Fig. 1: Study schematics.

a Schematics of the study and anatomical map of biospecimen collection for sequential and parallel multiregion analyses of the index TNBC patient. Timeline is provided in days from the diagnosis. The index patient presented here was a 49-year-old woman diagnosed with a stage III TNBC (T2N3, estrogen receptor (ER), progesterone receptor (PR) and HER2 0+ negative, Ki67 60%, grade 3) with a 3.5 cm right breast cancer mass and lymph node involvement, who underwent multiple systemic therapies due to recurrences and metastatic progression over 2033 days of clinical follow-up. She underwent neoadjuvant chemotherapy with anthracycline and taxane achieving a pathological complete response after mastectomy. The patient presented multiple clinical recurrences at the chest wall from day 373, and achieved complete response with cisplatin-based therapy, surgery, and local radiotherapy. A second chest wall recurrence occurred around day 666 with partial response to bevacizumab-based therapy. Immunotherapy employing anti-PD-L1 monoclonal antibody atezolizumab was administered on day 799 after diagnosis, followed by in a rapid disease progression. However, a long-lasting complete response of 22 months was evidenced after a re-challenge of cisplatin and gemcitabine (day-854 to day 1519), after a previous response to the same drug had been 2.2 fold shorter (day 373 to day 666). The anti-PD-L1 administration before the re-challenge with cisplatin, although culminating in a rapid disease progression, could have contributed to the subsequent long-lasting antitumor response to cisplatin, motivating our investigation of immune escape. Subsequently, the patient presented a progression at the chest wall, and received anti-PD-1 (pembrolizumab) and chemotherapy, with stable disease for 4 months. Then, upon chest wall progression, pembrolizumab plus toll-like receptor (TLR) 7 agonist (topical) was administered in the chest wall metastases with a transient local complete response that lasted around 50 days. The patient received other lines of systemic therapy (i.e. palbociclib followed by cyclophosphamide, pegylated liposomal doxorubicin, cisplatin plus gemcitabine, paclitaxel plus bevacizumab, eribulin) (Supplementary Table 1) and expired on day 2033. Sequential chest wall images illustrate the clinical evolution of a TNBC patient over time. Postmortem parallel multiregion metastases were synchronous, affecting the same metastasis or metastases affecting different anatomical sites (separated into 2 or 3 sites when indicated) within the index patient as indicated. aPD-L1 anti-programmed death-ligand 1 monoclonal antibody, aPD-1 anti-programmed cell death protein 1 monoclonal antibody, TNBC triple-negative breast cancer, CR complete response, cf circulating free (DNA), M metastasis, P primary tumor, PR partial response, PD progressive disease, Rec recurrence, SD stable disease, TLR7 Toll-like receptor 7. b Schematics of 11 TNBC patients cohort included in the study as validation cohort and subjected to re-analysis of their WES and bulk RNA-seq data for primary tumors (n = 10) and parallel multiregion metastases (n = 46). AD adrenal, BO bone, BR brain, BT breast [metastasis], CH chest, KI kidney, LN lymph node, LU lung, LI liver, ME meninges, PB primary breast, PE pleura, SP Spine, ST soft tissue, SK skin.