Titel Deelnemers "Korte inhoud" "Phase II Trial Assessing the Repeatability and Tumor Uptake of [68Ga]Ga-HER2 Single-Domain Antibody PET/CT in Patients with Breast Carcinoma" "Odrade Gondry, Vicky Caveliers, Catarina Xavier, Marian Vanhoeij, Guy Verfaillie, Christel Fontaine, Katrien Glorieus, Jacques De Grève, Sofie Joris, Ine Luyten, Karen Zwaenepoel, Frederik Vandenbroucke, Wim Waelput, Sheeno Thyparambil, Ilse Vaneycken, Julie Cousaert, Sophie Bourgeois, Nick Devoogdt, Lode Goethals, Hendrik Everaert, Frank De Geeter, Tony Lahoutte, Marleen Keyaerts, Laurens Raes" "Human epidermal growth factor receptor 2 (HER2) status is used for decision-making in breast carcinoma treatment. The status is obtained through immunohistochemistry or in situ hybridization. These two methods have the disadvantage of necessitating tissue sampling, which is prone to error due to tumor heterogeneity or interobserver variability. Whole-body imaging might be a solution to map HER2 expression throughout the body. Methods: Twenty patients with locally advanced or metastatic breast carcinoma (5 HER2-positive and 15 HER2-negative patients) were included in this phase II trial to assess the repeatability of uptake quantification and the extended safety of the [68Ga]Ga-NOTA-anti-HER2 single-domain antibody (sdAb). The tracer was injected, followed by a PET/CT scan at 90 min. Within 8 d, the procedure was repeated. Blood samples were taken for antidrug antibody (ADA) assessment and liquid biopsies. On available tissues, immunohistochemistry, in situ hybridization, and mass spectrometry were performed to determine the correlation of HER2 status with uptake values measured on PET. If relevant preexisting [18F]FDG PET/CT images were available (performed as standard of care), a comparison was made. Results: With a repeatability coefficient of 21.8%, this imaging technique was repeatable. No clear correlation between PET/CT uptake values and pathology could be established, as even patients with low levels of HER2 expression showed moderate to high uptake. Comparison with [18F]FDG PET/CT in 16 patients demonstrated that in 7 patients, [68Ga]Ga-NOTA-anti-HER2 shows interlesional heterogeneity within the same patient, and [18F]FDG uptake did not show the same heterogeneous uptake in all patients. In some patients, the extent of disease was clearer with the [68Ga]Ga-NOTA-anti-HER2-sdAb. Sixteen adverse events were reported but all without a clear relationship to the tracer. Three patients with preexisting ADAs did not show adverse reactions. No new ADAs developed. Conclusion: [68Ga]Ga-NOTA-anti-HER2-sdAb PET/CT imaging shows similar repeatability to [18F]FDG. It is safe for clinical use. There is tracer uptake in cancer lesions, even in patients previously determined to be HER2-low or -negative. The tracer shows potential in the assessment of interlesional heterogeneity of HER2 expression. In a subset of patients, [68Ga]Ga-NOTA-anti-HER2-sdAb uptake was seen in lesions with no or low [18F]FDG uptake. These findings support further clinical development of [68Ga]Ga-NOTA-anti-HER2-sdAb as a PET/CT tracer in breast cancer patients." "Comparison of Omentum and Subcutis as Implant Sites for Device-Encapsulated Human iPSC-Derived Pancreatic Endoderm in Nude Rats" "Jolien Nijns, Ines De Mesmaeker, Krista Suenens, Geert Stange, Kaat De Groot, Maria Marques de Lima, Marine R C Kraus, Bart Keymeulen, Wim Waelput, Daniel Jacobs-Tulleneers-Thevissen, Danny Pipeleers" "Subcutaneous implants of device-encapsulated stem cell-derived pancreatic endoderm (PE) can establish a functional beta cell mass (FBM) with metabolic control in immune-compromised mice. In a study with human-induced pluripotent stem cell-PE, this outcome was favored by a preformed pouch which allowed lesion-free insertion of devices in a pre-vascularized site. This was not reproduced in nude rats, known to exhibit a higher innate reactivity than mice and therefore relevant as preclinical model: a dense fibrotic capsule formed around subcutis (SC) implants with virtually no FBM formation. Placement in omentum (OM) of nude rats provided a less fibrous, better vascularized environment than SC. It resulted in less donor cell loss (56% recovery at post-transplant-PT week 3 versus 16% in SC) allowing FBM-formation. At PT week 30, 6/13 OM-recipients exhibited glucose-induced plasma hu-C-peptide to 0.1-0.4 ng/ml, versus 