Fibroblast-expressed LRRC15 is a receptor for SARS-CoV-2 spike and controls antiviral and antifibrotic transcriptional programs.

Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.

Autoimmunity Against Surfactant Protein B Is Associated with Pneumonitis During Checkpoint Blockade.

RATIONALE: Immune checkpoint inhibitor-related pneumonitis is a serious autoimmune event affecting up to 20% of patients with non-small cell lung cancer, yet the factors underpinning its development in some patients and not others are poorly understood. OBJECTIVES: To investigate the role of autoantibodies and autoreactive T cells against surfactant-related proteins in the development of pneumonitis. METHODS: The study cohort consisted of non-small cell lung cancer patients who gave blood samples before and during immune checkpoint inhibitor treatment. Serum was used for proteomics analyses and to detect autoantibodies present during pneumonitis. T cell stimulation assays and single-cell RNA sequencing were performed to investigate the specificity and functionality of peripheral autoreactive T cells. The findings were confirmed in a validation cohort comprising patients with non-small cell lung cancer and patients with melanoma. MEASUREMENTS AND MAIN RESULTS: Across both cohorts, patients who developed pneumonitis had higher pre-treatment levels of immunoglobulin G autoantibodies targeting surfactant protein-B. At the onset of pneumonitis, these patients also exhibited higher frequencies of CD4+ interferon-gamma-positive surfactant protein B-specific T cells, and expanding T cell clonotypes recognizing this protein, accompanied by a pro-inflammatory serum proteomic profile. CONCLUSIONS: Our data suggest that the co-occurrence of surfactant protein-B-specific immunoglobulin G autoantibodies and CD4+ T cells is associated with the development of pneumonitis during ICI therapy. Pre-treatment levels of these antibodies may represent a potential biomarker for elevated risk of developing pneumonitis and on-treatment levels may provide a diagnostic aid. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Pulmonary Surfactant Proteins Are Inhibited by Immunoglobulin A Autoantibodies in Severe COVID-19.

Rationale: Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome with fatal outcomes. Evidence suggests that dysregulated immune responses, including autoimmunity, are key pathogenic factors. Objectives: To assess whether IgA autoantibodies target lung-specific proteins and contribute to disease severity. Methods: We collected 147 blood, 9 lung tissue, and 36 BAL fluid samples from three tertiary hospitals in Switzerland and one in Germany. Severe COVID-19 was defined by the need to administer oxygen. We investigated the presence of IgA autoantibodies and their effects on pulmonary surfactant in COVID-19 using the following methods: immunofluorescence on tissue samples, immunoprecipitations followed by mass spectrometry on BAL fluid samples, enzyme-linked immunosorbent assays on blood samples, and surface tension measurements with medical surfactant. Measurements and Main Results: IgA autoantibodies targeting pulmonary surfactant proteins B and C were elevated in patients with severe COVID-19 but not in patients with influenza or bacterial pneumonia. Notably, pulmonary surfactant failed to reduce surface tension after incubation with either plasma or purified IgA from patients with severe COVID-19. Conclusions: Our data suggest that patients with severe COVID-19 harbor IgA autoantibodies against pulmonary surfactant proteins B and C and that these autoantibodies block the function of lung surfactant, potentially contributing to alveolar collapse and poor oxygenation.

Prolonged Response to Afatinib and Crizotinib in a Rare Case of EGFR-, HER2-, MET- and ROS1-Alterated Lung Adenocarcinoma.

