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Background. Postmortem tissue donation programs can importantly enhance sample access for translational research on metastatic disease. However, this post-mortem setting poses logistical and technical challenges in terms of preserving nucleic acid quality and in particular RNA. Here we present the results of an experiment within our breast cancer tissue donation program UPTIDER (NCT04531696), aiming at assessing RNA degradation rates and expression profile changes in function of tissue type and sample-specific postmortem interval (ssPMI). Patients & Methods. For 7 patients, bulk RNA sequencing was performed using the Lexogen protocol on fresh frozen samples from healthy or tumour tissues taken repeatedly (at 1.5h time intervals) during the autopsy. ssPMI was defined as the time between the death of the patient and the freezing of the sample. Quality threshold was set at 0.5 million (M) of assigned reads (AR). Other quality metrics included number of expressed genes, evolution of proliferation-, hypoxia-, stromal- and immune-related transcriptional signatures (PMID:18698033, 20087356) with increasing ssPMI. Associations between quality metrics and ssPMI were assessed by linear regressions for longitudinal data, with quality metrics as dependent variable, time as independent variable and accounting for the clustering of the data by patient and organ using the generalized estimating equation method. Three nested models - with constant, linear and non-linear relationship - were compared using ANOVA testing strategy. Non-linearity was rendered by a restrict cubic spline with three knots. All tests were performed by the Wald test on regression coefficients. Results. Ninety samples (67 healthy, 23 tumour) were analyzed. Median ssPMI was 7.50 hours (range: 3.07-11.12). Most (87%) samples passed quality thresholds, with median AR being 1.70M (interquartile range: [0.70M-3.57M]). No association was found between quality metrics and time in healthy samples. In tumor samples, regarding sequencing quality, negative associations with increasing time were found for AR and for number of expressed genes with an average decay of 242308 reads per hour (95 confidence interval (95CI): [94415.62-390200.50], p-value=.001) and 251 genes per hour (95CI: [17.30-485], p-value=.035), respectively. At the transcriptomic level, potential subtle changes were observed regarding immune (e.g. STAT1 signature: -0.02, 95CI: [-0.05;0.00]) and hypoxia-related signatures (e.g. PGAM1 signature: -0.03, 95CI [-0.05;-0.01]), while no effect of time was seen for the proliferation and stromal-related signatures. Sample size precluded organ specific analyses. Conclusion. A decrease in the number of AR and number of expressed genes with increasing ssPMI was found in tumor samples leading to subtle changes in few transcriptional programs. Healthy samples showed stable quality metrics over time possibly explained by a lower cell activity in healthy as compared to tumour cells. Knowledge derived from this study will be integrated in the upcoming transcriptomic analyses of the samples collected within UPTIDER. Citation Format: François Richard, Tatjana Geukens, Giuseppe Marano, Wouter Van Den Bogaert, Maxim De Schepper, Marion Maetens, Amena Mahdami, Karen Van Baelen, Ha-Linh Nguyen, Anirudh Pabba, Sophia Leduc, Edoardo Isnaldi, Maysam Hajipirloo, Imane Bachir, Evy Vanderheyden, Bram Boeckx, Diether Lambrechts, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Hans Wildiers, Elia Biganzoli, Giuseppe Floris, Christine Desmedt. Evaluation of changes in sequencing quality and transcriptomic profiles with increasing post-mortem interval: results from an optimization experiment within the UPTIDER tissue donation program [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-12.

Background: Liver metastases (LM) are ultimately present in ~50% of all patients with metastatic breast cancer (BC). Metastatic seeding to the liver relies on the interaction between cancer cells and the host microenvironment, resulting into two main histopathological growth patterns (HGPs): the replacement HGP (metastasis mimics the liver architecture and exploits liver vasculature) and the desmoplastic HGP (presence of a fibrotic rim separating the hepatocytes from the tumor cells). While the prognostic value of these HGPs is established for colorectal cancer, with the desmoplastic HGP being associated with better prognosis, investigation is needed for BC. It has also been reported that patients with LM have a poorer response to immune checkpoints inhibitors. A systematic evaluation of the HGP and LM-associated immune infiltrates (stromal tumor infiltrating lymphocytes, sTIL) is currently lacking. In this study, we aimed at: (i) investigating HGPs and sTIL in LM from patients with BC, and, (ii) evaluating the association of these HGPs and sTIL with standard variables and outcome. Patients and methods: The study currently includes clinical data and samples from: 1) a retrospective cohort of 122 patients from 7 hospitals with surgically resected LMs (represented by 548 hematoxylin and eosin (H&E)-sections), further referred to as ‘surgical cohort’ and, 2) LMs from 2 institutional post-mortem tissue donation studies for a total of 23 patients (97 H&E-sections). All available H&E-sections were used to assess HGP and sTIL. HGPs were scored according to a standardized method (PMIDs: 35650276) and categorized per patient as pure-replacement (rHGP, i.e. 100% of the tumor-liver interface is replacement) or any-desmoplastic (dHGP, i.e. at least 1% of the tumor-liver interface is desmoplastic). sTIL are expressed as the percentage of stromal area covered by mononuclear immune cells at the metastasis-liver interface. Associations were assessed using Fisher exact and Wilcoxon tests. Univariable and multivariable Cox regression analyses stratified by center were used to evaluate the role of HGP on progression-free (PFS) and overall survival (OS). Results: In the surgical cohort, 54 (44%) of patients displayed a rHGP and 68 (56%) a dHGP. Intra-patient, meaning inter-slide, heterogeneity of the HGP was observed in 24/122 (20%) of the patients suggesting that scoring multiple slides is needed for accurate assessment of the HGPs. We did not find any statistically significant association between HGP and clinico-pathological data. Higher sTIL were associated with dHGP (p=.003), as well as with a higher histological grade (p= .087), estrogen receptor-positivity (p=0.062) and ductal histology (p= .08) of the primary tumor. rHGP was associated with worse PFS and OS, both at the univariable and multivariable level (Table). In the post-mortem cohort, we observed a higher frequency of rHGP patients (19/23 patients, 83 %) and significantly lower levels of sTIL in the rHGP patients as compared to the rHGP patients from the surgical cohort (p= .009). Conclusion: This study represents the largest study evaluating HGPs and immune infiltrates of LMs from patients with BC. Approximately half of the surgically resected LMs have a dHGP, which is associated with higher sTIL and a better prognosis. The results from the LMs from the post-mortem cohort suggest that a more advanced stage of the disease is associated with an increase in rHGP and with a more immunosuppressed environment. Table 1: Univariable and multivariable analyses Citation Format: Sophia Leduc, Maxim De Schepper, Peter Vermeulen, Giuseppe Floris, Elia Biganzoli, Vincent Donckier, Ali Bohlok, Marco Gerling, François Richard, Marion Maetens, Joris Jaekers, Baki Topal, Emily Latacz, Karen Van Baelen, Tatjana Geukens, Ha Linh Nguyen, Luc Dirix, Denis Larsimont, Sophie Van Kerckhove, Rui Caetano Oliveira, Janina Kulka, Valerio Lucidi, Yannick Meyer, Cornelis Verhoef, Eva Santos, Ferenc Salamon, Lilla Madaras, A. Marcell Szasz, Székely Borbála, Kristòf Dede, Jennie Engstrand, Carlos Fernandez Moro, Christine Desmedt. Histopathological and immune characterization of liver metastases from patients with breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-06.

