PMC9732937_01
Representative histopathological results of small intestine metastasis. Small intestine metastasis was found to be diffuse infiltrating the submucosal muscularis propria and serosa (A) (HE staining, x200, scale bars: 200 microm). Immunohistochemical staining revealed positive expression of ER (B) and PR (C). Besides, p120 catenin was cytoplasmically positive (D). CK (E), CK7 (F), Mammaglobin (G) and GATA3 (H) were all found to be strongly positive, while PAX8 (I), S100 (J), CK20 (K) and Villin (L) were weakly expressed or negative. (immunohistochemical staining, x200, scale bars: 200 microm).
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Abdominal computed tomography (CT) revealed a thickened partial wall of the small intestine in the left lower abdomen, above which there was significant intestinal obstruction ( Figure S1 ). Tumor tissues were found to diffuse infiltrate into the submucosal muscularis propria and serosa, but no specific nerve or vascular infiltration nor residual tumor tissues were found on the upper or lower resection margins of the specimen ( Figure 1A ). The cells were strongly positive for estrogen receptor (ER) ( Figure 1B ) and progesterone receptor (PR) ( Figure 1C ). Besides, p120 catenin was cytoplasmically positive ( Figure 1D ). Cytokeratin (CK), CK7, mammaglobin, and GATA3 were found to be strongly positive ( Figures 1E-H ). PAX8 was weakly expressed ( Figure 1I ). No expression was observed for S100, CK20, or villin ( Figures 1J-L ).
Primary lesion and invasive metastasis in the breast. PET/CT (A-D) revealed multiple masses with increased glucose metabolism in the lateral quadrant of the left breast as well as nodules with slightly increased glucose metabolism in the lateral quadrant of the right breast, which indicated aggressive invasion of the primary tumor on the left. MRI (E, F) in axial sections of the patient's breast before chemotherapy showed the primary lesion in the left breast and multiple contralateral breast metastases.
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Further examination of positron emission tomography (PET)/CT ( Figures 2A-D ) revealed multiple masses with increased glucose metabolism in the lateral quadrant of the left breast, as well as nodules with slightly increased glucose metabolism in the lateral quadrant of the right breast, indicating left breast cancer and possible malignancy in the right breast. Magnetic resonance examination ( Figures 2E, F ) of the patient showed asymmetrical bilateral breasts, with enlargement of the left breast and a lobulated mass approximately 3.6x2.5 cm in size, with high diffusion-weighted imaging (DWI) signal and perfusion curves in washout type. Panorama with somatic copy number variations exhibited common copy number variations, with minor differences in the genome between the two tumor lesions ( Figure S2 ).
Representative histopathological results of the primary lesion in left breast. Needle biopsy revealed classic invasive lobular breast carcinoma (A). (A, D) show tumor of the resected left breast before (A) and after (D) chemotherapy. (HE staining, x200, scale bars: 200 microm) Immunohistochemical staining revealed strongly positive expression of ER (B) and PR (C) in the primary lesion, while medially or weakly expressed after chemotherapy (ER E, PR F). (immunohistochemical staining, x200, scale bars: 200 microm).
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I and no carcinoma in situ ( Figure 3A ). Immunohistochemistry (IHC) results revealed that the breast cancer cells expressed human epidermal growth factor receptor 2 (HER2) (2+), and a lack of HER2 amplification was confirmed by fluorescence in situ hybridization. The tumor cells strongly expressed ER (90%, strongly positive) ( Figure 3B ) and PR (90%, strongly positive) ( Figure 3C ), with Ki67 expression in approximately 15% of the tumor cells. Postoperative pathology suggested a 2.8x2.5x2-cm tumor bed in the lateral left papilla ( Figure 3D ). The cancer cells expressed HER2 (1+), ER (80%, medium to strong positivity) ( Figure 3E ), PR (60%, medium positivity) ( Figure 3F ), and Ki67 in the hotspots of approximately 5% of the cells. Most genes in the metastatic lesions were enriched in the regulation of circadian rhythm ( Figure S3A ), whereas in the primary lesions, most differentially expressed genes were enriched in protein localization to the M-band ( Figure S3B ). In addition, metastatic lesions of the small intestine showed significant enrichment in cancer pathways ( Figure S3C ), whereas osteoclast differentiation was the most enriched in the primary breast lesion ( Figure S3D ).
Analysis of subclones indicating evolutionary relations of clusters. All detected mutations of the two samples were clustered into 6 clones. The variant allele fraction (VAF) distribution of each detected mutation in different samples is shown in (A), and changes in the average VAF of detected mutations between subclones of the two samples are exhibited in the charts of (B). (C) indicates the evolutionary relations of clusters.
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Cluster number 4, which contains genes such as ARID1A, BCL9, BRWD1, CCDC116, CPD, CSNK1E, CYP1A1, and ERC1, was found to be the ancestral subclone when clonal evolutionary relationships were analyzed ( Figure 4 ).