Our work

Below you’ll find a highlight of our publications, illustrating the excellent scientific work of each research theme within the interfaculty RIMLS institute.

Felix Evers

Winner of the RIMLS Breakthrough Publication.

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Felix Evers

RIMLS awards annualy a prize for the best peer-reviewed publication. The publication should relate to a major scientific "breakthrough" that advances scientific understanding.

The winner of the RIMLS Breakthrough Publication: Felix Evers et al. Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum.


Infectious diseases and global health

Joep Joosten, Ezgi Taşköprü, Pascal W T C Jansen, Bas Pennings, Michiel Vermeulen, Ronald P Van Rij . PIWI proteomics identifies Atari and Pasilla as piRNA biogenesis factors in Aedes mosquitoes. Cell Reports 35:109073, 2021. PMID 33951430.

As in most arthropods, the PIWI-interacting RNA (piRNA) pathway in the vector mosquito Aedes aegypti is active in diverse biological processes in both soma and germline. To gain insights into piRNA biogenesis and effector complexes, we mapped the interactomes of the somatic PIWI proteins Ago3, Piwi4, Piwi5, and Piwi6 and identify numerous specific interactors as well as cofactors associated with multiple PIWI proteins. We describe the Piwi5 interactor AAEL014965, the direct ortholog of the Drosophila splicing factor pasilla. We find that Ae. aegypti Pasilla encodes a nuclear isoform and a cytoplasmic isoform, the latter of which is required for efficient piRNA production. In addition, we characterize a splice variant of the Tudor protein AAEL008101/Atari that associates with Ago3 and forms a scaffold for PIWI proteins and target RNAs to promote ping-pong amplification of piRNAs. Our study provides a useful resource for follow-up studies of somatic piRNA biogenesis, mechanism, and function in Aedes mosquitoes.

Emma J Kooistra, Aline H de Nooijer, Wout J Claassen, Inge Grondman, Nico A F Janssen, Mihai G Netea, Frank L van de Veerdonk, Johannes G van der Hoeven, Matthijs Kox, Peter Pickkers. A higher BMI is not associated with a different immune response and disease course in critically ill COVID-19 patients. International journal of obesity 45: 687-694, 2021. PMID 33495522.

Background/objectives Obesity appears to be an independent risk factor for ICU admission and a severe disease course in COVID-19 patients. An aberrant inflammatory response and impaired respiratory function have been suggested as underlying mechanisms. We investigated whether obesity is associated with differences in inflammatory, respiratory, and clinical outcome parameters in critically ill COVID-19 patients.

Subjects/methods Sixty-seven COVID-19 ICU patients were divided into obese (BMI ≥ 30 kg/m2, n = 18, 72% class I obesity, 28% class II obesity) and non-obese (BMI < 30 kg/m2, n = 49) groups. Concentrations of circulating interleukin (IL)-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interferon gamma-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, and IL-1 receptor antagonist (RA) were determined from ICU admission until 10 days afterward, and routine laboratory and clinical parameters were collected.

Results BMI was 32.6 [31.2-34.5] and 26.0 [24.4-27.7] kg/m2 in the obese and non-obese group, respectively. Apart from temperature, which was significantly lower in obese patients (38.1 [36.9-38.9] vs. 38.7 [38.0 -39.5] °C, p = 0.02), there were no between-group differences on ICU admission. Plasma cytokine concentrations declined over time (p < 0.05 for all), but no differences between obese and non-obese patients were observed. Also, BMI did not correlate with the cytokine response (IL-6 r = 0.09, p = 0.61, TNF-α r = 0.03, p = 0.99, IP-10 r = 0.28, p = 0.11). The kinetics of clinical inflammatory parameters and respiratory mechanics were also similar in both groups. Finally, no differences in time on ventilator, ICU length of stay or 40-day mortality between obese and non-obese patients were apparent.

Conclusions In COVID-19 patients requiring mechanical ventilation in the ICU, a higher BMI is not related to a different immunological response, unfavorable respiratory mechanics, or impaired outcome.

Intan M W Dewi, Cristina Cunha, Martin Jaeger, Mark S Gresnigt, Marina E Gkountzinopoulou, Fadel M Garishah, Cláudio Duarte-Oliveira, Cláudia F Campos, Lore Vanderbeke, Agustin Resendiz Sharpe, Roger J Brüggemann, Paul E Verweij, Katrien Lagrou, Greetje Vande Velde, Quirijn de Mast, Leo A B Joosten, Mihai G Netea, Andre J A M van der Ven, Joost Wauters, Agostinho Carvalho, Frank L van de Veerdonk. Neuraminidase and SIGLEC15 modulate the host defense against pulmonary aspergillosis. Cell Reports Medicine 2:100289, 2021. PMID 34095887.

Influenza-associated pulmonary aspergillosis (IAPA) has been reported increasingly since the advent of use of neuraminidase (NA) inhibitors following the 2009 influenza pandemic. We hypothesize that blocking host NA modulates the immune response against Aspergillus fumigatus. We demonstrate that NA influences the host response against A. fumigatus in vitro and that oseltamivir increases the susceptibility of mice to pulmonary aspergillosis. Oseltamivir impairs the mouse splenocyte and human peripheral blood mononuclear cell (PBMC) killing capacity of A. fumigatus, and adding NA restores this defect in PBMCs. Furthermore, the sialic acid-binding receptor SIGLEC15 is upregulated in PBMCs stimulated with A. fumigatus. Silencing of SIGLEC15 decrease PBMC killing of A. fumigatus. We provide evidence that host NA activity and sialic acid recognition are important for anti-Aspergillus defense. NA inhibitors might predispose individuals with severe influenza to invasive aspergillosis. These data shed light on the pathogenesis of invasive fungal infections and may identify potential therapeutic targets.

Reconstructive and regenerative medicine

Quan Xu, Georgios Georgiou, Siebren Frölich, Maarten van der Sande, Gert Jan C Veenstra, Huiqing Zhou, Simon J van Heeringen. ANANSE: an enhancer network-based computational approach for predicting key transcription factors in cell fate determination. Nucleic Acids  Research. 49: 7966-7985, 2021. PMID 34244796.

Proper cell fate determination is largely orchestrated by complex gene regulatory networks centered around transcription factors. However, experimental elucidation of key transcription factors that drive cellular identity is currently often intractable. Here, we present ANANSE (ANalysis Algorithm for Networks Specified by Enhancers), a network-based method that exploits enhancer-encoded regulatory information to identify the key transcription factors in cell fate determination. As cell type-specific transcription factors predominantly bind to enhancers, we use regulatory networks based on enhancer properties to prioritize transcription factors. First, we predict genome-wide binding profiles of transcription factors in various cell types using enhancer activity and transcription factor binding motifs. Subsequently, applying these inferred binding profiles, we construct cell type-specific gene regulatory networks, and then predict key transcription factors controlling cell fate transitions using differential networks between cell types. This method outperforms existing approaches in correctly predicting major transcription factors previously identified to be sufficient for trans-differentiation. Finally, we apply ANANSE to define an atlas of key transcription factors in 18 normal human tissues. In conclusion, we present a ready-to-implement computational tool for efficient prediction of transcription factors in cell fate determination and to study transcription factor-mediated regulatory mechanisms.

Xian Cheng, Dingpei Long, Lili Chen, John A Jansen, Sander C G Leeuwenburgh, Fang Yang. Electrophoretic deposition of silk fibroin coatings with pre-defined architecture to facilitate precise control over drug delivery. Bioactive Materials 6: 4243-4254, 2021. PMID 33997504.

The therapeutic precision and clinical applicability of drug-eluting coatings can be substantially improved by facilitating tunable drug delivery. However, the design of coatings which allows for precise control over drug release kinetics is still a major challenge. Here, a double-layered silk fibroin (SF) coating system was constructed by sequential electrophoretic deposition. A mixture of dissolved Bombyx mori SF (bmSF) molecules and pre-made bmSF nanospheres at different ratios was deposited as under-layer. Subsequently, this underlayer was covered by a top-layer comprising Antheraea pernyi SF (apSF) molecules (rich in arginylglycylaspartic acid, RGD) to improve the cellular response of the resulting double-layered coatings. Additionally, model drug doxycycline was either pre-mixed with dissolved bmSF molecules or pre-loaded into pre-made bmSF nanospheres at the same amount before their mixing and deposition. The thickness and nanosphere content of the under-layer architecture were proportional to the deposition time and nanosphere concentration in precursor mixtures, respectively. The surface topography, wettability, degradation rate and adhesion strength were comparable within the double-layered coating system. As expected, RGD-rich apSF top-layer improved cell adhesion, spreading and proliferation compared with bmSF top-layer. Furthermore, the amount and duration of drug release increased linearly with increasing nanosphere concentration at fixed deposition time, whereas drug release amount increased linearly with increasing deposition time. These results indicate that the dosage and kinetics of loaded drugs can be quantitatively tailored by altering nanosphere concentration and deposition time as main processing parameters. Overall, this study illustrates the strong potential of pre-defining coating architecture to facilitate control over drug delivery.