Background. Research in metastatic breast cancer is hampered by limited sample availability. Post-mortem tissue donation programs can help to overcome this problem but are logistically challenging and have thus far mainly focused on histopathological and genomic research. We here present the UPTIDER program (NCT04531696), aimed at the multilevel characterization of advanced breast cancer and generation of tumour models. Patients and Methods. Patients with stage IV breast cancer receiving their last line(s) of treatment are eligible for participation. Blood, urine and saliva samples are collected upon inclusion. Upon death, a post-mortem MRI (when possible) followed by a rapid autopsy is performed. Liquid biopsies from all body fluids and tissue samples from all macroscopically identified metastatic sites are collected. Samples are processed as mirrored biopsies in different conditions, such as fresh frozen for omics analyses, formalin fixed paraffin-embedded for histopathology, and slowly frozen in freezing medium or fresh for generation of xenograft and organoid models. Results. Since approval by the local Ethical Committee in November 2020, 22 patients have been enrolled and 15 autopsies have been performed. Mean interval between death and start of autopsy was 3h (range 2-6h), mean duration of the autopsies was 6h (4-9h). A post-mortem MRI was performed in 6 patients. Peripheral blood, central blood and bone marrow were collected from all patients; urine, ascites, cerebrospinal, pericardial and pleural fluid all in more than 2/3 of patients. On average, 232 (range 90-406) tissue samples of which 164 (45-303) pathological from 42 (15 – 79) metastases were collected for each patient. Most often sampled metastatic sites were lymph nodes, liver, bones, pleura and peritoneum. Samples from the primary tumour could be retrieved from all patients, either during the autopsy (n=6) or from historical archives. In total, 133 tumour samples were sent to collaborating partners for patient-derived xenograft creation. Already some have been successfully established and stored, including models derived from a patient with invasive lobular carcinoma (ILC) and one with metaplastic squamous cell carcinoma. When correlating microscopic and macroscopic findings, patients could largely be divided into three main categories. Eleven patients presented with overt and extensive disease burden, often characterized by diffuse visceral, pleural, peritoneal, bone and lymph node involvement. Two patients, both with ILC, presented with underestimated yet extensive disease burden. While gross examination and cross sectioning of organs did not reveal clear involvement, microscopical invasion of stomach and liver, amongst others, was found. Lastly, limited disease burden was seen in two patients, both with leptomeningeal involvement. In those patients, massive tumoral infiltration in the subarachnoid space and along the blood-brain barrier was seen microscopically, with no grey matter invasion. Conclusion. We successfully launched a new and comprehensive post-mortem tissue donation program for patients with metastatic breast cancer, enrolling ~ 1 patient per month. Post-mortem tumour samples already resulted in successful establishment of some patient-derived xenografts. From a clinical point of view, vast underestimation of the disease extent on imaging during life as well as macroscopically during the autopsy was observed in some patients with metastatic ILC. For patients with leptomeningeal metastasis, we showed that the highly aggressive nature of their disease might be explained by extensive meningeal infiltration disrupting the blood-brain barrier. Further insights into disease progression and heterogeneity will be generated by the ongoing multi-omics analyses. Citation Format: Tatjana Geukens, Maxim De Schepper, Karen Van Baelen, François Richard, Marion Maetens, Amena Mahdami, Ha-Linh Nguyen, Edoardo Isnaldi, Anirudh Pabba, Sophia Leduc, Imane Bachir, Maysam Hajipirloo, Emily Vanden Berghe, Sigrid Hatse, Eleonora Leucci, Maria Francesca Baietti, Georgios Sflomos, Cathrin Brisken, Patrick Derksen, Colinda Scheele, Vincent Vandecaveye, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Elia Biganzoli, Hans Wildiers, Wouter Van Den Bogaert, Giuseppe Floris, Christine Desmedt. Advancing research on metastatic breast cancer: the UPTIDER post-mortem tissue donation program [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-14.

Background. Trastuzumab deruxtecan (T-DXd) has shown promising activity in patients with HER2-low metastatic breast cancer. As the HER2-status can vary between the primary and its corresponding metastases, treatment decisions should ideally be based on HER2 assessment of a recent biopsy. However, limited data is available on intra-patient inter-metastatic heterogeneity in HER2-status, affecting representability of a single biopsy and potential therapeutic options and outcome. We therefore assessed HER2 status on multiple metastases from patients with primary ER-positive/HER2-non-amplified breast cancer in our prospective post-mortem tissue donation program UPTIDER (NCT04531696). Methods. Ninety-one metastatic samples retrieved during the autopsies of 6 patients (range: 13–16/patient) and their respective primary tumours were immunohistochemically (IHC) stained for HER2 (HercepTestTM, RTU, ISO-15189 accredited) in our institution. Consensus scoring was performed between two pathologists according to ASCO/CAP 2018 guidelines. The observers were blinded for patient ID. Reflex fluorescence in situ hybridization (FISH) testing was performed for samples with IHC score of 2+. HER2 status was categorized as HER2-zero (IHC 0), HER2-low (IHC 1+ or IHC 2+ with negative FISH), or HER2-positive (IHC 3+ or IHC 2+ with positive FISH). To assess stability of the performance of IHC scoring in the post-mortem setting, an additional 13 samples taken from 3 metastases at regular (every 1.5h) time intervals during the autopsy underwent HER2 IHC scoring. Results. Evaluation of HER2-status in the primary tumour showed 2 patients with HER2-zero disease and 4 with HER2-low disease. A discordance between HER2 status of the metastases and their respective primary was seen in all patients. Not a single lesion was found to be HER2-positive. For every patient, at least one HER2-low metastasis was observed, with the percentage being highly variable between patients and ranging between 7 and 100%. No association was observed between HER2 status and organ site: HER2-low as well as HER2-zero lesions were found in all organs evaluated in at least 4 patients (liver, bone, pleura, lymph nodes). For 5 patients, multiple lesions within the liver were evaluated: while HER2-zero versus HER2-low status was concordant in those lesions in 4 patients, a mix of HER2 IHC scores was seen in 3 of them. IHC scores were stable over time for tumour lesions assessed repeatedly. Discussion. Important inter-lesion heterogeneity in terms of HER2-low status was observed in patients with primary ER-positive/HER2-non-amplified breast cancer participating to our post-mortem tissue donation program. This observed heterogeneity is unlikely to be due to post-mortem changes in HER2 expression. HER2-low status was found in at least one distant lesion in all patients, complicating therapeutic decision-making based on a single biopsy. Of note, IHC 1+ and 2+ scores varied between metastases of each patient too, making assessment on a single biopsy less reliable for stratification in clinical trials. Further assessment on samples from UPTIDER-patients with ER-negative disease is currently ongoing and results will be available to be presented. Citation Format: Tatjana Geukens, Maxim De Schepper, François Richard, Marion Maetens, Karen Van Baelen, Amena Mahdami, Ha-Linh Nguyen, Edoardo Isnaldi, Sophia Leduc, Anirudh Pabba, Imane Bachir, Freya Mertens, Sara Vander Borght, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Hans Wildiers, Wouter Van Den Bogaert, Giuseppe Floris, Christine Desmedt. HER2-16 Inter-lesion heterogeneity of HER2-status in metastatic breast cancer: possible implications for treatment with anti-HER2 antibody-drug conjugates. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-16.

