The impact of molecular profile on the lymphatic spread pattern in stage III colon cancer

Abstract The anatomical spread of lymph node (LN) metastasis is of practical importance in the surgical management of colon cancer (CC). We examined the effect of KRAS, BRAF, and microsatellite instability (MSI) on LN count and anatomical spread pattern in stage III CC. We determined KRAS, BRAF, and MSI status from stage III CC patients. Biomarker status was correlated with LN count and anatomical spread pattern, which was classified as sequential or skipped. Relapse‐free survival (RFS) was estimated using Kaplan‐Meier method, and correlations were assessed using log‐rank and Cox regression analyses. We analyzed 369 stage III CC patients. The proportion of KRAS mutant (mt), BRAF mt, and MSI‐high (H) were 44.2% (163/344), 6.8% (25/344), and 6.8% (25/344), respectively. The mean number of metastatic LN was higher in microsatellite‐stable (MSS) compared with MSI patients (3.5 vs. 2.7, P = .0406), although no differences were observed in accordance with KRAS or BRAF status. Interestingly, patients with BRAF mt and MSI‐H were less likely to harbor skipped metastatic LN (9.3% vs 20% and 4% vs 10.5% compared with BRAF wild‐type (wt) and MSS, respectively), but KRAS status did not predict anatomical spread pattern. Patients with KRAS wt and MSI‐H showed superior RFS compared with KRAS mt and MSS patients, respectively, whereas BRAF status did not affect RFS. Differences exist in the anatomical pattern of invaded LN in accordance with the molecular status of stage III CC. Patients with MSI‐H CC have less invaded and skipped LN, suggesting that a tailored surgical approach is possible.


| INTRODUC TI ON
Colorectal cancer (CRC) is the third most common cancer worldwide, and accounts for approximately 1.7 million cases and 700 000 deaths per year. 1 Over the past few years, the treatment of metastatic CRC has been personalized based on molecular and biological factors. In patients with RAS wild-type (wt) CRC, anti-epidermal growth factor receptor (anti-EGFR) therapy has been used 2,3 and immune checkpoint inhibitors such as anti-PD-1 and anti-PDL-1 antibodies are effective in microsatellite instable (MSI-high) patients. 4 Recently, the efficacy of encorafenib, binimetinib, and cetuximab for BRAF mutant (mt) CRC was confirmed in a phase III trial. 5 However, current surgical treatment of stage III CRC does not incorporate the status of these molecular biomarkers.
Colon cancer spreads along hematogenous and lymphatic pathways, but the exact clinical relevance and anatomical pathways of lymphatic spread in CC are incompletely elucidated. [6][7][8][9] We previously reported that the anatomical pattern of lymphatic spread, and its prognostic implications, differed between right and left colon cancer. 8,9 Whether molecular biomarkers such as KRAS, BRAF and MSI status correlate with the extent and anatomical pattern of LN metastasis in stage III CC is currently unknown. 10,11 Insight into these relationships may allow tailoring of the surgical approach, and the extent of lymphadenectomy, based on the molecular status of the tumor.
Here, we investigated whether KRAS, BRAF, and MSI status were related to the extent of nodal involvement, the anatomical pattern of lymphatic spread, and RFS in a cohort of stage III CC patients who underwent extensive lymphadenectomy.

| Surgical treatment/chemotherapy
Open or laparoscopic colonic resection with Japanese D3 LN dissection was performed in all patients. For right-sided cancers, central vascular ligation was performed, removing draining LNs (stations 203, 213, and 223) along the superior mesenteric vein.
For left-sided cancer, either high ligation of the inferior mesenteric artery was performed, with removal of LNs at station 253, or the left colic artery was preserved, and the superior rectal artery divided at its origin. This technique is theoretically similar to CME with CVL. 13,14 Adjuvant therapy was administered based on local practice.

| Definition of LN spreading pattern and L level
According to the Japanese Society for Cancer of the Colon and Rectum (JSCCR), invaded LNs were classified into 3 groups, as carried out in our previous studies: 8  were used for evaluation as previously described. 15 We also analyzed the mutations of KRAS codon 12, KRAS codon 13, and BRAF codon 600 using Sanger sequencing. All analyses were performed at the Hyogo College of Medicine and Nippon Medical School Hospital.

