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).

中文翻译：

---

与匹配的原发性乳腺肿瘤相比，前哨淋巴结转移和多发淋巴结转移的遗传和免疫特征

背景

与多处淋巴结转移（≥3）的患者相比，单发淋巴结转移（来自前哨淋巴结）的乳腺癌患者的生存期延长。然而，关于区域淋巴结内乳腺癌转移的遗传和免疫学特征的信息很少，因为它们从下游的前哨淋巴结 (SLN) 进展到多个区域淋巴结 (MLN)。

方法

使用下一代测序面板进行基因组分析，涵盖 157 名女性乳腺癌患者的原发肿瘤和转移淋巴结中的 520 个癌症相关基因。我们在整个转录组分析中包括原发性肿瘤、转移性淋巴结和邻近的临床正常淋巴结（20 名来自 SLN 组的患者和 28 名来自 MLNs 组的患者）。

发现

 与 SLN 转移相比，下游转移淋巴结 ( P  = 0.029) 和原发性乳腺肿瘤 ( P = 0.011) 的 PIK3CA 突变频率更高。我们确定了来自单个前哨淋巴结转移的一组不同的 14 个突变和来自多个淋巴结转移的不同组的 15 个突变。只有 4 个不同的突变（PIK3CA、CDK4、NFKBIA 和 CDKN1B）在来自两种淋巴结设置的转移灶中得到保存。与配对原发性乳腺癌相比，单淋巴结转移的肿瘤突变负荷（TMB）显着降低（P  = 0.0021），而 MLNs 组的 TMB 下降未达到统计学意义（P = 0.083)。在基因集富集分析中，我们在 MLNs 组的原发性肿瘤和淋巴结转移中鉴定了 4 个上调特征，包括 3 个上皮-间质转化 (EMT) 特征和 1 个血管生成特征。与 MLNs 组的样本相比，SLN 单转移患者的相邻正常淋巴结的 CD8/Treg 比率和 CD8/EMT 比率均显着升高（分别为P  = 0.045 和P  = 0.023）。这表明来自 MLNs 患者的免疫防御可能具有不太有利的微环境，从而允许多个淋巴结转移。

解释

单淋巴结转移和多淋巴结转移在其分子谱和免疫谱上有显着差异。与前哨淋巴结单个转移的患者相比，这些发现与多发淋巴结转移患者的更具侵袭性的肿瘤特征和不太有利的免疫特征相关。

资金

该工作得到高水平医院建设项目（DFJH201921）、国家自然科学基金（81902828和82002928）、中央高校基本科研业务费专项资金（y2syD2192230）和广东省医学科研基金的资助省（B2019039）。