Immune-checkpoint profiles for T cells in bronchoalveolar lavage fluid of patients with immune-checkpoint inhibitor-related interstitial lung disease.

Immune-checkpoint inhibitors (ICIs) have improved clinical outcomes and are becoming a standard treatment for many cancer types. However, these drugs also induce immune-related adverse events, among which interstitial lung disease (ILD) is potentially fatal. The underlying mechanism of ILD induction by ICIs is largely unknown. With the use of flow cytometry, we determined the expression levels of the immune-checkpoint proteins PD-1, TIM-3, TIGIT, LAG-3 and PD-L1 in T cells of bronchoalveolar lavage fluid (BALF) from patients with ICI-related ILD and compared them with those for patients with sarcoidosis or with ILD related to connective tissue disease or cytotoxic drug use. The proportions of CD8+ T cells positive for both PD-1 and TIM-3 or for TIGIT in BALF were significantly higher for ICI-related ILD patients than for those with other types of ILD. A prominent increase in the proportion of PD-1+PD-L1+ cells among CD8+ T cells was also apparent in BALF of a patient with a fatal case of ICI-related ILD, and the proportion of such cells was positively correlated with the grade of ICI-related ILD. Our data reveal the immune-checkpoint profiles of T cells in ICI-related ILD and may provide mechanistic insight into the development of this adverse event.

Sessile serrated adenoma/polyp showed rapid malignant transformation in the final 13 months.

Based on the concept of the adenoma-carcinoma sequence, most colorectal cancers are considered to arise from conventional adenomas. However, recent studies suggested that a subset of colorectal cancers develop through the serrated neoplastic pathway. It has also been documented that serrated polyps can rapidly transform into invasive cancers even when they are small in size. We now describe a case of a sessile serrated adenoma/polyp which had been followed up for 4 years but eventually showed rapid transformation into an advanced cancer accompanied by a remarkable morphological change within only 13 months. Retrospective genetic and epigenetic analyses showed microsatellite instability, CpG island methylator phenotype-positive, and BRAF mutation in the lesion, suggesting the tumor had developed through the serrated neoplastic pathway. This case may provide valuable information about the natural history of sessile serrated adenoma/polyps which eventually progress to advanced cancers.

Subtypes of the Type II Pit Pattern Reflect Distinct Molecular Subclasses in the Serrated Neoplastic Pathway.

BACKGROUND: Colorectal serrated lesions (SLs) are important premalignant lesions whose clinical and biological features are not fully understood. AIMS: We aimed to establish accurate colonoscopic diagnosis and treatment of SLs through evaluation of associations among the morphological, pathological, and molecular characteristics of SLs. METHODS: A total of 388 premalignant and 18 malignant colorectal lesions were studied. Using magnifying colonoscopy, microsurface structures were assessed based on Kudo's pit pattern classification system, and the Type II pit pattern was subcategorized into classical Type II, Type II-Open (Type II-O) and Type II-Long (Type II-L). BRAF/KRAS mutations and DNA methylation of CpG island methylator phenotype (CIMP) markers (MINT1, - 2, - 12, - 31, p16, and MLH1) were analyzed through pyrosequencing. RESULTS: Type II-O was tightly associated with sessile serrated adenoma/polyps (SSA/Ps) with BRAF mutation and CIMP-high. Most lesions with simple Type II or Type II-L were hyperplastic polyps, while mixtures of Type II or Type II-L plus more advanced pit patterns (III/IV) were characteristic of traditional serrated adenomas (TSAs). Type II-positive TSAs frequently exhibited BRAF mutation and CIMP-low, while Type II-L-positive TSAs were tightly associated with KRAS mutation and CIMP-low. Analysis of lesions containing both premalignant and cancerous components suggested Type II-L-positive TSAs may develop into KRAS-mutated/CIMP-low/microsatellite stable cancers, while Type II-O-positive SSA/Ps develop into BRAF-mutated/CIMP-high/microsatellite unstable cancers. CONCLUSIONS: These results suggest that Type II subtypes reflect distinct molecular subclasses in the serrated neoplasia pathway and that they could be useful hallmarks for identifying SLs at high risk of developing into CRC.