 Corien Oostendorp, Paul J Geutjes, Frank Smit, Dorien M Tiemessen, Sjoerd Polman, Aya Abbawi, Katrien M Brouwer, Alex J Eggink, Wout F J Feitz, Willeke F Daamen, Toin H van Kuppevelt . Sustained postnatal skin regeneration upon prenatal application of functionalized collagen scaffolds. Tissue Engineering 27: 10-25, 2021. PMID 31971880.

Primary closure of fetal skin in spina bifida protects the spinal cord and improves clinical outcome, but is also associated with postnatal growth malformations and spinal cord tethering. In this study, we evaluated the postnatal effects of prenatally closed full-thickness skin defects in sheep applying collagen scaffolds with and without heparin/vascular endothelial growth factor/fibroblast growth factor 2, focusing on skin regeneration and growth. At 6 months, collagen scaffold functionalized with heparin, VEGF, and FGF2 (COL-HEP/GF) resulted in a 6.9-fold increase of the surface area of the regenerated skin opposed to 1.7 × for collagen only. Epidermal thickness increased 5.7-fold at 1 month, in line with high gene expression of S100 proteins, and decreased to 2.1 at 6 months. Increased adipose tissue and reduced scaffold degradation and number of myofibroblasts were observed for COL-HEP/GF. Gene ontology terms related to extracellular matrix (ECM) organization were enriched for both scaffold treatments. In COL-HEP/GF, ECM gene expression resembled native skin. Expression of hair follicle-related genes in COL-HEP/GF was comparable to native skin, and de novo hair follicle generation was indicated. In conclusion, in utero closure of skin defects using functionalized collagen scaffolds resulted in long-term skin regeneration and growth. Functionalized collagen scaffolds that grow with the child may be useful for prenatal treatment of closure defects like spina bifida. Impact statement Prenatal closure of fetal skin in case of spina bifida prevents damage to the spinal cord. Closure of the defect is challenging and may result in postnatal growth malformations. In this study, the postnatal effects of a prenatally applied collagen scaffold functionalized with heparin and vascular endothelial growth factor (VEGF)/fibroblast growth factor (FGF) were investigated. An increase of the surface area of regenerated skin ("growing with the child") and generation of hair follicles was observed. Gene expression levels resembled those of native skin with respect to the extracellular matrix and hair follicles. Overall, in utero closure of skin defects using heparin/VEGF/FGF functionalized collagen scaffolds results in long-term skin regeneration.


Cancer development and immune defense

Katarzyna W Kliza, Qiang Liu, Laura W M Roosenboom, Pascal W T C Jansen, Dmitri V Filippov, Michiel Vermeulen. Reading ADP-ribosylation signaling using chemical biology and interaction proteomics​. Molecular Cell 81: 4552-4567.e8, 2021. PMID 34551281.

ADP-ribose (ADPr) readers are essential components of ADP-ribosylation signaling, which regulates genome maintenance and immunity. The identification and discrimination between monoADPr (MAR) and polyADPr (PAR) readers is difficult because of a lack of suitable affinity-enrichment reagents. We synthesized well-defined ADPr probes and used these for affinity purifications combined with relative and absolute quantitative mass spectrometry to generate proteome-wide MAR and PAR interactomes, including determination of apparent binding affinities. Among the main findings, MAR and PAR readers regulate various common and distinct processes, such as the DNA-damage response, cellular metabolism, RNA trafficking, and transcription. We monitored the dynamics of PAR interactions upon induction of oxidative DNA damage and uncovered the mechanistic connections between ubiquitin signaling and ADP-ribosylation. Taken together, chemical biology enables exploration of MAR and PAR readers using interaction proteomics. Furthermore, the generated MAR and PAR interaction maps significantly expand our current understanding of ADPr signaling.

van Bergen MGJM, Marneth AE, Hoogendijk AJ, van Alphen F, van den Akker E, Laros-van Gorkom B, Hoeks M, Simons A, de Munnik S, Janssen JJ, Martens J, Jansen JH, Meijer AB, Van der Reijden BA. Specific proteome changes in platelets from individuals with GATA1-, GFI1B- and RUNX1-linked bleeding disorders. Blood 138: 86-90, 2021. PMID 33690840.

Mutations in the transcription factors GATA binding factor 1 (GATA1), growth factor independence 1B (GFI1B), and Runt-related transcription factor 1 (RUNX1) cause familial platelet and bleeding disorders. Mutant platelets exhibit common abnormalities including an α-granule reduction resulting in a grayish appearance in blood smears. This suggests that similar pathways are deregulated by different transcription factor mutations. To identify common factors, full platelet proteomes from 11 individuals with mutant GATA1R216Q, GFI1BQ287*, RUNX1Q154Rfs, or RUNX1TD2-6 and 28 healthy controls were examined by label-free quantitative mass spectrometry. In total, 2875 platelet proteins were reliably quantified. Clustering analysis of more than 300 differentially expressed proteins revealed profound differences between cases and controls. Among cases, 44 of 143 significantly downregulated proteins were assigned to platelet function, hemostasis, and granule biology, in line with platelet dysfunction and bleedings. Remarkably, none of these proteins were significantly diminished in all affected cases. Similarly, no proteins were commonly overrepresented in all affected cases compared with controls. These data indicate that the studied transcription factor mutations alter platelet proteomes in distinct largely nonoverlapping manners. This work provides the quantitative landscape of proteins that affect platelet function when deregulated by mutated transcription factors in inherited bleeding disorders.

Bettina Weigelin, Annemieke Th den Boer, Esther Wagena, Kelly Broen, Harry Dolstra, Rob J de Boer, Carl G Figdor, Johannes Textor, Peter Friedl. Cytotoxic T cells are able to efficiently eliminate cancer cells by additive cytotoxicity. Nature Communications 12: 5217, 2021. PMID 34471116.

Lethal hit delivery by cytotoxic T lymphocytes (CTL) towards B lymphoma cells occurs as a binary, "yes/no" process. In non-hematologic solid tumors, however, CTL often fail to kill target cells during 1:1 conjugation. Here we describe a mechanism of "additive cytotoxicity" by which time-dependent integration of sublethal damage events, delivered by multiple CTL transiting between individual tumor cells, mediates effective elimination. Reversible sublethal damage includes perforin-dependent membrane pore formation, nuclear envelope rupture and DNA damage. Statistical modeling reveals that 3 serial hits delivered with decay intervals below 50 min discriminate between tumor cell death or survival after recovery. In live melanoma lesions in vivo, sublethal multi-hit delivery is most effective in interstitial tissue where high CTL densities and swarming support frequent serial CTL-tumor cell encounters. This identifies CTL-mediated cytotoxicity by multi-hit delivery as an incremental and tunable process, whereby accelerating damage magnitude and frequency may improve immune efficacy.

Urological cancers

Slootbeek PHJ, Duizer ML, van der Doelen MJ, Kloots ISH, Kuppen MCP, Westgeest HM, Uyl-de Groot CA, Pamidimarri Naga S, Ligtenberg MJL, van Oort IM, Gerritsen WR, Schalken JA, Kroeze LI, Bloemendal HJ, Mehra N. Impact of DNA damage repair defects and aggressive variant features on response to carboplatin-based chemotherapy in metastatic castration-resistant prostate cancer. International Journal of Cancer 148: 385-395, 2021. PMID 32965028.

Platinum-based chemotherapy is not standard of care for unselected or genetically selected metastatic castration-resistant prostate cancer (mCRPC) patients. A retrospective assessment of 71 patients was performed on platinum use in the Netherlands. Genetically unselected patients yielded low response rates. For a predefined subanalysis of all patients with comprehensive next-generation sequencing, 30 patients were grouped based on the presence of pathogenic aberrations in genes associated with DNA damage repair (DDR) or aggressive variant prostate cancer (AVPC). Fourteen patients (47%) were DDR deficient (DDRd), of which seven with inactivated BRCA2 (BRCA2mut). Six patients classified as AVPC. DDRd patients showed beneficial biochemical response to carboplatin, largely driven by all BRCA2mut patients having >50% prostate-specific antigen (PSA) decline and objective radiographic response. In the wild-type BRCA2 subgroup, 35% had a >50% PSA decline (P = .006) and 16% radiographic response (P < .001). Median overall survival was 21 months for BRCA2mut patients vs 7 months (P = .041) for those with functional BRCA2. AVPC patients demonstrated comparable responses to non-AVPC, including a similar overall survival, despite the poor prognosis for this subgroup. In the scope of the registration of poly-(ADP)-ribose polymerase inhibitors (PARPi) for mCRPC, we provide initial insights on cross-resistance between PARPi and platinum compounds. By combining the literature and our study, we identified 18 patients who received both agents. In this cohort, only BRCA2mut patients treated with platinum first (n = 4), responded to both agents. We confirm that BRCA2 inactivation is associated with meaningful responses to carboplatin, suggesting a role for both PARPi and platinum-based chemotherapy in preselected mCRPC patients.

