Background The adipose tissue may serve as a source of energy supporting cancer growth and metastasis. Our understanding of the adipocytes which compose the adipose tissue in different anatomical locations of the body as well as potential microscopic tumor infiltration in patients with metastatic breast cancer remains limited. This study therefore investigates regional variations in adipocyte size and adipose tissue tumor infiltration in patients with metastatic breast cancer. Methods Within the UPTIDER rapid autopsy program, (NCT04531696), 94 adipose tissue samples from subcutaneous, visceral, retroperitoneal, and mammary depots of 22 patients with metastatic breast cancer were collected and analyzed. Distant adipocyte size was quantified using digital pathology, and tumor infiltration was assessed histologically. Linear mixed quantile regression analyzed the associations between adipocyte size, fat depot type and major histological subtypes. Results Distant adipocyte size did not significantly differ across fat depots. A trend towards smaller adipocytes in mammary fat at autopsy versus diagnosis was observed, suggesting potential age and/or treatment effects. Adipocyte size correlated positively with BMI at death, especially in subcutaneous and visceral fat. Visceral fat exhibited higher tumor infiltration, notably in patients with invasive lobular carcinoma (ILC). Conclusion This study highlights the relatively uniform adipocyte size across fat depots in patients with metastatic breast cancer, with potential changes in mammary adipocytes over the disease course. The microscopic tumor cell infiltration observed in the visceral fat, mainly for ILC, underscores the need to undertake additional research to understanding the contribution of the adipose tissue in breast cancer metastasis.

The immune landscape of hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer (HR+/HER2- mBC), the most common subtype of BC, remains understudied. This is mainly because of reduced sample acquisition opportunities from metastases as compared with primary tumors. In this study, we explored stromal tumor-infiltrating lymphocytes (sTIL) in metastatic samples collected through our post-mortem tissue donation program UZ/ KU Leuven Post-mortem Tissue Donation program to Enhance Research (NCT04531696). sTIL were scored as a continuous parameter according to the international guidelines on 427 metastases and 38 primary untreated tumors acquired from 20 patients with HR+/HER2- mBC. Estrogen receptor (ER) status was evaluated on 362 metastases with a cutoff value for positivity set at 1% according to the American Society of Clinical Oncology/College of American Pathologists guidelines. Our analyses show that 54% and 15% of metastases had sTIL levels of >= 1% and >= 5%, respectively. sTIL levels tended to be lower in metastases as compared with their respective primary tumors (estimate,-2.83; 95% CI,-5.77 to 0.11; P = .07). sTIL levels were lower in metastases from invasive lobular carcinoma than in metastases from invasive breast carcinoma of no special type (estimate,-1.67; 95% CI,-2.35 to-0.98; P < .001). A loss of ER expression was observed in 14% of all metastases, yet a negative ER status was not significantly associated with increased sTIL levels. Finally, sTIL levels were significantly higher in lung and axillary lymph node metastases compared with all metastases. Although these analyses were conducted on multiple metastases obtained at the end of life after several lines of treatment, the data provide novel and valuable insights into the state of immune infiltration in patients with HR+/HER2mBC.

Supplementary Figure 7 shows subgroup analyses of the association of pCR with clinicopathological and treatment variables.

Supplementary Figure 4 shows an overview of tumor emboli assessment.

