Genetic and immune characteristics of sentinel lymph node metastases and multiple lymph node metastases compared to their matched primary breast tumours

Abstract

Background: Patients with breast cancer presenting with single lymph node metastasis (from a sentinel node) experience prolonged survival compared to patients with multiple lymph node metastases (≥3). However, little information is available on the genetic and immunological characteristics of breast cancer metastases within the regional lymph nodes as they progress from the sentinel lymph node (SLN) downstream to multiple regional lymph nodes (MLNs).

Methods: Genomic profiling was performed using a next-generation sequencing panel covering 520 cancer-related genes in the primary tumour and metastatic lymph nodes of 157 female patients with breast cancer. We included primary tumours, metastatic lymph nodes and adjacent clinically normal lymph nodes (20 patients from the SLN group and 28 patients from the MLNs group) in the whole transcriptome analysis.

Findings: The downstream metastatic lymph nodes (P = 0.029) and the primary breast tumours (P = 0.011) had a higher frequency of PIK3CA mutations compared to the SLN metastasis. We identified a distinct group of 14 mutations from single sentinel node metastasis and a different group of 15 mutations from multiple nodal metastases. Only 4 distinct mutations (PIK3CA, CDK4, NFKBIA and CDKN1B) were conserved in metastases from both lymph node settings. The tumour mutational burden (TMB) was significantly lower in single nodal metastasis compared to the paired primary breast cancer (P = 0.0021), while the decline in TMB did not reach statistical significance in the MLNs group (P = 0.083). In the gene set enrichment analysis, we identified 4 upregulated signatures in both primary tumour and nodal metastases from the MLNs group, including 3 Epithelial-mesenchymal transition(EMT) signatures and 1 angiogenesis signature. Both the CD8/Treg ratio and the CD8/EMT ratio were significantly higher in adjacent normal lymph nodes from patients with a single metastasis in the SLN compared with samples from the MLNs group (P = 0.045 and P = 0.023, respectively). This suggests that the immune defence from the MLNs patients might have a less favourable microenvironment, thus permitting multiple lymph nodes metastasis.

Interpretation: Single lymph node metastases and multiple lymph node metastases have significant differences in their molecular profiles and immune profiles. The findings are associated with more aggressive tumour characteristics and less favourable immune charactoristics in patients with multiple nodal metastases compared to those with a single metastasis in the sentinel node.

Funding: This work was supported by funds from High-level Hospital Construction Project (DFJH201921), the National Natural Science Foundation of China (81902828 and 82002928), the Fundamental Research Funds for the Central Universities (y2syD2192230), and the Medical Scientific Research Foundation of Guangdong Province (B2019039).

Keywords: Breast cancer; Genome; Immune characteristics; Multiple lymph node metastases; Sentinel lymph node metastases.

Fig. 1

Molecular profiles of matched primary breast tumours and lymph node metastases. Oncoprint summarizing the genomic profiles of both sites of metastatic lymph nodes (SLNs and MLNs) and primary breast tumours. (a) In primary breast cancers, the mutation frequency of PIK3CA was higher in the MLNs group (34/72, 47.22%) than in the SLN group (15/61, 24.59%) (Chi-squared test,P =0.011). (b) In the metastatic tumours, the mutational frequency of PIK3CA was also higher in the MLNs group (31/72, 43.06%) than in the SLN group (15/61, 24.59%) (Chi-squared test,P =0.029). Oncoprint summarizing the genomic profiles of paired primary breast tumours and either (c) SLN or (d) MLN specimens. Only somatic alterations with a frequency of 4% or greater (≥4%) in the whole cohort are displayed. Stacked bar plot illustrating the distribution of mutation rates of various genes detected in either the primary breast tumour (breast) or lymph node (LN) specimens among the patients harbouring either (e) SLNs or (f) MLNs.

Fig. 2

Mutations unique to breast cancer lymph node metastases Eighteen genetic mutations in the single metastasis (SLN) group and 19 genetic mutations in multiple metastases (≥ 3) in the MLNs group were not identified in the matched primary breast tumour. Only 4 distinct mutations (PIK3CA, CDK4, NFKBIA and CDKN1B) were identified in metastases from both lymph node locations (in red).

Fig. 3

Distinct molecular features of paired primary breast tumours and either single nodal metastasis (SLN group) or multiple nodal metastases (MLNs group). Heat map summarizing the correlation between novel lymph node-associated mutations and the genetic or clinical features of patients with either (a) SLN or (b) MLNs (P-values were obtained using the Chi-squared test; only significant P-values are shown in the graph). Clinical and molecular features showing statistically significant correlations with novel lymph node-associated mutations are expanded in Fig.s C-E for patients with SLN and F-G for patients with MLNs. (c-e) Bar plot illustrating the significantly more novel lymph node-associated mutations among patients with (c) ERBB2-mutated (Chi-squared test,P=0.045) or (d) HER2+ (Chi-squared test,P=0.029) SLN. (e and g) Box plots illustrating the significantly higher TMB in the lymph nodes of patients with (e) SLN and (g) MLN who carried novel lymph node-associated mutations than those with non-novel mutations. (f) Bar plot illustrating the lower detection of novel lymph node-associated mutations among patients with MLNs who harbour AKT1 mutations (Chi-squared test,P=0.046).

Fig. 4

Genomic features of SLN- and MLN-positive breast cancer (a) TMB for primary tumours and metastatic lymph nodes from the SLN group and MLNs group (Chi-squared test). The TMB was remarkably reduced in metastatic LNs from the (b) SLN group (fold change = -1.20497 and paired Wilcoxon test P = 0.001), but not in the (c) MLNs group (fold change = -0.42248 and paired Wilcoxon test P = 0.05), and was analysed with paired Wilcoxon tests. Met means metastatic lymph nodes.

Fig. 5

Tumour cells in the MLNs group had an enhanced invasive capacity compared with the SLN group Functional enrichment was computed using a gene set enrichment analysis (GSEA). We observed an enrichment of invasive signatures in both (a and c) primary tumours and (b and d) metastatic lymph nodes derived from the MLNs group compared to the SLN group (Wilcoxon test, P < 0.05).

Fig. 6

Antitumour immune response was different in patients with SLN-positive and MLN-positive breast cancer. (a)Quantification of the activities of 28 immune cell subpopulations using the ssGSEA score. In primary tumours, (b) the CD8/Treg ratio and (c) the CD8/EMT ratio were not significantly different between the SLN group and the MLNs group. In metastatic lymph nodes, (d) CD8/Treg ratio showed no significant difference between SLN group and MLNs group, (e) while CD8/EMT ratio was higher in the MLNs group (Wilcoxon test, P < 0.001). Both the (f) CD8/Treg ratio and (g) CD8/EMT ratio were significantly decreased in normal lymph nodes from the MLNs group (Wilcoxon test, P < 0.05).