1044 Helicobacfer Pylori Infection in Patients with Gastric Carcinoma in Biopsy and Surgical Resection Specimens Tomoyuki Shibata, M.D. lchiro Imoto, Y.D. Yoshio Ohuchi, M.D. Yukiko Taguchi, M.D. Satoshi Takaji, M.D. Norihisa Ikemura, M.D. Kazuyuki Nakao, M.D. Teruo Shima, M.D. Third Department of Internal Medicine, Mie University School of Medicine, Mie Prefecture, Japan. BACKGROUND. The discrepancy between the high seropositivity for Helicobacfer pylori ( H . pylori) and the low diagnostic yield of H. pylori organism in gastric biopsies of patients with gastric carcinoma has yet to be clarified. The present study attempted to clarify this controversial point by performing a comparative evaluation between the detection rate of H. pylori in biopsy and in surgical specimens. METHODS. The presence of H. pylori in biopsy samples from 50 patients with gastric carcinoma and 50 age-matched controls was evaluated histologically. Six histologic sections were obtained from gastric noncancerous areas and the presence of H. pylori was evaluated in those H . p-vlori negative patients who underwent gastrectomy. RESULTS. H. pylori was positive in 35 of 50 controls (70%).I n biopsy samples, H. p.v/uri was detected in 29 of 37 patients (78.4%)with early gastric carcinoma, 7 of 13 (53.8%)with advanced carcinoma, 16 of 23 (69.6%)with intestinal type of gastric carcinoma, and 20 of 27 (74.1%)with diffuse type of carcinoma. Studies carried out in gastrectomy specimens increased the diagnostic yield of H. pylori to 33 (89.2%), 12 (92.3%),19 (82.6%), and 26 (96.3%)in patients with early, advanced, intestinal, and diffuse types of gastric carcinoma, respectively. Overall, H. pylori was positive in 36 biopsy specimens (72%) and 45 gastrectomy specimens (90%). Namely, the detection of H. pylori infection was significantly higher in patients with gastric carcinoma using gastrectomy specimens than in patients with gastric carcinoma using biopsy specimens only ( P < 0.05). CONCLUSIONS. These results indicate that the actual prevalence of H. pylori in patients with gastric carcinoma is considerably higher than that previously reported. Cancer 1996; 721044-9. 0 1996 Americari Cancer Sociefy KEYWORDS: Helicobacter p y M , gastric carcinoma, gastrectomy, histological studies. I Address for reprints: Shibata, M.D., Edobashi 2-chorne, 174 banchi, Tsu-city, Mie Prefecture, 514, Japan. Received July 24, 1995; revision received November 13.1995; accepted NovemberBO, 1995. 0 1996 American Cancer Society t is well-known that Helicobacter pylori ( H . pylori) is a causative factor of chronic antral gastritis.' Chronic active gastritis may progress to chronic atrophic gastritis with intestinal metaplasia and finally to dysplasia and gastric carcinoma.' Although the incidence a n d mortality rates of gastric cancer decreased over the past decades, gastric cancer is still the most common malignancy in Japan." The role of H. pylori infection in gastric carcinogenesis has recently become a subject of major concern. Some epidemiologic studies have shown the existence of a significant correlation between the incidence or mortality of gastric carcinoma and the prevalence of H. pylori seropositivity in China and C ~ l o r n b i a .Recently, ~,~ several serologic studies reported a strong association between H . pylori and gastric carcinoma."* However, despite the frequent detection of anti- Helicobacter pylori and Gastric CancerlShibata et at. 1045 TABLE 1 Helicobacter pylon’ Positivity In Control Subjects and in Patients with Gastric Carcinoma Subjects No. of subjects (do Controls Patients with Rastric carcinoma 50 (37113) 50 (37113) Mean age (Yd 61.8 62 H.pykvi positivity in biopsy specimens (%I H. pylori positivity in biopsy and resected specimens (46) 35 (70) 36 (721 45 (901“ m:male: I: female. ’ P < 0.05: comparedwith conlrol subjects. ’ P < 0.05; comparedwith positivity in biopsy specimens. body against H. pylori in patients with H. pylori infection, histologic diagnosis of H. pylori from biopsy specimens is low, particularly in those cases with diffuse types of cancer.”,” Namely, the actual prevalence of H. pylori infection in patients with gastric carcinoma remains still