Background:. Inflammatory breast cancer (IBC) represents a rare (~1-5% of all breast cancers, BC) and particularly aggressive type of BC, accounting for roughly 10% of BC-related mortality annually. So far IBC has been mainly characterized at the genomic level using samples from the primary tumor. Here, using publicly available data from two large cohorts, we compared the genomic alterations in primary and metastatic samples from patients with metastatic IBC to alterations in samples of patients with metastatic non-IBC. Patients and methods:. We retrieved publicly available clinical and genomic data from primary (P) and metastatic (M) samples from MSK-IMPACT (MSK) and the Metastatic Breast Cancer project (MBC) from cbioportal. IBC was defined as cT4d. We considered female metastatic patients with invasive BC of no special type (commonly referred to as invasive ductal BC): 34 patients with IBC and 602 with non-IBC. To focus on alterations with potential clinical relevance, we only retained the genomic alterations annotated as oncogenic by OncoKB. We applied Firth logistic regression adjusted for hormonal receptor status (positive vs negative), HER2 status (positive vs negative) stage (II vs I, III vs I, IV vs I) and cohort (MSK vs MBC) with genomic alterations or metastatic sites as outcome variables and IBC status as independent variable (yes vs no). A minimum of 5 patients harboring the alterations were required to perform the analysis. Chromosomal instability (CIN) score and Tumor Mutational Burden (TMB) were investigated by linear regression and quantile regression respectively, adjusted as described above and considering the median for the latest. P and M samples were analyzed separately, a patient was considered as ‘having’ the alteration if at least one of her samples was altered. All analyses were performed in R 4.0.2. Results:. Metastatic samples from chest wall and skin metastases were significantly more represented in patients with IBC as compared to patients with non-IBC (Adjusted Odds Ratio (ORadj): 6.16, 95% confidence interval (95CI): [1.94-18.38], p-value: .003 and ORadj: 7.60, 95CI: [1.66-30.05], p-value: .012 respectively), which is in line with the course of the IBC disease. Several studies have suggested IBC to have a higher TMB than non-IBC. Here we confirmed an increase in TMB in P and M IBC (Adjusted coefficient (coefadj): 3.00, 95CI: [1.60-4.20], p-value: .003 and coefadj: 4.00, 95CI: [0.85-4.15], p-value Table 1.Patients with primary samples. (279 patients with clinical information available - 279 samples)Patients with metastatic samples. (311 patients with clinical information available - 339 samples)AlterationAlteration countOR [95CI]p-valueAlteration countOR [95CI]p-valueTP53 mutation4120.18 [4.95-96.38] Citation Format: François Richard, Maxim De Schepper, Marion Maetens, Sophia Leduc, Edoardo Isnaldi, Tatjana Geukens, Karen Van Baelen, Ha-Linh Nguyen, Peter Vermeulen, Steven Van Laere, Luc Dirix, Giuseppe Floris, Elia Biganzoli, Christine Desmedt. Comparison of the genomic alterations in metastatic inflammatory and non-inflammatory breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-08-07.

