Abstract PD8-02: Phylogenetic reconstruction of advanced breast cancer reveals two different routes of metastatic dissemination associated with distinct clinical outcome
Authors: Venet David , Brown David , Fimereli Danai , Boeckx Bram , Maetens Marion , Majjaj Samira , Rouas Ghizlane , Capra Maria , Bonizzi Giuseppina , Contaldo Federica , Galant Christine , Piccart Martine , Pruneri Giancarlo , Larsimont Denis , Lambrechts Diether , Rothé Françoise , Desmedt Christine , Sotiriou Christos 
Published: March 2020

Abstract
Background: Local and regional lymph node metastases are associated with worse prognosis in a variety of cancers, including breast cancer. However, to date it still remains debated whether axillary lymph node metastases can seed distant metastases or whether they merely represent a surrogate marker for tumor aggressiveness. Here, using multiple matched samples from primary tumor, axillary lymph nodes and distant metastases from breast cancer patients, we explored to which extent lymph nodes are transit points for further dissemination events to distant organs and assessed their role in disease progression using copy number phylogenetic reconstruction.
Samples and Methods: We retrospectively identified 30 estrogen receptor-positive lymph node-positive metastatic breast cancer patients with 238 formalin-fixed paraffin-embedded tissue samples available together with well-annotated clinico-pathological and outcome data. Eligible patients had at least one primary tumor, one axillary lymph node metastasis, one distant metastatic sample as well as histologically normal tissue sample as germline reference. Low coverage whole genome sequencing was performed to infer somatic copy number aberrations. After rigorous filtering of the raw data, copy number phylogenetic reconstruction was performed with Integer Linear Program for the Copy-Number Tree Problem algorithm. Bootstrapping of the samples and of the segmented copy number data was performed to assess the robustness of the phylogenetic tree classification.
Results: Phylogenetic tree analyses revealed that two different routes of metastatic dissemination occur in breast cancer. Unexpectedly, in only 24% of the patients, lymph nodes and distant metastases shared a common origin implying that dissemination occurred via the axillary lymphatic system whereas in the majority of the cases (76%) lymph node and distant metastases showed a distinct origin suggesting a direct seeding from the primary tumor to a distant organ. Of note, histological grade was the only clinic-pathological feature significantly associated with the route of dissemination, low grade tumors being associated with the common origin dissemination route (p=0.04). Interestingly, the patients sharing a common origin dissemination route were found to have significantly worse prognosis than the remaining patients (p=5.3x10−4), despite being low grade. Bootstrapping analyses showed identical topology as the original phylogeny in about 80% of the cases demonstrating the robustness of our method.
Conclusions: In this study, we showed for the first time that the predominant route of metastatic dissemination operating in estrogen receptor-positive breast cancer is the direct seeding of tumor cells from the primary tumor to distant organ, independently of lymph node metastasis. These findings shed light on the routes on which tumor cells metastasize and could potentially improve the clinical management of lymph node-positive breast cancer patients.
Citation Format: David Venet, David Brown, Danai Fimereli, Bram Boeckx, Marion Maetens, Samira Majjaj, Ghizlane Rouas, Maria Capra, Giuseppina Bonizzi, Federica Contaldo, Christine Galant, Martine Piccart, Giancarlo Pruneri, Denis Larsimont, Diether Lambrechts, Françoise Rothé, Christine Desmedt, Christos Sotiriou. Phylogenetic reconstruction of advanced breast cancer reveals two different routes of metastatic dissemination associated with distinct clinical outcome [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD8-02.