Van Laere Research Articles

Phenotypic Variability Related to Mutations in Riboflavin Transporter in Brazilian Children: Pediatric Case Series

AbstractBrown–Vialetto–Van Laere syndrome or riboflavin transporter deficiency is a rare and genetically determined condition that results in a spectrum of neurological signs and symptoms from generalized muscle weakness to cranial nerve involvement with medullary symptoms and respiratory failure. Most patients have SLC52A3 gene biallelic variants, but some of them may have impairment of SLC52A2 gene, both related to the cell transport of riboflavin. We report the case of three unrelated Brazilian patients under 18 years of age with this diagnosis confirmed by molecular genetic sequencing. We observed that the clinical manifestations found were compatible with those already described in the literature by age group. Unusual findings of retinitis pigmentosa and immunodeficiency were identified related to pathogenic variants in the SLC52A2 gene. All patients received riboflavin replacement at a therapeutic dose without gastrointestinal intolerance and with clinical improvement after starting treatment.

The company premises as passport

In several academic disciplines that focus on (historical) building practice there is growing interest in building contractors and their employees. Their contribution and influence are increasingly the subject of research. After the Second World War the contractor’s field of work was defined by productivity, specialization and industrialization, which profoundly altered the professional character of this branch of industry. Social developments and the prevailing idea of an all-encompassing welfare state triggered top-down and bottom-up changes in this traditionally highly cyclical sector. This fostered diversity in the contracting world, yet those diverse profiles and identities often remain unrecognized today. This is compounded by the fact that, unlike architects for example, the source material left behind by contractors and their employees is usually less informative and often fragmented.
 This article seeks to demonstrate the potential of alternative source material by analysing company photos in order to better understand the profile and identity of two post-war Belgian contractors. During their existence, Van Coillie of Oostende (1919-1976) and Van Laere of Kruibeke (1938-1989) amassed extensive collections of photographs, which were recently made accessible by the Flanders Architecture Institute (VAi). The focus of this article is on a series of photos from the 1960s and ’70s depicting the headquarters of both companies. These company premises and buildings were the very heart of the businesses, which adopted a pragmatic or strategic design to reflect their profile and identity. The selected photos are compared with information gleaned from a series of in-depth interviews with ex-employees in order to place the companies in their particular historical context.
 The analysis is in two parts, the first of which focuses on the site, the company architecture and the implanting. This reveals information about the size and organization of the contractors, as well as about their company strategy and professional profile. For example, they used these elements to create a strong position in the building sector and in so doing cement their reputation. The second part analyses a few interiors of the 1961 Van Laere headquarters. A detailed visual examination reveals how these spaces reflected the internal organization and hierarchical structure and how Van Laere projected its company image both internally and externally. The pictures also chart the development of an organizational culture and a body of values and norms that in turn shaped the company identity. The company management, the internal organization and the social and local network in which the firms were embedded, consequently had a considerable effect on the position they occupied as individual players in building practice. The main finding from these two analyses is that these post-war construction players were highly diverse. A critical and biographical sketch in terms of activities, profile and identity is consequently a necessary starting point for a detailed analysis of their position and role within national and international post-war building practice. This article shows what a rich source photographic material can be in developing such a sketch.

Sudden Onset Auditory Neuropathy Spectrum Disorder: A Rare Case Report of Brown Vialetto Van Laere Syndrome.

Brown Vialetto Van Laere syndrome is a rare disorder characterized by progressive pontobulbar palsy with bilateral sensorineural hearing loss and lower cranial nerve palsies. Fifty-eight cases have been reported in the last hundred years. As the most common presenting complaint of this disorder is hearing loss, audiological evaluation plays a vital role in pointing towards and narrowing its diagnosis. We present a case report of a 12-year male child affected by this disorder.

Impact of the Regulatory Framework on Medical Device Software Manufacturers: Are the Guidance Documents Supporting the Practical Implementation? Comment on "Clinical Decision Support and New Regulatory Frameworks for Medical Devices: Are We Ready for It? - A Viewpoint Paper".

The increasing use in clinical practice of software such as mobile apps and clinical decision support (CDS) software has only recently been taken up by regulators around the world. Specifically, the European Commission and the US Food and Drug Administration (FDA) have updated their regulatory framework in the last years. Van Laere et al have given an extensive overview of the European and US approaches to regulate CDS software. This commentary further discusses regulatory differences between the two geographies and their impact on manufacturers of medical device software. We discuss the practical implementation of the regulatory framework for medical device software (especially CDS software) with a reference to the available international guidance documents and their limitations. Given the direction of stricter regulatory oversight in Europe, additional European guidelines/examples are desirable to enable a pragmatic regulatory approach ensuring continued access to innovative medical device software for European patients.