Background: High body mass index (BMI) is an established risk factor for developing breast cancer (BC), especially estrogen receptor (ER)-positive, and also has been associated with adverse survival. Still, patients with BC are currently treated independently of their BMI given limited understandings of the association between BC biology and patient adiposity. In this study, using retrospective data retrieved from two large BC studies, we aimed to identify genomic alterations of primary BC that are associated with BMI in the most common histological BC subtype - invasive carcinoma of no special type (NST). Patients, Data and Methods: Clinicopathological and genomic alteration data were retrieved from two study cohorts: METABRIC (Pereira et al. 2016) and ICGC (Nik-Zainal et al. 2016), with BMI recorded at the time of diagnosis and represented as either a continuous variable or a categorical variable of three categories - lean, overweight and obese. Stratification according to ER and HER2 status resulted in two focused subgroups: NST ER+/HER2- (n=392) and NST ER-/HER2- (n=152). Mutations classified as oncogenic using a set of predefined criteria were used to determine gene-level mutation status. Copy number alteration (CNA) calls were distinguished into three event types: amplification, hemizygous deletion and homozygous deletion. We used multivariable Firth’s logistic regression models with the presence of a genomic alteration as the response variable, BMI as the predicting variable of interest, and data cohort (METABRIC vs ICGC), age group (≤50 vs >50) and tumor grade (I & II vs III) as covariates, to assess the associations between BMI and recurrent gene-level genomic alterations, including gene mutations and CNAs. In a similar manner, we performed multivariable linear regression analysis, adjusting for age and tumor grade, to evaluate the associations of BMI with mutational signatures (MS) and tumor mutational burden in the ICGC NST subsets where these data are available. Results: Considering BMI as a categorical variable, we observed in the NST ER+/HER2- subgroup that PIK3CA was significantly less frequently mutated in obese compared to lean patients (33% vs 46%, odds ratio (OR) = 0.57 (95% confidence interval = (0.33, 0.97)), p = .039), while PTEN and TBX3 showed an increased frequency in overweight (6% vs 1%, OR = 4.14 (1.1, 22.34), p = .034) and obese (8% vs 1%, OR = 7.41 (1.82, 70.65), p = .008) patients, respectively. Regression analyses with BMI as a continuous variable revealed an increased prevalence of mutations in CDH1 and TBX3 genes as BMI increases by 1kg/m2 (OR = 1.14 (1.05, 1.24), p = .002, and OR = 1.13 (1.04, 1.22), p = .005, respectively) in patients with NST ER+/HER2- BC. No associations between BMI and oncogenic mutations was observed in the NST ER-/HER2- subgroup. Interrogation of gene-level CNAs in both subgroups demonstrated differences according to BMI in the prevalence of CNAs affecting a number of genes, many of which are known or have been presented with evidence to be involved in regulation of or regulated by hallmark pathways of BC, such as the MAPK/ERK, JAK/STAT and Wnt/β-catenin signaling pathways. We report a strong positive association between the single-base substitution signature 1 (SBS1), an age-correlated MS, and both continuous (coefficient (coef) = 18.3 (7.7, 28.9), p < .001) and categorical BMI (obese vs lean, coef = 336.3 (187.9, 484.8), p < .001) in the ICGC NST ER+/HER2- subgroup. Conclusion: This exploratory retrospective study suggests that the genomic profiles of primary BC may differ according to BMI. Clinical implications of these differences, especially the decreased prevalence of PIK3CA mutations in obese patients in the context of alpelisib, warrant further investigation. These results however indicate that patient adiposity should be taken into account in the era of personalized medicine. Citation Format: Ha-Linh Nguyen, Tatjana Geukens, Marion Maetens, Karen Van Baelen, Maxim De Schepper, Sophia Leduc, Edoardo Isnaldi, Sam Aparicio, Ake Borg, Jane Brock, Annegien Broeks, Carlos Caldas, Andrew Green, Hazem Khout, Eyfjörð Jórunn, Stian Knappskog, Savitri Krishnamurthy, Sunil Lakhani, Anita Langerod, John WM Martens, Leigh Murphy, Serena Nik-Zainal, Colin Purdie, Emad Rakha, Andrea Richardson, Anne Salomon, Peter Simpson, Christos Sotiriou, Paul Span, Benita Kiat-Tee Tan, Alastair Thompson, Stefania Tommasi, Marc Van de Vijver, Steven Van Laere, Alain Viari, Giuseppe Floris, Elia Biganzoli, François Richard, Christine Desmedt. The association between genomic alterations and body mass index in patients with early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-18.

Cancer cells outgrowing in distant organs of metastasis rewire their metabolism to fuel on the available nutrients. While this is often considered an adaptive pressure limiting metastasis formation, some nutrients available at the metastatic site naturally or through changes in organ physiology may inherently promote metastatic growth. We find that the lung, a frequent site of metastasis, is a lipid-rich environment. Moreover, we observe that pathological conditions such as pre-metastatic niche formation and obesity further increase the availability of the fatty acid palmitate in the lung. We find that targeting palmitate processing inhibits spheroid growthin vitroand metastasis formation in lean and obese mice. Mechanistically, we discover that breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a (CPT1a)-dependent manner. Lysine acetyltransferase 2a (KAT2a), whose expression is promoted by palmitate availability, relies on the available acetyl-CoA to acetylate the NF-κB subunit p65. This favors nuclear location of p65 and activates a pro-metastatic transcriptional program. Accordingly, deletion of KAT2a phenocopies CPT1a silencingin vitroas well asin vivoand patients with breast cancer show co-expression of both proteins in metastases across palmitate-rich metastatic sites. In conclusion, we find that palmitate-rich environments foster metastasis growth by increasing p65 acetylation resulting in elevated NF-κB signaling.

Adipocytes and cancer-associated adipocytes (CAAs) are poorly investigated cells in the tumor microenvironment. Different image analysis software exist for identifying and measuring these cells using scanned hematoxylin and eosin (H&E)-stained slides. It is however unclear which one is the most appropriate for breast cancer (BC) samples. Here, we compared three software (AdipoCount, Adiposoft, and HALO®). HALO® outperformed the other methods with regard to adipocyte identification, (> 96% sensitivity and specificity). All software performed equally good with regard to area and diameter measurement (concordance correlation coefficients > 0.97 and > 0.96, respectively). We then analyzed a series of 10 BCE samples (n = 51 H&E slides) with HALO®. Distant adipocytes were defined >2 mm away from cancer cells or fibrotic region, whereas CAAs as the first three lines of adipocytes close to the invasive front. Intra-mammary heterogeneity was limited, implying that measuring a single region of ∼500 adipocytes provides a reliable estimation of the distribution of their size features. CAAs had smaller areas (median fold-change: 2.62) and diameters (median fold-change: 1.64) as compared to distant adipocytes in the same breast (both p = 0.002). The size of CAAs and distant adipocytes was associated with the body mass index (BMI) of the patient (area: rho = 0.89, p = 0.001; rho = 0.71, p = 0.027, diameter: rho = 0.87 p = 0.002; rho = 0.65 p = 0.049, respectively). To conclude, we demonstrate that quantifying adipocytes in BC sections is feasible by digital pathology using H&E sections, setting the basis for a standardized analysis of mammary adiposity in larger series of patients., info:eu-repo/semantics/published

