Helicobacter pylori-negative Differentiated Adenocarcinoma of the Stomach

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1 CASE REPORT Helicobacter pylori-negative Differentiated Adenocarcinoma of the Stomach Tadanobu Nagaya 1,2, Naoki Tanaka 2,3, Yugo Iwaya 1,2, Yoko Jimbo 1, Toshiharu Tatai 1, Tetsuya Ito 1, Ayako Seki 1, Kenichi Suzawa 1, Yasuhide Ochi 1,EtsuoHara 1, Manabu Takata 4, Toshiaki Otsuki 5, Mai Iwaya 5, Noriko Hosaka 5, Norikazu Arakura 2, Eiji Tanaka 2 and Osamu Hasebe 1 Abstract A 58-year-old Japanese man was diagnosed with differentiated adenocarcinoma of the stomach. Histological findings of the resected specimen revealed well- to moderately-differentiated tubular adenocarcinoma (tub1, tub2), 13 mm in diameter, which invaded into the submucosa (SM1, 300 μm) and lymphovascular lumen (ly1). Serum antibody against Helicobacter pylori (Hp) and the 13 C-urea breath test were negative, and there were no atrophic changes in the tumor-adjacent mucosa. The immunohistochemical analysis showed that gastric mucin (MUC5AC) was strongly positive and intestinal mucin (MUC2) was weakly and partially positive. According to these results, the final diagnosis of Hp-negative well-differentiated early gastric cancer was made. Key words: early gastric cancer, Helicobacter pylori, differentiated adenocarcinoma, ESD, mucin phenotype () () Introduction Helicobacter pylori (Hp) infection plays an important role in the development of gastric cancer. It was previously reported that no gastric cancer developed in Hp-negative patients in a 9-year prospective study (1). Other factors including genetic factors, Epstein-Barr virus (EBV) infection, a high salt intake, duodenal reflex, and metabolic syndrome, are known to contribute to gastric carcinogenesis. Autoimmune gastritis is known to be a high-risk condition of gastric cancer, however, it is very rare among the Japanese population. The incidence of Hp-negative gastric cancer (HPNGC) is reported to be % (2-6). This great variation is likely derived from differing definitions of HPNGC employed among the previous studies. Despite several useful markers for detecting the presence of Hp infection, the false-negative rate cannot be ignored. Moreover, there is a possibility of natural clearance and incidental eradication of Hp. Therefore, confirming the existence of an atrophic change in the non-cancerous mucosa, in addition to various markers, is important for the accurate diagnosis of HPNGC. Matsuo et al. (3) defined HPNGC as: 1) a serum anti-hp antibody titer 10 U/mL; 2) the absence of Hp and gastritis by a histological examination; 3) the absence of an atrophic change in the gastric mucosa by an endoscopic examination; and 4) a negative urea breath test or rapid urease test. Additionally, Ono et al. (2) defined HPNGC as an adenocarcinoma arising from the gastric mucosa without histological atrophy, endoscopic atrophy, or serological atrophy. According to these strict and valid criteria, these two groups reported that the prevalence of HPNGC was 0.66% and 0.42% of all gastric cancer, respectively. Therefore, HPNGC is considered to be a rare subtype of gastric cancer in the Japanese population, however, its clinical and pathological features re- Department of Gastroenterology, Nagano Municipal Hospital, Japan, Department of Gastroenterology, Shinshu University School of Medicine, Japan, Department of Metabolic Regulation, Shinshu University Graduate School of Medicine, Japan, Department of Surgery, Nagano Municipal Hospital, Japan and Division of Clinical Pathology, Nagano Municipal Hospital, Japan Received for publication October 29, 2014; Accepted for publication March 3, 2015 Correspondence to Dr. Tadanobu Nagaya, nagaya@shinshu-u.ac.jp 2857

2 Table. Laboratory Data on Admission. Normal Normal Range Range White blood cell count (/ L) Na (mmol/l) Hemoglobin (g/dl) K (mmol/l) Platelet count ( 10 3 / L) Cl (mmol/l) Total protein (g/dl) Total cholesterol (mg/dl) Albumin (g/dl) Glucose (mg/dl) Bilirubin (mg/dl) CRP (mg/dl) < AST (IU/L) HBV surface antigen (-) (-) ALT (IU/L) anti-hcv antibody (-) (-) ALP (IU/L) CEA (ng/ml) <5 3.5 GT (IU/L) CA19-9 (U/mL) < LDH (IU/L) anti-h.pylori antibody (U/mL) <10 <3 BUN (mg/dl) C-urea breath test ( ) Creatinine (mg/dl) anti-parietal cell antibody (-) (-) AST: aspartate aminotransferase, ALT: alanine aminotransferase, ALP: alkaline phosphatase, GT: gamma-glutamyltransferase, LDH: lactate dehydrogenase, BUN: blood urea nitrogen, CRP: C-reactive protein, HBV: hepatitis B virus, HCV: hepatitis C virus, CEA: carcinoembryonic antigen, CA: carbohydrate antigen, H.pylori: Helicobacter pylori, (-): negative main undetermined. We herein present a rare case of differentiated early gastric carcinoma which arose from Hp-negative, non-atrophic