Supplementary Data

Supplementary Table 1. Clinicopathologic Information for Cases MBC 1-10 Case Post-Mortem Age at Stage at Primary Tumor Age at Interval Diagnosis Diagnosis Type and Grade Death 1 3 hours, 45 54 T2N0M0 ILC, grade 2 65 minutes 2 4 hours 33 T3N1M0 IDC, grade 3 37 3 2 hours 40 T1N1M0 IDC, grade 2 48 4 3 hours 44 T1miN0M0 IDC, grade 3 49 5 4 hours 59 T1N0M0 IDC, grade 2 68 6 3 hours, 30 56 T2N1M0 IDC, grade 2 61 minutes 7 1 hour, 15 51 T2N0M0 IDC, grade 3 53 minutes 8 3 hours 48 T2N3M0 IDC, grade 3 54 9 3 hours 38 T2NxM0 IDC, grade 3 43 10 3 hours 71 T2N0M0 IDC, grade 3 79 1 Supplementary Table 2: Sites of Metastasis. Case Liver Bone Lung Brain Adrenal GI Tract Mesentery Other MBC1 Extensive Rib +minimal - + Left Pancreas, Extensive gallbladder MBC2 + spine Extensive + Bilateral - + Axillary LN, ovary MBC3 Extensive Spine + + + Right - + Left ovary MBC4 Extensive Spine + + Bilateral Small bowel + Spleen MBC5 Extensive Spine + + +Right Pancreas, _ Fallopian tube, stomach breast, pericardium, pleura MBC6 Extensive Spine + - - Stomach, - Pleura, cecum pericardium MBC7 Extensive Spine - + Bilateral - + Mediastinal, axillary LN MBC8 Extensive Spine +minimal - - - - Diaphragm MBC9 - Spine, + NE - Pancreas - Abdominal femur LN, pleura MBC10 + NE + - +Left - + Mediastinum, pericardium, omentum, diaphragm NE = not examined at autopsy Bold = extensive LN=lymph node GI=gastrointestinal 1 Supplementary Table 3: Mesothelin Immunohistochemistry Case Primary Tumor Positive Metastases Negative Metastases MBC 1 Negative None Liver, Bone, Mesentery, Gallbladder, Rectum MBC 2 1+ Bone, Axillary LN, Lung, Adrenal, Brain (1+) Liver, Abdominal LN, Ovary MBC 3 Negative None Bone, Liver, Ovary, Brain, Mesentery, Lung MBC 4 N/A Lung Liver, Pericardium, Spleen, Small Bowel, Adrenal, Bone MBC 5 Negative None Lung, Liver, Pancreas, Fallopian Tube, Adrenal, Brain, Pericardium, Bone, Pleura MBC 6 Negative Pleura, Lung, Colon Liver, Axillary (1+) Lymph Node, Bone MBC 7 Positive (2+) Omentum, Diaphragm, Mediastinal LN(1+) Axillary Metastases, Adrenal, Bone, Liver MBC 8 Negative None Pleura, Liver, Bone, Axillary LN MBC 9 Negative None Bone, Abdominal LN, Adrenal MBC 10 Negative None Lung, Hilar LN, Liver, Pericardium, Adrenal, Omentum 1 Supplementary Table 4: Cox-2 Immunohistochemistry Case Primary Tumor Positive Metastases Negative Metastases MBC 1 Negative Liver, Mesentery Gallbladder, (2+), Bone (3+) Rectum MBC 2 1+ Left Adrenal Gland Brain , Bone, Liver, Abdominal LN, Lung, Ovary, Diaphragm (1+); Right Adrenal Gland, Axillary LN (2+) MBC 3 Negative None Bone, Liver, Ovary, Brain, Mesentery, Lung MBC 4 N/A Liver, Adrenal (1+) Lung, Pericardium, Spleen, Small Bowel, Bone MBC 5 Negative None Lung, Liver, Pancreas, Fallopian Tube, Adrenal, Brain, Pericardium, Bone, Pleura MBC 6 Negative Axillary Lymph Colon Node, Liver, Pleura, Lung, Bone (1+) MBC 7 Negative Omentum, Liver, Left Adrenal, Mediastinal LN, Bone Diaphragm, Right Adrenal (1+) MBC 8 Negative Bone (1+) Pleura, Liver, Axillary LN MBC 9 Positive (2+) None Bone, Abdominal LN, Adrenal MBC 10 Positive (2+) Liver, Mediastinal Omentum, LN, Adrenal, Lung Pericardium (1+); Hilar LN (2+) 1 Supplementary Table 5 : Percentage Promoter Methylation of RASSF1A, HIN1, CYCLIN D2, TWIST, RARβ, APC1, and ERα in lesions of primary and metastatic breast cancer. Scores for cumulative methylation were obtained by QM-MSP assay in liver , mesentery, gallbladder, bone, lung (lobes of right upper = RUL, right middle = RML, right lower = RLL, and left lower = LLL), ovary , adrenal , kidney, rib soft tissue, pericardium, omentum, diaphragm, and lymph node [abdominal (Abd) and mediastinal (Med)]. The % methylation = 100 X [ # copies methylated DNA / (# copies unmethylated + methylated DNA)] contained an aliquot produced from multiplex methylation-independent PCR wherein the entire gene panel was co-amplified before performing quantitative