Background: Invasive lobular carcinoma (ILC) accounts for 15% of all invasive breast cancers (BC) and has a peculiar metastatic spread in comparison to BC of no-special type. Since ILC is less likely to disrupt normal tissue architecture and is usually non-mass forming, imaging of ILC lesions entails many challenges. Insights into pathologic versus radiological metastatic invasion can lead to better diagnostic and monitoring tools for patients with ILC. Here, we compare microscopical findings during autopsy with clinical findings prior to death in patients with metastatic ILC included in our post-mortem tissue donation program UPTIDER (NCT04531696). Methods: UPTIDER was started in November 2020 in University Hospitals Leuven/KU Leuven with inclusion of patients with stage IV BC who were willing to participate. One of the predefined subgroups consisted of patients with primary pure (i.e. not mixed) ILC. Samples were taken from different macroscopically invaded and non-invaded sites. Clinical data on disease progression, imaging, biochemistry and treatment regimens were extracted from patient files. The number of samples that were preregistered as pathological served as a surrogate for lesions that were seen on imaging performed during the treatment of the patient. These samples are compared to microscopical findings of the autopsy. Results: Since the start of UPTIDER, an autopsy has been performed on 6 patients with pure ILC (26.1% of all autopsies). Median age at initial diagnosis was 52 years (range: 37 – 80 years). Three patients (50.0%) had stage IV disease at diagnosis, the others relapsed on average 162.7 months (range: 55 – 358 months) after initial diagnosis. The average time between clinical occurrence of metastases and death was 44.8 months (range: 15 – 83 months). Median number of treatment lines for stage IV disease was 5 (median endocrine lines 2; median chemotherapy regimens 3.5). To follow disease evolution, computed tomography of thorax and abdomen was used for 4 (66.7%) patients and whole-body diffusion-weighted magnetic resonance imaging (WB-DWI/MRI) for the remaining 2 (33.3%). Median time between last premortem imaging and death was 5.9 weeks (range: 1.6 – 16.6 weeks). At autopsy, a median of 26 unique metastases (range: 12-36) were sampled per patient. Table 1 gives an overview on the unique microscopically invaded metastases that were sampled per patient. Only 47.3% of the sampled unique metastases was preregistered. Of all unique metastases, 26.7% appeared macroscopically normal during autopsy. Tissues that appeared normal but turned out to be microscopically infiltrated included liver, stomach, adrenal glands, heart, pericardium, visceral and subcutaneous fat tissue. There were 2 patients with normal appearing, microscopically infiltrated livers. In these patients, elevation of γGT and transanimases was seen in the months prior to death. Conclusions: The disease burden of metastatic ILC reported on premortem imaging does not reflect the microscopical findings at autopsy. One of the priorities of metastatic ILC research should be the development of diagnostic tools to better estimate the extent of the disease. Future analyses of the performed postmortem MRIs in our study can aid in improving the interpretation of WB-DWI/MRI for patients with ILC. For now, clinicians should consider that unexplained clinical and/or biochemical findings might indicate progression of ILC. Table 1: patient overview on performed imaging and unique metastases sampled during autopsy CT = computed tomography; ER = estrogen receptor; HER2 = human epidermal growth factor receptor 2; pt = patient; WB-DWI MRI = whole-body diffusion-weighted magnetic resonance imaging *exclusion of samples of patient 2012 since no preregistration was performed for this patient due to unexpected death Citation Format: Karen Van Baelen, Gitte Zels, Maxim De Schepper, Marion Maetens, Josephine Van Cauwenberge, Tatjana Geukens, Kristien Borremans, François Richard, Amena Mahdami, Ha-Linh Nguyen, Sophia Leduc, Anirudh Pabba, Ann Smeets, Ines Nevelsteen, Patrick Neven, Hans Wildiers, Vincent Vandecaveye, Wouter Van Den Bogaert, Giuseppe Floris, Christine Desmedt. Underestimation of metastatic spread in patients with lobular breast cancer: results from the post-mortem tissue donation program UPTIDER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-06.

Association of pCR with DFS. A, Kaplan–Meier curves of DFS according to pCR. B, Forest plots showing the association of pCR and standard clinicopathologic and treatment variables with DFS quantified by Cox regression.

Supplementary Figure 9 shows potential non-linear association of RCB score with sTIL.