mucosa. Case Report A 58-year-old Japanese man was found to have a depressed lesion in the posterior wall of the gastric middle body while undergoing a routine health checkup endoscopic examination. A biopsy specimen revealed well-differentiated tubular adenocarcinoma (tub1). The patient was referred to our hospital for close examination and therapy. He had received cholecystectomy for gallbladder stones at 40 years of age. Hp eradication therapy was not provided in the past and proton-pump inhibitors and antibiotics were not prescribed at the time of diagnosis of the Hp infection. He had not taken habitual drugs, alcohol or smoking and his family history was unremarkable. The patient was asymptomatic, afebrile, and apparently healthy and his body weight was 76 kg (BMI 24.0 kg/m 2 ). The physical examination and laboratory data including serum anti-parietal cell antibody, anti-hp antibody, and the 13 C-urea breathe test (UBT) on admission showed no abnormalities (Table). Repeated endoscopic examination revealed no atrophic changes in the entire gastric mucosa and demonstrated a slightly depressed whitish area, approximately 10 mm in size, in the posterior wall of the gastric middle body (Fig. 1A, B). The lesion had a villous pattern and the borderlines of the white area could be clearly detected by magnified endoscopy with narrow band imaging (Fig. 1C, D). The biopsy specimen showed a differentiated adenocarcinoma. Furthermore, the cancer had no signs suggestive of deep invasion, such as deep depression, excavated lesion in the depression, or converged folds. Therefore, according to the endoscopic findings, this tumor was considered to be an intramucosal carcinoma, thus we did not perform an endoscopic ultrasound examination. Because no distant metastasis was detected by computed tomography, endoscopic submucosal dissection (ESD) was performed. ESD was successfully performed, and the histological findings of the resected tissues demonstrated a wellto moderately-differentiated adenocarcinoma (Fig. 2A-C), which invaded into the submucosa (SM1, 300 μm, Fig. 2D) and lymphovascular lumens (ly1) (Fig. 2E). Lymphovascular invasion was not detected by additional D2-40 immunostaining using continuous sections (Fig. 2F), however, we judged the invasion of lymphovascular lumens to be positive according to the findings of hematoxylin and eosin staining (Fig. 2E). Undifferentiated cancer cells were not found by the additional pathological evaluation using continuous sections. Moreover, no atrophic and dysplastic changes, intestinal metaplasia, inflammatory cell infiltration, or Hp infection by Giemsa staining were detected in the mucosa around the cancer. According to the Japanese classification system, the final pathological diagnosis was [M, Post., 0-IIc, 13 8 mm in mm, well- to moderately-differentiated adenocarcinoma (tub1, tub2), SM1 (300 μm), UL(-), ly1, v0, phm0, pvm0] (Fig. 2G). The tumor cells were positive for mucin (MUC)5AC and partially positive for MUC6 and MUC2 (Fig. 3) according to the immunohistochemistry analysis, thus the mucin phenotype was considered to be the mixed type. Additionally, immunostaining of pepsinogen (PG)IandH + /K + -ATPase was negative for the tumor cells (Fig. 3), indicating that this tumor was not derived from the fundic gland. In situ hybridization for the EBV genome (EBV-ISH) was also negative (Fig. 3). Due to the presence of lymphovascular involvement, additional fundusectomy was performed followed by the 2010 Japanese gastric cancer treatment guidelines, Ver.3. The surgical specimen revealed no residual cancer or lymph node metastasis. No postoperative intragastric recurrence or distant metastasis was observed. Discussion The incidence of HPNGC differs among previous reports (2-6), most likely due to the various definitions of 2858

3 A B C D Figure 1. Pre-treatment endoscopic examination. (A): A regular endoscopic examination revealed a slightly depressed white area, approximately 10 mm in size, with no deep invasion signs and no atrophic background mucosa in the posterior wall of the gastric middle body (yellow circle). A regular arrangement of collecting venules was seen in the tumor-adjacent mucosa. (B): Indigo-carmine staining. (C): NBI light endoscopy showed that the tumor had a villous pattern. (D): Magnified endoscopy with NBI revealed the borderlines of the lesion could be detected clearly. HPNGC. In some reports, the Hp status was assessed only by a histologic examination or serum antibody titer. Matsuo et al. (3) and Ono et al. (2) used the most stringent definitions of HPNGC, and in their reports, the prevalence of HPNGC was reported to be less than 1%. In the present case, serum antibodies against parietal cells and Hp and UBT were all negative, and no atrophic change was found