controversial. In this study, we investigated the prevalence of H. pylori infection in the same patients with gastric carcinoma using both biopsy and gastrectomy specimens. The histologic type of gastric carcinoma was classified in diffuse and intestinal types following the classification of Lauren.13 When two histologic types coexisted in the tissue, the diagnosis was based on the predominant histologic type. Three anatomical parts of the stomach were considered (according to the classification scheme of the lapanese Research Society for Gastric Cancer)’: C (upper third), M (middle third), and A (lower third). PATIENTS AND METHODS Endoscopic Examination Patients with Gastric Carcinoma and Control Subjects Upon endoscopic examination, four biopsy specimens were collected from each patient: two (one for histologic study and another for culture) from the lesser curvature of the antrum, and two (one for histology and another for culture) from the greater curvature of the corpus. Olympus endoscopes (Olympus, Tokyo, Japan) and biopsy forceps were washed and sterilized before the endoscopic procedure. Informed consent was obtained from all of the patients and control subjects before the initiation of the study. Fifty patients with histologically proven gastric carcinoma and 50 age-matched control subjects were examined. There were 37 males and 13 females and the mean age of the patients was 62 years. The control subjects were those referred to our hospital because of radiologic abnormalities of the stomach or duodenum found during health check, or because of gastrointestinal complaints without radiologic abnormalities. Subjects who were finally found not to have any localized disease in the stomach or the duodenum by endoscopy, and without any history of gastric surgery or of systemic disease, were grouped as control subjects. Classification of gastric carcinoma was done according to the pathologic stage, histologic type, and localization of the tumors. The pathologic staging of gastric carcinoma was done following the Japanese Gastroenterological Society Classification.’’ This differentiates two groups of gastric carcinoma; a group in early pathologic stage in which the tumoral lesion is confined within the gastric mucosa or submucosa regardless of the presence or absence of metastatic disease in the perigastric lymph node lesion, and a group in advanced stage characterized by tumoral extension within or beyond the muscularis propia. Early gastric carcinoma was further classified according to the gross appearance of the tumors into a protruded (I), superficially elevated (Ha), flat (IIb),superficially depressed (IIcj, and excavated types (111). Gastric carcinoma in advanced stage was also grouped according to the Borrmann’s classification (class 1, 2, 3, and 4).12 Histologic Examination The biopsy specimens obtained from two sites of the stomach were fixed in 10% formalin and embedded in paraffin. Paraffin sections were cut and stained with 0.01% acridine orange. The presence of H. pylori infection was assessed by examining the sections on light microscope ( ~ 1 0 0 0and ) by the growth of the bacteria in the culture. Six histologic specimens from noncancerous sites were taken from the resected portion of the stomach of patients previously diagnosed as not having H. pylori infection by biopsy study and the histologic presence of H. pylori was evaluated following the aforementioned method. We also investigated the presence of active chronic gastritis, atrophic gastritis, and intestinal metaplasia (IM) using hematoxylin & eosin (H & E) and periodic acidSchiff staining methods. Active chronic gastritis was diagnosed according to the description of Warren and Mar- 1046 CANCER March 15, 1996 / Volume 77 / Number 6 TABLE 2 Helicobacter pylon Positivity and Cancer Pathologlc Stage Pathologic stage Early Stage I ILA IIB IIC 111 Advanced Stage Bor. 1 Bor. 2 Bor. 3 Bor. 4 patients H. pylon positivity in biopsy specimens (W) 37 29 (78.4) 0 7 1 25 4 - No. of 13 0 3 0 22 4 7 (53.8) H. pylon positivity in biopsy and resected specimens (W) 33 (89.2) 4 0 25 4 TABLE 3 Helicobacter pjbn PositMty and Tumor Histology If. pylon positivity in Histologic No. of