Helicobacter pylori as a prognostic indicator after curative resection of gastric carcinoma: a prospective study

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Helicobacter pylori as a prognostic indicator after curative resection of gastric carcinoma: a prospective study Georgios Meimarakis, Hauke Winter, Ilka Assmann, Reinhard Kopp, Norbert Lehn, Manfred Kist, Manfred Stolte, Kalter-Walter Jauch, Rudolf A Hatz

Summary Background The effect of Helicobacter-pylori status on survival after curative resection for gastric adenocarcinoma is unknown. We aimed to follow-up patients who were positive or negative for infection with H pylori who had curative (ie, R0) resection for gastric adenocarcinoma to assess differences in relapse-free survival and overall survival. Methods Before surgery, we assessed the H pylori status of 166 patients who had R0 resection for gastric adenocarcinoma between 1992 and 2002 with bacterial culture, histological analyses (ie, staining with haematoxylin and eosin and with Warthin-Starry), and serological analyses. Findings At a median follow-up of 53·0 months (range 1–146), relapse-free survival was 56·7 months (95% CI 4·7–108·7) and overall survival was 61·9 months (13·0–110·9) in patients positive for H pylori, compared with 19·2 months (12·7–25·6) and 19·2 months (7·1–31·3), respectively, in patients negative for H pylori (p=0·0009 for difference in relapse-free survival between groups, and p=0·0017 for difference in overall survival between groups). In multivariate analyses, H pylori was an independent prognostic factor for relapse-free survival (hazard ratio 2·16 [95% CI 1·33–3·49]) and overall survival (2·00 [1·22–3·27]). Depth of tumour invasion (2·60 [1·66–4·08]), lymphnode metastasis (2·11 [1·25–3·57]), and patient age 67·5 years or older (1·75 [1·11–2·75]) were also independent prognostic factors for overall survival. Interpretation Tumour-specific immune responses might be downregulated in patients who are negative for H pylori, and these patients should be followed up carefully because of a poor outlook.

Introduction Gastric adenocarcinoma is not a homogeneous disease. Because patients with this disease commonly have a poor outlook, various attempts have been made to classify this disease to predict its outcome. However, neither the established Laurén1 or Borrmann classifications for this disease, nor the more recently defined classifications of Ming and Goseki have added reproducible additional information to the WHO system regarding patients’ outlook. The discovery of Helicobacter pylori2 has given a new tool for classification of gastric adenocarcinoma. The International Agency for Research on Cancer3 has classified H pylori as a class I carcinogen because of its epidemiological association with gastric cancer. However, although many patients with gastric cancer are positive for H pylori, a truly H-pylori-negative subgroup does exist.4 At present, there are no reliable data for differences in survival of patients who are positive for H pylori compared with those who are negative. To our knowledge, only two studies5,6 have suggested that patients with gastric adenocarcinoma who are positive for H pylori before surgery have higher overall survival compared with patients who are negative. However, in these studies,5,6 negative H-pylori status was significantly associated with infiltrative tumour types (ie, Borrmann stages III and IV), more-advanced tumour stage, and duodenal infiltration—findings that could explain the survival benefit noted for those who were H-pylori http://oncology.thelancet.com Vol 7 March 2006

positive. Moreover, the study designs5,6 were retrospective, patients who underwent non-curative resection (ie, R1 or R2) were included in statistical analyses,5,6 and serological analysis was the only method used to assess H-pylori status.5,6 We aimed to follow up patients who were positive or negative for infection with H pylori who had curative (ie, R0) resection for gastric adenocarcinoma to assess differences in relapse-free survival and overall survival. We planned to assess H-pylori status with a combination of bacteriological culture, serological analyses, and histological analyses.

Lancet Oncol 2006; 7: 211–22 Published Online February 15, 2006 DOI:10.1016/S1470-2045(06) 70586-1 University Hospitals Grosshadern, LudwigMaximilians-University Munich, Munich, Germany (Georgios Meimarakis MD, Hauke Winter MD, Ilka Assmann MD, Reinhard Kopp MD, Prof Karl-Walter Jauch MD, Prof Rudolf A Hatz MD); Institute for Medical Microbiology and Hygiene, University Regensburg, Regensburg, Germany (Prof Norbert Lehn MD); Institute for Medical Microbiology and Hygiene, University Freiburg, Freiburg, Germany (Prof Manfred Kist MD); and Institute for Pathology, Bayreuth Clinic, Bayreuth, Germany (Prof Manfred Stolte MD) Correspondence to: Dr Georgios Meimarakis, Department of Surgery, Klinikum Grosshadern, Ludwig Maximilians University Munich, Marchioninistrasse 15, 81377 Munich, Germany meimarakis@med. uni-muenchen.de

Methods Patients Between 1992 and 2002, 212 consecutive patients with gastric adenocarcinoma underwent curative resection at the University Hospitals Grosshadern, LudwigMaximilians-University, Munich, Germany. Patients with cardia and subcardia adenocarcinomas were included in the study, but Barrett’s oesophageal adenocarcinoma (ie, Siewert type I) was excluded. Oral and written informed consent was obtained from all patients enrolled in the study before gastroscopy and surgery; gastroscopy was done immediately before surgery. After approval from the ethics committee of Grosshadern Clinic for immunohistochemical analyses (ie, staining for tumour necrosis factor receptor superfamily member 4, OX40) of tumour tissue, written 211