in the non-tumorous mucosa by the histological and endoscopic examinations. Additionally, the possibility of a gastric fundic carcinoma or EBV-related cancer was excluded by the pathological evaluation. Therefore, the final diagnosis of Hp-unrelated differentiated adenocarcinoma developed from the normal gastric mucosa was made. In comparison with Hp-positive gastric cancers, strictlydefined HPNGC are diagnosed at an earlier age [mean age: 56.5 years (3)] with no gender predominance (2, 3). In the endoscopic appearance, the depressed lesion was characteristic for HPNGC (3), which is consistent with the present case. Previous studies demonstrated that approximately 70% of HPNGC was pathologically diagnosed as poorlydifferentiated or signet ring cell adenocarcinoma (3, 5). In the report from Ono et al. (2) 1 case (0.42%) was diagnosed as HPNGC in 240 cases of gastric cancer. Similarly, the report from Matsuo et al. (3) showed that 21 of 3161 gastric cancer patients (0.66%) were HPNGC while less than 7 cases had well- to moderately-differentiated HPNGC. Therefore, the present case is considered to be an extremely rare, differentiated adenocarcinoma in HPNGC which arose from the non-atrophic mucosa in the gastric body. Adenocarcinoma arising from the fundic gland (the chief cell predominant type) was recently proposed as a new and rare variant that may be involved in previous HPNGC cases. This type of gastric adenocarcinoma is confirmed by positive immunostaining against PG I or H + /K + -ATPase. The submucosal tumor-like shape, whitish color, dilated vessels with branching architecture, and non-atrophic background mucosa were reported to be common endoscopic features (7). Therefore, when we encounter HPNGC, immunostaining of PG I and H + /K + -ATPase should be added to exclude this type of tumor. However, in the present case, the tumor did not originate from the gastric fundic gland. In the former case, approximately one-half of all gastric cancer cells simultaneously expressed intestinal mucin despite the development from normal gastric mucosa without intestinal metaplasia (3-5). This finding suggests that intestinal metaplasia is not essential for the acquisition of the intestinal mucus phenotype. Yoshikawa et al. (8) revealed over 40% of early-stage Lauren s intestinal-type carcinomas consist of mainly gastric-type cancer cells, and a phenotypic shift from the gastric to intestinal type was evident with the 2859

4 A B Normal mucosa E Adenocarcinoma F Normal mucosa C G D tub1, 2 M tub1, 2 SM Figure 2. Appearance of the lesion resected by endoscopic submucosal dissection. (A-E) Hematoxylin and Eosin staining. The tumor was surrounded by normal mucosa without atrophic changes and inflammation (A and B). The atypical cells showed a well-differentiated tubular adenocarcinoma (tub1) in the surface layer of the mucosa and moderately-differentiated tubular adenocarcinoma (tub2) in the deep layer of mucosa (C). Invasion to the submucosa (D) and lymphovascular lumen (E) was detected. Lymphovascular invasion was not detected by additional D2-40 immunostaining using continuous sections (F). (G) Gross appearance of freshly-dissected specimen and mapping of the cancer. The tumor was slightly depressed, whitish, and 13 mm in diameter. Almost all parts of the tumor were limited in the mucosa (red lines), however, partially invaded in the submucosa (yellow lines). progression. The tumor cells in this case might acquire the intestinal phenotype with the progression and dedifferentiation. Hp infection and atrophic gastritis have been regarded as risk factors for gastric cancer. The combination of serum PG and Hp antibody (ABC stratification) has been suggested to serve as a useful predictive marker for patients with gastric cancer (9, 10). In these studies, an individual showing normal PG levels (PG I level 70 ng/ml and PG I/PG II ratio 3) and negative Hp antibody is classified into group A, which is judged as the absence of Hp infection and atrophic change. Although the serum PG levels were not measured on admission, the present case would be presumably included in group A. It was additionally reported that the incidence of gastric cancer development stratified by age (under 60 years old), sex (male), and ABC stratification (group A), was 0.1%/year (10). The present case demonstrates that gastric cancer is presumably in the group A individuals and should not be ignored, although the frequency is very rare. Further studies are needed to examine the prevalence and clinicopathological features of gastric cancer from group A patients. In the present case, an atypical submucosal invasion pattern was found. Although the cancer cells were histologically well- or moderately-differentiated, the mode of invasion appeared to be similar to that found in poorly- or undifferentiated cancer. We included an additional histological examination, however, we could not detect poorlydifferentiated or undifferentiated cancer components. The mechanism of invasion of gastric carcinoma is not currently 2860