Octaaf Van Laere – More than Keeper of the Eva Crane Historical Collection

Octaaf Van Laere – More than Keeper of the Eva Crane Historical Collection

The Challenges of Regulating Artificial Intelligence in Healthcare Comment on "Clinical Decision Support and New Regulatory Frameworks for Medical Devices: Are We Ready for It? - A Viewpoint Paper".

Regulation of health technologies must be rigorous, instilling trust among both healthcare providers and patients. This is especially important for the control and supervision of the growing use of artificial intelligence in healthcare. In this commentary on the accompanying piece by Van Laere and colleagues, we set out the scope for applying artificial intelligence in the healthcare sector and outline five key challenges that regulators face in dealing with these modern-day technologies. Addressing these challenges will not be easy. While artificial intelligence applications in healthcare have already made rapid progress and benefitted patients, these applications clearly hold even more potential for future developments. Yet it is vital that the regulatory environment keep up with this fast-evolving space of healthcare in order to anticipate and, to the extent possible, prevent the risks that may arise.

A riboflavin-responsive neuronopathy with unique characteristics: Brown-Vialetto- Van Laere syndrome

A riboflavin-responsive neuronopathy with unique characteristics: Brown-Vialetto- Van Laere syndrome

Abstract P1-04-07: Xiap expression is associated with infiltration of cd163+ tumor-associated macrophages in the tumor micro-environment of inflammatory breast cancer

Abstract BACKGROUND: Inflammatory breast cancer (IBC) is a rare, but aggressive type of locally advanced breast cancer for which a specific immune response seems to be an important driver (Van Laere et al., CCR 2013). The tumor immune micro-environment (TIME) of IBC is characterized by an influx of tumor-associated macrophages (TAMs) and patients with spatial colocalization of TAMs with tumor cells seem to have a worse prognosis (Van Berckelaer et al., SABCS 2019). In contrast with TAMs, the presence of CD8+ T cells was associated with a better prognosis in IBC. Cytotoxic T cells mediate killing of the tumor by inducing apoptosis after activation of effector caspases-3/-7, regulated by the X-Linked Inhibitor of Apoptosis protein (XIAP). Recently, we reported that XIAP in breast cancer is associated with shorter survival and resistance to chemotherapy (Devi et al, Cancers 2021). In IBC, XIAP is translationally upregulated during cellular stress and induces, via NFkB, an immunosuppressive signaling (Evan et al, Cancer Res 2018). Therefore, we investigated the interplay between tumor cell driven XIAP expression and the TIME. METHODOLOGY: IBC patient clinicopathological variables (n=75) along with Affymetrix data (n=30/75) were collected. Immunostainings for XIAP, CD8 and CD163 (Hematoxylin-DAB) were done according to validated protocols. All slides were digitized and evaluated in Visiopharm to quantify the number of DAB+ immune cells and the relative marker area (RMA: the DAB+ area/total area of interest). Spatial interactions were examined using an effector index (EI) that counts the number of immune cells in a circle with a radius of 30 mcm around a CD8+ T cell or a tumor cell. We evaluated correlations between XIAP protein and mRNA expression, immune cell signatures (using CIBERSORT) & gene-expression pathways as a validation of our IHC findings. RESULTS: High XIAP expression was detected in 45.3% of the IBC samples (n= 34/75). Although, XIAP expression was not associated with any clinicopathological parameters like grade, molecular subtype, or disease stage. High XIAP expressing tumors had more sTIL infiltration (P= 0.008), PDL1 expression (P= 0.012) and infiltration with TAMs (RMA: 1.03% (0.03% – 5.97%) vs. 2.73% (0.01% – 11.3%), P= 0.003). Furthermore, in these XIAP high samples TAMs were also located closer to the tumor cells (EI: 0.95 cells (0.06 – 3.48) vs. 2.04 cells (0.28 – 5.20), P< 0.001) and the CD8+ T cells (EI: 2.85 cells (0.26 – 8.85) vs. 5.13 cells (0.87 – 10.16), P< 0.001), while there was no correlation between XIAP and the total number of CD8+ cells. Although gene expression data analysis did not reveal any correlation between protein and mRNA expression of XIAP, there was a significant correlation between XIAP activation signature and protein expression (P<0.001). Furthermore, we discovered a strong association between XIAP protein and cellular stress response- & inflammation-related gene sets. Finally, in the CIBERSORT data, we observed the strongest correlation between XIAP expression and M2 macrophages & activated dendritic cells. CONCLUSION: This is the first study showing high XIAP expressing IBC tumors have increased infiltration of immunosuppressive TAMs, which co-localized near tumor and cytotoxic T cells. We also demonstrated an association between XIAP protein expression and inflammation related & cellular stress response gene signatures. This is relevant as XIAP function depends on translational upregulation during cellular stress which leads to immunosuppressive signaling and an aggressive breast cancer phenotype. XIAP antagonists are in clinical trials and these data suggest potential benefit in targeting XIAP to enhance immunotherapeutic efficacy in IBC therapy. FUNDING: DoDs-W81XWH-17-1-0297,W81XWH-20-1-0153, ACS Mission Boost Grants(GRD) Citation Format: Christophe Van Berckelaer, Steven Van Laere, Joseph Gerardts, Luc Dirix, Michael Morse, Mark Kockx, François Bertucci, Peter Van Dam, Gayathri R Devi. Xiap expression is associated with infiltration of cd163+ tumor-associated macrophages in the tumor micro-environment of 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 P1-04-07.

