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Chondrosarcomas are malignant cartilage-forming tumours that can arise centrally (in the medulla) or peripherally (at the surface) of the bone. They are classified into three histological grades which correspond to the clinical severity. Previous studies by our group have shown altered signal transduction of the fibroblast growth factor and Wnt signalling pathways during peripheral chondrosarcoma progression. Heparan sulphate (HS) is a glycosaminoglycan that facilitates receptor binding of multiple growth factors, in which the sulphation of 6O position plays a pivotal role. 6O-Sulphation occurs through three HS 6O-sulphotransferases (HS6ST1-3) and is fine-tuned by two endosulphatases (SULF1-2) that remove 6O-sulphate groups. We have investigated whether the expression of HS6STs and SULFs changes during chondrosarcoma progression and have determined 6O-sulphation levels in two chondrosarcoma cell lines. Immunohistochemistry on tissue microarrays of chondrosarcomas showed that HS6ST3 and SULF1 were highly expressed in most chondrosarcomas, whereas SULF2 expression was absent in most cases. HS6ST1 and HS6ST2 expression are significantly increased during chondrosarcoma progression, which suggest that 6O-sulphation is increased during progression. This was confirmed in one grade III chondrosarcoma cell line, which showed a dramatically increased 6O-sulphation compared to an articular chondrocyte cell line by HPLC; another cell line showed an increased expression of one 6O-sulphated HS disaccharide. In conclusion, our results show increased HS6ST1 and HS6ST2 expression during chondrosarcoma progression and increased HS 6O-sulphation in vitro. As 6O-sulphation plays an important role in signal transduction, altered HS6ST expression might be associated with changes in signal transduction pathways in chondrosarcoma progression. [ABSTRACT FROM AUTHOR]

Endochondral bone formation requires a cartilage template, known as the growth plate, and vascular invasion, bringing osteoblasts and osteoclasts. Endochondral chondrocytes undergo sequences of cell division, matrix secretion, cell hypertrophy, apoptosis, and matrix calcification/mineralisation. In this study, two critical steps of endochondral bone formation, the deposition of collagen X-rich matrix and blood vessel attraction/invasion, were investigated by immunohistochemistry. Fourteen multiple osteochondromas and six secondary peripheral chondrosarcomas occurring in patients with multiple osteochondromas were studied and compared to epiphyseal growth plate samples. Mutation analysis showed all studied patients (expect one) to harbour a germ-line mutations in either EXT1 or EXT2. Here, we described that homozygous mutations in EXT1/EXT2, which are causative for osteochondroma formation, are likely to affect terminal chondrocyte differentiation and vascularisation in the osteocartilaginous interface. Contrastingly, terminal chondrocyte differentiation and vascularisation seem to be unaffected in secondary peripheral chondrosarcoma. In addition, osteochondromas with high vascular density displayed a higher proliferation rate. A similar apoptotic rate was observed in osteochondromas and secondary peripheral chondrosarcomas. Recently, it has been shown that cells with functional EXT1 and EXT2 are outnumbering EXT1/EXT2 mutated cells in secondary peripheral chondrosarcomas. This might explain the increased type X collagen production and blood vessel attraction in these malignant tumours. [ABSTRACT FROM AUTHOR]

Purpose: Patients with cancer frequently undergo radiotherapy in their clinical management with unintended irradiation of blood vessels and copiously irrigated organs in which polymorphonuclear leukocytes circulate. Following the observation that such low doses of ionizing radiation are able to induce neutrophils to extrude neutrophil extracellular traps (NET), we have investigated the mechanisms, consequences, and occurrence of such phenomena in patients undergoing radiotherapy., Experimental Design: NETosis was analyzed in cultures of neutrophils isolated from healthy donors, patients with cancer, and cancer-bearing mice under confocal microscopy. Cocultures of radiation-induced NETs, immune effector lymphocytes, and tumor cells were used to study the effects of irradiation-induced NETs on immune cytotoxicity. Radiation-induced NETs were intravenously injected to mice for assessing their effects on metastasis. Circulating NETs in irradiated patients with cancer were measured using ELISA methods for detecting MPO-DNA complexes and citrullinated histone 3., Results: Irradiation of neutrophils with very low γ-radiation doses (0.5-1 Gy) elicits NET formation in a manner dependent on oxidative stress, NADPH oxidase activity, and autocrine IL8. Radiation-induced NETs interfere with NK cell and T-cell cytotoxicity. As a consequence, preinjection of irradiation-induced NETs increases the number of successful metastases in mouse tumor models. Increases in circulating NETs were readily detected in two prospective series of patients following the first fraction of their radiotherapy courses., Conclusions: NETosis is induced by low-dose ionizing irradiation in a neutrophil-intrinsic fashion, and radiation-induced NETs are able to interfere with immune-mediated cytotoxicity. Radiation-induced NETs foster metastasis in mouse models and can be detected in the circulation of patients undergoing conventional radiotherapy treatments. See related commentary by Mowery and Luke, p. 3965., (©2024 The Authors; Published by the American Association for Cancer Research.)

Background: Immunodeficient mice engrafted with peripheral blood mononuclear cells (PBMCs) are models to study new cancer immunotherapy agents. However, this approach is associated with xenograft-versus-host disease (xGVHD), which starts early after PBMC transfer and limits the duration and interpretation of experiments. Here, we explore different approaches to overcome xGVHD and better support the development of cancer immunotherapies., Methods: Immunodeficient NOD-scid IL2Rg null (NSG) mice were intravenously transferred with human PBMCs and subcutaneously co-engrafted with HT29 human colon carcinoma cells. Diverse strategies to reduce xGVHD while preserving the antitumor activity of human immune cells were evaluated: (1) ex vivo immune graft modification by depleting CD4 + T cells pre-transfer using magnetic beads, (2) post-transplantation cyclophosphamide administration to eliminate proliferating xenoreactive T-cell clones and (3) using major histocompatibility complex (MHC) class I and II-deficient NSG mice: (K b D b ) null (IA) null (MHC-dKO NSG). Body weight and plasma murine alanine aminotransferase levels were measured as indicators of xGVHD and tumor size was measured every 2-3 days to monitor antitumor activity. The antitumor effects and pharmacodynamics of nivolumab plus ipilimumab and an anti-epithelial cell adhesion molecule (EpCAM)/CD3 T-cell engager (αEpCAM/CD3 bispecific antibody (BsAb)) were evaluated in the model., Results: CD4 + T-cell depletion attenuates xGVHD but also abrogates the antitumor activity. Cyclophosphamide limits the antitumor response and does not substantially prevent xGVHD. In contrast, xGVHD was significantly attenuated in MHC-dKO NSG recipients, while the antitumor effect of human PBMCs was preserved. Furthermore, the administration of nivolumab plus ipilimumab caused exacerbated xGVHD in conventional NSG mice, thereby precluding the observation of their antitumor effects. Severe xGVHD did not occur in MHC-dKO NSG mice thus enabling the study of complete and durable tumor rejections. Similarly, NSG mice treated with an αEpCAM/CD3 BsAb showed complete tumor regressions, but died due to xGVHD. In contrast, MHC-dKO NSG mice on treatment with the αEpCAM/CD3 BsAb achieved complete tumor responses without severe xGVHD. A significant proportion of mice rendered tumor-free showed tumor rejection on rechallenge with HT29 cells without further treatment. Finally, tumor-infiltrating CD8 + T-cell number increase, activation and CD137 upregulation were observed on αEpCAM/CD3 BsAb treatment., Conclusion: Humanized MHC-dKO immunodeficient mice allow and refine the preclinical testing of immunotherapy agents for which experimentation is precluded in conventional immunodeficient mice due to severe xGVHD., Competing Interests: Competing interests: IE-S, EFdP, GC, PM-M, MC, DC, IA-B, IRL, RS-V, CEDA, LÁ-G and PB declare no conflicts of interest. IM reports grants and personal fees from Genmab during the conduct of the study, as well as grants and personal fees from Bristol Myers Squibb, Roche, AstraZeneca, and Pharmamar and personal fees from F-Star, Numab, Pieris, Boehringer Ingelheim, Gossamer, Alligator, Hotspot, Biolinerx, Bioncotech, Dompe, Highlight Therapeutics, Bright Peaks and Boston Therapeutics outside the submitted work. MFS reports grants from Bristol Myers Squibb and Roche during the conduct of the study, as well as grants and personal fees from Roche and Bristol Myers Squibb, and personal fees from Numab outside the submitted work., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

