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Abstract The diagnosis of lymphomas is challenging due to their diverse histological presentations and clinical manifestations. There is a need for inexpensive tools that require minimal expertise and are accessible for routine laboratories. Contrastingly, current conventional diagnostic methods are often found only in specialized environments. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy offers a nondestructive and user-friendly approach in the analysis of a wide range of samples. In this paper, we determined whether the technique coupled with machine learning can detect and differentiate lymphoma within lymphoid tissue samples. Tissue sections from 295 individuals diagnosed with lymphoma and 389 individuals without the disease were analyzed using ATR-FTIR spectroscopy. The resulting spectral dataset was split using a 70:30 train-test split. Partial least Squares Discriminant Analysis (PLS-DA) models were trained to distinguish non-malignant lymphoid tissue from lymphoma samples and to differentiate between subtypes. On the training set (n = 478), significant spectral differences were mainly identified in the 1800–900 cm–1 region, attributed to fundamental biochemical constituents like proteins, lipids, carbohydrates, and nucleic acids. On the independent test set (n = 206), the trained PLS-DA model achieved a promising AUC of 0.882 (95% CI: 0.881–0.884) in the differentiation between lymphoma and non-malignant lymphoid tissue. In addition, comparative analyses revealed spectral distinctions and notable clustering between the different lymphoma subtypes. This study provides valuable insights into the application of ATR-FTIR spectroscopy and machine learning in the field of lymphoma diagnosis as a non-destructive, rapid and inexpensive tool with the potential to be easily implemented in non-specialized laboratories.

Abstract Background As the spatial resolution of positron emission tomography (PET) scanners improves, understanding of radiotracer distributions in tissues at high resolutions is important. Hence, we propose a method for co-registration of high-resolution ex vivo specimen PET images, combined with computed tomography (CT) images, and the corresponding specimen histopathology. Methods We applied our co-registration method to breast cancer (BCa) specimens of patients who were preoperatively injected with 0.8 MBq/kg [ $$^{18}$$ 18 F]fluorodeoxyglucose ([18F]FDG). The method has two components. First, we used an image acquisition scheme that minimises and tracks tissue deformation: (1) We acquired sub-millimetre (micro)-PET-CT images of ±2 mm-thick lamellas of the fresh specimens, enclosed in tissue cassettes. (2) We acquired micro-CT images of the same lamellas after formalin fixation to visualise tissue deformation. (3) We obtained 1 hematoxylin and eosin (H&E) stained histopathology section per lamella of which we captured a digital whole slide image (WSI). Second, we developed an automatic co-registration algorithm to improve the alignment between the micro-PET-CT images and WSIs, guided by the micro-CT of the fixated lamellas. To estimate the spatial co-registration error, we calculated the distance between corresponding microcalcifications in the micro-CTs and WSIs. The co-registered images allowed to study standardised uptake values (SUVs) of different breast tissues, as identified on the WSIs by a pathologist. Results We imaged 22 BCa specimens, 13 cases of invasive carcinoma of no special type (NST), 6 of invasive lobular carcinoma (ILC), and 3 of ductal carcinoma in situ (DCIS). While the cassette framework minimised tissue deformation, the best alignment between the micro-PET-CT images and WSIs was achieved after deformable co-registration. We found an overall average co-registration error of 0.74 ± 0.17 mm between the micro-PET images and WSIs. (Pre)malignant tissue (including NST, ILC, and DCIS) generally showed higher SUVs than healthy tissue (including healthy glandular, connective, and adipose tissue). As expected, inflamed tissue and skin also showed high uptake. Conclusions We developed a method to co-register micro-PET-CT images of surgical specimens and WSIs with an accuracy comparable to the spatial resolution of the micro-PET images. While currently, we only applied this method to BCa specimens, we believe this method is applicable to a wide range of specimens and radiotracers, providing insight into distributions of (new) radiotracers in human malignancies at a sub-millimetre resolution.

Abstract Type I interferon (IFN) is a potent antitumoral drug, with an important history in the treatment of hematologic malignancies. However, its pleiotropic nature leads to severe dose-limiting toxicities that blunt its therapeutic potential. To achieve selective targeting of specific immune or tumor cells, AcTakines (Activity-on-Target Cytokines), i.e., immunocytokines utilizing attenuated cytokines, and clinically optimized A-Kines™ were developed. In syngeneic murine models, the CD20-targeted murine IFNα2-based AcTaferons (AFNs) have demonstrated clear antitumoral effects, with excellent tolerability. The current study explores the antitumoral potential of the humanized huCD20-Fc-AFN in 5 different humanized patient derived xenograft (PDX) models of huCD20+ aggressive B non-Hodgkin lymphomas (B-NHLs). The huCD20-Fc-AFN consists of a huCD20-specific single-domain antibody (VHH) linked through a heterodimeric ‘knob-in-hole’ human IgG1 Fc molecule to an attenuated huIFNα2 sequence. An in vitro targeting efficacy of up to 1.000-fold could be obtained, without detectable in vivo toxicities, except for selective (on-target) and reversible B cell depletion. Treatment with huCD20-Fc-AFN significantly increased the median overall survival (mOS) in both non-humanized (mOS 31 to 45 days; HR = 0.26; p = 0.001), and humanized NSG/NOG mice (mOS 34 to 80 days; HR = 0.37; p

