Galectin-10 in serum extracellular vesicles reflects asthma pathophysiology.

BACKGROUND: Novel biomarkers (BMs) are urgently needed for bronchial asthma (BA) with various phenotypes and endotypes. OBJECTIVE: We sought to identify novel BMs reflecting tissue pathology from serum extracellular vesicles (EVs). METHODS: We performed data-independent acquisition of serum EVs from 4 healthy controls, 4 noneosinophilic asthma (NEA) patients, and 4 eosinophilic asthma (EA) patients to identify novel BMs for BA. We confirmed EA-specific BMs via data-independent acquisition validation in 61 BA patients and 23 controls. To further validate these findings, we performed data-independent acquisition for 6 patients with chronic rhinosinusitis without nasal polyps and 7 patients with chronic rhinosinusitis with nasal polyps. RESULTS: We identified 3032 proteins, 23 of which exhibited differential expression in EA. Ingenuity pathway analysis revealed that protein signatures from each phenotype reflected disease characteristics. Validation revealed 5 EA-specific BMs, including galectin-10 (Gal10), eosinophil peroxidase, major basic protein, eosinophil-derived neurotoxin, and arachidonate 15-lipoxygenase. The potential of Gal10 in EVs was superior to that of eosinophils in terms of diagnostic capability and detection of airway obstruction. In rhinosinusitis patients, 1752 and 8413 proteins were identified from EVs and tissues, respectively. Among 11 BMs identified in EVs and tissues from patients with chronic rhinosinusitis with nasal polyps, 5 (including Gal10 and eosinophil peroxidase) showed significant correlations between EVs and tissues. Gal10 release from EVs was implicated in eosinophil extracellular trapped cell death in vitro and in vivo. CONCLUSION: Novel BMs such as Gal10 from serum EVs reflect disease pathophysiology in BA and may represent a new target for liquid biopsy approaches.

Antitumor effects of intracranial injection of B7-H3-targeted Car-T and Car-Nk cells in a patient-derived glioblastoma xenograft model.

BACKGROUND: Glioblastoma multiforme (GBM) is the most lethal primary brain tumor for which novel therapies are needed. Recently, chimeric antigen receptor (CAR) T cell therapy has been shown to be effective against GBM, but it is a personalized medicine and requires high cost and long time for the cell production. CAR-transduced natural killer (NK) cells can be used for "off-the-shelf" cellular immunotherapy because they do not induce graft-versus-host disease. Therefore, we aimed to analyze the anti-GBM effect of CAR-T or NK cells targeting B7-H3, which is known to be highly expressed in GBM. METHODS: CAR-T cells targeting B7-H3 were generated using previously reported anti-B7-H3 scFv sequences. Cord blood (CB)-derived NK cells transduced with the B7-H3 CAR were also generated. Their anti-GBM effect was analyzed in vitro. The antitumor effect of intracranial injection of the B7-H3 CAR-T or NK cells was investigated in an in vivo xenograft model with patient-derived GBM cells. RESULTS: Both B7-H3 CAR-T cells and CAR-NK cells exhibited marked cytotoxicity against patient-derived GBM cells in vitro. Furthermore, intracranial injection of CAR-T cells and CAR-NK cells targeting B7-H3 resulted in a significant antitumor effect against patient-derived GBM xenografts. CONCLUSION: Not only CAR-T cells but also CB-derived CAR-NK cells targeting B7-H3 may have the potential to eliminate GBM cells.

Multi-trait and cross-population genome-wide association studies across autoimmune and allergic diseases identify shared and distinct genetic component.

OBJECTIVES: Autoimmune and allergic diseases are outcomes of the dysregulation of the immune system. Our study aimed to elucidate differences or shared components in genetic backgrounds between autoimmune and allergic diseases. METHODS: We estimated genetic correlation and performed multi-trait and cross-population genome-wide association study (GWAS) meta-analysis of six immune-related diseases: rheumatoid arthritis, Graves' disease, type 1 diabetes for autoimmune diseases and asthma, atopic dermatitis and pollinosis for allergic diseases. By integrating large-scale biobank resources (Biobank Japan and UK biobank), our study included 105 721 cases and 433 663 controls. Newly identified variants were evaluated in 21 778 cases and 712 767 controls for two additional autoimmune diseases: psoriasis and systemic lupus erythematosus. We performed enrichment analyses of cell types and biological pathways to highlight shared and distinct perspectives. RESULTS: Autoimmune and allergic diseases were not only mutually classified based on genetic backgrounds but also they had multiple positive genetic correlations beyond the classifications. Multi-trait GWAS meta-analysis newly identified six allergic disease-associated loci. We identified four loci shared between the six autoimmune and allergic diseases (rs10803431 at PRDM2, OR=1.07, p=2.3×10-8, rs2053062 at G3BP1, OR=0.90, p=2.9×10-8, rs2210366 at HBS1L, OR=1.07, p=2.5×10-8 in Japanese and rs4529910 at POU2AF1, OR=0.96, p=1.9×10-10 across ancestries). Associations of rs10803431 and rs4529910 were confirmed at the two additional autoimmune diseases. Enrichment analysis demonstrated link to T cells, natural killer cells and various cytokine signals, including innate immune pathways. CONCLUSION: Our multi-trait and cross-population study should elucidate complex pathogenesis shared components across autoimmune and allergic diseases.

