Additional file 1 of N-glycolylneuraminic acid serum biomarker levels are elevated in breast cancer patients at all stages of disease

Additional file 1: Supplementary Information. Supplementary Methods. Figure S1. Glycan array analysis of SubB2M and SubBA12 using a Z-Biotech Neu5Ac/Neu5Gc array. A) Glycan array result of SubB2M and SubBA12 performed using the Z-Biotech Neu5Gc/Neu5Ac N-Glycan Array. Histogram represents the average relative fluorescent units of binding to each of the numbered structures shown in B. For structure ID see http://www.zbiotech.com/neu5gc-xenoantigen-microarray.html and http://nebula.wsimg.com/deda6829116ce09edb871bd7ce7cde6c?AccessKeyId=B5CD53DB37409833427C&disposition=0&alloworigin=1 for further information. Figure S2. Characterization of human CA125 O-glycosylation and bovine Alpha-1-acid glycoprotein (bAGP) by PGC-LC-MS/MS. Annotated Base Peak Chromatogram of the total A) O-glycome released from CA125 and Extracted ion chromatogram of m/z 681.32− (Neu5Gc) and 665.32− (Neu5Ac) and B) N-glycome released from bAGP and Extracted ion chromatogram of m/z 1127.42− (Neu5Gc) and 1111.42− (Neu5Ac). Confirmation of C) Neu5Gc (m/z 681.32−) and Neu5Ac (m/z 665.32−) containing O-glycan structures by MS/MS fragmentation and D) Neu5Gc (m/z 1127.42−) and Neu5Ac (m/z 1111.42−) containing glycan structures by MS/MS fragmentation. Figure S3. A representative Glycoprotein Units (GPUs) standard curve. Bovine AGP (MW = 41–43 kDa; ~ 50%/50% Neu5Ac/Neu5Gc; high total sialic acids) and human CA125 (MW = > 200 kDa, 5–10% Neu5Gc; low total sialic acid) were combined at starting concentrations of 15 μg/ml and 15 units/ml, respectively, in 0.5% normal human serum. This glycoprotein mixture was two-fold serially diluted down to 14.65 ng/ml and 0.0146515 units/ml, respectively, in 0.5% normal human serum. The Response Units (RUs) for each concentration of the standard mixture were determined by subtracting binding due to SubBA12 (flow cell 4) from binding due to SubB2M on flow cell 2 and flow cell 3. RUs obtained for the highest concentration standard was considered 100 GPUs. FC2 = flow cell 2; FC3 = flow cell 3. Figure S4. ROC curves depicting the ability of serum Neu5Gc levels determined by the optimized SubB2M-A12-SPR assay to distinguish Stage I – IV ovarian cancer patients from cancer-free (normal) individuals. Sensitivity% (true positive rate; ability to detect disease) is plotted against 100%-specificity% (false positive rate or 100%-true negative rate; ability to detect lack of disease). ROC analyses were performed with the data shown in Fig. 2B using Graphpad Prism 8.0. Figure S5. ROC curves depicting the ability of serum Neu5Gc levels to distinguish Stage I – IV breast cancer patients from normal (cancer-free) individuals. Sensitivity% (true positive rate; ability to detect disease) is plotted against 100%-specificity% (false positive rate or 100%-true negative rate; ability to detect lack of disease). ROC analyses were performed with the data shown in Fig. 3 using Graphpad Prism 8.0. Figure S6. Serum Neu5Gc levels determined by SubB2M-A12-SPR assay for A) relapse cases and B) remission cases from the Circ.BR cohort. The mean GPUs from duplicate analyses for each serum sample are shown. Error bars = ± 1 SD from the mean for each group. Two independent assays were performed with both showing the same trends. Results from one assay are shown. Clinical information for each patient is shown in the top right of each plot with treatment history and metastases overlaid. ALND: Axillary lymph node dissection, ILC: Invasive Lobular Carcinoma Mast: mastectomy, SNB: sentinel node biopsy, WLE: wide local excision, XRT: radiation therapy. Detailed information for each patient in the Circ.BR cohort are shown in Supplementary Table 3. Supplementary Table S1. Supplementary glycan microarray document based on MIRAGE guidelines DOI: https://doi.org/10.1093/glycob/cww118 . Supplementary Table S2. Details for each of the normal (cancer-free) individuals and breast cancer patients used in this study. Supplementary Table S3. Details for each of the breast cancer patients from the Circ.BR cohort used in this study. Supplementary Table 4. Optimal cut-off values, sensitivity and specificity for distinguishing Stage I, II, III and IV ovarian cancer patients from normal (cancer-free) individuals using serum Neu5Gc levels determined by optimized SubB2M-SPR assay before and after SubBA12 subtraction. Sensitivity and specificity were determined from the Receiver operating characteristic (ROC) curves (Fig. S4). Optimal cut-off values were selected to give the maximum sum of sensitivity and specificity. Supplementary Table 5. Optimal cut-off and area under the curve (AUC) values for distinguishing Stage I, II, III and IV breast cancer patients from normal (cancer-free) individuals using serum Neu5Gc levels. Sensitivity and specificity were determined from the receiver operating characteristic (ROC) curves (Fig. S5). Optimal cut-off values were selected to give the maximum sum of sensitivity and specificity.