Publications

Oncotopix Discovery, Publicly Sharable, paywall

AbstractObjective The standard of care for pre-malignant lesion risk assessment is dysplasia grading by histopathology. With significant overlap between dysplasia grades and high inter- and intra-observer variations, histopathology dysplasia grading alone is not a useful prognostic tool. Our aim is to investigate if a method for quantitatively assessing S100A7, a prognostic biomarker, using image analysis, can better predict clinical outcome in cases with oral dysplasia. Study Design Using Visiopharm image analysis system, we analyzed a cohort of 150 oral biopsy samples to build and test StraticyteTM, a model for an individualized assessment of the 5-year risk of progression of oral pre-cancerous lesions to invasive squamous cell carcinoma. Results Straticyte classified lesions more accurately than histopathological dysplasia grading for risk to progress to cancer over the following five years. The sensitivity of low-risk vs intermediate- and high-risk Straticyte groups was 95% compared to 75% for mild vs moderate and severe dysplasia. Further, the Negative Predictive Value for low-risk vs intermediate- and high-risk Straticyte groups was 78% compared to 59% for mild vs moderate and severe dysplasia. Conclusion By quantitatively assessing S100A7, Straticyte better defines the risk for developing oral squamous cell carcinoma then histopathological dysplasia grading alone.

Jason T K Hwang, Ying R Gu, Mi Shen, Ranju Ralhan, Paul G Walfish, Kenneth P H Pritzker, David Mock

Oncotopix Discovery, Publicly Sharable, nice

Alzheimer disease (AD) is characterized by deposition of amyloid-β (Aβ) plaques and neurofibrillary tangles in the brain, accompanied by synaptic dysfunction and neurodegeneration. Antibody-based immunotherapy against A? to trigger its clearance or mitigate its neurotoxicity has so far been unsuccessful. Here we report the generation of aducanumab, a human monoclonal antibody that selectively targets aggregated A?. In a transgenic mouse model of AD, aducanumab is shown to enter the brain, bind parenchymal Aβ, and reduce soluble and insoluble Aβ in a dose-dependent manner. In patients with prodromal or mild AD, one year of monthly intravenous infusions of aducanumab reduces brain Aβ in a dose- and time-dependent manner. This is accompanied by a slowing of clinical decline measured by Clinical Dementia Rating-Sum of Boxes and Mini Mental State Examination scores. The main safety and tolerability findings are amyloid-related imaging abnormalities. These results justify further development of aducanumab for the treatment of AD. Should the slowing of clinical decline be confirmed in ongoing phase 3 clinical trials, it would provide compelling support for the amyloid hypothesis.

Jeff Sevigny, Ping Chiao, Thierry Bussière, Paul H Weinreb, Leslie Williams, Marcel Maier, Robert W Dunstan, Stephen Salloway, Tianle Chen, Yan Ling, John OǴorman, Fang Qian, Mahin F Arastu, Mingwei Li, Sowmya Chollate, Melanie S Brennan, Omar Quintero-Monzon, Robert H Scannevin, H Moore Arnold, Thomas Engber, Kenneth J Rhodes, James Ferrero, Yaming Hang, Alvydas Mikulskis, Jan Grimm, Christoph Hock, Roger M Nitsch, Alfred Sandrock

Oncotopix Discovery, Publicly Sharable

Manual assessment of human epidermal growth factor receptor 2 (HER2) protein expression by immunohistochemistry (IHC) in gastric and gastroesophageal junction (GGEJ) adenocarcinomas is prone to interobserver variability and hampered by tumor heterogeneity and different scoring criteria. Equivocal cases are frequent, requiring additional in situ hybridization analysis. This study aimed to evaluate the accuracy of digital image analysis for the assessment of HER2 protein expression. In total, 110 GGEJ adenocarcinomas were included in tissue microarrays with 3 tissue cores per case. Two immunoassays, PATHWAY and HercepTest, and fluorescent in situ hybridization analysis were performed. The Visiopharm HER2-CONNECT Analysis Protocol Package was applied through the ONCOtopix digital image analysis software platform. HER2 membrane connectivity, calculated by the Analysis Protocol Package, was converted to standard IHC scores applying predetermined cutoff values for breast carcinoma as well as novel cutoff values. Cases with excessive cytoplasmic staining as well as HER2 amplified IHC negative cases were excluded from analysis. Applying HER2-CONNECT with connectivity cutoff values established for breast carcinoma resulted in 72.7% sensitivity and 100% specificity for the identification of HER2 positive gene amplified cases. By application of new cutoff values, the sensitivity increased to 100% without decreased specificity. With the new cutoff values, a 36% to 50% reduction of IHC equivocal cases was obtained. In conclusion, HER2-CONNECT with adjusted cutoff values seem to be an effective tool for standardized assessment of HER2 protein expression in GGEJ adenocarcinomas, decreasing the need for in situ hybridization analyzes.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/.

Sofie L. Nielsen, Soren Nielsen, Mogens Vyberg

Oncotopix Discovery, Publicly Sharable

Background: Abnormally sustained immune reactions drive B-cell proliferation in some cases of marginal zone lymphoma but the CD4+ T-cell subsets, which are likely to contribute to the B-cell responses in the tumour microenvironment, are not well characterised and neither has the spatial distribution of the different subsets in involved lymph nodes been investigated. Methods: Employing a workflow of multiplex semi-automated immunohistochemistry combined with image processing we investigated association between infiltrating T-cells and proliferating lymphoma B-cells. Results: Both total numbers of activating follicular helper (Tfh) cells (defined by high expression of PD1) and suppressive regulatory (Treg) T-cells (defined by FOXP3+ expression) and the Tfh:Treg ratio, assessed over relatively large areas of tissue, varied among cases of marginal zone lymphoma. We determined spatial distribution and demonstrated that PD1hi cells showed significantly more clustering than did FOXP3+. To investigate the association of infiltrating T-cells with lymphoma B-cells we employed Pearson correlation and Morisita-Horn index, statistical measures of interaction. We demonstrated that PD1hi cells were associated with proliferating B-cells and confirmed this by nearest neighbour analysis. Conclusions: The unexpected architectural complexity of T-cell infiltration in marginal zone lymphoma, revealed in this study, further supports a key role for Tfh cells in driving proliferation of lymphoma B-cells. We demonstrate the feasibility of digital analysis of spatial architecture of T-cells within marginal zone lymphoma and future studies will be needed to determine the clinical importance of these observations.

Katherine Wickenden, Nadia Nawaz, Sami Mamand, Deevia Kotecha, Amy L. Wilson, Simon D. Wagner, Matthew J. Ahearne

H&E, Oncotopix Discovery, Publicly Sharable

While the autoimmune character of T1D (type 1 diabetes) is being challenged, it is currently recognized that inflammation plays a key role in its development. We hypothesized that glucotoxicity could contribute to β-cell mass destruction through participation in islet inflammation. We evaluated the potential of empagliflozin (EMPA) and GABA (gamma-aminobutyric acid) to protect β-cell mass against glucotoxicity and to increase β-cell mass after diagnosis of T1D. Empagliflozin is a SGLT2 (sodium-dependent glucose cotransporter) inhibitor which thereby blocks glucose recapture by the kidney and promotes glucose excretion in urine. GABA is an inhibitory neurotransmitter, which stimulates α-to-β cell transdifferentiation. In streptozotocin-treated mice, empagliflozin and/or GABA were delivered for a period of five days or three weeks. As compared to untreated T1D mice, EMPA-treated T1D mice had decreased FFA (free fatty acid) levels and improved glucose homeostasis. EMPA-treated T1D mice had higher islet density, with preserved architecture, compared to T1D mice, and EMPA-treated T1D mice also differed from T1D mice by the total absence of immune cell infiltration within islets. Islets from EMPA-treated mice were also less subjected to ER (endoplasmic reticulum) stress and inflammation, as shown by qPCR analysis. Glucose homeostasis parameters and islet area/pancreas area ratio improved, as compared to diabetic controls, when T1D mice were treated for three weeks with GABA and EMPA. T1D EMPA+GABA mice had higher glucagon levels than T1D mice, without modifications of glucagon area/islet area ratios. In conclusion, empagliflozin and GABA, used in monotherapy in streptozotocin-induced diabetic mice, have positive effects on β-cell mass preservation or proliferation through an indirect effect on islet cell inflammation and ER stress. Further research is mandatory to evaluate whether empagliflozin and GABA may be a potential therapeutic target for the protection of β-cell mass after new-onset T1D.

Caroline Daems, Sophie Welsch, Hasnae Boughaleb, Juliette Vanderroost, Annie Robert, Etienne Sokal, Philippe A. Lysy

Oncotopix Discovery, Publicly Sharable, TissueAlign

Prostate cancer is a significant global health issue, and limitations to current patient management pathways often result in overtreatment or undertreatment. New ways to stratify patients are urgently needed. We conducted a feasibility study of such novel assessments, looking for associations between genomic changes and lymphocyte infiltration. An innovative workflow using an in-house targeted sequencing panel, immune cell profiling using an image analysis pipeline, RNA sequencing, and exome sequencing in select cases was tested. Gene fusions were profiled by RNA sequencing in 27 of 27 cases, and a significantly higher tumor-infiltrating lymphocyte (TIL) count was noted in tumors without a TMPRSS2:ERG fusion compared with those with the fusion (P = 0.01). Although this finding was not replicated in a larger validation set (n = 436) of The Cancer Genome Atlas images, there was a trend in the same direction. Differential expression analysis of TIL-high and TIL-low tumors revealed the enrichment of both innate and adaptive immune response pathways. Mutations in mismatch repair genes (MLH1 and MSH6 mutations in 1 of 27 cases) were identified. We describe a potential immune escape mechanism in TMPRSS2:ERG fusion-positive tumors. Detailed profiling, as shown herein, can provide novel insights into tumor biology. Likely differences with findings with other cohorts are related to methods used to define region of interest, but this warrants further study in a larger cohort.

Srinivasa R. Rao, Nasullah K. Alham, Elysia Upton, Stacey McIntyre, Richard J. Bryant, Lucia Cerundolo, Emma Bowes, Stephanie Jones, Molly Browne, Ian Mills, Alastair Lamb, Ian Tomlinson, David Wedge, Lisa Browning, Korsuk Sirinukunwattana, Claire Palles, Freddie C. Hamdy, Jens Rittscher, Clare Verrill

Oncotopix Discovery, Publicly Sharable

Current understanding of fibrosis remains incomplete despite the increasing burden of related diseases. Preclinical models are used to dissect the pathogenesis and dynamics of fibrosis, and to evaluate anti-fibrotic therapies. These studies require objective and accurate measurements of fibrosis. Existing histological quantification methods are operator-dependent, organ-specific, and/or need advanced equipment. Therefore, we developed a robust, minimally operator-dependent, and tissue-transposable digital method for fibrosis quantification. The proposed method involves a novel algorithm for more specific and more sensitive detection of collagen fibers stained by picrosirius red (PSR), a computer-assisted segmentation of histological structures, and a new automated morphological classification of fibers according to their compactness. The new algorithm proved more accurate than classical filtering using principal color component (red-green-blue; RGB) for PSR detection. We applied this new method on established mouse models of liver, lung, and kidney fibrosis and demonstrated its validity by evidencing topological collagen accumulation in relevant histological compartments. Our data also showed an overall accumulation of compact fibers concomitant with worsening fibrosis and evidenced topological changes in fiber compactness proper to each model. In conclusion, we describe here a robust digital method for fibrosis analysis allowing accurate quantification, pattern recognition, and multi-organ comparisons useful to understand fibrosis dynamics.

Guillaume E. Courtoy, Isabelle Leclercq, Antoine Froidure, Guglielmo Schiano, Johann Morelle, Olivier Devuyst, François Huaux, Caroline Bouzin

Oncotopix Discovery, Publicly Sharable

Metastasis causes ~90% of breast cancer mortality. However, standard prognostic tests based mostly on proliferation genes do not measure metastatic potential. Tumor MicroEnvironment of Metastasis (TMEM), an immunohistochemical biomarker for doorways on blood vessels that support tumor cell dissemination is prognostic for metastatic outcome in breast cancer patients. Studies quantifying TMEM doorways have involved manual scoring by pathologists utilizing static digital microscopy: a labor-intensive process unsuitable for use in clinical practice. We report here a validation study evaluating a new quantitative digital pathology (QDP) tool (TMEM-DP) for identification and quantification of TMEM doorways that closely mimics pathologists’ workflow and reduces pathologists’ variability to levels suitable for use in a clinical setting. Blinded to outcome, QDP was applied to a nested case-control study consisting of 259 matched case-control pairs. Sixty subjects of these were manually scored by five pathologists, digitally recorded using whole slide imaging (WSI), and then used for algorithm development and optimization. Validation was performed on the remainder of the cohort. TMEM-DP shows excellent reproducibility and concordance and reduces pathologist time from ~60 min to ~5 min per case. Concordance between manual scoring and TMEM-DP was found to be >0.79. These results show that TMEM-DP is capable of accurately identifying and scoring TMEM doorways (also known as MetaSite score) equivalent to pathologists.

David Entenberg, Maja H. Oktay, Timothy D’alfonso, Paula S. Ginter, Brian D. Robinson, Xiaonan Xue, Thomas E. Rohan, Joseph A. Sparano, Joan G. Jones, John S. Condeelis

Oncotopix Discovery, Publicly Sharable, Tissuealign

In cancer biology, epithelial-to-mesenchymal transition (EMT) is associated with tumorigenesis, stemness, invasion, metastasis, and resistance to therapy. Evidence of co-expression of epithelial and mesenchymal markers suggests that EMT should be a stepwise process with distinct intermediate states rather than a binary switch. In the present study, we propose a morphological approach that enables the detection and quantification of cancer cells with hybrid E/M states, i.e., which combine partially epithelial (E) and partially mesenchymal (M) states. This approach is based on a sequential immunohistochemistry technique performed on the same tissue section, the digitization of whole slides, and image processing. The aim is to extract quantitative indicators able to quantify the presence of hybrid E/M states in large series of human cancer samples and to analyze their relationship with cancer aggressiveness. As a proof of concept, we applied our methodology to a series of about a hundred urothelial carcinomas and demonstrated that the presence of cancer cells with hybrid E/M phenotypes at the time of diagnosis is strongly associated with a poor prognostic value, independently of standard clinicopathological features. Although validation on a larger case series and other cancer types is required, our data support the hybrid E/M score as a promising prognostic biomarker for carcinoma patients.

Louis Godin, Cédric Balsat, Yves Rémi Van Eycke, Justine Allard, Claire Royer, Myriam Remmelink, Ievgenia Pastushenko, Nicky D’Haene, Cédric Blanpain, Isabelle Salmon, Sandrine Rorive, Christine Decaestecker

Oncotopix Discovery, Publicly Sharable

Introduction: Microsatellite instability (MSI) predict response to anti-PD1 immunotherapy in colorectal cancer (CRC). CRCs with MSI have higher infiltration of immune cells related to a better survival. Elevated Microsatellite Alterations at Tetranucleotides (EMAST) is a form of MSI but its association with PD-L1 expression and immune-cell infiltration is not known. Methods: A consecutive, observational cohort of patients undergoing surgery for CRC. EMAST and clinicopathological characteristics were investigated against PD-L1, as well as CD3 and CD8 expression in the invasive margin or tumour centre (Immunoscore). Difference in survival between groups was assessed by log rank test. Results: A total of 149 stage I–III CRCs patients, with a median follow up of 60.1 months. Patients with PD-L1+ tumours (7%) were older (median 79 vs 71 years, p = 0.045) and had EMAST+ cancers (OR 10.7, 95% CI 2.2–51.4, p = 0.001). Recurrence-free survival was longer in cancers with PD-L1+ immune cells (HR 0.35, 95% CI 0.16–0.76, p = 0.008, independent of EMAST) and high Immunoscore (HR 0.10, 95% CI 0.01–0.72, p = 0.022). Patients expressing PD-L1 in immune cells had longer disease-specific survival (HR 0.28, 95% CI 0.10–0.77, p = 0.014). Conclusions: Higher Immunoscore (CD3/CD8 cells) and expression of tumour PD-L1 is found in CRCs with EMAST. Lymphocytic infiltrate and peritumoral PD-L1 expression have prognostic value in CRC.

Martin M. Watson, Dordi Lea, Einar Gudlaugsson, Ivar Skaland, Hanne R. Hagland, Kjetil Søreide

Oncotopix Discovery, Publicly Sharable, Tissuealign

B cells infiltrate pancreatic ductal adenocarcinoma (PDAC) and in preclinical cancer models, can suppress T cell immunosurveillance in cancer. Here, we conducted a pilot study to assess the safety and feasibility of administering lentiviral-transduced chimeric antigen receptor (CAR)-modified autologous T cells redirected against mesothelin to target tumor cells along with CART cells redirected against CD19 to deplete B cells. Both CARs contained 4-1BB and CD3ζ signaling domains. Three patients with chemotherapy-refractory PDAC received 1.5 g/m2 cyclophosphamide prior to separate infusions of lentiviral-transduced T cells engineered to express chimeric anti-mesothelin immunoreceptor SS1 (CART-Meso, 3 × 107/m2) and chimeric anti-CD19 immunoreceptor (CART-19, 3 × 107/m2). Treatment was well tolerated without dose-limiting toxicities. Best response was stable disease (1 of 3 patients). CART-19 (compared to CART-Meso) cells showed the greatest expansion in the blood, although persistence was transient. B cells were successfully depleted in all subjects, became undetectable by 7–10 days post-infusion, and remained undetectable for at least 28 days. Together, concomitant delivery of CART-Meso and CART-19 cells in patients with PDAC is safe. CART-19 cells deplete normal B cells but at the dose tested in these 3 subjects did not improve CART-Meso cell persistence.

Andrew H. Ko, Alexander C. Jordan, Evan Tooker, Simon F. Lacey, Renee B. Chang, Yan Li, Alan P. Venook, Margaret Tempero, Lloyd Damon, Lawrence Fong, Mark H. O'Hara, Bruce L. Levine, J. Joseph Melenhorst, Gabriela Plesa, Carl H. June, Gregory L. Beatty

Oncotopix Discovery, Publicly Sharable, TMA

Background: Statins have anticancer properties by acting as competitive inhibitors of the mevalonate pathway. They also have anti-inflammatory activity, but their role in suppressing inflammation in a cancer context has not been investigated to date. Methods: We have analyzed the relationship between statin use and tumor-Associated macrophages (TAMs) in a cohort of 262 surgically resected primary human lung adenocarcinomas. TAMs were evaluated by multiplex immunostaining for the CD68 pan-TAM marker and the CD163 protumorigenic TAM marker followed by digital slide scanning and partially automated quantitation. Links between statin use and tumor stage, virulence, and cancer-specific survival were also investigated in a wider cohort of 958 lung adenocarcinoma cases. All statistical tests were two-sided. Results: We found a statin dose-dependent reduction in protumorigenic TAMs (CD68 CD163 ) in both stromal (P .021) and parenchymal (P .003) compartments within regions of in situ tumor growth, but this association was lost in invasive regions. No statistically significant relationship between statin use and tumor stage was observed, but there was a statin dose-dependent shift towards lower histological grade as assessed by growth pattern (P .028). However, statin use was a predictor of slightly worse cancer-specific survival (P .032), even after accounting for prognostic variables in a multivariable Cox proportional hazards survival model (hazard ratio 1.38, 95% confidence interval 1.04 to 1.84). Conclusions: Statin use is associated with reduced numbers of protumorigenic TAMs within preinvasive lung adenocarcinoma and is related to reduced tumor invasiveness, suggesting a chemo-preventive effect in early tumor development. However, invasive disease is resistant to these effects, and no beneficial relationship between statin use and patient outcome is observed.

Esraa Al Dujaily, Juvenal Baena, Madhumita Das, Marco Sereno, Claire Smith, Tamihiro Kamata, Leah Officer, Catrin Pritchard, John Le Quesne

Oncotopix Discovery, Publicly Sharable

Purpose/Objective(s): T cell-mediated anti-tumor immune responses are gated in part by the relative abundance of cytotoxic T cells (Teffs) and regulatory (Tregs) in the tumor microenvironment (TME) whereby Treg may impair the efficacy of Teffs. Tregs can affect Teff function by direct cell-to-cell contact or by secretion of soluble mediators, consistent with the hypothesis that proximity of Tregs to Teff within tumor tissues may have prognostic significance. Here we used a novel chromogenic multiplex assay to investigate the spatial relationship of these functionally diverse T cell subsets in HPV positive and HPV negative head and neck squamous cell carcinomas (HNSCCs). Materials/Methods: Twenty head and neck cancer patients with primary tumors of the oropharynx or oral cavity were included in this study of which 10 were HPV positive and 10 HPV negative. Formalin-fixed tissues were stained with antibodies detecting CD8 (Teffs) and FoxP3 (Tregs). At least three different fields at the leading edge of tumor and adjacent stroma were evaluated. Stained tissue sections were digitally scanned at 40x magnification utilizing an iScan HT (Roche, Switzerland) whole-slide imaging scanner. Visiopharm (Visiopharm, Denmark) image analysis software was utilized by a pathologist to analyze the digitized slide images. Result(s): HPV positive tumor tissues contained >3-fold higher numbers of both CD8 and FoxP3 expressing cells when compared to HPV negative tumors whereas the ratios between these cell subsets (CD8/FoxP3) were comparable. The differential density of T-cells in the sampled areas was reflected in a significantly shorter mean distance between CD8+ and FoxP3+ cells in HPV positive (29.92 mum) tumors compared to that of HPV negative (52.56 mum) tumors (p=0.0045, 95% CI: 8.17-37.11). The mean frequency of distances <30mum measured in HPV positive tumors was 98.46 contrasted by 39.44 in HPV negative tumors; this difference was statistically different (p=0.0194). The mean frequency of distances >30mum measured in HPV positive tumors was 37.02 and in HPV negative tumors 36.06. Conclusion(s): Consistent with earlier reports, HPV positive lesions contained more CD8 and FoxP3 expressing T cells than HPV negative lesions. This difference was reflected in statistically significantly closer proximity of Teffs and Tregs in HPV positive lesions, potentially enhancing functional interaction of these T cell subsets in the tumor microenvironment of HPV positive lesions. The implications of these observations for prognosis and response to immunotherapeutic intervention remain to be investigated.Copyright © 2020

M. Stewart, R. Stapp, D. Amin, R. Ganti, U. Nwagu, T. Richa, M. Crippen, R. Zinner, A. Luginbuhl, J. Johnson, V. Bar-Ad, U. Martinez-Outschoorn, C. Solomides, U. Rodeck, J.M. Curry

Oncotopix Discovery, Phenoplex, Publicly Sharable, Tissuealign, nice

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Manuel Flores Molina, Thomas Fabre, Aurélie Cleret-Buhot, Geneviève Soucy, Liliane Meunier, Mohamed N. Abdelnabi, Nicolas Belforte, Simon Turcotte, Naglaa H. Shoukry

Oncotopix Discovery, Publicly Sharable, TMA

Hormone receptor expression patterns often correlate with infiltration of specific lymphocytes in tumors. Specifically, the presence of specific tumor-infiltrating lymphocytes (TILs) with particular hormone receptor expression is reportedly associated with breast cancer, however, this has not been revealed in epithelial ovarian cancer (EOC). Therefore, we investigated the association between hormone receptor expression and TILs in EOC. Here we found that ERα, AR, and GR expression increased in EOC, while PR was significantly reduced and ERβ expression showed a reduced trend compared to normal epithelium. Cluster analysis indicated poor disease-free survival (DFS) in AR+/GR+/PR+ subgroup (triple dominant group); while the Cox proportional-hazards model high-lighted the triple dominant group as an independent prognostic factor for DFS. In addition, significant upregulation of FoxP3+ TILs, PD-1, and PD-L1 was observed in the triple dominant group compared to other groups. NanoString analyses further suggested that tumor necrosis factor (TNF) and/or NF-κB signaling pathways were activated with significant upregulation of RELA, MAP3K5, TNFAIP3, BCL2L1, RIPK1, TRAF2, PARP1, and AKT1 in the triple dominant EOC group. The triple dominant subgroup correlates with poor prognosis in EOC. Moreover, the TNF and/or NF-κB signaling pathways may be responsible for hormone-mediated inhibition of the immune microenvironment.