Background: Inflammatory breast cancer (IBC) is a rare (1-5%), but aggressive form of breast cancer (BC), accounting for ~10% of BC mortality. In early setting (M0), standard of care is neoadjuvant chemotherapy (NACT), followed by surgery. Nevertheless, outcome is still relatively poor. Pathological complete response (pCR) after NACT is prognostic in BC in general, and can be predicted by a high percentage of stromal tumor infiltrating lymphocytes (sTIL) in the primary tumor. The predictive value of sTIL in IBC has only been sporadically investigated, often in smaller series. Our aim was to determine which variables, including sTIL, are associated with pCR and to determine the prognostic value of pCR in IBC in a large multicentric, retrospective cohort. Patients & Methods: We included patients with IBC treated with NACT+/- anti-Human Epidermal growth factor Receptor 2 (HER2) therapy, followed by surgery from 10/1996 to 10/2021 in 7 different European hospitals. Clinicopathological variables were collected and central pathological review was performed, including sTIL scoring. This study focused on M0 cases. Considered clinicopathological variables were: age, histology, tumor grade, estrogen receptor status (ER), HER2 status, focality (unifocal vs not), and baseline locoregional nodal status (Table 1). Associations between pCR, clinicopathological variables and sTIL were assessed using Firth’s logistic regression models: Model 1 was adjusted for center, Model 2 additionally included all variables of interest. Similarly, linear regression was used to investigate the association between sTIL and clinicopathological features. Univariable and multivariable Cox regression was used to evaluate the role of pCR on disease free survival (DFS), distant recurrence free survival (DRFS) and overall survival (OS). DFS and DRFS were analyzed considering death without the respective event as competing risk. Results: 494 patients were included. The distribution according to receptor status was: ER-/HER2- (24.3%), ER+/HER2- (34.4%), ER+/HER2+ (13%) and ER-/HER2+ (20.2%). pCR rate was 26% and per receptor status: ER-/HER2- (28%), ER+/HER2- (10%), ER+/HER2+ (42%) and ER-/HER2+ (45%). pCR was associated with grade (G3 vs G1/2, OR =2.79 (1.70 − 4.74), p < .001), ER-status (positive vs negative, OR = 0.39 (0.26 − 0.60), p < .001) and HER2 status (positive vs negative, OR = 3.74 (2.43 − 5.81), p < .001) in Model 1. Only the association with HER2 status remained significant in Model 2 (OR = 5.34 (2.83 − 10.47), p < .001). sTIL was scored for 385 patients. Median sTIL was 5.3% [IQR 2.0%;16.7%] and according to receptor status: ER-/HER2- (10%), ER+/HER2- (2.5%), ER+/HER2+ (6.7%) and ER-/HER2+ (8.3%). Higher sTIL was associated with NST (p = .032), grade 3 (p = .015), and ER-negativity (p = .007) in Model 1. This was no longer significant in Model 2, but the direction of the trends was preserved. sTIL was associated with pCR (5% increment, OR = 1.13 (1.05 − 1.22), p = .002), but no longer after adjustment. No association between pCR and sTIL was found stratifying by receptor status. The median FU was 9.4 years and multivariable Cox regression models revealed that ER+ and HER2+ status and achieving pCR were significantly associated with better DFS, DRFS, and OS (Table 1). Conclusion: Our results indicate that patients with HER2+ tumors have a higher probability of achieving pCR and that pCR has an independent prognostic role in IBC. This is the largest IBC study with centrally scored sTIL, demonstrating that sTIL is associated with pCR but its role as an independent predictor of pCR is still not certain. Citation Format: Maxim De Schepper, Ha-Linh Nguyen, François Richard, Florence Lerebours, Roman Vion, Florian Clatot, Anca Berghian, Marion Maetens, Sophia Leduc, Edoardo Isnaldi, Chiara Molinelli, Matteo Lambertini, Frederica Grillo, Gabriele Zoppoli, Luc Dirix, Hilde Wuyts, Kevin Punie, Hans Wildiers, Chantal Remmerie, Ann Smeets, Ines Nevelsteen, Patrick Neven, Anne Salomon, Denis Larsimont, Caroline Duhem, Patrice Viens, François BERTUCCI, Elia Biganzoli, Peter Vermeulen, Giuseppe Floris, Christine Desmedt. Stromal tumor infiltrating lymphocytes and pathological complete response in patients with inflammatory breast cancer treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-47.

Background. Liquid biopsies represent a less invasive alternative to tissue biopsy to characterize and possibly monitor the disease in patients with metastatic breast cancer. So far, blood remains the most frequently investigated body liquid in this context and the investigations mainly focus on the detection, quantification and characterization of the circulating tumor DNA (ctDNA). However, since blood might not capture the full disease profile, other sources of body liquids may have the potential to complement the information obtained from blood. The aims of the present study are therefore to assess whether: (i) ctDNA can be detected in different types of body liquids, and, (ii) the levels of ctDNA in a given liquid are associated with metastases in specific organs. Patients and methods. Twelve 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 negative, HER2 non-amplified (ER+/HER2-) (n=9), ER-/HER2- (n=2) and ER+/HER2+ (n=1). Median time between inclusion and death of the patient was 1.6 months (Interquartile range: [0.4-3.4]). 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, as well as saliva, urine, and ascites whenever possible) and at autopsy (except for saliva). In total, 108 liquid samples were collected and immediately centrifuged according to standard protocols. Cell free DNA (cfDNA) was extracted from the supernatant. All extracted cfDNA as well as germline DNA extracted from the 12 matched buffy coat samples underwent shallow whole genome sequencing. Log2 ratios were computed with CNVkit, and co-segmented per patient using the copynumber R package. Purity and ploidy were assessed by ABSOLUTE. Associations between organ involvement and ctDNA yield were assessed by Wilcoxon rank-sum tests. Samples at study inclusion and at autopsy were considered together unless otherwise specified. Results. At the sample level, ctDNA could be identified in 54% of the samples. At the patient level, the proportion of liquid types in which ctDNA was detected was highly variable (median: 58%, IQR: 34-77%, Table 1). CtDNA was detected in ascites of all patients when investigated, in 78% of PFL, 73% of CSF, 67% of blood and 37% of pericardial fluid. Only for one patient with invasive lobular carcinoma, ctDNA was detected in saliva and urine, the latter most likely explained by invasion of the bladder. Of note, in 4/12 patients ctDNA could not be identified in blood but was detected in at least one of the other fluids for 3 of these patients. At autopsy, ctDNA levels tended to be higher in PFL, ascites, and CSF in case of pleural, peritoneal, and central nervous system (CNS) metastases respectively, reaching statistical significance only for PFL. In CSF, two patients have CSF ctDNA detected with no documented involvement of the CNS. No brain autopsy was however performed for these patients. Conclusion. We have shown that ctDNA can be detected in all 7 different body liquids that were investigated in this study. The ctDNA levels in a given liquid can be associated with the presence of metastases in specific organs. Since ctDNA was not detected in 4 of our patients in blood but detectable for 3 of them in other liquids, the evaluation of additional sources of body fluids should be further investigated in patients with metastatic breast cancer. These results therefore open new avenues for the clinical monitoring and characterization of the disease. Table 1. Summary of ctDNA detection per liquid type at the patient level based on the 108 evaluated samples. Histo.= Histological, ILC= Invasive lobular carcinoma, NA= not available, nr= number, NST= non-special type 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 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, Elia Biganzoli, Giuseppe Floris, Christine Desmedt. ctDNA detection in seven different types of body liquids in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-06.