real-time MSP analyses of each gene. 1 ER Tissue RASSF1A HIN1 CYCLIN D2 TWIST RAR B APC1 ALPHA MBC001 Primary 6.840 0.000 0.000 na 0.000 43.179 0.000 Liver #1 11.352 0.000 2.924 18.483 4.422 28.052 0.000 Liver #4 24.005 0.000 13.767 53.007 3.454 77.666 0.000 Mesentery 24.047 0.000 60.268 86.402 32.348 85.684 0.091 Mesentery 26.789 0.065 36.007 75.656 28.135 79.247 0.032 Gall Bladder 26.882 0.987 30.472 68.654 28.884 65.913 0.051 Bone 23.029 0.000 46.280 92.201 0.000 99.635 0.000 MBC002 Primary 0.000 0.133 7.117 13.621 2.447 0.068 0.003 Lung, RML 0.027 0.040 0.000 72.768 1.402 1.889 0.000 Lung, RUL 0.000 0.000 0.000 62.985 2.342 1.131 0.000 Ovary 0.000 0.000 0.000 77.819 0.758 0.000 0.000 Lung, RLL 0.000 0.011 0.001 72.597 0.955 1.218 0.000 Adrenal 0.003 0.000 0.000 55.028 1.335 0.000 0.000 Kidney 0.000 0.000 0.000 69.925 3.191 0.001 0.000 Bone 0.002 0.018 0.000 45.917 0.128 0.000 0.013 MBC003 Primary 77.900 99.956 93.700 65.260 1.437 0.236 0.000 Liver #1 92.768 90.452 83.054 0.174 1.083 0.000 0.001 Liver #1 87.723 96.594 77.529 0.074 1.505 2.109 na Soft tissue rib 85.441 88.431 71.988 0.176 1.109 0.000 0.009 Liver #2 87.280 79.002 83.094 0.052 0.615 2.377 2.047 Lung, RLL 40.319 27.412 51.609 1.563 0.881 1.725 0.529 Bone 13.956 93.364 47.507 0.001 0.002 0.504 0.828 MBC004 Adrenal 1.461 0.028 0.000 0.010 0.090 29.379 0.0000 Pericardium 0.000 0.501 0.004 4.445 3.929 22.172 0.0780 Liver #2 1.805 6.158 0.076 1.164 0.162 51.272 0.1036 Liver #3 0.168 12.500 2.184 0.952 0.015 70.035 1.4547 Lung, RLL 0.000 15.081 0.062 2.190 0.369 32.282 0.0018 Lung, LLL 0.801 5.336 1.469 1.754 0.149 17.356 0.0000 MBC005 Primary 48.562 90.226 29.316 0.000 4.961 37.314 0.000 Lung, RUL 51.396 83.641 57.760 0.144 4.305 51.029 0.034 Pericardium 75.776 91.840 78.016 0.044 4.711 23.871 0.000 Liver, Left 66.513 89.146 77.099 0.207 5.140 0.447 0.429 Omentum 92.789 99.805 42.152 0.022 8.410 1.792 0.000 Liver, Right 74.196 90.749 76.298 0.012 4.274 0.884 1.531 Pancreas 60.214 75.503 66.476 0.117 5.765 0.749 0.737 MBC006 Primary 31.838 0.000 0.845 0.041 6.519 0.804 0.053 Liver, Right 1 25.977 0.189 2.135 0.058 10.062 27.669 1.953 Liver, Right 2 49.603 0.003 3.424 0.017 8.819 26.412 2.468 Liver, Left 28.579 0.000 10.492 0.049 9.802 10.370 0.503 Bone 0.032 0.078 0.000 0.098 0.032 0.000 0.000 MBC007 Primary 66.707 0.000 0.000 0.000 7.653 6.196 0.000 Omentum 69.271 0.000 6.283 0.098 6.778 0.000 0.080 Diaphragm 94.964 0.000 11.253 0.011 11.598 0.000 0.000 Liver, #1 30.161 0.000 11.832 0.395 12.710 34.493 3.830 Liver, #4 72.062 0.000 7.199 0.003 12.903 14.270 1.609 Adrenal 89.345 0.000 1.329 0.004 8.167 0.000 0.000 Bone 5.243 0.048 1.748 1.323 0.094 0.014 0.045 MBC008 Primary 64.470 100.000 45.061 48.206 14.680 0.000 0.000 Liver, Left #1 74.425 95.905 78.603 79.092 3.871 0.847 0.141 Liver, Right #1 86.350 99.975 91.810 95.276 8.232 1.812 0.790 Liver, Right # 2 72.102 98.485 70.680 72.958 4.528 2.084 0.002 Liver, Left #2 90.330 99.831 97.075 85.864 9.632 0.018 0.003 MBC009 Primary 53.572 69.797 29.090 58.917 4.894 0.900 0.0210 Adrenal 70.785 84.057 0.017 57.226 0.760 2.378 0.0004 Lung, RML 77.269 85.965 0.033 80.960 0.736 22.904 0.0000 Lymph node, Abd #1 78.182 76.728 0.000 69.305 0.520 25.000 0.0000 Lymph node, Abd #3 69.385 68.310 0.012 69.698 0.214 24.387 0.0000 Lung, RLL 40.298 43.007 0.005 50.759 0.439 25.171 0.0000 Lung, LUL 4.936 4.864 0.081 14.173 1.349 5.956 0.0000 MBC010 Primary 3.051 0.000 83.827 58.212 10.772 75.851 0.0000 Omentum 0.264 0.000 76.811 81.418 0.546 50.592 0.0000 Adrenal 1.110 0.000 60.031 50.048 0.531 27.777 0.0486 Lymph node, Med #1 2.859 13.451 74.165 75.244 0.218 43.717 0.0000 Lymph node, Med #5 1.892 0.000 69.487 76.638 0.639 32.946 0.0000 Lung, RLL 0.103 0.471 43.110 58.325 1.532 24.474 0.0000 Lung, LLL 0.000 0.063 61.754 66.961 5.267 74.767 0.0216 Liver 0.649 0.147 64.533 34.632 0.228 43.530 1.8245 2.

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