An association between type 2 diabetes (T2D) and breast cancer risk has been reported. This association can be potentially explained by alteration of the insulin/IGF system. Therefore, we aimed to prospectively investigate whether a previously reported Dietary-Based Diabetes Risk Score (DDS) inversely associated with T2D was also associated with breast cancer risk in the SUN ("Seguimiento Universidad de Navarra") cohort. We followed up 10,810 women (mean age = 35 years, SD = 11 years) for an average of 12.5 years during which 147 new cases of invasive breast cancer were diagnosed. A validated 136-item FFQ was administered at baseline and after 10 years of follow-up. The DDS (range: 11 to 55 points) positively weighted vegetables, fruit, whole cereals, nuts, coffee, low-fat dairy, fiber, PUFA; while it negatively weighted red meat, processed meats, and sugar-sweetened beverages. The DDS was categorized into tertiles. Self-reported medically diagnosed breast cancer cases were confirmed through medical records. We found a significant inverse association between the intermediate tertile of the DDS score and overall breast cancer risk (Hazard ratio, HR T2 vs. T1 = 0.55; 95% CI: 0.36-0.82) and premenopausal breast cancer risk (HR T2 = 0.26; 95% CI: 0.13-0.53), but not for the highest tertile. This association was stronger among women with a BMI < 25 kg/m 2 (p interaction : 0.029). In conclusion, moderate adherence to the DDS score was associated with a lower risk of breast cancer, especially among premenopausal women and women with a lower BMI. These findings underscore the importance of antidiabetic diet in reducing the risk of breast cancer., (© 2024. The Author(s).)

Background: Activating and inhibitory receptors of natural killer (NK) cells such as NKp, NKG2, or CLEC are highly relevant to cold tumors including glioblastoma (GBM). Here, we aimed to characterize the expression of these receptors in GBM to gain insight into their potential role as modulators of the intratumoral microenvironment., Methods: We performed a transcriptomic analysis of several NK receptors with a focus on the activating receptor encoded by KLRC2, NKG2C, among bulk and single-cell RNA sequencing GBM data sets. We also evaluated the effects of KLRC2-overexpressing GL261 cells in mice treated with or without programmed cell death protein-1 (PD-1) monoclonal antibody (mAb). Finally, we analyzed samples from two clinical trials evaluating PD-1 mAb effects in patients with GBM to determine the potential of NKG2C to serve as a biomarker of response., Results: We observed significant expression of several inhibitory NK receptors on GBM-infiltrating NK and T cells, which contrasts with the strong expression of KLRC2 on tumor cells, mainly at the infiltrative margin. Neoplastic KLRC2 expression was associated with a reduction in the number of myeloid-derived suppressor cells and with a higher level of tumor-resident lymphocytes. A stronger antitumor activity after PD-1 mAb treatment was observed in NKG2C high -expressing tumors both in mouse models and patients with GBM whereas the expression of inhibitory NK receptors showed an inverse association., Conclusions: This study explored the role of neoplastic NKG2C/ KLRC2 expression in shaping the immune profile of GBM and suggests that it is a predictive biomarker for positive responses to immune checkpoint inhibitor treatment in patients with GBM. Future studies could further validate this finding in prospective trials., Competing Interests: Competing interests: MER-R reports personal fees from BMS and grants from Highlight-Therapeutics and Roche outside the submitted work. She also has received speaker’s bureau honoraria from BMS and ROCHE. The rest of the authors declare that they have no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: The tissue immune microenvironment is associated with key aspects of tumor biology. The interaction between the immune system and cancer cells has predictive and prognostic potential across different tumor types. Spatially resolved tissue-based technologies allowed researchers to simultaneously quantify different immune populations in tumor samples. However, bare quantification fails to harness the spatial nature of tissue-based technologies. Tumor-immune interactions are associated with specific spatial patterns that can be measured. In recent years, several computational tools have been developed to increase our understanding of these spatial patterns., Topics Covered: In this review, we cover standard techniques as well as new advances in the field of spatial analysis of the immune microenvironment. We focused on marker quantification, spatial intratumor heterogeneity analysis, cell‒cell spatial interaction studies and neighborhood analyses., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: The aim of our study was to elucidate the impact of bevacizumab added to neoadjuvant chemotherapy (NACT) on the tumor immune microenvironment and correlate the changes with the clinical outcome of the patients., Experimental Design: IHC and multiplex immunofluorescence for lymphoid and myeloid lineage markers were performed in matched tumor samples from 23 patients with ovarian cancer enrolled in GEICO 1205/NOVA clinical study before NACT and at the time of interval cytoreductive surgery., Results: Our results showed that the addition of bevacizumab to NACT plays a role mainly on lymphoid populations at the stromal compartment, detecting a significant decrease of CD4+ T cells, an increase of CD8+ T cells, and an upregulation in effector/regulatory cell ratio (CD8+/CD4+FOXP3+). None of the changes observed were detected in the intra-epithelial site in any arm (NACT or NACT-bevacizumab). No differences were found in myeloid lineage (macrophage-like). The percentage of Treg populations and effector/regulatory cell ratio in the stroma were the only two variables significantly associated with progression-free survival (PFS)., Conclusions: The addition of bevacizumab to NACT did not have an impact on PFS in the GEICO 1205 study. However, at the cellular level, changes in CD4+, CD8+ lymphocyte populations, and CD8+/CD4+FOXP3 ratio have been detected only at the stromal site. On the basis of our results, we hypothesize about the existence of mechanisms of resistance that could prevent the trafficking of T-effector cells into the epithelial component of the tumor as a potential explanation for the lack of efficacy of ICI in the first-line treatment of advanced epithelial ovarian cancer. See related commentary by Soberanis Pina and Oza, p. 12., (©2023 The Authors; Published by the American Association for Cancer Research.)

Single cell spatial interrogation of the immune-structural interactions in COVID -19 lungs is challenging, mainly because of the marked cellular infiltrate and architecturally distorted microstructure. To address this, we develop a suite of mathematical tools to search for statistically significant co-locations amongst immune and structural cells identified using 37-plex imaging mass cytometry. This unbiased method reveals a cellular map interleaved with an inflammatory network of immature neutrophils, cytotoxic CD8 T cells, megakaryocytes and monocytes co-located with regenerating alveolar progenitors and endothelium. Of note, a highly active cluster of immature neutrophils and CD8 T cells, is found spatially linked with alveolar progenitor cells, and temporally with the diffuse alveolar damage stage. These findings offer further insights into how immune cells interact in the lungs of severe COVID-19 disease. We provide our pipeline [Spatial Omics Oxford Pipeline (SpOOx)] and visual-analytical tool, Multi-Dimensional Viewer (MDV) software, as a resource for spatial analysis., (© 2023. The Author(s).)

Multiplex immunofluorescence is a novel, high-content imaging technique that allows simultaneous in situ labeling of multiple tissue antigens. This technique is of growing relevance in the study of the tumor microenvironment, and the discovery of biomarkers of disease progression or response to immune-based therapies. Given the number of markers and the potential complexity of the spatial interactions involved, the analysis of these images requires the use of machine learning tools that rely for their training on the availability of large image datasets, extremely laborious to annotate. We present Synplex, a computer simulator of multiplexed immunofluorescence images from user-defined parameters: i. cell phenotypes, defined by the level of expression of markers and morphological parameters; ii. cellular neighborhoods based on the spatial association of cell phenotypes; and iii. interactions between cellular neighborhoods. We validate Synplex by generating synthetic tissues that accurately simulate real cancer cohorts with underlying differences in the composition of their tumor microenvironment and show proof-of-principle examples of how Synplex could be used for data augmentation when training machine learning models, and for the in silico selection of clinically relevant biomarkers. Synplex is publicly available at https://github.com/djimenezsanchez/Synplex.

Immune-mediated anti-tumoral responses, elicited by oncolytic viruses and augmented with checkpoint inhibition, may be an effective treatment approach for glioblastoma. Here in this multicenter phase 1/2 study we evaluated the combination of intratumoral delivery of oncolytic virus DNX-2401 followed by intravenous anti-PD-1 antibody pembrolizumab in recurrent glioblastoma, first in a dose-escalation and then in a dose-expansion phase, in 49 patients. The primary endpoints were overall safety and objective response rate. The primary safety endpoint was met, whereas the primary efficacy endpoint was not met. There were no dose-limiting toxicities, and full dose combined treatment was well tolerated. The objective response rate was 10.4% (90% confidence interval (CI) 4.2-20.7%), which was not statistically greater than the prespecified control rate of 5%. The secondary endpoint of overall survival at 12 months was 52.7% (95% CI 40.1-69.2%), which was statistically greater than the prespecified control rate of 20%. Median overall survival was 12.5 months (10.7-13.5 months). Objective responses led to longer survival (hazard ratio 0.20, 95% CI 0.05-0.87). A total of 56.2% (95% CI 41.1-70.5%) of patients had a clinical benefit defined as stable disease or better. Three patients completed treatment with durable responses and remain alive at 45, 48 and 60 months. Exploratory mutational, gene-expression and immunophenotypic analyses revealed that the balance between immune cell infiltration and expression of checkpoint inhibitors may potentially inform on response to treatment and mechanisms of resistance. Overall, the combination of intratumoral DNX-2401 followed by pembrolizumab was safe with notable survival benefit in select patients (ClinicalTrials.gov registration: NCT02798406)., (© 2023. The Author(s).)