Comparison of timing and decision-makers of do-not-resuscitate orders between thoracic cancer and non-cancer respiratory disease patients dying in a Japanese acute care hospital.

PURPOSE: The aim of the study was to compare timing and decision-makers of do-not-resuscitate (DNR) orders between patients with end-stage thoracic cancer and non-cancer respiratory diseases in a Japanese acute care hospital. METHODS: This study retrospectively reviewed the medical records of patients who died between January 2008 and March 2013 in the Department of Respiratory Medicine of Osaka Police Hospital, a teaching and acute care hospital. We compared the decision-making process, especially timing and decision-maker, of DNR orders between patients with thoracic cancer and patients with non-cancer respiratory diseases. RESULTS: There were 300 cancer patients and 147 non-cancer patients. Cancer patients were significantly younger, were hospitalized more frequently and for longer, were more likely to have a DNR order placed earlier and decided in advance of last admission, and were more likely to have normal cognitive function at the time of the DNR order than non-cancer patients. Spouses of cancer patients were more likely to participate in DNR discussion. Only approximately 6 % of patients participated in DNR discussion in both groups. Cancer patients less frequently received aggressive treatment at the end of life (EOL) and were more likely to die in general wards than in intensive care units. CONCLUSIONS: Our study found that most Japanese patients, with or without cancer, who died in an acute care respiratory department, were not included in DNR discussions and that familial surrogates usually made the DNR decision at the EOL.

Usefulness of net retrieval devices for central airway obstruction caused by blood clots during extracorporeal membrane oxygenation: Case series.

RATIONALE: Extracorporeal membrane oxygenation (ECMO) is the last trump card for severe respiratory failure. The main complications of ECMO are bleeding and thrombosis, both of which can be life-threatening. Large blood clots can cause central airway obstruction (CAO) during ECMO, and CAO should be removed as soon as possible because of asphyxiation. However, there is no comprehensive reports on its frequency and management. The purpose of this study is to share therapeutic experiences for rare and serious conditions and provide valuable insights. PATIENT CONCERNS: We report 3 patients placed on ECMO for severe respiratory failure. DIAGNOSIS: CAO due to large blood clots occurred during ECMO in all 3 patients. INTERVENTIONS: Large blood clots were removed using flexible bronchoscopy, grasping forceps, and net retrieval devices in all 3 patients. OUTCOMES: In all 3 patients, large blood clots were removed multiple times during ECMO. The patients' respiratory conditions improved and they were eventually weaned off the ECMO. LESSONS: CAO due to large blood clots during ECMO is rare. The frequency of CAO requiring bronchoscopic removal was estimated to be approximately 1,5%. When this occurs, clots should be removed as soon as possible. Net retrieval devices are useful tools for the collection of large blood clots.

Sema6D forward signaling impairs T cell activation and proliferation in head and neck cancer.

Immune checkpoint inhibitors (ICIs) are indicated for a diverse range of cancer types, and characterizing the tumor immune microenvironment is critical for optimizing therapeutic strategies, including ICIs. T cell infiltration and activation status in the tumor microenvironment greatly affects the efficacy of ICIs. Here, we show that semaphorin 6D (Sema6D) forward signaling, which is reportedly involved in coordinating the orientation of cell development and migration as a guidance factor, impaired the infiltration and activation of tumor-specific CD8+ T cells in murine oral tumors. Sema6D expressed by nonhematopoietic cells was responsible for this phenotype. Plexin-A4, a receptor for Sema6D, inhibited T cell infiltration and partially suppressed CD8+ T cell activation and proliferation induced by Sema6D stimulation. Moreover, mouse oral tumors, which are resistant to PD-1-blocking treatment in wild-type mice, showed a response to the treatment in Sema6d-KO mice. Finally, analyses of public data sets of human head and neck squamous cell carcinoma, pan-cancer cohorts, and a retrospective cohort study showed that SEMA6D was mainly expressed by nonhematopoietic cells such as cancer cells, and SEMA6D expression was significantly negatively correlated with CD8A, PDCD1, IFNG, and GZMB expression. Thus, targeting Sema6D forward signaling is a promising option for increasing ICI efficacy.