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informed consent was obtained from patients enrolled in the study. An independent reviewer assessed preoperative concentration of carcinoembryonic antigen (cut-off 3 g/L). Preoperative concentration of cancer antigen (CA) 19-9 (cut-off 37 U/mL) was assessed in 141 patients and CA 72-4 (cut-off 4 U/mL) in 140 patients. By contrast with carcinoembryonic antigen, analyses of CA19-9 and CA 72-4 were done routinely only for patients who were assessed at the clinic’s special surgical gastrointestinal outpatient unit before surgery (ie, not for patients who were referred from other hospitals). Surgery consisted of subtotal or total gastrectomy and D2 (ie, extended) lymph-node dissection in all patients. At University Hospitals Grossharden, an independent reviewer assessed the following after surgery for all patients: tumour size and localisation; depth of tumour invasion; lymph-node metastasis; histological grading; and tumour type according to Laurén classification.1 For all patients, MS assessed activity (ie, presence of granulocyte infiltrate) and chronicity (ie, presence of lymphocyte infiltrate) of gastritis according to Sydney classification;7 GM assessed history of ulcers. Between 1992 and 1997, nodal status was classified by the anatomical nodal site according to the 4th edition of the tumour-node-metastasis (TNM) classification; between 1998 and 2002, nodal status was classified by the number of positive nodes according to the 5th edition of TNM classification. 46 patients were excluded from the study because they had R1 resection (n=5), R2 resection (n=6), or metastatic disease (n=35). Thus, 166 patients were followed up, 127 of whom had total gastrectomy and 39 of whom (who had distal carcinoma, intestinal type) had subtotal gastrectomy. A treating oncologist gave the indication for adjuvant treatment based on pathological findings (ie, tumournode-metastasis staging), comorbidity, and compliance with treatment. 12 patients received adjuvant treatment: ten had intraoperative radiotherapy and two had postoperative chemotherapy with fluorouracil, cisplatin, and folinic acid. The decision to give adjuvant treatment was made independently and without knowledge of H-pylori status.

Tissue sampling, histopathological analyses, and H-pylori status At the Grossharden Clinic immediately after gastrectomy, RK obtained four biopsy samples for every patient from the antrum and two from the corpus mucosa. Samples were taken from macroscopically nontumorous mucosa at least 5 cm from the tumour. Samples from the corpus mucosa and two antral samples were placed immediately in 3·7% neutral formalin, and were embedded in paraffin by GM and RAH for histological analyses by MS at the Bayreuth Clinic. The two remaining samples from the antrum 212

were placed in H-pylori-specific transport medium (Portagerm pylori, BioMérieux, Marcy L’Etoile, France) and sent immediately to the Institute for Medical Microbiology and Hygiene in Regensburg (NL) and Freiburg (MK), Germany, for bacterial culture. A venous blood sample for serological testing was drawn from every patient by medical residents. Serum samples were stored at –20ºC until assay by MK at the Institute for Medical Microbiology and Hygiene, Freiburg. NL and MK assessed the H-pylori status of every patient after surgery by bacterial culture from nontumorous antrum mucosa (NL and MK), serological analyses (MK), and histological analyses (staining of non-tumorous samples of antrum and corpus mucosa with Warthin-Starry and haematoxylin and eosin; MS). Individuals were regarded negative for H pylori if they had no history of H pylori infection on questioning by GM before surgery and if they were negative in all three tests (ie, microbiological, serological, and histological analyses). Patients were regarded positive for H pylori if they had any reported history of H-pylori infection or if they were positive on one of the three tests. MS identified the presence and density of H pylori using the Warthin-Starry stain. To define the serological H-pylori status of patients, we used two enzyme-linked immunosorbent assay kits (Cobas Core®, Roche, Grenzach-Wyhlen, Germany, and Enzygnost®, Dade Behring, Marburg, Germany). These assays consist of antibodies (IgA and IgG) for H pylori, and have a cut-off for positive samples at 6 U/mL.8 The manufacturers of Cobas Core® cite a sensitivity of 94·4% and specificity of 90·6% for IgG; the manufacturers of Enzygnost® cite a sensitivity of 93·4% for IgG and 85·0% for IgA and specificity of 96·1% for IgG and IgA. MS did histopathological classification of gastritis on paraffin-embedded sections stained with haematoxylin and eosin; MS was unaware of patients’ clinical features. Classification was done in accordance with the updated Sydney system,7 and included assessment of atrophy grade, chronicity, activity, and intestinal metaplasia on a scale of 0 (absent) to 3 (high). Resected tumours were classified by tumour size, depth of invasion, lymph-node metastasis, and histological type according to the Laurén classification at Grosshadern Clinic.