Luna-Velez MV, Dijkstra JJ, Heuschkel MA, Smit FP, van de Zande G, Smeets D, Sedelaar JPM, Vermeulen M, Verhaegh GW, Schalken. Androgen receptor signalling confers clonogenic and migratory advantages in urothelial cell carcinoma of the bladder. Molecular Oncology 15: 1882-1900, 2021. PMID 33797847.

Bladder urothelial cell carcinoma (UCC) incidence is about three times higher in men compared with women. There are several indications for the involvement of hormonal factors in the aetiology of UCC. Here, we provide evidence of androgen signalling in UCC progression. Microarray and qPCR analysis revealed that the androgen receptor (AR) mRNA level is upregulated in a subset of UCC cases. In an AR-positive UCC-derived cell line model, UM-UC-3-AR, androgen treatment increased clonogenic capacity inducing the formation of big stem cell-like holoclones, while AR knockdown or treatment with the AR antagonist enzalutamide abrogated this clonogenic advantage. Additionally, blockage of AR signalling reduced the cell migration potential of androgen-stimulated UM-UC-3-AR cells. These phenotypic changes were accompanied by a rewiring of the transcriptome with almost 300 genes being differentially regulated by androgens, some of which correlated with AR expression in UCC patients in two independent data sets. Our results demonstrate that AR signals in UCC favouring the development of an aggressive phenotype and highlights its potential as a therapeutic target for bladder cancer.

Veltien A, van Asten J, Ravichandran N, de Graaf RA, De Feyter HM, Oosterwijk E, Heerschap A. Simultaneous recording of the uptake and conversion of glucose and choline in tumors by deuterium metabolic imaging. Cancers (Basel) 13: 4034, 2021. PMID 34439188.

Increased glucose and choline uptake are hallmarks of cancer. We investigated whether the uptake and conversion of [2H9]choline alone and together with that of [6,6'-2H2]glucose can be assessed in tumors via deuterium metabolic imaging (DMI) after administering these compounds. Therefore, tumors with human renal carcinoma cells were grown subcutaneously in mice. Isoflurane anesthetized mice were IV infused in the MR magnet for ~20 s with ~0.2 mL solutions containing either [2H9]choline (0.05 g/kg) alone or together with [6,6'-2H2]glucose (1.3 g/kg). 2H MR was performed on a 11.7T MR system with a home-built 2H/1H coil using a 90° excitation pulse and 400 ms repetition time. 3D DMI was recorded at high resolution (2 × 2 × 2 mm) in 37 min or at low resolution (3.7 × 3.7 × 3.7 mm) in 2:24 min. Absolute tissue concentrations were calculated assuming natural deuterated water [HOD] = 13.7 mM. Within 5 min after [2H9]choline infusion, its signal appeared in tumor spectra representing a concentration increase to 0.3-1.2 mM, which then slowly decreased or remained constant over 100 min. In plasma, [2H9]choline disappeared within 15 min post-infusion, implying that its signal arises from tumor tissue and not from blood. After infusing a mixture of [2H9]choline and [6,6'-2H2]glucose, their signals were observed separately in tumor 2H spectra. Over time, the [2H9]choline signal broadened, possibly due to conversion to other choline compounds, [[6,6'-2H2]glucose] declined, [HOD] increased and a lactate signal appeared, reflecting glycolysis. Metabolic maps of 2H compounds, reconstructed from high resolution DMIs, showed their spatial tumor accumulation. As choline infusion and glucose DMI is feasible in patients, their simultaneous detection has clinical potential for tumor characterization.

Tumors of the digestive tract

Femke Simmer, Ragna L A van der Linden, Marjolijn J L Ligtenberg, Bauke Ylstra, Rachel S van der Post, Iris D Nagtegaal. Multifocal colorectal cancer - do intraluminal metastases occur? Gastroenterology 160:1853-1855, 2021. PMID: 33316233.

Multifocal colorectal cancer (CRC) is diagnosed in approximately 5% of all CRC patients, either synchronous or metachronous.1, 2, 3, 4 There are several risk factors for the development of multifocal CRC, including genetic predisposition and chronic inflammation. In general, the second CRC is considered an independent primary and treated as such. However, other biological hypotheses might explain the occurrence of multifocal CRC, such as intraluminal spread due to tumor seeding.

Traditionally, the distinction between a primary tumor and a metastasis is based on the presence of a dysplastic precursor and the histologic growth pattern. However, growth of metastatic cancer along the intact basement membrane, so called colonization,5 can mimic a primary tumor and thus suggests that our traditional morphologic approach alone will not be sufficient to distinguish independent primaries and metastases.

Additional molecular methods can be used to establish clonal relationships between different CRCs. Here we demonstrate that intraluminal spreading of CRC occurs more often than is recognized in daily practice, which affects both diagnostics and treatment of patients with multifocal CRC.

Conclusion: We have shown that clonally related CRC is quite common in multifocal CRC. Sporadic intraluminal spread is one of the likely explanations for this phenomenon. The magnitude of clonally related CRC in patients with multifocal CRC illustrates the urgency of implementing further molecular testing in clinical practice to give the best treatment and to distinguish these patients from patients with multiple primaries who may be at high familial cancer risk due to genetic predisposition.

Richarda M de Voer, Illja J Diets, Rachel S van der Post, Robbert D A Weren, Eveline J Kamping, Tessa J J de Bitter, Lisa Elze, Rob H A Verhoeven, Elisa Vink-Börger, Astrid Eijkelenboom, Arjen Mensenkamp, Iris D Nagtegaal, Marjolijn C J Jongmans, Marjolijn J L Ligtenberg. Clinical, pathology, genetic, and molecular features of colorectal tumors in adolescents and adults 25 years or younger. Clinical Gastroenterology and Hepatology 19: 1642-1651.e8, 2021. PMID 32585361.

Background & aims Colorectal cancers (CRCs) are rare in adolescents and adults ages 25 years or younger. We analyzed clinical, pathology, and molecular features of colorectal tumors from adolescents and young adults in an effort to improve genetic counseling, surveillance, and, ultimately, treatment and outcomes.

Methods We analyzed clinical data and molecular and genetic features of colorectal tumor tissues from 139 adolescents or young adults (age, ≤25 y; median age, 23 y; 58% male), collected from 2000 through 2017; tumor tissues and clinical data were obtained from the nationwide network and registry of histopathology and cytopathology and The Netherlands Cancer Registry, respectively. DNA samples from tumors were analyzed for microsatellite instability, mutations in 56 genes, and genome-wide somatic copy number aberrations.

Results Mucinous and/or signet ring cell components were observed in 33% of tumor samples. A genetic tumor risk syndrome was confirmed for 39% of cases. Factors associated with shorter survival time included younger age at diagnosis, signet ring cell carcinoma, the absence of a genetic tumor risk syndrome, and diagnosis at an advanced stage of disease. Compared with colorectal tumors from patients ages 60 years or older in the Cancer Genome Atlas, higher proportions of tumors from adolescents or young adults were microsatellite stable with nearly diploid genomes, or contained somatic mutations in TP53 and POLE, whereas lower proportions contained mutations in APC.

Conclusions We found clinical, molecular, and genetic features of CRCs in adolescents or young adults to differ from those of patients older than age 60 years. In 39% of patients a genetic tumor risk syndrome was identified. These findings provide insight into the pathogenesis of CRC in young patients and suggest new strategies for clinical management. Performing genetic and molecular analyses for every individual diagnosed with CRC at age 25 years or younger would aid in this optimization.

Yonne Peters, Lotte J Huibertse, Ruud W M Schrauwen, Adriaan C Tan, Rachel S van der Post, Peter D Siersema. Increased risk of Barrett's oesophagus and related neoplasia in individuals with a positive family history. European Journal of Cancer 155:116-126, 2020. PMID 34375895.

Background Considering the poor prognosis of oesophageal adenocarcinoma (EAC), it is important to identify individuals at increased risk of developing EAC who may benefit from early detection and prevention strategies. We aimed to determine whether individuals with a positive family history of Barrett's oesophagus (BE) and EAC are at an increased risk of oesophageal neoplasia.

Methods In a multi-centre case-control study, BE patients with or without related oesophageal neoplasia and randomly selected population controls filled out a questionnaire to collect information on family history and other risk factors for BE and EAC. Positive family history was defined as having ≥1 first-degree relative with BE or EAC whose diagnosis was histologically confirmed in the Dutch nationwide histopathology database.

Findings We included 480 BE patients and 420 controls without BE who had a total of 6393 first-degree relatives. A pathologically confirmed positive family history was significantly higher in BE patients compared with controls (6.5% versus 0.9; p < 0.001). Positive family history was independently associated with an increased risk of BE (OR 5.04; 95% CI 1.45-17.58; p = 0.01) after adjusting for known risk factors, such as gastroesophageal reflux disease and body mass index, and family size.

Interpretation We found that familial clustering of BE and EAC is present in 6.5% of Dutch BE patients. Subjects with ≥1 first-degree relative with BE or EAC have a 5-fold increased risk of BE and EAC. These findings emphasize the importance of a detailed family history in patients with BE or EAC to identify individuals at increased risk who may benefit from early detection strategies to prevent EAC-related mortality.

