1. HOXC6 and HOXC8 are potentially novel prognosis predictors of esophageal squamous cell carcinoma Keneng Chen, M.D., PhD, RCSF Luyan Shen, M.D., PhD 2015/4/28 Department of Thoracic Surgery I, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Beijing Cancer Hospital, Peking University School of Oncology, Beijing, China

2. No potential conflicts of interest were disclosed

3. 02 03 04 01 The morbidity and mortality remains high long-term survival remains unsatisfied Lack of reports with long-term follow-up data from prospective database Lack of efficient biomarkers for helping to guide the clinical practice Several issues with the clinical practice of ESCC remain unsolved prognosis

4. Why we choose HOX genes ? 02 03 04 01 There is some similarity between embryogenesis and tumorigenesis AFP in liver cancer and CEA in colorectal cancer HOX gene family regulates embryogenesis HOX genes are deregulated in hematologic malignancy and many solid tumors

5. Regional expression of transcription factors in the developing gut Yuasa Y. Nat Rev Cancer. 2003.

6. Gene Positive cases in ESCC(%) Positive cases in noncancerous mucosa(%) HOXA103(8.3)0(0) HOXA1327(75)0(0) HOXB721(58.3)0(0) HOXC412(33.3)0(0) HOXC815(41.7)0(0) HOXD96(16.7)0(0) HOXD1015(41.7)0(0) HOXD1315(41.7)0(0) HOXA730(83.3)12(33.3) HOXA924(66.7)9(25) HOXC624(66.7)9(25) HOX genes were abnormally expressed in ESCC (n=36) Chen KN, et al. Clin Cancer Res. 2005. potential biomakers Expression of 11 HOX genes is deregulated in ESCC

7. CCK8 Assay Annexin v/PI Cell cycle & Apoptosis Proliferation Soft agar colony formation assay Ability of colony formation KYSE-450 IHC for HOXC6 、 HOXC8 protein detection Prognosis FFPE tissue specimen Lentivirus infection Knockdown of HOXC6/HOXC8 Tumor formation in nude mice Ability of tumorigenesis S Group (138 ) NC+S Group (136) S+AC Group (148) Study design and work flow diagram

8. Staining of IHC

9. The association of HOXC6/HOXC8 expression with overall survival in naive subgroup (n=138) ItemNo. (%) Median survival ， mo(95%CI) 5-y survival (%) p HOXC6 Low expression83(60.1)70.1(34.2-105.9) 51.5 0.070 High expression55(39.9)35.0(23.2-46.8) 32.9 HOXC8 Low expression85(61.6)110.3(35.5-185.3) 51.9 0.005 High expression53(38.4)30.3(17.7-42.9) 30.6 Du YB, et al. J Surg Res, 2014. p = 0.070p = 0.005 Surgery only Survival analysis in surgery subgroup

10. The association of HOXC6/HOXC8 expression with overall survival in neoadjuvant subgroup (n=136) ItemNo.(%)Median survival mo(95%CI) 5-y survival (%) p HOXC6 Low expression 31(22.8)82.7(-) 60 0.002 High expression105(77.2)27.8(16.7-38.8) 33 HOXC8 Low expression73(53.7)110.3(-) 49.9 0.004 High expression63(46.3)21.2(16.5-38.9) 25 NC+S p = 0.002p = 0.004 Survival analysis in neoadjuvant chemotherapy subgroup Du YB, et al. J Surg Res, 2014.

11. The association of HOXC6/HOXC8 expression with overall survival in neoadjuvant subgroup (n=148) ItemNo.(%)Median survival mo(95%CI) 5-y survival (%) p HOXC6 Low expression 81(54.7)61.0(21.8-112.2) 53.8 0.002 High expression67(45.3)33.4(20.5-46.3) 30.3 HOXC8 Low expression99(66.9)62.3(41.5-83.0) 53.0 0.001 High expression49(33.1)31.3(16.9-45.8) 21.7 S+AC p = 0.002 p = 0.001 Survival analysis in adjuvant chemotherapy subgroup

12. mRNA Protein HOXC6/HOXC8 Knock-down at mRNA and Protein level

13. Control HOXC8(-) HOXC6(-) shNC shHOXC6 shHOXC8 Tumor growth in nude mice Soft agar Colony formation CCK8 assay Down-regulation inhibited cell proliferation in vitro & in vivo

