cancers Article The Clinicopathological Characteristics and Genetic Alterations of Signet-Ring Cell Carcinoma in Gastric Cancer Kuo-Hung Huang 1,2 , Ming-Huang Chen 3, Wen-Liang Fang 1,2,*, Chien-Hsing Lin 4, Yee Chao 2,3 , Su-Shun Lo 2,5, Anna Fen-Yau Li 2,6, Chew-Wun Wu 1,2 and Yi-Ming Shyr 1,2 1 Division of General Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei 11217, Taiwan; [email protected] (K.-H.H.); [email protected] (C.-W.W.); [email protected] (Y.-M.S.) 2 School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan; [email protected] (Y.C.); [email protected] (S.-S.L.); [email protected] (A.F.-Y.L.) 3 Center of Immuno-Oncology, Department of Oncology, Taipei Veterans General Hospital, Taipei 11217, Taiwan; [email protected] 4 Genome Research Center, National Yang-Ming University, Taipei 11221, Taiwan; [email protected] 5 Department of Surgery, National Yang-Ming University Hospital, Yilan 26058, Taiwan 6 Department of Pathology, Taipei Veterans General Hospital, Taipei 11217, Taiwan * Correspondence: [email protected] Received: 12 July 2020; Accepted: 12 August 2020; Published: 17 August 2020 Abstract: Signet-ring cell carcinoma (SRC) in advanced gastric cancer (GC) is often associated with more invasiveness and a worse prognosis than other cell types. The genetic alterations associated with gastric carcinogenesis in SRC are still unclear. In this study, 441 GC patients receiving curative surgery for GC between 2005 and 2013 were enrolled. The clinicopathological characteristics and genetic alterations of GC patients with and without SRC were compared. Among the 441 GC patients, 181 had SRC. For early GC, patients with SRC had more tumors located in the middle and lower stomach, more infiltrating tumors and better overall survival (OS) rates than those without SRC. For advanced GC, patients with SRC had more scirrhous type tumors, more PIK3CA amplifications, fewer microsatellite instability-high (MSI-H) tumors, more peritoneal recurrences and worse 5-year OS rates than those without SRC. For advanced GC with SRC, patients with peritoneal recurrence tended to have PD-L1 expression. For advanced GC without SRC, patients with liver metastasis tended to have PD-L1 expression, PI3K/AKT pathway mutations, TP53 mutations and MSI-H tumors. For advanced GC, PD-L1 expression was associated with peritoneal recurrence in SRC tumors, while non-SRC tumors with liver metastasis were likely to have PI3K/AKT pathway mutations, TP53 mutations and PD-L1 expression; immunotherapy and targeted therapy may be beneficial for these patients. Keywords: signet-ring cell; genetic alterations; advanced GC; PD-L1; recurrence pattern; prognostic factor review; meta-analysis 1. Introduction Gastric cancer (GC) is the sixth most common cancer and the second most common cause of cancer death worldwide [1]. Signet-ring cell carcinoma (SRC) is characterized by malignant tumor cells with prominent mucin in the cytoplasm and eccentric crescent-shaped nuclei. According to the classification of the World Health Organization (WHO) [2], SRC is defined as being present in more than 50% of GC tumors. Compared to other types of GC, SRC tends to occur predominantly in those who are younger and female and has a higher frequency of lymph node metastasis and a poor prognosis [3]. Cancers 2020, 12, 2318; doi:10.3390/cancers12082318 www.mdpi.com/journal/cancers Cancers 2020, 12, 2318 2 of 17 A meta-analysis study [4] demonstrated that for early GC (T1), SRC was associated with a better prognosis than non-SRC, while for advanced GC (T2-T4), SRC had a worse prognosis than non-SRC, which was similar to the findings of our previous report [5]. We hypothesize that the genetic alterations between SRC and non-SRC in early and advanced GC may be different, and it deserves investigating whether these genetic alterations are associated with patient survival. To date, the molecular difference between SRC and non-SRC in GC is still unclear; further investigation of the genetic alterations may provide useful information to explain the phenomenon. The aim of the present study was to compare the clinicopathological characteristics and genetic alterations between patients with and without SRC in early and advanced GC. In addition, we investigated whether genetic alterations are associated with patient prognosis in SRC and non-SRC GC. 2. Results 2.1. Clinicopathological Features Among the 441 patients, 181 had SRC. Among the 260 non-SRC