Immunohistochemical analyses for OX40 expression Regulatory T cells have a central role in the regulation of antigen-specific antitumour immune responses. OX40 directly controls the suppression of the immune system that is mediated by regulatory T cells.9,10 Therefore, we analysed and compared the distribution of cells that express OX40 in the gastric-tumour tissue of 16 patients who were negative for H pylori and 17 patients who were positive. Groups did not differ in sex, age, histological features, or tumour markers. http://oncology.thelancet.com Vol 7 March 2006

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Immunohistochemical analyses were done between April, 2005, and June, 2005. Gastric tissues were fixed in phosphate-buffered formalin solution and embedded in paraffin (N van den Engel, University Hospitals Grosshadern). Paraffin sections of 4-m thickness were dried overnight at room temperature, fixed for 3 h at 60ºC, and deparaffinised with Roticlear® (Roth, Karlsruhe, Germany) for 10 min. Sections were hydrated through a series of alcohol bathes (two bathes at 5 min each in absolute ethanol, two bathes at 5 min each in 96% ethanol, and one bathe at 5 min in 70% ethanol). Sections were incubated for 15 min in 0·3% hydrogen peroxide and 70% ethanol to decrease endogenous peroxidase activity. Slides were washed with phosphate-buffered saline (pH 7·4) and incubated at 96ºC in 0·1 mol/L citrate buffer (pH 6·0) for epitope retrieval. After 20 min, slides were rinsed and incubated for 5 min at room temperature with Superblock (ZytoChemPlus HRP Broad Spectrum Kit, Zytomed, Berlin, Germany) to reduce non-specific binding. Slides were incubated with OX40 (CD134, Pharmingen, CA, San Diego; diluted 1:50 with phosphatebuffered saline) for 1 h in a humid chamber. Mouse IgG1 (Dako, Denmark, Glostrup; diluted 1:10 with phosphatebuffered saline) was used as an isotype control. The negative control was incubated phosphate-buffered saline instead of primary antibody. After three wash steps for 5 min with phosphate-buffered saline, sections were incubated with undiluted biotinylated secondary antibody (Zytomed) for 10 min. Subsequently, sections were incubated for 10 min with undiluted streptavidin-conjugated horseradish peroxidase (Zytomed) and washed three times. The horseradish-peroxidase reaction was visualised by use of 0·25 g/L 3-amino-9-ethylcarbazole (Sigma, St Louis, MO, USA) in 0·1 mol/L sodium acetate buffer, pH 4·9, containing 0·003% hydrogen peroxide. Slides were incubated for 8 min on a shaker and were washed once in distilled water. Sections were counterstained with undiluted Mayer’s hemalum solution (Merck, Darmstadt, Germany) for 15 s, rinsed for 10 min with tap water, and embedded with Kaiser’s glycerol gelatin (Merck). The frequency of OX40-positive cells were counted in 20 independent view fields at magnification 400 by two independent investigators (N van den Engel and HW) who were masked to the characteristics of the patients.

Serological analyses† Bacterial culture Histological analyses‡

Sensitivity

Specificity

Pearson’s coefficient

p*

100% 100% 100%

92·0% 51·6% 43·2%

0·8531 0·4380 0·4951

0·0001 0·0001 0·0001

*Pearson’s bivariate correlation coefficient. †Core® and Enzygnost®. ‡Warthin-Starry stain.

Table 1: Concordance analysis between serological, microbiological, and histological tests to define H-pylori status

The mean of the two separate analyses were used for statistical analyses.

Follow-up Patients were followed up at our hospital by GM, IA, and HW according to the recommendations of the Munich tumour registry centre. Within the first 2 years after surgery, follow-up every 3 months consisted of clinical examination, routine blood tests, assessment of Patients negative for H pylori (n=41) Patients Sex Women Men Gastrectomy Total Subtotal Adjuvant treatment No Yes History of gastric ulcer No Yes Histological analyses Tumour site Distal (ie, antrum or corpus) Proximal (ie, cardia or fundus) Tumour size 4 cm 4 cm Laurén classification Intestinal Diffuse Mixed type Grade Well differentiated Moderately differentiated Poorly differentiated Undifferentiated Depth of tumour invasion T1 T2 T3 T4 Lymph-node metastases N0 N1 N2 UICC stage IA IB II IIIA IIIB IV Grade of gastritis None Minimum Moderate Severe Activity of gastritis None Minimum Moderate Severe

Patients positive for H pylori (n=125)

p

0·0001 6 (15%) 35 (85%)

60 (48%) 65 (52%)

36 (88%) 5 (12%)

91 (73%) 34 (27%)

40 (98%) 1 (2%)

114 (91%) 11 (9%)

36 (88%) 5 (12%)

96 (77%) 29 (23%)

25 (61%) 16 (39%)

100 (79%) 25 (20%)

27 (66%) 14 (34%)

69 (55%) 56 (45%)

21 (51%) 13 (32%) 7 (17%)

56 (45%) 61 (49%) 8 (6%)

1 (2%) 14 (34%) 26 (63%) 0

1 (1%) 24 (19%) 99 (79%) 1 (1%)

5 (12%) 24 (59%) 11 (27%) 1 (2%)

24 (19%) 68 (54%) 29 (23%) 4 (3%)

16 (39%) 16 (39%) 9 (22%)

49 (39%) 38 (30%) 38 (30%)

5 (12%) 7 (17%) 14 (34%) 11 (27%) 3 (7%) 1 (2%)

20 (16%) 24 (19%) 34 (27%) 30 (24%) 13 (10%) 4 (3%)

0 27 (66%) 11 (27%) 3 (7%)

1 (1%) 21 (17%) 49 (39%) 54 (43%)

31 (76%) 10 (24%) 0 0

30 (24%) 22 (18%) 45 (36%) 28 (22%)

0·0492

0·2972

0·1297

0·0142

0·2306

0·1527

0·1681

0·7553

0·4760

0·9296

0·0001

0·0001

(continues)

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(continued) Tumour marker Carcinoembryonic antigen 3 g/L 3 g/L CA 19-9* 37 U/mL 37 U/mL CA 72-4† 4 U/mL 4 U/mL Outcome Status at last follow-up Alive Dead

0·0401 28 (68%) 13 (32%)

104 (83%) 21 (17%) 0·6696

29 (71%) 7 (17%)

81 (64%) 24 (19%)

24 (59%) 13 (32%)

75 (59%) 28 (22%)

17 (41%) 24 (59%)

68 (54%) 57 (46%)

0·3620

0·1504

Proportions might not add to 100% because of rounding. 2 test used to compare all variables, except gastrectomy (Fisher´s exact test) and age (unpaired t test); p refers to differences in proportions of variables between groups. *n=141. †n=140.