Treatment with a programmed cell death-1-specific antibody has little effect on afatinib- and naphthalene-induced acute pneumonitis in mice.

Although several antibodies developed to target programmed cell death-1 (PD-1) and its ligand (PD-L1) have demonstrated great promise for the treatment of non-small cell lung cancer (NSCLC), and other malignancies, these therapeutic antibodies can cause pneumonitis. Furthermore, epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-induced pneumonitis was reported after treatment with anti PD-1 antibodies. We previously demonstrated that mice with naphthalene-induced airway epithelial injury developed severe gefitinib-induced pneumonitis through a neutrophil-dependent mechanism. The present study aimed to investigate whether treatment with afatinib, an EGFR-TKI that effectively targets EGFR mutation-positive NSCLC, and anti PD-1 antibodies induces pneumonitis in mice. C57BL/6J mice were treated intraperitoneally with naphthalene (200 mg/kg) on day 0. Afatinib (20 mg/kg) was administered orally on days -1 to 13. An anti-PD-1 antibody (0.2 mg/mice) was also administered intraperitoneally every 3 days from day 1 until day 13. The bronchoalveolar lavage fluid (BALF) and lung tissues were sampled on day 14. As observed previously with gefitinib, afatinib significantly increased the severity of histopathologic findings and the level of protein in BALF on day 14, compared to treatment with naphthalene alone. A combined anti-PD-1 antibody and afatinib treatment after naphthalene administration had yielded the same histopathological grade of lung inflammation as did afatinib treatment alone. Our results suggest that anti-PD-1 antibody treatment has little effect on afatinib-induced lung injury.

Neutrophil elastase inhibitor sivelestat ameliorates gefitinib-naphthalene-induced acute pneumonitis in mice.

Gefitinib, an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), is an effective therapeutic agent for non-small cell lung cancer with EGFR mutations. It can cause severe acute pneumonitis in some patients. We previously demonstrated that mice with naphthalene-induced airway epithelial injury developed severe gefitinib-induced pneumonitis and that neutrophils played important roles in the development of the disease. This study aimed to investigate the effects of the neutrophil elastase inhibitor sivelestat on gefitinib-induced pneumonitis in mice. C57BL/6J mice received naphthalene (200 mg/kg) intraperitoneally on day 0. Gefitinib (250 or 300 mg/kg) was orally administered to mice from day -1 until day 13. Sivelestat (150 mg/kg) was administered intraperitoneally from day 1 until day 13. Bronchoalveolar lavage fluid (BALF) and lung tissues were sampled on day 14. Sivelestat treatment significantly reduced the protein level, neutrophil count, neutrophil elastase activity in BALF, and severity of histopathologic findings on day 14 for mice administered with 250 mg/kg of gefitinib. Moreover, sivelestat treatment significantly improved the survival of mice administered with 300 mg/kg of gefitinib. These results indicate that sivelestat is a promising therapeutic agent for severe acute pneumonitis caused by gefitinib.

Endoscopic and molecular characterization of colorectal sessile serrated adenoma/polyps with cytologic dysplasia.

BACKGROUND AND AIMS: Sessile serrated adenoma/polyps (SSA/Ps), which are precursor lesions of colorectal cancer (CRC) with BRAF mutation and the CpG island methylator phenotype (CIMP), develop cytologic dysplasia (CD) during the progression of colorectal tumorigenesis. In the present study we aimed to clarify the endoscopic and molecular signatures of SSA/Ps, with and without CD. METHODS: A series of 208 serrated lesions, including 41 hyperplastic polyps, 90 SSA/Ps, 33 SSA/Ps with CD, and 44 traditional serrated adenomas, were observed and resected using magnifying endoscopy. BRAF and KRAS mutations and methylation of CIMP markers (MINT1, MINT2, MINT12, MINT31, and p16) were analyzed through pyrosequencing. Molecular alterations were then compared with endoscopic and pathologic characteristics. RESULTS: Among SSA/Ps without CD, the Type II-Open pit pattern (Type II-O), BRAF mutation, and CIMP were tightly associated with a proximal colon location. SSA/Ps in the distal colon infrequently exhibited Type II-O and CIMP. By contrast, most SSA/Ps with CD showed Type II-O plus adenomatous pit patterns (Type III or IV), BRAF mutation, and CIMP, irrespective of their locations. CONCLUSIONS: Our results suggest that the Type II-O plus III/IV pit pattern is a common feature of SSA/Ps with CD in both the proximal and distal colon and that this pit pattern is a hallmark of serrated lesions at high risk of developing into CRCs.

