Abstract Background. In patients with metastatic breast cancer (BC), tumor cells can be observed in blood as well as in other fluids such as pleural fluid (PF), ascites (ASC), pericardial fluid (PERI) and cerebrospinal fluid (CSF). Here, we aimed at evaluating the association between presence of tumor cells in non-blood liquid biopsies (LB) and presence of metastases in surrounding tissues (aim 1), and assessing the heterogeneity of BC biomarkers (estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and KI67) in these non-blood LB (aim 2) from patients with metastatic BC included in our post-mortem tissue donation program, UPTIDER (NCT04531696, PMID: 38658604). Methods. 38 patients were included in aim 1. When possible, non-blood LB samples were collected during autopsy and instantly centrifuged. Cell pellets were formalin-fixed, paraffin-embedded, and evaluated histologically for tumor cellularity. Associations between the presence of tumor cells in LB and solid metastases in surrounding tissues were explored with a Fisher’s exact test. All LB with ≥5% tumor cellularity and primary tumor samples were considered for aim 2 and immunohistochemically stained for ER, PR, HER2 and KI67. Results. Aim 1. 155 samples (52 PF, 30 ASC, 34 PERI and 39 CSF) were analyzed with a median of 4 LB per patient (range 2-7). Malignant PF was observed in 29 out of 31 patients with collected PF. All except one patient (28/29) had pleural, lung and/or mediastinal metastases. Malignant ASC was observed in 18 from 27 patients with collected ASC. 17/18 had peritoneal, liver and/or gastro-intestinal metastases. Malignant CSF was detected in 6 out of 36 patients with collected CSF. Four (4/6) had brain and/or meningeal metastases. Malignant PERI was observed in 19 out of 34 patients with collected PERI, of whom 5/19 had pericardial or heart metastases. No statistical evidence was found for association of presence of tumor cells in PF, ASC or CSF and solid metastases in surrounding tissues (Fisher’s exact p-value (p) = 0.13, 1.00 and 0.37, respectively). However, a trend was seen for PERI and pericardial/heart involvement (p = 0.053). Aim 2 was evaluated on 69 samples from 27 patients. Among them, 20 presented with primary hormone receptor (HR)+/HER2- BC (18 ER+/PR+, 1 ER+/PR- and 1 ER-/PR+), and 7 patients had primary HR-/HER2- BC. Regarding intra-patient heterogeneity across different LB, the median variation in expression was 5% for ER, 0.5% for PR, 0% for HER2, and 13% for KI67 with maximum differences of 90%, 50%, 60% and 70% respectively. Of note, 10/19 (53%) patients diagnosed with primary ER+ BC, had at least 1 LB that lost ER-expression. Similarly, 14/19 (74%) patients with primary PR+ BC, lost PR-expression in at least 1 LB. All patients had HER2 non amplified primaries, but 11/27 (41%) had at least 1 LB with HER2-low and/or HER2-ultralow expression, while the other 16 patients (59%) showed no detectable HER2 expression in any of their LB. KI67 value from the primary was available for 17 patients. Twelve out of 17 patients presented with low KI67 (≤15%) at primary diagnosis of whom 5 had at least one LB with high KI67 (15%). The remaining 5 patients presenting with high KI67 at primary diagnosis all had at least one LB with low KI67. Conclusions. This study reports on the detection of tumor cells and expression of clinically relevant biomarkers in LB from patients with metastatic BC. Our findings suggest that a multi-fluid biopsy approach, when possible, may improve the characterization of the metastatic disease heterogeneity and aid in selecting the best next-line treatment options. Citation Format: C. Carette, G. Zels, A. Pabba, M. Maetens, K. Borremans, K. Van Baelen, J. Van Cauwenberge, T. Geukens, M. De Schepper, H. Nguyen, M. Nysen, H. Hoogsteyn, T. Van Assche, A. Mahdami, S. Leduc, P. Vermeulen, P. Neven, H. Wildiers, W. Van Den Bogaert, G. Floris, F. Richard, S. Hatse, C. Desmedt. Immunohistochemical characterization of the tumor cells present in four body fluids from patients with metastatic breast cancer in the context of the UPTIDER program abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-05-15.
Carette et al. (Tue,) studied this question.