We present the case of a 70-year-old never-smoking female patient with epidermal growth factor receptor (EGFR) p.L858R-mutated metastatic non-small cell lung cancer (NSCLC). After three months of first-line treatment with erlotinib, progression occurred and platinum/pemetrexed was initiated, followed by a response for more than two years. After the progression, the molecular testing of a vertebral metastasis revealed a ROS proto-oncogene 1 (ROS1) translocation and a human epidermal growth factor receptor 2 (HER2) p.S310F mutation, in addition to the known EGFR p.L858R mutation. Crizotinib then led to a durable response of 17 months. The molecular retesting of the tumour cells obtained from the recurrent pleural effusion revealed the absence of the ROS1 translocation, whereas the EGFR and HER2 mutations were still present. Afatinib was added to the crizotinib, and the combination treatment resulted in another durable response of more than two years. The patient died more than 7 years after the initial diagnosis of metastatic NSCLC. This case demonstrates that the repeated molecular testing of metastatic NSCLC may identify new druggable genomic alterations that can impact the patient management and improve the patient outcome.

Outcomes with immune checkpoint inhibitors for relapsed small-cell lung cancer in a Swiss cohort.

OBJECTIVES: Early clinical trials showed promising outcomes with immune-checkpoint inhibitors (ICI) in a subset of patients with relapsed small-cell lung carcinoma (SCLC). The aim of this retrospective analysis was to assess the efficacy and safety of ICI for relapsed SCLC in a real-world patient population. METHODS: Nine cancer centres in Switzerland contributed data to this cohort. Responses were assessed by the local investigators using standard RECIST v1.1 criteria. Progression-free survival (PFS) and overall survival (OS) were analysed by the Kaplan-Meier method. Associations between potential predictive markers and survival endpoints were probed by Cox proportional hazards. RESULTS: Forty-five patients were included in the analysis. Median age was 63 years, 73% were males and 18% had an ECOG performance status (PS) ≥ 2. ICIs were given as second-line treatment in 60%. Twenty-four patients (53%) received ipilimumab with nivolumab. Twenty-eight patients (62%) had undergone irradiation (RT) prior to or during ICI. Overall response rate (ORR) was 29% and median PFS and OS were 2.3 and 6.5 months, respectively. Median duration of response was 9 months (95% CI 2.8-NA). Five patients maintained their response for > 6 months, all of them receiving combination treatment. There were no new safety signals. CONCLUSION: This is the first report of "real-world" data on ICI in relapsed SCLC also including patients with poor PS. Promising durable responses were observed. No biological prognostic marker could be identified.

BP180-specific IgG is associated with skin adverse events, therapy response, and overall survival in non-small cell lung cancer patients treated with checkpoint inhibitors.

BACKGROUND: Anti-programmed cell death protein 1 (PD1)/programmed death-ligand 1(PD-L1) therapy frequently entails immune-related adverse events (irAEs), and biomarkers to predict irAEs are lacking. Although checkpoint inhibitors have been found to reinvigorate T cells, the relevance of autoantibodies remains elusive. OBJECTIVE: Our aim was to explore whether IgG autoantibodies directed against coexpressed antigens by tumor tissue and healthy skin correlate with skin irAEs and therapy outcome. METHODS: We measured skin-specific IgG via enzyme-linked immunosorbent assay in patients with non-small cell lung cancer (NSCLC) who received anti-PD1/PD-L1 treatment between July 2015 and September 2017 at the Kantonsspital St. Gallen. Sera were sampled at baseline and during therapy after 8 weeks. RESULTS: Analysis of publicly available tumor expression data revealed that NSCLC and skin coexpress BP180, BP230, and type VII collagen. A skin irAE developed in 16 of 40 recruited patients (40%). Only elevated anti-BP180 IgG at baseline significantly correlated with the development of skin irAEs (P = .04), therapy response (P = .01), and overall survival (P = .04). LIMITATIONS: The patients were recruited in a single tertiary care center. CONCLUSIONS: Our data suggest that the level of anti-BP180 IgG of NSCLC patients at baseline is associated with better therapy response and overall survival and with a higher probability to develop skin irAEs during anti-PD1/PD-L1 treatment.

Nano-analytical characterization of endogenous minerals in healthy placental tissue: mineral distribution, composition and ultrastructure.