Table 2: Characteristics of 166 patients with gastric carcinoma assessed for H-pylori status

concentration of tumour markers, and abdominal ultrasonography; endoscopy was done every 6 months for the first 2 years after surgery. For the next 3 years, patients were followed up every 6 months and underwent endoscopy every 12 months. At relapse (defined as local recurrence or metastasis at distant sites), all patients were staged fully to detect disease at other sites. Overall survival was defined as survival from gastric cancer (ie, only deaths from gastric cancer were counted). We calculated median survival unless otherwise stated. Additional data for follow-up were obtained by contacting family physicians.

Statistical analyses We used the 2 test to investigate the association between H-pylori status and other prognostic variables and clinicopathological features. For comparison of age at time of surgery we used the unpaired t test; the effect of gastrectomy (ie, subtotal vs total) was analysed by use of Fisher’s exact test. We assessed relapse-free survival in all patients who had not had local or distant relapse and who were alive, and assessed overall survival in all patients who were alive, irrespective of relapse status. We used the log-rank test in Kaplan-Meier survival analyses to assess the effect of variables on survival. We assessed the effect of H-pylori status on relapse-free survival and overall survival with Cox proportional-hazards regression using the forward stepwise (ie, Wald) method by including all significant variables in the univariate analysis and sex. We used the Mann-Whitney test to analyse differences between infiltration of OX40-expressing cells in histological sections. We used SPSS statistical package (version 12.0.1) for all analyses. For all statistical tests, we used an  level of 5%.

Role of the funding source The sponsors of the study had no role in the study design; in the collection, analysis, or interpretation of data; or in 214

the writing of the report. The corresponding author had full access to all data in the study and had final responsibility for the decision to submit for publication.

Results Table 1 shows results for concordance analysis of the tests used to define H-pylori status, and highlights that serological testing is the most specific and sensitive for detection of H-pylori infection. However, a test for H pylori might not always be accurate because patients can remain seropositive up to 12 months after eradication of H pylori. The low specificity of bacterial culture and Warthin-Starry stain could be due to the patchy distribution of H-pylori-associated gastritis in gastric mucosa.11 100 men and 66 women participated in the study; mean age of participants was 65·0 years (SD 11·2). Table 2 shows the characteristics of these patients. 34 patients ever had a gastric ulcer, 25 of whom had an ulcerated tumour. Whether such ulcerated tumours developed at the site of the previous ulcer is not known because most of these patients had endoscopy at other locations before admission to our hospital and endoscopy reports were not retrievable. The ratio of distal to proximal gastric carcinoma was 3 to 1. Of the 37 patients with gastric carcinoma located at the cardia, four had carcinoma located at the cardia and fundus. 35 patients had splenectomy in addition to gastrectomy. Perioperative morbidity was 7·8% (n=13, six patients who were H-pylori negative and seven who were H-pylori positive). Three patients developed an intra-abdominal abscess, two pneumonia, two an anastomosis insufficiency, one sepsis, one intraabdominal bleeding, one cerebral infarction, one severe cardiac arrhythmia, one pleural effusion, and one peritonitis. One patient died in the perioperative period. At the time of surgery, 125 patients were positive for H pylori whereas 41 were negative (table 2). Positivity for H pylori was associated with higher activity and chronicity of gastritis according to the Sydney classification, and with women. Although patients who were negative for H pylori had a significantly higher frequency of tumours in the cardia or fundus than did those who were positive for H pylori (table 2), this difference had no effect on relapse-free survival (p=0·62) or overall survival (p=0·51). On histological analysis, we noted that eight patients who were negative for H pylori and 27 patients who were positive had intestinal metaplasia. Before surgery, patients who were negative for H pylori had higher concentrations of carcinoembryonic antigen than did those who were positive (table 2; mean concentration 5·6 g/L [SD 12] vs 4·0 g/L [14]). Concentrations of CA 19-9 and CA 72-4 did not differ between groups (table 2). Follow-up data were available for all but one patient, whose address was unknown. Median follow-up was 53·0 months (range 1–146). At last follow-up, 83 patients http://oncology.thelancet.com Vol 7 March 2006

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Patients Sex Women Men Age at operation 67·5 years 67·5 years Gastrectomy Total Subtotal Adjuvant treatment No Yes Concomitant gastric ulcer No Yes Histological analyses Tumour site Distal (ie, antrum or corpus) Proximal (ie, cardia or fundus) Tumour size 4 cm 4 cm Laurén classification Diffuse Intestinal Mixed type Grade Well differentiated Moderately differentiated Poorly differentiated Undifferentiated Depth of tumour invasion T1 T2 T3 T4 Lymph-node metastases N0 N1 N2 UICC stage I–II III–IV Grade of gastritis None Minimum Moderate Severe Activity of gastritis None Minimum Moderate Severe Tumour marker Carcinoembryonic antigen 3 g/L 3 g/L CA 19-9 37 U/mL 37 U/mL CA 72-4 4 U/mL 4 U/mL

