Histopathological Phenotypes Research Articles

MODL-16. Generation of immunocompetent syngeneic allograft mouse models for pediatric diffuse midline glioma

Abstract Diffuse midline gliomas (DMG) are highly aggressive pediatric brain tumors with a grim prognosis. A lack of effective treatment options highlights the critical need to investigate new therapeutic strategies. This includes the use of immunotherapy, which has shown promise in other hard-to-treat tumors. To facilitate immunotherapeutic research in this field, and to complement the existing immunodeficient patient-derived DMG models, we developed three distinct immunocompetent mouse models representing different DMG subtypes, i.e., histone 3 wildtype and histone 3 K27M mutant DMG (H3.3K27M or H3.1K27M), that can be used for preclinical testing of new therapies. We first established primary tumor cell cultures from murine DMG tumors that were generated by brainstem-targeted intra-uterine electroporation (IUE). This method enabled the introduction of DMG-associated mutations within the intact developing brainstem, thereby generating DMG tumors in a spatially and temporally defined manner, while maintaining a genetically identical (isogenic) background. We then created allograft DMG mouse models by orthotopically implanting the established primary cell cultures into syngeneic (C57BL/6) mice. Herewith, we provide an allograft tool that is better suitable for large-scale therapeutic studies and more accessible to the scientific community. Importantly, we demonstrated that these allograft models recapitulate the histopathologic phenotype of human DMG, including their diffuse infiltrative growth and expression of DMG-associated antigens. Furthermore, CyTOF mass cytometry analysis indicated that these murine pontine tumors exhibit a tumor immune microenvironment (TIME) similar to human DMG, characterized by considerable myeloid cell infiltration and a paucity of T-lymphocytes and NK cells. As such, we provide a representative model to further delineate the immune landscape in DMG and to preclinically investigate novel (immuno)therapies. Currently, we are using these immunocompetent models to study the interaction between DMG cells and microglia, and we are investigating how we can modify the immune microenvironment to improve checkpoint inhibition in DMG.

A COL5A2 In-Frame Deletion in a Chihuahua with Ehlers-Danlos Syndrome.

Ehlers-Danlos syndrome (EDS) is a group of heterogeneous, rare diseases affecting the connective tissues. The main clinical signs of EDS are skin hyperextensibility, joint hypermobility, and skin fragility. Currently, the classification of EDS in humans distinguishes 13 clinical subtypes associated with variants in 20 different genes, reflecting the heterogeneity of this set of diseases. At present, variants in three of these genes have also been identified in dogs affected by EDS. The purpose of this study was to characterize the clinical and histopathological phenotype of an EDS-affected Chihuahua and to identify the causative genetic variant for the disease. The clinical examination suggested a diagnosis of classical EDS. Skin histopathology revealed an abnormally thin dermis, which is compatible with classical EDS. Whole-genome sequencing identified a heterozygous de novo 27 bp deletion in the COL5A2 gene, COL5A2:c.3388_3414del. The in-frame deletion is predicted to remove 9 amino acids in the triple-helical region of COL5A2. The molecular analysis and identification of a likely pathogenic variant in COL5A2 confirmed the subtype as a form of classical EDS. This is the first report of a COL5A2-related EDS in a dog.

FC080: A Systems Nephrology Framework for the Molecular Classification of Chronic Kidney Disease

Abstract BACKGROUND Conventional stratification by clinical and histopathological phenotypes only approximates the heterogeneity of chronic kidney disease (CKD) and is insufficient to drive discovery of disease-modifying therapies or predict clinical outcomes. Recent advances in molecular stratification offer a mechanistic approach to disease classification but are limited by the availability of rich patient cohort datasets [1]. The National Unified Renal Translational Research Enterprise (NURTuRE) is a unique prospective cohort study for CKD and idiopathic nephrotic syndrome that comprises bio-banked patient samples from a broad range of clinical diagnoses and kidney functional states. Access to diagnostic biopsies and biofluids enables in-depth histological and molecular analysis and offers unique opportunities for a mechanistic disease understanding. Importantly, anonymized rich clinical data are available through the UK Renal Registry, allowing for a comprehensive view on CKD heterogeneity. Here we used unsupervised molecular clustering and dimensionality reduction to integrate and visualize the complex relationship between molecular, morphological and clinical data in this large cohort. We aim to generate mechanistic disease understanding for patient-centric, integrated target and biomarker discovery that will enable the development of novel precision treatments. METHODS Diagnostic kidney biopsies were obtained from the NURTuRE biobank. Samples from 286 patients comprising 16 primary diagnoses were RNA-sequenced and passed extended control of library quality and composition. Self-organizing maps (SOM) as implemented in the OposSOM R package were used for unsupervised clustering of transcriptomes, after adjusting for batch effects and sex. We used PHATE dimensionality reduction to visualize global data structure with the aim to project clinical, morphological and molecular data onto the resulting nonlinear disease trajectories. RESULTS Unsupervised clustering of kidney transcriptomes inferred five groups with distinct molecular landscapes (F, E, C, A and AB) that were generally consistent with molecular clusters previously described for CKD.1 Correlation of metagenes revealed a highly polarized global data structure with cluster C connecting F and E with the opposing clusters A and AB. Gene set enrichment analysis suggested that the polarization results from strong opposing metabolic and immune signatures, likely reflecting tubular atrophy, immune response and infiltration (Figure 1A). Consistently, cluster identity was independent of primary diagnosis but aligned with disease progression as reflected by kidney function decline (eGFR), increasing inflammation (CRP) and age range (Figure 1B). PHATE dimensionality reduction revealed complex nonlinear relations of transcriptomes aligned with disease progression and interpreted as molecular disease trajectories partitioned by SOM clusters (Figure 1C). We are now integrating real-world data, including longitudinal kidney function and free-text histopathology reports, and are expanding the molecular analysis by proteomics and genomics. Initial exploration of histopathology and eGFR series suggests a nonrandom distribution of morphological risk factors and disease progression rates across molecular clusters and trajectories, highlighting unique opportunities for drug discovery. CONCLUSIONS Molecular stratification aligns with disease progression irrespective of clinical diagnosis, reflecting common cellular and molecular mechanisms of disease associated with kidney function decline and substantial morphological changes. Further integration of complementary datasets, including clinical time series, histopathology and multiomics, will enable mechanistic interpretation of molecular clusters and trajectories with the goal of identifying novel, targetable drivers of CKD in well-defined patient subsets.