Abstract Inflammatory breast cancer (IBC) is a rare (1%–5%), aggressive form of breast cancer, accounting for approximately 10% of breast cancer mortality. In the localized setting, standard of care is neoadjuvant chemotherapy (NACT) ± anti-HER2 therapy, followed by surgery. Here we investigated associations between clinicopathologic variables, stromal tumor-infiltrating lymphocytes (sTIL), and pathologic complete response (pCR), and the prognostic value of pCR. We included 494 localized patients with IBC treated with NACT from October 1996 to October 2021 in eight European hospitals. Standard clinicopathologic variables were collected and central pathologic review was performed, including sTIL. Associations were assessed using Firth logistic regression models. Cox regressions were used to evaluate the role of pCR and residual cancer burden (RCB) on disease-free survival (DFS), distant recurrence-free survival (DRFS), and overall survival (OS). Distribution according to receptor status was as follows: 26.4% estrogen receptor negative (ER−)/HER2−; 22.0% ER−/HER2+; 37.4% ER+/HER2−, and 14.1% ER+/HER2+. Overall pCR rate was 26.3%, being highest in the HER2+ groups (45.9% for ER−/HER2+ and 42.9% for ER+/HER2+). sTILs were low (median: 5.3%), being highest in the ER−/HER2− group (median: 10%). High tumor grade, ER negativity, HER2 positivity, higher sTILs, and taxane-based NACT were significantly associated with pCR. pCR was associated with improved DFS, DRFS, and OS in multivariable analyses. RCB score in patients not achieving pCR was independently associated with survival. In conclusion, sTILs were low in IBC, but were predictive of pCR. Both pCR and RCB have an independent prognostic role in IBC treated with NACT. Significance: IBC is a rare, but very aggressive type of breast cancer. The prognostic role of pCR after systemic therapy and the predictive value of sTILs for pCR are well established in the general breast cancer population; however, only limited information is available in IBC. We assembled the largest retrospective IBC series so far and demonstrated that sTIL is predictive of pCR. We emphasize that reaching pCR remains of utmost importance in IBC.

Research on metastatic cancer has been hampered by limited sample availability. Here we present the breast cancer post-mortem tissue donation program UPTIDER and show how it enabled sampling of a median of 31 (range: 5-90) metastases and 5-8 liquids per patient from its first 20 patients. In a dedicated experiment, we show the mild impact of increasing time after death on RNA quality, transcriptional profiles and immunohistochemical staining in tumor tissue samples. We show that this impact can be counteracted by organ cooling. We successfully generated ex vivo models from tissue and liquid biopsies from distinct histological subtypes of breast cancer. We anticipate these and future findings of UPTIDER to elucidate mechanisms of disease progression and treatment resistance and to provide tools for the exploration of precision medicine strategies in the metastatic setting.

Supplementary Figure 8 shows descriptive data of pCR rates according to several pathological features.

Abstract Background. Liquid biopsies represent a less invasive alternative to tissue biopsy to characterize the disease in patients with metastatic cancer. Blood remains the most frequently investigated body liquid with regards to detection, quantification and characterization of the circulating tumor DNA (ctDNA). However, it might not capture the full disease profile, and other sources of body liquids may be complementary. The aim of the present study is to compare ctDNA detection in different types of body liquids. Patients and methods. Sixteen patients from the post-mortem tissue donation program UPTIDER (NCT04531696) were included in this study. The receptor status of their primary tumor was: estrogen receptor positive, Human Epidermal Growth Factor Receptor 2 non-amplified (ER+/HER2-) (n=12), ER-/HER2- (n=3) and ER+/HER2+ (n=1). Seven types of liquids were collected: blood, saliva, ascites, pleural fluid (PFL), cerebrospinal fluid (CSF), pericardial fluid and urine. Fluids were collected at study inclusion (blood, saliva, urine, and ascites whenever possible) and at autopsy (except for saliva). In total, 281 liquid samples were collected and processed according to standard protocols. All extracted cfDNA (supernatant), and the 16 matched germline DNA (buffy coat) samples underwent shallow whole genome sequencing. Log2 coverage ratios were computed with CNVkit, and good quality profiles were co-segmented per patient using the copynumber R package. Purity and ploidy were assessed by ABSOLUTE and manually reviewed. Associations between organ involvement and ctDNA yield were assessed by Wilcoxon rank-sum tests. Results. At the patient level, the proportion of liquid types in which ctDNA was detected was highly variable (median: 62%, IQR: 25-89%). ctDNA was detected in ascites of all patients where investigation was possible, in 92% of PFL, 77% of CSF, 69% of blood, 30% of pericardial fluid and in 10% of urine samples. No ctDNA was detected in the saliva samples. At autopsy, ctDNA could not be identified in blood in 4 out of the 16 patients but was detected in at least one of the other fluids for 3 of these patients. ctDNA levels tended to be higher in PFL and CSF in case of pleural and central nervous system (CNS) metastases, respectively. 3 patients had CSF ctDNA detected with no documented involvement of the CNS. In ascites, ctDNA levels were independent of peritoneal invasion. Conclusion. ctDNA was detected in 6 out of the 7 investigated body liquids and its level was partially associated with metastases in surrounding organs, except for ascites. In 3 patients, blood was not contributive but other liquids were, encouraging the evaluation of additional body fluids, when possible, in patients with metastatic breast cancer. These results open new avenues for the clinical monitoring and characterization of the disease. Citation Format: François Richard, Tatjana Geukens, Maxim De Schepper, Amena Mahdami, Karen Van Baelen, Marion Maetens, Ha-Linh Nguyen, Anirudh Pabba, Sophia Leduc, Edoardo Isnaldi, Maysam Mohammadzadeh Hajipirloo, Emily Vanden Berghe, Imane Bachir, Sigrid Hatse, Peter Vermeulen, Evy Vanderheyden, Bram Boeckx, Diether Lambrechts, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Hans Wildiers, Wouter Van Den Bogaert, Jonas Demeulemeester, Elia Biganzoli, Giuseppe Floris, Christine Desmedt. Comparison of ctDNA detection in seven different types of body liquids from patients with metastatic breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A004.