Despite its crucial role, the placenta is the least understood human organ. Recent clinical studies indicate a direct association between placental calcification and maternal and offspring health. This study reveals distinct characteristics of minerals formed during gestational ageing using cutting-edge nano-analytical characterization and paves the way for investigations focused on the identification of potential markers for disease risks in a clinical setting based on atypical placental mineral fingerprints.

Endoscopic Treatment of Early Barrett's Adenocarcinoma and Dysplasia: Focus on Submucosal Cancer.

BACKGROUND/AIMS: Eradication of early Barrett's neoplasia by endoscopic resection and radiofrequency ablation is safe and effective. In T1b adenocarcinoma, standard of care remains controversial. We investigated the therapeutic outcome between high-grade dysplasia (HGD)/mucosal adenocarcinoma and submucosal adenocarcinoma in Barrett's patients. We hypothesised similar outcome in low-risk (LR) T1b compared to T1a/HGD. METHODS: Patients with endoscopically treated Barrett's esophagus were included in a Swiss tertiary center cohort study. Primary outcome parameter was complete eradication of early neoplasia. Secondary outcome parameters were recurrence-free survival and safety of endoscopic treatment. RESULTS: Forty-eight patients (1 female) with median Barrett's length C4M6 and mean age of 66 years were included. Complete endoscopic eradication of HGD/T1a was achieved in 33 out of 35 and in 11 out of 13 T1b adenocarcinoma. During a median follow-up of 41 (interquartile range 28-63) months no systemic recurrence was observed in endoscopically treated HGD/T1a and LR -T1b and one in a high-risk T1b adenocarcinoma after surgery. Local recurrences were amenable to surgical or endoscopic re-treatment. No lymphnode metastasis was detected in initial staging with esophageal endosonography/positron emission tomography-CT. CONCLUSION: Comparable endoscopic eradication and recurrence rate were observed in HGD/T1a and LR T1b adenocarcinoma. Carefully selected LR T1b cancer may receive endoscopic treatment in an expert center without any negative impact on recurrence.

CCL19-producing fibroblastic stromal cells restrain lung carcinoma growth by promoting local antitumor T-cell responses.

BACKGROUND: A particular characteristic of non-small cell lung cancer is the composition of the tumor microenvironment with a very high proportion of fibroblastic stromal cells (FSCs). OBJECTIVE: Lapses in our basic knowledge of fibroblast phenotype and function in the tumor microenvironment make it difficult to define whether FSC subsets exist that exhibit either tumor-promoting or tumor-suppressive properties. METHODS: We used gene expression profiling of lung versus tumor FSCs from patients with non-small cell lung cancer. Moreover, CCL19-expressing FSCs were studied in transgenic mouse models by using a lung cancer metastasis model. RESULTS: CCL19 mRNA expression in human tumor FSCs correlates with immune cell infiltration and intratumoral accumulation of CD8+ T cells. Mechanistic dissection in murine lung carcinoma models revealed that CCL19-expressing FSCs form perivascular niches to promote accumulation of CD8+ T cells in the tumor. Targeted ablation of CCL19-expressing tumor FSCs reduced immune cell recruitment and resulted in unleashed tumor growth. CONCLUSION: These data suggest that a distinct population of CCL19-producing FSCs fosters the development of an immune-stimulating intratumoral niche for immune cells to control cancer growth.

Organ-specific response to nivolumab in patients with non-small cell lung cancer (NSCLC).