FC080: A Systems Nephrology Framework for the Molecular Classification of Chronic Kidney Disease

Abstract BACKGROUND Conventional stratification by clinical and histopathological phenotypes only approximates the heterogeneity of chronic kidney disease (CKD) and is insufficient to drive discovery of disease-modifying therapies or predict clinical outcomes. Recent advances in molecular stratification offer a mechanistic approach to disease classification but are limited by the availability of rich patient cohort datasets [1]. The National Unified Renal Translational Research Enterprise (NURTuRE) is a unique prospective cohort study for CKD and idiopathic nephrotic syndrome that comprises bio-banked patient samples from a broad range of clinical diagnoses and kidney functional states. Access to diagnostic biopsies and biofluids enables in-depth histological and molecular analysis and offers unique opportunities for a mechanistic disease understanding. Importantly, anonymized rich clinical data are available through the UK Renal Registry, allowing for a comprehensive view on CKD heterogeneity. Here we used unsupervised molecular clustering and dimensionality reduction to integrate and visualize the complex relationship between molecular, morphological and clinical data in this large cohort. We aim to generate mechanistic disease understanding for patient-centric, integrated target and biomarker discovery that will enable the development of novel precision treatments. METHODS Diagnostic kidney biopsies were obtained from the NURTuRE biobank. Samples from 286 patients comprising 16 primary diagnoses were RNA-sequenced and passed extended control of library quality and composition. Self-organizing maps (SOM) as implemented in the OposSOM R package were used for unsupervised clustering of transcriptomes, after adjusting for batch effects and sex. We used PHATE dimensionality reduction to visualize global data structure with the aim to project clinical, morphological and molecular data onto the resulting nonlinear disease trajectories. RESULTS Unsupervised clustering of kidney transcriptomes inferred five groups with distinct molecular landscapes (F, E, C, A and AB) that were generally consistent with molecular clusters previously described for CKD.1 Correlation of metagenes revealed a highly polarized global data structure with cluster C connecting F and E with the opposing clusters A and AB. Gene set enrichment analysis suggested that the polarization results from strong opposing metabolic and immune signatures, likely reflecting tubular atrophy, immune response and infiltration (Figure 1A). Consistently, cluster identity was independent of primary diagnosis but aligned with disease progression as reflected by kidney function decline (eGFR), increasing inflammation (CRP) and age range (Figure 1B). PHATE dimensionality reduction revealed complex nonlinear relations of transcriptomes aligned with disease progression and interpreted as molecular disease trajectories partitioned by SOM clusters (Figure 1C). We are now integrating real-world data, including longitudinal kidney function and free-text histopathology reports, and are expanding the molecular analysis by proteomics and genomics. Initial exploration of histopathology and eGFR series suggests a nonrandom distribution of morphological risk factors and disease progression rates across molecular clusters and trajectories, highlighting unique opportunities for drug discovery. CONCLUSIONS Molecular stratification aligns with disease progression irrespective of clinical diagnosis, reflecting common cellular and molecular mechanisms of disease associated with kidney function decline and substantial morphological changes. Further integration of complementary datasets, including clinical time series, histopathology and multiomics, will enable mechanistic interpretation of molecular clusters and trajectories with the goal of identifying novel, targetable drivers of CKD in well-defined patient subsets.

Humanized mice reveal a macrophage-enriched gene signature defining human lung tissue protection during SARS-CoV-2 infection.