The incremental burden of pain in patients with depression: results of a Japanese survey.

BACKGROUND: Major depressive disorder (MDD) is a chronic mental illness which affects an estimated 3% of the Japanese population. Many patients with MDD report painful physical symptoms, and research outside of Japan suggests such patients may represent a subtype of depression which is more severe and difficult to treat. There is no evidence available about the characteristics or incremental burden of these patients in Japan. The objective of this study was to quantify the incremental burden of physical pain among individuals in Japan diagnosed with depression. METHODS: Data for individuals age 18 and older who reported a physician diagnosis of depression were obtained from the Japan National Health and Wellness Survey (NHWS). Respondents who also reported physical pain were matched to respondents who did not report pain using propensity scores and compared using bivariate statistics. Measures included Patient Health Questionnaire (PHQ-9) for depression severity, Medical Outcomes Study 12-Item Short Form Survey Instrument (SF-12v2) for health-related quality of life, the Work Productivity and Activity Impairment (WPAI) for work and activity impairment, and 6-month report of health care use. RESULTS: Individuals with depression who reported physical pain had higher PHQ-9 depression scores (14.3 vs. 11.1, p<0.001), lower health-related quality of life (Mental Component Summary score [MCS] 29.1 vs. 32.0, p<0.01; Physical Component Summary score [PCS] 43.0 vs. 47.2, p<0.001; health utility [SF-6D] 0.567 vs. 0.613, p<0.001), more presenteeism (46.3% vs. 36.8%, p<0.01), more overall work impairment (51.4% vs. 42.3%, p<0.01), more activity impairment (55.4% vs. 43.9%, p<0.001), and reported using more health care provider visits in the prior 6 months (17.7 vs. 12.8, p<0.01) as well as hospitalizations (1.7 vs. 0.8, p<0.05) relative to propensity-score matched controls without pain. Absenteeism (13.1% vs. 11.4%, p=0.51) and emergency room visits (0.31 vs. 0.35, p=0.76) were not significantly different between the two matched groups. CONCLUSIONS: Individuals whose depression is accompanied by physical pain have a higher burden of illness than those whose depression does not include physical pain. Clinicians should take the presence of pain into account and consider treating both the physical and emotional symptoms of these patients.

[Measurement of sitafloxacin MIC for Mycobacterium avium complex and application for treatment of pulmonary nontuberculous mycobacteriosis].

Treatment for pulmonary nontuberculous mycobacteriosis is difficult. Since current treatment has limitation, new application is needed. Fluoroquinolone is one of candidates. We have investigated the feasibility of sitafloxacin (STFX). At first, the drug of MIC (minimum inhibitory concentration) was determined by the methods based on BrothMIC NTM. The MICs of STFX, moxifloxacin (MFLX), gatifloxacin (GFLX) were low. On contrast, the MICs of garenoxacin (GRNX) and tosufloxacin (TFLX) were high. Two cases of pulmonary Mycobacterium avium-intracellulare complex (MAC) disease were treated by STFX-contained regimen. In all cases of pulmonary MAC disease, improve of symptoms and chest CT images were attained. Adverse events were slight. These MIC studies and case reports suggest that STFX might have excellent in vitro and in vivo antimicrobial activities against MAC and is considered to be a candidate for the medication against pulmonary MAC disease.

A case of inflammatory myofibroblastic tumor harboring EML4-ALK fusion with a brain metastasis responding to alectinib.

