CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity.

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.

In situ tumour arrays reveal early environmental control of cancer immunity.

The immune phenotype of a tumour is a key predictor of its response to immunotherapy1-4. Patients who respond to checkpoint blockade generally present with immune-inflamed5-7 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.

Oncolytic DNX-2401 Virus for Pediatric Diffuse Intrinsic Pontine Glioma.

BACKGROUND: Pediatric patients with diffuse intrinsic pontine glioma (DIPG) have a poor prognosis, with a median survival of less than 1 year. Oncolytic viral therapy has been evaluated in patients with pediatric gliomas elsewhere in the brain, but data regarding oncolytic viral therapy in patients with DIPG are lacking. METHODS: We conducted a single-center, dose-escalation study of DNX-2401, an oncolytic adenovirus that selectively replicates in tumor cells, in patients with newly diagnosed DIPG. The patients received a single virus infusion through a catheter placed in the cerebellar peduncle, followed by radiotherapy. The primary objective was to assess the safety and adverse-event profile of DNX-2401. The secondary objectives were to evaluate the effect of DNX-2401 on overall survival and quality of life, to determine the percentage of patients who have an objective response, and to collect tumor-biopsy and peripheral-blood samples for correlative studies of the molecular features of DIPG and antitumor immune responses. RESULTS: A total of 12 patients, 3 to 18 years of age, with newly diagnosed DIPG received 1×1010 (the first 4 patients) or 5×1010 (the subsequent 8 patients) viral particles of DNX-2401, and 11 received subsequent radiotherapy. Adverse events among the patients included headache, nausea, vomiting, and fatigue. Hemiparesis and tetraparesis developed in 1 patient each. Over a median follow-up of 17.8 months (range, 5.9 to 33.5), a reduction in tumor size, as assessed on magnetic resonance imaging, was reported in 9 patients, a partial response in 3 patients, and stable disease in 8 patients. The median survival was 17.8 months. Two patients were alive at the time of preparation of the current report, 1 of whom was free of tumor progression at 38 months. Examination of a tumor sample obtained during autopsy from 1 patient and peripheral-blood studies revealed alteration of the tumor microenvironment and T-cell repertoire. CONCLUSIONS: Intratumoral infusion of oncolytic virus DNX-2401 followed by radiotherapy in pediatric patients with DIPG resulted in changes in T-cell activity and a reduction in or stabilization of tumor size in some patients but was associated with adverse events. (Funded by the European Research Council under the European Union's Horizon 2020 Research and Innovation Program and others; EudraCT number, 2016-001577-33; ClinicalTrials.gov number, NCT03178032.).

Prophylactic TNF blockade uncouples efficacy and toxicity in dual CTLA-4 and PD-1 immunotherapy.

Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer1-3. However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients4. In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2-/-Il2rg-/- mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy.

Cell-Specific Dysregulation of Iron and Oxygen Homeostasis as a Novel Pathophysiology in PSP.

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.

Immunotherapy and lung cytopathology: Overview and possibilities.

Immunotherapy has become a promising cancer treatment in the past decade, and IHC is the most commonly used testing method for PDL-1/PD1 evaluation. In general, PD-L1 assays can be performed on both FFPE specimens and cytological samples. However, their use on smears is not yet well-established or validated. Nowadays, digital images and advanced algorithms can aid in interpreting PD-L1 in cytological samples. Understanding the immune environment of non-small cell lung cancer (NSCLC) is critical in developing successful anticancer immunotherapies. The use of a multiplexed immunofluorescence (mIF) assay on cytological samples obtained through minimally invasive methods appears to be a viable option for investigating the immune environment of NSCLC. This review aims to briefly summarize 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.

The association between the tumor immune microenvironments and clinical outcome in low-grade, early-stage endometrial cancer patients.

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.

Alpelisib decreases nevocytes of congenital melanocytic nevi.

BACKGROUND: Multiple, large or giant congenital melanocytic nevi (CMN) are uncommon and affected patients can show progressive growth and thickening, associate neurocutaneous melanocytosis or develop melanoma. Current treatment modalities are mostly complex surgeries that frequently do not solve the disease and its risks completely. Thus, investigation on new treatment options for CMN and its complications must continue. MAPK pathway inhibitors are being investigated, also targeting PI3K-AKT. Omipalisib (PI3K inhibitor, with no indications approved yet) has been studied for CMN in vitro and in mice with promising results. However, alpelisib, a PI3K inhibitor approved with an adequate safety profile for patients with severe manifestations of PROS (PIK3CA-Related Overgrowth Spectrum), had not yet been tested for CMN. OBJECTIVE: To evaluate the effect of alpelisib in nevocytes of congenital melanocytic nevi. METHODS: Nevomelanocytic tissue samples of 10 patients were collected prospectively and, following a previously reported preclinical ex vivo model, explants were placed in organotypic culture for 5 days, with or without alpelisib. Consecutively, tissue sections were stained and using scanned images with Qupath and ImageJ softwares, representative regions from the dermis were analysed (using Wilcoxon test and Spearman's correlation). RESULTS: When comparing alpelisib-treated explants with respect to control explants, we found a decrease in cell density (p = 0.0273), in density of SOX10+ -cells (p = 0.0391) and also in the % of S-100+ area (p = 0.0078), in alpelisib samples. The three markers showed a positive correlation (p < 0.05). CONCLUSIONS: This study provides first-time evidence that alpelisib induces nevocyte reduction in CMN from patient-derived explants, probably inducted by autophagy. Alpelisib is an approved drug with an adequate safety profile used in another mosaicism affecting PI3K (PROS). Further studies are needed to evaluate its efficacy in treating CMN and potentially, their complications, either with local or systemic administration, alone or in combination.