Gwan Hee Han, Ilseon Hwang, Hanbyoul Cho, Kris Ylaya, Jung A. Choi, Hyunja Kwon, Joon Yong Chung, Stephen M. Hewitt, Jae Hoon Kim

Oncotopix Discovery, Polaris, Publicly Sharable, RNAScope, Vectra

Background & Aims: Diligent side-by-side comparisons of how different methodologies affect growth efficiency and quality of intestinal colonoids have not been performed leaving a gap in our current knowledge. Here, we summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids. Methods: Mouse and human colon organoids were grown in four different media. Media-dependent long-term growth was measured by quantifying surviving organoids via imaging and a cell viability readout over five passages. The impact of diverse media on differentiation was assessed by quantifying the number of epithelial cell types using markers for enterocytes, stem cells, Goblet cells, and enteroendocrine cells by qPCR and histology upon removal of growth factors. Results: In contrast to Wnt3a-conditioned media, media supplemented with recombinant Wnt3a alone did not support long-term survival of human or mouse colon organoids. Mechanistically, this observation can be attributed to the fact that recombinant Wnt3a did not support stem cell survival or proliferation as demonstrated by decreased LGR5 and Ki67 expression. When monitoring expression of markers for epithelial cell types, the highest level of organoid differentiation was observed after combined removal of Wnt3a, Noggin, and R-spondin from Wnta3a-conditioned media cultures. Conclusion: Our study defined Wnt3a-containing conditioned media as optimal for growth and survival of human and mouse organoids. Furthermore, we established that the combined removal of Wnt3a, Noggin, and R-spondin results in optimal differentiation. This study provides a step forward in optimizing conditions for intestinal organoid growth to improve standardization and reproducibility of this model platform.

Sarah S. Wilson, Martha Mayo, Terry Melim, Heather Knight, Lori Patnaude, Xiaoming Wu, Lucy Phillips, Susan Westmoreland, Robert Dunstan, Edda Fiebiger, Sonia Terrillon

Oncotopix Discovery, Publicly Sharable, RNAScope

Background: The interleukin (IL)-23/IL-17 immune axis is of central importance in psoriasis. However, the impact of IL-17 family cytokines other than IL-17A in psoriasis has not been fully established. Objectives: To elucidate the contribution of IL-17 family cytokines in psoriasis. Methods: To address the expression and localization of IL-17 family cytokines, lesional and nonlesional skin samples from patients with psoriasis were analysed by several complementary methods, including quantitative polymerase chain reaction, immunoassays, in situ hybridization and immunohistochemistry. Mechanistic studies assessing the functional activity of IL-17 family cytokines were performed using ex vivo cultured human skin biopsies and primary human keratinocytes. Results: We demonstrated that IL-17A, IL-17F, IL-17A/F and IL-17C are expressed at increased levels in psoriasis lesional skin and induce overlapping gene expression responses in ex vivo cultured human skin that correlate with the transcriptomic signature of psoriasis skin. Furthermore, we showed that brodalumab, in contrast to ixekizumab, normalizes gene expression responses induced by the combination of IL-17A, IL-17F, IL-17A/F and IL-17C in human keratinocytes. Conclusions: Several IL-17 ligands signalling through IL-17RA are overexpressed in psoriasis skin and induce similar psoriasis-related inflammatory pathways demonstrating their relevance in relation to therapeutic intervention in psoriasis.

M. A.X. Tollenaere, J. Hebsgaard, D. A. Ewald, P. Lovato, S. Garcet, X. Li, S. D. Pilger, M. L. Tiirikainen, M. Bertelsen, J. G. Krueger, H. Norsgaard

Oncotopix Discovery, Publicly Sharable, RNAScope

Aims: Ulcerative colitis and Crohn's disease, collectively known as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the intestine for which key elements in disease initiation and perpetuation are defects in epithelial barrier integrity. Achieving mucosal healing is essential to ameliorate disease outcome and so new therapies leading to epithelial homeostasis and repair are under investigation. This study was designed to determine the mechanisms by which IL-22 regulates intestinal epithelial cell function. Main methods: Human intestinal organoids and resections, as well as mice were used to evaluate the effect of IL-22 on stem cell expansion, proliferation and expression of mucus components. IL-22 effect on barrier function was assessed in polarized T-84 cell monolayers. Butyrate co-treatments and organoid co-cultures with immune cells were performed to monitor the impact of microbial-derived metabolites and inflammatory environments on IL-22 responses. Key findings: IL-22 led to epithelial stem cell expansion, proliferation, barrier dysfunction and anti-microbial peptide production in human and mouse models evaluated. IL-22 also altered the mucus layer by inducing an increase in membrane mucus but a decrease in secreted mucus and goblet cell content. IL-22 had the same effect on anti-microbial peptides and membrane mucus in both healthy and IBD human samples. In contrast, this IL-22-associated epithelial phenotype was different when treatments were performed in presence of butyrate and organoids co-cultured with immune cells. Significance: Our data indicate that IL-22 promotes epithelial regeneration, innate defense and membrane mucus production, strongly supporting the potential clinical utility of IL-22 as a mucosal healing therapy in IBD.

Lori Patnaude, Martha Mayo, Regina Mario, Xiaoming Wu, Heather Knight, Kelly Creamer, Sarah Wilson, Valerie Pivorunas, Jozsef Karman, Lucy Phillips, Robert Dunstan, Rajesh V. Kamath, Bradford McRae, Sonia Terrillon

Oncotopix Discovery, Publicly Sharable, Tissuealign

Purpose: The CLASSICAL-Lung clinical trial tested the combination of pepinemab, an IgG4 humanized mAb targeting semaphorin 4D, with the PD-L1 inhibitor avelumab to assess the effects of coupling increased T-cell infiltration and reversal of immune suppression via pepinemab with sustained T-cell activation via checkpoint inhibition. Patients and Methods: This phase Ib/II, single-arm study was designed to evaluate the safety, tolerability, and efficacy of pepinemab in combination with avelumab in 62 patients with advanced non–small cell lung cancer (NSCLC), including immunotherapy-naïve (ION) patients and patients whose tumors progressed following anti-PD-1/L1 monotherapy (IOF). The main objectives were to evaluate safety/tolerability, establish a recommended phase 2 dose (RP2D), obtain a preliminary evaluation of antitumor activity, and investigate candidate biomarker activity. Results: The combination was well tolerated with no major safety signals identified. Pepinemab, 10 mg/kg with avelumab, 10 mg/kg, every 2 weeks, was selected as the RP2D. Among 21 evaluable ION patients, 5 patients experienced partial responses (PR), 4 patients evidenced clinical benefit ≥1 year, and the disease control rate (DCR) was 81%. Notably, overall response rate with the combination therapy was higher than previously reported for single-agent avelumab in the PD-L1-negative/low population. Among 29 evaluable IOF patients, the combination resulted in a DCR of 59%, including 2 PR and 7 patients with durable clinical benefit of ≥23 weeks. Biomarker analysis of biopsies demonstrated increased CD8 T-cell density correlating with RECIST response criteria. Conclusions: The combination of pepinemab with avelumab was well tolerated in NSCLC and showed signs of antitumor activity in immunotherapy-resistant and PD-L1-negative/low tumors.

Michael R. Shafique, Terrence L. Fisher, Elizabeth E. Evans, John E. Leonard, Desa Rae E. Pastore, Crystal L. Mallow, Ernest Smith, Vikas Mishra, Andreas Schröder, Kevin M. Chin, Joseph T. Beck, Megan A. Baumgart, Ramaswamy Govindan, Nashat Y. Gabrail, Alexander I. Spira, Nagashree Seetharamu, Yanyan Lou, Aaron S. Mansfield, Rachel E. Sanborn, Jonathan W. Goldman, Maurice Zauderer

Oncotopix Discovery, Publicly Sharable

Background: In colon cancer, the location and density of tumor-infiltrating lymphocytes (TILs) can classify patients into low and high-risk groups for prognostication. While a commercially available ‘Immunoscore®’ exists, the incurred expenses and copyrights may prevent universal use. The aim of this study was to develop a robust and objective quantification method of TILs in colon cancer. Methods: A consecutive, unselected series of specimens from patients with colon cancer were available for immunohistochemistry and assessment of TILs by automated digital pathology. CD3 + and CD8 + cells at the invasive margin and in tumor center were assessed on consecutive sections using automated digital pathology and image analysis software (Visiopharm®). An algorithm template for whole slide assessment, generated cell counts per square millimeters (cells/mm2), from which the immune score was calculated using distribution volumes. Furthermore, immune score was compared with clinical and histopathological characteristics to confirm its relevance. Results: Based on the quantified TILs numbers by digital image analyses, patients were classified into low (n = 83, 69.7%), intermediate (n = 14, 11.8%) and high (n = 22, 18.5%) immune score groups. High immune score was associated with stage I–II tumors (p = 0.017) and a higher prevalence of microsatellite instable (MSI) tumors (p = 0.030). MSI tumors had a significantly higher numbers of CD3 + TILs in the invasive margin and CD8 + TILs in both tumor center and invasive margin, compared to microsatellite stable (MSS) tumors. Conclusion: A digital template to quantify an easy-to-use immune score corresponds with clinicopathological features and MSI in colon cancer.

Dordi Lea, Martin Watson, Ivar Skaland, Hanne R. Hagland, Melinda Lillesand, Einar Gudlaugsson, Kjetil Søreide

Oncotopix Discovery, Publicly Sharable

A majority of breast cancers (BC) are age-related and we seek to determine what cellular and molecular changes occur in breast tissue with age that make women more susceptible to cancer initiation. Immune-epithelial cell interactions are important during mammary gland development and the immune system plays an important role in BC progression. The composition of human immune cell populations is known to change in peripheral blood with age and in breast tissue during BC progression. Less is known about changes in immune populations in normal breast tissue and how their interactions with mammary epithelia change with age. We quantified densities of T cells, B cells, and macrophage subsets in pathologically normal breast tissue from 122 different women who ranged in age from 24 to 74 years old. Donor-matched peripheral blood from a subset of 20 donors was analyzed by flow cytometry. Tissue immune cell densities and localizations relative to the epithelium were quantified in situ with machine learning-based image analyses of multiplex immunohistochemistry-stained tissue sections. In situ results were corroborated with flow cytometry analyses of peri-epithelial immune cells from primary breast tissue preparations and transcriptome analyses of public data from bulk tissue reduction mammoplasties. Proportions of immune cell subsets in breast tissue and donor-matched peripheral blood were not correlated. Density (cells/mm2) of T and B lymphocytes in situ decreased with age. T cells and macrophages preferentially localized near or within epithelial bilayers, rather than the intralobular stroma. M2 macrophage density was higher than M1 macrophage density and this difference was due to higher density of M2 in the intralobular stroma. Transcriptional signature analyses suggested age-dependent decline in adaptive immune cell populations and functions and increased innate immune cell activity. T cells and macrophages are so intimately associated with the epithelia that they are embedded within the bilayer, suggesting an important role for immune-epithelial cell interactions. Age-associated decreased T cell density in peri-epithelial regions, and increased M2 macrophage density in intralobular stroma suggests the emergence of a tissue microenvironment that is simultaneously immune-senescent and immunosuppressive with age.

Arrianna Zirbes, Jesuchristopher Joseph, Jennifer C. Lopez, Rosalyn W. Sayaman, Mudaser Basam, Victoria L. Seewaldt, Mark A. LaBarge

Oncotopix Discovery, Phenoplex, Publicly Sharable, Tissuealign

Despite their recognised role in HER2-positive (HER2+) breast cancer (BC), the composition, localisation and functional orientation of immune cells within tumour microenvironment, as well as its dynamics during anti-HER2 treatment, is largely unknown. We here investigate changes in tumour-immune contexture, as assessed by stromal tumour-infiltrating lymphocytes (sTILs) and by multiplexed spatial cellular phenotyping, during treatment with lapatinib-trastuzumab in HER2+ BC patients (PAMELA trial). Moreover, we evaluate the relationship of tumour-immune contexture with hormone receptor status, intrinsic subtype and immune-related gene expression. sTIL levels increase after 2 weeks of HER2 blockade in HR-negative disease and HER2-enriched subtype. This is linked to a concomitant increase in cell density of all four immune subpopulations (CD3+, CD4+, CD8+, Foxp3+). Moreover, immune contexture analysis showed that immune cells spatially interacting with tumour cells have the strongest association with response to anti-HER2 treatment. Subsequently, sTILs consistently decrease at the surgery in patients achieving pathologic complete response, whereas most residual tumours at surgery remain inflamed, possibly reflecting a progressive loss of function of T cells. Understanding the features of the resulting tumour immunosuppressive microenvironment has crucial implications for the design of new strategies to de-escalate or escalate systemic therapy in early-stage HER2+ BC.

G. Griguolo, G. Serna, T. Pascual, R. Fasani, X. Guardia, N. Chic, L. Paré, S. Pernas, M. Muñoz, M. Oliveira, M. Vidal, A. Llombart-Cussac, J. Cortés, P. Galván, B. Bermejo, N. Martínez, R. López, S. Morales, I. Garau, L. Manso, J. Alarcón, E. Martínez, P. Villagrasa, A. Prat, P. Nuciforo

Oncotopix Discovery, Publicly Sharable

Jiandong Huo, Halina Mikolajek, Jordan Clark, Parul Sharma, Anja Kipar, Joshua Dormon, Rosalind Franklin, Institute Chelsea, Norman Rosalind, Franklin Institute, Miriam Weckener, Daniel Clare, Karen Buttigieg, Francisco Salguero, Robert Watson, Daniel Knott

Oncotopix Discovery, Publicly Sharable

Simple Summary Hepatocellular carcinoma is the most common primary liver malignancy in the United States. Macrophages are immune cells that play a critical role in the promotion of cancer growth and configuration of the hepatic microenvironment. Studying intrahepatic macrophages is challenging because they are difficult to isolate, they transform their phenotype upon manipulation, and in vivo animal models poorly replicate the liver microenvironment. Understanding the complexity of intrahepatic macrophage populations is crucial because they coordinate antitumoral immunity. Application of novel methods that can detect immune cell phenotypes, along with their spatial co-localization in situ is critical and timely. Abstract The role of tumor-associated macrophages (TAMs) in the pathogenesis of hepatocellular carcinoma (HCC) is poorly understood. Most studies rely on platforms that remove intrahepatic macrophages from the microenvironment prior to evaluation. Cell isolation causes activation and phenotypic changes that may not represent their actual biology and function in situ. State-of-the-art methods provides new strategies to study TAMs without losing the context of tissue architecture and spatial relationship with neighboring cells. These technologies, such as multispectral imaging (e.g., Vectra Polaris), mass cytometry by time-of-flight (e.g., Fluidigm CyTOF), cycling of fluorochromes (e.g., Akoya Biosciences CODEX/PhenoCycler-Fusion, Bruker Canopy, Lunaphore Comet, and CyCIF) and digital spatial profiling or transcriptomics (e.g., GeoMx or Visium, Vizgen Merscope) are being utilized to accurately assess the complex cellular network within the tissue microenvironment. In cancer research, these platforms enable characterization of immune cell phenotypes and expression of potential therapeutic targets, such as PDL-1 and CTLA-4. Newer spatial profiling platforms allow for detection of numerous protein targets, in combination with whole transcriptome analysis, in a single liver biopsy tissue section. Macrophages can also be specifically targeted and analyzed, enabling quantification of both protein and gene expression within specific cell phenotypes, including TAMs. This review describes the workflow of each platform, summarizes recent research using these approaches, and explains the advantages and limitations of each. Keywords: cancer; CyTOF; HCC; liver; macrophages; spectral imaging; hepatic microenvironment; TAMs; TME; spatial genomics; scRNA-seq

DE Millian, OA Saldarriaga, T Wanninger, JK Burks - Cancers, undefined 2022

Oncotopix Discovery, Publicly Sharable

Around 15% of breast cancer patients are diagnosed as triple-negative (TNBC), which have significantly lower 5-year survival rates (77%) than other types of breast cancer (93%). Our study aimed at developing an image analysis-based biomarker to assess how the immune system interacts with the tumor and investigate the potential added value of stromal tumor-infiltrating lymphocytes (sTIL) for the prognosis of overall survival compared to the manual approach. In a large retrospective cohort of 257 patients, we found that our fully automated hematoxylin and eosin (H&E) image analysis pipeline can quantify sTIL density showing both high concordance with manual scoring and association with the prognosis of patients with TNBC. It also overcomes natural limitations of manual assessment that hinder clinical adoption of the immune biomarker. We conclude that sTIL scoring by automated image analysis has prognostic potential comparable to manual scoring and should be further investigated for future use in a clinical setting. Abstract Triple-negative breast cancer (TNBC) is an aggressive and difficult-to-treat cancer type that represents approximately 15% of all breast cancers. Recently, stromal tumor-infiltrating lymphocytes (sTIL) resurfaced as a strong prognostic biomarker for overall survival (OS) for TNBC patients. Manual assessment has innate limitations that hinder clinical adoption, and the International Immuno-Oncology Biomarker Working Group (TIL-WG) has therefore envisioned that computational assessment of sTIL could overcome these limitations and recommended that any algorithm should follow the manual guidelines where appropriate. However, no existing studies capture all the concepts of the guideline or have shown the same prognostic evidence as manual assessment. In this study, we present a fully automated digital image analysis pipeline and demonstrate that our hematoxylin and eosin (H&E)-based pipeline can provide a quantitative and interpretable score that correlates with the manual pathologist-derived sTIL status, and importantly, can stratify a retrospective cohort into two significant distinct prognostic groups. We found our score to be prognostic for OS (HR: 0.81 CI: 0.72–0.92 p = 0.001) independent of age, tumor size, nodal status, and tumor type in statistical modeling. While prior studies have followed fragments of the TIL-WG guideline, our approach is the first to follow all complex aspects, where appropriate, supporting the TIL-WG vision of computational assessment of sTIL in the future clinical setting. Keywords: deep learning; digital pathology; image analysis; prognostic biomarker; survival analysis; triple-negative breast cancer; tumor microenvironment (TME); tumor-infiltrating lymphocytes

Jeppe Thagaard, Elisabeth Specht Stovgaard, Line Grove Vognsen, Søren Hauberg, Anders Dahl, Thomas Ebstrup, Johan Doré, Rikke Egede Vincentz, Rikke Karlin Jepsen, Anne Roslind, Iben Kümler, Dorte Nielsen, Eva Balslev

Oncotopix Discovery, Publicly Sharable

Digital pathology evolved rapidly, enabling more systematic usage of image analysis and development of artificial intelligence (AI) applications. Here, combined AI models were developed to evaluate hepatocellular hypertrophy in rat liver, using commercial AI-based software on hematoxylin and eosin-stained whole slide images. In a first approach, deep learning-based identification of critical tissue zones (centrilobular, midzonal, and periportal) enabled evaluation of region-specific cell size. Mean cytoplasmic area of hepatocytes was calculated via several sequential algorithms including segmentation in microanatomical structures (separation of sinusoids and vessels from hepatocytes), nuclear detection, and area measurements. An increase in mean cytoplasmic area could be shown in groups given phenobarbital, known to induce hepatocellular hypertrophy when compared to control groups, in multiple studies. Quantitative results correlated with the gold standard: observation and grading performed by board-certified veterinary pathologists, liver weights, and gene expression. Furthermore, as a second approach, we introduce for the first time deep learning-based direct detection of hepatocellular hypertrophy with similar results. Cell hypertrophy is challenging to pick up, particularly in milder cases. Additional evaluation of mean cytoplasmic area or direct detection of hypertrophy, combined with histopathological observations and liver weights, is expected to increase accuracy and repeatability of diagnoses and grading by pathologists.