0/8 in SC-recipients. These levels are more than 10-fold lower than in a state of metabolic control. This shortcoming is not caused by inadequate glucose responsiveness of the beta cells but by their insufficient number. The size of the formed beta cell mass (0.4 ± 0.2 µl) was lower than that reported in mice receiving the same cell product subcutaneously; the difference is attributed to a lower expansion of pancreatic progenitor cells and to their lower degree of differentiation to beta cells. This study in the nude rat model demonstrates that OM provides a better environment for formation of beta cells in device-encapsulated PE-implants than SC. It also identified targets for increasing their dose-efficacy." "Discovery and 3D imaging of a novel ΔNp63-expressing basal cell type in human pancreatic ducts with implications in disease" "Sandrina Martens, Katarina Coolens, Mathias Van Bulck, Hediel Madhloum, Yves Heremans, Gunter Leuckx, Henry Heimberg, Luc Bouwens, Patrick Jacquemin, Diedert Luc De Paep, Pieter in 't Veld, Nicky D'haene, Wim Waelput, Pierre Lefesvre, Francisco X Real, Ilse Rooman" "OBJECTIVE: The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas.DESIGN: We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed.RESULTS: In normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme.CONCLUSION: In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models." "Undetectable viral RNA in follicular fluid, cumulus cells, and endometrial tissue samples in SARS-CoV-2-positive women" "Liese Boudry, Wafaa Essahib, Ileana Mateizel, Hilde Van De Velde, Deborah De Geyter, Denis Pierard, Wim Waelput, Valerie Uvin, Herman Tournaye, Michel De Vos, Michaël De Brucker" "OBJECTIVE: To study the presence of viral RNA in the follicular fluid, cumulus cells, and endometrial tissue samples in SARS-CoV-2-positive women undergoing assisted reproductive technology (ART).DESIGN: Prospective, single-center, observational study.SETTING: Tertiary hospital.PATIENT(S): A total of 16 patients undergoing transvaginal oocyte retrieval who had a positive SARS-CoV-2 RNA test" "Efficacy of a Flat Low Dose of Nivolumab in Advanced Cancer" "Sofie Joris, Christel Fontaine, Lore Decoster, Jacques Vanderauwera, Kris Thielemans, Wim Waelput, Jacques De Grève" "BACKGROUND/AIM: Immunotherapy with PD-1/PDL1 blocking monoclonal antibodies has improved survival compared to the standard-of-care chemotherapy for several malignancies at different stages of these malignancies. Due to several reasons, many cancer patients in medical need have no access to these drugs. In this study, we aimed to investigate whether a low dose of nivolumab could also lead to a therapeutic response.PATIENTS AND METHODS: Patients with advanced cancer were treated with a flat low dose of 10 mg of nivolumab IV every two weeks at no drug cost.RESULTS: Disease control was noted in nine of the 18 patients. Two patients achieved complete remission, two had prolonged partial remission, and five had stable disease, of these only two experienced adverse events.CONCLUSION: A flat low dose of nivolumab may have clinical activity and is a cheap therapeutic option in patients in medical need for whom standard-dose immune checkpoint inhibitors are not accessible for any reason." "Assessing Tumor-Infiltrating Lymphocytes in Breast Cancer" "Hanne Locy, Stefaan Verhulst, Wilfried Cools, Wim Waelput, Stefanie Brock, Louise Cras, Ann Schiettecatte, Jan Jonckheere, Leo A van Grunsven, Marian Vanhoeij, Kris Thielemans, Karine Breckpot" "Scoring of tumor-infiltrating lymphocytes (TILs) in breast cancer specimens has gained increasing attention, as TILs have prognostic and predictive value in HER2+ and triple-negative breast cancer. We evaluated the intra- and interrater variability when scoring TILs by visual inspection of hematoxylin and eosin-stained tissue sections. We further addressed whether immunohistochemical staining of these sections for immune cell surface markers CD45, CD3, CD4, and CD8 and combination with nanoString nCounter® gene expression analysis could refine TIL scoring. Formalin-fixed paraffin-embedded and fresh-frozen core needle biopsies of 12 female and treatment-naive breast cancer patients were included. Scoring of TILs was performed twice by three independent pathologists with a washout period of 3 days. Increasing intra- and interrater variability was observed with higher