PMC8056263_01
Histopathological examination of the primary and metastatic tumors. (A) Hematoxylin and eosin (H&E) staining of colonic primary tumor shows glandular differentiation and invasion into the submucosa (x20). (B) High-power view shows the malignant epithelial cells arranging in a glandular or cribriform manner (x400). (C) The lymphovascular invasion of colonic adenocarcinoma is indicated by an arrow (x200). (D) H&E staining shows intrahepatic adenocarcinoma (x100). Immunostaining for CK20 (E), x50 and CDX2 (F), x50 confirmed the tumor originated in colon.
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Table 1  and  Supplementary Figure 2 ). 3 liver metastatic tumors were identified in the left lobe of the liver, whereas the other 6 tumors were in the right lobe. The mean size of the metastatic tumors was 13 +- 2.055mm, ranging from 4mm-22mm ( Supplementary Table 1  and  Supplementary Figure 1B ). Pathological examination showed moderately differentiated adenocarcinoma of the colon ( Figures 1A-C ) and liver metastasis ( Figures 1D-F ). Table 1 ,  Supplementary Figures 1B ,  2 ). The CEA dropped to 105 ug/L, whereas CA199 remained normal ( Supplementary Figure 1A ). To further reduce the size and number of the tumors, enhanced treatment was recommended, and the combined treatment of EGFR inhibitor and chemotherapy was applied due to the fact that the patient harbored no RAS alteration according to the mutation profile of the patient. March 2020, his serum CEA dropped to 38.96 ug/L ( Supplementary Figure 1A ), and the CA199 remained normal. MRI scan on March 7th 2020 showed that 5 out of 18 liver metastatic tumors were reduced by 3 +- 0.55 mm ( Figure 2B ,  Supplementary T12, T13, T15-18 were identified as the metastases from the intestinal adenocarcinoma based on the immunohistochemistry staining results of CK20 and CDX2 ( Figures 1E, F ). T 11 and T14 After the treatment, 10 out of 18 tumors were eliminated, the rest 8 tumors were significantly reduced in size (10.38 +- 1.133 mm versus 6 +- 0.378 mm, p=0.008 ( Figure 2D ),  Supplementary Figure 1B ).
MRI scans illustrating the changes in liver metastatic tumors over time. (A) MRI scans on the 30th of December 2019 shows multiple liver metastases (red circles). (B) MRI scans on the 7th of March 2020 shows the size of some liver metastases was reduced (blue circles) following the combination treatment. (C) MRI scans on the 16th of April 2020 shows multiple liver metastases (red circles). (D) MRI scans on the 28th of June 2020 shows the sizes and numbers of liver metastasis were significantly reduced following the combined treatment.
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Table 1  and  Supplementary Figure 2 ). 3 liver metastatic tumors were identified in the left lobe of the liver, whereas the other 6 tumors were in the right lobe. The mean size of the metastatic tumors was 13 +- 2.055mm, ranging from 4mm-22mm ( Supplementary Table 1  and  Supplementary Figure 1B ). After chemotherapy, the sizes of 7/9 tumors were reduced, the average reduction was 2.778 +- 0.7027 mm ( Figure 2A ). Table 1 ,  Supplementary Figures 1B ,  2 ). The changes in tumor sizes and numbers after different treatments are shown in  Figures 2A-D , and the treatment records are shown in  Figure 3 . Table 1  and  Supplementary Figure 2 ), 11 tumors remained unchanged, and two tumors enlarged by 1mm. The average size of the metastatic tumors was 7.278 +- 0.885 mm, ranging from 4mm to 17 mm. The MRI review in April showed 18 metastases in the left lobe of the liver (segments II-VI), which possibly were recurrent tumors ( Figure 2C ). After the treatment, 10 out of 18 tumors were eliminated, the rest 8 tumors were significantly reduced in size (10.38 +- 1.133 mm versus 6 +- 0.378 mm, p=0.008 ( Figure 2D ),  Supplementary Figure 1B ).
Schematic diagram showing treatment record of the patient.
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The changes in tumor sizes and numbers after different treatments are shown in  Figures 2A-D , and the treatment records are shown in  Figure 3 . Table 3  and  Supplementary Figure 3 ).
The genetic heterogeneity of liver metastatic tumors. (A) A phylogenic tree showing the genomic similarity of the liver metastatic tumors and the primary intestinal adenoma. (B) Heatmap showing the frequencies and types of mutations of all mutated genes detected by targeted sequencing. T1-T15 were hepatic tumors and PB was the peripheral blood sample obtained in July 2020.
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T1 developed two more mutation sites FOXP2 and IKF1, which was followed by T2, T4, T5, T7, T12, and T14 who further developed 4 mutations. These tumors were located at the same branch of the phylogenic tree due to the fact that they exhibited the identical mutation profile, suggesting that they may be monoclonal and perhaps emerged at the same stage ( Figure 4B ). T10, T11 and T15 were evolutionally very close to T2, T4, T5, T7, T12, and T14, they further developed one or two mutations respectively.