Hannah Pischon, David Mason, Bettina Lawrenz, Olivier Blanck, Anna Lena Frisk, Frederic Schorsch, Valeria Bertani

Akoya, Oncotopix Discovery, Phenoplex, Polaris, Publicly Sharable, Tissuealign, Vectra

Introduction: Multiplex biomarker analysis of cytological body fluid specimens is often used to assist cytologists in distiguishing metastatic cancer cells from reactive meso-thelial cells. However, evaluating biomarker expression visually may be challenging, especially when the cells of interest are scant. Deep-learning algorithms (DLAs) may be able to assist cytologists in analyzing multiple biomarker expression at the single cell level in the multiplex fluores-cence imaging (MFI) setting. This preliminary study was performed to test the feasibility of using DLAs to identify immunofluorescence-stained metastatic adenocarcinoma cells in body fluid cytology samples. Methods: A DLA was developed to analyze MFI-stained cells in body fluid cyto-logical samples. A total of 41 pleural fluid samples, comprising of 20 positives and 21 negatives, were retrospectively collected. Multiplex immunofluorescence labeling for MOC31, BerEP4, and calretinin, were performed on cell block sections, and results were analyzed by manual analysis (manual MFI) and DLA analysis (MFI-DLA) independently. Results: All cases with positive original cytological diagnoses showed positive results either by manual MFI or MFI-DLA, but 2 of the 14 (14.3%) original cytologically negative cases had rare cells with positive MOC31 and/or BerEP4 staining in addition to calretinin. Manual MFI analysis and MFI-DLA showed 100% concordance. Conclusion: MFI combined with DLA provides a potential tool to assist in cytological diagnosis of metastatic malignancy in body fluid samples. Larger studies are warranted to test the clinical validity of the approach.

Weibo Yu, Elizabeth Rao, Curtis D Chin, Josephine S Aguilar-Jakthong, Yunfeng Li, Christine Chow, Shu Yu, Grace Wang, Jianyu Rao

Oncotopix Discovery, Publicly Sharable, nice

Amyloid beta (Aβ) plaque is a defining pathologic feature of Alzheimer disease (AD). Aducanumab, a monoclonal IgG1 that selectively binds aggregated species of Aβ, has been shown by amyloid positron emission tomography (Amyloid PET) to reduce Aβ plaques in patients with prodromal and mild AD. This is the first autopsy report of the AD neuropathology in a patient previously treated with aducanumab. The patient was an 84-year-old woman who was randomized to the placebo arm of the PRIME Phase 1b study (221AD103). The patient progressed to moderate dementia (MMSE = 14/30), beyond the targeted early AD treatment stage, before receiving aducanumab in the long-term extension (LTE). The patient then received 32 monthly doses of aducanumab, titrated up to 6 mg/kg, for a cumulative dose of 186 mg/kg. In the LTE, Amyloid PET scans demonstrated robust Aβ plaque reduction, from a composite standard uptake value ratio (SUVR) of 1.5 at screening to < 1.1 at 56 weeks post-aducanumab dosing. MRI examinations were negative for amyloid-related imaging abnormalities (ARIA). She passed away in hospice care 4 months after her last dose of aducanumab. The postmortem neuropathologic examination confirmed AD neuropathologic changes. Aβ and IBA1 immunohistochemistry assays demonstrated sparse residual Aβ plaque engaged by amoeboid reactive microglia. Phospho-Tau (pTau) immunohistochemistry demonstrated neocortical neurofibrillary degeneration (Braak stage V, NIA/AA Stage B3). However, the density of pTau neuropathology, including neuritic plaque pTau (NP-Tau), appeared lower in the PRIME LTE Patient compared to a reference cohort of untreated Braak stage V–VI, NIA/AA Stage B3 AD cases. Taken together, this case report is the first to provide Amyloid PET and neuropathologic evidence substantiating the impact of aducanumab to reduce Aβ plaque neuropathology in a patient with AD. Furthermore, this report underscores the critical importance of autopsy neuropathology studies to augment our understanding of aducanumab’s mechanism of action and impact on AD biomarkers.

Edward D. Plowey, Thierry Bussiere, Raj Rajagovindan, Jennifer Sebalusky, Stefan Hamann, Christian von Hehn, Carmen Castrillo-Viguera, Alfred Sandrock, Samantha Budd Haeberlein, Christopher H. van Dyck, Anita Huttner

Oncotopix Discovery, Publicly Sharable

Colorectal cancer (CRC) is the third most common cancer worldwide. Although clinical outcome varies among patients diagnosed within the same TNM stage it is the cornerstone in treatment decisions as well as follow-up programmes. Tumor-infiltrating lymphocytes have added value when evaluating survival outcomes. The aim of this study was to develop a fully automated method for quantification of subsets of T lymphocytes in the invasive margin and central tumor in patients with CRC based on Deep Learning powered artificial intelligence. The study cohort consisted of 163 consecutive patients with a primary diagnosis of CRC followed by a surgical resection. Double-labeling immunohistochemical staining with cytokeratin in combination with CD3 or CD8, respectively, was performed on 1 representative slide from each patient. Visiopharm Quantitative Digital Pathology software was used to develop Application Protocol Packages for visualization of architectural details (background, normal epithelium, cancer epithelium, surrounding tissue), identification of central tumor and invasive margin as well as subsequent quantitative analysis of immune cells. Fully automated counts for CD3 and CD8 positive T cells were obtained in 93% and 92% of the cases, respectively. In the remaining cases, manual editing was required. In conclusion, the development of a fully automated method for counting CD3+and CD8+lymphocytes in a cohort of patients with CRC provided excellent results eliminating not only observer variability in lymphocyte counts but also in identifying the regions of interest for the quantitative analysis. Validation of the performance of the Application Protocol Packages including clinical correlation is needed.

Anne Marie K. Fiehn, Bjoern Reiss, Mikail Gögenur, Michael Bzorek, Ismail Gögenur

Oncotopix Discovery, Publicly Sharable

Background Hepatocellular carcinoma (HCC) is a highly lethal cancer and the second leading cause of cancer-related deaths worldwide. As demonstrated in other solid neoplasms and HCC, infiltrating CD8 + T cells seem to be related to a better prognosis, but the mechanisms affecting the immune landscape in HCC are still mostly unknown. Necroptosis is a programmed, caspase-independent cell death that, unlike apoptosis, evokes immune response by releasing damage-associated molecular factors. However, in HCC, the relationship between the necroptotic machinery and the tumor-infiltrating lymphocytes has not been fully investigated so far. Methods We investigated the association between the main necroptosis-related genes, that is, RIPK1, RIPK3, MLKL-p, and CD3 + /CD8 + tumor-infiltrating T cell by RNA-seq data analysis in 371 patients with primary HCC from The Cancer Genome Atlas and then by immunohistochemistry in two independent cohorts of HCC patients from Italy (82) and Japan (86). Results Our findings highlighted the immunogenetic role of necroptosis and its potential prognostic role in HCC: RIPK1, RIPK3 and MLKL-p were found significantly associated with intratumoral CD3 + and CD8 + T cells. In addition, multivariate survival analysis showed that the expression of RIPK1, RIPK3 and MLKL-p was associated with better overall survival in the two independent cohorts. Conclusions Our results confirmed the immunogenetic properties of necroptosis (NCP) in human HCC, showing that tumor-infiltrating lymphocytes (TILs) and, specifically, CD8+ T cells accumulate in tumors with higher expression of the necroptosis-related genes. These results suggest the importance of further studies to better assess the specific composition, as well as the functional features of the immune environment associated with a necroptotic signature in order to explore new possible diagnostic and immunotherapeutic scenarios.

Lorenzo Nicolè, Tiziana Sanavia, Rocco Cappellesso, Valeria Maffeis, Jun Akiba, Akihiko Kawahara, Yoshiki Naito, Claudia Maria Radu, Paolo Simioni, Davide Serafin, Giuliana Cortese, Maria Guido, Giacomo Zanus, Hirohisa Yano, Ambrogio Fassina

Oncotopix Discovery, Publicly Sharable

Digital pathology has recently been more broadly deployed, fueling artificial intelligence (AI) application development and more systematic use of image analysis. Here, two different AI models were developed to evaluate follicular cell hypertrophy in hematoxylin and eosin-stained whole-slide-images of rat thyroid gland, using commercial AI-based-software. In the first, mean cytoplasmic area measuring approach (MCA approach), mean cytoplasmic area was calculated via several sequential deep learning (DL)-based algorithms including segmentation in microanatomical structures (separation of colloid and stroma from thyroid follicular epithelium), nuclear detection, and area measurements. With our additional second, hypertrophy area fraction predicting approach (HAF approach), we present for the first time DL-based direct detection of the histopathological change follicular cell hypertrophy in the thyroid gland with similar results. For multiple studies, increased output parameters (mean cytoplasmic area and hypertrophic area fraction) were shown in groups given different hypertrophy-inducing reference compounds in comparison to control groups. Quantitative results correlated with the gold standard of board-certified veterinary pathologists’ diagnoses and gradings as well as thyroid hormone dependent gene expressions. Accuracy and repeatability of diagnoses and grading by pathologists are expected to be improved by additional evaluation of mean cytoplasmic area or direct detection of hypertrophy, combined with standard histopathological observations.

Valeria Bertani, Olivier Blanck, Davy Guignard, Frederic Schorsch, Hannah Pischon

Oncotopix Discovery, Publicly Sharable, Tissuealign

Objective: Salivary glands of primary SS (pSS) patients characteristically harbour periductal infiltrates, in which lymphoepithelial lesions (LELs) can develop. LELs are composed of hyperplastic ductal epithelium with infiltrating lymphocytes and may assist in the challenging diagnostic process of pSS. As manual identification of LELs remains difficult, we aimed to identify LELs by using an objective digital image analysis (DIA) algorithm that detects intraepithelial lymphocytes. Methods: A virtual triple-staining technique developed for this study was used to count intraepithelial lymphocytes in consecutive slides stained for CD3 (T-lymphocytes), high-molecular-weight cytokeratin (hmwCK) (striated ducts) and CD20 (B-lymphocytes) in labial and parotid gland biopsies in a diagnostic cohort of 109 sicca patients. Patients were classified as having pSS or non-SS according to the ACR-EULAR classification criteria. Results: T-lymphocytes were detected in almost all analysed ducts of pSS and non-SS sicca patients, whereas intraepithelial B-lymphocytes were present in 59-68% of labial and parotid gland biopsies of pSS patients, against only 2-3% of patients classified as non-SS. Intraepithelial B-lymphocytes were found in almost all striated ducts with hyperplasia (LELs). Remarkably, ∼25% of analysed striated ducts without hyperplasia of pSS patients also contained B-lymphocytes (precursor-LELs). Furthermore, presence of intraepithelial B-lymphocytes was associated with clinical parameters of pSS (i.e. serology). Conclusion: The presence of intraepithelial B-lymphocytes in salivary gland biopsies of sicca patients is a clear indicator of pSS and can be used as an objective alternative to LEL scoring. Therefore, identification of B-lymphocyte-containing ducts should be added to the diagnostic histopathological work-up of patients suspected of pSS.

Martha S. Van Ginkel, Tineke Van Der Sluis, Marian L.C. Bulthuis, Henk J. Buikema, Erlin A. Haacke, Suzanne Arends, Stine Harder, Fred K.L. Spijkervet, Hendrika Bootsma, Arjan Vissink, Frans G.M. Kroese, Bert Van Der Vegt

Oncotopix Discovery, Publicly Sharable, nice

A frequently used treatment strategy in locally advanced rectal cancer (RC) is neoadjuvant therapy followed by surgery. Patients treated with neoadjuvant therapy achieve varying pathological response, and currently, predicting the degree of response is challenging. This study examined the association between digitally assessed histopathological features in the diagnostic biopsies and pathological response to neoadjuvant therapy, aiming to find potential predictive biomarkers. 50 patients with RC treated with neoadjuvant chemotherapy and/or radiotherapy followed by surgery were included. Deep learning-based digital algorithms were used to assess the epithelium tumor area percentage (ETP) based on H&E-stained slides, and to quantify the density of CD3+ and CD8+ lymphocytes, as well as the CD8+/CD3+ lymphocyte percentage, based on immunohistochemically stained slides, from the diagnostic tumor biopsies. Pathological response was assessed according to the Mandard method. A good pathological response was defined as tumor regression grade (TRG) 1-2, and a complete pathological response was defined as Mandard TRG 1. Associations between the ETP and lymphocyte densities in the diagnostic biopsies and the pathological response were examined. The density of CD8+ lymphocytes, and the CD8+/CD3+ lymphocyte percentage, were associated with both good and complete response to neoadjuvant therapy, while the density of CD3+ lymphocytes was associated with complete response. The ETP did not correlate with response to neoadjuvant therapy. It is well-known that infiltration of lymphocytes in colorectal cancer is a prognostic biomarker. However, assessment of CD8+ and CD3+ lymphocytes in the diagnostic tumor biopsies of patients with RC may also be useful in predicting response to neoadjuvant therapy.

Dea Natalie Munch Jepsen, Henrik Høeg, Michael Bzorek, Adile Orhan, Jens Ole Eriksen, Ismail Gögenur, Björn Reiss, Anne-Marie Kanstrup Fiehn

HE, Oncotopix Discovery, Publicly Sharable

Type 2 diabetes (T2D) and its complications can have debilitating, sometimes fatal consequences for afflicted individuals. The disease can be difficult to control, and therapeutic strategies to prevent T2D-induced tissue and organ damage are needed. Here we describe the results of administering a potent and selective inhibitor of Protein Kinase C (PKC) family members PKCα and PKCβ, Cmpd 1, in the ZSF1 obese rat model of hyperphagia-induced, obesity-driven T2D. Although our initial intent was to evaluate the effect of PKCα/β inhibition on renal damage in this model setting, Cmpd 1 unexpectedly caused a marked reduction in the hyperphagic response of ZSF1 obese animals. This halted renal function decline but did so indirectly and indistinguishably from a pair feeding comparator group. However, above and beyond this food intake effect, Cmpd 1 lowered overall animal body weights, reduced liver vacuolation, and reduced inguinal adipose tissue (iWAT) mass, inflammation, and adipocyte size. Taken together, Cmpd 1 had strong effects on multiple disease parameters in this obesity-driven rodent model of T2D. Further evaluation for potential translation of PKCα/β inhibition to T2D and obesity in humans is warranted.

Ju Wang, Agustin Casimiro-Garcia, Bryce G. Johnson, Jennifer Duffen, Michael Cain, Leigh Savary, Stephen Wang, Prashant Nambiar, Matthew Lech, Shanrong Zhao, Li Xi, Yutian Zhan, Jennifer Olson, James A. Stejskal, Hank Lin, Baohong Zhang, Robert V. Martinez, Katherine Masek-Hammerman, Franklin J. Schlerman, Ken Dower

Oncotopix Discovery, Publicly Sharable, TMA

Purpose: The number of M1-like and M2-like tumour-associated macrophages (TAMs) and their ratio can play a role in breast cancer development and progression. Early clinical trials using macrophage targeting compounds are currently ongoing. However, the most optimal detection method of M1-like and M2-like macrophage subsets and their clinical relevance in breast cancer is still unclear. We aimed to optimize the assessment of TAM subsets in different breast cancer subtypes, and therefore related TAM subset numbers and ratio to clinicopathological characteristics and clinical outcome. Methods: Tissue microarrays of 347 consecutive primary Luminal-A, Luminal-B, HER2-positive and triple-negative tumours of patients with early-stage breast cancer were serially sectioned and immunohistochemically stained for the pan-macrophage marker CD68 and the M2-like macrophage markers CD163, CSF-1R and CD206. TAM numbers were quantified using a digital image analysis algorithm. M1-like macrophage numbers were calculated by subtracting M2-like TAM numbers from the total TAM number. Results: M2-like markers CD163 and CSF-1R showed a moderate positive association with each other and with CD68 (r ≥ 0.47), but only weakly with CD206 (r ≤ 0.06). CD68 + , CD163 + and CSF-1R + macrophages correlated with tumour grade in Luminal-B tumours (P < 0.001). Total or subset TAM numbers did not correlate with disease outcome in any breast cancer subtype. Conclusion: In conclusion, macrophages and their subsets can be detected by means of a panel of TAM markers and are related to unfavourable clinicopathological characteristics in Luminal-B breast cancer. However, their impact on outcome remains unclear. Preferably, this should be determined in prospective series.

Mieke C. Zwager, Rico Bense, Stijn Waaijer, Si Qi Qiu, Hetty Timmer-Bosscha, Elisabeth G.E. de Vries, Carolien P. Schröder, Bert van der Vegt

Oncotopix Discovery, Publicly Sharable

Endometrial hyperplasia is a precursor to endometrial cancer, characterized by excessive proliferation of glands that is distinguishable from normal endometrium. Current classifications define 2 types of EH, each with a different risk of progression to endometrial cancer. However, these schemes are based on visual assessments and, therefore, subjective, possibly leading to overtreatment or undertreatment. In this study, we developed an automated artificial intelligence tool (ENDOAPP) for the measurement of morphologic and cytologic features of endometrial tissue using the software Visiopharm. The ENDOAPP was used to extract features from whole-slide images of PAN-CK+-stained formalin-fixed paraffin-embedded tissue sections from 388 patients diagnosed with endometrial hyperplasia between 1980 and 2007. Follow-up data were available for all patients (mean = 140 months). The most prognostic features were identified by a logistic regression model and used to assign a low-risk or high-risk progression score. Performance of the ENDOAPP was assessed for the following variables: images from 2 different scanners (Hamamatsu XR and S60) and automated placement of a region of interest versus manual placement by an operator. Then, the performance of the application was compared with that of current classification schemes: WHO94, WHO20, and EIN, and the computerized-morphometric risk classification method: D-score. The most significant prognosticators were percentage stroma and the standard deviation of the lesser diameter of epithelial nuclei. The ENDOAPP had an acceptable discriminative power with an area under the curve of 0.765. Furthermore, strong to moderate agreement was observed between manual operators (intraclass correlation coefficient: 0.828) and scanners (intraclass correlation coefficient: 0.791). Comparison of the prognostic capability of each classification scheme revealed that the ENDOAPP had the highest accuracy of 88%-91% alongside the D-score method (91%). The other classification schemes had an accuracy between 83% and 87%. This study demonstrated the use of computer-aided prognosis to classify progression risk in EH for improved patient treatment.

Emma Rewcastle, Einar Gudlaugsson, Melinda Lillesand, Ivar Skaland, Jan P.A. Baak, Emiel A.M. Janssen

Oncotopix Discovery, Publicly Sharable

In colorectal cancer liver metastases (CRLM), the density of tumor-infiltrating lymphocytes, the expression of class I major histocompatibility complex (MHC-I), and the pathological response to preoperative chemotherapy have been associated with oncological outcomes after complete resection. However, the prognostic significance of the heterogeneity of these features in patients with multiple CRLMs remains under investigation. We used a TMA tissue microarray of 220 mismatch repair-gene proficient CRLMs resected in 97 patients followed prospectively to quantify CD3+ T cells and MHC-I by immunohistochemistry. Histopathological response to preoperative chemotherapy was assessed using standard scoring systems. We tested associations between clinical, immunological, and pathological features with oncologic outcomes. Overall, 29 patients (30.2%) had CRLMs homogeneous for CD3+ T cell infiltration and MHC-I. Patients with immune homogeneous compared to heterogeneous CRLMs had longer median time to recurrence (TTR) (30 vs. 12 months, p = .0018) and disease-specific survival (DSS) (not reached vs. 48 months, p = .0009). At 6 years, 80% of the patients with immune homogeneous CRLMs were still alive. Homogeneity of response to preoperative chemotherapy was seen in 60 (61.9%) and 69 (80.2%) patients according to different grading systems and was not associated with TTR or DSS. CD3 and MHC-I heterogeneity was independent of response to pre-operative chemotherapy and of other clinicopathological variables for their association with oncological outcomes. In patients with multiple CRLMs resected with curative intent, similar adaptive immune features seen across metastases could be more informative than pathological response to pre-operative chemotherapy in predicting oncological outcomes.

David Henault, David Stephen, Pierre Antoine St-Hilaire, Nouredin Messaoudi, Franck Vandenbroucke-Menu, Eve Simoneau, Zhixia Rong, Marylène Plasse, Richard Létourneau, André Roy, Michel Dagenais, Réal Lapointe, Bich Nguyen, Anne Marie Mes-Masson, G. Soucy, Simon Turcotte

Oncotopix Discovery, Publicly Sharable

Background Hepatic immune tolerance might contribute to the development of therapeutic resistance to immunotherapy. However, addressing this issue is challenging since the efficacy of immunotherapy in the context of liver metastasis (LM) remains poorly studied. Here, we aimed to establish an LM common immune feature (LMCIF) score to quantify the characteristics of LM immunotolerance across cancer types for assisting clinical disease management. Methods Large-scale clinical data were collected to identify the prognosis of LM. Multi-omics datasets of metastatic cancers with LM special immune-related pathways (LMSIPs) from the Molecular Signatures Database (MSigDB) were used to obtain an LMCIF cluster. Based on differential expression genes (DEGs), a novel LMCIF score for the LMCIF cluster was constructed. In addition, multi-omics, and immunohistochemistry (IHC) data from the public and in-house cohorts were used to explore the features of LM, and LMCIF score. Results Patients with LM had a worse prognosis and significantly lower infiltration of immune cells than patients with metastasis to other organs when analyzed with combined clinical and RNA sequencing data. After extracting the LMCIF cluster from 373 samples by utilizing 29 LMSIPs and validating them in a microarray cohort, an LMCIF score was established to confirm the role of the immunosuppressive environment as a contributor to the poor prognosis of LM across cancer types. Moreover, this LMCIF score could be used to predict the immune response of cancer patients undergoing immunotherapy. Finally, we identified that the majority of the 31 LMCIF genes exhibited a negative correlation with TME cells in LM patients, one of them, KRT19, which possessed the strongest positive correlation with LMCIF score, was confirmed to have an immunosuppressive effect through IHC analysis. Conclusions Our results suggest that LM across cancer types share similar immunological profiles that induce immunotolerance and escape from immune monitoring. The novel LMCIF score represents a common liver metastasis immune cluster for predicting immunotherapy response, the results of which might benefit clinical disease management.