The human immunological mechanisms defining the clinical outcome of SARS-CoV-2 infection remain elusive. This knowledge gap is mostly driven by the lack of appropriate experimental platforms recapitulating human immune responses in a controlled human lung environment. Here, we report a mouse model (i.e., HNFL mice) co-engrafted with human fetal lung xenografts (fLX) and a myeloid-enhanced human immune system to identify cellular and molecular correlates of lung protection during SARS-CoV-2 infection. Unlike mice solely engrafted with human fLX, HNFL mice are protected against infection, severe inflammation, and histopathological phenotypes. Lung tissue protection from infection and severe histopathology associates with macrophage infiltration and differentiation and the upregulation of a macrophage-enriched signature composed of 11 specific genes mainly associated with the type I interferon signaling pathway. Our work highlights the HNFL model as a transformative platform to investigate, in controlled experimental settings, human myeloid immune mechanisms governing lung tissue protection during SARS-CoV-2 infection.

Somatic Mosaicism of a PDGFRB Activating Variant in Aneurysms of the Intracranial, Coronary, Aortic, and Radial Artery Vascular Beds.

BackgroundActivating variants in platelet‐derived growth factor receptor beta (PDGFRB), including a variant we have previously described (p.Tyr562Cys [g.149505130T>C [GRCh37/hg19]; c.1685A>G]), are associated with development of multiorgan pathology, including aneurysm formation. To investigate the association between the allele fraction genotype and histopathologic phenotype, we performed an expanded evaluation of post‐mortem normal and aneurysmal tissue specimens from the previously published index patient.Methods and ResultsFollowing death due to diffuse subarachnoid hemorrhage in a patient with mosaic expression of the above PDGFRB variant, specimens from the intracranial, coronary, radial and aortic arteries were harvested. DNA was extracted and alternate allele fractions (AAF) of PDGFRB were determined using digital droplet PCR. Radiographic and histopathologic findings, together with genotype expression of PDGFRB were then correlated in aneurysmal tissue and compared to non‐aneurysmal tissue. The PDGFRB variant was identified in the vertebral artery, basilar artery, and P1 segment aneurysms (AAF: 28.7%, 16.4%, and 17.8%, respectively). It was also identified in the coronary and radial artery aneurysms (AAF: 22.3% and 20.6%, respectively). In phenotypically normal intracranial and coronary artery tissues, the PDGFRB variant was not present. The PDGFRB variant was absent from lymphocyte DNA and normal tissue, confirming it to be a non‐germline somatic variant. Primary cell cultures from a radial artery aneurysm localized the PDGFRB variant to CD31‐, non‐endothelial cells.ConclusionsConstitutive expression of PDGFRB within the arterial wall is associated with the development of human fusiform aneurysms. The role of targeted therapy with tyrosine kinase inhibitors in fusiform aneurysms with PDGFRB mutations should be further studied.

CD147 expression lacks prognostic relevance in esophageal cancer

IntroductionThe role of CD147 as an important indicator of tumor prognosis remains controversially discussed in literature. We focused on the prognostic significance of CD147 expression in esophageal cancer patients. While some studies report that CD147 is an unfavorable prognostic factor in esophageal squamous cell carcinoma, others showed no significant correlation. However, only one study draws attention to the significance of CD147 in esophageal adenocarcinoma, which is one of the most rapidly increasing neoplasms in the western world.MethodsTo finally clarify the impact of CD147 as a prognostic factor, especially for esophageal adenocarcinomas, we analyzed CD147 expression in a tissue microarray of 359 esophageal adenocarcinomas and 254 esophageal squamous cell cancer specimens. For the immuno-histochemical analysis, we used a primary antibody specific for CD147. Staining intensity and proportion of positive tumor cells were scored (negative, weak, moderate, strong staining). These findings were compared to normal esophageal tissue and correlated to the histopathological tumor phenotype and survival data.ResultsCD147 expression was detectable in weak intensities in benign esophageal tissue (85.78%) and expressed in predominately moderate to strong intensities in esophageal cancer (88.34%). Strong CD147 immunostaining was linked to increased infiltration depth (p = 0.015) and differentiation (p = 0.016) in esophageal squamous cell cancer but revealed no significant correlation with histopathology of adenocarcinoma. Moreover, CD147 intensity was unrelated to overall survival in this collective for both subtypes of esophageal cancer.ConclusionThus, our data show that CD147 has no prognostic value, neither in esophageal adenocarcinoma nor squamous cell carcinoma.

Generation of immunocompetent syngeneic allograft mouse models for pediatric diffuse midline glioma.