BACKGROUND: Response to immune checkpoint inhibitors depends on tumor intrinsic properties and also on host factors in the tumour microenvironment including the presence of immune cells (IC). We hypothesized that nivolumab efficacy varies across different metastatic sites. METHODS: We retrospectively analyzed computed tomography scans of patients with metastatic non-small cell lung carcinoma (NSCLC) receiving nivolumab. RECIST 1.1 criteria were applied to assess the overall response rate (ORR) and organ-specific response rate (OSRR). RESULTS: We analyzed 52 patients including 44% females, 58% adenocarcinoma and 8% never smokers. Involved organs had target-lesions in the lung (42%), liver (25%), lymph nodes (56%) and soft tissue (13%) and non-target lesions in the bones (23%). ORR and disease control rate (DCR) were 20% and 45%, respectively. Median overall survival, progression-free survival and duration of response were 11.9, 2.3 and 10.3 months. OSRR and organ-specific DCR (OSDCR) were 28% and 90% in lymph nodes, 8% and 54 in the liver, and 9% and 55% in lung metastases. Nine out of 12 patients with bone metastases had progressive lesions. The cumulative incidence probability of organ-specific progression at 6 months was 14% in lymph nodes, 42% in the liver, 36% in lung metastases and 26% in the primary tumor, 29% in soft tissue and 33% in adrenal metastases. CONCLUSION: In conclusion, the efficacy of immunotherapy is dependent on the metastatic location. Treatment appears more active in lymph nodes compared to other organ sites such as liver, adrenals and bone. Future strategies may include additional local treatment in case of oligoprogression in these organs in patients with otherwise sustained treatment benefit.

Tumor infiltrating lymphocytes in lymph node metastases of stage III melanoma correspond to response and survival in nine patients treated with ipilimumab at the time of stage IV disease.

Prognosis of metastatic melanoma improved with the development of checkpoint inhibitors. The role of tumor infiltrating lymphocytes (TILs) in lymph node metastases of stage III melanoma remains unclear. We retrospectively characterized TILs in primary melanomas and matched lymph node metastases (stage III melanoma) of patients treated with the checkpoint inhibitor ipilimumab. Tumor infiltrating lymphocytes were characterized for CD3, CD4, and CD8 expressions by immunohistochemistry. 4/9 patients (44%) responded to treatment with ipilimumab (1 complete and 2 partial remissions, 1 stable disease). All responders exhibited CD4 and CD8 T-cell infiltration in their lymph node metastases, whereas all non-responders did not show an infiltration of the lymph node metastasis with TILs. The correlation between the presence and absence of TILs in responders vs. non-responders was statistically significant (p = 0.008). Median distant metastases free survival, i.e., progression from stage III to stage IV melanoma, was similar in responders and non-responders (22.1 vs. 19.3 months; p = 0.462). Median progression free and overall survival show a trend in favor of the patients having TIL rich lymph node metastases (6.8 vs. 3.3 months, p = 0.09; and all alive at last follow-up vs. 8.2 months, respectively, p = 0.08). Our data suggest a correlation between the T-cell infiltration of the lymph node metastases in stage III melanoma and the response to ipilimumab once these patients progress to stage IV disease.

The prognostic value of signet ring cell histology in stage I/II colon cancer-a population-based, propensity score-matched analysis.

BACKGROUND: Previous research associated signet ring cell histology in colon cancer patients with poor survival outcomes. The aim of this study was to analyze the prognostic significance of signet ring cell histology on overall and cancer-specific survival in patients with localized colon cancer. METHODS: Stage I and II colon cancer patients treated with surgical resection between 2004 and 2015 were identified in the Surveillance, Epidemiology, and End Results (SEER) database. Overall survival (OS) and cancer-specific survival (CSS) were assessed using risk-adjusted Cox proportional hazards regression models and propensity score methods. RESULTS: Eighty-eight thousand nine hundred fifty-eight stage I-II colon cancer patients were identified. Overall, 446 (0.5%) showed signet ring cell histology. In unadjusted analyses, the 5-year OS and CSS rates of patients with signet ring cell histology were 65.8 and 83.1%, respectively, compared with 74.3 and 88.7% in patients with non-signet ring cell adenocarcinoma (p values: OS, p < 0.001; CSS, p < 0.001). Neither in risk-adjusted Cox proportional hazard regression analysis (OS: hazard ratio (HR), 0.96 (95% CI, 0.82-1.12%) p = 0.616; CSS: HR, 1.01 (95% CI, 0.79-1.28%) p = 0.946) nor with propensity score matching (OS: HR, 0.96 (95% CI, 0.82-1.14%) p = 0.669 and CSS: HR: 1.09 (95% CI: 0.84-1.40%) p = 0.529), a survival disadvantage was found for signet ring cell histology. CONCLUSION: This is the first propensity score-adjusted population-based investigation on exclusively stage I and II colon cancer patients providing compelling evidence that signet ring cell histology does not negatively impact survival in stage I and II colon cancer after risk-adjusting for known prognostic factors. Therefore, standard treatment strategies can be applied in these patients.