Yuzhen Gao, Shipeng Chen, Hao Wang, Chenghao Wu, Rui An, Guoli Li, Min Yang, Ying Zhou, Yundong Zhou, Xinyou Xie, Hong Yu, Jun Zhang

Oncotopix Discovery, Publicly Sharable

The upper limit for partial hepatectomy (PH) in rats is 90%, which is associated with an increased risk of post-hepatectomy liver failure (PHLF), correlating with high mortality. Sixty-eight rats were randomized to 90% PH, sham operation, or no surgery. Further block randomization was performed to determine the time of euthanasia, whether 12, 24, or 48 h after surgery. A general distress score (GDS) was calculated to distinguish between rats with reversible (GDS < 10) and irreversible PHLF (GDS ≥ 10). At euthanasia, the liver remnant and blood were collected. Liver-specific biochemistry and regeneration ratio were measured. Hepatocyte proliferation and volume were estimated using stereological methods. All rats subjected to 90% experienced biochemical PHLF. The biochemical and morphological liver responses did not differ between the groups until 48 h after surgery. At 48 h, liver regeneration and function were significantly improved in survivors. The peak mean regeneration ratio was 15% for rats with irreversible PHLF compared to 26% for rats with reversible PHLF. The 90% PH rat model was associated with PHLF and high mortality. Irreversible PHLF was characterized by impaired liver regeneration capacity and an insufficient ability to metabolize ammonia.

Andrea Lund, Kasper Jarlhelt Andersen, Michelle Meier, Marie Ingemann Pedersen, Anders Riegels Knudsen, Jakob Kirkegård, Frank Viborg Mortensen, Jens Randel Nyengaard

H&E, Oncotopix Discovery, Publicly Sharable

Chikungunya virus (CHIKV) infection has been associated with severe cardiac manifestations, yet, how CHIKV infection leads to heart disease remains unknown. Here, we leveraged both mouse models and human primary cardiac cells to define the mechanisms of CHIKV heart infection. Using an immunocompetent mouse model of CHIKV infection as well as human primary cardiac cells, we demonstrate that CHIKV directly infects and actively replicates in cardiac fibroblasts. In immunocompetent mice, CHIKV is cleared from cardiac tissue without significant damage through the induction of a local type I interferon response from both infected and non-infected cardiac cells. Using mice deficient in major innate immunity signaling components, we found that signaling through the mitochondrial antiviral-signaling protein (MAVS) is required for viral clearance from the heart. In the absence of MAVS signaling, persistent infection leads to focal myocarditis and vasculitis of the large vessels attached to the base of the heart. Large vessel vasculitis was observed for up to 60 days post infection, suggesting CHIKV can lead to vascular inflammation and potential long-lasting cardiovascular complications. This study provides a model of CHIKV cardiac infection and mechanistic insight into CHIKV-induced heart disease, underscoring the importance of monitoring cardiac function in patients with CHIKV infections. Mosquito-borne viruses are serious global public health threats associated with severe atypical cardiovascular manifestations. Here, the authors dissect how chikungunya virus directly infects cardiac tissue leading to heart disease and define key host pathways involved in viral cardiac persistence and tissue damage.

Maria G. Noval, Sophie N. Spector, Eric Bartnicki, Franco Izzo, Navneet Narula, Stephen T. Yeung, Payal Damani-Yokota, M. Zahidunnabi Dewan, Valeria Mezzano, Bruno A. Rodriguez-Rodriguez, Cynthia Loomis, Kamal M. Khanna, Kenneth A. Stapleford

Oncotopix Discovery, Publicly Sharable

The respiratory system is the main target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 19 (COVID-19) where acute respiratory distress syndrome is considered the leading cause of death. Changes in pulmonary blood vessels, among which an endothelialitis/endotheliitis has been particularly emphasized, have been suggested to play a central role in the development of acute lung injury. Similar vascular changes are also observed in animal models of COVID-19. The present study aimed to determine whether the latter are specific for SARS-CoV-2 infection, investigating the vascular response in the lungs of mice infected with SARS-CoV-2 and other respiratory viruses (influenza A and murine gammaherpesvirus) by in situ approaches (histology, immunohistology, morphometry) combined with RNA sequencing and bioinformatic analysis. Non-selective recruitment of monocytes and T and B cells from larger muscular veins and arteries was observed with all viruses, matched by a comparable transcriptional response. There was no evidence of endothelial cell infection in any of the models. Both the morphological investigation and the transcriptomics approach support the interpretation that the lung vasculature in mice mounts a stereotypic response to alveolar and respiratory epithelial damage. This may have implications for the treatment and management of respiratory disease in humans.

Simon De Neck, Rebekah Penrice-Randal, Jordan J. Clark, Parul Sharma, Eleanor G. Bentley, Adam Kirby, Daniele F. Mega, Ximeng Han, Andrew Owen, Julian A. Hiscox, James P. Stewart, Anja Kipar

Akoya, Oncotopix Discovery, Publicly Sharable, Vectra Polaris

The arachidonic acid pathway participates in immunosuppression in various types of cancer. Our previous observation detailed that microsomal prostaglandin E2 synthase 1 (mPGES-1), an enzyme downstream of cyclooxygenase 2 (COX-2), limited antitumor immunity in melanoma; in addition, genetic depletion of mPGES-1 specifically enhanced immune checkpoint blockade therapy. The present study set out to distinguish the roles of mPGES-1 from those of COX-2 in tumor immunity and determine the potential of mPGES-1 inhibitors for reinforcing immunotherapy in melanoma. Genetic deletion of mPGES-1 showed different profiles of prostaglandin metabolites from that of COX-2 deletion. In our syngeneic mouse model, mPGES-1–deficient cells exhibited similar tumorigenicity to that of COX-2–deficient cells, despite a lower ability to suppress PGE2 synthesis by mPGES-1 depletion, indicating the presence of factors other than PGE2 that are likely to regulate tumor immunity. RNA sequencing analysis revealed that mPGES-1 depletion reduced the expressions of collagen-related genes, which have been found to be associated with immunosuppressive signatures. In our mouse model, collagen was reduced in mPGES-1–deficient tumors, and phenotypic analysis of tumor-infiltrating lymphocytes indicated that mPGES-1-deficient tumors had fewer TIM3+ exhausted CD8+ T cells compared with COX-2–deficient tumors. CAY10678, an mPGES-1 inhibitor, was equivalent to celecoxib, a selective COX-2 inhibitor, in reinforcing anti–PD-1 treatment. Our study indicates that mPGES-1 inhibitors represent a promising adjuvant for immunotherapies in melanoma by reducing collagen deposition and T cell exhaustion.

Yasunari Fukuda, Sun-Hee Kim, Matias A. Bustos, Sung-Nam Cho, Jason Roszik, Jared K. Burks, Hong Kim, Dave S.B. Hoon, Elizabeth A. Grimm, Suhendan Ekmekcioglu

Oncotopix Discovery, Publicly Sharable, Straticyte

While a 3-tier oral epithelial dysplasia grading system has been utilized for decades, it is widely recognized as a sub-optimal risk indicator for transformation to cancer. A 2-tier grading system has been proposed, although not yet validated. In this study, the 3-tier and 2-tier dysplasia grading systems, and an S100A7 immunohistochemical signature-based grading system were compared to assess prediction of risk of transformation to oral cancer. Formalin-fixed, paraffin-embedded biopsy specimens with known clinical outcomes were obtained retrospectively from a cohort of 48 patients. Hematoxylin and eosin-stained slides were used for the 2-and 3-tier dysplasia grading, while S100A7 for biomarker signature-based assessment was based on immunohistochemistry. Inter-observer variability was determined using Cohen's kappa (K) statistic with Cox regression disease free survival analysis used to determine if any of the methods were a predictor of transformation to oral squamous cell carcinoma. Both the 2-and 3-tier dysplasia grading systems ranged from slight to substantial inter-observer agreement (Kw between 0.093 to 0.624), with neither system a good predictor of transformation to cancer (at least P = 0.231; (P > > > 0.05). In contrast, the S100A7 immunohistochemical signature-based grading system showed almost perfect inter-observer agreement (Kw = 0.892) and was a good indicator of transformation to cancer (P = 0.047 and 0.030). The inherent grading challenges with oral epithelial dysplasia grading systems and the lack of meaningful prediction of transformation to carcinoma highlights the significant need for a more objective, quantitative, and reproducible risk assessment tool such as the S100A7 immunohistochemical signature-based system.

Mark Roger Darling, Jason T K Hwang, Benjamin J Dickson, Jean-Claude Cutz, Samih Salama, Christina Mccord, Kenneth P H Pritzker, David Mock, Lester D R Thompson

ISH, Oncotopix Discovery, Publicly Sharable, RNAScope

Background and aims: Tumour necrosis factor-α (TNF) antagonists have improved the management of inflammatory bowel disease (IBD), however, their usage and administration persist to be suboptimal. Here, we examined the relationship between tissue-specific TNF mRNA expression in mucosal biopsies from IBD patients and anti-TNF treatment response. Methods: Archived tissue samples from patients with luminal IBD that had all been or were in treatment with anti-TNF were included (18 adults and 24 paediatric patients). Patients were stratified into three groups according to anti-TNF response: responders, primary non-responders (PNR) and secondary loss of response (SLOR). TNF mRNA was detected using RNAscope in situ hybridisation (ISH) and the expression was quantified using image analysis. Results: The ISH analysis showed varying occurrence of TNF mRNA positive cells located in lamina propria and often with increased density in lymphoid follicles (LF). Consequently, expression estimates were obtained in whole tissue areas with and without LF. Significantly higher TNF mRNA expression levels were measured in adults compared to paediatric patients in both the analyses with and without LF (p = .015 and p = .016, respectively). Considering the relation to response, the adult and paediatric patients were evaluated separately. In adults, the TNF expression estimates were higher in PNRs compared to responders with and without LF (p = .017 and p = .024, respectively). Conclusion: Our data indicate that adult PNR have significantly higher TNF mRNA levels than responders. This suggests that higher anti-TNF dose may be considered for IBD patients with high TNF mRNA expression estimates from the start of treatment. Keywords: Image analysis; Paediatric IBD; Tumour necrosis factor; inflammation; lymphoid follicle; mucosal TNF mRNA; quantitative in situ hybridisation; treatment non-response.

Jaslin P James, Schnack Nielsen, Ebbe Langholz, Mikkel Malham, Estrid Høgdall, & Lene, Buhl Riis, Boye Schnack Nielsen, Lene Buhl Riis

Oncotopix Discovery, Publicly Sharable

The prognostic value of conventional histopathological parameters in the sinonasal intestinal-type adenocarcinoma (ITAC) has been debated and novel variables should be investigated. Increasing evidence demonstrated that the evolution of cancer is strongly dependent upon the complex interactions within tumor microenvironment. The aim of this retrospective study was to assess the features of immune microenvironment in terms of CD3+ and CD8+ cells in a series of ITAC and explore their prognostic role, as well as their relations with clinicopathological variables. A computer-assisted image analysis of CD3+ and CD8+ tumor-infiltrating lymphocytes (TIL) density was conducted on surgical specimens of 51 patients with ITAC that underwent a curative treatment including surgery. ITAC displays variable TIL density, which is associated with OS. In a univariate model, the density of CD3+ TIL was significantly related to OS (p = 0.012), whereas the association with CD8+ TIL density resulted in being non-significant (p = 0.056). Patients with intermediate CD3+ TIL density were associated with the best outcome, whereas 5-year OS was the lowest for intermediate CD8+ TIL density. CD3+ TIL density maintained a significant association with OS in the multivariable analysis. TIL density was not significantly related to demographic and clinicopathological variables. CD3+ TIL density was independently associated with OS in a non-linear fashion and patients with intermediate CD3+ TIL density had the best outcome. Though based on a preliminary analysis on a relatively small series of patients, this finding makes TIL density a potential independent prognostic factor of ITAC.

Marco Ferrari, Lara Alessandrini, Enrico Savietto, Diego Cazzador, Gloria Schiavo, Stefano Taboni, Andrea L. C. Carobbio, Leonardo Calvanese, Giacomo Contro, Piergiorgio Gaudioso, Enzo Emanuelli, Marta Sbaraglia, Elisabetta Zanoletti, Gino Marioni, Angelo P. Dei Tos, Piero Nicolai

Oncotopix Discovery, Publicly Sharable

T cell factor 1 (Tcf-1) expressing CD8+ T cells exhibit stem-like self-renewing capacity, rendering them key for immune defense against chronic viral infection and cancer. Yet, the signals that promote the formation and maintenance of these stem-like CD8+ T cells (CD8+SL) remain poorly defined. Studying CD8+ T cell differentiation in mice with chronic viral infection, we identified the alarmin interleukin-33 (IL-33) as pivotal for the expansion and stem-like functioning of CD8+SL as well as for virus control. IL-33 receptor (ST2)-deficient CD8+ T cells exhibited biased end differentiation and premature loss of Tcf-1. ST2-deficient CD8+SL responses were restored by blockade of type I interferon signaling, suggesting that IL-33 balances IFN-I effects to control CD8+SL formation in chronic infection. IL-33 signals broadly augmented chromatin accessibility in CD8+SL and determined these cells' re-expansion potential. Our study identifies the IL-33-ST2 axis as an important CD8+SL-promoting pathway in the context of chronic viral infection.

Anna-Friederike Marx, Sandra M. Kallert, Tobias M. Brunner, José A. Villegas, Florian Geier, Jonas Fixemer, Tiago Abreu-Mota, Peter Reuther, Weldy V. Bonilla, Jelizaveta Fadejeva, Mario Kreutzfeldt, Ingrid Wagner, Patricia Aparicio-Domingo, Leo Scarpellino, Mélanie Charmoy, Daniel T. Utzschneider, Claudia Hagedorn, Min Lu, Karen Cornille, Karsten Stauffer, Florian Kreppel, Doron Merkler, Dietmar Zehn, Werner Held, Sanjiv A. Luther, Max Löhning, Daniel D. Pinschewer

Oncotopix Discovery, Publicly Sharable, TMA

Gwan Han, Hee Yun, Joon-Yong Chung, Jae-Hoon Kim, Hanbyoul Cho

Oncotopix Discovery, Publicly Sharable

Ovarian tissue transplantation can restore fertility in young cancer survivors, however the detrimental loss of follicles following transplantation of cryopreserved ovarian tissue is hampering the efficiency of the procedure. This study investigates whether needle puncturing prior to transplantation can enhance revascularization and improve follicle survival in xenotransplanted human ovarian cortex. Cryopreserved human ovarian cortex pieces (N = 36) from 20 women aged 24–36 years were included. During the thawing process, each piece of tissue was cut in halves; one half serving as the untreated control and the other half was punctured approximately 150–200 times with a 29-gauge needle. The cortex pieces were transplanted subcutaneously to immunodeficient mice for 3, 6 and 10 days (N = 8 patients) and for 4 weeks (N = 12 patients). After 3, 6 and 10 days, revascularization of the ovarian xenografts were assessed using immunohistochemical detection of CD31 and gene expression of angiogenic factors (Vegfα, Angptl4, Ang1, and Ang2), and apoptotic factors (BCL2 and BAX) were performed by qPCR. Follicle density and morphology were evaluated in ovarian xenografts after 4 weeks. A significant increase in the CD31 positive area in human ovarian xenografts was evident from day 3 to 10, but no significant differences were observed between the needle and control group. The gene expression of Vegfα was consistently higher in the needle group compared to control at all three time points, but not statistically significant. The expression of Ang1 and Ang2 increased significantly from day 3 to day 10 in the control group (p < 0.001, p = 0.0023), however, in the needle group this increase was not observed from day 6 to 10 (Ang2 p = 0.027). The BAX/BCL2 ratio was similar in the needle and control groups. After 4-weeks xenografting, follicle density (follicles/mm3, mean ± SEM) was higher in the needle group (5.18 ± 2.24) compared to control (2.36 ± 0.67) (p = 0.208), and a significant lower percentage of necrotic follicles was found in the needle group (19%) compared to control (36%) (p = 0.045). Needle puncturing of human ovarian cortex prior to transplantation had no effect on revascularization of ovarian grafts after 3, 6 and 10 days xenotransplantation. However, needle puncturing did affect angiogenic genes and improved follicle morphology.

Hanna Ørnes Olesen, Susanne Elisabeth Pors, Cristina Subiran Adrados, Mette Christa Zeuthen, Linn Salto Mamsen, Anette Tønnes Pedersen, Stine Gry Kristensen

Oncotopix Discovery, Publicly Sharable

Contrasting the antigen-presenting dendritic cells (DCs) in the conducting airways, the alveolar DC populations in human lungs have remained poorly investigated. Consequently , little is known about how alveolar DCs are altered in diseases such as chronic obstructive pulmonary disease (COPD). This study maps multiple tissue DC categories in the distal lung across COPD severities. Specifically, single-multiplex immunohistochemistry was applied to quantify langerin/ CD207 + , CD1a + , BDCA2 + , and CD11c + subsets in distal lung compartments from patients with COPD (GOLD stage I-IV) and never-smoking and smoking controls. In the alveolar pa-renchyma, increased numbers of CD1a + langerin − (p < 0.05) and BDCA-2 + DCs (p < 0.001) were observed in advanced COPD compared with controls. Alveolar CD11c + DCs also increased in advanced COPD (p < 0.01). In small airways, lan-gerin + and BDCA-2 + DCs were also significantly increased. Contrasting the small airway DCs, most alveolar DC subsets frequently extended luminal protrusions. Importantly, alve-olar and small airway langerin + DCs in COPD lungs displayed site-specific marker profiles. Further, multiplex immunohis-tochemistry with single-cell quantification was used to specifically profile langerin DCs and reveal site-specific expression patterns of the maturation and activation markers S100, fascin, MHC2, and B7. Taken together, our results show that clinically advanced COPD is associated with increased levels of multiple alveolar DC populations exhibiting features of both adaptive and innate immunity phenotypes. This expansion is likely to contribute to the distal lung immunopathol-ogy in COPD patients.

Michiko Mori, Carl-Magnus Clausson, Caroline Sanden, Jimmie Jönsson, Cecilia K Andersson, Premkumar Siddhuraj, Medya Shikhagaie, Karolina Åkesson, Anders Bergqvist, Claes-Göran Löfdahl, Jonas S Erjefält

Oncotopix Discovery, Publicly Sharable

Background Development of reliable disease activity biomarkers is critical for diagnostics, prognostics, and novel drug development. Although computed tomography (CT) is the gold-standard for quantification of bone erosions, there are no consensus approaches or rationales for utilization of specific outcome measures of erosive arthritis in complex joints. In the case of preclinical models, such as sexually dimorphic tumor necrosis factor transgenic (TNF-Tg) mice, disease severity is routinely quantified in the ankle through manual segmentation of the talus or small regions of adjacent bones primarily due to the ease in measurement. Herein, we sought to determine the particular hindpaw bones that represent reliable biomarkers of sex-dependent disease progression to guide future investigation and analysis. Methods Hindpaw micro-CT was performed on wild-type (n = 4 male, n = 4 female) and TNF-Tg (n = 4 male, n = 7 female) mice at monthly intervals from 2–5 (females) and 2-8-months (males) of age, since female TNF-Tg mice exhibit early mortality from cardiopulmonary disease at approximately 5-6-months. Further, 8-month-old WT (n = 4) and TNF-Tg males treated with anti-TNF monoclonal antibodies (n = 5) or IgG placebo isotype controls (n = 6) for 6-weeks were imaged with micro-CT every 3-weeks. For image analysis, we utilized our recently developed high-throughput and semi-automated segmentation strategy in Amira software. Synovial and osteoclast histology of ankle joints was quantified using Visiopharm. Results First, we demonstrated that the accuracy of automated segmentation, determined through analysis of ~9000 individual bones by a single user, was comparable in wild-type and TNF-Tg hindpaws before correction (79.2±8.9% vs 80.1±5.1%, p = 0.52). Compared to other bone compartments, the tarsal region demonstrated a sudden, specific, and significant bone volume reduction in female TNF-Tg mice, but not in males, by 5-months (4-months 4.3± 0.22 vs 5-months 3.4± 0.62 mm3, p<0.05). Specifically, the cuboid showed significantly reduced bone volumes at early timepoints compared to other tarsals (i.e., 4-months: Cuboid -24.1±7.2% vs Talus -9.0±5.9% of 2-month baseline). Additional bones localized to the anterolateral region of the ankle also exhibited dramatic erosions in the tarsal region of females, coinciding with increased synovitis and osteoclasts. In TNF-Tg male mice with severe arthritis, the talus and calcaneus exhibited the most sensitive response to anti-TNF therapy measured by effect size of bone volume change over treatment period. Conclusions We demonstrated that sexually dimorphic changes in arthritic hindpaws of TNF-Tg mice are bone-specific, where the cuboid serves as a reliable early biomarker of erosive arthritis in female mice. Adoption of automated segmentation approaches in pre-clinical or clinical models has potential to translate quantitative biomarkers to monitor bone erosions in disease and evaluate therapeutic efficacy.

H Mark Kenney, Kiana L Chen, Lindsay Schnur Id, Jeffrey I Fox Id, Ronald W Wood Id, Lianping Xing, Christopher T Ritchlin, Homaira Rahimi, Edward M Schwarz Id, Hani A Awad Id

Oncotopix Discovery, Publicly Sharable

Purpose To test the hypothesis that Pressure Enabled Drug Delivery (PEDD) with a TriNav device (TNV-21120-35, TriSalus Life Sciences, Westminster, CO) would improve the delivery of surrogate therapeutic glass microspheres (GM) via hepatic artery infusion (HAI) to liver tumors when compared to a conventional endhole microcatheter. Materials and Methods The study was conducted in transgenic pigs (Oncopigs) with induced liver tumors. Tumors were infused intra-arterially with fluorescently labeled GM. PEDD with a TriNav device was compared to conventional endhole delivery in both lobar and selective infusions. Near-Infrared (nearIR, NIR) imaging was used to detect GM fluorescent signal in tumors. Image analysis with a custom Deep Learning algorithm (Visiopharm A/S) was used to quantitate signal intensity in relation to the tumor border. Results With lobar infusions, significant increases in GM signal intensity were observed in and around tumors after PEDD (n=10) when compared to conventional delivery (n=7), with PEDD increasing penetration into the tumor by 117% (p = 0.004). In selective infusions, PEDD (n=9) increased penetration into the tumor by 39% relative to conventional delivery (n=8, p =0.032). Lobar PEDD delivery of GM to the tumor was statistically equivalent to conventional selective delivery (p=0.497). Conclusions PEDD with a TriNav device significantly improved GM uptake in liver tumors relative to conventional infusion in both lobar and selective procedures. Lobar GM delivery with PEDD was equivalent to conventional selective delivery with an endhole device, suggesting that proximal PEDD infusions may enable effective delivery without selection of distal target vessels.

