1. Volume 141, Issue 3, Pages 837-845.e7 (September 2011)
Tests for Serum Levels of Trefoil Factor Family Proteins Can Improve Gastric Cancer Screening 
Susumu Aikou, Yasukazu Ohmoto, Toshiaki Gunji, Nobuyuki Matsuhashi, Hiroshi Ohtsu, Hirona Miura, Kensuke Kubota, Yukinori Yamagata, Yasuyuki Seto, Atsushi Nakajima, James R. Goldenring, Michio Kaminishi, Sachiyo Nomura 
Gastroenterology 
Volume 141, Issue 3, Pages e7 (September 2011)
DOI: /j.gastro
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2. Figure 1
Serum TFF levels were measured by ELISA. Serum TFF levels in patients with gastric cancer were significantly higher than in the control group without H pylori infection. Serum TFF levels in the control group with H pylori infection were also significantly higher than in the control group without H pylori infection. Serum TFF2 and TFF3 levels in patients with gastric cancer were also higher than in the control group with H pylori infection.
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3. Figure 2
(A) ROC curves for serum TFF level to distinguish individuals with H pylori infection. The area under the curve of TFF1, TFF2, and TFF3 was 0.952, 0.811, and 0.587, respectively. TFF1 correlates very well with H pylori infectious status. (B) ROC curves for serum TFF level to distinguish patients with gastric cancer. The area under the curve of TFF1, TFF2, TFF3, and pepsinogen I/II ratio was 0.835, 0.735, 0.886, and for all the analyzed patients, respectively. The positive predictive value of TFF1, TFF2, TFF3, and pepsinogen I/II ratio was 0.648, 0.665, 0.709, and 0.608, respectively, and the negative predictive value was 0.918, 0.810, 0.895, and 0.874, respectively. (C) For H pylori–negative patients, all of the markers show good ROC curves. The positive predictive value of TFF1, TFF2, TFF3, and pepsinogen I/II ratio was 0.919, 0.843, 0.784, and 0.814, respectively, and the negative predictive value was 0.934, 0.865, and (D) However, for H pylori–positive patients, only TFF3 shows a good ROC curve. The positive predictive value of TFF1, TFF2, TFF3, and pepsinogen I/II ratio was 0.618, 0.812, 0.776, and 0.606, respectively, and the negative predictive value was 0.68, 0.587, 0.805, and
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4. Figure 3
Sensitivity, specificity, and odds ratio of TFFs, pepsinogen test, and anti–H pylori IgG for detecting gastric cancer. TFF1 and TFF3 show significantly higher odds ratios than the pepsinogen test.
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5. Figure 4
The ROC curves of pepsinogen I/II (PG I/II), TFF3, and TFF3 plus PG I/II are shown. TFF3 plus PG I/II can provide the best gastric cancer screening marker among them.
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6. Figure 5
(A–B) The serum TFF1 and TFF2 levels of the patients with undifferentiated cancer were significantly higher than that in the patients with differentiated cancer (TFF1: P = .018; TFF2: P = .016). (C) The serum TFF3 levels of the patients with differentiated gastric cancer and with undifferentiated gastric cancer did not differ significantly (P = .312).
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7. Figure 6
Serum TFF levels before and after gastrectomy. Serum TFF1 and TFF2 levels were significantly decreased after operation for gastric cancer. There was no significant change in serum TFF3 level between before and after operation.
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8. Supplementary Figure 1
Purity of human TFFs (hTFFs) expressed by E coli and native hTFFs expressed by COS cells is shown. These peptides were used for immunizing rabbits. (A) Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) of hTFF2 expressed by E coli, with and without 2-mercaptethanol (ME). Without ME, hTFF2 assembles multimers. (B) SDS-PAGE of TFF1 and TFF3 expressed by E coli, with and without ME. Without ME, hTFF1 forms monomers and dimers. (C) SDS-PAGE of purified hTFFs-His expressed by COS cells is shown. Molecular weight size marker was applied in lane M. Lanes 1–3, lanes 4–5, and lanes 7–9 were elutions of hTFF1, hTFF2, and hTFF3, respectively. Lanes 1, 4, and 7 were flow-through, lanes 2, 5, and 8 were elution 1, and lanes 3, 6, and 9 were elution 2. Recombinant hTFF proteins were purified from the culture supernatants with Ni-NTA agarose gel.
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9. Supplementary Figure 2
Specificity of antibodies was confirmed by Western blot against commercially available hTFFs. The hTFF1 and hTFF3 were purchased from Abnova (Taipei, Taiwan), and hTFF2 was purchased from PeproTech (Rocky Hill, NZ). Lane 1, molecular weight marker; lane 2, hTFF1-GST; lane 3, hTFF2; lane 4, hTFF3-GST. All antibodies worked specifically against each TFF. (A) OPP22203 antibody against hTFF1. (B) OPP22205 antibody against hTFF1. (C) OPP20602 antibody against hTFF2. (D) OPP20601 antibody against hTFF2. (E) OPP22303 antibody against hTFF3. (F) OPP22305 antibody against hTFF3.
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10. Supplementary Figure 3
Specificity of ELISA system was confirmed with native TFFs expressed by COS cells. ELISA system was specific for each native TFF. (A) hTFF1 ELISA, (B) hTFF2 ELISA, and (C) hTFF3 ELISA.
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11. Supplementary Figure 4
Human urine separated by high-performance liquid chromatography was analyzed by ELISA systems, and specific detection for appropriate molecular weight was confirmed. (A) hTFF1 ELISA, (B) hTFF2 ELISA, and (C) hTFF3 ELISA.
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12. Supplementary Figure 5
Immunohistochemistry of (A, C, and E) normal fundic gland and gastric mucosa with (B) foveolar hyperplasia, (D) SPEM, and (F) intestinal metaplasia for (A and B) TFF1, (C and D) TFF2, and (E and F) TFF3. Foveolar cells were positive for TFF1, mucous neck cells in fundic gland were positive for TFF2, and TFF3 was negative in normal gastric mucosa. TFF1-positive foveolar cells expand in foveolar hyperplasia, TFF2 was strongly positive in SPEM, and TFF3 was positive in intestinal metaplasia.
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13. Supplementary Figure 6
Serum TFF3 levels of the second cohort of patients with gastric cancer, aged in their 30s to 60s, was compared with age-matched control individuals. The TFF3 level was significantly higher in the group of patients with gastric cancer (P < .001).
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14. Supplementary Figure 7
Serum TFF levels more than 3 months after resection of stomach. Serum TFF value before resection was set as 1. TFF1 and TFF2 remained low after resection of the stomach. However, TFF3 remained high even 3 months after resection. (A) TFF1, (B) TFF2, (C) TFF3.
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