Abstract P5-08-14: Maternal embryonic leucine zipper kinase is associated with metastasis in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) has high rates of relapse and metastasis. It has a high proportion of cancer stem-like cells (CSCs), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, plays a critical role in promoting CSC maintenance, cell proliferation, and malignant transformation. Here, we assessed the role of MELK in TNBC aggressiveness. Methods: The clinical relevance of MELK in TNBC was analyzed using the World IBC Consortium dataset (n = 314). The effects of MELK knockdown (siRNA), knockout (CRISPR-Cas9), and MELK inhibition with a novel selective small molecule inhibitor MELK-In-7 on migration and invasion were determined using the migration and invasion assays, respectively. MELK’s role in regulating CSC phenotypes was assessed using the mammosphere assay and flow cytometry. The impact of MELK knockdown on epithelial-to-mesenchymal transition (EMT) was determined using Western blotting. MELK’s protumorigenic role was determined in vitro using soft agar assay and in vivo using TNBC xenograft mouse models. Results: MELK mRNA expression level was higher in TNBC tumors than in tumors expressing hormone receptor, HER2, or both (P < 0.0001). In univariate analysis, breast cancer patients with high-MELK-expressing tumors had worse overall survival (OS, P < 0.001) and distant metastasis-free survival (DMFS, P < 0.01) than patients with low-MELK-expressing tumors, suggesting a prognostic impact of MELK in breast cancer. MELK knockdown using siRNA or MELK inhibition using MELK-In-7 significantly reduced migration, invasion, mammosphere formation, CD24-/CD44+ subpopulations, and ALDH activity, and reversed EMT in TNBC cells. This result suggests a role for MELK in regulating CSCs and EMT, which play crucial roles in tumor invasion and metastasis. Nude mice injected with MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and significantly improved OS compared with the mice injected with parental MDA-MB-231 cells (P < 0.05). Further, MELK-selective inhibitor (MELK-In-7) suppressed the growth of 4T1 mouse TNBC tumors in a dose-dependent manner in syngeneic BALB/c mice (P < 0.001). These results indicate an essential role for MELK in promoting TNBC tumor growth and metastasis. To uncover the underlying molecular mechanism of MELK’s regulation of TNBC metastasis, we performed microarray analysis and identified SERPINE1, a serine protease inhibitor known to regulate metastasis, as a downstream target of MELK. We validated that mRNA levels of SERPINE1 were reduced in MELK-knockout MDA-MB-231 cells and MELK-In-7-treated MDA-MB-231 cells. Moreover, SERPINE1 knockdown using siRNA reduced migration and invasion of TNBC cells. These results suggest that MELK regulates TNBC cell motility through SERPINE1. Conclusion: This work confirms MELK as a driver of aggressiveness and metastasis in TNBC. The mechanisms underlying MELK’s regulation of TNBC metastasis via SERPINE1 require further studies. Citation Format: Xuemei Xie, Gaurav B. Chauhan, Ramakrishna Edupuganti, Takahiro Kogawa, Jihyun Park, Moises T Tacam, Fnu Vidhu, Diane D. Liu, Juliana M. Taliaferro, Mary Kathryn Pitner, Yu Shen, Naoto T. Ueno, Savitri Krishnamurthy, Gabriel N. Hortobagyi, Debu Tripathy, Steven J. Van Laere, Geoffrey Bartholomeusz, Kevin Dalby, Chandra Bartholomeusz. Maternal embryonic leucine zipper kinase is associated with metastasis in triple-negative 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 P5-08-14.

Abstract P3-09-18: The association between genomic alterations and body mass index in patients with early breast cancer

Abstract 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.

Abstract P3-14-18: Adaptive stress response genes associated with breast cancer subtypes and survival outcomes reveal race-related differences