The immune phenotype of a tumour is a key predictor of its response to immunotherapy 1-4 . Patients who respond to checkpoint blockade generally present with immune-inflamed 5-7 tumours that are highly infiltrated by T cells. However, not all inflamed tumours respond to therapy, and even lower response rates occur among tumours that lack T cells (immune desert) or that spatially exclude T cells to the periphery of the tumour lesion (immune excluded) 8 . Despite the importance of these tumour immune phenotypes in patients, little is known about their development, heterogeneity or dynamics owing to the technical difficulty of tracking these features in situ. Here we introduce skin tumour array by microporation (STAMP)-a preclinical approach that combines high-throughput time-lapse imaging with next-generation sequencing of tumour arrays. Using STAMP, we followed the development of thousands of arrayed tumours in vivo to show that tumour immune phenotypes and outcomes vary between adjacent tumours and are controlled by local factors within the tumour microenvironment. Particularly, the recruitment of T cells by fibroblasts and monocytes into the tumour core was supportive of T cell cytotoxic activity and tumour rejection. Tumour immune phenotypes were dynamic over time and an early conversion to an immune-inflamed phenotype was predictive of spontaneous or therapy-induced tumour rejection. Thus, STAMP captures the dynamic relationships of the spatial, cellular and molecular components of tumour rejection and has the potential to translate therapeutic concepts into successful clinical strategies., (© 2023. The Author(s).)

Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular carcinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co-expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4 + anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addition of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive T cell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 regimens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance T cell infiltration and the intratumoral performance of T lymphocytes., Competing Interests: Declaration of interests I.M. acknowledges grants from Roche, Alligator, Genmab, BMS, AstraZeneca, Pharmamar, and Bioncotech, as well as consultancy fees from BMS, Roche, Genmab, Numab, F-Star, Biolinerx, Pierre Fabre, Sanofi, Gossamer, Alligator, AstraZeneca, and Pharmamar. B.S. received consulting fees from Adaptimmune, AstraZeneca, Bayer, BMS, BTG, Eisai, Exelixis, Eli-Lilly, IPSEN, Merck, Onxeo, Roche, and Sirtex; lecture fees from AstraZeneca, Bayer, BMS, Eisai, Eli-Lilly, Incyte, IPSEN, Roche, and Sirtex; and institutional research grants from BMS and Sirtex., (Copyright © 2023. Published by Elsevier Inc.)

Predicting recurrence in low-grade, early-stage endometrial cancer (EC) is both challenging and clinically relevant. We present a weakly-supervised deep learning framework, NaroNet, that can learn, without manual expert annotation, the complex tumor-immune interrelations at three levels: local phenotypes, cellular neighborhoods, and tissue areas. It uses multiplexed immunofluorescence for the simultaneous visualization and quantification of CD68 + macrophages, CD8 + T cells, FOXP3 + regulatory T cells, PD-L1/PD-1 protein expression, and tumor cells. We used 489 tumor cores from 250 patients to train a multilevel deep-learning model to predict tumor recurrence. Using a tenfold cross-validation strategy, our model achieved an area under the curve of 0.90 with a 95% confidence interval of 0.83-0.95. Our model predictions resulted in concordance for 96,8% of cases (κ = 0.88). This method could accurately assess the risk of recurrence in EC, outperforming current prognostic factors, including molecular subtyping., (© 2023. The Author(s).)

Objective: Progressive supranuclear palsy (PSP) is a 4R-tauopathy showing heterogeneous tau cytopathology commencing in the globus pallidus (GP) and the substantia nigra (SN), regions also associated with age-related iron accumulation. Abnormal iron levels have been extensively associated with tau pathology in neurodegenerative brains, however, its role in PSP pathogenesis remains yet unknown. We perform the first cell type-specific evaluation of PSP iron homeostasis and the closely related oxygen homeostasis, in relation to tau pathology in human postmortem PSP brains., Methods: In brain regions vulnerable to PSP pathology (GP, SN, and putamen), we visualized iron deposition in tau-affected and unaffected neurons, astroglia, oligodendrocytes, and microglia, using a combination of iron staining with immunolabelling. To further explore molecular pathways underlying our observations, we examined the expression of key iron and oxygen homeostasis mRNA transcripts and proteins., Results: We found astrocytes as the major cell type accumulating iron in the early affected regions of PSP, highly associated with cellular tau pathology. The same regions are affected by dysregulated expression of alpha and beta hemoglobin and neuroglobin showing contrasting patterns. We discovered changes in iron and oxygen homeostasis-related gene expression associated with aging of the brain, and identified dysregulated expression of rare neurodegeneration with brain iron accumulation (NBIA) genes associated with tau pathology to distinguish PSP from the healthy aging brain., Interpretation: We present novel aspects of PSP pathophysiology highlighting an overlap with NBIA pathways. Our findings reveal potential novel targets for therapy development and have implications beyond PSP for other iron-associated neurodegenerative diseases. ANN NEUROL 2023;93:431-445., (© 2022 American Neurological Association.)

IL12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs, and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding IL12 and IL18. Moreover, we have used a form of IL18 [decoy-resistant IL18 (DR-18)] which has preserved bioactivity but does not bind to the IL18 binding protein decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically cotransferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant noninjected lesions. Cotreatment was conducive to the presence of more activated CD8+ T cells in the treated and noninjected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL12 plus DR-18 local mRNA coinjection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy., (©2022 American Association for Cancer Research.)

Severe lung damage resulting from COVID-19 involves complex interactions between diverse populations of immune and stromal cells. In this study, we used a spatial transcriptomics approach to delineate the cells, pathways, and genes present across the spectrum of histopathological damage in COVID-19-affected lung tissue. We applied correlation network-based approaches to deconvolve gene expression data from 46 areas of interest covering more than 62,000 cells within well-preserved lung samples from 3 patients. Despite substantial interpatient heterogeneity, we discovered evidence for a common immune-cell signaling circuit in areas of severe tissue that involves crosstalk between cytotoxic lymphocytes and pro-inflammatory macrophages. Expression of IFNG by cytotoxic lymphocytes was associated with induction of chemokines, including CXCL9, CXCL10, and CXCL11, which are known to promote the recruitment of CXCR3+ immune cells. The TNF superfamily members BAFF (TNFSF13B) and TRAIL (TNFSF10) were consistently upregulated in the areas with severe tissue damage. We used published spatial and single-cell SARS-CoV-2 data sets to validate our findings in the lung tissue from additional cohorts of patients with COVID-19. The resulting model of severe COVID-19 immune-mediated tissue pathology may inform future therapeutic strategies.