Inflammatory diseases

Joyce Aarts, Debbie M Roeleveld, Monique M Helsen, Birgitte Walgreen, Elly L Vitters, Jay Kolls, Fons A van de Loo, Peter L van Lent, Peter M van der Kraan, Marije I Koenders. TSystemic overexpression of interleukin-22 induces the negative immune-regulator SOCS3 and potently reduces experimental arthritis in mice. Reumatology (Oxford) 60: 1974-1983, 2021. PMID 33197269.

Objective High levels of IL-22 are present in serum and synovial fluid of patients with RA. As both pro- and anti-inflammatory roles for IL-22 have been described in studies using animal models of RA, its exact function in arthritis remains poorly defined. With this study we aimed to further unravel the mechanism by which IL-22 exerts its effects and to decipher its therapeutic potential by overexpression of IL-22 either locally or systemically during experimental arthritis.

Methods CIA was induced in DBA-1 mice by immunization and booster injection with type II collagen (col II). Before arthritis onset, IL-22 was overexpressed either locally by intra-articular injection or systemically by i.v. injection using an adenoviral vector and clinical arthritis was scored for a period of 10 days. Subsequently, joints were isolated for histological analysis of arthritis severity and mRNA and protein expression of various inflammatory mediators was determined in the synovium, spleen and serum.

Results Local IL-22 overexpression did not alter arthritis pathology, whereas systemic overexpression of IL-22 potently reduced disease incidence, severity and pathology during CIA. Mice systemically overexpressing IL-22 showed strongly reduced serum cytokine levels of TNF-α and macrophage inflammatory protein 1α that correlated significantly with the enhanced expression of the negative immune regulator SOCS3 in the spleen.

Conclusion With this study, we revealed clear anti-inflammatory effects of systemic IL-22 overexpression during CIA. Additionally, we are the first to show that the protective effect of systemic IL-22 during experimental arthritis is likely orchestrated via upregulation of the negative regulator SOCS3.

R Wiegertjes, N G M Thielen, A P M van Caam, M van Laar, H M van Beuningen, M I Koenders, P L E M van Lent, P M van der Kraan, F A J van de Loo, E N Blaney Davidson. Increased IL-6 receptor expression and signaling in ageing cartilage can be explained by loss of TGF-β-mediated IL-6 receptor suppression. Osteoarthritis and Cartilage 29: 773-782, 2021. PMID 33617971.

Objective Osteoarthritis (OA) development is strongly associated with ageing, possibly due to age-related changes in transforming growth factor-β (TGF-β) signaling in cartilage. Recently, we showed that TGF-β suppresses interleukin (IL)-6 receptor (IL-6R) expression in chondrocytes. As IL-6 is involved in cartilage degeneration, we hypothesized that age-related loss of TGF-β signaling results in increased IL-6R expression and signaling in ageing cartilage.

Design Bovine articular cartilage was collected and immediately processed to study age-related changes in IL-6R expression using qPCR and IHC (age-range: 0.5-14 years). Moreover, cartilage from young and aged cows was stimulated with rhIL-6 and/or rhTGF-β1 to measure IL-6-induced p-STAT3 using Western blot. Expression of STAT3-responsive genes was analyzed using qPCR.

Results Expression of IL-6 receptor (bIL-6R) significantly increased in cartilage upon ageing (slope: 0.32, 95%CI: 0.20-0.45), while expression of glycoprotein 130 (bGP130) was unaffected. Cartilage stimulation with IL-6 showed increased induction of p-STAT3 upon ageing (slope: 0.14, 95%CI: 0.08-0.20). Furthermore, IL-6-mediated induction of STAT3-responsive genes like bSOCS3 and bMMP3 was increased in aged compared to young cartilage. Interestingly, the ability of TGF-β to suppress bIL6R expression in young cartilage was lost upon ageing (slope: 0.21, 95%CI: 0.13-0.30). Concurrently, an age-related loss in TGF-β-mediated suppression of IL-6-induced p-STAT3 and bSOCS3 expression was observed.

Conclusions Ageing results in enhanced IL-6R expression and subsequent IL-6-induced p-STAT3 signaling in articular cartilage. This is likely caused by age-related loss of protective TGF-β signaling, resulting in loss of TGF-β-mediated IL-6R suppression. Because of the detrimental role of IL-6 in cartilage, this mechanism may be involved in age-related OA development.

Pieter Langerhorst, Arie B Brinkman, Martijn M VanDuijn, Hans J C T Wessels, Patricia J T A Groenen, Irma Joosten, Alain J van Gool, Jolein Gloerich, Blanca Scheijen, Joannes F M Jacobs. Clonotypic features of rearranged immunoglobulin genes yield personalized biomarkers for minimal residual disease monitoring in multiple myeloma. Clinical Chemistry 67: 867-875, 2021. PMID 33709101.

Background Due to improved treatment, more patients with multiple myeloma (MM) reach a state of minimal residual disease (MRD). Different strategies for MM MRD monitoring include flow cytometry, allele-specific oligonucleotide-quantitative PCR, next-generation sequencing, and mass spectrometry (MS). The last 3 methods rely on the presence and the stability of a unique immunoglobulin fingerprint derived from the clonal plasma cell population. For MS-MRD monitoring it is imperative that MS-compatible clonotypic M-protein peptides are identified. To support implementation of molecular MRD techniques, we studied the presence and stability of these clonotypic features in the CoMMpass database.

Methods An analysis pipeline based on MiXCR and HIGH-VQUEST was constructed to identify clonal molecular fingerprints and their clonotypic peptides based on transcriptomic datasets. To determine the stability of the clonal fingerprints, we compared the clonal fingerprints during disease progression for each patient.

Results The analysis pipeline to establish the clonal fingerprint and MS-suitable clonotypic peptides was successfully validated in MM cell lines. In a cohort of 609 patients with MM, we demonstrated that the most abundant clone harbored a unique clonal molecular fingerprint and that multiple unique clonotypic peptides compatible with MS measurements could be identified for all patients. Furthermore, the clonal immunoglobulin gene fingerprints of both the light and heavy chain remained stable during MM disease progression.

Conclusions Our data support the use of the clonal immunoglobulin gene fingerprints in patients with MM as a suitable MRD target for MS-MRD analyses.


Metabolic disorders

Felix Evers, Alfredo Cabrera-Orefice, Dei M Elurbe, Mariska Kea-Te Lindert, Sylwia D Boltryk, Till S Voss, Martijn A Huynen, Ulrich Brandt, Taco W A Kooij. Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum. Nature Communications 12: 3820, 2021. PMID 34155201.

Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs.

René G Feichtinger, Andreas Hüllen, Andreas Koller, Dieter Kotzot, Valerian Grote, Erdmann Rapp, Peter Hofbauer, Karin Brugger, Christian Thiel, Johannes A Mayr, Saskia B Wortmann. A spoonful of L-fucose-an efficient therapy for GFUS-CDG, a new glycosylation disorder. EMBO Molecular Medicine 13: e14332, 2021. PMID 34468083.

Congenital disorders of glycosylation are a genetically and phenotypically heterogeneous family of diseases affecting the co- and posttranslational modification of proteins. Using exome sequencing, we detected biallelic variants in GFUS (NM_003313.4) c.[632G>A];[659C>T] (p.[Gly211Glu];[Ser220Leu]) in a patient presenting with global developmental delay, mild coarse facial features and faltering growth. GFUS encodes GDP-L-fucose synthase, the terminal enzyme in de novo synthesis of GDP-L-fucose, required for fucosylation of N- and O-glycans. We found reduced GFUS protein and decreased GDP-L-fucose levels leading to a general hypofucosylation determined in patient's glycoproteins in serum, leukocytes, thrombocytes and fibroblasts. Complementation of patient fibroblasts with wild-type GFUS cDNA restored fucosylation. Making use of the GDP-L-fucose salvage pathway, oral fucose supplementation normalized fucosylation of proteins within 4 weeks as measured in serum and leukocytes. During the follow-up of 19 months, a moderate improvement of growth was seen, as well as a clear improvement of cognitive skills as measured by the Kaufmann ABC and the Nijmegen Pediatric CDG Rating Scale. In conclusion, GFUS-CDG is a new glycosylation disorder for which oral L-fucose supplementation is promising.

Udo Fh Engelke, Rianne E van Outersterp, Jona Merx, Fred Amg van Geenen, Arno van Rooij, Giel Berden, Marleen Cdg Huigen, Leo Aj Kluijtmans, Tessa Ma Peters, Hilal H Al-Shekaili, Blair R Leavitt, Erik de Vrieze, Sanne Broekman, Erwin van Wij, Laura A Tseng, Purva Kulkarni, Floris Pjt Rutjes, Jasmin Mecinović, Eduard A Struys, Laura A Jansen, Sidney M Gospe Jr, Saadet Mercimek-Andrews, Keith Hyland, Michèl Aap Willemsen, Levinus A Bok, Clara Dm van Karnebeek, Ron A Wevers, Thomas J Boltje, Jos Oomens, Jonathan Martens, Karlien Lm Coene. Untargeted metabolomics and infrared ion spectroscopy identify biomarkers for pyridoxine-dependent epilepsy. The Journal of Clinical Investigation 131: e148272, 2021. PMID: 34138754.