14. Apoptosis rate （ % ） shHOXC6 32.8±0.29 * shHOXC8 36.1±0.35 * ShNC12.7±0.31 Apoptosis rate variation when HOXC6 or HOXC8 was knock- down * p ＜ 0.050 Down-regulation induced cell apoptosis

15. G2+S(%)G1(%) shHOXC6 45.38±2.29 * 54.62±2.29 * shHOXC8 28.04±3.1 * 71.9±3.1 * shNC64.87±3.1735.1±3.17 Knockdown of HOXC6 and HOXC8 Affects the Cell Cycle * p ＜ 0.050 HOXC6(-) HOXC8(-) Control Down-regulation arrested cell cycle in G1 phase

16. 0101 0202 HOXC6, HOXC8 might be biomarker for evaluating prognosis of ESCC HOXC6/HOXC8 promotes the esophageal cancer cell proliferation Conclusion

17. Acknowledgment Dr. Chen’s LAB: Yabing Du Bin Dong Wanpu Yan Liang Dai Xiaozheng Kang Zhen Liang Hongchao Xiong

19. 13 12 11 10 9 8 7 6 5 4 3 2 1 D13 D12 D11 D10 D9 D8 D4 D3 D1 C13 C12 C11 C10 C9 C8 C6 C5 C4 B13 B9 B8 B7 B6 B5 B4 B3 B2 B1 A13 A11 A10 A9 B9 A6 A5 A4 A3 A2 A1 cervicalthoracic lumbo-sacral 3' 5' Anterior Early Posterior Late Schematics representation of four HOX loci 7p15.3 17p21.3 12q13.3 2q31 HOX genes present the expression pattern of temporal and spatial colinearity Cillo C, et al. J cell physiol, 2001

20. Clinicopathologic Characteristics HOXC6 expression No.(%) PHOXC8 expression No.(%) P HighLow HighLow Age ≦ 60 74(62.7)44(37.3)0.21953(44.9)65(55.1)0.309 ＞ 60 87(55.0)71(45.0) 63(39.9)95(60.1) Sex Male121(59.0)84(41.0)0.89188(42.9)117(57.1)0.608 Female 40(56.3)31(43.7) 28(39.4) 43(60.6) G stage G110(41.7)14(58.3)0.0108(33.3)16(66.7)0.222 G225(39.0)39(61.0) 29(45.3)35(54.7) G320(40.0)30(60.0) 16(32.0)34(68.0) Tumor location(L) L015(62.5)9(37.5)0.1019(37.5)15(62.5)0.524 L138(67.9)18(32.1) 26(46.4)30(53.6) L275(59.1)52(40.9) 57(44.9)70(55.1) L330(46.2)35(53.8) 23(35.4)42(64.6) Tumor invasion(T) T133(47.1)37(52.9)0.01119(27.1)51(72.9)<0.001 T233(50.0) 22(34.9)41(65.1) T368(63.0)40(37.0) 52(48.1)56(51.9) T426(78.8)7(21.2) 23(69.7)10(30.3) Lymph node metastasis (N) N096(53.9)82(46.1)0.03165(36.5)113(63.5)0.004 N133(58.9)23(41.1) 32(57.1)24(42.9) N220(71.4)8(28.6) 10(35.7)18(64.3) N311(91.7)1(8.30) 9(75.0)3(25.0) TNMG stage I79(45.7)94(54.3)<0.00159(34.1)114(65.9)0.001 II41(73.2)15(26.8) 28(50.0) III41(87.2)6(12.8) 29(61.7)18(38.3) Association between HOXC6 or HOXC8 expression and clinical characteristics (n=274) HOXC6/HOXC8 expression was related with TNM stage

21. HOXC6 expression No.(%)pHOXC8 expression No.(%)p Low HighLow High TRG TRG1/215(48.4) 27(30.9 ） 0.01635(47.9) 7(11.1)<0.001 TRG3/416(51.6)78(69.1) 38(52.1)56(88.9) The association of HOXC6 and HOXC8 expression with TRG (n=136) NC+S Tumor Regression Grade (TRG) Du YB, et al. J Surg Res, 2014.