Low tissue levels of miR-125b predict malignancy in solitary fibrous tumors of the pleura.

BACKGROUND: Solitary fibrous tumors of the pleura (SFTP) are rare neoplasia of the chest. A subset of SFTP follows a malignant course, sometimes several years after complete resection. Traditional scoring systems based on clinical and histological features are poor predictors of biological behavior. This study aimed to investigate tumor-associated miRNAs expression as novel biomarkers to predict the clinical behavior of SFTP. METHODS: Formalin-fixed and paraffin-embedded SFTP tissues blocks from patients surgically resected between 1992 and 2013 at two tertiary care teaching hospitals were included. SFTP tumors were categorized as either malignant or benign variants according to the WHO classification. Following miRNAs levels were measured: let-7a, miR-16b, miR-17, miR-21, miR-31, miR-34a, miR-92a, miR-125a, miR-125b, miR-195-5b, miR-203a, and miR-223. Differential gene expressions which were calculated with the threshold cycle (Ct) method were compared among the two variants. RESULTS: Thirty-eight patients (40% male, mean age 62.2 (±10.9) years) were included. Expression levels of miR-125b showed a significant difference between benign compared to malignant variants (-3.08 ± 0.93 vs. -2.22 ± 1.36, p = 0.0068). Furthermore, lower levels of miR-125b were found to be associated with increased tumor size (p = 0.0414). Thus, downregulation of miR-125b indicates malignant transformation. All other investigated miRNAs were not associated with grading of SFTP. CONCLUSIONS: Our data suggest a potential role of miR-125b in the pathogenesis of tumor growth and malignant transformation of SFTP, respectively. Further studies have to address the potential use of miRNA-125b as a biomarker or therapeutic target in SFTP.

Prognostic value of MIB-1 proliferation index in solitary fibrous tumors of the pleura implemented in a new score - a multicenter study.

BACKGROUND: Although the majority of solitary fibrous tumors of the pleura (SFTP) follow a benign course, 10-25% of patients suffer from recurrence or metastatic disease. Several scoring models have been proposed to predict the outcome. However, none of these included immunohistochemical (IHC) markers as possible prognosticators. METHODS: In this multicenter study, we collected clinical data and formalin-fixed and paraffin-embedded (FFPE) tissue blocks of patients with histologically proven SFTP which had been surgically resected between 2000 und 2015. After systematic and extensive IHC staining on tissue microarrays, the results were analyzed and compared to histomorphological and clinical data for their possible prognostic value. RESULTS: In total, 78 patients (mean age 61 ± 11 years) were included. Of these, 9 patients (11%) had an adverse outcome including SFTP recurrence (n = 6) or SFTP-related death (n = 3). Mean overall survival was 172 ± 13 months. 1 and 10-year event-free survival rates were 99% and 93%. In the multivariable analysis only MIB-1 proliferation index (Ki-67) ≥10% (HR 12.3, CI 1.1-139.5, p = 0.043), ≥4 mitoses per 10 high power fields (HR 36.5, CI 1.2-1103.7, p = 0.039) and tumor size larger than 10 cm (HR 81.8, CI 1.7-4016.8, p = 0.027) were independently associated with adverse outcome. CONCLUSION: A high proliferation rate by MIB-1 IHC was associated with impaired outcome. Upon this, we established a new score using mitosis, necrosis, size of the tumor and MIB-1, which performed better than the traditional scores in our data set. This prognostic score could help to better evaluate outcome of SFTP, but requires external validation.