Number of patients (n=166)

Number relapsed (n=83)

Actuarial 2-year survival, % (SE)

Number of patients at risk at 2 years

Actuarial 5-year survival, % (SE)

Number of patients at risk at 5 years

66 100

32 51

72 (6) 58 (5)

42 47

45 (7) 44 (6)

13 25

83 83

33 50

72 (5) 56 (6)

48 41

57 (6) 33 (6)

24 14

127 39

62 21

63 (5) 67 (8)

63 26

43 (5) 48 (9)

28 10

154 12

79 4

63 (4) 82 (12)

80 9

42 (5) 73 (13)

34 4

132 34

69 14

61 (5) 75 (8)

65 24

41 (5) 57 (9)

26 12

125 41

62 21

64 (5) 64 (8)

69 20

46 (5) 37 (9)

32 6

96 70

44 39

57 (6) 72 (5)

40 49

46 (6) 44 (6)

18 20

77 74 15

39 36 8

68 (6) 63 (6) 49 (14)

47 37 5

44 (7) 45 (7) 39 (14)

16 21 1

2 38 125 1

1 16 66 0

50 (35) 68 (8) 63 (5) 100

1 21 66 1

50 (35) 43 (11) 44 (5) NA

1 4 33 0

29 92 40 5

5 42 31 5

93 (5) 68 (5) 39 (8) 40 (22)

21 53 12 20

77 (9) 51 (6) 16 (7) NA

9 24 5 0

65 54 47

20 33 30

79 (5) 60 (7) 49 (8)

42 27 20

64 (7) 32 (7) 33 (8)

23 9 6

104 62

40 43

74 (4) 48 (7)

64 25

56 (6) 26 (6)

32 6

1 48 60 57

0 23 29 31

100 56 (8) 66 (6) 68 (6)

1 21 32 35

100 43 (8) 45 (7) 43 (7)

1 10 14 13

61 32 45 28

29 15 27 12

59 (7) 69 (9) 66 (7) 68 (9)

28 17 28 16

45 (7) 41 (11) 43 (8) 46 (11)

15 5 12 6

134 32

61 22

70 (4) 42 (9)

78 12

49 (5) 24 (9)

34 4

110 31

51 17

64 (5) 77 (8)

57 20

47 (5) 41 (10)

23 7

99 41

45 23

68 (5) 62 (8)

58 19

46 (6) 45 (9)

21 10

P*

0·5488

0·0183

0·6788

0·1559

0·0717

0·6196

0·5458

0·8248

0·7543

0·0001

0·0003

0·0001

0·8185

0·8137

0·0027 0·8350

0·1576

(continues)

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(continued) Microbiological analysis H-pylori status Negative Positive

0·0009 41 125

25 58

43 (9) 70 (4)

11 78

25 (9) 50 (5)

5 33

NA=not available. *Log-rank p for difference between variables in median relapse-free survival.

Table 3: Univariate analysis of predictive factors for relapse-free survival in 166 patients with gastric carcinoma

had relapsed (72 with local recurrence and 11 with metastases at distant sites). 25 patients who were negative for H pylori relapsed compared with 58 who were positive (p=0·0009). Relapse-free survival was 56·7 months (95% CI 4·7–108·7) in patients positive for H pylori, compared with 19·2 months (12·7–25·6) in patients negative for H pylori (p=0·0009 for difference in relapsefree survival between groups) Univariate analyses showed an association between relapse-free survival and negative H-pylori status, age, stage according to the International Union Against Cancer (UICC), tumour depth, nodal status, and concentration of carcinoembryonic antigen (table 3). In multivariate analyses, negative H-pylori status was an independent prognostic factor for poor relapse-free survival (table 4). Depth of invasion, lymph-node involvement, and age were also prognostic factors for relapse-free survival (table 4); sex, carcinoembryonic antigen, and UICC stage were not significantly associated with relapse-free survival in multivariate analyses (data not shown). By March 5, 2005, 81 patients had died of local recurrence (n=24) or distant metastases (n=57, table 2 and figure 1); two people were alive with local recurrence. Table 5 shows that data for overall survival were much the same as those for relapse-free survival. Overall survival was 61·9 months (13·0–110·9) in patients positive for H pylori, compared with 19·2 months (7·1–31·3) in patients negative for H pylori (p=0·0017 for difference in overall survival between groups). Multivariate analyses show that negative H-pylori status, increased depth of invasion, lymph-node involvement, and age older than 67·5 years were independent prognostic markers for poor overall survival (table 6). For patients with early-stage gastric cancer (ie, T1 and T2), we recorded a significant difference in overall survival between those who were positive for H pylori and those who were negative; however, we found no such association between H-pylori status and overall survival

Depth of tumour invasion Lymph-node metastases Negative H-pylori status Age 67·5 years

Hazard ratio (95% CI)

p

2·49 (1·60–3·88) 2·33 (1·38–3·94) 2·16 (1·33–3·49) 1·94 (1·23–3·06)

0·0001 0·0015 0·0019 0·0043

Table 4: Predictive factors for relapse-free interval in multivariate Cox proportional-hazard analysis of 166 patients with gastric carcinoma

