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Gastroesophageal junction (GEJ) cancer is a tumor that occurs at the junction of stomach and esophagus anatomically. GEJ cancer frequently metastasizes to lymph nodes, however the heterogeneity and clonal evolution process are unclear. This study is the first of this kind to use single cell DNA sequencing to determine genomic variations and clonal evolution related to lymph node metastasis. Multiple Annealing and Looping Based Amplification Cycles (MALBAC) and bulk exome sequencing were performed to detect single cell copy number variations (CNVs) and single nucleotide variations (SNVs) respectively. Four GEJ cancer patients were enrolled with two (Pt.3, Pt.4) having metastatic lymph nodes. The most common mutation we found happened in the TTN gene, which was reported to be related with the tumor mutation burden in cancers. Significant intra-patient heterogeneity in SNVs and CNVs were found. We identified the SNV subclonal architecture in each tumor. To study the heterogeneity of CNVs, the single cells were sequenced. The number of subclones in the primary tumor was larger than that in lymph nodes, indicating the heterogeneity of primary site was higher. We observed two patterns of multi-station lymph node metastasis: one was skip metastasis and the other was to follow the lymphatic drainage. Taken together, our single cell genomic analysis has revealed the heterogeneity and clonal evolution in GEJ cancer.

Genomic Heterogeneity and Clonal Evolution in Gastroesophageal Junction Cancer Revealed by Single Cell DNA Sequencing