Abstract Background & Hypothesis: The death rate is higher in Black (B) patients (40%), and, in particular, is two-fold higher among young B women (<50 years) compared to White (W) women with breast cancer. Aggressive breast cancer variants, like hormone receptor-negative and inflammatory breast cancer (IBC), contribute to a race-related survival gap and poor clinical outcomes in African American (AA) patients compared to Whites (W) in the United States. We previously identified the dominant role of XIAP in a tumor cell adaptive stress response (ASR) that promotes nuclear transcription factor (NFκB)-mediated proliferative, invasive, and immunosuppressive signaling in breast cancer progression. Our current work sought to address the urgent need to interrogate molecular mechanisms driving the disparity in both AA and IBC tumor biology by investigating gene expression differences between AA and White tumors and Triple-negative breast cancer (TNBC) and non-TNBC tumors, thereby identifying common genes which may be modifiable risk factors and targets that can be modulated for therapeutic benefits. Methods: We used The Cancer Genome Atlas (TCGA) breast cancer data to conduct a comparative analysis of the expression of 226 ASR genes in breast cancer molecular subtypes, normal-adjacent tissues, and in AA and W patients. The ASR gene list includes 101 XIAP-related, 11 NFκB targets, 10 MNK targets, 33 Oxidative Stress Response (OSR)-related, 13 TGFβ-related, 6 JAG1-Notch targets, and 52 immune-related, in addition to 14 genes that belong to more than one ASR set. In the present study, we focused on primary tumor samples [1090 Primary Solid Tumor] and normal tissue samples adjacent to the tumors [113 Solid Tissue Normal]. Within these, our sample set included 559 lumA, 207 lumB, 82 Her2, 190 Basal, and 40 Normal-like. Racial designations in TCGA are based on patient self-identification. In the present study, the race-related analysis focused on AA and W breast cancer patient datasets from TCGA (179 AA and 744 W). It is important to note that among the 113 Normal-adjacent samples, there are 105 W and only 6 AA samples. Results: Analysis of the ASR gene score in different Breast cancer subtypes revealed that ASR genes associated with oxidative stress and immune response pathways score highly in the basal breast cancer subtype. We also observed differential scores between samples of AA or W. The differential gene expression analysis identified 46-88 genes in the ASR set to be differentially expressed (≥2 fold-change and adjusted p-value <0.05) in breast cancer subtypes when comparing each subtype to normal-adjacent tissue or comparing the subtypes to each other. Pathway analysis of the enriched genes revealed the involvement of the cell cycle, the DNA damage response, the signal transduction, and the regulation of cell death-related processes. Moreover, on average, 20% of the ASR genes showed race-related differential expression. Furthermore, a number of ASR that were differentially expressed in breast cancer subtypes and significantly associated (hazard ratio >1.5 and p-value <0.05) with AA and/or W patient survival. For example, CDC45, MCM3, and CDKN2D were uniquely associated with poor survival in AA, but not in W. Conclusion: Within invasive breast cancer, factors that regulate tumor cell ASR reveal subtype and race-specific differences in expression. Understanding their function and downstream signaling will elucidate the intrinsic mechanisms that drive racial disparities in breast cancer outcomes and have the potential to reveal race-related biomarkers and therapeutic targets. Support: NCI-P20 NCCU-Duke Cancer Disparities Translational Research Partnership (SP; GRD, KPW, JF), DoD-W81XWH-17-1-0297 (GRD), Duke Bridge Funds (GRD). Citation Format: Muthana Al Abo, Larisa Gearhart-Serna, Steven Van Laere, Jennifer Freedman, Steven Patierno, Eun-Sil Hwang, Savitri Krishnamurthy, Kevin Williams, Gayathri Devi. Adaptive stress response genes associated with breast cancer subtypes and survival outcomes reveal race-related differences [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 P3-14-18.

Abstract PS17-13: The prognostic role of tumor-infiltrating lymphocytes after treatment with neo-adjuvant chemotherapy in inflammatory breast cancer patients