BackgroundDiffuse midline gliomas (DMG) are highly malignant incurable pediatric brain tumors. A lack of effective treatment options highlights the need to investigate novel therapeutic strategies. This includes the use of immunotherapy, which has shown promise in other hard-to-treat tumors. To facilitate preclinical immunotherapeutic research, immunocompetent mouse models that accurately reflect the unique genetic, anatomical, and histological features of DMG patients are warranted.MethodsWe established cell cultures from primary DMG mouse models (C57BL/6) that were generated by brainstem targeted intra-uterine electroporation (IUE). We subsequently created allograft DMG mouse models by orthotopically implanting these tumor cells into syngeneic mice. Immunohistochemistry and -fluorescence, mass cytometry, and cell-viability assays were then used to verify that these murine tumors recapitulated human DMG.ResultsWe generated three genetically distinct allograft models representing histone 3 wildtype (H3WT) and K27M-mutant DMG (H3.3K27M and H3.1K27M). These allograft models recapitulated the histopathologic phenotype of their human counterparts, including their diffuse infiltrative growth and expression of DMG-associated antigens. These murine pontine tumors also exhibited an immune microenvironment similar to human DMG, characterized by considerable myeloid cell infiltration and a paucity of T-lymphocytes and NK cells. Finally, we show that these murine DMG cells display similar sensitivity to histone deacetylase (HDAC) inhibition as patient-derived DMG cells.ConclusionsWe created and validated an accessible method to generate immunocompetent allograft models reflecting different subtypes of DMG. These models adequately recapitulated the histopathology, immune microenvironment, and therapeutic response of human DMG, providing useful tools for future preclinical studies.

Comprehensive Imaging Characterization of Colorectal Liver Metastases

Colorectal liver metastases (CRLM) have heterogenous histopathological and immunohistochemical phenotypes, which are associated with variable responses to treatment and outcomes. However, this information is usually only available after resection, and therefore of limited value in treatment planning. Improved techniques for in vivo disease assessment, which can characterise the variable tumour biology, would support further personalization of management strategies. Advanced imaging of CRLM including multiparametric MRI and functional imaging techniques have the potential to provide clinically-actionable phenotypic characterisation. This includes assessment of the tumour-liver interface, internal tumour components and treatment response. Advanced analysis techniques, including radiomics and machine learning now have a growing role in assessment of imaging, providing high-dimensional imaging feature extraction which can be linked to clinical relevant tumour phenotypes, such as a the Consensus Molecular Subtypes (CMS). In this review, we outline how imaging techniques could reproducibly characterize the histopathological features of CRLM, with several matched imaging and histology examples to illustrate these features, and discuss the oncological relevance of these features. Finally, we discuss the future challenges and opportunities of CRLM imaging, with a focus on the potential value of advanced analytics including radiomics and artificial intelligence, to help inform future research in this rapidly moving field.

Unbiased profiling of miR-132 targetome with implications in adult hippocampal neurogenesis and Alzheimer's disease.

MicroRNA-132 (miR-132), a potent neuroimmune regulator, is robustly downregulated in the brain of Alzheimer's Disease (AD) patients and its deficiency has been functionally linked to amyloid deposition, TAU hyperphosphorylation and memory decline, in both human and rodents. Of note, miR-132 can explain a higher percentage of the observed variance in histopathological AD phenotypes than APOE e4, a major risk factor for AD. This suggests that miR-132-dependent network-based gene regulation in human AD brain converges onto biological pathways that can drive disease endophenotypes. miR-132 regulates several targets in molecular pathways involved in AD pathophysiology, such as memory formation, neuronal morphogenesis, synaptic plasticity, immune homeostasis, neuronal survival and cell death. Recently, we reported miR-132 as a potent regulator of adult hippocampal neurogenesis (AHN) in healthy and AD brain. Direct miR-132 infusion in the AD mouse brain can counteract several aspects of pathology and memory deficits, suggesting its putative therapeutic relevance. microRNA multi-targeting entails the risk of on- and off- target toxicity, highlighting the importance of systematic genome-wide target validation. While specific miR-132 targets with direct implications in amyloid and TAU pathologies have been previously identified, the complex multicellular miR-132 targetome involved in miR-132-dependent regulation of AHN in AD remains elusive. In order to identify the mechanisms underpinning the positive regulatory effects of miR-132 in AHN and in AD in general, we employed a single-cell RNA-sequencing approach to assess miR-132-specific transcriptomic responses in AHN-relevant cells, such as adult neural stem cells in vivo. We identify novel miR-132 targets with putative neuroimmune functions involved in AHN and possibly in AD pathophysiology. Our results shed light on previously unknown molecular regulators of AHN in brain physiology and AD, provide possible novel therapeutic targets, and further add to the systematic profiling of miR-132 targetome, a prerequisite for successful translation of miR-132 supplementation in AD.

Chronic pharmacological antagonism of the GM-CSF receptor in mice does not replicate the pulmonary alveolar proteinosis phenotype but does alter lung surfactant turnover.