Abstract Objectives Cytogenetic abnormalities are predictors of poor prognosis in multiple myeloma (MM). This paper aims to build and validate a multiparametric conventional and functional whole-body MRI-based prediction model for cytogenetic risk classification in newly diagnosed MM. Methods Patients with newly diagnosed MM who underwent multiparametric conventional whole-body MRI, spinal dynamic contrast-enhanced (DCE-)MRI, spinal diffusion-weighted MRI (DWI) and had genetic analysis were retrospectively included (2011–2020/Ghent University Hospital/Belgium). Patients were stratified into standard versus intermediate/high cytogenetic risk groups. After segmentation, 303 MRI features were extracted. Univariate and model-based methods were evaluated for feature and model selection. Testing was performed using receiver operating characteristic (ROC) and precision-recall curves. Models comparing the performance for genetic risk classification of the entire MRI protocol and of all MRI sequences separately were evaluated, including all features. Four final models, including only the top three most predictive features, were evaluated. Results Thirty-one patients were enrolled (mean age 66 ± 7 years, 15 men, 13 intermediate-/high-risk genetics). None of the univariate models and none of the models with all features included achieved good performance. The best performing model with only the three most predictive features and including all MRI sequences reached a ROC-area-under-the-curve of 0.80 and precision-recall-area-under-the-curve of 0.79. The highest statistical performance was reached when all three MRI sequences were combined (conventional whole-body MRI + DCE-MRI + DWI). Conventional MRI always outperformed the other sequences. DCE-MRI always outperformed DWI, except for specificity. Conclusions A multiparametric MRI-based model has a better performance in the noninvasive prediction of high-risk cytogenetics in newly diagnosed MM than conventional MRI alone. Critical relevance statement An elaborate multiparametric MRI-based model performs better than conventional MRI alone for the noninvasive prediction of high-risk cytogenetics in newly diagnosed multiple myeloma; this opens opportunities to assess genetic heterogeneity thus overcoming sampling bias. Key points • Standard genetic techniques in multiple myeloma patients suffer from sampling bias due to tumoral heterogeneity. • Multiparametric MRI noninvasively predicts genetic risk in multiple myeloma. • Combined conventional anatomical MRI, DCE-MRI, and DWI had the highest statistical performance to predict genetic risk. • Conventional MRI alone always outperformed DCE-MRI and DWI separately to predict genetic risk. DCE-MRI alone always outperformed DWI separately, except for the parameter specificity to predict genetic risk. • This multiparametric MRI-based genetic risk prediction model opens opportunities to noninvasively assess genetic heterogeneity thereby overcoming sampling bias in predicting genetic risk in multiple myeloma. Graphical Abstract

Abstract Ovarian cancer is the most lethal gynecologic malignancy, mainly due to late-stage diagnosis, frequent recurrences, and eventually therapy resistance. To identify potentially actionable genetic variants, sequencing data of 351 Belgian ovarian cancer patients were retrospectively captured from electronic health records. The cohort included 286 (81%) patients with high-grade serous ovarian cancer, 17 (5%) with low-grade serous ovarian cancer, and 48 (14%) with other histotypes. Firstly, an overview of the prevalence and spectrum of the BRCA1/2 variants highlighted germline variants in 4% (11/250) and somatic variants in 11% (37/348) of patients. Secondly, application of a multi-gene panel in 168 tumors revealed a total of 214 variants in 28 genes beyond BRCA1/2 with a median of 1 (IQR, 1–2) genetic variant per patient. The ten most often altered genes were (in descending order): TP53, BRCA1, PIK3CA, BRCA2, KRAS, ERBB2 (HER2), TERT promotor, RB1, PIK3R1 and PTEN. Of note, the genetic landscape vastly differed between the studied histotypes. Finally, using ESCAT the clinical evidence of utility for every genetic variant was scored. Only BRCA1/2 pathogenic variants were classified as tier-I. Nearly all patients (151/168; 90%) had an ESCAT tier-II variant, most frequently in TP53 (74%), PIK3CA (9%) and KRAS (7%). In conclusion, our findings imply that although only a small proportion of genetic variants currently have direct impact on ovarian cancer treatment decisions, other variants could help to identify novel (personalized) treatment options to address the poor prognosis of ovarian cancer, particularly in rare histotypes.

Introduction: This case report demonstrates the possibility of successful eye and vision-sparing therapy for caruncular melanoma. Case Presentation: We present an atypical presentation of a caruncular melanoma. After excisional biopsy, residual flat conjunctival melanosis resolved using topical chemotherapy (5-fluorouracil), which was well tolerated. Relapse of the melanoma was treated with external beam radiotherapy, but the tumor grew despite treatment. Eighteen months after complete excision of the relapsed melanoma, the patient remains tumor-free while the eye and its function remain preserved. Conclusion: This case report suggests that aggressive eye-sparing therapy for caruncular melanoma combining surgery, adjuvant topical chemotherapy, and external beam radiotherapy, can be an alternative for primary orbital exenteration.