Background Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine catabolism that presents with refractory epilepsy in newborns. Biallelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase/antiquitin, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important cofactor pyridoxal-5'-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but intellectual disability may still develop. Early diagnosis and treatment, preferably based on newborn screening, could optimize long-term clinical outcome. However, no suitable PDE-ALDH7A1 newborn screening biomarkers are currently available.MethodsWe combined the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy to discover and identify biomarkers in plasma that would allow for PDE-ALDH7A1 diagnosis in newborn screening.

Results We identified 2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) as a PDE-ALDH7A1 biomarker, and confirmed 6-oxopiperidine-2-carboxylic acid (6-oxoPIP) as a biomarker. The suitability of 2-OPP as a potential PDE-ALDH7A1 newborn screening biomarker in dried bloodspots was shown. Additionally, we found that 2-OPP accumulates in brain tissue of patients and Aldh7a1-knockout mice, and induced epilepsy-like behavior in a zebrafish model system.

Conclusion This study has opened the way to newborn screening for PDE-ALDH7A1. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients.

Funding Society for Inborn Errors of Metabolism for Netherlands and Belgium (ESN), United for Metabolic Diseases (UMD), Stofwisselkracht, Radboud University, Canadian Institutes of Health Research, Dutch Research Council (NWO), and the European Research Council (ERC)



Valentina Palacio-Castañeda, Simon Dumas, Philipp Albrecht, Thijmen J Wijgers, Stéphanie Descroix, Wouter P R Verdurmen. A Hybrid In Silico and Tumor-on-a-Chip Approach to Model Targeted Protein Behavior in 3D Microenvironments). Cancers (Basel) 13: 2461, 2021. PMID: 34070171.

To rationally improve targeted drug delivery to tumor cells, new methods combining in silico and physiologically relevant in vitro models are needed. This study combines mathematical modeling with 3D in vitro co-culture models to study the delivery of engineered proteins, called designed ankyrin repeat proteins (DARPins), in biomimetic tumor microenvironments containing fibroblasts and tumor cells overexpressing epithelial cell adhesion molecule (EpCAM) or human epithelial growth factor receptor (HER2). In multicellular tumor spheroids, we observed strong binding-site barriers in combination with low apparent diffusion coefficients of 1 µm2·s-1 and 2 µm2 ·s-1 for EpCAM- and HER2-binding DARPin, respectively. Contrasting this, in a tumor-on-a-chip model for investigating delivery in real-time, transport was characterized by hindered diffusion as a consequence of the lower local tumor cell density. Finally, simulations of the diffusion of an EpCAM-targeting DARPin fused to a fragment of Pseudomonas aeruginosa exotoxin A, which specifically kills tumor cells while leaving fibroblasts untouched, correctly predicted the need for concentrations of 10 nM or higher for extensive tumor cell killing on-chip, whereas in 2D models picomolar concentrations were sufficient. These results illustrate the power of combining in vitro models with mathematical modeling to study and predict the protein activity in complex 3D models.

Alexander H van Asbeck, Jürgen Dieker, Rik Oude Egberink, Lennard van den Berg, Johan van der Vlag, Roland Brock. Protein Expression Correlates Linearly with mRNA Dose over Up to Five Orders of Magnitude In Vitro and In Vivo. Biomedicines 9(5):511, 2021. PMID: 34063094.

Messenger RNA is rapidly gaining significance as a therapeutic modality. Here, we address the dependence of dose-response functions on the type of delivery vehicle, cell line, and incubation time. Knowledge of these characteristics is crucial for the application of mRNA. As delivery vehicles, a lipid-based formulation and the cell-penetrating peptide Pepfect14 (PF14) were employed. As cell lines, we included a glomerular endothelial cell line (mGEnC) as a model for differentiated cells, HeLa cells, and SKOV-3 ovarian carcinoma cells. Uptake and expression were detected by flow cytometry, using a Cy5-labelled mRNA coding for enhanced green fluorescent protein (EGFP). There was a linear correlation of dose, uptake, and expression, and this correlation was maintained for over up to 72 h. Through application of a multistep kinetic model, we show that differences in expression levels can already be explained by the number of mRNAs packaged per delivery vehicle. Using luciferase as a reporter protein, linearity of expression was observed over 5 orders of magnitude in vitro and 3 orders of magnitude in vivo. Overall, the results demonstrate that mRNA provides excellent quantitative control over protein expression, also over extended periods of time..

Ward Vleeshouwers, Koen van den Dries, Sandra de Keijzer, Ben Joosten, Diane S Lidke, Alessandra Cambi. Characterization of the signaling modalities of prostaglandin E2 receptors EP2 and EP4 reveals crosstalk and a role for microtubules. Front Immunology 11: 613286, 2021. PMID: 33643295.

Prostaglandin E2 (PGE2) is a lipid mediator that modulates the function of myeloid immune cells such as macrophages and dendritic cells (DCs) through the activation of the G protein-coupled receptors EP2 and EP4. While both EP2 and EP4 signaling leads to an elevation of intracellular cyclic adenosine monophosphate (cAMP) levels through the stimulating Gαs protein, EP4 also couples to the inhibitory Gαi protein to decrease the production of cAMP. The receptor-specific contributions to downstream immune modulatory functions are still poorly defined. Here, we employed quantitative imaging methods to characterize the early EP2 and EP4 signaling events in myeloid cells and their contribution to the dissolution of adhesion structures called podosomes, which is a first and essential step in DC maturation. We first show that podosome loss in DCs is primarily mediated by EP4. Next, we demonstrate that EP2 and EP4 signaling leads to distinct cAMP production profiles, with EP4 inducing a transient cAMP response and EP2 inducing a sustained cAMP response only at high PGE2 levels. We further find that simultaneous EP2 and EP4 stimulation attenuates cAMP production, suggesting a reciprocal control of EP2 and EP4 signaling. Finally, we demonstrate that efficient signaling of both EP2 and EP4 relies on an intact microtubule network. Together, these results enhance our understanding of early EP2 and EP4 signaling in myeloid cells. Considering that modulation of PGE2 signaling is regarded as an important therapeutic possibility in anti-tumor immunotherapy, our findings may facilitate the development of efficient and specific immune modulators of PGE2 receptors.


Vascular damage

Regina E Konst, Suzette E Elias-Smale, Dario Pellegrini, Mariëlle Hartzema-Meijer, Bas J C van Uden, Tijn P J Jansen, Priya Vart, Helmut Gehlmann, Angela H E M Maas, Niels van Royen, Peter Damman. Absolute coronary blood flow measured by continuous thermodilution in patients with ischemia and nonobstructive disease. Journal of the American College of Cardiology 77: 728-741, 2021. PMID 33573743.

Background Intracoronary continuous thermodilution is a novel technique to quantify absolute coronary flow (Q) and resistance (R) and has potential advantages over current methods such as coronary flow reserve (CFR) and index of microvascular resistance (IMR). However, no data are available in patients with ischemia and nonobstructive coronary artery disease (INOCA).

Objectives This study aimed to assess the relationship of Q and R with the established CFR/IMR in INOCA patients, to explore the potential of absolute Q, and to predict self-reported angina.

Methods Consecutive INOCA patients (n = 84; 87% women; mean age 56 ± 8 years) underwent coronary function testing, including acetylcholine (ACH) provocation testing, adenosine (ADE) testing (CFR/IMR), and continuous thermodilution (absolute Q and R) with saline-induced hyperemia.

Results ACH testing was abnormal (ACH+) in 87%, and ADE testing (ADE+) in 38%. The median absolute Q was 198 ml/min, and the median absolute R was 416 WU. The absolute R was higher in patients with ADE+ versus ADE- (495 WU vs. 375 WU; p = 0.04) but did not differ between patients with ACH+ versus ACH- (421 WU vs. 409 WU; p = 0.74). Low Q and high R were associated with severe angina (odds ratio: 3.09; 95% confidence interval: 1.16 to 8.28; p = 0.03; and odds ratio: 2.60; 95% confidence interval: 0.99 to 6.81; p = 0.05), respectively.

Conclusions In this study, absolute R was higher in patients with abnormal CFR/IMR, whereas both Q and R were unrelated to coronary vasospasm. Q and R were associated with angina, although their exact predictive value should be determined in larger studies.

Margo Dona, Selma Waaijers, Susan Richter, Graeme Eisenhofer, Jeroen Korving, Sarah M Kamel, Jeroen Bakkers, Elena Rapizzi, Richard J Rodenburg, Jan Zethof, Marnix Gorissen, Gert Flik, Peter M T De, Henri J L M Timmers. Loss of sdhb in zebrafish larvae recapitulates human paraganglioma characteristics. Endocrine-related Cancer 28: 65-77, 2021. PMID 33156815.