Metastatic inflammatory myofibroblastic tumor (IMT) is very rare and detailed reports on diagnosis and treatment are limited. Here, we report a case of metastatic IMT with ALK rearrangement. A 73-year-old woman was diagnosed with IMT involving a brain metastasis. Next generation sequencing (NGS) panel testing with Oncomine dx target test revealed that her tumor was positive for EML4-ALK. Treatment with alectinib was initiated, resulting in remarkable shrinkage of both the primary tumor and the brain metastasis. This report is the first to identify ALK rearrangement in IMT using a commercially available NGS panel testing, followed by treatment with alectinib. This case suggests that NGS panel testing may be useful in the diagnosis and treatment of patients with metastatic IMT.

Survival and acute exacerbation for patients with idiopathic pulmonary fibrosis (IPF) or non-IPF idiopathic interstitial pneumonias: 5-year follow-up analysis of a prospective multi-institutional patient registry.

OBJECTIVE: Few prospective cohort studies with relatively large numbers of patients with non-idiopathic pulmonary fibrosis (non-IPF) of idiopathic interstitial pneumonia (IIP) have been described. We aimed to assess disease progression and cause of death for patients with non-IPF IIPs or IPF under real-life conditions. METHODS: Data were analysed for a prospective multi-institutional cohort of 528 IIP patients enrolled in Japan between September 2013 and April 2016. Diagnosis of IPF versus non-IPF IIPs was based on central multidisciplinary discussion, and follow-up surveillance was performed for up to 5 years after patient registration. Survival and acute exacerbation (AE) were assessed. RESULTS: IPF was the most common diagnosis (58.0%), followed by unclassifiable IIPs (35.8%) and others (6.2%). The 5-year survival rate for non-IPF IIP and IPF groups was 72.8% and 53.7%, respectively, with chronic respiratory failure being the primary cause of death in both groups. AE was the second most common cause of death for both non-IPF IIP (24.1%) and IPF (23.5%) patients. The cumulative incidence of AE did not differ significantly between the two groups (p=0.36), with a 1-year incidence rate of 7.4% and 9.0% in non-IPF IIP and IPF patients, respectively. We found that 30.2% and 39.4% of non-IPF IIP and IPF patients, respectively, who experienced AE died within 3 months after an AE event, whereas 55.8% and 66.7% of such patients, respectively, died within 5 years after registration. CONCLUSION: Closer monitoring of disease progression and palliative care interventions after AE are important for non-IPF IIP patients as well as for IPF patients.

Identifying risk factors for refractory febrile neutropenia in patients with lung cancer.

Information about the development of febrile neutropenia in patients with solid tumors remains insufficient. In this study, we tried to identify the risk factors for refractory febrile neutropenia in patients with lung cancer. A total of 59 neutropenic fever episodes associated with anti-tumor chemotherapy for lung cancer were retrospectively analyzed. We compared patient characteristics according to their initial response to treatment with antibiotics. For 34 of 59 (58%) episodes a response to initial antibiotics was obtained whereas 25 of 59 (42%) were refractory to treatment. Multivariate analysis demonstrated independent risk factors for refractory febrile neutropenia with lung cancer. These risk factors were the severity of febrile neutropenia (odds ratio (OR) 6.11; 95% confidence interval (CI) 1.85-20.14) and C-reactive protein more than 10 mg/dl (OR 4.39; 95% CI 1.22-15.74). These factors could predict outcome for patients with lung cancer who develop refractory febrile neutropenia.

The clinical efficacy and safety of a fluoroquinolone-containing regimen for pulmonary MAC disease.