Purpose: To describe the clinical presentation and management of severe capecitabine-induced corneal toxicity. Observations: A 71-year-old woman presented with severe bilateral vision loss. Four months earlier, capecitabine was initiated for a metastatic invasive ductal carcinoma. Biomicroscopy revealed bilateral whorl-like corneal epitheliopathy accompanied by metaplasia, keratinization and subepithelial fibrosis. After consulting the treating oncologist, capecitabine treatment was discontinued. Initially, a non-surgical approach was adopted and intensive topical dexamethasone treatment was applied. Despite capecitabine discontinuation and topical steroid treatment, visual acuity progressively declined. Bilateral corneal scraping and bandage contact lens fitting was performed. This resulted in significant improvement of visual acuity, corneal surface regularity and quality of life. Conclusion and importance: We report the first case of severe visual impairment due to capecitabine-induced corneal toxicity. Early corneal scraping, especially when confronted with profound vision loss, may yield better outcomes compared to relying on spontaneous recovery after capecitabine discontinuation. Patients experiencing ocular discomfort and vision loss, while receiving capecitabine therapy, should be referred for semi-urgent ophthalmological examination.

AbstractBurn injuries can result in a significant inflammatory response, often leading to hypertrophic scarring (HTS). Local drug therapies e.g. corticoid injections are advised to treat HTS, although they are invasive, operator-dependent, extremely painful and do not permit extended drug release. Polymer-based microneedle (MN) arrays can offer a viable alternative to standard care, while allowing for direct, painless dermal drug delivery with tailorable drug release profile. In the current study, we synthesized photo-crosslinkable, acrylate-endcapped urethane-based poly(ε-caprolactone) (AUP-PCL) toward the fabrication of MNs. Physico-chemical characterization (1H-NMR, evaluation of swelling, gel fraction) of the developed polymer was performed and confirmed successful acrylation of PCL-diol. Subsequently, AUP-PCL, and commercially available PCL-based microneedle arrays were fabricated for comparative evaluation of the constructs. Hydrocortisone was chosen as model drug. To enhance the drug release efficiency of the MNs, Brij®35, a nonionic surfactant was exploited. The thermal properties of the MNs were evaluated via differential scanning calorimetry. Compression testing of the arrays confirmed that the MNs stay intact upon applying a load of 7 N, which correlates to the standard dermal insertion force of MNs. The drug release profile of the arrays was evaluated, suggesting that the developed PCL arrays can offer efficient drug delivery for up to two days, while the AUP-PCL arrays can provide a release up to three weeks. Finally, the insertion of MN arrays into skin samples was performed, followed by histological analysis demonstrating the AUP-PCL MNs outperforming the PCL arrays upon providing pyramidical-shaped perforations through the epidermal layer of the skin.

Background and purpose: Chemoradiotherapy followed by brachytherapy is the standard of care for locally advanced cervical cancer (LACC). In this study, we postulate that omitting an iconographical unaffected uterus (+12 mm distance from the tumour) from the treatment volume is safe and that no tumour will be found in the non-targeted uterus (NTU) leading to reduction of high-dose volumes of surrounding organs at risk (OARs) Material and Methods: In this single-arm phase 2 study, two sets of target volumes were delineated: one standard-volume (whole uterus) and an EXIT-volume (exclusion of non-tumour-bearing parts of the uterus with a minimum 12 mm margin from the tumour). All patients underwent chemoradiotherapy targeting the EXIT-volume, followed by completion hysterectomy. In 15 patients, a plan comparison between two treatment plans (PTV vs PTV_EXIT) was performed. The primary endpoint was the pathological absence of tumour involvement in the non-targeted uterus (NTU). Secondary endpoints included dosimetric impact of target volume reduction on OARs, acute and chronic toxicity, overall survival (OS), locoregional recurrence-free survival (LRFS), and progression-free survival (PFS). Results: In all 21 (FIGO stage I: 2; II: 14;III: 3; IV: 2) patients the NTU was pathologically negative. Ssignificant reductions in Dmean in bladder, sigmoid and rectum; V15Gy in sigmoid and rectum, V30Gy in bladder, sigmoid and rectum; V40Gy and V45Gy in bladder, bowel bag, sigmoid and rectum; V50Gy in rectum were achieved. Median follow-up was 54 months (range 7–79 months). Acute toxicity was mainly grade 2 and 5 % grade 3 urinary. The 3y- OS, PFS and LRFS were respectively 76,2%, 64,9% and 81 %. Conclusion: MRI-based exclusion of the non-tumour-bearing parts of the uterus at a minimum distance of 12 mm from the tumour out of the target volume in LACC can be done without risk of residual disease in the NTU, leading to a significant reduction of the volume of surrounding OARS treated to high doses.