David B. Jaroch, Yujia Liu, Alexander Y. Kim, Steven C. Katz, Bryan F. Cox, Thomas G. Hullinger

Oncotopix Discovery, Publicly Sharable

To compare the effects of organic selenium nanoparticles (SeNPs, Se0) and inorganic sodium selenite (NaSe, Na2SeO3, Se4+) on the antioxidant response in maternal and fetal rat liver, pregnant females were treated with two forms of selenium (Se) at equivalent doses during gestation (0.5 mg SeNPs or 0.5 mg NaSe/kg body weight/day). Structural parameters of the liver of gravid females and their fetuses were examined in a sex-specific manner. The oxidative stress parameters superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione S-transferase (GST), total glutathione (GSH) and sulfhydryl groups (SH) were established. In addition, the Se concentration was determined in the blood, liver, urine and feces of the gravid females and in the liver of the fetuses. The structure of the liver of gravid females remained histologically the same after supplementation with both forms of Se, while the oxidative stress in the liver was significantly lower after the use of SeNPs compared to NaSe. Immaturity of fetal antioxidant defenses and sex specificity were demonstrated. This study provides a detailed insight into the differences in the bioavailability of the nano form of Se compared to sodium selenite in the livers of pregnant females and fetuses. antioxidant response; selenium nanoparticles; sodium selenite; pregnancy; fetuses; liver; histology; oxidative stress parameters; sex differences

Milica Manojlovi´cmanojlovi´c-Stojanoski, Slavica Borkovi´c, Borkovi´c-Miti´c Miti´c, Nataša Nestorovi´c Nestorovi´c, Nataša Risti´c Risti´c, Radomir Stefanovi´cstefanovi´c, Magdalena Stevanovi´c Stevanovi´c, Nenad Filipovi´cfilipovi´c, Aleksandar Stojsavljevi´c Stojsavljevi´c, Sla ¯ Dan, Pavlovi´c Pavlovi´c

Oncotopix Discovery, Phenoplex, Publicly Sharable

Background: Neutrophils are key mediators of inflammation during acute liver injury (ALI). Emerging evidence suggests that they also contribute to injury resolution and tissue repair. However, the different neutrophil subsets involved in these processes and their kinetics are undefined. Herein, we characterized neutrophil kinetics and heterogeneity during ALI. Methods: We used the carbon tetrachloride model of ALI and employed flow cytometry, tissue imaging, and quantitative RT-PCR to characterize intrahepatic neutrophils during the necroinflammatory early and late repair phases of the wound healing response to ALI. We FACS sorted intrahepatic neutrophils at key time points and examined their transcriptional profiles using RNA-sequencing. Finally, we evaluated neutrophil protein translation, mitochondrial function and metabolism, reactive oxygen species content, and neutrophil extracellular traps generation. Results: We detected 2 temporarily distinct waves of neutrophils during (1) necroinflammation (at 24 hours after injury) and (2) late repair (at 72 hours). Early neutrophils were proinflammatory, characterized by: (1) upregulation of inflammatory cytokines, (2) activation of the noncanonical NF-κB pathway, (3) reduction of protein translation, (4) decreased oxidative phosphorylation, and (5) higher propensity to generate reactive oxygen species and neutrophil extracellular traps. In contrast, late neutrophils were prorepair and enriched in genes and pathways associated with tissue repair and angiogenesis. Finally, early proinflammatory neutrophils were characterized by the expression of a short isoform of C-X-C chemokine receptor 5, while the late prorepair neutrophils were characterized by the expression of C-X-C chemokine receptor 4. Conclusions: This study underscores the phenotypic and functional heterogeneity of neutrophils and their dual role in inflammation and tissue repair during ALI.

Yousef Maali, Manuel Flores Molina, Omar Khedr, Mohamed N. Abdelnabi, Jessica Dion, Ghada S. Hassan, Naglaa H. Shoukry

Oncotopix Discovery, Publicly Sharable

BACKGROUND The influence of Helicobacter-pylori (H. pylori) infection and the characteristics of gastric cancer (GC) on tumor-infiltrating lymphocyte (TIL) levels has not been extensively studied. Analysis of infiltrating-immune-cell subtypes as well as survival is necessary to obtain comprehensive information. AIM To determine the rates of deficient mismatch-repair (dMMR), HER2-status and H. pylori infection and their association with TIL levels in GC. METHODS Samples from 503 resected GC tumors were included and TIL levels were evaluated following the international-TILs-working-group recommendations with assessment of the intratumoral (IT), stromal (ST) and invasive-border (IB) compartments. The density of CD3, CD8 and CD163 immune cells, and dMMR and HER2-status were determined by immunohistochemistry (IHC). H. pylori infection was evaluated by routine histology and quantitative PCR (qPCR) in a subset of samples. RESULTS dMMR was found in 34.4%, HER2+ in 5% and H. pylori-positive in 55.7% of samples. High IT-TIL was associated with grade-3 (P = 0.038), while ST-TIL with grade-1 (P < 0.001), intestinal-histology (P < 0.001) and no-recurrence (P = 0.003). dMMR was associated with high TIL levels in the ST (P = 0.019) and IB (P = 0.01) compartments, and ST-CD3 (P = 0.049) and ST-CD8 (P = 0.05) densities. HER2- was associated with high IT-CD8 (P = 0.009). H. pylori-negative was associated with high IT-TIL levels (P = 0.009) when assessed by routine-histology, and with high TIL levels in the 3 compartments (P = 0.002-0.047) and CD8 density in the IT and ST compartments (P = 0.001) when assessed by qPCR. A longer overall survival was associated with low IT-CD163 (P = 0.003) and CD8/CD3 (P = 0.001 in IT and P = 0.002 in ST) and high IT-CD3 (P = 0.021), ST-CD3 (P = 0.003) and CD3/CD163 (P = 0.002). CONCLUSION TIL levels were related to dMMR and H. pylori-negativity. Low CD8/CD3 and high CD163/CD3 were associated with lower recurrence and longer survival. Key Words: Lymphocytes, Macrophages, Gastric cancer, Helicobacter pylori, HER2, Mismatch repair Castaneda CA, Castillo M, Bernabe LA, Sanchez J, Fassan M, Tello K, Wistuba II, Chavez Passiuri I, Ruiz E, Sanchez J, Barreda F, Valdivia D, Bazan Y, Abad-Licham M, Mengoa C, Fuentes H, Montenegro P, Poquioma E, Alatrista R, Flores CJ, Taxa L. Association between Helicobacter pylori infection, mismatch repair, HER2 and tumor-infiltrating lymphocytes in gastric cancer. World J Gastrointest Oncol 2024; 16(6): 2487-2503 [DOI: 10.4251/wjgo.v16.i6.2487]

Carlos A Castaneda, Miluska Castillo, Luis A Bernabe, Joselyn Sanchez, Matteo Fassan, Katherine Tello, Ignacio Ivan Wistuba, Ivan Chavez Passiuri, Eloy Ruiz, Juvenal Sanchez, Fernando Barreda, Daniel Valdivia, Yaqueline Bazan, Milagros Abad-Licham, Claudio Mengoa, Hugo Fuentes, Paola Montenegro, Ebert Poquioma, Raul Alatrista, Claudio J Flores, Luis Taxa

Oncotopix Discovery, Publicly Sharable

Zika virus (ZikV) infection during pregnancy can cause congenital Zika syndrome (CZS) and neurodevelopmental delay in infants, of which the pathogenesis remains poorly understood. We utilize an established female pigtail macaque maternal-to-fetal ZikV infection/exposure model to study fetal brain pathophysiology of CZS manifesting from ZikV exposure in utero. We find prenatal ZikV exposure leads to profound disruption of fetal myelin, with extensive downregulation in gene expression for key components of oligodendrocyte maturation and myelin production. Immunohistochemical analyses reveal marked decreases in myelin basic protein intensity and myelinated fiber density in ZikV-exposed animals. At the ultrastructural level, the myelin sheath in ZikV-exposed animals shows multi-focal decompaction, occurring concomitant with dysregulation of oligodendrocyte gene expression and maturation. These findings define fetal neuropathological profiles of ZikV-linked brain injury underlying CZS resulting from ZikV exposure in utero. Because myelin is critical for cortical development, ZikV-related perturbations in oligodendrocyte function may have long-term consequences on childhood neurodevelopment, even in the absence of overt microcephaly. Pathogenesis of neurodevelopmental delays in infants during maternal Zika virus infection is not well understood. Here, the authors show that congenital Zika virus infection in macaques disrupts fetal neurodevelopment through myelin decompaction and suppression of myelin gene expression.

Jennifer Tisoncik-Go, Caleb Stokes, Leanne S. Whitmore, Daniel J. Newhouse, Kathleen Voss, Andrew Gustin, Cheng-Jung Sung, Elise Smith, Jennifer Stencel-Baerenwald, Edward Parker, Jessica M. Snyder, Dennis W. Shaw, Lakshmi Rajagopal, Raj P. Kapur, Kristina M. Adams Waldorf, Michael Gale

Oncotopix Discovery, Publicly Sharable

Background and Purpose Only limited therapeutic agents have been developed for non-alcoholic steatohepatitis (NASH). Glabridin, a promising anti-obesity candidate, has only limited druggability due to its low in vivo chemical stability and bioavailability. Therefore, we developed vutiglabridin (VUTI), which is based on a glabridin backbone, and investigated its mechanism of action in treating NASH in animal models. Experimental Approach Anti-NASH effects of VUTI were determined in in vitro fatty liver models, spheroids of primary human hepatocytes and L02 normal liver cell lines. To identify VUTI possible cellular target/s, biotin-labelled VUTI was synthesized and underwent chemical proteomic analysis. Further, the evaluation of VUTI therapeutic efficacy was carried out using an amylin-NASH and high-fat (HF) diet-induced obese (DIO) mouse models. This was carried out using transcriptomic, lipidomic and proteomic analyses of the livers from the amylin-NASH mouse model. Key Results VUTI treatment markedly reduces hepatic steatosis, fibrosis and inflammation by promoting lipid catabolism, activating autophagy and improving mitochondrial dysfunction, all of which are hallmarks of effective NASH treatment. The cellular target of VUTI was identified as paraoxonase 2 (PON2), a newly proposed protein target for the treatment of NASH, VUTI enhanced PON2 activity. The results using PON2 knockdown cells demonstrated that PON2 is important for VUTI- activation of autophagy, promoting mitochondrial function, decreasing oxidative stress and alleviating lipid accumulation under lipotoxic condition. Conclusion and Implications Our data demonstrated that VUTI is a promising therapeutic for NASH. Targeting PON2 may be important for improving liver function in various immune-metabolic diseases including NASH.

Gu-Choul Shin, | Hyeong, Min Lee, Nayeon Kim, Jihyeon Hur, Sang-Ku Yoo, | Yun, Sun Park, | Hyung, Soon Park, Dongryeol Ryu, Min-Ho Park, | Jung, Hee Park, Sang-Uk Seo, | Leo, Sungwong Choi, | Martin, Rønn Madsen, Michael Feigh, | Kwang, Pyo Kim, Kyun-Hwan Kim

Oncotopix Discovery, Publicly Sharable

Improving prognostication to direct personalised therapy remains an unmet need. This study prospectively investigated promising CT, genetic, and immunohistochemical markers to improve the prediction of colorectal cancer recurrence. This multicentre trial (ISRCTN 95037515) recruited patients with primary colorectal cancer undergoing CT staging from 13 hospitals. Follow-up identified cancer recurrence and death. A baseline model for cancer recurrence at 3 years was developed from pre-specified clinicopathological variables (age, sex, tumour-node stage, tumour size, location, extramural venous invasion, and treatment). Then, CT perfusion (blood flow, blood volume, transit time and permeability), genetic (RAS, RAF, and DNA mismatch repair), and immunohistochemical markers of angiogenesis and hypoxia (CD105, vascular endothelial growth factor, glucose transporter protein, and hypoxia-inducible factor) were added to assess whether prediction improved over tumour-node staging alone as the main outcome measure. Three hundred twenty-six of 448 participants formed the final cohort (226 male; mean 66 ± 10 years. 227 (70%) had ≥ T3 stage cancers; 151 (46%) were node-positive; 81 (25%) developed subsequent recurrence. The sensitivity and specificity of staging alone for recurrence were 0.56 [95% CI: 0.44, 0.67] and 0.58 [0.51, 0.64], respectively. The baseline clinicopathologic model improved specificity (0.74 [0.68, 0.79], with equivalent sensitivity of 0.57 [0.45, 0.68] for high vs medium/low-risk participants. The addition of prespecified CT perfusion, genetic, and immunohistochemical markers did not improve prediction over and above the clinicopathologic model (sensitivity, 0.58–0.68; specificity, 0.75–0.76). A multivariable clinicopathological model outperformed staging in identifying patients at high risk of recurrence. Promising CT, genetic, and immunohistochemical markers investigated did not further improve prognostication in rigorous prospective evaluation. A prognostic model based on clinicopathological variables including age, sex, tumour-node stage, size, location, and extramural venous invasion better identifies colorectal cancer patients at high risk of recurrence for neoadjuvant/adjuvant therapy than stage alone.

Vicky Goh, Susan Mallett, Victor Boulter, Robert Glynne-Jones, Saif Khan, Sarah Lessels, Dominic Patel, Davide Prezzi, Manuel Rodriguez-Justo, Stuart A. Taylor, Richard Beable, Margaret Betts, David J. Breen, Ingrid Britton, John Brush, Peter Correa, Nicholas Dodds, Joanna Dunlop, Sofia Gourtsoyianni, Nyree Griffin, Antony Higginson, Andrew Lowe, Andrew Slater, Madeline Strugnell, Damian Tolan, Ian Zealley, Steve Halligan, David J. Breen, John Brush, Peter Correa, Nicholas Dodds, Nyree Griffin, Andrew Lowe, Susan Mallett, Michelle McDermaid, Amjad Mohammad, Colin Oliver, Madeline Strugnell, Stuart A. Taylor, Damian Tolan, Biju Thomas, Ian Zealley

Covid, Oncotopix Discovery, Publicly Sharable

The Omicron strains of SARS-CoV-2 pose a significant challenge to the development of effective antibody-based treatments as immune evasion has compromised most available immune therapeutics. Therefore, in the ‘arms race’ with the virus, there is a continuing need to identify new biologics for the prevention or treatment of SARS-CoV-2 infections. Here, we report the isolation of nanobodies that bind to the Omicron BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized with either the Wuhan or Beta spike proteins. The structure and binding properties of three of these nanobodies (A8, H6 and B5-5) have been characterized in detail providing insight into their binding epitopes on the Omicron spike protein. Trimeric versions of H6 and B5-5 neutralized the SARS-CoV-2 variant of concern BA.5 both in vitro and in the hamster model of COVID-19 following nasal administration. Thus, either alone or in combination could serve as starting points for the development of new anti-viral immunotherapeutics.

Katy Cornish, Jiandong Huo, Luke Jones, Parul Sharma, Joseph W. Thrush, Sahar Abdelkarim, Anja Kipar, Siva Ramadurai, Miriam Weckener, Halina Mikolajek, Sai Liu, Imogen Buckle, Eleanor Bentley, Adam Kirby, Ximeng Han, Stephen M. Laidlaw, Michelle Hill, Lauren Eyssen, Chelsea Norman, Audrey Le Bas, John Clarke, William James, James P. Stewart, Miles Carroll, James H. Naismith, Raymond J. Owens

Oncotopix Discovery, Publicly Sharable

Environmental enteric dysfunction (EED) is a subclinical enteropathy challenging to diagnose due to an overlap of tissue features with other inflammatory enteropathies. EED subjects (n=52) from Pakistan, controls (n=25), and a validation EED cohort (n=30) from Zambia were used to develop a machine-learning-based image analysis classification model. We extracted histologic feature representations from the Pakistan EED model and correlated them to transcriptomics and clinical biomarkers. In-silico metabolic network modeling was used to characterize alterations in metabolic flux between EED and controls and validated using untargeted lipidomics. Genes encoding beta-ureidopropionase, CYP4F3, and epoxide hydrolase 1 correlated to numerous tissue feature representations. Fatty acid and glycerophospholipid metabolism-related reactions showed altered flux. Increased phosphatidylcholine, lysophosphatidylcholine (LPC), and ether-linked LPCs, and decreased ester-linked LPCs were observed in the duodenal lipidome of Pakistan EED subjects, while plasma levels of glycine-conjugated bile acids were significantly increased. Together, these findings elucidate a multi-omic signature of EED.

F Zulqarnain, X Zhao, K D R Setchell, Y Sharma, P Fernandes, S Srivastava, A Shrivastava, L Ehsan, V Jain, S Raghavan, C Moskaluk, Y Haberman, L A Denson, K Mehta, N T Iqbal, N Rahman, K Sadiq, Z Ahmad, R Idress, J T Iqbal, S Ahmed, A Hotwani, F Umrani, B Amadi, P Kelly, D E Brown, S R Moore, S A Ali, S Syed

Oncotopix Discovery, Publicly Sharable

STUDY QUESTION: Are maternal levels of moderate-to-vigorous physical activity (MVPA) and sedentary time (ST) in obese pregnant women associated with placental structural adaptations for facilitating oxygen delivery to the fetus? SUMMARY ANSWER: Higher maternal MVPA and ST are associated with a higher density of villi, a proxy measure of placental surface area for oxygen delivery to the fetus, without further added placental vessels. WHAT IS KNOWN ALREADY: Physical activity during pregnancy intermittently reduces uterine blood flow, potentially limiting placental and fetal oxygen supply. The placenta can mount several adaptive responses, including enlargement of the surface area of villi and/or feto-placental vessels to accommodate fetal needs. Early research on the morphology and growth of the placenta with exercise interventions has shown inconsistencies and is lacking, particularly in non-lean pregnant women. STUDY DESIGN, SIZE, DURATION: This study is a secondary longitudinal analysis of the vitamin D and lifestyle intervention for ges-tational diabetes prevention (DALI) randomized controlled trial. The prospective study was conducted between 2012 and 2015 in nine European countries at 11 different sites. In this analysis, 92 pregnant women with a BMI ≥ 29 kg/m 2 were combined into one cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: MVPA and percentage of time spent sedentary (% ST) were measured with accel-erometers during gestation. Placental sections were immunostained for endothelial cell-specific CD34. Artificial intelligence (AI)-based stereology assessed villous density, number, and cross-sectional area of vessels on whole-slide images and in selected regions comprising peripheral villi only, where the majority of vascular adaptations occur. Expression of pro-and anti-angiogenic factors was quantified using molecular counting analysis. MAIN RESULTS AND THE ROLE OF CHANCE: In multivariable regression, higher levels of maternal MVPA (min/day) were associated with a higher density of villi in both whole-slide images (beta 0.12; 95% CI 0.05, 0.2) and selected regions (0.17; CI 0.07, 0.26). Unexpectedly, ST was also positively associated with density of villi (0.23; CI 0.04, 0.43). MVPA and ST were not associated with vessel count/mm 2 villous area, vessel area, or pro-and anti-angiogenic factor mRNA expression. All estimates and statistical significance of the sensitivity analyses excluding smokers, women who developed gestational diabetes or pre-eclampsia and/or pregnancy-induced hypertension were similar in the main analysis. LIMITATIONS, REASONS FOR CAUTION: The placenta is a complex organ undergoing dynamic changes. While various adjustments were made to account for different maternal contributing factors, in addition to the outcome measures, various other factors could impact oxygen delivery to the fetus. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, we evaluated the association between placental structures quantified using an AI-based approach with objectively measured physical activity and ST at multiple time points in pregnant women with obesity. The observed adaptations contribute to the advancement of our understanding of the hemodynamics and adaptations of the placental unit in response to MVPA and ST. However, our results might not be generalizable to lean pregnant women.

Saghi Zafaranieh, Monika Siwetz, Barbara Leopold-Posch, Daniel Kummer, Berthold Huppertz, Gernot Desoye, Mireille van Poppel, DALI Core Investigator Group, Gernot Desoye, David Simmons, Rosa Corcoy, Juan M Adelantado Perez, Alexandra Kautzky-Willer, Jürgen Harreiter, Peter Damm, Elizabeth Mathiesen, Dorte M Jensen, Lise Lotte T Andersen, Fidelma Dunne, Annunziata Lapolla, Maria G Dalfra, Alessandra Bertolotto, Mireille van Poppel, Judith G M Jelsma, Sander Galjaard, Ewa Wender-Oegowska, Agnieszka Zawiejska, David J Hill, Roland Devlieger, Frank J Snoek

Oncotopix Discovery, Publicly Sharable, TMA, Tissuealign

Background Conventional clinicopathological characteristics insufficiently predict prognosis in oral squamous cell carcinoma (OSCC). We aimed to assess the added predictive value of tumor microenvironment immune cell composition (TMICC) in addition to conventional clinicopathological characteristics. Methods Primary tumor samples of 290 OSCC patients were immunohistochemically stained for CD4, CD8, CD20, CD68, CD163, CD57, FoxP3 and Programmed cell Death Ligand 1. Additionally, clinicopathological characteristics were obtained from patients’ medical files. Predictive models were trained and validated by conducting Least Absolute Shrinkage and Selection Operator (LASSO) regression analyses with cross-validation. To quantify the added predictive power of TMICC within models, receiver operating characteristic (ROC) analyses were used. Results Recurrence occurred in 74 patients (25.5%). Conventional clinicopathological characteristics (tumor localization, pathological T-stage, pathological N-stage, extracapsular spread, resection margin, differentiation grade, perineural invasion, lymphovascular invasion) and treatment modality, were used to build a LASSO logistic regression-based predictive model. Addition of TMICC to the model resulted in a comparable AUC of respectively 0.79 (±0.01) and 0.76 (±0.1) in the training and test sets. The model indicated that high numbers of CD4+ T cells protected against recurrence. Lymph node metastasis, extracapsular spread, perineural invasion, positive surgical margins and reception of adjuvant treatment were associated with increased risk for recurrence. Conclusions The TMICC, specifically the number of CD4+ T cells, is an independent predictor , however, addition to conventional clinicopathological characteristics does not improve the performance of a predictive model for recurrence in OSCC.