CD98 heavy chain protein is overexpressed in non-small cell lung cancer and is a potential target for CAR T-cell therapy.

Chimeric antigen receptor (CAR) T cells are effective against hematological cancers, but are less effective against solid tumors such as non-small cell lung cancer (NSCLC). One of the reasons is that only a few cell surface targets specific for NSCLC cells have been identified. Here, we report that CD98 heavy chain (hc) protein is overexpressed on the surface of NSCLC cells and is a potential target for CAR T cells against NSCLC. Screening of over 10,000 mAb clones raised against NSCLC cell lines showed that mAb H2A011 bound to NSCLC cells but not normal lung epithelial cells. H2A011 recognized CD98hc. Although CAR T cells derived from H2A011 could not be established presumably due to the high level of H2A011 reactivity in activated T cells, those derived from the anti-CD98hc mAb R8H283, which had been shown to lack reactivity with CD98hc glycoforms expressed on normal hematopoietic cells and some normal tissues, were successfully developed. R8H283 specifically reacted with NSCLC cells in six of 15 patients. R8H283-derived CAR T cells exerted significant anti-tumor effects in a xenograft NSCLC model in vivo. These results suggest that R8H283 CAR T cells may become a new therapeutic tool for NSCLC, although careful testing for off-tumor reactivity should be performed in the future.

SFTPB in serum extracellular vesicles as a biomarker of progressive pulmonary fibrosis.

Progressive pulmonary fibrosis (PPF), defined as the worsening of various interstitial lung diseases (ILDs), currently lacks useful biomarkers. To identify novel biomarkers for early detection of patients at risk of PPF, we performed a proteomic analysis of serum extracellular vesicles (EVs). Notably, the identified candidate biomarkers were enriched for lung-derived proteins participating in fibrosis-related pathways. Among them, pulmonary surfactant-associated protein B (SFTPB) in serum EVs could predict ILD progression better than the known biomarkers, serum KL-6 and SP-D, and it was identified as an independent prognostic factor from ILD-gender-age-physiology index. Subsequently, the utility of SFTPB for predicting ILD progression was evaluated further in 2 cohorts using serum EVs and serum, respectively, suggesting that SFTPB in serum EVs but not in serum was helpful. Among SFTPB forms, pro-SFTPB levels were increased in both serum EVs and lungs of patients with PPF compared with those of the control. Consistently, in a mouse model, the levels of pro-SFTPB, primarily originating from alveolar epithelial type 2 cells, were increased similarly in serum EVs and lungs, reflecting pro-fibrotic changes in the lungs, as supported by single-cell RNA sequencing. SFTPB, especially its pro-form, in serum EVs could serve as a biomarker for predicting ILD progression.

A case of CD74-ROS1-positive lung adenocarcinoma diagnosed by next-generation sequencing achieved long-term survival with pemetrexed regimens.

Previously, cytotoxic drugs were the only option for patients with non-small cell lung cancer (NSCLC) and the prognosis was poor. However, molecularly targeted therapies and immune checkpoint inhibitors represent a breakthrough in the treatment of advanced NSCLC and have improved survival rates. In addition, advances in next-generation sequencing (NGS) have revealed the landscape of genomic alterations in patients with different cancers, aiding in the development of new molecularly targeted drugs. The patient reported here was a 54-year-old woman with left lower lung adenocarcinoma. The lung cancer was staged as T2aN3M1a stageIVA 11 years ago. She had received seven regimens of chemotherapy for 11 years. Among these, pemetrexed (PEM) regimens particularly showed long-term effects totaling more than 5 years. We performed NGS after disease progression of the seventh treatment. NGS revealed CD74-ROS1 fusion and she was treated with entrectinib. She has been taking entrectinib for over 20 months now. Herein, we report a rare case of CD74-ROS1-positive lung adenocarcinoma diagnosed by NGS that achieved long-term survival with cytotoxic drugs, especially PEM regimens. In patients showing favorable clinical response to PEM regimens, physicians should consider testing for ROS1/ALK rearrangement.

Identification of glioblastoma-specific antigens expressed in patient-derived tumor cells as candidate targets for chimeric antigen receptor T cell therapy.