| Statistical analysis
The chi-square or Fisher exact test was used to evaluate differences between proportions, and Student t test or Mann-Whitney U test was used to evaluate differences between means, as appropriate. RFS were estimated from the date of surgery until recurrence or death from any cause. The Kaplan-Meier method and log-rank test were used for survival analysis. Multivariate Cox proportional hazards regression was used to identify independent prognostic variables. The presence of pathological lymphatic invasion and venous invasion was classified in accordance with the JSCCR system: 0; no invasion, 1; mild invasion, 2; moderate invasion, and 3; severe invasion. 16 Pathological lymphatic and venous invasions were analyzed as categorical covariates. All analyses were performed with IBM® SPSS® Statistics, version 25. P-values were reported and interpreted in accordance with recent guidelines from the American Statistical Association. 17

| RE SULTS
In total, data from 369 stage III CC patients were collected and analyzed. The median follow-up time was 53 mo. Patient characteristics are shown in Table 1. The proportion of KRAS mt, BRAF mt, and MSI-H patients was 44.2% (163/206), 6.8% (25/344), and 6.8% The anatomical pattern of LN metastasis is shown in Table 2.
Mutational status of KRAS did not affect the proportion of patients with skipped LN metastases (9.8% in KRAS mt and 10.2% skipped in KRAS wt). However, the proportion of BRAF mt and MSI-H patients with a skipped lymphatic spread pattern tended to be lower compared with that of BRAF wt and MSS, (9.3% vs 20%; P = .0901 and 4% vs 10.5%, P = .259, respectively).
Survival based on biomarker status is shown in Figure 2. KRAS wt patients showed better RFS (  was observed between the sequential and skipped patterns in KRAS wt patients ( Figure 3B; P = .82). Conversely, RFS of KRAS mt patients with a sequential pattern was better compared with those with a skipped pattern ( Figure 3C; P = .018). Univariate analysis of RFS based on the maximal level of LN invasion showed a significant difference in RFS between L1, L2, and L3 ( Figure 4A; P < .001). This difference remained unchanged after correction for KRAS status in a Cox model ( Figure 4B; Table 3).

| D ISCUSS I ON
Although the anatomical pathways of lymphatic spread in CC have been discussed for several decades, the exact mechanisms are not fully understood. Historically, 2 theoretical models have been advocated (Figure 1) 20 -22 We have previously reported the patterns of LN spread in stage III CC, but molecular data were not incorporated. 8,20 It is likely that the temporal and anatomical patterns of lymphatic spread are governed by the molecular and genetic properties of the primary cancer. This is the first analysis of the association between the anatomical pattern of LN spread and selected molecular biomarkers in CC.
The results of the present study confirmed that the pattern of LN metastasis in stage III CC is related to biomolecular properties.
First, we found striking differences in the proportion of patients with a skipped metastatic pattern in accordance with MSI and BRAF status. In the entire cohort, the proportion of patients with a skipped metastatic LN pattern was comparable with previous studies, ranging between 0% and 18%. 8,9,23 Patients with a deficient mismatch repair had a much lower proportion of skipped LN stations (4% vs 10.5%), although this difference was not statistically significant due to the relatively small number of MSI-H patients.
Previous studies have shown that mismatch repair deficient tumors are characterized by extensive infiltration by activated T-cells. [24][25][26][27][28][29][30] Little information is known on the effect of mismatch repair (MMR) status on the microenvironment of locoregional LN. One study found that, compared with MSS, LN from MSI-H tumors tended to have more follicular hyperplasia and very prominent paracortical hyperplasia. 31 As the paracortex is a T cell zone, this phenotype may reflect regional infiltration of T lymphocytes, which may prevent metastatic cells to effectively transfer node station and cause a skipped metastatic LN pattern. Similarly, we found that patients harboring a BRAF mutation were significantly less likely to display a skipped metastatic nodal pattern (9.3% vs 20%). This finding may be related to the fact that a subset of patients with BRAF mutations is known to display a high stromal CD8-positive cell infiltration and to have a prognosis that is similar to wt patients. This is supported by the fact that we did not find a survival difference in accordance with BRAF mutational status, in contrast with other published reports. Therefore, our findings may not be applicable to all patients with a BRAF mutation.
In addition, we found that the total number of invaded LN in

Wim Ceelen is a senior clinical investigator from the Fund for
Scientific Research -Flanders (FWO).

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to disclose.