Sangeeta K. Bisheshar, Martine F. van der Kamp, Julius de Vries, Lorian Slagter-Menkema, Ed M.D. Schuuring, Gerton A. Lunter, Gyorgy B. Halmos, Bert van der Vegt

#10113, Oncotopix Discovery, Publicly Sharable

Objective Blockade of activin 2 receptor (ACVR2) signaling has been shown to improve insulin sensitivity and aid in weight loss. Inhibition of ACVR2 signaling restores cardiac function in multiple heart failure models. However, its potential in the treatment of obesity-related cardiometabolic disease remains unknown. Here, we investigated targeting ACVR2 signaling in cardiometabolic disease manifested with metabolic dysfunction–associated steatotic liver disease (MASLD). Methods Mice were fed a high-fat, high-sugar diet combined with the administration of nitric oxide synthase inhibitor L-NAME in drinking water, which causes hypertensive stress. For the last eight weeks, the mice were treated with the soluble ACVR2B decoy receptor (sACVR2B-Fc). Results sACVR2B-Fc protected against the development of comorbidities associated with cardiometabolic disease. This was most pronounced in the liver where ACVR2 blockade attenuated the development of MASLD including cessation of pro-fibrotic activation. It also significantly reduced total plasma cholesterol levels, impeded brown adipose tissue whitening, and improved cardiac diastolic function. In vitro, ACVR2 ligands activin A, activin B and GDF11 induced profibrotic signaling and the proliferation of human cardiac fibroblasts. Conclusions Blockade of ACVR2B exerts broad beneficial effects for therapy of cardiometabolic disease. By reducing obesity, ameliorating cardiovascular deterioration and restraining MASLD, blockade of ACVR2B signaling proves a potential target in MASLD and its comorbidities.

Julia Swan, Zoltán Szabó, Juliana Peters, Outi Kummu, Anna Kemppi, Lea Rahtu-Korpela, Anja Konzack, Jukka Hakkola, Arja Pasternack, Olli Ritvos, Risto Kerkelä, Johanna Magga

Oncotopix Discovery, Publicly Sharable, TMA

RAB3B is essential for the transportation and secretion within cells. Its increased expression is linked to the development and progression of various malignancies. However, understanding of RAB3B’s involvement in carcinogenesis is mostly limited to specific cancer subtypes. Hence, exploring RAB3B's regulatory roles and molecular mechanisms through comprehensive cancer datasets might offer innovative approaches for managing clinical cancer. To examine the potential involvement of RAB3B in the development of cancer, we analyzed data from various sources including The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression Project (GTEx), cBioPortal, HPA, UALCAN, and tissue microarray (TMA). Using bioinformatics techniques, we examined the correlation between RAB3B expression and prognosis, tumor heterogeneity, methylation modifications, and immune microenvironment across different cancer types. Our findings indicate that elevated RAB3B expression can independently predict prognosis in many tumors and has moderate accuracy for diagnosing most cancers. In most cancer types, we identified RAB3B mutations that showed a significant correlation with tumor mutational burden (TMB), mutant-allele tumor heterogeneity (MATH), and microsatellite instability (MSI). Abnormal DNA methylation patterns were also observed in most cancers compared to normal tissues. Additionally, we found significant correlations between RAB3B expression, immune cell infiltration, and immune scores across various cancers. Through pan-cancer analysis, we observed significant differences in RAB3B expression levels between tumors and normal tissues, making it a potential primary factor for cancer diagnosis and prognosis. The IHC results revealed that the expression of RAB3B in six types of tumors was consistent with the results of the pan-cancer analysis of the database. Furthermore, RAB3B showed potential associations with tumor heterogeneity and immunity. Thus, RAB3B can be utilized as an auxiliary diagnostic marker for early tumor detection and a prognostic biomarker for various tumor types.

Xu-Sheng Liu, Ya-Lan Chen, Yu-Xuan Chen, Rui-Min Wu, Fan Tan, Ya-Lan Wang, Zi-Yue Liu, Yan Gao, Zhi-Jun Pei

Oncotopix Discovery, Publicly Sharable

Among the several animal models of α-synucleinopathies, the well-known viral vector-mediated delivery of wild-type or mutated (A53T) α-synuclein requires new tools to increase the lesion in mice and follow up in vivo expression. To this end, we developed a bioluminescent expression reporter of the human A53T-α-synuclein gene using the NanoLuc system into an AAV2/9, embedded or not in a fibroin solution to stabilise its expression in space and time. We first verified the expression of the fused protein in vitro on transfected cells by bioluminescence and Western blotting. Next, two groups of C57Bl6Jr mice were unilaterally injected with the AAV-NanoLuc-human-A53T-α-synuclein above the substantia nigra combined (or not) with fibroin. We first show that the in vivo cerebral bioluminescence signal was more intense in the presence of fibroin. Using immunohistochemistry, we find that the human-A53T-α-synuclein protein is more restricted to the ipsilateral side with an overall greater magnitude of the lesion when fibroin was added. However, we also detected a bioluminescence signal in peripheral organs in both conditions, confirmed by the presence of viral DNA corresponding to the injected AAV in the liver using qPCR.

Claire Mazzocco, Coralie Genevois, Qin Li, Evelyne Doudnikoff, Nathalie Dutheil, Thierry Leste-Lasserre, Marie-Laure Arotcarena, Erwan Bezard

Oncotopix Discovery, Publicly Sharable, RNAScope

Background/Objectives Median survival of pancreatic ductal adenocarcinoma (PDAC) is around eight months and new prognostic tools are needed. Circular RNAs (circRNAs) have gained interest in different types of cancer. However, only a few studies have evaluated their potential in PDAC. We aimed to identify the most differentially expressed circRNAs in PDAC compared to controls and to explore their potential as prognostic markers. Methods Using frozen specimens with PDAC and controls, we performed RNA sequencing and identified 20,440 unique circRNAs. A custom code set of capture- and reporter probes for NanoString nCounter analysis was designed to target 152 circRNAs, based on abundancy, differential expression and literature study. Expression of these 152 circRNAs was examined in 108 formalin-fixed and paraffin-embedded surgical PDAC specimens and controls. The spatial expression of one of the most promising candidates, ciRS-7 (hsa_circ_0001946), was evaluated by chromogenic in situ hybridization (CISH) of multi-punch tissue microarrays (TMAs) and digital imaging analysis. Results Based on circRNA expression profiles, we identified different PDAC subclusters. The 30 most differentially expressed circRNAs showed log2 fold changes from -3.43 to 0.94, where circNRIP1 (hsa_circ_0004771), circMBOAT2 (hsa_circ_0007334) and circRUNX1 (hsa_circ_0002360) held significant prognostic value in multivariable analysis. CiRS-7 was absent in PDAC cells but highly expressed in tumor microenvironment. Conclusions We identified several new circRNAs with biomarker potential, three of which showed an independent prognostic value in surgically treated PDAC. We also found that ciRS-7 is absent in cancer cells but abundant in tumor microenvironment and may have potential as marker of activated stroma. Keywords circRNA pancreatic ductal adenocarcinoma RNA sequencing prognosis survival

Siri Vreim Ørbeck, Theresa Jakobsen, Juan Luis García-Rodríguez, Mark Burton, Lukas Gammelgaard Rasmussen, Jesper Dupont Ewald, Claus Wilki Fristrup, Per Pfeiffer, Michael Bau Mortensen, Lasse Sommer Kristensen, Sönke Detlefsen

Oncotopix Discovery, Publicly Sharable

Background: There are limited data on the role of multigene tests and their correlation with immuno-histochemistry (IHC), especially on core biopsy. MammaTyper is a quantitative conformite Eur-opeeanne (CE) marked, National Institute for Health and Care excellence (NICE) approved, in in vitro diagnostic quantitative real-time polymerase chain reaction (RT-qPCR) test for assessment of mRNA expression of four biomarkers (ESR1, PGR, ERBB2, MKI67). Methods: We evaluated the concordance of Mamma-Typer with oestrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 by IHC on 133 core needle biopsies of breast cancer. HER2 was positive if IHC 3+ or 2+ and fluorescence in situ hybridization (FISH)-amplified. Global and hotspot Ki67 expression was analysed using a cutoff of ≥20% assessed manually and by digital image analysis. Agreements were expressed as overall percent agreement (OPA), positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa. Results: RT-qPCR results of ESR1 were highly con-cordant with IHC with OPA of 94.7% using 1% cutoff and 91.7% when the low ER-positive category was included. The PPA and NPA between RT-qPCR and IHC for PR was 91.5% and 88.0%, respectively, when using the 1% cutoff. For ERBB2/HER2, the OPA was 95% and the PPA was 84.6%. 40 of 72 HER2 IHC score 0 tumours were classified as ERBB2 low. Best concordance between MKI67 by MammaTyper and Ki67 IHC was achieved using hotspot digital image analysis (OPA: 87.2%, PPA: 90.6%, NPA: 80%). Conclusion: RT-qPCR-based assessment of the mRNA expression of ESR1, PGR, ERBB2, and MKI67 showed high concordance with IHC, suggesting that the Mam-maTyper test on core needle biopsies represents a reliable, efficient, and reproducible alternative for breast cancer classification and refining HER2 low categorisation. aJoint senior authors. Abbreviations: CI, confidence interval; CISH, chromogenic in situ hybridisation; ER, oestrogen receptor alpha protein; ERBB2, ERB-B2 tyrosine kinase 2/human epidermal growth factor receptor 2 gene; ESR1, oestrogen receptor 1 gene; FFPE, formalin-fixed paraffin-embedded; FISH, fluorescent in situ hybridisation; HER2, ERB-B2 tyrosine kinase 2/human epidermal growth factor receptor 2 protein; IHC, immunohistochemistry; Ki67, marker of proliferation Ki67 protein; MKI67, marker of proliferation Ki67 gene; MT, MammaTyper; PA, percent agreement; PGR, progesterone receptor gene; PR, progesterone receptor protein.

Nahla M Badr, Mohamed Zaakouk, Qi Zhang, Daniel Kearns, Anthony Kong, Abeer M Shaaban, Badr N M, Shaaban A M

Oncotopix Discovery, Publicly Sharable

The acute exudative phase of acute respiratory distress syndrome (ARDS), a severe form of respiratory failure, is characterized by alveolar damage, pulmonary oedema, and an exacerbated inflammatory response. There is no effective treatment for this condition, but based on the major contribution of inflammation, anti‐inflammatory strategies have been evaluated in animal models and clinical trials, with conflicting results. In COVID‐19 ARDS patients, interleukin (IL)‐1 and IL‐6 receptor antagonists (IL‐1Ra and IL‐6Ra, kineret and tocilizumab, respectively) have shown some efficacy. Moreover, we have previously developed novel peptides modulating IL‐1R and IL‐6R activity (rytvela and HSJ633, respectively) while preserving immune vigilance and cytoprotective pathways. We aimed to assess the efficacy of these novel IL‐1Ra and IL‐6Ra, compared to commercially available drugs (kineret, tocilizumab) during the exudative phase (day 7) of bleomycin‐induced acute lung injury (ALI) in mice. Our results first showed that none of the IL‐1Ra and IL‐6Ra compounds attenuated bleomycin‐induced weight loss and venous increase. Histological analyses and lung water content measurements also showed that these drugs did not improve lung injury scores or pulmonary oedema, after the bleomycin challenge. Finally, IL‐1Ra and IL‐6Ra failed to alleviate the inflammatory status of the mice, as indicated by cytokine levels and alveolar neutrophil infiltration. Altogether, these results indicate a lack of beneficial effects of IL‐1R and IL‐6R antagonists on key parameters of ALI in the bleomycin mouse model.

Émilie Meunier, Mélissa Aubin Vega, Damien Adam, Anik Privé, Mohammad Ali, Mohammad Nezhady, Isabelle Lahaie, Christiane Quiniou, Sylvain Chemtob, Emmanuelle Brochiero

Oncotopix Discovery, Publicly Sharable, Stereology

Pre-injured lungs are prone to injury progression in response to mechanical ventilation. Heterogeneous ventilation due to (micro)atelectases imparts injurious strains on open alveoli (known as volutrauma). Hence, recruitment of (micro)atelectases by positive end-expiratory pressure (PEEP) is necessary to interrupt this vicious circle of injury but needs to be balanced against acinar overdistension. In this study, the lung-protective potential of alveolar recruitment was investigated and balanced against overdistension in pre-injured lungs. Mice, treated with empty vector (AdCl) or adenoviral active TGF-β1 (AdTGF-β1) were subjected to lung mechanical measurements during descending PEEP ventilation from 12 to 0 cmH2O. At each PEEP level, recruitability tests consisting of two recruitment maneuvers followed by repetitive forced oscillation perturbations to determine tissue elastance (H) and damping (G) were performed. Finally, lungs were fixed by vascular perfusion at end-expiratory airway opening pressures (Pao) of 20, 10, 5 and 2 cmH2O after a recruitment maneuver, and processed for design-based stereology to quantify derecruitment and distension. H and G were significantly elevated in AdTGF-β1 compared to AdCl across PEEP levels. H was minimized at PEEP = 5–8 cmH2O and increased at lower and higher PEEP in both groups. These findings correlated with increasing septal wall folding (= derecruitment) and reduced density of alveolar number and surface area (= distension), respectively. In AdTGF-β1 exposed mice, 27% of alveoli remained derecruited at Pao = 20 cmH2O. A further decrease in Pao down to 2 cmH2O showed derecruitment of an additional 1.1 million alveoli (48%), which was linked with an increase in alveolar size heterogeneity at Pao = 2–5 cmH2O. In AdCl, decreased Pao resulted in septal folding with virtually no alveolar collapse. In essence, in healthy mice alveoli do not derecruit at low PEEP ventilation. The potential of alveolar recruitability in AdTGF-β1 exposed mice is high. H is optimized at PEEP 5–8 cmH2O. Lower PEEP folds and larger PEEP stretches septa which results in higher H and is more pronounced in AdTGF-β1 than in AdCl. The increased alveolar size heterogeneity at Pao = 5 cmH2O argues for the use of PEEP = 8 cmH2O for lung protective mechanical ventilation in this animal model.

Franziska Roeder, Tina Röpke, Lara-Kristin Steinmetz, Martin Kolb, Ulrich A. Maus, Bradford J. Smith, Lars Knudsen

Oncotopix Discovery, Phenoplex, Publicly Sharable

Focal cortical dysplasia (FCD) represents a group of diverse localized cortical lesions that are highly epileptogenic and occur due to abnormal brain development caused by genetic mutations, involving the mammalian target of rapamycin (mTOR). These somatic mutations lead to mosaicism in the affected brain, posing challenges to unravel the direct and indirect functional consequences of these mutations. To comprehensively characterize the impact of mTOR mutations on the brain, we employed here a multimodal approach in a preclinical mouse model of FCD type II (Rheb), focusing on spatial omics techniques to define the proteomic and lipidomic changes. Mass Spectrometry Imaging (MSI) combined with fluorescence imaging and label free proteomics, revealed insight into the brain's lipidome and proteome within the FCD type II affected region in the mouse model. MSI visualized disrupted neuronal migration and differential lipid distribution including a reduction in sulfatides in the FCD type II-affected region, which play a role in brain myelination. MSI-guided laser capture microdissection was conducted on FCD type II and control regions, followed by label free proteomics, revealing changes in myelination pathways by oligodendrocytes. Surgical resections of FCD type IIb and postmortem human cortex were analyzed by bulk transcriptomics to unravel the interplay between genetic mutations and molecular changes in FCD type II. Our comparative analysis of protein pathways and enriched Gene Ontology pathways related to myelination in the FCD type II-affected mouse model and human FCD type IIb transcriptomics highlights the animal model's translational value. This dual approach, including mouse model proteomics and human transcriptomics strengthens our understanding of the functional consequences arising from somatic mutations in FCD type II, as well as the identification of pathways that may be used as therapeutic strategies in the future. Focal cortical dysplasia Mass spectrometry imaging Proteomics Transcriptomics Lipidomics Spatial omics

Isabeau Vermeulen, Natalia Rodriguez-Alvarez, Liesbeth François, Delphine Viot, Fariba Poosti, Eleonora Aronica, Stefanie Dedeurwaerdere, Patrick Barton, Berta Cillero-Pastor, Ron M.A. Heeren

Oncotopix Discovery, Publicly Sharable

Background Tralokinumab is a monoclonal antibody that specifically neutralizes interleukin (IL)-13, a key driver of skin inflammation and barrier abnormalities in atopic dermatitis (AD). This study evaluated early and 2-year impacts of IL-13 neutralization on skin and serum biomarkers following tralokinumab treatment in adults with moderate-to-severe AD. Methods Skin biopsies and blood samples were evaluated from a subset of patients enrolled in the Phase 3 ECZTRA 1 (NCT03131648) and the long-term extension ECZTEND (NCT03587805) trials. Gene expression was assessed by RNA sequencing; protein expression was assessed by immunohistochemistry and immunoassay. Results Tralokinumab improved the transcriptomic profile of lesional skin by Week 4. Mean improvements in the expression of genes dysregulated in AD were 39% at Week 16 and 85% at 2 years with tralokinumab, with 15% worsening at Week 16 with placebo. At Week 16, tralokinumab significantly decreased type 2 serum biomarkers (CCL17/TARC, periostin, and IgE), reduced epidermal thickness versus placebo, and increased loricrin coverage versus baseline. Two years of tralokinumab treatment significantly reduced expression of genes in the Th2 (IL4R, IL31, CCL17, and CCL26), Th1 (IFNG), and Th17/Th22 (IL22, S100A7, S100A8, and S100A9) pathways as well as increased expression of epidermal differentiation and barrier genes (CLDN1 and LOR). Tralokinumab also shifted atherosclerosis signaling pathway genes (SELE, IL-37, and S100A8) toward non-lesional expression. Conclusion Tralokinumab treatment improved epidermal pathology, reduced systemic markers of type 2 inflammation, and shifted expression of key AD biomarkers in skin towards non-lesional levels, further highlighting the key role of IL-13 in the pathogenesis of AD.

Emma Guttman-Yassky, Kenji Kabashima, Delphine Staumont-Salle, | Walter, K Nahm, Sylvia Pauser, Joel Correa, Da Rosa, | Britta, Cathrina Martel, | Daniel, Elenius Madsen, Mads Røpke, Petra Arlert, Louise Steffensen, Andrew Blauvelt, Kristian Reich

Oncotopix Discovery, Publicly Sharable, Tissuealign

Cancer remains one of the leading causes of mortality worldwide, leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade, CD103+ T cells have been associated with better clinical prognosis in patients with cancer. However, the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here, we show an unexpected and transient CD61 expression, which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling, improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically, the presence of CD61+ tumor-infiltrating T lymphocytes is associated with improved clinical outcomes, mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion, this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells, which potentiates a new target for immune-based cellular therapies. CD103+ T cells are associated with control over tumors but how this is mediated is unclear. Here the authors show that CD61 colocalizes and functionally combines with CD103 in the T cell synaptic response to promote antitumor T cell responses.

Megat H. B. A. Hamid, Pablo F. Cespedes, Chen Jin, Ji-Li Chen, Uzi Gileadi, Elie Antoun, Zhu Liang, Fei Gao, Renuka Teague, Nikita Manoharan, David Maldonado-Perez, Nasullah Khalid-Alham, Lucia Cerundolo, Raul Ciaoca, Svenja S. Hester, Adán Pinto-Fernández, Simeon D. Draganov, Iolanda Vendrell, Guihai Liu, Xuan Yao, Audun Kvalvaag, Delaney C. C. Dominey-Foy, Charunya Nanayakkara, Nikolaos Kanellakis, Yi-Ling Chen, Craig Waugh, Sally-Ann Clark, Kevin Clark, Paul Sopp, Najib M. Rahman, Clare Verrill, Benedikt M. Kessler, Graham Ogg, Ricardo A. Fernandes, Roman Fisher, Yanchun Peng, Michael L. Dustin, Tao Dong

Oncotopix Discovery, Publicly Sharable

Objectives To compare focus score (FS) and other histopathological features between paired labial and parotid salivary gland biopsies in a diagnostic cohort of suspected Sjögren’s disease (SjD) patients. Methods Labial and parotid salivary gland biopsies were simultaneously obtained from patients with sicca complaints, suspected of having SjD. Biopsies were formalin fixed and paraffin embedded. Sections were stained with haematoxylin & eosin (H&E) and for CD3, CD20, CD45, cytokeratin, CD21, Bcl6, activation induced deaminase (AID), and IgA/IgG. FS and other histopathological features characteristic for SjD were analysed. Results Based on the expert opinion of three experienced rheumatologists, 36 patients were diagnosed as SjD and 63 as non-SjD sicca patients. When taking all patients together, absolute agreement of various histopathological features between labial and parotid biopsies was high and varied between 80% (FS) and 93% ((pre-)lymphoepithelial lesions (LELs)). More labial gland biopsies had a FS ≥ 1 compared with their parotid counterpart. Accordingly, the area of infiltrate was larger in labial gland biopsies. When considering only SjD patients, labial glands contained significantly less B-lymphocytes, GCs/mm2 and less severe LELs compared with parotid glands. Conclusion Labial and parotid glands from SjD patients contain similar histopathological key features, and thus both glands can be used for diagnosis and classification of SjD. However, parotid salivary glands reveal more evident B-lymphocyte related features, while labial glands exhibit more inflammation, which may be partially unrelated to SjD. Sjögren’s disease, histopathology, gland biopsy, sicca, labial gland, parotid salivary gland Topic: biopsy sjogren's syndrome lip parotid gland salivary glands histopathology tests salivary gland biopsy

Uzma Nakshbandi, Martha S Van Ginkel, Gwenny M P J Verstappen, Fred K L Spijkervet, Suzanne Arends, Erlin A Haacke, Silvia C Liefers, Arjan Vissink, Hendrika Bootsma, Frans G M Kroese, Bert Van Der Vegt, U Nakshbandi

Oncotopix Discovery, Publicly Sharable

Objective The Visiopharm artificial intelligence (AI) algorithm for oestrogen receptor (ER) immunohistochemistry (IHC) in whole slide images (WSIs) has been successfully validated in surgical pathology. This study aimed to assess its efficacy in cytology specimens. Methods The study cohort comprised 105 consecutive cytology specimens with metastatic breast carcinoma. ER IHC WSIs were seamlessly integrated into the Visiopharm platform from the Image Management System (IMS) during our routine digital workflow, and an AI algorithm was employed for analysis. ER AI scores were compared with pathologists' manual consensus scores. Optimization steps were implemented and evaluated to reduce discordance. Results The overall concordance between pathologists' scores and AI scores was excellent (99/105, 94.3%). Six cases exhibited discordant results, including two false-negative (FN) cases due to abundant histiocytes incorrectly counted as negatively stained tumour cells by AI, two FN cases owing to weak staining, and two false-positive (FP) cases where pigmented macrophages were erroneously counted as positively stained tumour cells by AI. The Pearson correlation coefficient of ER-positive percentages between pathologists' and AI scores was 0.8483. Optimization steps, such as lowering the cut-off threshold and additional training using higher input magnification, significantly improved accuracy. Conclusions The automated ER AI algorithm demonstrated excellent concordance with pathologists' assessments and accurately differentiated ER-positive from ER-negative metastatic breast carcinoma cytology cases. However, precision in identifying tumour cells in cytology specimens requires further enhancement.