TIL numbers. The highest reproducibility was observed on tissue sections stained for CD3 and CD8. The latter TIL scores correlated well with the TIL scores obtained through nanoString nCounter® gene expression analysis. Gene expression analysis also revealed 104 and 62 genes that are positively and negatively related to both TIL scores. In conclusion, integration of immunohistochemistry and gene expression analysis is a valuable strategy to refine TIL scoring in breast tumors." "Comprehensive genome-wide analysis of routine non-invasive test data allows cancer prediction" "Liesbeth Lenaerts, Nathalie Brison, Charlotte Maggen, Leen Vancoillie, Huiwen Che, Peter Vandenberghe, Daan Dierickx, Lucienne Michaux, Barbara Dewaele, Patrick Neven, Giuseppe Floris, Thomas Tousseyn, Lore Lannoo, Tatjana Jatsenko, Isabelle Vanden Bempt, Kristel Van Calsteren, Vincent Vandecaveye, Luc Dehaspe, Koenraad Devriendt, Eric Legius, Kris Van den Bogaert, Joris Robert Vermeesch, Frédéric Amant" "Background: Implausible false positive results in non-invasive prenatal testing (NIPT) have been occasionally associated with the detection of occult maternal malignancies. Hence, there is a need for approaches allowing accurate prediction of whether the NIPT result is pointing to an underlying malignancy, as well as for organized programs ensuring efficient downstream clinical management of these cases.Methods: Using a data set of 88,294 NIPT performed at University Hospital Leuven (Belgium) between November 2013 and March 2020, we retrospectively evaluated the positive predictive value (PPV) of our NIPT approach for cancer detection. In this approach, whole-genome cell-free DNA (cfDNA) data from NIPT were scrutinized for the presence of (sub)chromosomal copy number alterations (CNAs) predictive for a malignancy, using an unbiased NIPT analysis pipeline coined GIPSeq. For suspected cases, the presence of a maternal cancer was evaluated via subsequent multidisciplinary clinical follow-up examinations. The cancer-specificity of the identified CNAs in cfDNA was assessed through genetic analyses of a tumor biopsy.Findings: Fifteen women without a cancer history were identified with a GIPSeq result suggestive of a malignant process. Their cfDNA profiles showed either genome-wide aberrations or a single trisomy 8. Upon clinical examinations, a solid or hematological cancer was identified in 4 and 7 cases, respectively. Three women were identified as having a clonal mosaicism. For one case no underlying condition was found. These numbers add to a PPV of 73%. Based on this experience, we presented a multidisciplinary care path for efficient clinical management of these cases.Interpretation: The presented approach for analysing NIPT results has a high PPV, yet unknown sensitivity, for detecting asymptomatic malignancies upon routine NIPT. Given the complexity of diagnosing a pregnant woman with cancer, clinical follow-up should occur in a well-designed multidisciplinary setting, such as via the care model that we presented here.Funding: This work was supported by Research Foundation Flanders and KU Leuven funding." "Overcoming the Challenges of High Quality RNA Extraction from Core Needle Biopsy" "Hanne Locy, Rohann J.M. Correa, Dorien Autaers, Ann Schiettecatte, Jan Jonckheere, Wim Waelput, Louise Cras, Stefanie Brock, Stefaan Verhulst, Keith Kwan, Marian Vanhoeij, Kris Thielemans, Karine Breckpot" "The use of gene expression profiling (GEP) in cancer management is rising, as GEP can be used for disease classification and diagnosis, tailoring treatment to underlying genetic determinants of pharmacological response, monitoring of therapy response, and prognosis. However, the reliability of GEP heavily depends on the input of RNA in sufficient quantity and quality. This highlights the need for standard procedures to ensure best practices for RNA extraction from often small tumor biopsies with variable tissue handling. We optimized an RNA extraction protocol from fresh-frozen (FF) core needle biopsies (CNB) from breast cancer patients and from formalin-fixed paraffin-embedded (FFPE) tissue when FF CNB did not yield sufficient RNA. Methods to avoid ribonucleases andto homogenize or to deparaffinize tissues and the impact of tissue composition on RNA extraction were studied. Additionally, RNA’s compatibility with the nanoString nCounter® technology was studied. This