Pheochromocytomas and paragangliomas (PPGLs) caused by mutations in the B-subunit of the succinate dehydrogenase (SDHB) have the highest metastatic rate among PPGLs, and effective systemic therapy is lacking. To unravel underlying pathogenic mechanisms, and to evaluate therapeutic strategies, suitable in vivo models are needed. The available systemic Sdhb knock-out mice cannot model the human PPGL phenotype: heterozygous Sdhb mice lack a disease phenotype, and homozygous Sdhb mice are embryonically lethal. Using CRISPR/cas9 technology, we introduced a protein-truncating germline lesion into the zebrafish sdhb gene. Heterozygous sdhb mutants were viable and displayed no obvious morphological or developmental defects. Homozygous sdhb larvae were viable, but exhibited a decreased lifespan. Morphological analysis revealed incompletely or non-inflated swim bladders in homozygous sdhb mutants at day 6. Although no differences in number and ultrastructure of the mitochondria were observed. Clear defects in energy metabolism and swimming behavior were observed in homozygous sdhb mutant larvae. Functional and metabolomic analyses revealed decreased mitochondrial complex 2 activity and significant succinate accumulation in the homozygous sdhb mutant larvae, mimicking the metabolic effects observed in SDHB-associated PPGLs. This is the first study to present a vertebrate animal model that mimics metabolic effects of SDHB-associated PPGLs. This model will be useful in unraveling pathomechanisms behind SDHB-associated PPGLs. We can now study the metabolic effects of sdhb disruption during different developmental stages and develop screening assays to identify novel therapeutic targets in vivo. Besides oncological syndromes, our model might also be useful for pediatric mitochondrial disease caused by loss of the SDHB gene.

Marlies P Noz, Adelina S Plachokova, Esther M M Smeets, Erik H J G Aarntzen, Siroon Bekkering, Prya Vart, Leo A B Joosten, Mihai G Netea, Niels P Riksen. An explorative study on monocyte reprogramming in the context of periodontitis in vitro and in vivo. Frontiers Immunology 12: 695227, 2021. PMID 34484192.

Aims Periodontitis is an independent risk factor for cardiovascular disease, but the mechanistic link is not fully understood. In atherosclerotic cardiovascular disease, monocytes can adopt a persistent hyperresponsive phenotype, termed trained immunity. We hypothesized that periodontitis-associated bacteria can induce trained immunity in monocytes, which subsequently accelerate atherosclerosis development.

Materials and methods We combined in vitro experiments on human primary monocytes and in vivo techniques in patients with periodontitis to test this hypothesis. Adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with lipopolysaccharide (LPS) or Pam3CysK4 (P3C) six days later, to measure interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) production. In an exploratory observational study, patients with severe periodontitis (63 ± 6 years, n=14) and control subjects with no-to-mild periodontitis (54 ± 10 years, n=14) underwent venipuncture and 2'-deoxy-2'-[18F]fluoro-D-glucose positron-emission-tomography ([18F]FDG PET/CT) scanning.

Results When adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with LPS or P3C six days later, IL-6 and TNFα production was significantly increased (TNFα/P3C, p<0.01). Circulating leukocytes, IL-6 and interleukin-1 receptor antagonist (IL-1Ra) concentrations were generally higher in patients compared to controls (leukocytes: p<0.01; IL-6: p=0.08; IL-1Ra: p=0.10). Cytokine production capacity in PBMCs after 24h stimulation revealed no differences between groups. [18F]FDG PET/CT imaging showed a trend for increased [18F]FDG-uptake in the periodontium [mean standard uptake value (SUVmean), p=0.11] and in femur bone marrow (SUVmean, p=0.06), but no differences were observed for vascular inflammation. Positive correlations between severity of periodontitis, measured by The Dutch Periodontal Screening Index and pocket depth, with circulating inflammatory markers and tissue inflammation were found.

Conclusions P. gingivalis induces long-term activation of human monocytes in vitro (trained immunity). Patients with severe periodontitis did have signs of increased systemic inflammation and hematopoietic tissue activation. However, their circulating monocytes did not show a hyperresponsive phenotype. Together we suggest that trained immunity might contribute to local periodontal inflammation which warrants further investigation.

Women's cancers

Julie E M Swillens, Quirinus J M Voorham, Iris D Nagtegaal, Rosella P M G Hermens. Improving interdisciplinary communication: Barriers and facilitators for implementation of standardized structured reporting in oncology. Journal of the National Comprehensive Cancer Network 1-11, 2021. PMID 34653965.

Background Standardized structured reporting (SSR) improves quality of diagnostic cancer reporting and interdisciplinary communication in multidisciplinary team (MDT) meetings, resulting in more adequate treatment decisions and better health outcomes. However, use of SSR varies widely among pathologists, but might be encouraged by MDT members (MDTMs). Our objectives were to identify barriers and facilitators (influencing factors) for SSR implementation in oncologic pathology from the perspective of MDTMs and their determinants.

Methods In a multimethod design, we identified influencing factors for SSR implementation related to MDT meetings, using 5 domains: (1) innovation factors, (2) individual professional factors, (3) social setting factors, (4) organizational factors, and (5) political and legal factors. Four focus groups with MDTMs in urologic, gynecologic, and gastroenterologic oncology were conducted. We used an eSurvey among MDTMs to quantify the qualitative findings and to analyze determinants affecting these influencing factors.

Results Twenty-three MDTMs practicing in 9 oncology-related disciplines participated in the focus groups and yielded 28 barriers and 28 facilitators in all domains. The eSurvey yielded 211 responses. Main barriers related to lack of readability of SSR: difficulties with capturing nuances (66%) and formulation of the conclusion (43%); lack of transparency in the development (50%) and feedback processes of SSR templates (38%); and lack of information exchange about SSR between pathologists and other MDTMs (45%). Main facilitators were encouragement of pathologists' SSR use by MDTMs (90%) and expanding the recommendation of SSR use in national guidelines (80%). Oncology-related medical discipline and MDT type were the most relevant determinants for SSR implementation barriers.

Conclusions Although SSR makes diagnostic reports more complete, this study shows important barriers in implementing SSR in oncologic pathology. The next step is to use these factors for developing and testing implementation tools to improve SSR implementation.

Tanya D Geertse, Wikke Setz-Pels, Daniëlle van der Waal, Joost Nederend, Bram Korte, Eric Tetteroo, Ruud M Pijnappel, Mireille J M Broeders, Lucien E M Duijm. Added value of prereading screening mammograms for breast cancer by radiologic technologists on early screening outcomes. Radiology 302: 276-283, 2021. PMID 34751612.

Background In the Dutch breast cancer screening program, mammograms are preread by technologists to identify possible abnormalities, leading to "warning signals" (an audible and visual alert if the technologist observed an abnormality suspicious for cancer) for radiologists. The best moment to present these warning signals is unknown. Purpose To determine the effect that blinding of technologists' warning signals has on radiologists' early screening outcome measures during interpretation of mammograms. Materials and Methods In this prospective study from September 2017 to May 2019, on alternating months, radiologists were either blinded or nonblinded to the warning signals of the technologist when interpreting screening mammograms for breast cancer. All discrepancies between radiologists and technologists were reviewed during quality assurance sessions every 6 weeks, which could result in secondary recalls. The outcome measures of this study were recall rate, cancer detection rate, and positive predictive value of recall. A χ2 test was used to test for differences between the two groups. Results During the study period, 109 596 women (mean age, 62 years ± 7 [standard deviation]), including 53 291 in the blinded and 56 305 in the nonblinded groups, participated. The overall recall rate (including secondary recalls) was lower for women in the blinded group than in the nonblinded group (blinded: 1140 of 53 291 women [2.1%], nonblinded: 1372 of 56 305 women [2.4%]; P = .001). There was no evidence of cancer detection rate differences between the groups (blinded: 349 of 53 291 women [6.5 per 1000 screening examinations], nonblinded: 360 of 56 305 women [6.4 per 1000 screening examinations]; P = .75). The blinded group thus had a higher positive predictive value of recall (blinded: 349 of 1140 women [30.6%], nonblinded: 360 of 1372 women [26.2%]; P = .02). Conclusion While interpreting screening mammograms for breast cancer, radiologists blinded to technologists' warning signals had lower recall rates with higher positive predictive values than nonblinded radiologists, yet cancer detection rates seemed to remain unchanged. See also the editorial by Hofvind and Lee in this issue.

Miranda P Steenbeek, Marline G Harmsen, Nicoline Hoogerbrugge, Marieke Arts de Jong, Angela H E M Maas, Judith B Prins, Johan Bulten, Steven Teerenstra, Majke H D van Bommel, Helena C van Doorn, Marian J E Mourits, Marc van Beurden, Ronald P Zweemer, Katja N Gaarenstroom, Brigitte F M Slangen, Monique M A Brood-van Zanten, M Caroline Vos, Jurgen M J Piek, Luc R C W van Lonkhuijzen, Mirjam J A Apperloo, Sjors F P J Coppus, Leon F A G Massuger, Joanna IntHout, Rosella P M G Hermens, Joanne A de Hullu. Association of salpingectomy with delayed oophorectomy versus salpingo-oophorectomy with quality of life in BRCA1/2 pathogenic variant carriers: A nonrandomized controlled trial. JAMA Oncology 7:1203-1212, 2021. PMID 34081085.