Brenna C. Li, Scott Hammond, Anil V. Parwani, Rulong Shen

Oncotopix Discovery, Publicly Sharable

Chronic watery diarrhea is a frequent symptom. In approximately 10% of the patients, a diagnosis of microscopic colitis (MC) is established. The diagnosis relies on specific, but sometimes subtle, histopathological findings. As the histology of normal intestinal mucosa vary, discriminating subtle features of MC from normal tissue can be challenging and therefore auxiliary stainings are increasingly used. The aim of this study was to determine the variance in number of intraepithelial lymphocytes (IELs) and presence of a subepithelial band in normal ileum and colonic mucosa, according to different stains and digital assessment. Sixty-one patients without diarrhea referred to screening colonoscopy due to a positive feacal blood test and presenting with endoscopically normal mucosa were included. Basic histological features, number of IELs, and thickness of a subepithelial band was manually evaluated and a deep learning-based algorithm was developed to digitally determine the number of IELs in each of the two compartments; surface epithelium and cryptal epithelium, and the density of lymphocytes in the lamina propria compartment. The number of IELs was significantly higher on CD3-stained slides compared with slides stained with Hematoxylin-and-Eosin (HE) (p<0.001), and even higher numbers were reached using digital analysis. No significant difference between right and left colon in IELs or density of CD3-positive lymphocytes in lamina propria was found. No subepithelial band was present in HE-stained slides while a thin band was visualized on special stains. Conclusively, in this cohort of prospectively collected ileum and colonic biopsies from asymptomatic patients, the range of IELs and detection of a subepithelial collagenous band varied depending on the stain and method used for assessment. As assessment of biopsies from patients with diarrhea constitute a considerable workload in the pathology departments digital image analysis is highly desired. Knowledge provided by the present study highlight important differences that should be considered before introducing this method in the clinic.

Anne-Marie Kanstrup Fiehn, Peter Johan Heiberg Engel, Ulla Engel, Dea Natalie Munch Jepsen, Thomas Blixt, Julie Rasmussen, Signe Wildt, Wojciech Cebula, Andreea-Raluca Diac, Lars Kristian Munck

Oncotopix Discovery, Publicly Sharable

1 Macrophage and osteoclast proliferation, differentiation and survival are regulated by colony-2 stimulating factor-1 receptor (CSF1R) signaling. Osteopetrosis associated with Csf1 and Csf1r 3 mutations has been attributed to the loss of osteoclasts and deficiency in bone resorption. Here we 4 demonstrate that homozygous Csf1r mutation in rat leads to delayed postnatal skeletal ossification 5 associated with substantial loss of osteal macrophages (osteomacs) in addition to osteoclasts. 6 Osteosclerosis and site-specific skeletal abnormalities were reversed by intraperitoneal transfer of 7 wild-type bone marrow cells (BMT) at weaning. Following BMT, IBA1 + macrophages were 8 detected before TRAP + osteoclasts at sites of ossification restoration. These observations extend 9 evidence that osteomacs independently contribute to bone anabolism and are required for normal 10 postnatal bone growth and morphogenesis. 11 12

Lena Batoon, Sahar Keshvari, Katharine M Irvine, Eileen Ho, Melanie Caruso, Anuj Sehgal, Susan M Millard, David A Hume, Allison R Pettit

Oncotopix Discovery, Publicly Sharable

Article Interrogation of endothelial and mural cells in brain metastasis reveals key immune-regulatory mechanisms Graphical abstract Highlights d We performed single-cell and bulk RNA-seq of human BrM and healthy brain vascular cells d BrMs are defined by pathological endothelial and mural cell subtypes d We created a preclinical platform by integrating human data with mouse BrM models d This interspecies analysis revealed CD276 as a potential therapeutic target for BrM The brain tumor-associated vasculature is a key component of the tumor microenvironment in brain metastasis, protecting cancer cells from immune attack and interfering with the delivery of therapeutic agents into the brain. In this study, Bejarano et al. unravel the vascular heterogeneity in human BrM and further construct a preclinical platform to design novel vascular-targeted therapeutic strategies for the treatment of these aggressive tumors. SUMMARY Brain metastasis (BrM) is a common malignancy, predominantly originating from lung, melanoma, and breast cancers. The vasculature is a key component of the BrM tumor microenvironment with critical roles in regulating metastatic seeding and progression. However, the heterogeneity of the major BrM vascular components, namely endothelial and mural cells, is still poorly understood. We perform single-cell and bulk RNA-sequencing of sorted vascular cell types and detect multiple subtypes enriched specifically in BrM compared to non-tumor brain, including previously unrecognized immune regulatory subtypes. We integrate the human data with mouse models, creating a platform to interrogate vascular targets for the treatment of BrM. We find that the CD276 immune checkpoint molecule is significantly upregulated in the BrM vasculature, and anti-CD276 blocking antibodies prolonged survival in preclinical trials. This study provides important insights into the complex interactions between the vasculature, immune cells, and cancer cells, with translational relevance for designing therapeutic interventions.

Leire Bejarano, Annamaria Kauzlaric, Eleni Lamprou, Roy T Daniel, Monika E Hegi, Johanna A Joyce Correspondence, Joao Lourenco, Nadine Fournier, Michelle Ballabio, Roberto Colotti, Roeltje Maas, Sabine Galland, Matteo Massara, Klara Soukup, Johanna Lilja, Jean-Philippe Brouland, Andreas F Hottinger, Johanna A Joyce

Oncotopix Discovery, Publicly Sharable, RNAScope, TMA

Cyclophilin B (CypB), encoded by peptidylprolyl isomerase B (PPIB), is involved in cellular transcriptional regulation, immune responses, chemotaxis, and proliferation. Recent studies have shown that PPIB/CypB is associated with tumor progression and chemoresistance in various cancers. However, the clinicopathologic significance and mechanism of action of PPIB/CypB in non-small cell lung cancer (NSCLC) remain unclear.

Ilseon Hwang, Joon Seon Song, Eunho Cho, Kwon-Ho Song, Sang Hyun Ra, Chang-Min Choi, Tae Woo Kim, Sung-Han Kim, Jeong Won Kim, Joon-Yong Chung

Oncotopix Discovery, Publicly Sharable

The global incidence of human papillomavirus (HPV) associated head and neck carcinoma is on the rise, in response to this a tetravalent therapeutic vaccine named Qβ-HPVag was developed. This vaccine, utilizing virus-like particles (VLPs) loaded with toll-like receptor ligands and chemically coupled to four HPV16-derived peptides, demonstrated strong anti-tumor effects in a murine head and neck cancer model. Qβ-HPVag impeded tumor progression, increased infiltration of HPV-specific T cells, and significantly improved survival. The vaccine`s efficacy was associated with immune repolarization in the tumor microenvironment, characterized by expanded activated dendritic cell subsets (cDC1, cDC2, DC3). Notably, mice responding to treatment exhibited a higher percentage of migratory DC3 cells expressing CCR7. These findings suggest promising prospects for optimized VLP-based vaccines in treating HPV-associated head and neck cancer.

Romano Josi, Daniel E. Speiser, Simone de Brot, Anne Cathrine Vogt, Eva M. Sevick-Muraca, Genrich V. Tolstonog, Martin F. Bachmann, Mona O. Mohsen

Oncotopix Discovery, Publicly Sharable

Chimeric antigen receptor T cell (CAR-T) therapy is an emerging strategy to improve treatment outcomes for recurrent high-grade glioma, a cancer that responds poorly to current therapies. Here we report a completed phase I trial evaluating IL-13Rα2-targeted CAR-T cells in 65 patients with recurrent high-grade glioma, the majority being recurrent glioblastoma (rGBM). Primary objectives were safety and feasibility, maximum tolerated dose/maximum feasible dose and a recommended phase 2 dose plan. Secondary objectives included overall survival, disease response, cytokine dynamics and tumor immune contexture biomarkers. This trial evolved to evaluate three routes of locoregional T cell administration (intratumoral (ICT), intraventricular (ICV) and dual ICT/ICV) and two manufacturing platforms, culminating in arm 5, which utilized dual ICT/ICV delivery and an optimized manufacturing process. Locoregional CAR-T cell administration was feasible and well tolerated, and as there were no dose-limiting toxicities across all arms, a maximum tolerated dose was not determined. Probable treatment-related grade 3+ toxicities were one grade 3 encephalopathy and one grade 3 ataxia. A clinical maximum feasible dose of 200 × 106 CAR-T cells per infusion cycle was achieved for arm 5; however, other arms either did not test or achieve this dose due to manufacturing feasibility. A recommended phase 2 dose will be refined in future studies based on data from this trial. Stable disease or better was achieved in 50% (29/58) of patients, with two partial responses, one complete response and a second complete response after additional CAR-T cycles off protocol. For rGBM, median overall survival for all patients was 7.7 months and for arm 5 was 10.2 months. Central nervous system increases in inflammatory cytokines, including IFNγ, CXCL9 and CXCL10, were associated with CAR-T cell administration and bioactivity. Pretreatment intratumoral CD3 T cell levels were positively associated with survival. These findings demonstrate that locoregional IL-13Rα2-targeted CAR-T therapy is safe with promising clinical activity in a subset of patients. ClinicalTrials.gov Identifier: NCT02208362 . In a large trial of patients with recurrent high-grade gliomas, IL-13Rα2-targeting CAR-T cells were feasible to manufacture and well tolerated when delivered via intratumoral and/or intraventricular routes.

Christine E. Brown, Jonathan C. Hibbard, Darya Alizadeh, M. Suzette Blanchard, Heini M. Natri, Dongrui Wang, Julie R. Ostberg, Brenda Aguilar, Jamie R. Wagner, Jinny A. Paul, Renate Starr, Robyn A. Wong, Wuyang Chen, Noah Shulkin, Maryam Aftabizadeh, Aleksandr Filippov, Ammar Chaudhry, Julie A. Ressler, Julie Kilpatrick, Paige Myers-McNamara, Mike Chen, Leo D. Wang, Russell C. Rockne, Joseph Georges, Jana Portnow, Michael E. Barish, Massimo D’Apuzzo, Nicholas E. Banovich, Stephen J. Forman, Behnam Badie

Oncotopix Discovery, Publicly Sharable

Objective To assess the expression pattern of X-linked inhibitor of apoptosis protein (XIAP), a cellular stress sensor, and delineate the associated changes in the tumor immune microenvironment (TiME) for prognostic value and new therapeutic targets in inflammatory breast cancer (IBC). Methods Immunohistochemistry was conducted to assess the spatial localization of immune subsets, XIAP, and PDL1 expression in IBC and non-inflammatory breast cancer (nIBC) pretreatment tumors (n = 142). Validation and further exploration were performed by gene expression analysis of patient tumors along with signaling studies in a co-culture model. Results High XIAP in 37/81 IBC patients correlated significantly with high PD-L1, increased infiltration of FOXP3+ Tregs, CD163+ tumor-associated macrophages (TAMs), low CD8/CD163 ratio in both tumor stroma (TS) and invasive margins (IM), and higher CD8+ T cells and CD79α+ B cells in the IM. Gene set enrichment analysis identified cellular stress response- and inflammation-related genes along with tumor necrosis factor receptor 1 (TNFR1) expression in high-XIAP IBC tumors. Induction of TNFR1 and XIAP was observed when patient-derived SUM149 IBC cells were co-cultured with human macrophage-conditioned media simulating TAMs, further demonstrating that the TNF-α signaling pathway is a likely candidate governing TAM-induced XIAP overexpression in IBC cells. Finally, addition of Birinapant, a pan IAP antagonist, induced cell death in the pro-survival cytokine-enriched conditions. Conclusion Using immunophenotyping and gene expression analysis in patient biospecimens along with in silico modeling and a preclinical model with a pan-IAP antagonist, this study revealed an interplay between increased TAMs, TNF-α signaling, and XIAP activation during (immune) stress in IBC. These data demonstrate the potential of IAP antagonists as immunomodulators for improving IBC therapeutic regimens.

Christophe Van Berckelaer, Steven Van Laere, Seayoung Lee, Michael A Morse, Joseph Geradts, Luc Dirix, Mark Kockx, François Bertucci, Peter Van Dam, Gayathri R Devi

Oncotopix Discovery, Publicly Sharable

Previously we reported that a novel αvβ6-specific peptide-drug conjugate (SG3299) could eliminate established human pancreatic ductal adenocarcinoma (PDAC) xenografts. However the development of effective therapies for PDAC, which is an essential need, must show efficacy in relevant immunocompetent animals. Previously we reported that the KPC mouse transgenic PDAC model that closely recapitulates most stages of development of human PDAC, unlike in humans, failed to express αvβ6 on their tumours or metastases. In this study we have taken the KPC-derived PDAC line TB32043 and engineered a variant line (TB32043mb6S2) that expresses mouse integrin αvβ6. We report that orthotopic implantation of the αvβ6 over-expressing TB32043mb6S2 cells promotes shorter overall survival and increase in metastases. Moreover, systemic treatment of mice with established TB32043mb6S2 tumours in the pancreas with SG2399 lived significantly longer (p < 0.001; mean OS 48d) compared with PBS or control SG3511 (mean OS 25.5d and 26d, respectively). Thus SG3299 is confirmed as a promising candidate therapeutic for the therapy of PDAC.

Nicholas F. Brown, Elizabeth R. Murray, Lauren C. Cutmore, Philip Howard, Luke Masterson, Francesca Zammarchi, John A. Hartley, Patrick H. van Berkel, John F. Marshall

Oncotopix Discovery, Publicly Sharable

In obesity, sustained adipose tissue (AT) inflammation constitutes a cellular memory that limits the effectiveness of weight loss interventions. Yet, the impact of fasting regimens on the regulation of AT immune infiltration is still elusive. Here we show that intermittent fasting (IF) exacerbates the lipid-associated macrophage (LAM) inflammatory phenotype of visceral AT in obese mice. Importantly, this increase in LAM abundance is strongly p53 dependent and partly mediated by p53-driven adipocyte apoptosis. Adipocyte-specific deletion of p53 prevents LAM accumulation during IF, increases the catabolic state of adipocytes, and enhances systemic metabolic flexibility and insulin sensitivity. Finally, in cohorts of obese/diabetic patients, we describe a p53 polymorphism that links to efficacy of a fasting-mimicking diet and that the expression of p53 and TREM2 in AT negatively correlates with maintaining weight loss after bariatric surgery. Overall, our results demonstrate that p53 signalling in adipocytes dictates LAM accumulation in AT under IF and modulates fasting effectiveness in mice and humans. Adipose tissue (AT) inflammation is strongly associated with obesity and constitutes an obesogenic memory upon weight loss. Here, the authors show that intermittent fasting leads to an adipocyte p53-signaling dependent emergence of lipid-associated macrophages in visceral AT of obese mice which limits the systemic fasting response.

Isabel Reinisch, Helene Michenthaler, Alba Sulaj, Elisabeth Moyschewitz, Jelena Krstic, Markus Galhuber, Ruonan Xu, Zina Riahi, Tongtong Wang, Nemanja Vujic, Melina Amor, Riccardo Zenezini Chiozzi, Martin Wabitsch, Dagmar Kolb, Anastasia Georgiadi, Lisa Glawitsch, Ellen Heitzer, Tim J. Schulz, Michael Schupp, Wenfei Sun, Hua Dong, Adhideb Ghosh, Anne Hoffmann, Dagmar Kratky, Laura C. Hinte, Ferdinand von Meyenn, Albert J. R. Heck, Matthias Blüher, Stephan Herzig, Christian Wolfrum, Andreas Prokesch

Oncotopix Discovery, Publicly Sharable

PDE6H encodes PDE6γ′, the inhibitory subunit of the cGMP-specific phosphodiesterase 6 in cone photoreceptors. Inhibition of PDE6, which has been widely studied for its role in light transduction, increases cGMP levels. The purpose of this study is to characterise the role of PDE6H in cancer cell growth. From an siRNA screen for 487 genes involved in metabolism, PDE6H was identified as a controller of cell cycle progression in HCT116 cells. Role of PDE6H in cancer cell growth and metabolism was studied through the effects of its depletion on levels of cell cycle controllers, mTOR effectors, metabolite levels, and metabolic energy assays. Effect of PDE6H deletion on tumour growth was also studied in a xenograft model. PDE6H knockout resulted in an increase of intracellular cGMP levels, as well as changes to the levels of nucleotides and key energy metabolism intermediates. PDE6H knockdown induced G1 cell cycle arrest and cell death and reduced mTORC1 signalling in cancer cell lines. Both knockdown and knockout of PDE6H resulted in the suppression of mitochondrial function. HCT116 xenografts revealed that PDE6H deletion, as well as treatment with the PDE5/6 inhibitor sildenafil, slowed down tumour growth and improved survival, while sildenafil treatment did not have an additive effect on slowing the growth of PDE6γ′-deficient tumours. Our results indicate that the changes in cGMP and purine pools, as well as mitochondrial function which is observed upon PDE6γ′ depletion, are independent of the PKG pathway. We show that in HCT116, PDE6H deletion replicates many effects of the dark retina response and identify PDE6H as a new target in preventing cancer cell proliferation and tumour growth.

Ceren Yalaz, Esther Bridges, Nasullah K. Alham, Christos E. Zois, Jianzhou Chen, Karim Bensaad, Ana Miar, Elisabete Pires, Ruth J. Muschel, James S. O. McCullagh, Adrian L. Harris

Oncotopix Discovery, Publicly Sharable

Background: In metastatic breast cancer (MBC), [18F]fluorodeoxyglucose positron emission tomography/computed tomography ([18F]FDG-PET/CT) can be used for staging. We evaluated the correlation between BC histopathological characteristics and [18F]FDG uptake in corresponding metastases. Patients and Methods: Patients with non-rapidly progressive MBC of all subtypes prospectively underwent a baseline histological metastasis biopsy and [18F]FDG-PET. Biopsies were assessed for estrogen, progesterone, and human epidermal growth factor receptor 2 (ER, PR, HER2); Ki-67; and histological subtype. [18F]FDG uptake was expressed as maximum standardized uptake value (SUVmax) and results were expressed as geometric means. Results: Of 200 patients, 188 had evaluable metastasis biopsies, and 182 of these contained tumor. HER2 positivity and Ki-67 ≥ 20% were correlated with higher [18F]FDG uptake (estimated geometric mean SUVmax 10.0 and 8.8, respectively; p = 0.0064 and p = 0.014). [18F]FDG uptake was lowest in ER-positive/HER2-negative BC and highest in HER2-positive BC (geometric mean SUVmax 6.8 and 10.0, respectively; p = 0.0058). Although [18F]FDG uptake was lower in invasive lobular carcinoma (n = 31) than invasive carcinoma NST (n = 146) (estimated geometric mean SUVmax 5.8 versus 7.8; p = 0.014), the metastasis detection rate was similar. Conclusions: [18F]FDG-PET is a powerful tool to detect metastases, including invasive lobular carcinoma. Although BC histopathological characteristics are related to [18F]FDG uptake, [18F]FDG-PET and biopsy remain complementary in MBC staging (NCT01957332).

Jorianne Boers, Bertha Eisses, Mieke C. Zwager, Jasper J. L. van Geel, Frederike Bensch, Erik F. J. de Vries, Geke A. P. Hospers, Andor W. J. M. Glaudemans, Adrienne H. Brouwers, Martijn A. M. den Dekker, Sjoerd G. Elias, Evelien J. M. Kuip, Carla M. L. van Herpen, Agnes Jager, Astrid A. M. van der Veldt, Daniela E. Oprea-Lager, Elisabeth G. E. de Vries, Bert van der Vegt, Willemien C. Menke-van der Houven van Oordt, Carolina P. Schröder

Oncotopix Discovery, Publicly Sharable

Introduction Accurate grading of pancreatic neuroendocrine tumors (PanNETs) relies on the assessment of Ki-67 immunohistochemistry (IHC). While digital imaging analysis (DIA) has been employed for Ki-67 IHC assessment in surgical specimens, its applicability to cytologic specimens remains underexplored. This study aimed to evaluate an automated DIA for assessing Ki-67 IHC on PanNET cell blocks. Materials and methods The study included 61 consecutive PanNETs and 5 pancreatic neuroendocrine carcinomas. Ki-67 IHC slides from cell blocks were digitally scanned into whole slide images using Philips IntelliSite Scanners and analyzed in batches using the Visiopharm Ki-67 App in a digital workflow. Ki-67 scores obtained through DIA were compared to pathologists' manual scores. Results The Pearson correlation coefficient of the percentage of Ki-67-stained nuclei between DIA reads and the originally reported reads was 0.9681. Concordance between DIA Ki-67 grades and pathologists’ Ki-67 grades was observed in 92.4% (61/66) of cases with the calculated Cohen's Kappa coefficient of 0.862 (almost perfect agreement). Discordance between DIA and pathologists’ consensus reads occurred in 5 PanNET cases which were upgraded from G1 to G2 by DIA due to contaminated Ki-67-stained inflammatory cells. Conclusions DIA demonstrated excellent concordance with pathologists’ assessments, with only minor grading discrepancies. However, the essential role of pathologists in confirming results is emphasized to enhance overall accuracy.