Baseline clinical and pathologic characteristics of patients in the entire cohort and in each of the breast cancer subgroups

Abstract Background: Obesity is a risk factor for postmenopausal hormone receptor-positive (HR+) breast cancer (BC) and is associated with poorer outcomes. “Obesity-paradox”, a phenomenon where obesity (BMI ≥ 30) is associated with better outcomes in patients treated with immune checkpoint inhibitors (ICI), has been observed in several cancer types. It is however under-explored in BC, especially HR+ BC. Emerging data from recent trials (KEYNOTE-756 and CheckMate 7FL) have shown the potential benefits of ICI for treating early HR+ BC. This prompted the need for better insights into the biological link between obesity and HR+ BC, particularly its tumor immune microenvironment (TIME). Patients & Methods: A monocentric cohort of 33 patients (11 lean, 11 overweight, 11 obese) with early HR+ BC was selected for the study, whose tumor tissues were collected and subjected to snRNA-seq. Unsupervised clustering and cluster annotation were performed on the processed data leading to identification of cell types and subtypes. We then performed differential gene expression (DGEA) and gene set enrichment analyses (GSEA) according to BMI category for each of the immune cell types. Results: We identified a total of 76,773 immune cells belonging to six major cell types, namely B/plasma cell, dendritic cell (DC), mast cell, monocyte/macrophage (mono/macro), T cell, and natural killer (NK) cell. Their proportions varied between patients, however immune cellular composition was not associated with BMI. DGEA and GSEA revealed highly cell type-specific obesity-associated differences in the expression profile of immune cells. Notably, an enrichment of B cell-mediated immune activities, substantiated by the upregulation of Ig genes, was observed in B cells and plasma cells. This was however coupled with an overexpression of the checkpoint BTLA in B cells. CD8+ and CD4+ T cells showed a lower level of activation (higher LEF1, TCF7, lower ANXA1, IL4R), and NK cells a decreased cytokine production (CD96, CD226) and conflicting differential expression of cytotoxic molecules (lower NKG7, higher granzymes). Checkpoint genes such as PDCD1, CTLA4, TIGIT, LAG3, HAVCR2 did not show significant changes in their expression in T/NK cells. Mast cells displayed a downregulation of antigen presenting genes (CD74, HLA-A/B/E). DC and mono/macro populations also exhibited both up- and down-regulation of genes involved in different immune pathways. Conclusion: Our current data suggested that obesity was associated with multi-directional changes in the immune activities, possibly implying an unresolved inflammation in the HR+ BC tissue. Further investigation incorporating other cell types and their interaction with immune cells in both tumor and matched normal mammary tissues is warranted and on-going, which could provide more understanding of HR+ breast TIME and lead to speculation on therapeutic implications. Citation Format: Ha-Linh Nguyen, Tatjana Geukens, Maxim De Schepper, Karen Van Baelen, Marion Maetens, Gitte Zels, Amena Mahdami, Edoardo Isnaldi, Sigrid Hatse, Joke Verbeke, Christophe Matthys, Bernard Thienpont, Evy Vanderheyden, Thomas Van Brussel, Rogier Schepers, Gino Philips, Bram Boeckx, Diether Lambrechts, Giuseppe Floris, François Richard, Christine Desmedt. Heterogeneity of the hormone receptor-positive breast tumor immune microenvironment according to patient’s body mass index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1541.