Background: New therapies for glioblastoma (GBM) are urgently needed because the disease prognosis is poor. Chimeric antigen receptor (CAR)-T cell therapy that targets GBM-specific cell surface antigens is a promising therapeutic strategy. However, extensive transcriptome analyses have uncovered few GBM-specific target antigens. Methods: We established a library of monoclonal antibodies (mAbs) against a tumor cell line derived from a patient with GBM. We identified mAbs that reacted with tumor cell lines from patients with GBM but not with nonmalignant human brain cells. We then detected the antigens they recognized using expression cloning. CAR-T cells derived from a candidate mAb were generated and tested in vitro and in vivo. Results: We detected 507 mAbs that bound to tumor cell lines from patients with GBM. Among them, E61 and A13 reacted with tumor cell lines from most patients with GBM, but not with nonmalignant human brain cells. We found that B7-H3 was the antigen recognized but E61. CAR-T cells were established using the antigen-recognition domain of E61-secreted cytokines and exerted cytotoxicity in co-culture with tumor cells from patients with GBM. Conclusions: Cancer-specific targets for CAR-T cells were identified using a mAb library raised against primary GBM tumor cells from a patient. We identified a GBM-specific mAb and its antigen. More mAbs against various GBM samples and novel target antigens are expected to be identified using this strategy.

Tumor-derived semaphorin 4A improves PD-1-blocking antibody efficacy by enhancing CD8+ T cell cytotoxicity and proliferation.

Immune checkpoint inhibitors (ICIs) have caused revolutionary changes in cancer treatment, but low response rates remain a challenge. Semaphorin 4A (Sema4A) modulates the immune system through multiple mechanisms in mice, although the role of human Sema4A in the tumor microenvironment remains unclear. This study demonstrates that histologically Sema4A-positive non-small cell lung cancer (NSCLC) responded significantly better to anti-programmed cell death 1 (PD-1) antibody than Sema4A-negative NSCLC. Intriguingly, SEMA4A expression in human NSCLC was mainly derived from tumor cells and was associated with T cell activation. Sema4A promoted cytotoxicity and proliferation of tumor-specific CD8+ T cells without terminal exhaustion by enhancing mammalian target of rapamycin complex 1 and polyamine synthesis, which led to improved efficacy of PD-1 inhibitors in murine models. Improved T cell activation by recombinant Sema4A was also confirmed using isolated tumor-infiltrating T cells from patients with cancer. Thus, Sema4A might be a promising therapeutic target and biomarker for predicting and promoting ICI efficacy.

Proposal of a novel pipeline involving precise bronchoscopy of distal peripheral pulmonary lesions for genetic testing.

Next-generation sequencing (NGS) has become increasingly more important for lung cancer management. We now expect biopsies to be sensitive, safe, and yielding sufficient samples for NGS. In this study, we propose ultraselective biopsy (USB) with sample volume adjustment (SVA) as a novel method that integrates an ultrathin bronchoscope, radial probe endobronchial ultrasound, and the direct oblique method for ultraselective navigation, and adjustment of sample volume for NGS. Our purpose was to estimate the diagnostic potential and the applicability of USB-SVA for amplicon-based NGS analysis. The diagnostic yield of bronchoscopy in forty-nine patients with malignant peripheral pulmonary lesions (PPLs) was retrospectively analyzed, and amplicon-based NGS analysis was performed on samples from some patients using USB. The diagnostic yields of distal PPLs in the USB group were significantly higher than those in the non-USB group (90.5% vs. 50%, respectively, p = 0.015). The extracted amounts of nucleic acids were at least five times the minimum requirement and the sequence quality met the criteria for the Oncomine™ Target Test. Only the tumor cell content of some samples was insufficient. The feasibility of the pipeline for USB, SVA, and amplicon-based NGS in distal PPLs was demonstrated.

Selective targeting of multiple myeloma cells with a monoclonal antibody recognizing the ubiquitous protein CD98 heavy chain.

Cancer-specific cell surface antigens are ideal therapeutic targets for monoclonal antibody (mAb)-based therapy. Here, we report that multiple myeloma (MM), an incurable hematological malignancy, can be specifically targeted by an mAb that recognizes a ubiquitously present protein, CD98 heavy chain (hc) (also known as SLC3A2). We screened more than 10,000 mAb clones raised against MM cells and identified R8H283, an mAb that bound MM cells but not normal hematopoietic or nonhematopoietic cells. R8H283 specifically recognized CD98hc. R8H283 did not react with monomers of CD98hc; instead, it bound CD98hc in heterodimers with a CD98 light chain (CD98lc), a complex that functions as an amino acid transporter. CD98 heterodimers were abundant on MM cells and took up amino acids for constitutive production of immunoglobulin. Although CD98 heterodimers were also present on normal leukocytes, R8H283 did not react with them. The glycoforms of CD98hc present on normal leukocytes were distinct from those present on MM cells, which may explain the lack of R8H283 reactivity to normal leukocytes. R8H283 exerted anti-MM effects without damaging normal hematopoietic cells. These findings suggested that R8H283 is a candidate for mAb-based therapies for MM. In addition, our findings showed that a cancer-specific conformational epitope in a ubiquitous protein, which cannot be identified by transcriptome or proteome analyses, can be found by extensive screening of primary human tumor samples.