H. pybd positivity in type patients biopsy specimens (%) Intestinal Early Advanced 23 19 4 14 (60.9) Diffuse 27 Early Advanced 18 9 16 (69.6) 2 (8.7) 20 (74.1) 15 (55.6) 5 (18.5) biopsy and resected specimens (%) 19 (82.6) 15 (65.2) 4 (17.4) 26 (96.3) 18 (66.7) 8 (29.6) 12 (92.3) 4 6 TABLE 4 3 H e l i c o h r pjhri Positivity and Cancer Location Bor.: Borrmann. H. pylori positivity in No. of shalLi4 Atrophic gastritis and IM were diagnosed according to the classification of Whitehead et aLL5 Culture Methods Biopsy specimens for culture were plated directly onto trypticase soy agar containing 5% sheep's blood (Becton Dickinson Co., Tokyo, Japan) and incubated at 37°C under microaerophilic environment for 5 days. The culture was considered positive for H. pylori if one or more colonies of gram-negative, oxidase, catalase, and urease-positive grew in the medium. Enzyme-Linked lmmunosorbent Assay Anti-H. pylori antibody in the serum of 6 patients with gastric carcinoma was measured by enzyme-linked immunoadsorbent assay (ELISA) using stocked sera preserved at less than -2OOC. The measurements were carried out using the HM-CAP immunoassay (Enteric Products Inc. Westbury, NY). Statistical Analysis Statistical analysis was performed using the Student's t test, and the cell chi-square test with Yates' correction. A P < 0.05 was considered a significant difference. RESULTS Biopsy studies revealed a frequency of H. pylori infection in patients with gastric carcinoma of 72% (36 of 50) and in control subjects of 70% (35 of 50). The frequency of H. pylori infection between these two groups was not significantly different (Table 1). The incidence of H. pylori infection in patients with different clinical stages of gastric carcinoma is summarized in Table 2. The mean age of patients with early Location patients C 5 26 19 M A H. pylon positivity in biopsy specimens (96) 3 (60) 18 (69.2) 15 (78.9) biopsy and resected specimens (%) 4 (801 25 (96.2)' 16 (84.2) C upper third of stomach; M: middle third of sromach; A: lower third of stomach P < 0.02: comoared with wsitive bioosv soecimen. and advanced gastric carcinoma was 62.1 and 61.6 years, respectively. H. pylori was positive in 29 of 37 patients (78.4%)with early gastric carcinoma, and in 7 of 13 patients (53.8%)with advanced cancer. Eight patients with early gastric carcinoma were negative for H. pylori in biopsy specimens. Among these patients, in 4 of 5 (80%) undergoing surgical therapy, the surgical specimen was positive for H. pylori. Three non-operated cases in this stage were endoscopically treated. All six patients with advanced cancer judged as H. pylori negative by biopsy study underwent surgical treatment. H. pylori was detected in the surgical specimen in 5 of 6 patients (83.3%). Overall, H. pylori was positive in 89.2% of the patients with early gastric carcinoma and in 92.3%of the patients with advanced cancer (Table 2). Twenty-three patients presented intestinal type of cancer and 27 presented diffuse type. The mean age of these two groups of patients was 66.9 and 57.9 years, respectively. Biopsy studies were positive in 69.6% and in 74.1% of the patients with intestinal and diffuse types, respectively. The inclusion of positive cases of H. pylori in the surgical specimen yielded a total positivity for H. pylori of 82.6% and 96.3% in the intestinal and diffuse types of gastric carcinoma, respectively (Table 3). The relationship between H. pylori positivity and cancer location is summarized in Table 4. The detection rate of H. pylori increased significantly from 69.2% to He/imbactef py/ofi and Gastric CancerlShibata et al. 1047 TABLE 5 TABLE 6 Features of Gastritis in Biopsy Samples of Helhbacfer pylon Negative Patients Features of Gastritis in Resected Specimens of Helicobacter pylon Negative Patients corpus Number of ACC (96) Number of AC (%I Number of IM (96) 12/14 (85.7) 11/14 (78.6) 6/14 (42.9) Antnun 9/14 (64.3) 14/14 (100) 11/14 (78.6) ACC: active chronic eastritis:AG: atroDhic easlritis: IM: intestinal rnetaDlasia 96.2%( P < 0.02) in the M group after including the posi- tive cases diagnosed by evaluating the surgical specimens (Table 41. The features of gastritis from