Multiplexed immunofluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides allow the identification of multiple cell phenotypes while retaining spatial and morphological context. Multiplex immunofluorescence protocols have already been developed and validated for mouse tissues. Immunophenotyping analysis reliably depicts the immune landscape of cancer tissues that has been demonstrated to influence cancer development and progression as well as to have an impact on therapy responsiveness and resistance. Here, we describe a method for multiplexed fluorescence image analysis, enabling analysis of mouse cancer morphology and cell phenotypes in FFPE sections., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Aims: Gene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated., Methods: Cell lines harbouring EML4 (13) -ALK (20) and SLC34A2 (4) -ROS1 (32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides., Results: Four (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms., Conclusions: Reference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches., Competing Interests: Competing interests: UM has received personal fees (as consultant and/or speaker bureau) from Boehringer Ingelheim, Roche, MSD, Amgen, Thermo Fisher Scientifics, Eli Lilly, Diaceutics, GSK, Merck and Astra Zeneca, unrelated to the current work. Lukas Bubendorf has a consulting or advisory role with Astra Zeneca, AbbVie, Bayer, Boehringer Ingelheim, Eli Lilly, MSD, Pfizer, Takeda and F. Hoffmann-La Roche and has received research funding (institution) from F. Hoffmann-La Roche, MSD and Sanofi. PH reports personal fees (as advisor) from Roche, Astrazeneca, BMS, MSD, Pfizer, Bayer, Amgen, Illumina, Qiagen, Thermo Fisher Scientific, Biocartis, Ed Lilly, unrelated to the current work. SM-B has received personal fees (as consultant and/or speaker bureau) from Astra Zeneca, Roche, BMS, Novartis, GSK, MSD, Targos, Merck, unrelated to the current work. Spasenija Savic Prince received personal fees from MSD, Astra Zeneca, Boehringer Ingelheim, Roche, Pfizer, Bristol-Myers Squibb and Thermo Fisher Scientific, unrelated to the submitted work. EV has received personal fees (as consultant and/or speaker bureau) from Diaceutics, unrelated to the current work. GTr reports personal fees (as speaker bureau or advisor) from Roche, MSD, Pfizer, Boehringer Ingelheim, Eli Lilly, BMS, GSK, Menarini, AstraZeneca, Amgen and Bayer, unrelated to the current work. The other authors have nothing to disclose., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Endometrial tumors show substantial heterogeneity in their immune microenvironment. This heterogeneity could be used to improve the accuracy of current outcome prediction tools. We assessed the immune microenvironment of 235 patients diagnosed with low-grade, early-stage endometrial cancer. Multiplex quantitative immunofluorescence was carried out to measure CD8, CD68, FOXP3, PD-1, and PD-L1 markers, as well as cytokeratin (CK), on tissue microarrays. Clustering results revealed five robust immune response patterns, each associated with specific immune populations, cell phenotypes, and cell spatial clustering. Most samples (69%) belonged to the immune-desert subtype, characterized by low immune cell densities. Tumor-infiltrating lymphocyte (TIL)-rich samples (4%) displayed high CD8 + T-cell infiltration, as well as a high percentage of CD8/PD-1 + cells. Immune-exclusion samples (19%) displayed the lowest CD8 + infiltration combined with high PD-L1 expression levels in CK + tumor cells. In addition, they demonstrated high tumor cell spatial clustering as well as increased spatial proximity of CD8 + /PD-1 + and CK/PD-L1 + cells. FOXP3 and macrophage-rich phenotypes (3% and 4% of total samples) displayed relatively high levels of FOXP3 + regulatory T-cells and CD68 + macrophages, respectively. These phenotypes correlated with clinical outcomes, with immune-exclusion tumors showing an association with tumor relapse. When compared with prediction models built using routine pathological variables, models optimized with immune variables showed increased outcome prediction capacity (AUC = 0.89 versus 0.78) and stratification potential. The improved prediction capacity was independent of mismatch repair protein status and adjuvant radiotherapy treatment. Further, immunofluorescence results could be partially recapitulated using single-marker immunohistochemistry (IHC) performed on whole tissue sections. TIL-rich tumors demonstrated increased CD8 + T-cells by IHC, while immune-exclusion tumors displayed a lack of CD8 + T-cells and frequent expression of PD-L1 in tumor cells. Our results demonstrate the capability of the immune microenvironment to improve standard prediction tools in low-grade, early-stage endometrial carcinomas. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Background: Despite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear., Methods: To better understand the role of TILs in EC, we analyzed the phenotypic traits of CD8 + and CD4 + EC - resident T cells from 47 primary tumors by high-dimensional flow cytometry. In addition, CD8 + and CD4 + TIL subpopulations were isolated based on the differential expression of programmed cell death protein-1 (PD-1) (negative, dim and high) and CD39 (positive or negative) by fluorescence activated cell sorting (FACS), expanded in vitro, and screened for autologous tumor recognition. We further investigated whether phenotypic markers preferentially expressed on CD8 + and CD4 + tumor-reactive TIL subsets were associated with the four distinct molecular subtypes of EC, tumor mutational burden and patient survival., Results: We found that CD8 + TILs expressing high levels of PD-1 (PD-1hi) co-expressed CD39, TIM-3, HLA-DR and CXCL13, as compared with TILs lacking or displaying intermediate levels of PD-1 expression (PD-1 - and PD-1 dim , respectively). Autologous tumor reactivity of sorted and in vitro expanded CD8+ TILs demonstrated that the CD8 + PD-1 dim CD39 + and PD-1 hi CD39 + T cell subsets both contained tumor-reactive TILs and that a higher level of PD-1 expression was associated with increased CD39 and a superior frequency of tumor reactivity. With respect to CD4 + T conventional (Tconv) TILs, co-expression of inhibitory and activation markers was more apparent on PD-1 hi compared with PD-1 - or PD-1 dim T cells, and in fact, it was the CD4 + PD-1 hi subpopulation that accumulated the antitumor T cells irrespective of CD39 expression. Most importantly, detection of CD8 + PD-1 hi CD39+ and CD4 + PD-1 hi tumor-reactive T-cell subsets, but also markers specifically expressed by these subpopulations of TILs, that is, PD-1 hi , CD39, CXCL13 and CD103 by CD8 + TILs and PD-1 hi and CXCL13 by CD4 + Tconv TILs, correlated with prolonged survival of patients with EC., Conclusions: Our results demonstrate that EC are frequently infiltrated by tumor-reactive TILs, and that expression of PD-1 hi and CD39 or PD-1 hi can be used to select and expand CD8 + and CD4 + tumor-reactive TILs, respectively. In addition, biomarkers preferentially expressed on tumor-reactive TILs, rather than the frequency of CD3 + , CD8 + and CD4 + lymphocytes, hold prognostic value suggesting their protective role in antitumor immunity., Competing Interests: Competing interests: AG is a member of the scientific advisory board (SAB) of Achilles Therapeutics, SingulaBIO, RootPath, and BioNTech SE, and consults for PACT Pharma, and Instil BIo. AG is co-inventor of patents licensed and with royalties related to this work E-059-2013/0, E-085-2013/0, E-149-2015/0. IM reports grants from Roche, BMS, AstraZeneca and Genmab, as well as consultancy activities for Roche, BMS, AZ, Phamamar, F-sar, Merus, Amunix, Pieris, Numab, Third Rock and Highlight therapeutics. The authors declare that no other conflict of interest exists., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Excessive inflammation is pathogenic in the pneumonitis associated with severe COVID-19. Neutrophils are among the most abundantly present leukocytes in the inflammatory infiltrates and may form neutrophil extracellular traps (NETs) under the local influence of cytokines. NETs constitute a defense mechanism against bacteria, but have also been shown to mediate tissue damage in a number of diseases., Research Question: Could NETs and their tissue-damaging properties inherent to neutrophil-associated functions play a role in the respiratory failure seen in patients with severe COVID-19, and how does this relate to the SARS-CoV-2 viral loads, IL-8 (CXCL8) chemokine expression, and cytotoxic T-lymphocyte infiltrates?, Study Design and Methods: Sixteen lung biopsy samples obtained immediately after death were analyzed methodically as exploratory and validation cohorts. NETs were analyzed quantitatively by multiplexed immunofluorescence and were correlated with local levels of IL-8 messenger RNA (mRNA) and the density of CD8+ T-cell infiltration. SARS-CoV-2 presence in tissue was quantified by reverse-transcriptase polymerase chain reaction and immunohistochemistry analysis., Results: NETs were found in the lung interstitium and surrounding the bronchiolar epithelium with interindividual and spatial heterogeneity. NET density did not correlate with SARS-CoV-2 tissue viral load. NETs were associated with local IL-8 mRNA levels. NETs were also detected in pulmonary thrombi and in only one of eight liver tissues. NET focal presence correlated negatively with CD8+ T-cell infiltration in the lungs., Interpretation: Abundant neutrophils undergoing NETosis are found in the lungs of patients with fatal COVID-19, but no correlation was found with viral loads. The strong association between NETs and IL-8 points to this chemokine as a potentially causative factor. The function of cytotoxic T-lymphocytes in the immune responses against SARS-CoV-2 may be interfered with by the presence of NETs., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1β upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1β induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1β inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated., Significance: IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1β are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents. This article is highlighted in the In This Issue feature, p. 2007., (©2022 American Association for Cancer Research.)