Abstract Craniofacial abnormalities account for approximately one third of birth defects. The regulatory programs that build the face require precisely controlled spatiotemporal gene expression, achieved through tissue-specific enhancers. Clusters of coactivated enhancers and their target genes, known as superenhancers, are important in determining cell identity but have been largely unexplored in development. In this study we identified superenhancer regions unique to human embryonic craniofacial tissue. To demonstrate the importance of such regions in craniofacial development and disease, we focused on an ~600 kb noncoding region located between NPVF and NFE2L3. We identified long range interactions with this region in both human and mouse embryonic craniofacial tissue with the anterior portion of the HOXA gene cluster. Mice lacking this superenhancer exhibit perinatal lethality, and present with highly penetrant skull defects and orofacial clefts phenocopying Hoxa2-/- mice. Moreover, we identified two cases of de novo copy number changes of the superenhancer in humans both with severe craniofacial abnormalities. This evidence suggests we have identified a critical noncoding locus control region that specifically regulates anterior HOXA genes and copy number changes are pathogenic in human patients.

Abstract Background Long-term drug evaluation heavily relies upon rodent models. Drug discovery methods to reduce animal models in oncology may include three-dimensional (3D) cellular systems that take into account tumor microenvironment (TME) cell types and biomechanical properties. Methods In this study we reconstructed a 3D tumor using an elastic polymer (acrylate-endcapped urethane-based poly(ethylene glycol) (AUPPEG)) with clinical relevant stiffness. Single cell suspensions from low-grade serous ovarian cancer (LGSOC) patient-derived early passage cultures of cancer cells and cancer-associated fibroblasts (CAF) embedded in a collagen gel were introduced to the AUPPEG scaffold. After self-organization in to a 3D tumor, this model was evaluated by a long-term (> 40 days) exposure to a drug combination of MEK and HSP90 inhibitors. The drug-response results from this long-term in vitro model are compared with drug responses in an orthotopic LGSOC xenograft mouse model. Results The in vitro 3D scaffold LGSOC model mimics the growth ratio and spatial organization of the LGSOC. The AUPPEG scaffold approach allows to test new targeted treatments and monitor long-term drug responses. The results correlate with those of the orthotopic LGSOC xenograft mouse model. Conclusions The mechanically-tunable scaffolds colonized by a three-dimensional LGSOC allow long-term drug evaluation and can be considered as a valid alternative to reduce, replace and refine animal models in drug discovery. Graphical Abstract

The development of neuroblastoma (NB) is regulated by multiple core transcription factors. Here, SOX11 is identified as a potential epigenetic master regulator upstream of the core regulatory circuitry in adrenergic high-risk neuroblastoma.

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) are rare aggressive hematologic malignancies. Current treatment consists of intensive chemotherapy leading to 80% overall survival but is associated with severe toxic side effects. Furthermore, 10-20% of patients still die from relapsed or refractory disease providing a strong rationale for more specific, targeted therapeutic strategies with less toxicities. Here, we report a novel MYH9::PDGFRB fusion in a T-LBL patient, and demonstrate that this fusion product is constitutively active and sufficient to drive oncogenic transformation in vitro and in vivo. Expanding our analysis more broadly across T-ALL, we found a T-ALL cell line and multiple patient-derived xenograft models with PDGFRB hyperactivation in the absence of a fusion, with high PDGFRB expression in TLX3 and HOXA T-ALL molecular subtypes. To target this PDGFRB hyperactivation, we evaluated the therapeutic effects of a selective PDGFRB inhibitor, CP-673451, both in vitro and in vivo and demonstrated sensitivity if the receptor is hyperactivated. Altogether, our work reveals that hyperactivation of PDGFRB is an oncogenic driver in T-ALL/T-LBL, and that screening T-ALL/T-LBL patients for phosphorylated PDGFRB levels can serve as a biomarker for PDGFRB inhibition as a novel targeted therapeutic strategy in their treatment regimen.

IntroductionDiffuse large B-cell lymphoma (DLBCL) and primary mediastinal B-cell lymphoma (PMBCL) are aggressive histological subtypes of non-Hodgkin’s lymphoma. Improved understanding of the underlying molecular pathogenesis has led to new classification and risk stratification tools, including the development of cell-free biomarkers through liquid biopsies. The goal of this study was to investigate cell-free RNA (cfRNA) biomarkers in DLBCL and PMBCL patients.Materials and methodsBlood plasma samples (n=168) and matched diagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples (n=69) of DLBCL patients, PMBCL patients and healthy controls were collected between 2016-2021. Plasma samples were collected at diagnosis, at interim evaluation, after treatment, and in case of refractory or relapsed disease. RNA was extracted from 200 µl plasma using the miRNeasy serum/plasma kit and from FFPE tissue using the miRNeasy FFPE kit. RNA was subsequently sequenced on a NovaSeq 6000 instrument using the SMARTer Stranded Total RNA-seq pico v3 library preparation kit.ResultsHigher cfRNA concentrations were demonstrated in lymphoma patients compared to healthy controls. A large number of differentially abundant genes were identified between the cell-free transcriptomes of DLBCL patients, PMBCL patients, and healthy controls. Overlap analyses with matched FFPE samples showed that blood plasma has a unique transcriptomic profile that significantly differs from that of the tumor tissue. As a good concordance between tissue-derived gene expression and the immunohistochemistry Hans algorithm for cell-of-origin (COO) classification was demonstrated in the FFPE samples, but not in the plasma samples, a 64-gene cfRNA classifier was developed that can accurately determine COO in plasma. High plasma levels of a 9-gene signature (BECN1, PRKCB, COPA, TSC22D3, MAP2K3, UQCRHL, PTMAP4, EHD1, NAP1L1 pseudogene) and a 5-gene signature (FTH1P7, PTMAP4, ATF4, FTH1P8, ARMC7) were significantly associated with inferior progression-free and overall survival in DLBCL patients, respectively, independent of the NCCN-IPI score.ConclusionTotal RNA sequencing of blood plasma samples allows the analysis of the cell-free transcriptome in DLBCL and PMBCL patients and demonstrates its unexplored potential in identifying diagnostic, cell-of-origin, and prognostic cfRNA biomarkers.