Abstract Introduction: Inflammatory breast cancer (IBC) is a rare, but aggressive form of breast cancer. Increasing evidence indicates that immune cells in the tumor micro-environment play an important role in IBC progression: infiltration with stromal Tumor Infiltrating Lymphocytes (sTIL) is associated with a better response to neo-adjuvant chemotherapy (NACT) and longer overall survival in IBC patients. However, the prognostic role of sTIL in patients without a complete pathological response (pCR) after NACT remains unclear. In this study we evaluated the effect of NACT on sTIL in IBC and locally advanced non-inflammatory breast cancer (LABC) and the prognostic impact of sTIL in IBC. Methodology: In this retrospective case-control study we evaluated sTIL in patients with IBC (n=60) and LABC (n= 134) that received anthracyclin-taxane based NACT. Tumor tissue was sampled as part of the routine work-up, before (diagnostic biopsy) and after (resection specimen) NACT. sTIL scoring was performed on Haematoxylin & Eosin stained 5-µm sections of formalin-fixed paraffin-embedded tumor tissue by two different researchers according to the recommendations by the International TILs Working Group. In case of discrepancy, the sample was scored by a third observer in order to obtain a consensus score. sTIL difference between pre-treatment and post-treatment specimen was called δsTIL. Cellularity of the residual cancer after NACT was evaluated according to the MD Anderson Residual Cancer Burden (RCB) method. Results: Most of the IBC patients presented with a hormone receptor (HR) positive carcinoma (n=35/60, 58.3%) and 25 patients had pCR after NACT (42%); the latter patients had less than 1% sTIL in the tumor bed area. Besides having histologically more poorly differentiated tumors (P= 0.033), no significant clinicopathological differences between the IBC and LABC cohort were observed. There was no significant difference in the pre-treatment median sTIL score between IBC (12.5%, range: 1% - 80%) and LABC (10%, range: 1% - 85%). Furthermore, both in the IBC (median δsTIL: -4.5%, P= 0.01) and in the LABC (median δsTIL: -1%, P= 0.06) cohort the number of sTIL was lower after NACT. This decrease was significantly greater in the IBC cohort (P= 0.04). In a multivariate model - including HR and HER2 status, histological differentiation grade, nodal status and IBC/LABC phenotype - IBC disease correlated with a stronger decrease of sTILs after NACT (OR: 0.31, 95% CI 0.10 - 0.89, P= 0.04). As we previously demonstrated, higher sTIL score before NACT was associated with better overall survival (OS) (P= 0.006) in IBC. IBC patients without pCR, but with a stronger decrease (> 4.5 %) of the number of sTIL after NACT had a better OS (P= 0.04) and disease-free survival (DFS) (P= 0.04). There was a significant correlation between δsTIL and both higher sTIL score before NACT (P< .001) and lower residual cancer cellularity (P= 0.02). However, in a multivariate model - including sTIL before NACT, δsTIL and residual cancer cellularity - both a strong decrease of sTIL (HR: 0.32; 95% CI 0.10 - 1.00; P= 0.05) and a low residual cancer cellularity (HR: 0.22; 95% CI 0.07 - 0.68; P= 0.009) were independent predictors of a better DFS. Conclusion: Both the IBC and LABC cohort had the same number of sTIL before NACT. However, the IBC phenotype was associated with a stronger decrease of sTIL after NACT independent of molecular subtype and nodal stage. Furthermore, a strong decrease in sTIL was an independent predictor of better prognosis in IBC. Citation Format: Christophe Van Berckelaer, Leonie Vercauteren, Iris Vermeiren, Glenn Broeckx, Steven Van Laere, Luc Dirix, Cecile Colpaert, Peter Van Dam. The prognostic role of tumor-infiltrating lymphocytes after treatment with neo-adjuvant chemotherapy in inflammatory breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-13.

Functional Study of the Human Riboflavin Transporter 2 Using Proteoliposomes System.

Riboflavin is essential for cell viability. The biologically active forms of riboflavin, FMN and FAD, participate in many biochemical redox reactions including the metabolism of carbohydrates, amino acids, and lipids. Differently from bacteria, fungi, and plants which synthesize riboflavin, higher organisms have lost the ability to synthesize the vitamin and must absorb it from food and intestinal microflora production. The riboflavin flux through cell membranes occurs via specific transporters belonging to the SLC52 family. Three members of this family have been identified so far which show poor homology with the riboflavin transporters of Saccharomyces cerevisiae or bacteria. Alterations of RFVTs are causative of severe diseases. Indeed, under pathological stress, humans are susceptible of developing riboflavin deficiency. Such a deficiency in pregnancy induces fetus abnormalities, and has been indicated as a risk factor for anemia, cancer, cardiovascular diseases, and neurodegeneration. Moreover, inherited diseases are also of interest; the most well-described is the Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset sensorineural deafness and pontobulbar palsy. Numerous polymorphisms of Slc52a2 and Slc52a3 genes associated with this syndrome have been discovered. In spite of their important metabolic role and their relevance to human health, the riboflavin transporters are still poorly characterized. Bacterial overexpression, purification, and protein reconstitution in liposomes represent an up-to-date methodology for obtaining functional data information. The methodology for reconstituting the RFVT2 into proteoliposomes and performing transport assay is described. These methods will be suitable for investigating the functional defects of the variants of RFVTs associated with human pathologies.

Imaging Acute Metabolic Changes in Patients with Mild Traumatic Brain Injury Using Hyperpolarized [1-13C]Pyruvate.

SummaryTraumatic brain injury (TBI) involves complex secondary injury processes following the primary injury. The secondary injury is often associated with rapid metabolic shifts and impaired brain function immediately after the initial tissue damage. Magnetic resonance spectroscopic imaging (MRSI) coupled with hyperpolarization of 13C-labeled substrates provides a unique opportunity to map the metabolic changes in the brain after traumatic injury in real-time without invasive procedures. In this report, we investigated two patients with acute mild TBI (Glasgow coma scale 15) but no anatomical brain injury or hemorrhage. Patients were imaged with hyperpolarized [1-13C]pyruvate MRSI 1 or 6 days after head trauma. Both patients showed significantly reduced bicarbonate (HCO3–) production, and one showed hyperintense lactate production at the injured sites. This study reports the feasibility of imaging altered metabolism using hyperpolarized pyruvate in patients with TBI, demonstrating the translatability and sensitivity of the technology to cerebral metabolic changes after mild TBI.