Background: One of the main difficulties of adoptive cell therapies with chimeric antigen receptor (CAR)-T cells in solid tumors is the identification of specific target antigens. The tumor microenvironment can present suitable antigens for CAR design, even though they are not expressed by the tumor cells. We have generated a CAR specific for the splice variant extra domain A (EDA) of fibronectin, which is highly expressed in the tumor stroma of many types of tumors but not in healthy tissues., Methods: EDA expression was explored in RNA-seq data from different human tumor types and by immunohistochemistry in paraffin-embedded tumor biopsies. Murine and human anti-EDA CAR-T cells were prepared using recombinant retro/lentiviruses, respectively. The functionality of EDA CAR-T cells was measured in vitro in response to antigen stimulation. The antitumor activity of EDA CAR-T cells was measured in vivo in C57BL/6 mice challenged with PM299L-EDA hepatocarcinoma cell line, in 129Sv mice-bearing F9 teratocarcinoma and in NSG mice injected with the human hepatocarcinoma cell line PLC., Results: EDA CAR-T cells recognized and killed EDA-expressing tumor cell lines in vitro and rejected EDA-expressing tumors in immunocompetent mice. Notably, EDA CAR-T cells showed an antitumor effect in mice injected with EDA-negative tumor cells lines when the tumor stroma or the basement membrane of tumor endothelial cells express EDA. Thus, EDA CAR-T administration delayed tumor growth in immunocompetent 129Sv mice challenged with teratocarcinoma cell line F9. EDA CAR-T treatment exerted an antiangiogenic effect and significantly reduced gene signatures associated with epithelial-mesenchymal transition, collagen synthesis, extracellular matrix organization as well as IL-6-STAT5 and KRAS pathways. Importantly, the human version of EDA CAR, that includes the human 41BB and CD3ζ endodomains, exerted strong antitumor activity in NSG mice challenged with the human hepatocarcinoma cell line PLC, which expresses EDA in the tumor stroma and the endothelial vasculature. EDA CAR-T cells exhibited a tropism for EDA-expressing tumor tissue and no toxicity was observed in tumor bearing or in healthy mice., Conclusions: These results suggest that targeting the tumor-specific fibronectin splice variant EDA with CAR-T cells is feasible and offers a therapeutic option that is applicable to different types of cancer., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Aim: Next generation sequencing (NGS) represents a key diagnostic tool to identify clinically relevant gene alterations for treatment-decision making in cancer care. However, the complex manual workflow required for NGS has limited its implementation in routine clinical practice. In this worldwide study, we validated the clinical performance of the TargetPlex FFPE-Direct DNA Library Preparation Kit for NGS analysis. Impressively, this new assay obviates the need for separate, labour intensive and time-consuming pre-analytical steps of DNA extraction, purification and isolation from formalin-fixed paraffin embedded (FFPE) specimens in the NGS workflow., Methods: The TargetPlex FFPE-Direct DNA Library Preparation Kit, which enables NGS analysis directly from FFPE, was specifically developed for this study by TargetPlex Genomics Pleasanton, California. Eleven institutions agreed to take part in the study coordinated by the Molecular Cytopathology Meeting Group (University of Naples Federico II, Naples, Italy). All participating institutions received a specific Library Preparation Kit to test eight FFPE samples previously assessed with standard protocols. The analytical parameters and mutations detected in each sample were then compared with those previously obtained with standard protocols., Results: Overall, 92.8% of the samples were successfully analysed with the TargetPlex FFPE-Direct DNA Library Preparation Kit on Thermo Fisher Scientific and Illumina platforms. Altogether, in comparison with the standard workflow, the TargetPlex FFPE-Direct DNA Library Preparation Kit was able to detect 90.5% of the variants., Conclusion: The TargetPlex FFPE-Direct DNA Library Preparation Kit combined with the SiRe panel constitutes a convenient, practical and robust cost-saving solution for FFPE NGS analysis in routine practice., Competing Interests: Competing interests: UM has received personal fees (as consultant and/or speaker bureau) from Boehringer Ingelheim, Roche, MSD, Amgen, Thermo Fisher Scientifics, Diaceutics, GSK, Merck and AstraZeneca, unrelated to the current work. LB is has a consulting or advisory role with AstraZeneca, AbbVie, Bayer, Boehringer Ingelheim, Eli Lilly, MSD, Pfizer, Takeda and F. Hoffmann-La Roche, and has received research funding (institution) from F. Hoffmann-La Roche, MSD and Sanofi. PH reports personal fees (as advisor) from Roche, Astrazeneca, BMS, MSD, Pfizer, Bayer, Amgen, Illumina, Qiagen, Thermo Fisher Scientific, Biocartis, Ed Lilly, unrelated to the current work. MI reports personal fees (as speaker bureau) from Roche, Merck & Co, AstraZeneca, Bristol-Myers Squibb and Boehringer-Ingelheim, unrelated to the current work. GR is the coinventor of the intellectual property used in the background noise suppression aspects of the TargePlex FFPE-Direct NGS Library Preparation Kit developed in collaboration with SenseCare Medicals. AR reports personal fees from Bristol, Pfizer, Bayer, Kyowa Kirin, Ambrosetti for advisory board activity and speaker honorarium from Roche Diagnostic speaker honorarium, unrelated to the submitted work. SS received personal fees from MSD, Astra Zeneca, Boehringer Ingelheim, Roche, Pfizer, Bristol-Myers Squibb and Thermo Fisher Scientific, unrelated to the submitted work. TX is an employee and affiliate with SenseCare Medicals. GTroncone reports personal fees (as speaker bureau or advisor) from Roche, MSD, Pfizer and Bayer, unrelated to the current work.The other authors have nothing to disclose., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Abstract: Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1/CD203a (ENPP1) to be closely associated with LRF. ENPP1hi circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of polymorphonuclear myeloid-derived suppressor cells and neutrophil extracellular trap (NET) formation. Genetic and pharmacologic ENPP1 inhibition or NET blockade extends relapse-free survival. Furthermore, in combination with fractionated irradiation, ENPP1 abrogation obliterates LRF. Mechanistically, ENPP1-generated adenosinergic metabolites enhance haptoglobin (HP) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse., Significance: CTC exploit the ENPP1/HP axis to promote local recurrence post-surgery and post-irradiation by subduing myeloid suppressor cells in breast tumors. Blocking this axis impairs tumor engraftment, impedes immunosuppression, and obliterates NET formation, unveiling new opportunities for therapeutic intervention to eradicate local relapse and ameliorate patient survival. This article is highlighted in the In This Issue feature, p. 1171., (©2022 The Authors; Published by the American Association for Cancer Research.)

Understanding the spatial interactions between the elements of the tumor microenvironment -i.e. tumor cells. fibroblasts, immune cells- and how these interactions relate to the diagnosis or prognosis of a tumor is one of the goals of computational pathology. We present NaroNet, a deep learning framework that models the multi-scale tumor microenvironment from multiplex-stained cancer tissue images and provides patient-level interpretable predictions using a seamless end-to-end learning pipeline. Trained only with multiplex-stained tissue images and their corresponding patient-level clinical labels, NaroNet unsupervisedly learns which cell phenotypes, cell neighborhoods, and neighborhood interactions have the highest influence to predict the correct label. To this end, NaroNet incorporates several novel and state-of-the-art deep learning techniques, such as patch-level contrastive learning, multi-level graph embeddings, a novel max-sum pooling operation, or a metric that quantifies the relevance that each microenvironment element has in the individual predictions. We validate NaroNet using synthetic data simulating multiplex-immunostained images where a patient label is artificially associated to the -adjustable- probabilistic incidence of different microenvironment elements. We then apply our model to two sets of images of human cancer tissues: 336 seven-color multiplex-immunostained images from 12 high-grade endometrial cancer patients; and 382 35-plex mass cytometry images from 215 breast cancer patients. In both synthetic and real datasets, NaroNet provides outstanding predictions of relevant clinical information while associating those predictions to the presence of specific microenvironment elements., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors, and patient survival has not changed despite many therapeutic efforts, emphasizing the urgent need for effective treatments. Here, we evaluated the anti-DIPG effect of the oncolytic adenovirus Delta-24-ACT, which was engineered to express the costimulatory ligand 4-1BBL to potentiate the antitumor immune response of the virus. Delta-24-ACT induced the expression of functional 4-1BBL on the membranes of infected DIPG cells, which enhanced the costimulation of CD8+ T lymphocytes. In vivo, Delta-24-ACT treatment of murine DIPG orthotopic tumors significantly improved the survival of treated mice, leading to long-term survivors that developed immunological memory against these tumors. In addition, Delta-24-ACT was safe and caused no local or systemic toxicity. Mechanistic studies showed that Delta-24-ACT modulated the tumor-immune content, not only increasing the number, but also improving the functionality of immune cells. All of these data highlight the safety and potential therapeutic benefit of Delta-24-ACT the treatment of patients with DIPG.