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for patients with advanced cancer (ie, T3 and T4; figure 2). Findings were much the same on stratification of patients by other classifications for early versus advanced disease: patients who were positive for H pylori had significantly higher survival than did those who were negative only for those with no nodal involvement and only for those with UICC stage I or II disease (figure 2). For patients with advanced proximal gastric cancer (ie, T3 or T4 tumours located in the cardia, fundus, or both), overall survival for the eight people who were positive for H pylori was 21·1 months (95% CI 0·1–47·8) and for the five people who were negative was 17·6 months (0·1–36·1; p=0·5053). For patients with early-stage distal cancer (ie, T1 or T2 tumours in the antrum, corpus, or both), mean overall survival for the 75 people who were positive was 100·7 months (85·6–115·8; p=0·0002) and for the 18 people who were negative for H pylori was 35·8 months (17·5–54.2). Figure 3 shows that patients who were positive for H pylori had significantly fewer cells that expressed OX40 in gastric-tumour tissue compared with those who were negative (p=0·0273).

Discussion We have shown that infection with H pylori is associated with higher relapse-free survival and overall survival in patients with gastric adenocarcinoma who have curative resection without residual, local, or metastasised tumour. We identified H pylori as an independent, beneficial prognostic factor, the effect of which was most pronounced in patients with early-stage cancer. Gastric cancer is a disease with poor prognosis, despite the opportunity for curative resection. Therefore, predictive factors are needed to identify patients who are at risk of early recurrence and death from this disease. Several independent prognostic factors for survival have been reported for patients with gastric cancer who have had curative resection—eg, immunoreactivity for ERBB212 and expression of vascular endothelial growth factor,13,14 which both have a role in lymphatic spread of tumour cells. Furthermore, the association between improved survival from gastric cancer and endopeptidases such as matrix metalloproteinases,15 cathepsin D,16 and the urokinase-type plasminogen activator system17 is thought to be a result of the role of these molecules in maintenance of the basement membrane and subsequent prevention of spread of http://oncology.thelancet.com Vol 7 March 2006

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B

A 100

100 p0·0001

p=0·0017

Survival (%)

T1 (n=29)

80

80

60

60 H-pylori positive (n=125)

T2 (n=92) 40

40

20

20

H-pylori negative (n=41)

T3 (n=40)

T4 (n=5) 0

0 0

Numbers at risk H-pylori positive 125 H-pylori negative 41

30

60

90

120

150

73 11

35 6

15 3

10 0

0 0

Numbers at risk T1 T2 T3 T4

0

30

60

90

120

150

29 92 40 5

20 51 12 1

9 27 5 0

4 12 2 0

3 6 1 0

0 0 0 0

D

C

100

100 p=0·0007

p=0·0005 80

80

Carcinoembryonic antigen3 g/L (n=132)

Survival (%)

N0 (n=65) 60

60

N1 (n=54)

40

20

40

20

N2 (n=47)

Carcinoembryonic antigen 3 g/L (n=34)

0

0 0

30

60

90

120

150

0

30

Time after operation (months) Numbers at risk 65 NO 54 N1 47 N2

41 26 17

23 12 6

12 4 2

60

90

120

150

Time after operation (months) 8 1 1

0 0 0

Numbers at risk Carcinoembryonic 132 antigen 3 g/L Carcinoembryonic 34 antigen 3 g/L

74

36

17

10

0

10

5

1

1

0

Figure 1: Effect of H-pylori status (A), extent of tumour invasion (B), lymph-node metastasis (C), and concentration of carcinoembryonic antigen (D) on overall survival—univariate analyses

malignant cells into systemic circulation. However, these factors are difficult to assess, and standardised tests for clinical use are not readily available. Few clinicopathological factors have been shown to affect survival in patients with gastric cancer who have had curative resection. Recording of the extent of lymphnode dissection18 and positive cytology on peritoneal http://oncology.thelancet.com Vol 7 March 2006

washing19,20—especially detection of aneuploid tumour cells21—have been suggested in addition to tumournode-metastasis classification. By contrast, two prospective studies22,23 noted increased morbidity and mortality after D2 lymphadenectomy compared with that of D1 (ie, limited) lymphadenectomy, without a benefit in 5-year survival. 217

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Patients Sex Women Men Age at operation 67·5 years 67·5 years Gastrectomy Total Subtotal Adjuvant treatment No Yes Concomitant gastric ulcer No Yes Histological analyses Tumour site Distal (ie, antrum or corpus) Proximal (ie, cardia or fundus) Tumour size 4 cm 4 cm Laurén classification Diffuse Intestinal Mixed type Grade Well differentiated Moderately differentiated Poorly differentiated Undifferentiated Depth of tumour invasion T1 T2 T3 T4 Lymph-node metastasis N0 N1 N2 UICC stage I–II III–IV Grade of gastritis None Minimum Moderate Severe Activity of gastritis None Minimum Moderate Severe Tumour marker Carcinoembryonic antigen 3 g/L 3 g/L Cancer antigen 19-9 37 U/mL 37 U/mL Cancer antigen 72-4 4 U/mL 4 U/mL

Number of patients (n=166)

Number of deaths (n=81)

Actuarial 2-year survival, % (SE)

Number of patients at risk at 2 years

Actuarial 5-year survival, % (SE)

Number of patients p* at risk at 5 years

66 100

32 49

74 (6) 61 (5)

43 50

43 (7) 48 (6)

13 28

83 83

33 48

73 (5) 60 (6)

49 44

59 (6) 35 (6)

25 16

127 39

61 20

65 (5) 69 (7)