The impact of adiposity on the efficacy of endocrine treatment in patients with estrogen receptor positive breast cancer is poorly investigated. Here, we retrospectively investigated in a cohort of 56 patients whether body mass index and/or mammary adiposity are associated with anti-proliferative response in the neoadjuvant setting. Anti-proliferative response was defined as high Ki67 at baseline (Ki67bl) and low Ki67 at surgery (Ki67srg), using the 14% cut-off. Mammary adipocyte size was assessed on hematoxylin and eosin slides from the surgical samples using digital pathology. A higher proportion of tumors with an anti-proliferative response was observed in patients with obesity (54.5%) as compared to patients with normal weight (9.0%) and patients with overweight (40.0%) (p = 0.031), confirmed by multivariable regression analysis adjusted for baseline Ki67 (OR, obese vs normal weight: 13.76, 95%CI: 1.49–207.63, p = 0.020). Larger adipocyte diameter was identified as predictor of anti-proliferative response (OR per increase in diameter of 5 μm for adipocytes distant from the tumor: 2.24, 95%CI: 1.01–14.32, p = 0.046). This study suggests that anti-proliferative response to neoadjuvant letrozole might be more frequent in patients with increased systemic or mammary adiposity.

Currently, there are no markers to identify patients with liver-only or liver-dominant metastases that would benefit from hepatic surgery. Here we characterized histopathological growth patterns (HGPs) of liver metastases in a consecutive series of 36 breast cancer patients who underwent hepatic surgery. Survival analyses showed that the presence of a desmoplastic HGP in the liver metastases (a rim of fibrous tissue separating cancer cells from the liver parenchyma, present in 20 (56%) patients) is independently associated with favorable progression-free and overall survival when compared with the replacement HGP (cancer cells growing into the liver parenchyma, present in 16 (44%) patients)., SCOPUS: ar.j, info:eu-repo/semantics/published

Introduction: Decision making in refractory colorectal cancer (rCRC) is challenging, with limited data available to predict patient outcome. We conducted a study to assess the pace of cancer progression as a potential prognostic and decision tool. Methods: CORIOLAN was a prospective, single-center, single-arm trial recruiting refractory CRC patients with an ECOG performance status of &le, 1 and an estimated life expectancy of &ge, 12 weeks. 18 fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) scan and blood sample collection were carried out at baseline and after 2 weeks with no cancer treatment given between these timepoints. The primary objective was to evaluate the association between pace of cancer progression as defined by changes of the whole-body metabolically active tumor volume (WB-MATV) and overall survival (OS). Exploratory objectives included evaluation of the prognostic value of circulating cell-free DNA (cfDNA), circulating tumor cells (CTCs) and carcinoembryonic antigen (CEA). Results: 47 eligible patients who had received a median number of 5 (range 2&ndash, 8) prior treatments were enrolled. At the time of analysis, 45 deaths had occurred, with 26% of patients dying within 12 weeks. The median OS was 6.3 months (range 0.4&ndash, 14.3). The median relative delta between WB-MATV at baseline and 2 weeks was +21%. Changes of WB-MATV, however, failed to predict OS (hazard ratio (HR) 1.3, p = 0.383). Similarly, no association was observed between changes of any of the circulating biomarkers investigated and prognosis. By contrast, high WB-MATV (4.2 versus 9.4 months, HR 3.1, p = 0.003), high CEA (4.4 versus 7.0 months, HR 1.9, p = 0.053), high cfDNA (4.7 versus 7.0 months, HR 2.2, p = 0.015) and high CTC count (3.3 versus 7.5 months, HR 6.5, p &lt, 0.001) at baseline were associated with worse OS. Conclusions: In this study, approximately 1 out of 4 refractory CRC patients who were judged to have a life expectancy &gt, 12 weeks actually died within 12 weeks. Baseline assessment of WB-MATV, cfDNA, CTCs and CEA, but not early change evaluation of the same, may help to refine patient prognostication and guide management decisions.

High-throughput sequencing technologies are increasingly used in research but limited data are available on the feasibility and value of these when routinely adopted in clinical practice.We analyzed all consecutive cancer patients for whom genomic testing by a 48-gene next-generation sequencing (NGS) panel (Truseq Amplicon Cancer Panel, Illumina) was requested as part of standard care in one of the largest Belgian cancer networks between 2014 and 2019. Feasibility of NGS was assessed in all study patients, while the impact of NGS on the decision making was analyzed in the group of gastrointestinal cancer patients.Tumor samples from 1064 patients with varying tumor types were tested, the number of NGS requests increasing over time (Our findings confirm that NGS is feasible in the clinical setting with acceptably low failure rates and rapid turnaround time. In gastrointestinal cancers, however, NGS-based multiple-gene testing adds very little to standard targeted sequencing, and in routine practice the clinical impact of NGS panels including genes which are not routinely recommended by international guidelines remains limited.

Contains fulltext : 283492.pdf (Publisher’s version ) (Closed access) The first consensus guidelines for scoring the histopathological growth patterns (HGPs) of liver metastases were established in 2017. Since then, numerous studies have applied these guidelines, have further substantiated the potential clinical value of the HGPs in patients with liver metastases from various tumour types and are starting to shed light on the biology of the distinct HGPs. In the present guidelines, we give an overview of these studies, discuss novel strategies for predicting the HGPs of liver metastases, such as deep-learning algorithms for whole-slide histopathology images and medical imaging, and highlight liver metastasis animal models that exhibit features of the different HGPs. Based on a pooled analysis of large cohorts of patients with liver-metastatic colorectal cancer, we propose a new cut-off to categorise patients according to the HGPs. An up-to-date standard method for HGP assessment within liver metastases is also presented with the aim of incorporating HGPs into the decision-making processes surrounding the treatment of patients with liver-metastatic cancer. Finally, we propose hypotheses on the cellular and molecular mechanisms that drive the biology of the different HGPs, opening some exciting preclinical and clinical research perspectives.

