1. An inverse association between Helicobacter pylori infection and esophageal squamous cell carcinoma risk in Taiwan I-Chen Wu 1,2, Deng-Chyang Wu 1, Ming-Tsang Wu 3 1 Division of Gastroenterology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 2 Graduate Institute of Medicine, College of Medicine, 3 Graduate Institute of Occupational Safety and Health, Kaohsiung Medical University, Kaohsiung, Taiwan

2. Background Most epidemiological studies have indicated an inverse association between Helicobacter pylori (Hp) infection and esophageal adenocarcinoma (EA) risk. However, its association with esophageal squamous cell carcinoma (ESCC) is still inconclusive.

3. Aim This study aims to examine (1) the association between Hp infection and ESCC risk, and (2) whether Hp could cause apoptosis in one ESCC cell line.

4. Patients and Controls 317 newly diagnosed ESCC patients (301 males and 16 females) were recruited from two medical centers in the Kaohsiung in southern Taiwan 1 st hospital-based control: age, sex-matched subjects from Department of Preventive Medicine of the same two hospitals in Kaoshiung 2 nd hospital-based control: healthy subjects from Department of Preventive Medicine of National Taiwan University Hospital, Taipei (not matched) Community-based control: healthy residents of Kaohsiung metropolitan environ (not matched)

5. Hp serology and CagA status Immunochromatographic test (Chembio H. pylori STAT-PAK, Chembio Diagnostic System, Inc., NY, USA): utilizing a combination of Hp antigen coated particles and anti-human IgG to detect Hp antibodies in serum Helori CTX IgG (ravo Diagnostika GmbH, Freiburg, Germany) was used to detect the IgG antibodies against serum CagA by enzyme immunoassay Information about personal history and substance use was collected by using a questionnaire

6. In-vitro studies Cell Lines: –one ESCC cell line: CE 81T/VGH cells –one gastric adenocarcinoma cell line: AGS cells Hp strain: CagA- and VacA-positive Apoptosis assays: –Flow cytometric analysis: Annexin-V-FITC stains early apoptotic cells PI stains both apoptotic and necrotic cells –Terminal transferase-mediated dUTP nick end labeling (TUNEL) assay and cell morphology –Western blot analysis for caspase-3

7. Results and Conclusion Hp seropositivity was inversely associated with ESCC risk (adjusted odds ratio (AOR): 0.315-0.472; all p- values < 0.05) compared to the three control groups (Table 1-3). In in vitro studies, increased apoptosis was found in CE 81T/VGH, but not in AGS cells, after being co-cultured with Hp at a concentration ratio of 1 Hp and 100 cancer cells for 36 hrs (Figure 1-3). There is an inverse association between Hp seropositivity and ESCC risk in Taiwan, possibly due to Hp-induced apoptosis in ESCC cells

8. Table 1. Demographic characteristics of ESCC patients and controls.

9. Table 2. Serum status of Hp and CagA in ESCC patients and three comparison groups *CagA status was determined among the subjects with Hp seropositivity. (No CagA data was available in 3 ESCC and 1 community-based control group).

10. Table 3. Association between Hp seropositivity and ESCC risk *OR: odds ratio **Compared to first and second hospital-based control groups: adjusting for age (continuous), gender (female vs. male), educational levels (  primary school vs. > high school; junior and high schools vs. > high school), cigarette smoking (yes vs. vs. no), alcohol consumption (yes vs. no) and betel nut chewing (yes vs. no); Compared to community-based control group: adjusting for the above factors except for betel nut chewing

11. Figure 1. Flow cytometric analysis Legend: most of the intact cells from controls are categorized into a double-negative group (left lower quadrate). The percentage of apoptotic cells (right lower quadrate) in CE 81T/VGH cells increased from 0.95% in control to 14.05% in 1/100 Hp–treated group after 36 hours. The AGS cells remained intact before and after co-culturing with Hp.

12. Figure 2. Cell morphology and TUNEL staining. Legend: cells were treated with Hp with a MOI of 0, 1/400 and 1/100. A: cell morphology. B: DAPI (blue) stained cellular DNA C: positive TUNEL staining (green) indicated fragmented DNA in apoptotic CE 81T/VGH cells after treated with Hp with a MOI of /100 (arrow head).

13. Figure 3. Protein contents of caspase-3 by Western blot analysis Legend: the amount of the activated caspase-3 (19/17 kDa) increased in CE 81T/VGH cells, but not in AGS cells, after co-culturing with Hp at a MOI of 1/100 for 36 hours