Background: BO-112 is a nanoplexed form of polyinosinic:polycytidylic acid that acting on toll-like receptor 3 (TLR3), melanoma differentiation-associated protein 5 (MDA5) and protein kinase RNA-activated (PKR) elicits rejection of directly injected transplanted tumors, but has only modest efficacy against distant untreated tumors. Its clinical activity has also been documented in early phase clinical trials. The 5,6-dimethylxanthenone-4-acetic acid (DMXAA) stimulator of interferon genes (STING) agonist shows a comparable pattern of efficacy when used via intratumoral injections., Methods: Mice subcutaneously engrafted with bilateral MC38 and B16.OVA-derived tumors were treated with proinflammatory immunotherapy agents known to be active when intratumorally delivered. The combination of BO-112 and DMXAA was chosen given its excellent efficacy and the requirements for antitumor effects were studied on selective depletion of immune cell types and in gene-modified mouse strains lacking basic leucine zipper ATF-like transcription factor 3 (BATF3), interferon-α/β receptor (IFNAR) or STING. Spatial requirements for the injections were studied in mice bearing three tumor lesions., Results: BO-112 and DMXAA when co-injected in one of the lesions of mice bearing concomitant bilateral tumors frequently achieved complete local and distant antitumor efficacy. Synergistic effects were contingent on CD8 T cell lymphocytes and dependent on conventional type 1 dendritic cells, responsiveness to type I interferon (IFN) and STING function in the tumor-bearing host. Efficacy was preserved even if BO-112 and DMXAA were injected in separate lesions in a manner able to control another untreated third-party tumor. Efficacy could be further enhanced on concurrent PD-1 blockade., Conclusion: Clinically feasible co-injections of BO-112 and a STING agonist attain synergistic efficacy able to eradicate distant untreated tumor lesions., Competing Interests: Competing interests: MA, CM, CEDA, MF, MV, JGG, MCO, JGV, AT, FA, MER, FX, WHS and PB declare no competing interests. MFS reports receiving research funding from Roche. MQ is full-time employee of Highlight Therapeutics. IM reports receiving commercial research grants from BMS, Highlight Therapeutics, Alligator, Pfizer Genmab and Roche; has received speakers bureau honoraria from MSD; and is a consultant or advisory board member for BMS, Roche, AstraZeneca, Genmab, Pharmamar, F-Star, Bioncotech, Bayer, Numab, Pieris, Gossamer, Alligator and Merck Serono., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) disease (COVID-19) pandemic has caused millions of deaths worldwide. Genome-wide association studies identified the 3p21.31 region as conferring a twofold increased risk of respiratory failure. Here, using a combined multiomics and machine learning approach, we identify the gain-of-function risk A allele of an SNP, rs17713054G>A, as a probable causative variant. We show with chromosome conformation capture and gene-expression analysis that the rs17713054-affected enhancer upregulates the interacting gene, leucine zipper transcription factor like 1 (LZTFL1). Selective spatial transcriptomic analysis of lung biopsies from patients with COVID-19 shows the presence of signals associated with epithelial-mesenchymal transition (EMT), a viral response pathway that is regulated by LZTFL1. We conclude that pulmonary epithelial cells undergoing EMT, rather than immune cells, are likely responsible for the 3p21.31-associated risk. Since the 3p21.31 effect is conferred by a gain-of-function, LZTFL1 may represent a therapeutic target., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Neutrophil extracellular traps (NETs) are webs of extracellular nuclear DNA extruded by dying neutrophils infiltrating tissue. NETs constitute a defence mechanism to entrap and kill fungi and bacteria. Tumours induce the formation of NETs to the advantage of the malignancy via a variety of mechanisms shown in mouse models. Here, we investigated the presence of NETs in a variety of human solid tumours and their association with IL-8 (CXCL8) protein expression and CD8 + T-cell density in the tumour microenvironment. Multiplex immunofluorescence panels were developed to identify NETs in human cancer tissues by co-staining with the granulocyte marker CD15, the neutrophil marker myeloperoxidase and citrullinated histone H3 (H3Cit), as well as IL-8 protein and CD8 + T cells. Three ELISA methods to detect and quantify circulating NETs in serum were optimised and utilised. Whole tumour sections and tissue microarrays from patients with non-small cell lung cancer (NSCLC; n = 14), bladder cancer (n = 14), melanoma (n = 11), breast cancer (n = 31), colorectal cancer (n = 20) and mesothelioma (n = 61) were studied. Also, serum samples collected retrospectively from patients with metastatic melanoma (n = 12) and NSCLC (n = 34) were ELISA assayed to quantify circulating NETs and IL-8. NETs were detected in six different human cancer types with wide individual variation in terms of tissue density and distribution. At least in NSCLC, bladder cancer and metastatic melanoma, NET density positively correlated with IL-8 protein expression and inversely correlated with CD8 + T-cell densities. In a series of serum samples from melanoma and NSCLC patients, a positive correlation between circulating NETs and IL-8 was found. In conclusion, NETs are detectable in formalin-fixed human biopsy samples from solid tumours and in the circulation of cancer patients with a considerable degree of individual variation. NETs show a positive association with IL-8 and a trend towards a negative association with CD8 + tumour-infiltrating lymphocytes. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland., (© 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.)

Objective: Understanding the immune environment of non-small cell lung cancer (NSCLC) is important for designing effective anticancer immunotherapies. We describe the use of multiplex immunofluorescence (mIF) assays to enable characterisation of the tumour-infiltrating immune cells and their interactions, both across and within immune subtypes., Methods: Six cytological samples of NSCLC taken by transoesophageal ultrasound-guided fine needle aspiration were tested with an mIF assay designed to detect the expression of key immune cell markers such as CD3, CD8, CD20, CD11b, and CD68. Pan-cytokeratin was used to detect the NSCLC cells. Fluorescence images were acquired on a Vectra-Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences)., Results: MIF assay was able to reliably detect and quantify the myeloid cell markers CD11b, CD68, CD3+ and CD8+ T cells, and CD20+ B lymphocytes on cytological samples of NSCLC. Whole-tissue analysis and its correlation with the corresponding H&E stains allowed a better understanding of the tissue morphology and the relationship between tumour and stroma compartments. Additionally, a uniform, specific, and correct staining pattern was seen for every immune marker., Conclusion: The implementation of mIF assay on cytological samples taken with minimally invasive methods seems feasible and can be used to explore the immune environment of NSCLC., (© 2021 John Wiley & Sons Ltd.)

With a growing number of predictive biomarkers needed to manage patients with non-small cell lung cancer (NSCLC), there has been a paradigm shift in care and handling of diagnostic samples. Among the various testing methods, immunohistochemistry (IHC) is the most cost- effective and widely available. Furthermore, over the past decade immunotherapy has emerged as one of the most promising cancer treatments. In this scenario IHC is the most used testing method available for PDL-1/PD1 immunotherapy. Several monoclonal antibodies targeting programmed death 1 (PD-1)/programmed death ligand-1 (PD-L1) pathways have been integrated into standard-of-care treatments of a wide range of cancer types, once provided evidence of PD-L1 expression in tumor cells by immunohistochemistry (IHC). Since currently available PD-L1 assays have been developed on formalin-fixed paraffin embedded (FFPE) histological specimens, a growing body of research is being dedicated to confirm the feasibility of applying PDL-1 assays also to cytological samples. Albeit promising results have been reported, several important issues still need to be addressed. Among these are the type of cytological samples, pre-analytical issues, cyto-histological correlation, and inter-observer agreement. This review briefly summarizes the knowledge of the role of cytopathology in the analysis of PD-L1 by immunocytochemistry (ICC) and future directions of cytopathology in the immunotherapy setting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright 2021 Tejerina, Garca Tobar, Echeveste, de Andrea, Vigliar and Lozano.)

One of the most important mechanisms by which cancer fosters its own development is the generation of an immune microenvironment that inhibits or impairs antitumor immune responses. A cancer permissive immune microenvironment is present in a large proportion of the patients with cancer who do not respond to immunotherapy approaches intended to trigger preexisting antitumor immune responses, for instance, immune checkpoint blockade. High circulating levels of IL8 in patients with cancer quite accurately predict those who will not benefit from checkpoint-based immunotherapy. IL8 has been reported to favor cancer progression and metastases via different mechanisms, including proangiogenesis and the maintenance of cancer stem cells, but its ability to attract and functionally modulate neutrophils and macrophages is arguably one of the most important factors. IL8 does not only recruit neutrophils to tumor lesions, but also triggers the extrusion of neutrophil extracellular traps (NET). The relevance and mechanisms underlying the contribution of both neutrophils and NETs to cancer development and progression are starting to be uncovered and include both direct effects on cancer cells and changes in the tumor microenvironment, such as facilitating metastasis, awakening micrometastases from dormancy, and facilitating escape from cytotoxic immune cells. Blockade of IL8 or its receptors (CXCR1 and CXCR2) is being pursued in drug development, and clinical trials alone or in combination with anti-PD-L1 checkpoint inhibitors are already ongoing., (©2020 American Association for Cancer Research.)