5 MUC5AC MUC6 MUC2 CD10 Pepsinogen I H + /K + -ATPase EVB-ISH Figure 3. Immunohistochemical and in situ hybridization analyses of the tumor cells. The adenocarcinoma cells were positive for MUC5AC and partially positive for MUC6 and MUC2, but negative for CD10, pepsinogen I, H + /K + -ATPase, and EBV genome. clear. However, cell adhesion is one of the important steps in invasion and metastasis (11). It has been previously reported that the aberrant expression of proteins associated with cell adhesion is a possible marker of submucosal invasion in differentiated early gastric cancer and may be a useful predictor of lymph node metastasis (12). The breakdown of cell-cell contact may be associated with the invasion of gastric cancer cells into the submucosa and lymphovascular 2861

6 lumens. Compared with advanced gastric cancer, lymph node metastasis is the only independent prognostic factor for early gastric cancer (13, 14). In this case, we performed gastrectomy and lymph node dissection, however, no local recurrences and lymph node metastases were observed in the resected specimen. Careful screening is necessary because the clinical course and recurrence rate of HPNGC are unclear. In summary, we herein reported a rare case of differentiated HPNGC. Because the prevalence of persistent Hp infection and the related gastric cancer is estimated to be decreased in Japan due to the establishment of an early screening system and Hp eradiation strategy (15), HPNGC will most likely become a more frequent type of gastric cancer in the future. Therefore, the accumulation of HPNGC cases is important to clarify the clinicopathological characteristics of HPNGC. The authors state that they have no Conflict of Interest (COI). References 1. Uemura N, Okamoto S, Yamamoto S, et al. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med 345: , Ono S, Kato M, Suzuki M, et al. Frequency of Helicobacter pylori -negative gastric cancer and gastric mucosal atrophy in a Japanese endoscopic submucosal dissection series including histological, endoscopic and serological atrophy. Digestion 86: 59-65, Matsuo T, Ito M, Takata S, Tanaka S, Yoshihara M, Chayama K. Low prevalence of Helicobacter pylori-negative gastric cancer among Japanese. Helicobacter 16: , Kakinoki R, Kushima R, Matsubara A, et al. Re-evaluation of histogenesis of gastric carcinomas: a comparative histopathological study between Helicobacter pylori-negative and H. pylori-positive cases. Dig Dis Sci 54: , Kato S, Matsukura N, Tsukada K, et al. Helicobacter pylori infection-negative gastric cancer in Japanese hospital patients: incidence and pathological characteristics. Cancer Sci 98: , Wu MS, Hung HW, Wang JT, et al. Helicobacter pyloriseronegative gastric carcinoma: a subset of gastric carcinoma with distinct clinicopathologic features. Hepatogastroenterology 45: , Ueyama H, Matsumoto K, Nagahara A, Hayashi T, Yao T, Watanabe S. Gastric adenocarcinoma of the fundic gland type (chief cell predominant type). Endoscopy 46: , Yoshikawa A, Inada Ki K, Yamachika T, Shimizu N, Kaminishi M, Tatematsu M. Phenotypic shift in human differentiated gastric cancers from gastric to intestinal epithelial cell type during disease progression. Gastric Cancer 1: , Kudo T, Kakizaki S, Sohara N, et al. Analysis of ABC (D) stratification for screening patients with gastric cancer. World J Gastroenterol 17: , Watabe H, Mitsushima T, Yamaji Y, et al. Predicting the development of gastric cancer from combining Helicobacter pylori antibodies and serum pepsinogen status: a prospective endoscopic cohort study. Gut 54: , Chu YQ, Ye ZY, Tao HQ, Wang YY, Zhao ZS. Relationship between cell adhesion molecules expression and the biological behavior of gastric carcinoma. World J Gastroenterol 14: , Tanaka M, Kitajima Y, Edakuni G, Sato S, Miyazaki K. Abnormal expression of E-cadherin and beta-catenin may be a molecular marker of submucosal invasion and lymph node metastasis in early gastric cancer. Br J Surg 89: , Kikuchi S, Katada N, Sakuramoto S, et al. Survival after surgical treatment of early gastric cancer: surgical techniques and longterm survival. Langenbecks Arch Surg 389: 69-74, Zu H, Wang H, Li C, Xue Y. Clinicopathologic characteristics and prognostic value of various histological types in advanced gastric cancer. Int J Clin Exp Pathol 7: , Ito M, Takata S, Tatsugami M, et al. Clinical prevention of gastric cancer by Helicobacter pylori eradication therapy: a systematic review. J Gastroenterol 44: , The Japanese Society of Internal Medicine