Despite recent advances in chemotherapy, the treatment of pulmonary Mycobacterium avium complex (MAC) disease remains unsatisfactory. Judging from its MIC, fluoroquinolones including gatifloxacin (GFLX) are expected to demonstrate efficacy against MAC disease. However, there have been few clinical studies using fluoroquinolones. Therefore, a prospective study to evaluate the clinical efficacy and safety of a fluoroquinolone-containing regimen for the treatment of pulmonary MAC disease was conducted. In this trial, patients with pulmonary MAC disease received protocol-guided combined chemotherapy with rifampin (RFP) and ethambutol (EB) plus either GFLX or clarithromycin (CAM). Adult patients who fulfilled the criteria of the ATS definition of pulmonary MAC disease were enrolled in this study. The patients provided their informed consent, and treatments were administered for 1 year. Of 27 patients enrolled from three facilities, 14 patients were treated with the CAM-containing regimen and 13 patients were treated with the GFLX-containing regime. Four patients did not complete the 1-year treatment because of adverse events. Nine patients (64.3%) in the CAM group and 11 patients (84.6%) in the GFLX group achieved eradication of pathogens. Adverse events were observed more frequently in the GFLX group than in the CAM group. However, there were no severe adverse events in either group. The long-term results showed a similar relapse rate between the CAM and GFLX groups. The fluoroquinolone-containing regimen demonstrated both high efficacy and relative safety for pulmonary MAC disease that was similar to that of the CAM-containing regimen, which is considered to be the standard regimen.

The clinical efficacy and safety of micafungin-itraconazole combination therapy in patients with pulmonary aspergilloma.

In spite of recent advances in chemotherapy, the treatment of pulmonary aspergilloma remains unsatisfactory. To evaluate the clinical efficacy and safety of combination therapy for pulmonary aspergilloma, we conducted a multi-institutional prospective study using micafungin (MCFG) and itraconazole (ITCZ). Adult patients who fulfilled the criteria for pulmonary aspergilloma were enrolled in this study. After patient consent had been obtained, intravenous MCFG 150 mg/day and an oral capsule of ITCZ 200 mg/day were administered for at least 1 month. The primary endpoint was the response assessed using an algorithm that incorporated the levels of improvement by evaluating clinical symptoms and signs, mycological and serological tests, and diagnostic imaging. A total of 17 patients were enrolled from three facilities. The response rate to the combination therapy was 58.8% (10/17). The long-term control in the group of patients who responded to treatment was better than that in the group of patients who did not respond. Adverse events occurred in 6 of the 17 patients (35.3%), but there were no severe adverse events. MCFG-ITCZ combination therapy appeared to be relatively safe and effective in patients with pulmonary aspergilloma.

Scedosporium apiospermum lung disease in a patient with nontuberculous mycobacteria.

Although tuberculosis is a major underlying cause of pulmonary mycetoma due to Scedosporium apiospermum, little is known about coinfection with nontuberculous mycobacteria (NTM) and S. apiospermum. A 67-year-old man with NTM presented with recurrent haemoptysis. Computed tomography of the chest revealed pulmonary mycetoma in the left upper lobe of the lung, and culture of bronchial washing fluid yielded S. apiospermum. Oral voriconazole therapy ameliorated both haemoptysis and mycetoma findings. As far as we are aware, this is the first reported case of S. apiospermum lung disease in a patient with NTM but without tuberculosis. The possibility of S. apiospermum infection should thus be considered in the differential diagnosis of pulmonary mycetoma. Although S. apiospermum mycetoma resembles aspergilloma, antifungal strategies for these two conditions differ, with the collection of culture specimens such as by bronchoscopy being compulsory for accurate diagnosis and appropriate management of S. apiospermum infection.

Imaging Changes and Immune-Checkpoint Expression on T Cells in Bronchoalveolar Lavage Fluid from Patients with Pulmonary Sarcoidosis.

Sarcoidosis is a systemic, granulomatous disease caused by unknown immunological abnormalities. The organs most vulnerable to sarcoidosis are the lungs. Patients often resolve spontaneously, but the lungs can also be severely affected. Although details regarding prognostic factors in sarcoidosis patients with lung involvement remain unclear, several reports have suggested that immune checkpoint molecules are involved in the pathogenesis of sarcoidosis. In this study, we divided sarcoidosis patients into two groups based on chest computed tomography (CT) findings and compared immune checkpoint molecules expressed on T cells in bronchoalveolar lavage fluid (BALF) in the two groups, using flow cytometry. We found elevated programmed cell death 1 (PD-1) or T cell immunoglobulin- and mucin-domain-containing molecule-3 (TIM-3) expression on T cells in BALF in patients with spontaneous improvement in CT findings, compared with those in patients without improvement in CT findings. In conclusion, our study implies that PD-1 or TIM-3 expression on T cells in BALF may be a prognostic factor for pulmonary lesions in sarcoidosis.