67 27

44 (5) 51 (9)

30 11

154 12

77 4

65 (4) 82 (12)

84 9

44 (5) 72 (13)

37 4

132 34

68 13

63 (5) 78 (7)

68 25

41 (5) 64 (9)

27 14

125 41

60 21

65 (5) 70 (8)

71 22

49 (5) 35 (10)

35 6

96 70

43 38

60 (5) 74 (5)

43 50

47 (6) 47 (6)

19 22

77 74 15

37 36 8

70 (5) 64 (6) 56 (14)

49 38 6

49 (6) 44 (7) 38 (14)

19 21 1

2 38 125 1

1 16 64 0

50 (35) 73 (7) 64 (5) 100

1 23 68 0

50 (35) 42 (11) 46 (5) NA

1 4 37 0

29 92 40 5

5 40 31 5

92 (5) 72 (5) 41 (8) 40 (22)

21 56 14 2

76 (10) 55 (6) 16 (6) 0

9 27 5 0

65 54 47

20 31 30

79 (6) 62 (7) 56 (8)

42 29 23

64 (7) 40 (7) 30 (8)

23 12 6

104 62

38 43

75 (5) 53 (7)

65 28

60 (5) 23 (7)

35 6

1 48 60 57

0 23 28 30

100 60 (8) 68 (6) 70 (6)

1 23 33 36

100 43 (8) 47 (7) 48 (7)

1 10 15 15

61 32 45 28

29 14 26 12

63 (7) 69 (9) 68 (7) 72 (9)

30 17 29 17

45 (7) 44 (12) 45 (8) 52 (11)

15 6 13 7

134 32

57 24

72 (4) 45 (9)

79 14

51 (5) 27 (8)

36 5

110 31

50 13

64 (5) 80 (7)

58 21

47 (6) 50 (10)

24 9

99 41

44 22

69 (5) 67 (8)

59 21

48 (6) 48 (9)

25 11

0·7003

0·0405

0·6082

0·1807

0·0555

0·5146

0·5723

0·7282

0·8223

0·0001

0·0007

0·0001

0·8036

0·9000

0·0005

0·9716

0·2314

(continues)

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(continued) Microbiological analyses H-pylori status Negative Positive

0·0017 41 125

24 57

46 (9) 73 (4)

12 81

28 (9) 51 (5)

6 35

NA=not available. *Log-rank p for difference in median overall survival between variables.

Table 5: Univariate analysis of predictive factors for overall survival in 166 patients with gastric carcinoma

We recorded a higher frequency of infection with H pylori in women with gastric cancer than in men with gastric cancer, by contrast with most studies, which have showed infection to be much the same between men and women. A possible explanation could be that inclusion of patients only after curative resection of the gastric tumour led to selection of patients. Data for the role of tumour markers as prognostic factors for outcome of gastric cancer have been inconsistent. However, preoperative assessment of concentrations of carcinoembryonic antigen24,25 and doubling time of this marker26 have been reported as independent prognostic factors for outcome after gastric cancer. CA 19-9 and CA 72-4 have been identified as important tumour markers for patients with gastrointestinal cancer. Multivariate analyses27,28 have shown that concentrations of these markers are independent prognostic factors of survival in patients with metastatic gastric cancer. Therefore carcinoembryonic antigen, CA 19-9, and CA 72-4 are assessed routinely in our department before surgery to define baseline and postoperative values in patients with gastric cancer for early detection of progressive disease or relapse. The identification of H-pylori status as an independent prognostic factor could be a useful tool in clinical practice to distinguish effectively between patients at high risk and low risk for adverse disease outcome. H-pylori status can be assessed easily in the clinic before resection. Thus, our study might contribute to a new classification of gastric cancer and have a substantial effect on disease outcome. However, the reasons for our recorded differences between patients who are positive for H pylori and those who are not are not known. Because of its role in carcinogenesis, H pylori might affect the biological behaviour of gastric carcinoma. Although it was postulated29 that eradication of H pylori reduces the risk of developing gastric carcinoma, many controlled studies30 did not show an improvement in atrophy and intestinal metaplasia after eradication treatment, and these patients have a high risk of developing gastric cancer and a poor prognosis. We suggest that the tumour-specific immune response is more beneficial in early-stage gastric cancer as a result of an increased ratio between tumour-specific T cells and tumour cells, or because of an increased number of micrometastases in advanced-stage gastric cancer. http://oncology.thelancet.com Vol 7 March 2006

Two studies4,31 investigated molecular differences in gastric cancer as a result of H-pylori status. Gastric carcinomas that were positive and negative for H pylori did not differ in expression oncogenes and tumoursuppressor genes.31 Although molecular profiling showed differences between early and advanced gastric cancers and histological type according to Laurén classification, no association was noted for H-pylori status.4,31 Kim and colleagues32 reported that H pylori decreases DNA mismatch-repair proteins, suggesting that infection with H pylori increases the risk of mutation in gastric mucosa cells and risk of gastric cancer. Data for the frequency of microsatellite instability, which might be associated with improved outlook in gastric cancer, are conflicting.33 Some investigators34,35 have shown a significant relation between H-pylori status—both positive34 and negative35—and microsatellite instability, whereas others36–38 have not confirmed these findings. Assessment of BAT-26 as a marker for a mutator phenotype in gastric cancer showed that microsatellite changes were associated with higher frequency of H-pylori infection and with higher survival after surgery39—findings that might be due to the relation between H pylori and microsatellite instability. By contrast, differences in patients’ antitumour immune responses might affect outlook in gastric cancer. In vaccination experiments40 with cholera toxoid in human beings, H-pylori infection acts as an adjuvant for the induction of a local B-cell response in gastric mucosa. Furthermore, presentation of tumour antigens in the setting of inflammation might induce stronger immune responses in the presence of H pylori, and the cellular immune response caused by H pylori displays a type-1 T-helper-cell (Th1) type.41 Thus, if the relation between a type 1 response and antitumour activity is confirmed by further studies, H pylori might contribute to an improved immune response against the tumour.