Background: Adoptive immunotherapy with tumour-infiltrating lymphocytes (TIL) may benefit from the use of selective markers, such as PD-1, for tumour-specific T-cell enrichment, and the identification of predictive factors that help identify those patients capable of rendering tumour-reactive TILs. We have investigated this in ovarian cancer (OC) patients as candidates for TIL therapy implementation., Methods: PD-1 - and PD-1 + CD8 TILs were isolated from ovarian tumours and expanded cells were tested against autologous tumour cells. Baseline tumour samples were examined using flow cytometry, multiplexed immunofluorescence and Nanostring technology, for gene expression analyses, as well as a next-generation sequencing gene panel, for tumour mutational burden (TMB) calculation., Results: Tumour-reactive TILs were detected in half of patients and were exclusively present in cells derived from the PD-1 + fraction. Importantly, a high TIL density in the fresh tumour, the presence of CD137 + cells within the PD-1 + CD8 + TIL subset and their location in the tumour epithelium, together with a baseline T-cell-inflamed genetic signature and/or a high TMB, are features that identify patients rendering tumour-reactive TIL products., Conclusion: We have demonstrated that PD-1 identifies ovarian tumour-specific CD8 TILs and has uncovered predictive factors that identify OC patients who are likely to render tumour-specific cells from PD-1 + TILs.

Introduction: The use of immune-checkpoint inhibitors has drastically improved the management of patients with non-small cell lung cancer (NSCLC), but innate and acquired resistances are hurdles needed to be solved. Immunomodulatory drugs that can reinvigorate the immune cytotoxic activity, in combination with antiprogrammed cell death 1 (PD-1) antibody, are a great promise to overcome resistance. We evaluated the impact of the SRC family kinases (SFKs) on NSCLC prognosis, and the immunomodulatory effect of the SFK inhibitor dasatinib, in combination with anti-PD-1, in clinically relevant mouse models of NSCLC., Methods: A cohort of patients from University Clinic of Navarra (n=116) was used to study immune infiltrates by multiplex immunofluorescence (mIF) and YES1 protein expression in tumor samples. Publicly available resources (TCGA, Km Plotter, and CIBERSORT) were used to study patient's survival based on expression of SFKs and tumor infiltrates. Syngeneic NSCLC mouse models 393P and UNSCC680AJ were used for in vivo drug testing., Results: Among the SFK members, YES1 expression showed the highest association with poor prognosis. Patients with high YES1 tumor levels also showed high infiltration of CD4+/FOXP3+ cells (regulatory T cells (Tregs)), suggesting an immunosuppressive phenotype. After testing for YES1 expression in a panel of murine cell lines, 393P and UNSCC680AJ were selected for in vivo studies. In the 393P model, dasatinib+anti-PD-1 treatment resulted in synergistic activity, with 87% tumor regressions and development of immunological memory that impeded tumor growth when mice were rechallenged. In vivo depletion experiments further showed that CD8+ and CD4+ cells are necessary for the therapeutic effect of the combination. The antitumor activity was accompanied by a very significant decrease in the number of Tregs, which was validated by mIF in tumor sections. In the UNSCC680AJ model, the antitumor effects of dasatinib+anti-PD-1 were milder but similar to the 393P model. In in vitro assays, we demonstrated that dasatinib blocks proliferation and transforming growth factor beta-driven conversion of effector CD4+ cells into Tregs through targeting of phospholymphocyte-specific protein tyrosine kinase and downstream effectors pSTAT5 and pSMAD3., Conclusions: YES1 protein expression is associated with increased numbers of Tregs in patients with NSCLC. Dasatinib synergizes with anti-PD-1 to impair tumor growth in NSCLC experimental models. This study provides the preclinical rationale for the combined use of dasatinib and PD-1/programmed death-ligand 1 blockade to improve outcomes of patients with NSCLC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Low-grade, early-stage endometrial carcinoma (EC) is the most frequent malignant tumor of the uterine corpus. However, the molecular alterations that underlie these tumors are far from being fully understood. The purpose of this study is to describe dysregulated molecular pathways from EC patients. Sixteen samples of tumor tissue and paired healthy controls were collected and both were subjected to mass spectrometry (MS)/MS proteomic analysis. Gene ontology and pathway analysis was performed to discover dysregulated pathways and/or proteins using different databases and bioinformatic tools. Dysregulated pathways were cross-validated in an independent external cohort. Cell signaling, immune response, and cell death-associated pathways were robustly identified. The SLIT/ROBO signaling pathway demonstrated dysregulation at the proteomic and transcriptomic level. Necroptosis and ferroptosis were cell death-associated processes aberrantly regulated, in addition to apoptosis. Immune response-associated pathways showed a dominance of innate immune responses. Tumor immune infiltrates measured by immunofluorescence demonstrated diverse lymphoid and myeloid populations. Our results suggest a role of SLIT/ROBO, necroptosis, and ferroptosis, as well as a prominent role of innate immune response in low-grade, early-stage EC. These results could guide future research in this group of tumors.

The precise nature of the local immune responses in lung tuberculosis (TB) granulomas requires a comprehensive understanding of their environmental complexities. At its most basic level, a granuloma is a compact, organized immune aggregate of macrophages surrounded by myeloid, B and T cells. We established two complementary multiplex immunolabeling panels to simultaneously evaluate the myeloid and lymphocytic contexture of 14 human lung TB granulomas in formalin-fixed paraffin-embedded tissue samples. We observed diverse CD3+ and CD8+ T-cell and CD20+ B lymphocyte compositions of the granuloma immune environment and a relatively homogeneous distribution of all myeloid cells. We also found significant associations between CD8+ T-cell densities and the myeloid marker CD11b and phagocytic cell marker CD68. In addition, significantly more CD68+ macrophages and CD8+ T cells were found in Mycobacterium tuberculosis-infected granulomas, as detected by Ziehl-Neelsen staining. FOXP3 expression was predominately found in a small subset of CD4+ T cells in different granulomas. As the success or failure of each granuloma is determined by the immune response within that granuloma at a local and not a systemic level, we attempted to identify the presence of reactive T cells based on expression of the T-cell activation marker CD137 (4-1BB) and programmed cell death-1 (PD-1). Only a small fraction of the CD4+ and CD8+ T cells expressed PD-1. CD137 expression was found only in a very small fraction of the CD4+ T cells in two granulomas. Our results also showed that multinucleated giant cells showed strong PD-L1 but not CTLA-4 membrane staining. This study offers new insights into the heterogeneity of immune cell infiltration in lung TB granulomas, suggesting that each TB granuloma represents a unique immune environment that might be independently influenced by the local adaptive immune response, bacterial state, and overall host disease status.

In December 2019, an outbreak of severe acute respiratory syndrome associated to SARS-CoV2 was reported in Wuhan, China. To date, little is known on histopathological findings in patients infected with the new SARS-CoV2. Lung histopathology shows features of acute and organising diffuse alveolar damage. Subtle cellular inflammatory infiltrate has been found in line with the cytokine storm theory. Medium-size vessel thrombi were frequent, but capillary thrombi were not present. Despite the elevation of biochemical markers of cardiac injury, little histopathological damage could be confirmed. Viral RNA from paraffin sections was detected at least in one organ in 90% patients., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Macrophage polarization is relevant for tumor biology. M2 polarized macrophages favor tumor growth and survival, while M1 macrophages support tumor destruction and antigen presentation. Markers identifying M1/M2 polarization are a subject of debate. We conducted a systematic review and meta-analysis to investigate the association of proposed macrophage markers with prognosis across epithelial tumors and melanoma. The Medline search engine was used and 195 articles were recovered for full review. Only articles which measured markers using immunohistochemistry or immunofluorescence and had overall survival (OS) as the primary endpoint were included. One hundred and thirteen articles were finally accepted for analysis. CD68 was associated with worse survival across tumors (hazard ratio (HR) = 1.24, 95% CI = 1.11-1.37). Tumor anatomical location influenced this association. Colorectal tumors showed an inverse association between CD68 and OS in contrast to the rest of cancer types (HR = 0.56 vs. 1.34). The approach taken to measure CD68 had an impact on prognosis; when macrophages were measured at the tumor invasion front prognosis was more favorable than when they were measured intratumorally (HR = 0.94 vs. 1.4). CD163, CD204, and CD206 showed a robust association with worse OS (HR = 1.63, 1.95, 1.65, respectively). Tumors arising in the lung and the liver showed a weaker association between CD163 and OS as compared with other locations (β = -0.5401 for the lung and -0.5940 for the liver compared with other anatomical locations). The counting strategy also had an impact on CD163 association with OS, with hot-spot counting having higher HRs compared with averaging macrophage counts across spots or absolute cell counting (β = -0.4678). In conclusion, proposed M2 markers are associated with worse survival across epithelial tumors and melanoma. The anatomical origin of tumors influences this association. The compartment where the macrophages were scored and counting strategy influenced the association with OS of CD68 and CD163, respectively.