technology platform enables GEP using small RNA fragments. After optimization of the protocol, RNA of high quality and sufficient quantity was obtained from FF CNB in 92% of samples. For the remaining 8% of cases, FFPE material prepared by the pathology department was used for RNA extraction. Both resulting RNA end products are compatible with the nanoString nCounter® technology." "Evaluation of an online training tool for scoring programmed cell death ligand-1 (PD-L1) diagnostic tests for lung cancer" "Bharat Jasani, Gudrun Bänfer, Rebecca Fish, Wim Waelput, Yves Sucaet, Craig Barker, Jessica L. Whiteley, Jill Walker, Rudy Hovelinck, Rolf Diezko" "BACKGROUND: Numerous studies indicate that higher tumour programmed cell death ligand-1 (PD-L1) expression is associated with greater response to anti-programmed cell death-1 (PD-1)/PD-L1 immunotherapy in non-small cell lung cancer (NSCLC). In the era of precision medicine, there is a need to provide reliable, standardised training for pathologists to improve their accuracy of interpretation and scoring, as the results are used directly to inform clinical decisions. Here we present findings regarding reader reproducibility of PD-L1 tumour cell (TC) staining scoring for NSCLC using a PD-L1 e-trainer tool as part of a PD-L1 immunohistochemistry reader training course.METHODS: The PD-L1 training course was developed based on the use of VENTANA PD-L1 (SP263) and Dako PD-L1 IHC PharmDx 22C3 stained NSCLC samples in combination with a PD-L1 e-trainer tool. Five-hundred formalin-fixed, paraffin-embedded archival samples were obtained from commercial sources and stained for PD-L1. Slides were scored by two expert pathologists, then scanned to produce digital images and re-scored. Thirty-three cases were selected and sorted into three sets: a training set and two self-assessment tests (pre-test and 'competence' test). Participants (all selected board-certified pathologists) received face-to-face training including use of an e-trainer tool. Statistical analyses were performed using the competence test set. Overall percentage agreement (OPA) was assessed between the participant pathologists' registered scores and the reference scores assigned by expert pathologists at clinically relevant PD-L1 cut-offs (≥1%, ≥25% and ≥ 50%).RESULTS: Seven sessions were held and 69 participant pathologists completed the training. Inter-reader concordance indicated high OPA (85-95%) for PD-L1 TC scoring at clinically relevant cut-offs, with Fleiss' Kappa > 0.5.CONCLUSIONS: Use of this web-based training tool incorporated into classroom-style training was associated with an overall moderately good level of inter-reader reproducibility at key cut-offs for TC PD-L1 expression testing in NSCLC. Overall, the online training tool offers a means of standardised training for practising pathologists in a clinical setting." "Evaluation of Distance Metrics and Spatial Autocorrelation in Uniform Manifold Approximation and Projection Applied to Mass Spectrometry Imaging Data" "Tina Smets, Marc Claesen, Thomas Tousseyn, Wim Waelput" "In this work, uniform manifold approximation and projection (UMAP) is applied for nonlinear dimensionality reduction and visualization of mass spectrometry imaging (MSI) data. We evaluate the performance of the UMAP algorithm on MSI data sets acquired in mouse pancreas and human lymphoma samples and compare it to those of principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), and the Barnes-Hut (BH) approximation of t-SNE. Furthermore, we compare different distance metrics in (BH) t-SNE and UMAP and propose the use of spatial autocorrelation as a means of comparing the resulting low-dimensional embeddings. The results indicate that UMAP is competitive with t-SNE in terms of visualization and is well-suited for the dimensionality reduction of large (>100000 pixels) MSI data sets. With an almost fourfold decrease in runtime, it is more scalable in comparison with the current state-of-the-art: t-SNE or the Barnes-Hut approximation of t-SNE. In what seems to be the first application of UMAP to MSI data, we assess the value of applying alternative distance metrics, such as the correlation, cosine, and the Chebyshev metric, in contrast to the traditionally used Euclidean distance metric. Furthermore, we propose ""histomatch"" as an additional custom distance metric for the analysis of MSI data."