Depth of tumour invasion Lymph-node metastasis Negative H-pylori status Age 67·5 years

Hazard ratio (95% CI)

p

2·60 (1·66–4·08) 2·11 (1·25–3·57) 2·00 (1·22–3·27) 1·75 (1·11–2·75)

0·0001 0·0053 0·0057 0·0162

Table 6: Predictive factors for overall survival in a multivariate Cox proportional-hazard analysis of 166 patients with gastric carcinoma

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B

T1 or T2

A

T3 or T4

100

100

80

80 H-pylori positive (n=92)

Survival (%)

60

60 p=0·0012

p=0·4809

40

40

H-pylori positive (n=33)

H-pylori negative (n=29)

20

20 H-pylori negative (n=12) 0

0 Numbers at risk H-pylori positive H-pylori negative C

0

30

60

90

120

150

0

30

60

90

120

150

92 29

62 9

31 5

13 3

9 0

0 0

33 12

11 2

4 1

2 0

1 0

0 0

N0

N1 or N2

D 100

100

80

80

Survival (%)

H-pylori positive (n=49) 60

60

p=0·0532

p=0·02073

H-pylori positive (n=76)

40

40 H-pylori negative (n=16)

20

20

H-pylori negative (n=25) 0

0 Numbers at risk H-pylori positive H-pylori negative

0

30

60

90

120

150

0

30

60

90

120

150

49 16

37 4

21 2

10 2

8 0

0 0

76 25

36 7

14 4

5 1

2 0

0 0

UICC I or II

E

UICC III or IV

F

100

100

80

80 H-pylori positive (n=78) 60

Survival (%)

60

p=0·6513

p=0·0003 40

40

H-pylori positive (n=47) H-pylori negative (n=26) 20

20

0

0

H-pylori negative (n=15) 0 Numbers at risk H-pylori positive H-pylori negative

30

60

90

120

150

0

30

78 26

57 6

31 4

14 3

60

90

120

150

Time after operation (months)

Time after operation (months) 10 0

0 0

47 15

16 5

5 2

1 0

0 0

0 0

Figure 2: Effect of H-pylori status according to extent of tumour invasion (A, B), lymph-node metastasis (C, D), and UICC (E, F) stage on overall survival

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Articles

Because our results suggest that patients with gastric cancer who are negative for H pylori have a poor outlook, these patients need careful follow-up. Moreover, on elucidation of the mechanisms of the reported differences between individuals who are positive and negative for H pylori, our findings could lead to more aggressive treatment of patients with H-pylori-negative gastric adenocarcinoma.

OX40-positive cells per view

15

10 p=0·0273

5

Contributors G Meimarakis, H Winter, and I Assmann contributed to the design of the study. All authors contributed to the interpretation of results and writing of the report. H Winter contributed to the assessment of OX40-positive infiltrating T cells. I Assmann contributed to collection and assessment of patient data. R Kopp did most of the endoscopies. N Lehn contributed to microbiological assessment of H pylori. M Kist contributed to serological investigations and microbiological assessments of H pylori. M Stolte contributed to histological assessment of gastric tissue from patients.

0

Conflict of interest We declare no conflicts of interest. H-pylori positive

H-pylori negative

Figure 3: Mean expression of OX40 in tumour tissue from patients positive and negative for H pylori Horizontal bars show mean.

CD4-positive and CD25-positive regulatory T cells downregulate antigen-specific immune responses.42 Gene-expression analyses43 have shown high expression of the gene for OX40 in CD4-positive and CD25-positive regulatory T cells compared with CD4-positive and CD25-negative T cells. OX40 is a member of the tumour necrosis factor receptor superfamily, and is transiently expressed after ligation to the T-cell receptor.44,45 OX40 directly controls immune suppression mediated by regulatory T cells. Takeda and co-workers9 have shown that suppression of T-cell responses by regulatory T cells is significantly impaired in the absence of OX40. Furthermore, experiments46 with OX40-deficient mice showed a central role for OX40 in the development and maintenance of regulatory T cells. Because OX40 is expressed mainly on regulatory T cells, and because patients who were negative for H pylori had higher numbers of cells that expressed OX40 in tumour tissue than did those who were positive, we suggest that regulatory T cells infiltrate tumour tissue in patients who are negative for H pylori. Thus, we postulate that tumour-specific immune responses are downregulated in patients with gastric carcinoma who are negative for H pylori, possibly as a result of increased infiltration of OX40-positive regulatory T cells. However, this idea needs further confirmation in larger studies. To investigate the effect of H pylori on the survival of patients with gastric cancer after curative resection, we chose a longitudinal study design. However, because our data are from one centre, the results need to be validated further. We have started to cooperate with other medical centres in Germany to do an external and temporal validation of our findings in a larger cohort of patients. http://oncology.thelancet.com Vol 7 March 2006

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