Paraffin-embedded blocks of gastric cancer tissues were used for pathologic diagnosis, IHC, and molecular analysis. The tumor showed an infiltrative growth pattern on gross examination (Figure 1A). Histologically, the tumor was poorly differentiated. It had a solid pattern with necrosis and was heterogeneous with glandular differentiation and prominent tumor-infiltrating lymphocytes (Figure 1B and C). The tumor cells were positive for low-molecular-weight cytokeratin (AE1/AE3, Ventana) (Figure 1D). The combined positive score (CPS) of programmed death-ligand 1 (PD-L1, 22C3, Dako) was high (Figure 1E). In situ hybridization of EBV-encoded small RNA was negative (Figure 1F). MMR proteins, including MLH1 (M1, Ventana), PMS2 (A16-4, Ventana), MSH2 (G219-1129, Ventana), and MSH6 (SP93, Ventana), were analyzed using IHC. All four proteins were negative in the tumor cells but positive in the positive control, stromal, and inflammatory cells. IHC results coincided with those of other paraffin blocks (Figure 2). HER2 (4B5, Ventana) was 1+, and Ki-67 (30-9, Ventana) was 60%.
Figure 1 Pathological characteristics.
A: Gross appearance of the tumor; B: Microscopic appearance of the tumor with necrosis; C: Microscopic appearance of the tumor with solid pattern and lymphatic stroma; D: The tumor cells were positive for cytokeratin AE1/AE3 by immunohistochemical staining; E: High expression of PDL1 (22C3) CPS>1; F: Epstein–Barr virus encoded small RNA was negative (figures at 100-200 × magnification).
Figure 2 Expression of mismatch repair-related proteins by immunohistochemical staining.
The tumor cells were completely negative for all four proteins. A: MLH1; B: PMS2; C: MSH2; D: MSH6 (all images at 200 × magnification).
Subsequently, a methylation-specific polymerase chain reaction assay of the MLH1 promoter region was performed, which revealed promoter hypermethylation of MLH1.
Next-generation sequencing (NGS) of genes including MLH1, MSH2, MSH6, and PMS2 was performed. In addition, 37 genes, including gastrointestinal tumor-related genes such as AKT1, ATM, BRCA1, BRCA2, CDH1, EGFR, ERBB2, HRAS, KIT, MET, PDGFRA, PIK3CA, PMS1, PTCH1, SDHB, SDHC, and SDHD; genes related to drug metabolism toxicity such as CYP2D6, DPYD, and UGT1A1; and genes related to gastrointestinal therapy, prognosis, and inheritance such as APC, BLM, BMPR1A, CHEK2, EPCAM, GALNT12, GREM1, MUTYH, POLD1, POLE, PTEN, SMAD4, STK11, TP53, KRAS, NRAS, and BRAF, were identified.
NGS demonstrated that MLH1, MSH2, MSH6, and PMS2 genes exhibited changes. There was a mutation in the splicing region of exon 12 of MLH1 (c.1039-13_1039-8del), a missense mutation in exon 11 of PMS2 (c.1799T>C, p.Met600Thr), a missing copy number in exon 14 of MSH2 (Figure 3), and a missense mutation in exon 4 of MSH6 (c.2693C>A, p.Pro898His). This mutation of MSH2 may lead to a shift in the subsequent coding frame of MSH2, leading to the loss of protein expression. Mutation analysis using peripheral blood showed no germline mutations in these four genes. The presence of MLH1 promoter methylation and mutation of MSH2 confirmed MSI-H. The patient had no personal or family tumor history, indicating that this MMR deficiency was highly likely sporadic in nature. Thus, there were no clinical management implications for his family.
Figure 3 Results of next-generation sequencing.
The mutation of MSH2 and amplification of PIK3CA were observed.
The tumor showed several other mutations, including copy number amplification of the PIK3CA gene (Figure 3), possibly leading to the upregulation of PIK3CA expression. The PTEN gene had a frameshift mutation in exon 7 (c.800del, p.Lys267fs) and a nonsense mutation in exon 5 (c.388C>T, p.Arg130). The exon began to shift from the amino acid residue 267 (lysine) in exon 7. Terminators are likely to be introduced into the new reading frame. The amino acid residue 130 encoded by exon 5 was mutated from arginine to the terminator. These two premature terminators may lead to meaningless mRNA degradation, resulting in protein loss. There was a missense mutation of p.g245s in exon 7 of the TP53 gene and mutations in the splicing region of exon 6 of the ATM gene (c.497-5_497-4del) and exon 12 of the MET gene (c. 2584-13_2584-9del).