biopsy samples of H. pylori negative patients are shown in Table 5. Atrophic gastritis was found in all specimens of the antrum. In the group with a high detection rate of H. pylori in resected specimens, the frequency of IM tended to be lower than in the group with a low positive rate for H. pylori. (Table 6).The number of cases with positive H. pylori in resected specimens was 28, and that with negative H. pylori was 38. (Table 7). The frequency rate of atrophic gastritis was higher in negative specimens than in positive specimens for H. pylori infection. The frequency of IM was significantly higher in the negative specimens than that in the positive samples for H. pylori (Table 7). Among 14 H. pylori negative patients diagnosed only by biopsy, 11 underwent surgery and of those, 9 had H. pylori infection (81.8%). The positivity of H. pylori in gastric carcinoma was 44/47 in patients undergoing surgical treatment (93.6%).Surgical specimen studies increased the final positivity of H. pylori to 90%. The frequency of H. pylori in patients with gastric carcinoma using surgical specimens was significantly higher than that in the matched control subjects and higher than that obtained in those patients in whom the detection of H. pylori was assessed by biopsy study alone. (90%vs. 70% and 72%, P < 0.05) (Table 1). In six patients with H. pylori positive in histologic specimens, the serum anti-H. pylori antibody was also found to be positive. DISCUSSION The mortality for gastric carcinoma remains still high in Japan (40:100,000).Recent studies suggested that patients infected with H. pylori might have an increased risk of developing gastric c a r c i n ~ m a . ~This ~ ' ~ ~assumption is based on previous observations that superficial chronic gastritis caused by H. pylori may lead potentially to the development of atrophic gastritis and intestinal metaplasia.16-20Many previous reports have shown this potential association of H. pylori infection with the occurrence of Patient no. No. of H. pylori positive specimens Presence Presence Presence of ACG of AG of IM 1 2 3 4 5 6 7 8 9 10 11 016 016 116 216 216 2/6 316 416 416 416 516 216 6/6 6/6 516 416 316 616 4/6 616 616 5/6 616 616 6/6 616 6/6 616 416 416 616 6/6 216 616 6/6 6/6 516 616 6/6 4/6 416 416 616 216 ACC: active chronic eastritis:A G anoohic eastritb: IM: intestinal metaolasia. TABLE 7 Features of Gastritis in Resected Specimens of HeliGo6ucter pylon Negative Patients No. of specimens Presence of ACC (W) Presence of AG (%I Presence of 28 25/28 (89.3) 20128 (71.4) 18128 (64.3) 38 28/38 (73.7) 38138 (100) 38138 (100)' IM (W H. pylori positive specimens H. pylon' negative specimens ACG: active chronic gastritis;AC: atrophic gaslrilis; IM:inteslinal metaplasia. ' P < 0.05; comDared with Dositkt soecimens. gastric carcinoma based on the detection of anti-H. pylori antibodies in the serum or by detecting the bacteria in surgical or biopsy specimens of patients with gastric carcinoma.& 1021.22 Comparative study between biopsy and surgically resected specimens can not always be performed. However, in this study, we investigated the H. pylori infection in surgical specimens of patients who were operated on and judged as H. pylori negative by biopsy study. The present investigation is the first to demonstrate an association between H. pylori and gastric carcinoma using biopsy and surgical specimens from the same patients. The positivity of H. pylori was evaluated in biopsy samples and then the actual prevalence of H. pylori infection was reassessed histologically in resected specimens. The detection of H. pylori in gastric carcinoma based on biopsy specimens was found to be lower than H. pylori serologic positivity. The initial detection rate of H. pylori in advanced cancer may also be common based on histologic methods. Previous studies have shown a lower detection 1048 CANCER March 15,1696 / Volume 77 I Number 6 rate of H. pylori using histologic methods as compared with serologic studies in a proportion of 19%to 89%.9.22,23 This discrepancy may be due, in part, to the disappearance of the H. pylori infection during the process of atrophy and subsequent gastric carcinoma, while the seropositivity