Importance Most women with a BRCA1/2 pathogenic variant undergo premature menopause with potential short- and long-term morbidity due to the current method of ovarian carcinoma prevention: risk-reducing salpingo-oophorectomy (RRSO). Because the fallopian tubes play a key role in ovarian cancer pathogenesis, salpingectomy with delayed oophorectomy may be a novel risk-reducing strategy with benefits of delaying menopause.

Objective To compare menopause-related quality of life after risk-reducing salpingectomy (RRS) with delayed oophorectomy with RRSO in carriers of the BRCA1/2 pathogenic variant.

Design, setting, and participants A multicenter nonrandomized controlled preference trial (TUBA study), with patient recruitment between January 16, 2015, and November 7, 2019, and follow-up at 3 and 12 months after surgery was conducted in all Dutch university hospitals and a few large general hospitals. In the Netherlands, RRSO is predominantly performed in these hospitals. Patients at the clinical genetics or gynecology department between the ages of 25 and 40 years (BRCA1) or 25 to 45 years (BRCA2) who were premenopausal, had completed childbearing, and were undergoing no current treatment for cancer were eligible.

Interventions Risk-reducing salpingo-oophorectomy at currently recommended age or RRS after completed childbearing with delayed oophorectomy. After RRSO was performed, hormone replacement therapy was recommended for women without contraindications.

Main outcomes and measures Menopause-related quality of life as assessed by the Greene Climacteric Scale, with a higher scale sum (range, 0-63) representing more climacteric symptoms. Secondary outcomes were health-related quality of life, sexual functioning and distress, cancer worry, decisional regret, and surgical outcomes.

Results A total of 577 women (mean [SD] age, 37.2 [3.5] years) were enrolled: 297 (51.5%) were pathogenic BRCA1 variant carriers and 280 (48.5%) were BRCA2 pathogenic variant carriers. At the time of analysis, 394 patients had undergone RRS and 154 had undergone RRSO. Without hormone replacement therapy, the adjusted mean increase from the baseline score on the Greene Climacteric Scale was 6.7 (95% CI, 5.0-8.4; P < .001) points higher during 1 year after RRSO than after RRS. After RRSO with hormone replacement therapy, the difference was 3.6 points (95% CI, 2.3-4.8; P < .001) compared with RRS.

Conclusions and relevance Results of this nonrandomized controlled trial suggest that patients have better menopause-related quality of life after RRS than after RRSO, regardless of hormone replacement therapy. An international follow-up study is currently evaluating the oncologic safety of this therapy.


Rare cancers

Thomas Crezee, Marika H Tesselaar, James Nagarajah, Willem E Corver, Johannes Morreau 4, Catrin Pritchard, Shioko Kimura, Josephina G Kuiper, Ilse van Engen-van Grunsven, Jan W A Smit, Romana T Netea-Maier, Theo S Plantinga. Digoxin treatment reactivates in vivo radioactive iodide uptake and correlates with favorable clinical outcome in non-medullary thyroid cancer. Cellular Oncology 44: 611-625, 2021. PMID 33534128.

Purpose Non-medullary thyroid cancer (NMTC) treatment is based on the ability of thyroid follicular cells to accumulate radioactive iodide (RAI). However, in a subset of NMTC patients tumor dedifferentiation occurs, leading to RAI resistance. Digoxin has been demonstrated to restore iodide uptake capacity in vitro in poorly differentiated and anaplastic NMTC cells, termed redifferentiation. The aim of the present study was to investigate the in vivo effects of digoxin in TPO-Cre/LSL-BrafV600E mice and digoxin-treated NMTC patients.

Methods Mice with thyroid cancer were subjected to 3D ultrasound for monitoring tumor growth and 124I PET/CT for measurement of intratumoral iodide uptake. Post-mortem analyses on tumor tissues comprised gene expression profiling and measurement of intratumoral autophagy activity. Through PALGA (Dutch Pathology Registry), archived tumor material was obtained from 11 non-anaplastic NMTC patients who were using digoxin. Clinical characteristics and tumor material of these patients were compared to 11 matched control NMTC patients never treated with digoxin.

Results We found that in mice, tumor growth was inhibited and 124I accumulation was sustainably increased after short-course digoxin treatment. Post-mortem analyses revealed that digoxin treatment increased autophagy activity and enhanced expression of thyroid-specific genes in mouse tumors compared to vehicle-treated mice. Digoxin-treated NMTC patients exhibited significantly higher autophagy activity and a higher differentiation status as compared to matched control NMTC patients, and were associated with favourable clinical outcome.

Conclusions These in vivo data support the hypothesis that digoxin may represent a repositioned adjunctive treatment modality that suppresses tumor growth and improves RAI sensitivity in patients with RAI-refractory NMTC.

Massis Krekorian, Kimberley R G Cortenbach, Milou Boswinkel, Annemarie Kip, Gerben M Franssen, Andor Veltien, Tom W J Scheenen, René Raavé, Nicolaas Koen van Riessen, Mangala Srinivas, Ingrid Jolanda M de Vries, Carl G Figdor, Erik H J G Aarntzen, Sandra Heskamp. In Vivo PET imaging of monocytes labeled with [ 89 Zr]Zr-PLGA-NH 2 nanoparticles in tumor and staphylococcus aureus infection models. Cancers (Basel) 13:5069, 2021. PMID 34680219.

The exponential growth of research on cell-based therapy is in major need of reliable and sensitive tracking of a small number of therapeutic cells to improve our understanding of the in vivo cell-targeting properties. 111In-labeled poly(lactic-co-glycolic acid) with a primary amine endcap nanoparticles ([111In]In-PLGA-NH2 NPs) were previously used for cell labeling and in vivo tracking, using SPECT/CT imaging. However, to detect a low number of cells, a higher sensitivity of PET is preferred. Therefore, we developed 89Zr-labeled NPs for ex vivo cell labeling and in vivo cell tracking, using PET/MRI. We intrinsically and efficiently labeled PLGA-NH2 NPs with [89Zr]ZrCl4. In vitro, [89Zr]Zr-PLGA-NH2 NPs retained the radionuclide over a period of 2 weeks in PBS and human serum. THP-1 (human monocyte cell line) cells could be labeled with the NPs and retained the radionuclide over a period of 2 days, with no negative effect on cell viability (specific activity 279 ± 10 kBq/106 cells). PET/MRI imaging could detect low numbers of [89Zr]Zr-THP-1 cells (10,000 and 100,000 cells) injected subcutaneously in Matrigel. Last, in vivo tracking of the [89Zr]Zr-THP-1 cells upon intravenous injection showed specific accumulation in local intramuscular Staphylococcus aureus infection and infiltration into MDA-MB-231 tumors. In conclusion, we showed that [89Zr]Zr-PLGA-NH2 NPs can be used for immune-cell labeling and subsequent in vivo tracking of a small number of cells in different disease models.

Martin Jaeger, Yvette J E Sloot, Rob Ter Horst, Xiaojing Chu, Hans J P M Koenen, Valerie A C M Koeken, Simone J C F M Moorlag, Charlotte J de Bree, Vera P Mourits, Heidi Lemmers, Helga Dijkstra, Marco Medici, Antonius E van Herwaarden, Irma Joosten, Leo A B Joosten, Yang Li, Johannes W A Smit, Mihai G Netea, Romana T Netea-Maier. Thyrotrophin and thyroxine support immune homeostasis in humans. Immunology 163: 155-168, 2021. PMID 33454989.

The endocrine and the immune systems interact by sharing receptors for hormones and cytokines, cross-control and feedback mechanisms. To date, no comprehensive study has assessed the impact of thyroid hormones on immune homeostasis. By studying immune phenotype (cell populations, antibody concentrations, circulating cytokines, adipokines and acute-phase proteins, monocyte-platelet interactions and cytokine production capacity) in two large independent cohorts of healthy volunteers of Western European descent from the Human Functional Genomics Project (500FG and 300BCG cohorts), we identified a crucial role of the thyroid hormone thyroxin (T4) and thyroid-stimulating hormone (TSH) on the homeostasis of lymphocyte populations. TSH concentrations were strongly associated with multiple populations of both effector and regulatory T cells, whereas B-cell populations were significantly associated with free T4 (fT4). In contrast, fT4 and TSH had little impact on myeloid cell populations and cytokine production capacity. Mendelian randomization further supported the role of fT4 for lymphocyte homeostasis. Subsequently, using a genomics approach, we identified genetic variants that influence both fT4 and TSH concentrations and immune responses, and gene set enrichment pathway analysis showed enrichment of fT4-affected gene expression in B-cell function pathways, including the CD40 pathway, further supporting the importance of fT4 in the regulation of B-cell function. In conclusion, we show that thyroid function controls the homeostasis of the lymphoid cell compartment. These findings improve our understanding of the immune responses and open the door for exploring and understanding the role of thyroid hormones in the lymphocyte function during disease.