Granulocyte macrophage colony stimulating factor (GM-CSF) is a key participant in, and a clinical target for, the treatment of inflammatory diseases including rheumatoid arthritis (RA). Therapeutic inhibition of GM-CSF signalling using monoclonal antibodies to the α-subunit of the GM-CSF receptor (GMCSFRα) has shown clear benefit in patients with RA, giant cell arteritis (GCAs) and some efficacy in severe SARS-CoV-2 infection. However, GM-CSF autoantibodies are associated with the development of pulmonary alveolar proteinosis (PAP), a rare lung disease characterised by alveolar macrophage (AM) dysfunction and the accumulation of surfactant lipids. We assessed how the anti-GMCSFRα approach might impact surfactant turnover in the airway. Female C57BL/6J mice received a mouse-GMCSFRα blocking antibody (CAM-3003) twice per week for up to 24 weeks. A parallel, comparator cohort of the mouse PAP model, GM-CSF receptor β subunit (GMCSFRβ) knock-out (KO), was maintained up to 16 weeks. We assessed lung tissue histopathology alongside lung phosphatidylcholine (PC) metabolism using stable isotope lipidomics. GMCSFRβ KO mice reproduced the histopathological and biochemical features of PAP, accumulating surfactant PC in both broncho-alveolar lavage fluid (BALF) and lavaged lung tissue. The incorporation pattern of methyl-D9-choline showed impaired catabolism and not enhanced synthesis. In contrast, chronic supra-pharmacological CAM-3003 exposure (100 mg/kg) over 24 weeks did not elicit a histopathological PAP phenotype despite some changes in lung PC catabolism. Lack of significant impairment of AM catabolic function supports clinical observations that therapeutic antibodies to this pathway have not been associated with PAP in clinical trials.

Abstract 13021: Post-Myocardial Infarction Differences in Cardiac Histopathologic Phenotypes Across Clinical Categories

Introduction: Defining tissue-level phenotypic factors occurring in people with different clinical profiles may yield insights into pathogenesis of post-MI cardiac dysfunction. Methods: Two trained adjudicators analyzed reports from autopsies performed on patients with clinical diagnoses of MI who underwent autopsy at a large urban medical center between 10/31/2002 and 4/1/2021 (N=275). Standardized criteria were used to extra whether patients had the following histopathological criteria: 1) cardiomegaly; 2) coronary or large artery atherosclerosis; 3) severe coronary atherosclerosis; histopathologic features of 4) remote and/or 5) acute MI; 6) and myocardial fibrosis; and/or 7) myocardial inflammation. Interobserver agreement for histopathologic phenotypes was 0.929. Chi-squared and ANOVA were used to determine differences in histopathologic phenotypes by demographic and clinical variables. A Bonferroni-corrected p-value of 0.001 was used to determine statistical significance. Results: Among patients with clinical diagnoses of MI who underwent autopsy (N=275), not all had evidence of MI observed on histopathology; 185 (67%) had histopathologic evidence of remote MI, 86 (31%) had histopathologic evidence of acute MI (including 50 who had evidence of both acute and remote MI), and 55 (20%) had no histopathologic evidence of prior MI (Table). Patients with MI diagnoses who had chronic inflammatory diseases were significantly more likely than those without chronic inflammatory diseases to have myocardial tissue inflammation at autopsy (78% vs. 23%; p=0.0008). Patients with LVEF >=50% were substantially less likely than those with LVEF <40% and 40 to <50% to have histopathologic evidence of remote MI (51%, 69%, and 87% in each group, respectively; p=0.0002). Conclusion: Chronic inflammatory disease status and LVEF strata were associated with different myocardial tissue-level phenotypes at autopsy among patients with clinical diagnoses of MI.

Abstract 13019: Association of Histopathologic Phenotypes With Demographics and Cardiovascular Risks in People With Clinically Diagnosed Heart Failure

Introduction: Heart failure phenotypes are often defined based on imaging diagnoses and clinical risk factors without corroborative tissue-level investigation. Defining clinical factors associated with specific tissue phenotypes among people with HF may yield important insights. Methods: We analyzed cardiac autopsies performed on people with clinical diagnoses of HF (N=526) at a large urban medical center between 4/6/2002 and 04/01/2021. We determined whether or not patients had histopathologic evidence of the following: (1) cardiomegaly; (2) atherosclerosis (any); (3) severe/obstructive coronary atherosclerosis; (4) remote MI; (5) acute/recent MI (<14 days old pathologically); (6) myocardial fibrosis; and/or (7) myocardial inflammation. We determined associations of demographics and clinical factors with histopathologic phenotypes among people with a clinical diagnosis of HF using Chi-square and ANOVA. A Bonferroni-corrected p-value of <0.00102 was considered statistically significant to correct for multiple comparisons. Results: We observed significant differences by sex and several clinical factors in cardiac histopathology among patients with clinical diagnoses of HF (Table). Men were significantly more likely than women to have histopathologically-defined fibrosis (73% vs. 59%; p=0.0008) and cardiomegaly (76% vs. 60%; p=0.0002). HF patients with atrial fibrillation were significantly more likely to have cardiac fibrosis than those without atrial fibrillation (73% vs. 58%; p=0.0005). HF patients with LVEF >=50% were significantly less likely to have pathologic evidence of prior MI than patients with LVEF 40 to <50% and LVEF <40% (respectively, 24%, 50%, and 48% of patients in these groups; p<0.00001). Conclusions: Among patients with HF, sex, atrial fibrillation, and LVEF were associated with distinct cardiac histopathology at autopsy. Future tissue-level analyses in humans to define underlying mechanisms are needed.

