Origins of lymphatic and distant metastases in human colorectal cancer

Abstract

The spread of cancer cells from primary tumors to regional lymph nodes is often associated with reduced survival. One prevailing model to explain this association posits that fatal, distant metastases are seeded by lymph node metastases. This view provides a mechanistic basis for the TNM staging system and is the rationale for surgical resection of tumor-draining lymph nodes. Here we examine the evolutionary relationship between primary tumor, lymph node, and distant metastases in human colorectal cancer. Studying 213 archival biopsy samples from 17 patients, we used somatic variants in hypermutable DNA regions to reconstruct high-confidence phylogenetic trees. We found that in 65% of cases, lymphatic and distant metastases arose from independent subclones in the primary tumor, whereas in 35% of cases they shared common subclonal origin. Therefore, two different lineage relationships between lymphatic and distant metastases exist in colorectal cancer.

Fig. 1. Tracing tumor evolution through indels in hypermutable DNA.

(A) Study design schematic. DNA samples from primary tumor (P), distant metastases (M), lymph node metastases (L) and normal tissue/germ line (N/GL) from 19 colorectal cancer patients were genotyped across 20-43 hypermutable polyguanine repeats. The genetic divergence between two samples is the average distance across all markers. Pairwise distances between all samples from a patient were used as input for phylogenetic reconstruction with the neighbor-joining algorithm. (B) Anatomical sketch and raw data example for a cancer (C38) with microsatellite instability. Mutations can be present in varying percentages of cells within a sample. Therefore, the distance of a tumor sample to the normal reference is a continuous value. The heatmap shows tumor-normal distances across all samples and markers. Dark green, deletions. Purple, insertions. Note that the heatmap only shows a small part of the full data set for a patient. Pairwise distances between all samples are used for phylogenetic reconstruction. (C) Anatomical sketch and raw data example for a microsatellite stable cancer (C58). Heatmaps for all patients are provided in Fig. S13.

Fig. 2. Common versus distinct origins of lymph node and distant metastases.

(A) Clonal expansions of single intestinal stem cells (ISCs) from children (age<15, n=12) have fewer polyguanine indels than clonal expansions from a 66-year old adult (n=6). Data are mean +/- standard error of the mean, two-tailed t-test. (B) Clonal mutation frequency in cancers (defined as JSD => 0.11 in 95% of tumor biopsies) is correlated with patient age at diagnosis. Normal ISCs on average have fewer polyguanine indels than age-matched cancers, but the two distributions overlap. Lines indicate the mean. (C) Most lymph node metastases are more closely related to the primary tumor than to distant metastases. The plot shows d(L-M)/d(L-P)-1, the distance of each lymph node metastasis (L) to its closest distant metastasis (M), divided by its distance to its closest primary tumor sample (P), minus one. Yellow, closest neighbor is a metastasis. Dark blue, closest neighbor is a primary tumor sample. (D) Analogous plot for distant metastases, showing d(M-L)/d(M-P)-1 (E) Classification of patients into cases with common or distinct origins of lymphatic and distant metastases. (F) Bootstrap values reflecting origin classification confidence for each patient. (Bootstrap n=1000).

Fig. 3. Phylogenetic trees of cancers with common origin of lymphatic and distant metastases.

All trees except C38 (MSI case) are drawn to scale and were constructed with the neighbor-joining method. Seeding events (internal node/common ancestor and branches) that gave rise to distant metastases are shaded in red; events that gave rise to lymph node metastases are shaded in blue. Clinical information boxes show whether a patient received neoadjuvant therapy, whether primary tumor resection was complete (all margins unaffected), whether distant metastases occurred synchronously or metachronously, what percentage of suitable lymph nodes (i.e. those that were large and pure enough) was sampled, and the origin classification bootstrap value. Timelines summarize treatment and known lifespan for each patient. VI, venous invasion. Sat, satellite nodule. Lower case letters after sample numbers (a,b,c) indicate multiple biopsies from the same tumor mass. SOC, standard of care.

Fig. 4. Phylogenetic trees of cancers with distinct origins of lymphatic and distant metastases.

All trees except C12 (MSI case) are drawn to scale and were constructed with the neighbor-joining method. Shading and clinical information as in Figure 3.