The impact of aging on the immune landscape of luminal breast cancer (Lum-BC) is poorly characterized. Understanding the age-related dynamics of immune editing in Lum-BC is anticipated to improve the therapeutic benefit of immunotherapy in older patients. To this end, here we applied the 'multiple iterative labeling by antibody neo-deposition' (MILAN) technique, a spatially resolved single-cell multiplex immunohistochemistry method. We created tissue microarrays by sampling both the tumor center and invasive front of luminal breast tumors collected from a cohort of treatment-na & iuml;ve patients enrolled in the prospective monocentric IMAGE (IMmune system and AGEing) study. Patients were subdivided into three nonoverlapping age categories (35-45 = 'young', n = 12; 55-65 = 'middle', n = 15; >= 70 = 'old', n = 26). Additionally, depending on localization and amount of cytotoxic T lymphocytes, the tumor immune types 'desert' (n = 22), 'excluded' (n = 19), and 'inflamed' (n = 12) were identified. For the MILAN technique we used 58 markers comprising phenotypic and functional markers allowing in-depth characterization of T and B lymphocytes (T&B-lym). These were compared between age groups and tumor immune types using Wilcoxon's test and Pearson's correlation. Cytometric analysis revealed a decline of the immune cell compartment with aging. T&B-lym were numerically less abundant in tumors from middle-aged and old compared to young patients, regardless of the geographical tumor zone. Likewise, desert-type tumors showed the smallest immune-cell compartment and were not represented in the group of young patients. Analysis of immune checkpoint molecules revealed a heterogeneous geographical pattern of expression, indicating higher numbers of PD-L1 and OX40-positive T&B-lym in young compared to old patients. Despite the numerical decline of immune infiltration, old patients retained higher expression levels of OX40 in T helper cells located near cancer cells, compared to middle-aged and young patients. Aging is associated with important numerical and functional changes of the immune landscape in Lum-BC. (c) 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Association of RCB with clinicopathologic features and treatment in all patients. A–C, Forest plots showing the association of RCB, either as RCB class or continuous RCB score, with standard clinicopathologic and treatment variables (A), with sTILs (continuous %; B), and with sTILs (categorical; C) evaluated by regression analyses in all patients. Analyses were performed on the subset of patients diagnosed and treated at UZ Leuven, Belgium.

Supplementary Table 2 shows clinicopathological characteristics of the entire cohort and the Leuven sub-cohort.

Quantification of PSMA expression via PSMA PET is well-established, however quantification of PSMA via immunohistochemistry (IHC) is not standardized. Our aim was to determine the most optimal PSMA IHC scoring system to quantify PSMA expression with PSMA PET as reference standard. Primary intermediate- and high-risk prostate cancer patients received an [18F]PSMA-1007 PET/MRI followed by radical prostatectomy. SUVmax, SUVmean and Ki of the prostate tumor was determined. Prostate tumors were stained with anti-PSMA antibodies and scored by 2 readers via 10 IHC scoring systems: histochemical score (H-score), immunoreactivity scorepredominant intensity (IRSpredominant intensity), IRS classificationpredominant intensity, IRSmean intensity, IRS classificationmean intensity, Allred score, predominant expression pattern, Shannon diversity index (SDI), percentage negatively stained cells and total percentage positively stained cells. Spearman’s rank correlation coefficients (ρ) were calculated between PET parameters and IHC scoring systems. Interreader agreement for the IHC scoring systems was measured by the intraclass correlation coefficient (ICC). Fifty tumors in 46 patients were analysed. H-score had the best correlation with SUVmax (ρ 0.615 p < 0.0001) and SUVmean (ρ 0.570, p < 0.0001) and the second best correlation with Ki (ρ 0.411, p = 0.0030). SDI had the best correlation with Ki (ρ -0.440, p = 0.0014) and the second best correlation with SUVmax (ρ -0.516, p = 0.0001) and SUVmean (ρ -0.490, p = 0.0003). A moderate interreader agreement was observed for H-score (ICC 0.663, 95

Association of pCR with OS. A, Kaplan–Meier curves of OS according to pCR. B, Forest plots showing the association of pCR and standard clinicopathologic and treatment variables with OS quantified by Cox regression.

Supplementary Figure 14 shows analyses of the association of sTIL with DRFS.

Supplementary Figure 1 shows examples of sTIL scoring in skin on H&E images.