Self-perceived health problems and unmet care needs of homeless people in the Netherlands : the need for pro-active integrated care

Homeless people suffer from bad health and encounter barriers in accessing services, and live in difficult social circumstances. However, no insight exists in how homeless people in the Netherlands themselves experience their health and unmet care needs in relation to their social Marieke Verheul MSc MD Department of Primary and Community Care Radboud university medical center, Nijmegen, the Netherlands Igor van Laere PhD MD Netherlands Streetdoctors Group, the Netherlands 22 Journal of Social Intervention: Theory and Practice – 2020 – Volume 29, Issue 1 Self-perceived health problems and unmet care needs of homeless people in the Netherlands circumstances. In order to tailor medical and other services to their needs, this study aims to gain insight into the self-perceived health problems, and unmet care needs in relation to the social circumstances of homeless people in the Netherlands.

Abstract P6-06-08: Transcriptome analysis of patient derived xenograft mouse models of inflammatory breast cancer to gain insights into the contribution of tumor microenvironment

Abstract Purpose: Inflammatory breast cancer (IBC) is an aggressive variant of breast cancer characterized by visible skin symptoms on the breast at the time of presentation. We demonstrated mesenchymal stem cells (MSC) promote skin symptoms in the SUM149 xenograft model. Here, we extend this finding to IBC patient-derived xenograft (PDX) lines and performed RNA-sequencing analysis of tumors from IBC patient-derived xenograft (PDX) lines by presence of skin symptoms. We hypothesize host MSC variation may explain sporadic skin symptoms in IBC PDX tumors across generations. Methods: MSC were co-injected at the time of tumor initiation in PDX tumors (IBC-3, 5, 6, and 7) and skin symptoms assessed visually and at the time of gross resection. A priori analysis plan was to group PDX by treatment group irrespective of PDX line. IBC PDX models spontaneously but inconsistently develop skin symptoms after multiple passages. RNA sequencing was performed on IBC-PDX tumors (n=33) and non-IBC PDX tumors (n=12) across generations from IBC PDX lines 5, 6, and 7 and non-IBC PDX lines 2, 3, and 4. Four samples were discarded due to poor quality. TSNE was applied using the top differential expressed genes. Xenome analysis was performed to examine host stromal expression by skin involvement. Samples from PDX lines 5 were compared with lines 6 and 7 and the top differentially expressed genes were compared with the Molecular Signatures Databases (MSigDB). CIBERSORT was performed to deconvolute cell type composition. Results: Co-injecting MSC with PDX transplant increased skin symptoms (57% of PDX animals (cohort from IBC5, IBC6, IBC7 and non-IBC4) with MSC vs. 0% without (cohort from IBC5, IBC6, IBC7, non-IBC2 and non-IBC4) (P = 0.0007). TSNE analysis of IBC PDX split samples into three subgroups distinguished by PDX lines and skin invasion. Xenome was used to split reads from human or mouse: samples show high percentage of reads coming from human (mean±SD, 97.44%± 8.98%). Seventy-four genes show FDR ≤ 0.2 between PDX line 5 and line 6 and comparing these top 74 genes with MSigDb for interaction revealed pathways such as GPCR, signal transduction, and sensory perception. CIBERSORT revealed no enrichment for MSC in IBC PDX with skin invasion, however significant differences in CD8 expression in these immunosuppressed PDX models casts doubt on the robustness of this finding. Conclusions: Exogenous MSC enhance skin symptoms in a cohort of IBC and non-IBC PDX. Skin symptoms segregate IBC PDX tumors by expression suggesting specific biology drives this presentation. However, we are unable to demonstrate evidence for spontaneous MSC signaling in these models. Future work is warranted to link top skin symptom related signaling to stromal signals and unravel the unexpected CD8 signaling identified in these models. Citation Format: Omar Rahal, Jie Yang, Li Li, Richard Larson, Steven Van Laere, Naoto Ueno, Erik Sulman, Wendy Woodward. Transcriptome analysis of patient derived xenograft mouse models of inflammatory breast cancer to gain insights into the contribution of tumor microenvironment [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 P6-06-08.

