Keisuke Goto et al

19 Instruction: The human amphoterin-induced gene and open reading frame (AMIGO) was 20 identified as a novel cell adhesion molecule of type I transmembrane protein. AMIGO2 is 21 one of three members of the AMIGO family (AMIGO1, 2, and 3), and the similarity 22 between them is approximately 40% at the amino acid level. We have previously shown that 23 AMIGO2 functions as a driver of liver metastasis. Immunohistochemical analysis of 24 AMIGO2 expression in colorectal cancer (CRC) using a commercially available 25 anti-AMIGO2 mouse monoclonal antibody clone sc-373699 (sc mAb) correlated with liver 26 metastasis and poor prognosis. However, the sc mAb was found to be cross-reactive with all 27 three molecules in the AMIGO family. 28 Methods: We generated a rat monoclonal antibody clone rTNK1A0012 (rTNK mAb) for 29 human AMIGO2. The rTNK mAb was used to re-evaluate the association between 30 AMIGO2 expression and liver metastases/clinical outcomes using the same CRC tissue 31 samples previously reported with sc mAb. 32 Results: Western blot analysis revealed that a rTNK mAb was identified as being specific 33 for AMIGO2 protein and did not cross-react with AMIGO1 and AMIGO3. The rTNK mAb 34 and sc mAb showed higher AMIGO2 expression, which correlates with a high frequency of 35 liver metastases (65.3% and 47.5%, respectively), while multivariate analysis showed that 36 AMIGO2 expression was an independent prognostic factor for liver metastases (p = 37 7.930E-10 and p = 1.707E-5). The Kaplan-Meier analyses showed that the rTNK mAb (p = 38 0.004), but not sc mAb (p = 0.107), predicted worse overall survival in patients with high 39 AMIGO2 expression. The relationship between AMIGO2 expression and poor 40 disease-specific survival showed a higher level of significance for rTNK mAb (p = 0.00004) 41 compared to sc mAb (p = 0.001). 42 Keisuke Goto et al. 3 Conclusion: These results indicate that the developed rTNK1A0012 mAb is an antibody 43 that specifically recognizes AMIGO2 by immunohistochemistry and can be a more reliable 44 and applicable method for the diagnostic detection of liver metastases and worse prognosis 45 in patients with high AMIGO2-expressing CRC. 46

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