laterally spreading tumors.

Figure 2

Immunoreactive scores for MUC2, MUC5AC, MUC6 and CD10 in all (A, D, G, J), proximal (B, E, H, K) and distal (C, F, I, L) laterally spreading tumors. Gr, Gr-LST (black bar); NGr, NGr-LST (white bar). IRS: Immunoreactive score; LGD: Adenoma with low grade dysplasia; HGD: Adenoma with high grade dysplasia; INV: Adenocarcinoma invading into submucosa; LSTs: laterally spreading tumors; Gr: Granular; NGr: Non-granular. Data are mean ± SD; ^{a, c, e, g, i, k, m, o, q, s, u, w, y}P < 0.05, MUC2: all NGr-HGD vs NGr-INV; proximal colon NGr-LGD vs NGr-HGD or NGr-INV; Gr-LGD vs Gr-HGD; distal colon Gr-LGD vs Gr-INV; NGr-HGD vs NGr-INV; MUC5AC: all Gr-LGD vs Gr-INV; distal colon NGr-INV vs NGr-HGD or NGr-LGD; MUC6: proximal colon NGr-INV vs NGr-LGD or NGr-HGD; CD10: all NGr-LGD vs NGr-HGD; proximal colon NGr-LGD vs NGr-HGD; ^{b, d, f, h, j, l, n, p, r, t}P < 0.01, MUC2: all Gr-LGD vs Gr-HGD or Gr-INV; NGr-LGD vs NGr-INV; proximal colon Gr-LGD vs Gr-INV; distal colon Gr-LGD vs Gr-HGD; MUC5AC: all Gr-LGD vs NGr-LGD; Gr-LGD vs Gr-HGD; proximal colon Gr-type vs NGr-type; Gr-LGD vs NGr-LGD; Gr-LGD vs Gr-INV.

Figure 3

Immunoreactive scores for p53 (A) and nuclear β-catenin (B), and labeling indices for Ki-67 in the lower zone (C) in all laterally spreading tumors. Gr, Gr-LST (black bar); NGr, NGr-LST (white bar); LSTs: laterally spreading tumors. Data are mean ± SD; ^{a, c, e}P < 0.05, p53: Gr-HGD vs Gr-INV; nuclear β-catenin: Gr-type vs NGr-type; Ki-67, Gr-type vs NGr-type; ^{b, d, f, h, j, l, n}P < 0.01, p53: NGr-LGD vs NGr-INV; Gr-LGD vs Gr-HGD or Gr-INV; nuclear β-catenin: Gr-LGD vs Gr-HGD or Gr-INV: Gr-LGD vs NGr-LGD; Ki-67, Gr-LGD vs NGr-INV. IRSs: Immunoreactive scores; LGD: Adenoma with low grade dysplasia; HGD: Adenoma with high grade dysplasia; INV: Adenocarcinoma invading into submucosa; Gr: Granular; NGr: Non-granular; LI: Labeling indice.

Figure 4

Histology and immunohistochemistry of laterally spreading tumors. Immunoreactive scores (IRSs) for Gr-LGD: MUC2, 4 points; MUC5AC, 2 points; MUC6, 0 points; CD10, 0 points; p53, 0 points; nuclear β-catenin, 0 points. IRSs for NGr-LGD: MUC2, 3 points; MUC5AC, 1 points; MUC6, 0 points, CD10, 0 points; p53, 0 points; nuclear β-catenin, 1 points. IRSs for Gr-HGD: MUC2, 1 points; MUC5AC, 1 points; MUC6, 0 points, CD10, 1 points; p53, 1 points; nuclear β-catenin, 1 points. IRSs for NGr-HGD: MUC2, 2 points; MUC5AC, 1 points; MUC6, 1 points, CD10, 1 points; p53, 1 points; nuclear β-catenin, 2 points. IRSs for Gr-INV: MUC2, 1 points; MUC5AC, 1 points; MUC6, 0 points, CD10, 1 points; p53, 4 points; nuclear β-catenin, 1 points. IRSs for NGr-INV: MUC2, 1 points; MUC5AC, 0 points; MUC6, 2 points, CD10, 2 points; p53, 2 points; nuclear β-catenin, 2 points (H and E, ×80; immunoperoxidase: MUC2, MUC5AC and MUC6, ×100; CD10 and p53, ×120; β-catenin, ×300); LGD: Adenoma with low grade dysplasia; HGD: Adenoma with high grade dysplasia; INV: Adenocarcinoma invading into submucosa; Gr: Granular; NGr: Non-granular.

Figure 5

v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog and v-raf murine sarcoma viral oncogene homologue B1 mutational patterns in laterally spreading tumors. D: Aspartic acid; E: Glutamic acid; G: Glycine; S: Serine; V: Valine; Black box: Mutated case; White box: Non-mutated case; LGD: Adenoma with low grade dysplasia; HGD: Adenoma with high grade dysplasia; INV: Adenocarcinoma invading into submucosa; Gr: Granular; NGr: Non-granular; KRAS: v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; BRAF: v-raf murine sarcoma viral oncogene homologue B1.

Figure 6

Alterations of expression of mucin core protein, p53 and β-catenin, cell proliferation and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog mutations in malignant transformation of laterally spreading tumors. Large arrow: Marked upregulation; Medium arrow: Moderate upregulation; Small arrow: Mild upregulation; ++: Frequently mutated; +: Infrequently mutated; -: Not mutated; LGD: Adenoma with low grade dysplasia; HGD: Adenoma with high grade dysplasia; INV: Adenocarcinoma invading into submucosa; KRAS: v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; Gr: Granular; NGr: Non-granular.