Untangling the clinicopathological significance of MRE11-RAD50-NBS1 complex in sporadic breast cancers

The MRE11–RAD50–NBS1 (MRN) complex is critical for genomic stability. Although germline mutations in MRN may increase breast cancer susceptibility, such mutations are extremely rare. Here, we have conducted a comprehensive clinicopathological study of MRN in sporadic breast cancers. We have protein expression profiled for MRN and a panel of DNA repair factors involved in double-strand break repair (BRCA1, BRCA2, ATM, CHK2, ATR, Chk1, pChk1, RAD51, γH2AX, RPA1, RPA2, DNA-PKcs), RECQ DNA helicases (BLM, WRN, RECQ1, RECQL4, RECQ5), nucleotide excision repair (ERCC1) and base excision repair (SMUG1, APE1, FEN1, PARP1, XRCC1, Pol β) in 1650 clinical breast cancers. The prognostic significance of MRE11, RAD50 and NBS1 transcripts and their microRNA regulators (hsa-miR-494 and hsa-miR-99b) were evaluated in large clinical datasets. Expression of MRN components was analysed in The Cancer Genome Atlas breast cancer cohort. We show that low nuclear MRN is linked to aggressive histopathological phenotypes such as high tumour grade, high mitotic index, oestrogen receptor- and high-risk Nottingham Prognostic Index. In univariate analysis, low nuclear MRE11 and low nuclear RAD50 were associated with poor survival. In multivariate analysis, low nuclear RAD50 remained independently linked with adverse clinical outcomes. Low RAD50 transcripts were also linked with reduced survival. In contrast, overexpression of hsa-miR-494 and hsa-miR-99b microRNAs was associated with poor survival. We observed large-scale genome-wide alterations in MRN-deficient tumours contributing to aggressive behaviour. We conclude that MRN status may be a useful tool to stratify tumours for precision medicine strategies.

A PUZZLING CASE OF RECURRENT PNEUMONIA: PLEUROPARENCHYMAL FIBROELASTOSIS

TOPIC: Lung Pathology TYPE: Medical Student/Resident Case Reports INTRODUCTION: Pleuroparenchymal fibroelastosis (PPFE) is a fibroelastic process mainly affecting the visceral pleura and subpleural spaces with a propensity towards the upper lobes. It has unique clinical, pathological and radiological features1. CASE PRESENTATION: An 80 year old female with history of recurrent pneumonias and remote granulomatous disease presented with progressive dyspnea and fatigue. She denied other symptoms including chronic cough or sputum production or fever or chills. Chest X-ray showed extensive parenchymal and pleural fibrotic changes of the upper lobes. Pulmonary function tests suggested a restrictive pattern with reduced diffusion capacity (DLCO). Echocardiogram showed no significant abnormalities. Serial CT scans of the chest done over two years showed worsening pleuroparenchymal fibrotic changes of the upper lobes in a pattern very consistent with PPFE, with no evidence of honeycombing. This was likely idiopathic but potentially related to prior pneumonias and felt less likely to be related to her remote minimal parakeet exposure associated hypersensitivity pneumonitis. Her ANA titers were mildly elevated at 1:160 but further workup for auto-antibodies were negative including RF and CCP antibodies. ESR and CRP were minimally elevated. A bronchoscopy was offered to evaluate for infectious or other etiologies but she opted to wait. Her CT findings were however very consistent with PPFE and a provisional diagnosis of the same was made. DISCUSSION: PPFE is a rare but under-diagnosed type of interstitial lung disease. It is mostly idiopathic in nature but sometimes occurs as a late complication after lung transplantation. Although similar findings may be sometimes described in patients with autoimmune or connective tissue disease, it usually presents as a late complication and in certain patients with hypersensitivity pneumonitis, the idiopathic ones are best classified as PPFE1. PPFE is mostly seen in patients aged 40 to 70 years and presents as progressive dyspnea with or without cough. PPFE has to be differentiated from other upper lobe diseases like hypersensitivity pneumonia, sarcoidosis, idiopathic interstitial pneumonitis, non-tuberculous mycobacterial infection, pneumoconiosis, malignancy and apical pleural cap. High resolution CT scan remains the best diagnostic modality to show diffuse fibroelastic process predominantly affecting pleura and upper lobe parenchyma1. Pulmonary function test shows a restrictive pattern with reduced DLCO. No specific laboratory tests are diagnostic2. Surgical biopsies although not required for diagnosis can demonstrate intra-alveolar fibrosis and elastosis2. CONCLUSIONS: The prognosis of PPFE remains poor as no treatment is proven to be effective except therapeutic lung transplantation1. Further studies to understand pathogenesis, diagnostic markers and treatment modalities for this rare progressive disease are much warranted. REFERENCE #1: Chua F, Desai SR, Nicholson AG, et al. Pleuroparenchymal fibroelastosis. A review of clinical, radiological and pathological characteristics. Ann Am Thorac Soc 2019;16:1351-9. REFERENCE #2: Reddy TL, Tominaga M, Hansell DM, von der Thusen J, Rassl D, Parfrey H, Guy S, Twentyman O, Rice A, Maher TM, Renzoni EA, Wells AU, Nicholson AG. Pleuroparenchymal fibroelastosis: a spectrum of histopathological and imaging phenotypes. Eur Respir J. 2012 Aug;40(2):377-85. DISCLOSURES: No relevant relationships by Ansaam Daoud, source=Web Response No relevant relationships by Palak Rath, source=Web Response No relevant relationships by Lokesh Venkateshaiah, source=Web Response