patients, there were 5 papillary adenocarcinomas (1.9%), 240 tubular adenocarcinomas (92.3%) and 15 mucinous adenocarcinomas (5.8%), and no patient was diagnosed with undifferentiated adenocarcinoma. We compared the clinicopathological characteristics between patients with and without SRC (Table1). Univariate analysis showed that patients with SRC were younger and more likely to be female and had more tumors located in the middle stomach, more scirrhous stromal reaction, a more infiltrating type, fewer microsatellite instability-high (MSI-H) tumors, more PIK3CA amplifications, less PD-L1 expression, fewer PI3K/AKT pathway mutations and more advanced Tumor, Node, Metastasis (TNM) stages than those without SRC. Multiple testing correction logistic regression demonstrated that patients with SRC were more likely to be female and had more scirrhous stromal reaction, a more infiltrating type, fewer MSI-H tumors and more PIK3CA amplifications than those without SRC. With regard to early GC (Table2), univariate analysis showed that patients with SRC were more likely to be female and had more tumors located in the middle stomach, a poorer differentiation and a more infiltrating type than those without SRC. Multiple testing correction logistic regression demonstrated that patients with SRC had more tumors located in the middle stomach and a more infiltrating type than those without SRC. With regard to advanced GC (Table3), patients with SRC were younger and more likely to be female and had more tumors located in the middle stomach, more scirrhous stromal reactions, a more infiltrating type, fewer MSI-H tumors, more PIK3CA amplifications, less PD-L1 expression, fewer ARID1A mutations and more advanced TNM stages than those without SRC. Multiple testing correction logistic regression demonstrated that patients with SRC had more scirrhous stromal reactions, fewer MSI-H tumors and more PIK3CA amplifications than those without SRC. With regard to the combined positive score (CPS) of PD-L1 expression, there was no significant difference in CPS between patients with SRC and without SRC (0.49 1.48 vs. 0.92 2.56, p = 0.166), ± ± which was observed in both early GC (0.40 1.13 vs. 0.43 1.30, p = 0.915) and advanced GC ± ± (0.54 1.65 vs. 1.09 2.87, p = 0.199). ± ± Cancers 2020, 12, 2318 3 of 17 Table1. Clinical profile of gastric cancer (GC) patients with and without signet-ring cell carcinoma (SRC). Multiple Testing Correction Logistic Univariate Analysis Regression Variables Non-SRC SRC Confidence n = 260 n = 181 p-Value Odds Ratio p-Value Interval n (%) n (%) Age <0.001 0.099 <65 years 82 (31.5) 96 (53.0) 1.00 65 years 178 (68.5) 85 (47.0) 0.65 0.394–1.083 ≥ Gender <0.001 0.013 Male 203 (78.1) 108 (59.7) 1.00 Female 57 (21.9) 73 (40.3) 2.02 1.163–3.493 Tumor size 0.165 <5cm 109 (41.9) 64 (35.4) 5 cm 151 (58.1) 117 (64.6) ≥ Tumor location <0.001 0.311 Upper stomach 55 (21.2) 25 (13.8) 1.00 Middle stomach 70 (26.9) 76 (42.0) 2.40 1.187–4.843 Lower stomach 133 (51.2) 71 (39.2) 1.07 0.543–2.097 Whole stomach 4.36 0.681–27.877 Stromal reaction type <0.001 <0.001 Medullary 47 (18.1) 18 (9.9) 1.00 Intermediate 181 (69.6) 57 (31.5) 0.54 0.237–1.216 Scirrhous 32 (12.3) 106 (58.6) 4.28 1.748–10.500 Ming’s classification <0.001 <0.001 Expanding 99 (38.1) 14 (7.7) 1.00 Infiltrating 161 (61.9) 167 (92.3) 3.96 1.938–8.105 Extent of lymphadenectomy 0.751 Limited (D1+) 57 (21.9) 42 (23.2) Extended (D2) 203 (78.1) 139 (76.8) Lymphovascular invasion 187 (71.9) 124 (68.5) 0.439 Lymphoid stroma 38 (14.6) 25 (13.8) 0.813 MSI status 0.001 0.008 MSI-L/S 229 (88.1) 175 (96.7) 1.00 MSI-H 31 (11.9) 6 (3.3) 0.23 0.076–0.684 HP infection 90 (34.6) 67 (37.0) 0.604 PIK3CA amplification 106 (40.8) 100 (55.2) 0.003 1.71 1.025–2.855 0.040 PD-L1 expression 68 (26.2) 28 (15.5) 0.007 0.68 0.352–1.299 0.240 Genetic mutation PI3K/AKT pathway 49 (18.9) 21 (11.6) 0.039 0.82 0.389–1.708 0.589 ARID1A 37 (14.2) 16 (8.8) 0.087 TP53 32 (12.3) 30 (16.6) 0.205 KRAS 9 (3.5) 1 (0.6) BRAF 3 (1.2) 0 Pathologic TNM Stage 0.022 0.774 I 60 (23.1) 28 (15.5) 1.00 II 80 (30.8) 46 (25.4) 1.13 0.516–2.456 III 120 (46.2) 107 (59.1) 1.29 0.607–2.741 GC—gastric cancer; SRC—signet-ring cell; MSI—microsatellite instability; MSI-H—microsatellite instability-high; MSI-L/S—microsatellite instability-low/stable; HP—Helicobacter pylori; EBV—Epstein–Barr virus; bold—statistically significant. Cancers 2020, 12, 2318 4 of 17 Table 2. Clinical profile in early GC patients with and without SRC.