Defects in primary or motile cilia result in a variety of human pathologies, and retinal degeneration is frequently associated with these so-called ciliopathies. We found that homozygosity for a truncating variant in CEP162, a centrosome and microtubule-associated protein required for transition zone assembly during ciliogenesis and neuronal differentiation in the retina, caused late-onset retinitis pigmentosa in 2 unrelated families. The mutant CEP162-E646R*5 protein was expressed and properly localized to the mitotic spindle, but it was missing from the basal body in primary and photoreceptor cilia. This impaired recruitment of transition zone components to the basal body and corresponded to complete loss of CEP162 function at the ciliary compartment, reflected by delayed formation of dysmorphic cilia. In contrast, shRNA knockdown of Cep162 in the developing mouse retina increased cell death, which was rescued by expression of CEP162-E646R*5, indicating that the mutant retains its role for retinal neurogenesis. Human retinal degeneration thus resulted from specific loss of the ciliary function of CEP162.

Here the authors provide a detailed virological analysis of thirteen postmortem COVID-19 cases, including presence of replication-competent SARS-CoV-2 in extrapulmonary organs and tissue-specific patterns of SARS-CoV-2 genome diversity of an immunocompromised patient.

Abstract DNA mismatch repair deficiency (dMMR) testing is crucial for diagnosing Lynch syndrome and detection of microsatellite unstable (MSI) tumors eligible for immunotherapy. The aim of this study was to compare the relative diagnostic performance of three molecular MSI assays: polymerase chain reaction (PCR), MSI testing by Idylla and next-generation-sequencing (NGS) on 49 tumor samples (28 colorectal and 21 endometrial adenocarcinomas) versus immunohistochemistry (IHC). Discrepancies were investigated by MLH1 methylation analysis and integrated with germline results if available. Overall, the molecular assays achieved equivalent diagnostic performance for MSI detection with area under the ROC curves (AUC) of respectively 0.91 for Idylla and PCR, and 0.93 for NGS. In colorectal cancers with tumor cell percentages ≥ 30% all three molecular assays achieved 100% sensitivity and specificity (AUC = 1) versus IHC. Also, in endometrial cancers, all three molecular assays showed equivalent diagnostic performance, albeit at a clearly lower sensitivity ranging from 58% for Idylla to 75% for NGS, corresponding to negative predictive values from 78 to 86%. PCR, Idylla and NGS show similar diagnostic performance for dMMR detection in colorectal and endometrial cancers. Molecular MSI analysis has lower sensitivity for dMMR detection in endometrial cancer indicating that combined use of both IHC and molecular methods is recommended. Clinical Trial Number/IRB: B1172020000040, Ethical Committee, AZ Delta General Hospital.

The consensus in aging is that inflammation, cellular senescence, free radicals, and epigenetics are contributing factors. Skin glycation through advanced glycation end products (AGEs) has a crucial role in aging. Additionally, it has been suggested that their presence in scars leads to elasticity loss. This manuscript reports fructosamine-3-kinase (FN3K) and fructosyl-amino acid oxidase (FAOD) in counteracting skin glycation by AGEs. Skin specimens were obtained (n = 19) and incubated with glycolaldehyde (GA) for AGE induction. FN3K and FAOD were used as monotherapy or combination therapy. Negative and positive controls were treated with phosphate-buffered saline and aminoguanidine, respectively. Autofluorescence (AF) was used to measure deglycation. An excised hypertrophic scar tissue (HTS) (n = 1) was treated. Changes in chemical bonds and elasticity were evaluated using mid-infrared spectroscopy (MIR) and skin elongation, respectively. Specimens treated with FN3K and FAOD in monotherapy achieved an average decrease of 31% and 33% in AF values, respectively. When treatments were combined, a decrease of 43% was achieved. The positive control decreased by 28%, whilst the negative control showed no difference. Elongation testing of HTS showed a significant elasticity improvement after FN3K treatment. ATR-IR spectra demonstrated differences in chemical bounds pre- versus post-treatment. FN3K and FAOD can achieve deglycation and the effects are most optimal when combined in one treatment.

Abstract This report highlights the importance of integrating clinical, radiological, genetic, and pathological laboratory findings to make a correct diagnosis especially with challenging and rare entities.