PLEUROPARENCHYMAL FIBROELASTOSIS: A RARE INTERSTITIAL LUNG DISEASE

TOPIC: Diffuse Lung Disease TYPE: Medical Student/Resident Case Reports INTRODUCTION: Pleuroparenchymal fibroelastosis (PPFE) is a rare interstitial lung disease (ILD) characterized by upper lobe predominant pleural and parenchymal fibrosis. We report a patient with clinical and radiographic findings consistent with PPFE with no other explanation for her ILD. CASE PRESENTATION: A 65 year-old female with history of depression, hypothyroidism, and irritable bowel syndrome presented to the pulmonary clinic with chronic dry cough and exertional dyspnea. She also endorsed malaise and fatigue, but denied orthopnea, paroxysmal nocturnal dyspnea, chest pain, or any other symptoms. Physical exam was only notable for a few scattered fine crackles at the upper lung fields. Pulmonary Function Tests revealed a mildly reduced DLCO with no other abnormalities. A high resolution CT scan (HRCT) of the chest performed due to clinical suspicion for interstitial lung disease demonstrated bi-apical consolidative-type changes upper lobe volume loss and peripherally-based reticular opacities (also involving the superior segments of the lower lobes). A transthoracic echocardiogram showed normal left and right ventricular size and function. Our patient did not have any occupational or medication exposures. An extensive rheumatological workup was negative. The patient declined lung biopsy. Given the patient's progressive pulmonary symptoms, the decision was made to initiate antifibrotic therapy with pirfenidone, as the patients' irritable bowel syndrome with diarrhea precluded her from nintedanib therapy. DISCUSSION: PPFE is a chronic, progressive lung fibrotic disorder with unclear etiology, but may be associated with chemotherapy, lung or bone marrow transplantation, or recurrent pulmonary infections. Symptoms include dry cough, dyspnea, weight loss or pneumothorax. A thorough history and evaluation is critical to exclude other potential interstitial lung disorders. Platythorax, a reduction of the anterior-posterior diameter of the thorax relative to the transverse diameter, has been shown to be a clinical sign and a potential marker of disease progression. HRCT typically shows bilateral apical pleural thickening with architectural distortion, dense subpleural consolidation and traction bronchiectasis, with upper lobe volume loss. Definitive diagnosis is established by surgical lung biopsy. There is presently no approved therapy for PPFE, although there is limited evidence to support the use of antifibrotic agents. CONCLUSIONS: Given the rarity of PPFE, it is important for providers to recognize the existence of this lung condition as it has both prognostic and therapeutic implications. REFERENCE #1: Chua, Felix. Annals of the American Thoracic Society, 30 May 2019, www.atsjournals.org/doi/full/10.1513/AnnalsATS.201902-181CME. REFERENCE #2: Reddy, Taryn L., et al. "Pleuroparenchymal Fibroelastosis: a Spectrum of Histopathological and Imaging Phenotypes." European Respiratory Society, European Respiratory Society, 1 Aug. 2012, erj.ersjournals.com/content/40/2/377. REFERENCE #3: Redondo, M.T., et al. "Idiopathic Pleuroparenchymal Fibroelastosis: A Rare but Increasingly Recognized Entity." Pulmonology, Elsevier, 1 Jan. 2015, www.journalpulmonology.org/pt-idiopathic-pleuroparenchymal-fibroelastosis-a-rare-articulo-X0873215915856150. DISCLOSURES: No relevant relationships by Raymond Foley, source=Web Response No relevant relationships by Simrina Sabharwal, source=Web Response No relevant relationships by Evan Wasserman, source=Web Response

Increased sensitivity to SMAC mimetic LCL161 identified by longitudinal ex vivo pharmacogenomics of recurrent, KRAS mutated rectal cancer liver metastases