Introduction Mucinous carcinoma, a special histological subtype of breast cancer (BC) which accounts for ˜2% of all invasive breast tumors, is characterized by the presence of extracellular mucin, typically expresses the estrogen receptor (ER), and lacks HER2 amplification. The majority of patients with mucinous BC are older at diagnosis, rarely present axillary lymph node metastases, and are associated with a better prognosis as compared to invasive ductal cancer patients (IDC, formally referred to as “breast cancer of no special type”). So far, it is unknown what is driving the mucinous phenotype of these tumors. Here we interrogated the genomics, transcriptomics, immune infiltration and epigenetics profiles of these tumors, using a retrospective institutional series and a publicly available dataset. Patients and methods After central pathology review and DNA extraction, a total of 31 pure mucinous cases from Institut J. Bordet Biobank were included (referred to as IJB cohort). Tumor infiltrating lymphocytes (TILs) were assessed on hematoxylin and eosin (H&E). Low pass whole genome sequencing was conducted to assess ploidy and to detect copy number aberrations (CNAs). A second cohort was analyzed from a publicly available dataset (referred to as BRCA560 cohort), with available centrally reviewed histology subtyping and DNA methylation profiles (Nik-Zainal et al. Nature 2016). 207 ER+ primary BC, 14 of which were mucinous, had HumanMethylation450K methylation profiles. RNAseq was available for a subset of 145 cancers, 13 of which were mucinous. DNA methylation data was processed in R using the minfi package. Beta-values were normalized (preprocessQuantile) and dmpFinder was used to identify differentially methylated positions. Results In the IJB cohort, whole genome sequencing revealed that all but two tumors were diploid. The most frequently deleted cancer genes were RB1 (38,1%), CDH1 (23,8%), BRCA2 (38,1%),TP53 (23,8%), MAP2K4 (23,8%), EGFR (28,6%) and PGR (23,8%). In terms of amplifications, only ZNF217 (19,4%) and FGFR1/ZNF703 (9,5%) were observed. These mucinous tumors generally displayed low TIL levels (median=5%). In the BRCA560 cohort, there was no significant difference in the clinico-pathological features of ER+ IDC vs mucinous subtypes. At the genomic level, we identified a lower frequency of PIK3CA mutations as compared to ER+ IDC from the same cohort. At the DNA methylation level, we identified 8013 differentially methylated CpGs (q-val < .05) between IDC and mucinous tumors. The top differentially methylated CpG mapped to MUC2, an extracellular mucin. MUC2 was significantly demethylated in mucinous tumors as compared to IDC (q-val Conclusion To the best of our knowledge, this study is the first to report hypomethylation of MUC2 in mucinous carcinoma of the breast, as a possible mechanism of extracellular production of mucin in these tumors. Furthermore, it highlights the minor immune infiltration of these cancers, possibly prevented by the mucin, and sheds light on the few genomic alterations present in these overall stable cancer genomes. Citation Format: Desmedt C, Veys I, Nguyen B, Leduc S, Bareche Y, Majjaj S, Boeckx B, Lambrechts D, Sotiriou C. Molecular characterization of mucinous breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-07-04.

Background: The impact of obesity on the efficacy of endocrine treatment (ET) in patients with estrogen receptor positive (ER+) breast cancer (BC) is poorly investigated. Several neoadjuvant trials, using changes in Ki67 as biomarker of anti-proliferative response, have demonstrated that neoadjuvant ET improves surgical and clinical outcomes. A recent study has shown that the subgroup of patients with an anti-proliferative response to neoadjuvant aromatase inhibition has a better prognosis as compared to the other patients (Smith et al., The Lancet 2020). However, it is unclear if obesity, and mammary adiposity are associated with anti-proliferative response. Material and Methods: We included 56 post-menopausal patients with ER+ BC that were treated with 4 months of neoadjuvant letrozole in a single institution. Body mass index (BMI) was categorized according to the WHO criteria and considered both as categorical and continuous variable. Centralized Ki67 assessment was performed as per study protocol. Consistently with previous neoadjuvant clinical trials, Ki67 index was dichotomized ( Citation Format: Edoardo Isnaldi, François Richard, Maxim De Schepper, Sophia Leduc, Marion Maetens, Tatjana Geukens, Karen Van Baelen, Ha-Linh Nguyen, Ghizlane Rouas, Gabriele Zoppoli, Fatima Cardoso, Christos Sotiriou, Denis Larsimont, Giuseppe Floris, Elia Biganzoli, Christine Desmedt. The association between adiposity and anti-proliferative response to neoadjuvant endocrine therapy with letrozole in post-menopausal patients with estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-02.

Although invasive ductal breast cancer (IDC) represents the most common histological type of breast cancer, minor subtypes exist such as mucinous breast cancer (MuBC). MuBC are distinguished by tumor cells floating in extracellular mucin. MuBC patients are generally older and associated with a favorable prognosis. To unravel the molecular architecture of MuBC, we applied low-pass whole-genome sequencing and microscopic evaluation of stromal tumor infiltrating lymphocytes to 30 MuBC from a retrospective institutional cohort. We further analyzed two independent datasets from the International Cancer Genomics Consortium and The Cancer Genome Atlas. Genomic data (n = 26 MuBC, n = 535 estrogen receptor [ER] positive/HER2-negative IDC), methylation data (n = 28 MuBC, n = 529 ER-positive/HER2-negative IDC), and transcriptomic data (n = 27 MuBC, n = 467 ER-positive/HER2-negative IDC) were analyzed. MuBC was characterized by low tumor infiltrating lymphocyte levels (median = 0.0%, average = 3.4%, 95% confidence interval = 1.9% to 4.9%). Compared with IDC, MuBC had a lower genomic instability (P =. 01, two-sided Mann-Whitney U test) and a decreased prevalence of PIK3CA mutations (39.7% in IDC vs 6.7% in MuBC, P =. 01 in the International Cancer Genomics Consortium; and 34.8% vs 0.0%, P =. 02 in The Cancer Genome Atlas, two-sided Fisher's exact test). Finally, our report identifies aberrant DNA methylation of MUC2 as a possible cause of extracellular production of mucin in MuBC., SCOPUS: ar.j, info:eu-repo/semantics/published

