Author: Cheng-Chi Wang, Chi-Huey Wong Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer Author: Cheng-Chi Wang, Chi-Huey Wong Presenter: Bernard Shen

Introduction Breast cancer ranks as one of the leading cancer type in the world. Development of a rapid and reliable method will help decrease the mortality rate in woman Scientists seek molecular targets of cancer cells that are absent from normal cells

Globo H A hexasaccharide and a member of cancer-associated carbohydrate antigens Globo H expressed on the cancer cell surface, as a glycolipid and possibly as a glycoprotein. Globo H is overexpressed on a variety of epithelial cell tumors such as lung and breast cancers, with the used of anti- Globo H monoclonal antibodies MBr1 or VK-9.

Question need to further investigate associated with Globo H? (i) Are Globo H and Gb5 differentially expressed in disease stages? (ii) Can the glycan array be used for the quantitative and high-sensitivity detection of anti-Globo H antibody present in the serum as an indication of cancer? (iii) Can the array be used to measure the activity of fucosyltransferase in breast cancer cells at different stages, to screen inhibitors of the enzyme and to monitor the immune response to the Globo H-based vaccine?

Binding of monoclonal antibodies to Globo H and its analogues

Binding curves for Globo H printed at different concentrations

Glycan Microarray of Globo H Good minic of cell surface expressing multiple glcans very small amount of material is required more appropriate and more sensitive system than the traditional solution- phase ELISA analysis

IgG ratios of Globo H analogs to Gb5 from breast cancer patients GH/Gb5 Bb4/Gb5 Bb3/Gb5 Bb2/Gb5 Healthy Cancer Mean 26.92 58.31 11.91 17.84 15.22 22.54 78.02 120.8 SD 18.49 31.14 20.54 19.60 23.12 23.22 63.63 92.26 P P < 0.0001*** 0.1360 (ns) 0.1106 (ns) 0.0063** IgM ratios of Globo H analogs to Gb5 from breast cancer patients IgM ratio, % GH/Gb5 Bb4/Gb5 Bb3/Gb5 Bb2/Gb5 Healthy Cancer Mean 27.82 53.98 15.73 22.74 15.36 40.51 16.19 30.36 SD 23.42 35.41 21.60 23.98 23.30 59.76 18.83 35.44 P P < 0.0001*** 0.1259* 0.0043** 0.0105*

ELISA (Enzyme-linked immunosorbent assay) used mainly in immunology to detect the presence of an antibody in a sample useful tool for determining serum antibody concentrations

The comparison of glycan micorarray and ELISA The antibody levels were detectable up to 1:1,920 dilution method in Glycan microarray 1:240 dilution in ELISA working concentration of Globo H in array is 4 orders of magnitude lower than ELISA

The comparison of sensitivity of glycan microarray and ELISA Dilution Increase fold of signal Glycan microarray ELISA 30 374.7 ± 87.83 4.01 ± 1.58 120 188.4 ± 78.93 1.92 ± 0.75 240 102.2 ± 44.21 1.08 ± 0.48 480 44.86 ± 17.05 0.20 ± 0.10 960 12.13 ± 4.08 0.30 ± 0.14 1,920 3.203 ± 1.048 ND

Conclusion Quantitative analysis of the breast cancer- associated carbohydrate Globo H with minimizing the amount of serum and carbohydrate Significant difference in anti-Globo H levels between normal and patient plasma Demonstrate the superiority of our glycan microarray over conventional ELISA Offer new way to analysis of fucosyltransferase activity

Improvement and further direction The number of mice (4 in the experiment ) that use to gather data for the comparison of glycan microarray and ELISA should be larger