Abstract Background In early-stage breast cancer, the cornerstone of treatment is surgery. After breast-conserving surgery, adjuvant radiotherapy has shown to improve locoregional control and overall survival rates. The use of breast radiotherapy in the preoperative (preop) setting is far less common. Nevertheless, it might improve disease-free survival as compared to postoperative radiotherapy. There is also a possibility of downsizing the tumour which might lead to a lower need for mastectomy. There are some obstacles that complicate its introduction into daily practice. It may complicate surgery or lead to an increase in wound complications or delayed wound healing. Another fear of preop radiotherapy is delaying surgery for too long. At Ghent University Hospital, we have experience with a 5-fraction radiotherapy schedule allowing radiotherapy delivery in a very short time span. Methods Twenty female breast cancer patients with non-metastatic disease receiving preop chemotherapy will be randomized between preop or postoperative radiotherapy. The feasibility of preop radiotherapy will be evaluated based on overall treatment time. All patients will be treated in 5 fractions of 5.7 Gy to the whole breast with a simultaneous integrated boost to the tumour/tumour bed of 5 × 6.2 Gy. In case of lymph node irradiation, the lymph node regions will receive a dose of 27 Gy in 5 fractions of 5.4 Gy. The total duration of therapy will be 10 to 12 days. In the preop group, overall treatment time is defined as the time between diagnosis and the day of last surgery, in the postop group between diagnosis and last irradiation fraction. Toxicity related to surgery, radio-, and chemotherapy will be evaluated on dedicated case-report forms at predefined time points. Tumour response will be evaluated on the pathology report and on MRI at baseline and in the interval between chemotherapy and surgery. Discussion The primary objective of the trial is to investigate the feasibility of preop radiotherapy. Secondary objectives are to search for biomarkers of response and toxicity and identify the involved cell death mechanisms and the effect of preop breast radiotherapy on the in-situ immune micro-environment.

Abstract Peritoneal spread indicates poor prognosis in patients with serous ovarian carcinoma (SOC) and is generally treated by surgical cytoreduction and chemotherapy. Novel treatment options are urgently needed to improve patient outcome. Clinically relevant cell lines and patient-derived xenograft (PDX) models are of critical importance to therapeutic regimen evaluation. Here, a PDX model was established, by orthotopic engraftment after subperitoneal tumor slurry injection of low-grade SOC, resulting in an early-stage transplantable peritoneal metastasis (PM)-PDX model. Histology confirmed the micropapillary and cribriform growth pattern with intraluminal tumor budding and positivity for PAX8 and WT1. PM-PDX dissociated cells show an epithelial morphotype with a 42 h doubling time and 40% colony forming efficiency, they are low sensitive to platinum derivatives and highly sensitive to paclitaxel (IC50: 6.3 ± 2.2 nM, mean ± SEM). The patient primary tumor, PM, PM-PDX and derived cell line all show a KRAS c.35 G > T (p.(Gly12Val)) mutation and show sensitivity to the MEK inhibitor trametinib in vitro (IC50: 7.2 ± 0.5 nM, mean ± SEM) and in the PM mouse model. These preclinical models closely reflecting patient tumors are useful to further elucidate LGSOC disease progression, therapy response and resistance mechanisms.

Abstract Background Accurate lung cancer classification is crucial to guide therapeutic decisions. However, histological subtyping by pathologists requires tumor tissue—a necessity that is often intrinsically associated with procedural difficulties. The analysis of circulating tumor DNA present in minimal-invasive blood samples, referred to as liquid biopsies, could therefore emerge as an attractive alternative. Methods Concerning adenocarcinoma, squamous cell carcinoma, and small cell carcinoma, our proof of concept study investigates the potential of liquid biopsy-derived copy number alterations, derived from single-end shallow whole-genome sequencing (coverage 0.1–0.5×), across 51 advanced stage lung cancer patients. Results Genomic abnormality testing reveals anomalies in 86.3% of the liquid biopsies (16/20 for adenocarcinoma, 13/16 for squamous cell, and 15/15 for small cell carcinoma). We demonstrate that copy number profiles from formalin-fixed paraffin-embedded tumor biopsies are well represented by their liquid equivalent. This is especially valid within the small cell carcinoma group, where paired profiles have an average Pearson correlation of 0.86 (95% CI 0.79–0.93). A predictive model trained with public data, derived from 843 tissue biopsies, shows that liquid biopsies exhibit multiple deviations that reflect histological classification. Most notably, distinguishing small from non-small cell lung cancer is characterized by an area under the curve of 0.98 during receiver operating characteristic analysis. Additionally, we investigated how deeper paired-end sequencing, which will eventually become feasible for routine diagnosis, empowers tumor read enrichment by insert size filtering: for all of the 29 resequenced liquid biopsies, the tumor fraction could be increased in silico, thereby “rescuing” three out of five cases with previously undetectable alterations. Conclusions Copy number profiling of cell-free DNA enables histological classification. Since shallow whole-genome sequencing is inexpensive and often fully operational at routine molecular laboratories, this finding has current diagnostic potential, especially for patients with lesions that are difficult to reach.