Background. Liver is the 3rd most common site for metastatic disease in BC. The proportion of patients with BC LM presenting with so-called oligometastatic disease is estimated to be ~10%. Some of these patients have an indolent disease course after hepatic surgery. However, as of today, it is impossible to identify the ideal candidates for hepatic surgery based on standard clinico-pathological characteristics. Two main HGP have previously been identified in LM from colorectal cancer patients1: (i) the desmoplastic HGP with angiogenesis, inflammation and a rim of fibrous tissue that separates the cancer cells from the liver parenchyma, and (ii) the replacement HGP, with vessel co-option, no angiogenesis, and cancer cells growing into the liver parenchyma while replacing the hepatocytes, with minimal inflammation. This replacement HGP was associated with worse survival and lack of benefit from bevacizumab in patients operated for colorectal LM2. Here, we aimed at characterizing HGP of LM from BC patients who underwent hepatic surgery. Patients and methods. We considered a series of 36 consecutive patients with invasive BC from Institut Jules Bordet and Hôpital Erasme, Brussels, who underwent surgical resection of LM. Patients were operated between April 2000 and October 2017, and the median follow-up was 10.7 years. The HGPs of resected LM were scored according to international guidelines on H&E stained section by light microscopy3, by a pathologist (PV) blinded to outcome data. All sections of all LM were evaluated for each patient. Associations between HGP and clinico-pathological characteristics were assessed using the Fisher exact test, and associations with progression-free (PFS) and overall survival (OS) using Cox proportional hazard regressions considering date of hepatic surgery as the starting time. Age at hepatic surgery, ER and HER2 status of the LM, and presence of extra-hepatic metastasi(e)s were considered as adjustment variables and centre as stratification factor. Results. In 16 patients (44%) the LM exclusively presented the replacement HGP (pure-replacement group) while in the remaining 20 patients (56%) the LM were at least partly desmoplastic (any-desmoplastic group). There was no association of HGP with ER or HER2 status of primary BC or LM. LM subtypes were as follows: 2 ER-/HER2- (7%), 11 HER2+ (38%), 16 ER+/HER2- (55%)- information is currently missing for 7 patients. In 10/30 (33.3%) patients, ER-status changed in the LM compared with the primary BC (8 losses, 2 gains). Significantly more patients in the any-desmoplastic group had a primary BC without lymph node metastases (56% versus 21% in the replacement group, p=.02). Survival analyses showed that any-desmoplastic HGP was independently associated with increased PFS after liver surgery when compared with pure-replacement HGP (Hazard Ratio, HR, 0.34; 95% CI: 0.12-0.96; p=.041). All patients with pure-replacement HGP relapsed within the first two years after hepatic surgery. The other factors in the model were not significantly associated with PFS. Similar results were observed for OS (adjusted HR for HGP: 0.26; 0.07-1.01, p=.052). Conclusion. In this study, we showed evidence that the pure-replacement HGP of BC LM is related to worse outcome after hepatic surgery, corroborating our findings in patients with colorectal LM1. These findings, which are currently being validated in additional series, suggest that HGP of LM could represent a promising candidate marker of oligometastatic progression in BC. Refs: 1. doi:10.1007/s10456-019-09661-5; 2. doi:10.1038/s41571-019-0181-9; 3. doi:10.1038/bjc.2017.334. Citation Format: Peter B Vermeulen, Ali Bohlok, Sophia Leduc, François Richard, Lara Botzenhart, Michail Ignatiadis, Philippe Aftimos, Christos Sotiriou, Martine Piccart, Alain Hendlisz, Steven Van Laere, Luc Y Dirix, Jean-Christophe Noël, Valerio Lucidi, Elia Biganzoli, Denis Larsimont, Christine Desmedt, Vincent Donckier. Association between the histopathological growth patterns (HGP) of liver metastases (LM) and survival after hepatic surgery in patients with oligometastatic breast cancer (BC) [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 P3-01-13.

e16012 Background: Decision making in rCRC patients (pts) who have exhausted all available therapies is challenging. Clinical trials, treatment re-challenge and best supportive care are possible options. There are limited data, however, to predict pt outcome and guide treatment choices. Methods: CORIOLAN was a prospective, single-centre, single-arm trial. Eligible pts had to have an ECOG PS of ≤1, a life expectancy of ≥12 weeks (wks) and had to have received prior standard chemotherapy and targeted agents. No cancer treatment was allowed within 4 wks prior to study entry. 18F-FDG PET scan and blood sample collection were carried out at baseline and after 2 wks. No treatment was given between these timepoints, while further management was left to the treating physician. The primary objective was to evaluate the association between tumour metabolic progression index as defined by changes of the whole-body metabolically active tumour volume (WB-MATV) and OS. Exploratory objectives included evaluation of the prognostic value of circulating biomarkers such as cell-free DNA (cfDNA), circulating tumour cells (CTCs, CellTracks Analyzer II, CellSearch) and CEA. Validated cut-off values, Kaplan-Meier method, Cox proportional hazards model and logrank tests were used (R version 3.5.1). Results: From 2012 to 2018, 47 eligible pts were enrolled. Median age: 65 years (38-82). ECOG PS 1: 64%. Males: 53%. Left-sided tumours: 71%. Metastases: lung (83%), liver (66%), lymph-nodes (45%), peritoneum (30%), bone (21%). Known RAS/BRAF mutations: 57%/0%. Median number of prior treatments: 5 (2-8). At the time of analysis, 45 deaths had occurred, with 26% of pts dying within 12 wks. The median relative delta between WB-MATV at baseline and after 2 wks was +21%. Changes of WB-MATV, however, failed to predict OS (p≥0.383). Similarly, no association was observed between changes of any of the circulating biomarkers investigated and prognosis. By contrast, low WB-MATV (9.4 vs 4.2 months, HR 0.65, p = 0.003), low cfDNA (7.0 vs 4.7 months, HR 0.58, p = 0.015), low CTC count (7.5 vs 3.3 months, HR 0.67, p < 0.001) and low CEA (7.0 vs 4.4 months, HR 0.58, p = 0.053) at baseline were associated with longer OS. Conclusions: This study confirms the inaccuracy of life expectancy estimation for rCRC pts based on a physician judgement largely relying on conventional criteria. Baseline assessment of WB-MATV, cfDNA, CTCs and CEA, but not early change evaluation of the same, may help to refine pt prognostication and guide management decisions. Clinical trial information: NCT01591590 .

SUMMARYMetastasizing cancer cells must adapt their metabolism to the nutrient environment of distant organs. We performed a loss-of-function CRISPR screen against Solute Carrier Transporters (SLCs) in a breast cancer mouse model to define nutrient requirements for lung and liver metastases. Most SLC transporter dependencies of metastases were organ-specific such as the mitochondrial iron importer Slc25a37 (mitoferrin-1, Mfrn1), whose loss inhibited liver but not lung metastasis. Accordingly, SLC25A37 was highly expressed in liver compared to lung metastases of breast cancer patients. Functionally,Slc25a37expression was induced by HIF1α to support heme synthesis. This enabled cancer cells to grow in hypoxic liver regions because they utilized heme to synthesize the lipophilic antioxidant bilirubin. Treating mice with a ferroptosis inhibitor fully restored the capacity ofSlc25a37deficient cancer cells to grow in the liver. Thus, we defined the nutrient transport liabilities of lung and liver metastases and identified Slc25a37-dependent bilirubin synthesis as a requirement of liver metastasis to resist ferroptosis.Abstract Figure