Abstract P5-04-04: The spatial interactions between FOXP3+ Tregs, CD8+ cytotoxic T cells and tumor cells predict response to therapy and prognosis in inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare and aggressive type of locally advanced breast cancer. A 79-gene signature, reported by our lab, is shaped by specific immune response programs and discriminates between IBC and non-IBC (nIBC). However, it remains an enigma how infiltrating immune cells are able to determine the IBC phenotype. Furthermore, the presence of immune cells like FOXP3+ Tregs and CD8+ cytotoxic T cells is associated with outcome in proliferative subtypes of breast cancer and the interaction between these cells plays a role in the functional immune response. Therefore, we aimed to assess the spatial associations between immune cells in IBC. Additionally, we used deep-learning to examine interactions between cancer and immune cells. Methodology: Immunostainings (Hematoxylin-DAB, H-DAB) were done according to well-validated protocols for CD8 (cytotoxic T-cells), FOXP3 (Tregs) and CD163 (tumor-associated macrophages, TAMs) in a large population of 134 IBC patients. All slides were digitalized and evaluated using VISIOPHARM® software, allowing virtual multiplexing. We quantified the number of DAB+ immune cells and each positive immune cell was located using XY coordinates. Spatial co-localization was examined using statistics developed for ecological studies based on point pattern and quadrant analysis. TILs were scored according to the TIL working group guidelines on H&E slides. Tumor cell coordinates were collected using a deep-learning algorithm applied to the CD8 stained slide. To perform deep-learning, we aligned two consecutive slides: one PanCK stained slide and one slide stained for CD8 (H-DAB). Using virtual multiplexing and the PanCK staining, we determined the tumor regions on the H-DAB stained slide. Subsequently, 18 images were incorporated to train the algorithm with more than 150.000 iterations (Deeplabv3+), after which the algorithm was evaluated in a test set of 12 images, approved and applied to all images to locate the tumor cells. Results: Most of the patients presented with a hormone receptor (HR) positive carcinoma (60.6%, n= 82/132). The presence of distant disease, HR status and TIL score were associated with overall survival (OS), but the density of the different immune cells or the CD8/FOXP3 ratio was not. However, using an effector index we demonstrated that patients with more FOXP3+ cells in a radius of 30 μm surrounding a CD8+ cell had a significant worse outcome (Median OS: 2.7 vs. 6.3 years, P= .01) and this remained significant in a multivariate model (HR: 2.85, P< .001). Complete pathological response (pCR) after neo-adjuvant chemotherapy was achieved by 28.7% (n= 27/94) of the patients with initially localized disease. Infiltration with CD8+ T cells (P= .005), TAMs (P= .008) and TILs (P= .002) predicted pCR, but a likelihood ratio test showed no difference between a model using CD8+ cells, TAMs or TILs. Interestingly, pCR was less often achieved in patients that had colocalization of FOXP3+ cells near the tumor cells (P= .003). This colocalization was determined using a Morista-Horn index and independent of the number of Tregs, CD8+ cells or TILs. The deep-learning algorithm had a mean dice coefficient in the test set of 0.83, indicating a good overlap between the tumor area determined by the AI on the CD8 slides and the area on the PanCK stained slides. Conclusions: We have created a deep learning algorithm that adequately detects IBC tumor cells on H-DAB stained images and show, in a large cohort of 132 patients, that the negative impact of Tregs appears to depend on the spatial arrangement. While solely the number of Tregs is not associated with pCR or OS, patients with FOXP3+ Tregs that cluster together near CD8+ cytotoxic T cells had a worse outcome and pCR was achieved more often in patients with less Tregs near the tumor cells. Citation Format: Christophe Van Berckelaer, Charlotte Rypens, Steven Van Laere, Koen Marien, Pieter-Jan van Dam, Peter Vermeulen, Luc Dirix, Mark Kockx, Cecile Colpaert, Peter van Dam. The spatial interactions between FOXP3+ Tregs, CD8+ cytotoxic T cells and tumor cells predict response to therapy and prognosis in inflammatory breast cancer [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 P5-04-04.

Abstract P3-01-13: Association between the histopathological growth patterns (HGP) of liver metastases (LM) and survival after hepatic surgery in patients with oligometastatic breast cancer (BC)

Abstract 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.

Abstract P3-03-03: Inflammatory breast cancer exhibits amplification and transcriptional activation of MYC in conjunction with NOTCH and TGFβ signaling