Nada Shaker, Rulong Shen, Abberly Lott Limbach, Swati Satturwar, Peter Kobalka, Saman Ahmadian, Shaoli Sun, Wei Chen, Giovanni Lujan, Ashwini Esnakula, Anil Parwani, Zaibo Li

Oncotopix Discovery, Publicly Sharable

Background: Hepatocellular senescence may be a causal factor in the development and progression of non-alcoholic steatohepatitis (NASH). The most effective currently available treatment for NASH is lifestyle intervention, including dietary modification. This study aimed to evaluate the effects of dietary intervention on hallmarks of NASH and molecular signatures of hepatocellular senescence in the Gubra-Amylin NASH (GAN) diet-induced obese (DIO) and biopsy-confirmed mouse model of NASH. Methods: GAN DIO-NASH mice with liver biopsy-confirmed NASH and fibrosis received dietary intervention by switching to chow feeding (chow reversal) for 8, 16 or 24 weeks. Untreated GAN DIO-NASH mice and chow-fed C57BL/6J mice served as controls. Pre-to-post liver biopsy histology was performed for within-subject evaluation of NAFLD Activity Score and fibrosis stage. Terminal endpoints included blood/liver biochemistry, quantitative liver histology, mitochondrial respiration and RNA sequencing. Results: Chow-reversal promoted substantial benefits on metabolic outcomes and liver histology, as demonstrated by robust weight loss, complete resolution of hepatomegaly, hypercholesterolemia, elevated transaminase levels and hepatic steatosis in addition to attenuation of inflammatory markers. Notably, all DIO-NASH mice demonstrated ≥ 2 point significant improvement in NAFLD Activity Score following dietary intervention. While not improving fibrosis stage, chow-reversal reduced quantitative fibrosis markers (PSR, collagen 1a1, α-SMA), concurrent with improved liver mitochondrial respiration, complete reversal of p21 overexpression, lowered γ-H2AX levels and widespread suppression of gene expression markers of hepatocellular senescence. Conclusions: Dietary intervention (chow reversal) substantially improves metabolic, biochemical and histological hallmarks of NASH and fibrosis in GAN DIO-NASH mice. These benefits were reflected by progressive clearance of senescent hepatocellular cells, making the model suitable for profiling potential senotherapeutics in preclinical drug discovery for NASH.

Mathias Flensted-Jensen, Denise Oró, Emma A. Rørbeck, Chen Zhang, Martin Rønn Madsen, Andreas Nygaard Madsen, Jenny Norlin, Michael Feigh, Steen Larsen, Henrik H. Hansen

Oncotopix Discovery, Publicly Sharable

Tyrosine kinase inhibitors (TKIs) may be combined with radiation therapy (RT) to enhance tumor control; however, increased incidences of gastrointestinal (GI) toxicity have been reported with this combination. We hypothesize that toxicity is due to compromised intestinal healing caused by inhibition of vascular repair and proliferation pathways. This study explores underlying tissue toxicity associated with abdominal RT and concurrent sunitinib in a mouse model. Four groups of CD-1 mice were treated with 12 Gy abdominal RT, oral sunitinib, abdominal RT + sunitinib, or sham treatment. Mice received oral sunitinib or the vehicle via gavage for 14 days. On day 7, mice were irradiated with 12 Gy abdominal RT or sham treated. Mice were euthanized on day 14 and intestinal tract was harvested for semiquantitative histopathologic evaluation and immunohistochemical quantification of proliferation (Ki67 Ki-67) and vascular density (CD31). Non-irradiated groups had stable weights while abdominal irradiation resulted in weight loss, with mice receiving RT + SUN having greater weight loss than mice receiving RT alone. Semiquantitative analysis showed significant increases in inflammation in irradiated groups. The difference in the density of CD31+ cells was significantly increased in RT alone compared to SUN alone. Ki67+ density was not significant. In summary, we identify a lack of angiogenic response in irradiated GI tissues when abdominal RT is combined with a TKI, which may correlate with clinical toxicities seen in canine and human patients receiving combined treatment.

Melpo Christofidou-Solomidou, Amber R Prebble, Bailey Latka, Braden Burdekin, Del Leary, Mac Harris, Daniel Regan, Mary-Keara Boss

Oncotopix Discovery, Publicly Sharable, Stereology

Children born to obese mothers are at increased risk of developing mood disorders and cognitive impairment. Experimental studies have reported structural changes in the brain such as the gliovascular unit as well as activation of neuroinflammatory cells as a part of neuroinflammation processing in aged offspring of obese mothers. However, the molecular mechanisms linking maternal obesity to poor neurodevelopmental outcomes are not well established. The ephrin system plays a major role in a variety of cellular processes including cell–cell interaction, synaptic plasticity, and long-term potentiation. Therefore, in this study we determined the impact of maternal obesity in pregnancy on cortical, hippocampal development, vasculature and ephrin-A3/EphA4-signaling, in the adult offspring in mice. Maternal obesity was induced in mice by a high fat/high sugar Western type of diet (HF/HS). We collected brain tissue (prefrontal cortex and hippocampus) from 6-month-old offspring of obese and lean (control) dams. Hippocampal volume, cortical thickness, myelination of white matter, density of astrocytes and microglia in relation to their activity were analyzed using 3-D stereological quantification. mRNA expression of ephrin-A3, EphA4 and synaptic markers were measured by qPCR in the brain tissue. Moreover, expression of gap junction protein connexin-43, lipocalin-2, and vascular CD31/Aquaporin 4 were determined in the hippocampus by immunohistochemistry. Volume of hippocampus and cortical thickness were significantly smaller, and myelination impaired, while mRNA levels of hippocampal EphA4 and post-synaptic density (PSD) 95 were significantly lower in the hippocampus in the offspring of obese dams as compared to offspring of controls. Further analysis of the hippocampal gliovascular unit indicated higher coverage of capillaries by astrocytic end-feet, expression of connexin-43 and lipocalin-2 in endothelial cells in the offspring of obese dams. In addition, offspring of obese dams demonstrated activation of microglia together with higher density of cells, while astrocyte cell density was lower. Maternal obesity affects brain size, impairs myelination, disrupts the hippocampal gliovascular unit and decreases the mRNA expression of EphA4 and PSD-95 in the hippocampus of adult offspring. These results indicate that the vasculature–glia cross-talk may be an important mediator of altered synaptic plasticity, which could be a link between maternal obesity and neurodevelopmental/neuropsychiatric disorders in the offspring.

Seyedeh Marziyeh, Jabbari Shiadeh, Fanny Goretta, Pernilla Svedin, Thomas Jansson, Carina Mallard, Maryam Ardalan

Oncotopix Discovery, Publicly Sharable

Aims Pathologists often use immunohistochemical staining of the proliferation marker Ki67 in their diagnostic assessment of melanocytic lesions. However, the interpretation of Ki67 can be challenging. We propose a new workflow to improve the diagnostic utility of the Ki67-index. In this workflow, Ki67 is combined with the melanocytic tumour-cell marker SOX10 in a Ki67/SOX10 double nuclear stain. The Ki67-index is then quantified automatically using digital image analysis (DIA). The aim of this study was to optimise and test three different multiplexing methods for Ki67/SOX10 double nuclear staining. Methods Multiplex immunofluorescence (mIF), multiplex immunohistochemistry (mIHC), and multiplexed immunohistochemical consecutive staining on single slide (MICSSS) were optimised for Ki67/SOX10 double nuclear staining. DIA applications were designed for automated quantification of the Ki67-index. The methods were tested on a pilot case-control cohort of benign and malignant melanocytic lesions (n = 23). Results Using the Ki67/SOX10 double nuclear stain, malignant melanocytic lesions could be completely distinguished from benign lesions by the Ki67-index. The Ki67-index cut-offs were 1.8% (mIF) and 1.5% (mIHC and MICSSS). The AUC of the automatically quantified Ki67-index based on double nuclear staining was 1.0 (95% CI: 1.0;1.0), whereas the AUC of conventional Ki67 single-stains was 0.87 (95% CI: 0.71;1.00). Conclusions The novel Ki67/SOX10 double nuclear stain highly improved the diagnostic precision of Ki67 interpretation. Both mIHC and mIF were useful methods for Ki67/SOX10 double nuclear staining, whereas the MICSSS method had challenges in the current setting. The Ki67/SOX10 double nuclear stain shows potential as a valuable diagnostic aid for melanocytic lesions. Previous article in issue

Mette Bak Brogård, Patricia Switten Nielsen, Kristina Bang Christensen, Jeanette Bæhr Georgsen, Anne Wandler, Johanne Lade-Keller, Torben Steiniche

Oncotopix Discovery, Publicly Sharable

The rates of alcohol use disorder among women are growing, yet little is known about how the female brain is affected by alcohol. The neuroimmune system, and specifically microglia, have been implicated in mediating alcohol neurotoxicity, but most preclinical studies have focused on males. Further, few studies have considered changes to the microglial phenotype when examining the effects of ethanol on brain structure and function. Therefore, we quantified microglial reactivity in female rats using a binge model of alcohol dependence, assessed through morphological and phenotypic marker expression, coupled with regional cytokine levels. In a time- and region-dependent manner, alcohol altered the microglial number and morphology, including the soma and process area, and the overall complexity within the corticolimbic regions examined, but no significant increases in the proinflammatory markers MHCII or CD68 were observed. The majority of cytokine and growth factor levels examined were similarly unchanged. However, the expression of the proinflammatory cytokine TNFα was increased, and the anti-inflammatory IL-10, decreased. Thus, female rats showed subtle differences in neuroimmune reactivity compared to past work in males, consistent with reports of enhanced neuroimmune responses in females across the literature. These data suggest that specific neuroimmune reactions in females may impact their susceptibility to alcohol neurotoxicity and other neurodegenerative events with microglial contributions.

Jennifer K. Melbourne, Jessica I. Wooden, Erika R. Carlson, Chinchusha Anasooya Shaji, Kimberly Nixon

NASH, Oncotopix Discovery, Publicly Sharable

Objective: The metabolic benefits of GLP-1 receptor (GLP-1R) agonists on glycemic and weight control are well established as therapy for type 2 diabetes and obesity. Glucagon's ability to increase energy expenditure is well described, and the combination of these mechanisms-of-actions has the potential to further lower hepatic steatosis in metabolic disorders and could therefore be attractive for the treatment for non-alcoholic steatohepatitis (NASH). Here, we have investigated the effects of a dual GLP-1/glucagon receptor agonist NN1177 on hepatic steatosis, fibrosis, and inflammation in a preclinical mouse model of NASH. Having observed strong effects on body weight loss in a pilot study with NN1177, we hypothesized that direct engagement of the hepatic glucagon receptor (GCGR) would result in a superior effect on steatosis and other liver related parameters as compared to the GLP-1R agonist semaglutide at equal body weight. Methods: Male C57Bl/6 mice were fed a diet high in trans-fat, fructose, and cholesterol (Diet-Induced Obese (DIO)-NASH) for 36 weeks. Following randomization based on the degree of fibrosis at baseline, mice were treated once daily with subcutaneous administration of a vehicle or three different doses of NN1177 or semaglutide for 8 weeks. Hepatic steatosis, inflammation and fibrosis were assessed by immunohistochemistry and morphometric analyses. Plasma levels of lipids and liver enzymes were determined, and hepatic gene expression was analyzed by RNA sequencing. Results: NN1177 dose-dependently reduced body weight up to 22% compared to vehicle treatment. Plasma levels of ALT, a measure of liver injury, were reduced in all treatment groups with body weight loss. The dual agonist reduced hepatic steatosis to a greater extent than semaglutide at equal body weight loss, as demonstrated by three independent methods. Both the co-agonist and semaglutide significantly decreased histological markers of inflammation such as CD11b and Galectin-3, in addition to markers of hepatic stellate activation (αSMA) and fibrosis (Collagen I). Interestingly, the maximal beneficial effects on above mentioned clinically relevant endpoints of NN1177 treatment on hepatic health appear to be achieved with the middle dose tested. Administering the highest dose resulted in a further reduction of liver fat and accompanied by a massive induction in genes involved in oxidative phosphorylation and resulted in exaggerated body weight loss and a downregulation of a module of co-expressed genes involved in steroid hormone biology, bile secretion, and retinol and linoleic acid metabolism that are also downregulated due to NASH itself. Conclusions: These results indicate that, in a setting of overnutrition, the liver health benefits of activating the fasting-related metabolic pathways controlled by the glucagon receptor displays a bell-shaped curve. This observation is of interest to the scientific community, due to the high number of ongoing clinical trials attempting to leverage the positive effects of glucagon biology to improve metabolic health.

Thomas Monfeuga, Jenny Norlin, Anne Bugge, Elisabeth D. Gaalsgaard, Cesar A. Prada-Medina, Markus Latta, Sanne S. Veidal, Pia S. Petersen, Michael Feigh, Dorte Holst

Oncotopix Discovery, Publicly Sharable, TMA

Genomic instability, as caused by oncogene-induced replication stress, can lead to the activation of inflammatory signaling, involving the cGAS-STING and JAK-STAT pathways. Inflammatory signaling has been associated with pro-tumorigenic features, but also with favorable response to treatment, including to immune checkpoint inhibition. In this study, we aim to explore relations between inflammatory signaling, markers of replication stress, and immune cell infiltration in breast cancer. Expression levels of cGAS-STING signaling components (STING, phospho-TBK1, and phospho-STAT1), replication stress markers (γH2AX and pRPA), replication stress-related proto-oncogenes (Cyclin E1 and c-Myc) and immune cell markers (CD20, CD4, and CD57) are determined immunohistochemically on primary breast cancer samples (n = 380). RNA-sequencing data from TCGA (n = 1082) and METABRIC (n = 1904) are used to calculate cGAS-STING scores. pTBK1, pSTAT1 expression and cGAS-STING pathway scores are all increased in triple-negative breast cancers compared to other subtypes. Expression of γH2AX, pRPA, Cyclin E1, c-Myc, and immune cell infiltration positively correlate with p-STAT1 expression ( P 0.001). Additionally, we observe significant positive associations between expression of pTBK1 and γH2AX, pRPA, c-Myc, and number of CD4+ cells and CD20+ cells. Also, cGAS-STING scores are correlated with genomic instability metrics, such as homologous recombination deficiency ( P  < 0.001) and tumor mutational burden ( P  < 0.01). Moreover, data from the I-SPY2 clinical trial (n = 71) confirms that higher cGAS-STING scores are observed in breast cancer patients who responded to immunotherapy combined with chemotherapy. In conclusion, the cGAS-STING pathway is highly expressed in TNBCs and is correlated with genomic instability and immune cell infiltration.

Mengting Chen, Shibo Yu, Tineke van der Sluis, Mieke C. Zwager, Carolien P. Schröder, Bert van der Vegt, Marcel A. T. M. van Vugt

Oncotopix Discovery, Publicly Sharable

Non-muscle invasive papillary urothelial carcinoma is a prevalent disease with a high recurrence tendency. Good prognostic and reproducible biomarkers for tumor recurrence and disease progression are lacking. Currently, WHO grade and tumor stage are essential in risk stratification and treatment decision-making. Here we present the prognostic value of proliferation markers (Ki67, mitotic activity index (MAI) and PPH3) together with p53, CD25 and CK20 immunohistochemistry (IHC). In this population-based retrospective study, 349 primary non-muscle invasive bladder cancers (NMIBC) were available. MAI and PPH3 were calculated manually according to highly standardized previously described methods, Ki-67 by the semi-automated QPRODIT quantification system, p53 and CD25 by the fully automated digital image analysis program Visipharm® and CK20 with the help of the semi-quantitative immunoreactive score (IRS). Survival analyses with log rank test, as well as univariate and multivariate Cox regression analyses were performed for all investigated variables. Age and multifocality were the only significant variables for tumor recurrence. All investigated variables, except gender, were significantly associated with stage progression. In multivariate analysis, MAI was the only prognostic variable for stage progression (p<0.001).

Vebjørn Kvikstad, Melinda Lillesand, Einar Gudlaugsson, Ok Målfrid Mangrud, Emma Rewcastle, Ivar Skaland, Jan P. A. Baak, Emiel A. M. Janssen

Oncotopix Discovery, Publicly Sharable

PD-1/PD-L1 play a crucial role as immune checkpoint inhibitors in various types of cancer. Although our previous study revealed that NPM1 was a novel transcriptional regulator of PD-L1 and stimulated the transcription of PD-L1, the underlying regulatory mechanism remains incompletely characterized. Various human cancer cell lines were used to validate the role of NPM1 in regulating the transcription of PD-L1. The acetyltransferase NAT10 was identified as a facilitator of NPM1 acetylation by coimmunoprecipitation and mass spectrometry. The potential application of combined NAT10 inhibitor and anti-CTLA4 treatment was evaluated by an animal model. We demonstrated that NPM1 enhanced the transcription of PD-L1 in various types of cancer, and the acetylation of NPM1 played a vital role in this process. In particular, NAT10 facilitated the acetylation of NPM1, leading to enhanced transcription and increased expression of PD-L1. Moreover, our findings demonstrated that Remodelin, a compound that inhibits NAT10, effectively reduced NPM1 acetylation, leading to a subsequent decrease in PD-L1 expression. In vivo experiments indicated that Remodelin combined with anti-CTLA-4 therapy had a superior therapeutic effect compared with either treatment alone. Ultimately, we verified that the expression of NAT10 exhibited a positive correlation with the expression of PD-L1 in various types of tumors, serving as an indicator of unfavorable prognosis. This study suggests that the NAT10/NPM1 axis is a promising therapeutic target in malignant tumors.

Ge Qin, Fan Bai, Huabin Hu, Jianwei Zhang, Weixiang Zhan, Zehua Wu, Jianxia Li, Yang Fu, Yanhong Deng

Oncotopix Discovery, Publicly Sharable, Tissuealign

Tumor cell intravasation is essential for metastatic dissemination, but its exact mechanism is incompletely understood. We have previously shown that in breast cancer, the direct and stable association of a tumor cell expressing Mena, a Tie2 hi /Vegf hi macrophage, and a vascular endothelial cell, creates an intravasation portal, called a "tumor microenvironment of metastasis" (TMEM) doorway, for tumor cell intravasation, leading to dissemination to distant sites. The density of TMEM doorways, also called TMEM doorway score, is a clinically validated prognostic marker of distant metastasis in breast cancer patients. Although we know that tumor cells utilize TMEM doorway-associated transient vascular openings to intravasate, the precise signaling mechanisms involved in TMEM doorway function are only partially understood. Using two mouse models of breast cancer and an in vitro assay of intravasation, we report that CSF-1 secreted by the TMEM doorway tumor cell stimulates local secretion of VEGF-A from the Tie2 hi TMEM doorway macrophage, leading to the dissociation of endothelial junctions between TMEM doorway associated endothelial cells, supporting tumor cell intravasation. Acute blockage of CSF-1R signaling decreases macrophage VEGF secretion as well as TMEM doorway-associated vascular opening, tumor cell trans-endothelial migration, and dissemination. These new insights into signaling events regulating TMEM doorway function should be explored further as treatment strategies for metastatic disease.

Chinmay R Surve, Camille L Duran, Xianjun Ye, Xiaoming Chen, Yu Lin, Allison S Harney, Yarong Wang, Ved P Sharma, E Richard Stanley, John C Mcauliffe, David Entenberg, Maja H Oktay, John S Condeelis

Oncotopix Discovery, Publicly Sharable

Summary

Background

X-linked myotubular myopathy (XLMTM) is a rare, life-threatening congenital muscle disease caused by mutations in the MTM1 gene that result in profound muscle weakness, significant respiratory insufficiency, and high infant mortality. There is no approved disease-modifying therapy for XLMTM. Resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) is an investigational adeno-associated virus (AAV8)-mediated gene replacement therapy designed to deliver MTM1 to skeletal muscle cells and achieve long-term correction of XLMTM-related muscle pathology. The clinical trial ASPIRO (NCT03199469) investigating resamirigene bilparvovec in XLMTM is currently paused while the risk:benefit balance associated with this gene therapy is further investigated.

Methods

Muscle biopsies were taken before treatment and 24 and 48 weeks after treatment from ten boys with XLMTM in a clinical trial of resamirigene bilparvovec (ASPIRO; NCT03199469). Comprehensive histopathological analysis was performed.

Findings

Baseline biopsies uniformly showed findings characteristic of XLMTM, including small myofibres, increased internal or central nucleation, and central aggregates of organelles. Biopsies taken at 24 weeks post-treatment showed marked improvement of organelle localisation, without apparent increases in myofibre size in most participants. Biopsies taken at 48 weeks, however, did show statistically significant increases in myofibre size in all nine biopsies evaluated at this timepoint. Histopathological endpoints that did not demonstrate statistically significant changes with treatment included the degree of internal/central nucleation, numbers of triad structures, fibre type distributions, and numbers of satellite cells. Limited (predominantly mild) treatment-associated inflammatory changes were seen in biopsy specimens from five participants.

Interpretation

Muscle biopsies from individuals with XLMTM treated with resamirigene bilparvovec display statistically significant improvement in organelle localisation and myofibre size during a period of substantial improvements in muscle strength and respiratory function. This study identifies valuable histological endpoints for tracking treatment-related gains with resamirigene bilparvovec, as well as endpoints that did not show strong correlation with clinical improvement in this human study.

Funding

Astellas Gene Therapies (formerly Audentes Therapeutics, Inc.).

Michael W. Lawlor, Benedikt Schoser, Marta Margeta, Caroline A. Sewry, Karra A. Jones, Perry B. Shieh, Nancy L. Kuntz, Barbara K. Smith, James J. Dowling, Wolfgang Müller-Felber, Carsten G. Bönnemann, Andreea M. Seferian, Astrid Blaschek, Sarah Neuhaus, A. Reghan Foley, Dimah N. Saade, Etsuko Tsuchiya, Ummulwara R. Qasim, Margaret Beatka, Mariah J. Prom, Emily Ott, Susan Danielson, Paul Krakau, Suresh N. Kumar, Hui Meng, Mark Vanden Avond, Clive Wells, Heather Gordish-Dressman, Alan H. Beggs, Sarah Christensen, Edward Conner, Emma S. James, Jun Lee, Chanchal Sadhu, Weston Miller, Bryan Sepulveda, Fatbardha Varfaj, Suyash Prasad, Salvador Rico