Glucose metabolism in Legionella pneumophila: dependence on the Entner-Doudoroff pathway and connection with intracellular bacterial growth.

Glucose metabolism in Legionella pneumophila was studied by focusing on the Entner-Doudoroff (ED) pathway with a combined genetic and biochemical approach. The bacterium utilized exogenous glucose for synthesis of acid-insoluble cell components but manifested no discernible increase in the growth rate. Assays with permeabilized cell preparations revealed the activities of three enzymes involved in the pathway, i.e., glucokinase, phosphogluconate dehydratase, and 2-dehydro-3-deoxy-phosphogluconate aldolase, presumed to be encoded by the glk, edd, and eda genes, respectively. Gene-disrupted mutants for the three genes and the ywtG gene encoding a putative sugar transporter were devoid of the ability to metabolize exogenous glucose, indicating that the pathway is almost exclusively responsible for glucose metabolism and that the ywtG gene product is the glucose transporter. It was also established that these four genes formed part of an operon in which the gene order was edd-glk-eda-ywtG, as predicted by genomic information. Intriguingly, while the mutants exhibited no appreciable change in growth characteristics in vitro, they were defective in multiplication within eukaryotic cells, strongly indicating that the ED pathway must be functional for the intracellular growth of the bacterium to occur. Curiously, while the deficient glucose metabolism of the ywtG mutant was successfully complemented by the ywtG(+) gene supplied in trans via plasmid, its defect in intracellular growth was not. However, the latter defect was also manifested in wild-type cells when a plasmid carrying the mutant ywtG gene was introduced. This phenomenon, resembling so-called dominant negativity, awaits further investigation.

Methylation-associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect.

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that the silencing of some miRNAs is associated with CpG island hypermethylation. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by treatment with 5-aza-2'-deoxycytidine and 4-phenylbutyrate. We found that miR-34b and miR-34c are epigenetically silenced in GC and that their downregulation is associated with hypermethylation of the neighboring CpG island. Methylation of the miR-34b/c CpG island was frequently observed in GC cell lines (13/13, 100%) but not in normal gastric mucosa from Helicobacter pylori-negative healthy individuals. Transfection of a precursor of miR-34b and miR-34c into GC cells induced growth suppression and dramatically changed the gene expression profile. Methylation of miR-34b/c was found in a majority of primary GC specimens (83/118, 70%). Notably, analysis of non-cancerous gastric mucosae from GC patients (n = 109) and healthy individuals (n = 85) revealed that methylation levels are higher in gastric mucosae from patients with multiple GC than in mucosae from patients with single GC (27.3 versus 20.8%; P < 0.001) or mucosae from H. pylori-positive healthy individuals (27.3 versus 20.7%; P < 0.001). These results suggest that miR-34b and miR-34c are novel tumor suppressors frequently silenced by DNA methylation in GC, that methylation of miR-34b/c is involved in an epigenetic field defect and that the methylation might be a predictive marker of GC risk.

Mycobacterium avium pleuritis with multiple nodules in the pleura.

As opposed to tuberculosis, pleurisy hardly develops in patients with nontuberculous mycobacteria (NTM) infection. In spite of increasing prevalence of NTM infection, little is known about thoracoscopic or pathological findings of the NTM-infected pleura. We now report the first case of NTM pleuritis with multiple granulomatous nodules in the pleura. A 74-year-old woman was admitted to our hospital due to massive effusion of the left thoracic cavity. The analysis of pleural fluid showed lymphocytic exudative effusions with increased levels of adenosine deaminase, although culture of the pleural fluid was negative. The patient accordingly underwent thoracoscopy, which revealed multiple pleural nodules. Biopsy of the nodules demonstrated epithelioid cell granulomas without caseous necrosis. In addition, culture of the biopsy specimens confirmed infection by Mycobacterium avium. As culture of pleural fluid often fails to detect NTM pathogens, demonstration of pleural nodules during thoracoscopy can contribute to prompt diagnosis and treatment of NTM pleuritis.