Reproducible Tissue Homogenization and Protein Extraction for Quantitative Proteomics Using MicroPestle-Assisted Pressure-Cycling Technology.

The reproducible and efficient extraction of proteins from biopsy samples for quantitative analysis is a critical step in biomarker and translational research. Recently, we described a method consisting of pressure-cycling technology (PCT) and sequential windowed acquisition of all theoretical fragment ions-mass spectrometry (SWATH-MS) for the rapid quantification of thousands of proteins from biopsy-size tissue samples. As an improvement of the method, we have incorporated the PCT-MicroPestle into the PCT-SWATH workflow. The PCT-MicroPestle is a novel, miniaturized, disposable mechanical tissue homogenizer that fits directly into the microTube sample container. We optimized the pressure-cycling conditions for tissue lysis with the PCT-MicroPestle and benchmarked the performance of the system against the conventional PCT-MicroCap method using mouse liver, heart, brain, and human kidney tissues as test samples. The data indicate that the digestion of the PCT-MicroPestle-extracted proteins yielded 20-40% more MS-ready peptide mass from all tissues tested with a comparable reproducibility when compared to the conventional PCT method. Subsequent SWATH-MS analysis identified a higher number of biologically informative proteins from a given sample. In conclusion, we have developed a new device that can be seamlessly integrated into the PCT-SWATH workflow, leading to increased sample throughput and improved reproducibility at both the protein extraction and proteomic analysis levels when applied to the quantitative proteomic analysis of biopsy-level samples.

Minimal sample requirement for highly multiplexed protein quantification in cell lines and tissues by PCT-SWATH mass spectrometry.

The amount of sample available for clinical and biological proteomic research is often limited and thus significantly restricts clinical and translational research. Recently, we have integrated pressure cycling technology (PCT) assisted sample preparation and SWATH-MS to perform reproducible proteomic quantification of biopsy-level tissue samples. Here, we further evaluated the minimal sample requirement of the PCT-SWATH method using various types of samples, including cultured cells (HeLa, K562, and U251, 500 000 to 50 000 cells) and tissue samples (mouse liver, heart, brain, and human kidney, 3-0.2 mg). The data show that as few as 50 000 human cells and 0.2-0.5 mg of wet mouse and human tissues produced peptide samples sufficient for multiple SWATH-MS analyses at optimal sample load applied to the system. Generally, the reproducibility of the method increased with decreasing tissue sample amounts. The SWATH maps acquired from peptides derived from samples of varying sizes were essentially identical based on the number, type, and quantity of identified peptides. In conclusion, we determined the minimal sample required for optimal PCT-SWATH analyses, and found smaller sample size achieved higher quantitative accuracy.

Transfer studies of polystyrene nanoparticles in the ex vivo human placenta perfusion model: key sources of artifacts.

Nanotechnology is a rapidly expanding and highly promising new technology with many different fields of application. Consequently, the investigation of engineered nanoparticles in biological systems is steadily increasing. Questions about the safety of such engineered nanoparticles are very important and the most critical subject with regard to the penetration of biological barriers allowing particle distribution throughout the human body. Such translocation studies are technically challenging and many issues have to be considered to obtain meaningful and comparable results. Here we report on the transfer of polystyrene nanoparticles across the human placenta using an ex vivo human placenta perfusion model. We provide an overview of several challenges that can potentially occur in any translocation study in relation to particle size distribution, functionalization and stability of labels. In conclusion, a careful assessment of nanoparticle properties in a physiologically relevant milieu is as challenging and important as the actual study of nanoparticle-cell interactions itself.