Impact of sarcopenia in patients with advanced non-small cell lung cancer treated with PD-1 inhibitors: A preliminary retrospective study.

The aim of this study was to investigate the clinical impact of sarcopenia on the efficacy of programmed death (PD)-1 inhibitors. We retrospectively reviewed the medical records of all patients treated with nivolumab or pembrolizumab between January 2016 and September 2018 for previously treated advanced non-small cell lung cancer (NSCLC). The cross-sectional area of the psoas muscle at the level of the third lumbar vertebra on baseline computed tomography was assessed to calculate the psoas muscle index (PMI). Sarcopenia was defined based on PMI cut-off values for Asian adults (6.36 cm2/m2 for males and 3.92 cm2/m2 for females). A total of 42 patients were analysed. The prevalence of sarcopenia was 52.4%. Sarcopenia was significantly associated with poorer progression-free survival (PFS) (median, 2.1 vs. 6.8 months, p = 0.004). Compared to patients with sarcopenia, those without sarcopenia had a higher overall response rate (40.0% vs. 9.1%, p = 0.025) and 1-year PFS rate (38.1% vs. 10.1%). In conclusion, sarcopenia at baseline as determined using computed tomography is a significant predictor of worse outcome in patients with advanced NSCLC receiving PD-1 blockade. Screening for sarcopenia may help identify patients more likely to achieve a long-term response in routine clinical practice.

Phase II trial of induction chemotherapy of pemetrexed plus split-dose cisplatin followed by pemetrexed maintenance for untreated non-squamous non-small-cell lung cancer.

PURPOSE: We conducted a phase II trial to evaluate the efficacy and safety of induction chemotherapy of pemetrexed plus split-dose cisplatin followed by pemetrexed maintenance for advanced non-squamous non-small-cell lung cancer (NSCLC). METHODS: Patients with advanced or recurrent untreated non-squamous NSCLC received split-dose cisplatin (40 mg/m2, days 1 and 8) plus pemetrexed (500 mg/m2, day 1) tri-weekly. After four cycles of induction, patients without disease progression received pemetrexed maintenance until disease progression or unacceptable toxicity. The primary endpoint was the 1-year survival rate. The secondary endpoints were progression-free survival (PFS), overall survival (OS), response in induction phase, and safety. RESULTS: From February 2012 to September 2014, 53 assessable patients were enrolled in this study. Thirty-eight (71.7%) patients completed induction therapy, while 35 (66.0%) received maintenance therapy. The 1-year survival rate was 67.7%. The median PFS and OS were 5.3 and 18.6 months, respectively. The response rate and disease control rate (DCR) during the induction phase were 37.7 and 86.8%, respectively. Eight patients (15.1%) discontinued the therapy due to adverse events (AEs) during the induction phase, but both hematological and non-hematological AEs were infrequent. CONCLUSIONS: Treatment with induction chemotherapy of pemetrexed plus split-dose cisplatin showed a promising 1-year survival rate, DCR, and transition rate into maintenance phase. This regimen is feasible and well-tolerated. A phase III study comparing this regimen with conventional tri-weekly regimen is warranted.

Amylase-Producing Lung Cancer with a Positive Epidermal Growth Factor Receptor Mutation Treated With Gefitinib: A Case Report.

A 60-year-old woman was diagnosed with metastatic pulmonary adenocarcinoma (c-stage IV) with an L858R point mutation in the gene encoding epidermal growth factor receptor (EGFR). Serum amylase levels were elevated (1,531 IU/L) with the salivary-type enzyme dominating. First-line chemotherapy using carboplatin plus paclitaxel reduced serum amylase levels, although second-line gefitinib eventually failed to control tumor growth and hyperamylasemia after 4.5 months of treatment. The cancer cells harbored a positive EGFR mutation and secreted amylase. The number of amylase-producing cancer cells and the immunochemical staining intensity for amylase were significantly reduced after gefitinib treatment. This was a rare case of a lung cancer that expressed amylase and harbored a positive EGFR mutation.