Checkpoint inhibitors have improved the survival of patients with advanced tumors and show a manageable toxicity profile. However, auto-immune colitis remains a relevant side effect, and combinations of anti-PD1/PDL1 and anti-CTLA-4 increase its incidence and severity. Here, we report the case of a 50-year-old patient diagnosed with stage IV cervical cancer that relapsed following radical surgery, external radiation/brachytherapy and standard chemotherapy. She was subsequently treated with Nivolumab and Ipilimumab combination and developed grade 2 colitis presenting a dissociation between endoscopic and pathological findings. At cycle 10 the patient reported grade 3 diarrhea and abdominal discomfort, without blood or mucus in the stools. Immunotherapy was withheld and a colonoscopy was performed, showing normal mucosa in the entire colon. Puzzlingly, histologic evaluation of randomly sampled mucosal biopsy of the distal colon showed an intense intraepithelial lymphocyte infiltration with crypt loss and some regenerating crypts with a few apoptotic bodies set in a chronically inflamed lamina propria, consistent with the microscopic diagnosis of colitis. Treatment with methylprednisolone 2 mg/kg was initiated which led to a decrease in the number of stools to grade 1. Additional investigations to exclude other causes of diarrhea rendered negative results. The patient experienced a major partial response and, following the resolution of diarrhea, she was re-challenged again with immunotherapy, with the reappearance of grade 2 diarrhea, leading to permanent immunotherapy interruption. We conclude and propose that performing random colonic biopsies should be considered in cases of immune checkpoint-associated unexplained diarrhea, even when colonoscopy shows macroscopically normal colonic mucosa inflammatory lesions., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Activation-induced cell death (AICD) is a complex immunoregulatory mechanism that causes the demise of a fraction of T-lymphocytes upon antigen-driven activation. In the present study we investigated the direct role of TNF in AICD of CD8 T lymphocytes. Methods : Human peripheral mononuclear cells were isolated from healthy donors and fresh tumor-infiltrating lymphocytes were obtained from cancer patients undergoing surgery. T cells were activated with anti-CD3/CD28 mAbs or with a pool of virus peptides, in combination with clinical-grade TNF blocking agents. Results : A portion of CD8 T cells undergoes apoptosis upon CD3/CD28 activation in a manner that is partially prevented by the clinically used anti-TNF agents infliximab and etanercept. TNF-mediated AICD was also observed upon activation of virus-specific CD8 T cells and tumor-infiltrating CD8 T lymphocytes. The mechanism of TNF-driven T cell death involves TNFR2 and production of mitochondrial oxygen free radicals which damage DNA. Conclusion : The use of TNF blocking agents reduces oxidative stress, hyperpolarization of mitochondria, and the generation of DNA damage in CD8 T celss undergoing activation. The fact that TNF mediates AICD in human tumor-reactive CD8 T cells suggests that the use of TNF-blocking agents can be exploited in immunotherapy strategies., Competing Interests: Competing Interests: I.M. reports advisory roles with Roche-Genentech, Bristol-Myers Squibb, CYTOMX, Incyte, MedImmune, Tusk, F-Star, Genmab, Molecular Partners, Alligator, Bioncotech, MSD, Merck Serono, Boehringer Ingelheim, Astra Zeneca, Numab, Catalym, and Bayer, and research funding from Roche, BMS, Alligator, and Bioncotech. P.B. reports advisory roles with Tusk and Moderna, research funding from Sanofi, and Bavarian Nordic and speaker honoraria from BMS, MSD, Novartis and AstraZeneca. The rest of the authors have no conflict of interest to declare., (© The author(s).)

Despite the advent of immunotherapy, metastatic melanoma represents an aggressive tumor type with a poor survival outcome. The successful application of immunotherapy requires in-depth understanding of the biological basis and immunosuppressive mechanisms within the tumor microenvironment. In this study, we conducted spatially explicit analyses of the stromal-immune interface across 400 melanoma hematoxylin and eosin (H&E) specimens from The Cancer Genome Atlas. A computational pathology pipeline (CRImage) was used to classify cells in the H&E specimen into stromal, immune, or cancer cells. The estimated proportions of these cell types were validated by independent measures of tumor purity, pathologists' estimate of lymphocyte density, imputed immune cell subtypes, and pathway analyses. Spatial interactions between these cell types were computed using a graph-based algorithm (topological tumor graphs, TTG). This approach identified two stromal features, namely stromal clustering and stromal barrier, which represented the melanoma stromal microenvironment. Tumors with increased stromal clustering and barrier were associated with reduced intratumoral lymphocyte distribution and poor overall survival independent of existing prognostic factors. To explore the genomic basis of these TTG-derived stromal phenotypes, we used a deep learning approach integrating genomic (copy number) and transcriptomic data, thereby inferring a compressed representation of copy number-driven alterations in gene expression. This integrative analysis revealed that tumors with high stromal clustering and barrier had reduced expression of pathways involved in naïve CD4 signaling, MAPK, and PI3K signaling. Taken together, our findings support the immunosuppressive role of stromal cells and T-cell exclusion within the vicinity of melanoma cells. SIGNIFICANCE: Computational histology-based stromal phenotypes within the tumor microenvironment are significantly associated with prognosis and immune exclusion in melanoma., (©2019 American Association for Cancer Research.)

Harnessing the immune system by blocking the programmed cell death protein 1 (PD-1) pathway has been a major breakthrough in non-small-cell lung cancer treatment. Nonetheless, many patients fail to respond to PD-1 inhibition. Using three syngeneic models, we demonstrate that short-term starvation synergizes with PD-1 blockade to inhibit lung cancer progression and metastasis. This antitumor activity was linked to a reduction in circulating insulin-like growth factor 1 (IGF-1) and a downregulation of IGF-1 receptor (IGF-1R) signaling in tumor cells. A combined inhibition of IGF-1R and PD-1 synergistically reduced tumor growth in mice. This effect required CD8 cells, boosted the intratumoral CD8/T reg ratio and led to the development of tumor-specific immunity. In patients with non-small-cell lung cancer, high plasma levels of IGF-1 or high IGF-1R expression in tumors was associated with resistance to anti-PD-1-programmed death-ligand 1 immunotherapy. In conclusion, our data strongly support the clinical evaluation of IGF-1 modulators in combination with PD-1 blockade., (© 2020. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objective: Alzheimer disease (AD) is the leading cause of dementia, and although its etiology remains unclear, it seems that type 2 diabetes mellitus (T2DM) and other prediabetic states of insulin resistance could contribute to the appearance of sporadic AD. As such, we have assessed whether tau and β-amyloid (Aβ) deposits might be present in pancreatic tissue of subjects with AD, and whether amylin, an amyloidogenic protein deposited in the pancreas of T2DM patients, might accumulate in the brain of AD patients., Methods: We studied pancreatic and brain tissue from 48 individuals with no neuropathological alterations and from 87 subjects diagnosed with AD. We examined Aβ and tau accumulation in the pancreas as well as that of amylin in the brain. Moreover, we performed proximity ligation assays to ascertain whether tau and/or Aβ interact with amylin in either the pancreas or brain of these subjects., Results: Cytoplasmic tau and Aβ protein deposits were detected in pancreatic β cells of subjects with AD as well as in subjects with a normal neuropathological examination but with a history of T2DM and in a small cohort of control subjects without T2DM. Furthermore, we found amylin deposits in the brain of these subjects, providing histological evidence that amylin can interact with Aβ and tau in both the pancreas and hippocampus., Interpretation: The presence of both tau and Aβ inclusions in pancreatic β cells, and of amylin deposits in the brain, provides new evidence of a potential overlap in the mechanisms underlying the pathogenesis of T2DM and AD. ANN NEUROL 2019;86:539-551., (© 2019 American Neurological Association.)