Renal disorders

Lynette J Oost, Amber A W A van der Heijden, Emma A Vermeulen, Caro Bos, Petra J M Elders, Roderick C Slieker, Steef Kurstjens, Miranda van Berkel, Joost G J Hoenderop, Cees J Tack, Joline W J Beulens, Jeroen H F de Baaij. Serum magnesium is inversely associated with heart failure, atrial fibrillation, and microvascular complications in type 2 diabetes. Diabetics Care 44: 1757-1765, 2021. PMID 34385344.

Objective We investigated whether serum magnesium (Mg2+) was prospectively associated with macro- or microvascular complications and mediated by glycemic control (hemoglobin A1c [HbA1c]), in type 2 diabetes (T2D).

Research design and methods We analyzed in 4,348 participants the association of serum Mg2+ with macrovascular disease and mortality (acute myocardial infarction [AMI], coronary heart disease [CHD], heart failure [HF], cerebrovascular accident [CVA], and peripheral arterial disease [PAD]), atrial fibrillation (AF), and microvascular complications (chronic kidney disease [CKD], diabetic retinopathy, and diabetic foot) using Cox regression, adjusted for confounders. Mediation analysis was performed to assess whether HbA1c mediated these associations.

Results The average baseline serum Mg2+ concentration was 0.80 ± 0.08 mmol/L. During 6.1 years of follow-up, serum Mg2+ was inversely associated with major macrovascular, 0.87 (95% CI 0.76; 1.00); HF, 0.76 (95% CI 0.62; 0.93); and AF, 0.59 (95% CI 0.49; 0.72). Serum Mg2+ was not associated with AMI, CHD, CVA, and PAD. During 5.1 years of follow-up, serum Mg2+ was inversely associated with overall microvascular events, 0.85 (95% CI 0.78; 0.91); 0.89 (95% CI 0.82; 0.96) for CKD, 0.77 (95% CI 0.61; 0.98) for diabetic retinopathy, and 0.85 (95% CI 0.78; 0.92) for diabetic foot. HbA1c mediated the associations of serum Mg2+ with HF, overall microvascular events, diabetic retinopathy, and diabetic foot.

Conclusions Serum Mg2+ concentration is inversely associated with the risk to develop HF and AF and with the occurrence of CKD, diabetic retinopathy, and foot complications in T2D. Glycemic control partially mediated the association of serum Mg2+ with HF and microvascular complications.

Lia C M J Goltstein, Karina V Grooteman, Alba Rocco, Grainne Holleran, Santiago Frago, Paulo S Salgueiro, Thomas Aparicio, Giuseppe Scaglione, Stefania Chetcuti Zammit, Raul Prados-Manzano , Robert Benamouzig, Gerardo Nardone, Deirdre McNamara, Mourad Benallaoua, Spyridon Michopoulos, Reena Sidhu, Wietske Kievit, Joost P H Drenth, Erwin J M van Geenen. Effectiveness and predictors of response to somatostatin analogues in patients with gastrointestinal angiodysplasias: a systematic review and individual patient data meta-analysis. The Lancet Gastroenterology & Hepatology 6: 922-932, 2021. PMID 34508668.

Background Gastrointestinal angiodysplasias are vascular malformations that often cause red blood cell transfusion-dependent anaemia. Several studies suggest that somatostatin analogues might decrease rebleeding rates, but the true effect size is unknown. We therefore aimed to investigate the efficacy of somatostatin analogues on red blood cell transfusion requirements of patients with gastrointestinal angiodysplasias and to identify subgroups that might benefit the most from somatostatin analogue therapy.

Methods We did a systematic review and individual patient data meta-analysis. We searched MEDLINE, Embase, and Cochrane on Jan 15, 2016, with an updated search on April 25, 2021. All published randomised controlled trials and cohort studies that reported on somatostatin analogue therapy in patients with gastrointestinal angiodysplasias were eligible for screening. We excluded studies without original patient data, single case reports, small case series (ie, <10 participants), studies in which patients had a specific aetiology of gastrointestinal angiodysplasias, and studies in which somatostatin analogue therapy was initiated simultaneously with other treatment modalities. Authors of eligible studies were invited to share individual patient data. Aggregated data was used if individual patient data were not provided. The primary outcome was the mean reduction in the number of red blood cell transfusions during somatostatin analogue therapy, compared with baseline, expressed as the incidence rate ratio (IRR) and absolute mean decrease. We defined patients as either good responders (≥50% reduction in the number of red blood cell transfusions) or poor responders (<50% reduction). A mixed-effects negative binomial regression was used to account for clustering of patients and skewness in data. This study was registered in the International Prospective Register of Systematic Reviews (PROSPERO), number CRD42020213985.

Findings We identified 11 eligible studies (one randomised controlled trial and ten cohort studies) of moderate-to-high quality and obtained individual patient data from the authors of nine (82%) studies. The remaining two (18%) studies provided sufficient information in the published manuscript to extract individual patient data. In total, we analysed data from 212 patients. Somatostatin analogues reduced the number of red blood cell transfusions with an IRR of 0·18 (95% CI 0·14-0·24; p<0·0001) during a median treatment duration of 12 months (IQR 6·0-12·0) and follow-up period of 12 months (12·0-12·0), correlating with a mean absolute decrease in the number of red blood cell transfusions from 12·8 (95% CI 10·4-15·8) during baseline to 2·3 (1·9-2·9) during follow-up-ie, a reduction of 10·5 red blood cell transfusions (p<0·0001). 177 (83%) of 212 patients had a good response to somatostatin analogue therapy (defined as at least a 50% reduction in the number of red blood cell transfusions). Heterogeneity across studies was moderate (I2=53%; p=0·02). Location of gastrointestinal angiodysplasias in the stomach compared with angiodysplasias in the small bowel and colon (IRR interaction 1·92 [95% CI 1·13-3·26]; p=0·02) was associated with worse treatment response. Octreotide was associated with a better treatment response than lanreotide therapy (IRR interaction 2·13 [95% CI 1·12-4·04]; p=0·02). The certainty of evidence was high for the randomised controlled trial and low for the ten cohort studies. Adverse events occurred in 38 (18%) of 212 patients receiving somatostatin analogue therapy, with ten (5%) discontinuing this therapy because of adverse events. The most common adverse events were loose stools (seven [3%] of 212), cholelithiasis (five [2%]), flatulence (four [2%]), and administration site reactions (erythema, five [2%]).

Interpretation Somatostatin analogue therapy is safe and effective in most patients with red blood cell transfusion-dependent bleeding due to gastrointestinal angiodysplasias. Somatostatin analogue therapy is more effective in patients with angiodysplasias located in the small bowel and colon, and octreotide therapy seems to be more effective than lanreotide therapy.

Funding The Netherlands Organisation for Health Research and Development and the Radboud University Medical Center.

Mandy M T van Leent, Thijs J Beldman, Yohana C Toner, Marnix A Lameijer, Nils Rother, Siroon Bekkering, Abraham J P Teunissen, Xianxiao Zhou, Roy van der Meel, Joost Malkus, Sheqouia A Nauta, Emma D Klein, Francois Fay, Brenda L Sanchez-Gaytan, Carlos Pérez-Medina, Ewelina Kluza, Yu-Xiang Ye, Gregory Wojtkiewicz, Edward A Fisher, Filip K Swirski, Matthias Nahrendorf, Bin Zhang, Yang Li, Bowen Zhang, Leo A B Joosten, Gerard Pasterkamp, Arjan Boltjes, Zahi A Fayad, Esther Lutgens, Mihai G Netea, Niels P Riksen, Willem J M Mulder, Raphaël Duivenvoorden. Prosaposin mediates inflammation in atherosclerosis. Science Translational Medicine 13: eabe1433, 2021. PMID 33692130.

Macrophages play a central role in the pathogenesis of atherosclerosis. The inflammatory properties of these cells are dictated by their metabolism, of which the mechanistic target of rapamycin (mTOR) signaling pathway is a key regulator. Using myeloid cell-specific nanobiologics in apolipoprotein E-deficient (Apoe -/-) mice, we found that targeting the mTOR and ribosomal protein S6 kinase-1 (S6K1) signaling pathways rapidly diminished plaque macrophages' inflammatory activity. By investigating transcriptome modifications, we identified Psap, a gene encoding the lysosomal protein prosaposin, as closely related with mTOR signaling. Subsequent in vitro experiments revealed that Psap inhibition suppressed both glycolysis and oxidative phosphorylation. Transplantation of Psap -/- bone marrow to low-density lipoprotein receptor knockout (Ldlr -/-) mice led to a reduction in atherosclerosis development and plaque inflammation. Last, we confirmed the relationship between PSAP expression and inflammation in human carotid atherosclerotic plaques. Our findings provide mechanistic insights into the development of atherosclerosis and identify prosaposin as a potential therapeutic target.