continues for at least a few months as it was recently demonstrated by Karnes et aLZ4Also, H. pylori infection can not persist for a long time in a mucosa with intestinal metaplasia. Since gastric mucosal atrophy is severe in advanced cancer or intestinal type of cancer, various biopsy examples may show false negative results for H. pylori infection. In this study, the features of gastritis in both biopsy and resected samples may support this theory. Namely, detection rate of H. pylori decreased when the prevalence of IM increased. Similar tendency was observed in the frequency of atrophic gastritis and in the detection rate of H. pylori. Another explanation for the diagnostic yield discrepancy between histologic and serologic studies may be the patchy distribution of H. pylori in the gastric ~ L I C O S Fixed ~ . ~ ~biopsy studies may yield more frequently false negative results due to this type of distribution. The final detection rate of H. pylori in this study was relatively high compared with that obtained in previous studies. The final detection rate in advanced cases increased to 92.3%. This percentage is higher than that reported in previous histologic studies using biopsy specimens alone. The small number of patients in this study may explain this high rate. However, the addition of results in nonoperated patients did not change the overall diagnostic yield of H. pylori. Consequently, the actual prevalence of H. pylori infection in gastric carcinoma obtained by histologic studies was almost similar to that obtained using serologic methods. The detection of H. pylori in diffuse type of cancer was high even in biopsy specimens. This result is different from that of previous studies carried out in non-Japanese populations,6 and from that obtained in Japanese populations with early stage gastric carcinoma.'o The different methods used for the detection of H. pylori may explain the different results obtained in each study. Furthermore, ethnic factors may also account for this discrepancy. Cancerous lesions located in the M lesion showed an interesting result. Although the initial detection rate of H. pylori was 69.2%, the final positivity was 96.2%. This statistically significant increase suggests that the frequency of H. pylori infection in patients with tumor located in M lesion may be associated with body mucosal atrophy. The reason why there was a high rate of false negative for H. pylori infection may be due to the fact that most of the patients had advanced cancer or because they were older than age 60 years. Concerning the relationship of H. pylori detection in biopsy and surgical specimens with the result of serum anti-H. pylori antibodies, except in one patient, all patients who were found to be positive for H. pylori in histologic specimens, also had anti-H. pylori antibody in their sera. Further, the patient in whom H. pylori was not detected in the histologic specimen has a very low level of serum anti-H. pylori antibody. From the foregoing data, there seems to be a correlation between serum anti-H. pylori antibodies and the presence of H. pylon in histologic specimens. However, further studies in a larger population are required to confirm these data. In conclusion, in patients with gastric carcinoma, a sampling error can easily occur, especially in advanced and intestinal types of gastric carcinoma and in tumors originated in the M lesion of the stomach. Therefore, the actual prevalence of H. pylori infection may be considerably higher than the previously reported frequency based on biopsy studies alone. The detection rate obtained in the current study is similar to that obtained using serologic methods. H. pylori infection is possibly associated with all types of gastric carcinoma, though in previous studies the association between H. pylori and gastric carcinoma was not described in all types of gastric carcinoma. We concluded that to ascertain the presence of H. pylori in patients with gastric carcinoma it is necessary to carry out serologic studies and histologic methods using both biopsy and surgical resected specimens. 1. 2. 3. 4. Morris A, Nicholson G. 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