Green synthesis of gold nanoparticles (AuNPs) using microorganisms has been generally studied aiming for high-yield production and morphologies appropriated for various applications, such as bioremediation, (bio)sensors, and (bio)catalysis. Numerous approaches showed the individual effect of factors influencing the synthesis of AuNPs with limited analysis of the governing factors enhancing the production and desired quality of the precipitates. This study proposes a fractional-factorial design to investigate the simultaneous influence of seven environmental factors (cell concentration, temperature, anoxic/oxic conditions, pH, gold concentration, electron donor type, and bacterial species) on the recovery yield and synthesis of targeted AuNPs. Various sizes and morphologies of the AuNPs were obtained by varying the environmental factors studied. The factors with significant effects (i.e., 0.2 mM Au and pH 5) were selected according to statistical analysis for optimal removal of 88.2 ± 3.5% of gold and with the production of valuable 50 nm AuNPs, which are known for their enhanced sensitivity. Implications of the cytochrome-C on the bacterial mechanisms and the provision of electron donors via an electrochemical system are further discussed. This study helps develop gold recovery and nanoparticle synthesis methods, focusing on the determining factor(s) for efficient, low-cost, green synthesis of valuable materials.

We assess the performance of mRNA capture sequencing to identify fusion transcripts in FFPE tissue of different sarcoma types, followed by RT-qPCR confirmation. To validate our workflow, six positive control tumors with a specific chromosomal rearrangement were analyzed using the TruSight RNA Pan-Cancer Panel. Fusion transcript calling by FusionCatcher confirmed these aberrations and enabled the identification of both fusion gene partners and breakpoints. Next, whole-transcriptome TruSeq RNA Exome sequencing was applied to 17 fusion gene-negative alveolar rhabdomyosarcoma (ARMS) or undifferentiated round cell sarcoma (URCS) tumors, for whom fluorescence in situ hybridization (FISH) did not identify the classical pathognomonic rearrangements. For six patients, a pathognomonic fusion transcript was readily detected, i.e., PAX3-FOXO1 in two ARMS patients, and EWSR1-FLI1, EWSR1-ERG, or EWSR1-NFATC2 in four URCS patients. For the 11 remaining patients, 11 newly identified fusion transcripts were confirmed by RT-qPCR, including COPS3-TOM1L2, NCOA1-DTNB, WWTR1-LINC01986, PLAA-MOB3B, AP1B1-CHEK2, and BRD4-LEUTX fusion transcripts in ARMS patients. Additionally, recurrently detected secondary fusion transcripts in patients diagnosed with EWSR1-NFATC2-positive sarcoma were confirmed (COPS4-TBC1D9, PICALM-SYTL2, SMG6-VPS53, and UBE2F-ALS2). In conclusion, this study shows that mRNA capture sequencing enhances the detection rate of pathognomonic fusions and enables the identification of novel and secondary fusion transcripts in sarcomas.

Inactivating variants as well as a missense variant in the centrosomal CEP78 gene have been identified in autosomal recessive cone-rod dystrophy with hearing loss (CRDHL), a rare syndromic inherited retinal disease distinct from Usher syndrome. Apart from this, a complex structural variant (SV) implicating CEP78 has been reported in CRDHL. Here we aimed to expand the genetic architecture of typical CRDHL by the identification of complex SVs of the CEP78 region and characterization of their underlying mechanisms. Approaches used for the identification of the SVs are shallow whole-genome sequencing (sWGS) combined with quantitative polymerase chain reaction (PCR) and long-range PCR, or ExomeDepth analysis on whole-exome sequencing (WES) data. Targeted or whole-genome nanopore long-read sequencing (LRS) was used to delineate breakpoint junctions at the nucleotide level. For all SVs cases, the effect of the SVs on CEP78 expression was assessed using quantitative PCR on patient-derived RNA. Apart from two novel canonical CEP78 splice variants and a frameshifting single-nucleotide variant (SNV), two SVs affecting CEP78 were identified in three unrelated individuals with CRDHL: a heterozygous total gene deletion of 235 kb and a partial gene deletion of 15 kb in a heterozygous and homozygous state, respectively. Assessment of the molecular consequences of the SVs on patient’s materials displayed a loss-of-function effect. Delineation and characterization of the 15-kb deletion using targeted LRS revealed the previously described complex CEP78 SV, suggestive of a recurrent genomic rearrangement. A founder haplotype was demonstrated for the latter SV in cases of Belgian and British origin, respectively. The novel 235-kb deletion was delineated using whole-genome LRS. Breakpoint analysis showed microhomology and pointed to a replication-based underlying mechanism. Moreover, data mining of bulk and single-cell human and mouse transcriptional datasets, together with CEP78 immunostaining on human retina, linked the CEP78 expression domain with its phenotypic manifestations. Overall, this study supports that the CEP78 locus is prone to distinct SVs and that SV analysis should be considered in a genetic workup of CRDHL. Finally, it demonstrated the power of sWGS and both targeted and whole-genome LRS in identifying and characterizing complex SVs in patients with ocular diseases.