Abstract Inflammatory breast cancer (IBC) is an aggressive and poorly characterized breast cancer subtype with few targets available for molecular therapy. Several attempts aimed at characterizing the disease at the genomic, transcriptomic and proteomic level. Together, these studies suggest that MYC is central to the biology of IBC. Here, we analyzed data sets from patient samples and preclinical models to corroborate this hypothesis. Genomic and transcriptomic profiles were obtained using next-generation sequencing and Affymetrix GeneChip analysis on a series of 101 and 137 samples from patients with IBC. In addition, non-IBC (nIBC) control samples were included from our own repositories (N=252) as well as from publicly available data sets (i.e. TCGA and METABRIC). Finally, RNA-sequencing and peptide phosphorylation data from preclinical IBC and nIBC models, both with and without TGFβ treatment, were explored. Apart from TP53 and ERBB2, MYC is the 3rd most frequently mutated gene with single nucleotide variants, indels and amplifications found in 22% of 101 patients. Furthermore, MYC mutations are mutually exclusive with alterations in the NOTCH genes (i.e. NOTCH1, -2, -3, and -4 and CREBBP). At the gene expression level, a MYC activation score is elevated in IBC as compare to nIBC and correlates with a NOTCH activation signature. Genes overexpressed in IBC are enriched for various sets of MYC target genes and are associated with MYC amplifications in TCGA and METABRIC samples. In preclinical models, MYC target genes are particularly responsive to TGFβ treatment in IBC cells but not in nIBC cells and analysis of peptide phosphorylation patterns suggest this interaction may be regulated via PTK2B. The present data suggest that MYC could be an important driver of IBC biology, which corroborates with the recent observation that MYC is an important hub in the signal transduction network of SUM149 and -190 cells. Furthermore, our data reveal that MYC activation is linked to NOCTH and TGFβ signaling. In earlier studies it was demonstrated that lymphovascular tumor emboli in the Mary-X mouse model of IBC exhibit NOTCH3 addition with downstream MYC overexpression, indicating that NOTCH/MYC signaling could be crucial for metastatic progression in IBC. In this context, it should be noted that an allele associated with metastatic risk in IBC patients resides at the 8q24 locus that harbors the MYC gene. Citation Format: Charlotte Rypens, François Bertucci, Patrice Viens, John WM Martens, Marcel Smid, Carolien Schröder, Naoto T Ueno, Massimo Cristofanilli, Piet Dirix, Peter Vermeulen, Luc Dirix, Steven Van Laere. Inflammatory breast cancer exhibits amplification and transcriptional activation of MYC in conjunction with NOTCH and TGFβ signaling [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 P3-03-03.

Abstract P3-01-08: Infiltrating immune cells in the tumor emboli of inflammatory breast cancer

Abstract INTRODUCTION: Inflammatory breast cancer (IBC) is a rare and aggressive type of locally advanced breast cancer. The hallmark of IBC is the presence of E-Cadherin positive tumor emboli within the dermal and parenchymal lymphatic vessels. These tumor emboli are thought to be responsible for the clinical presentation by the physical obstruction of draining lymph vessels. In addition, the intravascular growth of tumor emboli may account for the typical diffuse growth pattern and the fast local spread associated with IBC. Tumor emboli can result from encircling lymphovasculogenesis or lymphovascular invasion and little is known about the infiltrating immune cells in these emboli. This is the first study to investigate the immune infiltrate in the tumor emboli of patients with IBC. METHODOLOGY: To analyze the immune composition of the emboli, four slides of 24 IBC patients were stained according to a validated protocol and scored using the VISIOPHARM® software that makes virtual multiplexing possible after the alignment of different digital slides. We used four validated antibodies: CD79α (B-cell lineage), CD8 (cytotoxic T-cells), FOXP3 (Tregs) and CD163 (Tumor associated macrophages; TAMs), combined with a standard H&E stained section to mark the tumor area and tumor emboli on pretreatment biopsy sections. RESULTS: Half of the IBC patients presented with a hormone receptor positive carcinoma (54.2% - n= 13/24) and 8 out of 21 patients with initially localized disease achieved complete pathological response (pCR) after neo-adjuvant chemotherapy (NACT). Our results are summarized in table 1. Table 1: For every staining we report the number of patients that have infiltrated emboli, the median density in the invasive carcinoma (IC), the median density in the emboli (E) and the ratio between the invasive tumor and emboli in table 1.# patientsDensity (IC) #/μm2Density (E) #/μm2Ratio Density E/ICP-valueCD812/24 (50 %)19.62.40.11< .001CD16319/23 (83 %)35.412.80.43.02CD79α11/24 (46 %)18.23.50.31.01FOXP34/23 (17 %)5.10.30.04.003 Patients with more immune cells in the invasive carcinoma (IC) also had more immune cells in the emboli (E). This correlation was significant for all stainings (Table 1). CD163+ macrophages are the most abundant immune cells in emboli, especially when compared to the invasive carcinoma, whereas CD8+ cells were uncommon in the emboli. The density of CD8+ cells in the invasive carcinoma or emboli did not predict pCR. Interestingly, patients with a higher IC/E CD8 density ratio achieved more often pCR (Kruskal-Wallis χ = 3.6, P = 0.05). Only 20.8% (n= 5/24) had invasive carcinoma directly surrounding the intravascular emboli, however, median overall survival in these patients was significantly shorter (P= 0.003). CONCLUSIONS: This is the first study looking at infiltrating immune cells in IBC. Although based on a small discovery cohort of only 24 patients, we showed that there is a strong correlation between the density of immune cells in the emboli and the density in the invasive carcinoma. Furthermore, we demonstrate that TAMs are the predominant immune cell type in IBC emboli. Cytotoxic T cells are less common and having more CD8+ cells in the invasive carcinoma compared to the emboli was associated with pCR. Patients with invasive carcinoma directly surrounding the emboli had a worse prognosis. Citation Format: Christophe Van Berckelaer, Charlotte Rypens, Peter Vermeulen, Steven Van Laere, Peter Van Dam, Cecile Colpaert. Infiltrating immune cells in the tumor emboli of inflammatory breast cancer [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 P3-01-08.