The possibility to individuate those clones with a predilection to seed lymph nodes versus those with a proclivity to cause distant metastases could have an impact on clinical practice, especially for early- stage CRC patients. Recent population-based studies on metastases in CRC showed that between 40% 9 and 63% 10 of metachronous metastases develop in patients without lymph nodes metastases, demonstrating that distant metastases can develop independently of the presence of lymph nodes metastases 11. These findings are consistent with a model of metastasis where multiple waves of metastatic cells escape the primary tumor over time and seed lymph nodes and other metastatic sites during tumor progression 8. Additionally, they could show that a single lymph node can harbor subclones that arise in different geographic regions of the primary tumor. 8, only recently, found that lymph nodes metastases are polyclonal and differ considerably from one lymph node to another. Zhang and colleagues showed that skip spreading of tumor cells within the lymphatic network occurs frequently 7. To date, only a few studies have investigated the molecular profile and the mutational heterogeneity of lymph nodes metastases. Interestingly, it has been shown by the The Cancer Genome Atlas (TCGA) consortium that exon mutations do not differ between colon cancers that do and those that do not metastasize 1, 6. This model is consistent with previous studies demonstrating that somatic exonic mutations in a colon cancer patient’s liver metastases are not only identical to those found in the patient’s lymph nodes deposits but are also found in samples from the primary tumor site 5. In the vast majority of cases, they could show that independent subclones are responsible for lymphatic and hematogenic spreading. They have modeled a phylogenetic tree of subclones spreading to lymph nodes and/or distant organs using in -depth evaluation of hypermutable DNA regions. However, it is still unclear whether a single metastatic subclone evolves in the primary tumor, subsequently spreading to lymph nodes and distant sites, or whether multiple subclones in the primary tumor become dispersed and independently seed lymph nodes and distant metastases.Ī study published by Naxerova and colleagues takes the issue one important step further 4. This view provides a mechanistic basis for the TNM staging system and is often the rationale for surgical resection of tumor-draining lymph nodes. One hypothesis to explain this association proposes that distant metastases are seeded by lymph nodes metastases. While stage 1 disease the 5-year overall survival (OS) approaches 90%, in the metastatic stage the 5-year OS rate decreases to approximately 10–15% 3. Spreading of cancer cells from primary colorectal tumors to regional lymph nodes and to distant organs is associated with reduced survival. Our findings are hypothesis generating and need to be examined in prospective studies.Ĭolorectal cancer (CRC) is a highly heterogeneous disease caused by multiple genetic and epigenetic triggers 1, 2. Our data support the hypothesis that lymphatic and distant metastases harbor different mutational landscape. Our cohort of 30 paired samples confirmed the molecular heterogeneity between primaries, LNs, and distant metastases. Overall, LNs showed significantly different rates of mutations in APC, KRAS, PI3KCA, KDM6A, and BRIP1 ( p < 0.01) vs primaries, while presenting a distinct molecular profile compared to distant metastases. TMB-high is significantly more common in LNs vs distant metastases and primaries ( P < 0.0001), regardless MSI-H status. LNs showed a higher mean TMB (13 mut/MB) vs distant metastases (9 mut/MB, p < 0.0001). In total, 11,871 samples were examined, comprising primaries ( N = 5862), distant ( N = 5605) and LNs metastases ( N = 404). Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations, and microsatellite instability (MSI) was evaluated by NGS of known MSI loci. Samples were analyzed using next generation sequencing (NGS, MiSeq on 47 genes, NextSeq on 592 genes) and immunohistochemistry. Herein, we characterized the molecular landscape and the differences between LNs, distant metastases and primary colorectal cancers (CRCs). Lymph nodes (LNs) and distant metastases can arise from independent subclones of the primary tumor.