Shallow-depth sequencing of cell-free DNA, an inexpensive and standardized approach to obtain molecular information on tumors non-invasively, has been insufficiently explored for the diagnosis of lymphoma and disease follow-up. This study collected 318 samples, including longitudinal liquid and paired solid biopsies, from a prospectively- recruited cohort of 38 Hodgkin lymphoma (HL) and 85 aggressive B-cell non-HL patients, represented by 81 diffuse large B-cell lymphoma (DLBCL) cases. Following sequencing, copy number alterations and viral read fractions were derived and analyzed. At diagnosis, liquid biopsies showed detectable copy number alterations in 84.2% of HL patients (88.6% for classical HL) and 74.1% of DLBCL patients. Of the DLBCL patients, copy number profiles between liquid-solid pairs were highly concordant (r=0.815±0.043); and, compared to tissue, HL liquid biopsies had abnormalities with higher amplitudes (P=0.010). This implies that tumor DNA is more abundant in plasma. Additionally, 39.5% of HL and 13.6% of DLBCL cases had a significantly elevated number of plasma Epstein-Barr virus DNA fragments, achieving a sensitivity of 100% compared to the current standard. A longitudinal analysis determined that, when detectable, copy number patterns were similar across (re)staging moments in refractory or relapsed patients. Further, the overall profile anomaly correlated highly with the total metabolic tumor volume (P

Uterus transplantation is an experimental infertility treatment for women with uterine factor infertility. During donor uterus retrieval and subsequent storage, ischemia and other stressors are likely to occur, resulting in the delayed restoration of organ function and increased graft rejection. The uterus expresses connexin-based hemichannels, the opening of which can promote ischemic cell death, as well as gap junctions that may expand cell death by bystander signaling. We investigated if connexin channel inhibition with connexin channel inhibitor Gap27 could protect the uterus against cell death during the storage period. The study involved 9 female patients undergoing gender-change surgery. Before uterus removal, it was exposed to in situ warm ischemia with or without reperfusion. Uterus biopsies were taken before, during, and after ischemia, with or without reperfusion, and were subsequently stored under cold (4ᵒC) or warm (37ᵒC) conditions. TUNEL cell death assay was done at various time points along the combined in vivo/ex vivo experimental timeline. We found that Gap27 protected against storage-related cell death under cold but not warm conditions when the uterus had experienced in situ ischemia/reperfusion. For in situ brief ischemia without reperfusion, Gap27 reduction of cell death was delayed and significantly less, suggesting that protection critically depends on processes initiated when the organ was still in the donor. Thus, the inclusion of the connexin channel inhibitor Gap27 during cold storage protects the uterus against cell death, and the degree of protection depends on the history of exposure to warm ischemia. Gap27 protection may be indicated for uteri from deceased donors, in which ischemia is likely because life-saving organs have retrieval priority.

Abstract Background Previous studies on cervical cancer reported a worse outcome for adenocarcinoma (AC) compared with squamous cell carcinoma (SCC). Nevertheless, standard treatment remains identical. Insight in the impact of histological types on biological behavior and pathological complete response rates might result in a treatment paradigm shift. Methods Clinicopathological characteristics, survival rates and relapse patterns were compared between AC (n = 36) and SCC (n = 143) cervical cancer patients. Pathological response to treatment was evaluated in the patient subgroup treated with neo-adjuvant chemoradiation followed by surgery (NA-CRT group; n = 84). Results In the entire cohort, 5y Disease Specific Survival (DSS) was 97.1 and 84% for AC and SCC respectively (p = 0.150). In the NA-CRT group 5y DSS was 100 and 75.5% for AC and SCC respectively (p = 0.059). Relapse patterns did not differ significantly between AC and SCC in the entire cohort, or in the NA-CRT group. Adenocarcinoma patients treated with NA-CRT showed significantly less pathological complete response compared with SCC patients (AC = 7%, SCC = 43%, p = 0.027). Conclusions There were no statistically significant differences regarding relapse and DSS rates between SCC and AC in the entire cohort, or the NA-CRT group. However, a trend to better 5y DSS of AC in the NA-CRT group was observed. This analysis showed significant differences in treatment responses after NA-CRT: patients with AC responded remarkably less to chemoradiation, resulting in a significantly lower pathological complete response rate. These findings imply a need for a paradigm shift in the treatment of cervical AC patients.

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, is of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are rescued by expressing wild-type Nup88 but not the disease-linked mutant forms of Nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.

The classification of soft tissue tumors has evolved considerably in the last decade, largely due to advances in understanding the pathogenetic basis of many of these, sometimes rare, tumors. Deletion of Retinoblastoma 1 (RB1), a well-known tumor suppressor gene, has been implicated in the tumorigenesis of a particular group of soft tissue neoplasms. This group of so-called “RB1-deleted soft tissue tumors” has been rapidly expanding in recent years, currently consisting of spindle cell/pleomorphic lipoma, atypical spindle cell/pleomorphic lipomatous tumor, pleomorphic liposarcoma, myofibroblastoma, cellular angiofibroma, and acral fibromyxoma. Most of these neoplasms, except pleomorphic liposarcoma, are considered benign entities and are mainly described in the older adult population. This article will review the currently known morphological, immunohistochemical, and molecular features of this heterogeneous group of mesenchymal tumors with an emphasis on differential diagnosis.