Tumor heterogeneity is a primary cause of treatment failure. However, changes in drug sensitivity over time are not well mapped in cancer. Patient-derived organoids (PDOs) may predict clinical drug responses ex vivo and offer an opportunity to evaluate novel treatment strategies in a personalized fashion. Here we have evaluated spatio-temporal functional and molecular dynamics of five PDO models established after hepatic re-resections and neoadjuvant combination chemotherapies in a patient with microsatellite stable and KRAS mutated metastatic rectal cancer. Histopathological differentiation phenotypes of the PDOs corresponded with the liver metastases, and ex vivo drug sensitivities generally reflected clinical responses and selection pressure, assessed in comparison to a reference data set of PDOs from metastatic colorectal cancers. PDOs from the initial versus the two recurrent metastatic settings showed heterogeneous cell morphologies, protein marker expression, and drug sensitivities. Exploratory analyses of a drug screen library of 33 investigational anticancer agents showed the strongest ex vivo sensitivity to the SMAC mimetic LCL161 in PDOs of recurrent disease compared to those of the initial metastasis. Functional analyses confirmed target inhibition and apoptosis induction in the LCL161 sensitive PDOs from the recurrent metastases. Gene expression analyses indicated an association between LCL161 sensitivity and tumor necrosis factor alpha signaling and RIPK1 gene expression. In conclusion, LCL161 was identified as a possible experimental therapy of a metastatic rectal cancer that relapsed after hepatic resection and standard systemic treatment.

Polymyositis: does it really exist as a distinct clinical subset?

To summarize information on polymyositis; diagnosis, definitions, published data and opinions. Polymyositis originally referred to inflammatory muscle diseases presenting with muscle weakness and inflammatory cell infiltrates on muscle tissue visible by microscopy. Over time and with improved technology to immunophenotype infiltrating inflammatory cells and characterize muscle fibres, the meaning of polymyositis changed and became more specific. There is ongoing controversy over the term polymyositis, with proponents for a strict definition based on histopathological and immunohistochemical features on muscle biopsies whereas others advocate for a broader clinical and histopathological phenotype. Over the past decades, the discovery of several myositis-specific autoantibodies together with distinct histopathological features have enabled the identification of new subsets previously labelled as polymyositis notably the antisynthetase syndrome and the immune-mediated necrotizing myopathies thus reducing the number of patients classified as polymyositis. There are still a small number of patients among the idiopathic inflammatory myopathies that can be classified as polymyositis as discussed in this review but the entity is now considered relatively rare.

NGS Analysis Confirms Common TP53 and RB1 Mutations, and Suggests MYC Amplification in Ocular Adnexal Sebaceous Carcinomas.

Ocular adnexal (OA) sebaceous carcinomas generally demonstrate more aggressive clinical and histopathological phenotypes than extraocular cases, but the molecular drivers implicated in their oncogenesis remain poorly defined. A retrospective review of surgical and ocular pathology archives identified eleven primary resection specimens of OA sebaceous carcinomas with adequate tissue for molecular analysis; two extraocular cases were also examined. Next-generation sequencing was used to evaluate mutations and copy number changes in a large panel of cancer-associated genes. Fluorescence in situ hybridization (FISH) confirmed MYC copy number gain in select cases, and immunohistochemistry to evaluate MYC protein expression. The commonest mutations occurred in TP53 (10/13) and RB1 (7/13). Additional mutations in clinically actionable genes, or mutations with a frequency of at least 25%, included the NF1 (3/12), PMS2 (4/12), ROS1 (3/12), KMT2C (4/12), MNX1 (6/12), NOTCH1 (4/12), PCLO (3/12), and PTPRT (3/12) loci. Low level copy number gain suggestive of amplification of the MYC locus was seen in two cases, and confirmed using FISH. MYC protein expression, as assessed by immunohistochemistry, was present in almost all sebaceous carcinoma cases. Our findings support the concept that alterations in TP53 and RB1 are the commonest alterations in sebaceous carcinoma, and suggest that MYC may contribute to the oncogenesis of these tumors.

Gastric neoplasms in patients with familial adenomatous polyposis: endoscopic and clinicopathologic features

Gastric neoplasms in patients with familial adenomatous polyposis (FAP) occur at a high rate and can cause death. The endoscopic findings of gastric neoplasms in these patients are characteristic but not well recognized. To identify the relevant characteristics to enable early detection, we retrospectively investigated endoscopic findings of gastric neoplasms in patients with FAP and then compared the clinical, histopathologic, and genetic features among subgroups. Of 234 patients with 171 pedigrees at 2 institutes, 56 cases (24%, 133 gastric neoplasms) with 44 pedigrees were examined. Immunostaining was performed for histopathologic evaluation by 1 blinded pathologist. According to the endoscopic findings, gastric neoplasms were divided into 4 types based on location (L: antrum and pylorus, UM: the rest of the stomach) and color (W: white, T: translucent, R: reddish) and their clinicopathologic features examined. Of the cases, 93% could be classified into a single type. Among histologic phenotypes, high-grade dysplasia was present in 26% (type L), 41% (type UM-W), 0% (type UM-T), and 22% (type UM-R). The immunologic phenotype comprised the gastric type in 69% (93% in Type UM) and the intestinal phenotype, including the mixed type, in 31% (61% in type L). Moreover, 96% of patients had concurrent duodenal neoplasms. Adenomatous polyposis coli gene status was identified in 93% of patients; the pathogenic variant was detected in 98% but did not influence any endoscopic features. Gastric neoplasms in patients with FAP were stratified into 4 types according to their endoscopic findings. The endoscopic phenotype was related to the histopathologic phenotype but not to germline variants.