Heterogenous presence of neutrophil extracellular traps in human solid tumours is partially dependent on IL-8.

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.

High IGKC-Expressing Intratumoral Plasma Cells Predict Response to Immune Checkpoint Blockade.

Resistance to Immune Checkpoint Blockade (ICB) constitutes the current limiting factor for the optimal implementation of this novel therapy, which otherwise demonstrates durable responses with acceptable toxicity scores. This limitation is exacerbated by a lack of robust biomarkers. In this study, we have dissected the basal TME composition at the gene expression and cellular levels that predict response to Nivolumab and prognosis. BCR, TCR and HLA profiling were employed for further characterization of the molecular variables associated with response. The findings were validated using a single-cell RNA-seq data of metastatic melanoma patients treated with ICB, and by multispectral immunofluorescence. Finally, machine learning was employed to construct a prediction algorithm that was validated across eight metastatic melanoma cohorts treated with ICB. Using this strategy, we have unmasked a major role played by basal intratumoral Plasma cells expressing high levels of IGKC in efficacy. IGKC, differentially expressed in good responders, was also identified within the Top response-related BCR clonotypes, together with IGK variants. These results were validated at gene, cellular and protein levels; CD138+ Plasma-like and Plasma cells were more abundant in good responders and correlated with the same RNA-seq-defined fraction. Finally, we generated a 15-gene prediction model that outperformed the current reference score in eight ICB-treated metastatic melanoma cohorts. The evidenced major contribution of basal intratumoral IGKC and Plasma cells in good response and outcome in ICB in metastatic melanoma is a groundbreaking finding in the field beyond the role of T lymphocytes.

The mutational load and a T-cell inflamed tumour phenotype identify ovarian cancer patients rendering tumour-reactive T cells from PD-1+ tumour-infiltrating lymphocytes.

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.

Utilisation of cytological samples for multiplex immunofluorescence assay.

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.

Histopathological findings in fatal COVID-19 severe acute respiratory syndrome: preliminary experience from a series of 10 Spanish patients.

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.

Prognostic value of macrophage polarization markers in epithelial neoplasms and melanoma. A systematic review and meta-analysis.

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 (ß = -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 (ß = -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.

Diverse immune environments in human lung tuberculosis granulomas assessed by quantitative multiplexed immunofluorescence.

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.

Amylin as a potential link between type 2 diabetes and alzheimer disease.

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 ß-amyloid (Aß) 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 Aß 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 Aß interact with amylin in either the pancreas or brain of these subjects. RESULTS: Cytoplasmic tau and Aß protein deposits were detected in pancreatic ß 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 Aß and tau in both the pancreas and hippocampus. INTERPRETATION: The presence of both tau and Aß inclusions in pancreatic ß 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.

Cell cycle deregulation and mosaic loss of Ext1 drive peripheral chondrosarcomagenesis in the mouse and reveal an intrinsic cilia deficiency.

Peripheral chondrosarcoma (PCS) develops as malignant transformation of an osteochondroma, a benign cartilaginous outgrowth at the bone surface. Its invasive, lobular growth despite low-grade histology suggests a loss of chondrocyte polarity. The known genetics of osteochondromagenesis include mosaic loss of EXT1 or EXT2 in both hereditary and non-hereditary cases. The most frequent genetic aberrations in human PCS also include disruptions of CDKN2A or TP53. In order to test the sufficiency of either of these to drive progression of an osteochondroma to PCS, we added conditional loss of Trp53 or Ink4a/Arf in an Ext1-driven mouse model of osteochondromagenesis. Each additional tumour suppressor silencing efficiently drove the development of growths that mimic human PCS. As in humans, lobules developed from both Ext1-null and Ext1-functional clones within osteochondromas. Assessment of their orientation revealed an absence of primary cilia in the majority of mouse PCS chondrocytes, which was corroborated in human PCSs. Loss of primary cilia may be responsible for the lost polarity phenotype ascribed to PCS. Cilia deficiency blocks proliferation in physeal chondrocytes, but cell cycle deregulation is sufficient to rescue chondrocyte proliferation following deciliation. This provides a basis of selective pressure for the frequent cell-cycle regulator silencing observed in peripheral chondrosarcomagenesis. Mosaic loss of Ext1 combined with loss of cell cycle regulators promotes peripheral chondrosarcomagenesis in the mouse and reveals deficient ciliogenesis in both the model and the human disease, explaining biological behaviour including lobular and invasive growth.

GNAS mutations are not detected in parosteal and low-grade central osteosarcomas.

Parosteal osteosarcoma, low-grade central osteosarcoma, and fibrous dysplasia share similar histological features that may pose a diagnostic challenge. The detection of GNAS mutations in primary bone tumors has been useful in clinical practice for diagnosing fibrous dysplasia. However, the recent report of GNAS mutations being detected in a significant proportion of parosteal osteosarcoma challenges the specificity of this mutation. As the number of cases reported in this study was small we set out to determine if these results could be reproduced. We studied 97 formalin-fixed paraffin-embedded low-grade osteosarcomas from 90 patients including 62 parosteal osteosarcomas, of which MDM2 amplification was detected in 79%, 11 periosteal osteosarcomas and 24 low-grade central osteosarcoma samples. The mutational status of GNAS was analyzed in codons p.R201, p.Q227, and other less common GNAS alterations by bidirectional Sanger sequencing and/or next generation sequencing using the Life Technologies Ion Torrent platform. GNAS mutations were not detected in any of the low-grade osteosarcomas from which informative DNA was extracted. Our findings therefore support prior observations that GNAS mutations are highly specific for fibrous dysplasia and occur rarely, if ever, in parosteal and other low-grade osteosarcomas.