Novel therapeutic strategies targeting bypass pathways and mitochondrial dysfunction to combat resistance to RET inhibitors in NSCLC.

RET fusion is an oncogenic driver in 1-2 % of patients with non-small cell lung cancer (NSCLC). Although RET-positive tumors have been treated with multikinase inhibitors such as vandetanib or RET-selective inhibitors, ultimately resistance to them develops. Here we established vandetanib resistance (VR) clones from LC-2/ad cells harboring CCDC6-RET fusion and explored the molecular mechanism of the resistance. Each VR clone had a distinct phenotype, implying they had acquired resistance via different mechanisms. Consistently, whole exome-seq and RNA-seq revealed that the VR clones had unique mutational signatures and expression profiles, and shared only a few common remarkable events. AXL and IGF-1R were activated as bypass pathway in different VR clones, and sensitive to a combination of RET and AXL inhibitors or IGF-1R inhibitors, respectively. SMARCA4 loss was also found in a particular VR clone and 55 % of post-TKI lung tumor tissues, being correlated with higher sensitivity to SMARCA4/SMARCA2 dual inhibition and shorter PFS after subsequent treatments. Finally, we detected an increased number of damaged mitochondria in one VR clone, which conferred sensitivity to mitochondrial electron transfer chain inhibitors. Increased mitochondria were also observed in post-TKI biopsy specimens in 13/20 cases of NSCLC, suggesting a potential strategy targeting mitochondria to treat resistant tumors. Our data propose new promising therapeutic options to combat resistance to RET inhibitors in NSCLC.

Patient-derived spheroids and patient-derived organoids simulate evolutions of lung cancer.

Cancer cells harbor many genetic mutations and gene expression profiles different from normal cells. Patient-derived cancer cells (PDCC) are preferred materials in cancer study. We established patient-derived spheroids (PDSs) and patient-derived organoids (PDOs) from PDCCs isolated from the malignant pleural effusion in 8 patients. The morphologies suggested that PDSs may be a model of local cancer extensions, while PDOs may be a model of distant cancer metastases. The gene expression profiles differed between PDSs and PDOs: Gene sets related to inflammatory responses and EMT were antithetically regulated in PDSs or in PDOs. PDSs demonstrated an attenuation of the pathways that contribute to the enhancement of transforming growth factor beta (TGF-ß) induced epithelial mesenchymal transition (EMT), while PDOs demonstrated an attenuation of it. Taken together, PDSs and PDOs have differences in both the interaction to the immune systems and to the stroma. PDSs and PDOs will provide a model system that enable intimate investigation of the behavior of cancer cells in the body.

Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer.

BACKGROUND: Bronchoscopy is a minimally invasive procedure for establishing the diagnosis of lung cancer. It sometimes fails to obtain tissue samples but readily collects cytological samples. METHODS: We developed PNA-LNA dual-PCR (PLDP), which amplified mutant sequences by a high-fidelity DNA polymerase in the presence of a peptide nucleic acid (PNA) oligomer having a wild-type sequence. Mutations are detected either by locked nucleic acid (LNA) probes for quick detection of a limited number of mutations, which are EGFR, KRAS, and BRAF mutations in the current study, or by direct sequencing for a comprehensive screening. In a total of 233 lung cancer samples, the results for cytological samples by PLDP were compared with those for tissue samples by cobas® EGFR mutation test (cobas) or by the PNA-LNA PCR clamp method (P-LPC). Moreover, the performance of PLDP using cell-free DNA (cfDNA) was investigated. RESULTS: Peptide nucleic acid-LNA dual-PCR was able to detect each synthesized mutant sequence with high sensitivity. PLDP detected EGFR mutations in 80 out of 149 clinical samples, while the cobas or the P-LPC detected in 66 matched. The correctness of PLDP was confirmed both by clinical response and by the results of sequencing using a next-generation sequencer. PLDP detected mutations from cfDNA in approximately 70% of patients who harbors mutations in the tumor. CONCLUSIONS: Peptide nucleic acid-LNA dual-PCR exhibited an excellent performance, even using cytological samples. PLDP is applicable for the investigation of cfDNA. The combination of bronchoscopy and PLDP is attractive and will expand the utility of bronchoscopy in clinical practice.

Cytological detection of metastatic chordoma cells in pleural effusions: A case report.

Cytological detection of chordoma cells in the serosal cavity is challenging because of its rare presentation. Herein, we report a case of chordoma showing malignant pleural effusion accompanied by pleuropulmonary metastases in a 68-year-old woman. Cytological analysis was performed using pleural fluid obtained following thoracentesis. Conventional cytological staining demonstrated few clusters of large, atypical cells characterized by epithelial cell-like connectivity and rich cytoplasm with foamy and/or multivacuolar changes. The nuclei of these atypical cells were large and either round or oval with no conspicuous irregularities in the nuclear membrane. Periodic acid-Schiff staining of these atypical cells revealed fine granules in the cytoplasm. Giemsa staining showed foamy and/or multivacuolar cytoplasm in these cells, with metachromatic mucoid stroma in the surroundings. Immunocytochemistry analysis using cellblock showed these cells to be positive for broad cytokeratins, epithelial membrane antigen, S100 protein, vimentin, and Brachyury. To the best of our knowledge, this is the first case report in which chordoma cells were cytologically detected in pleural effusions. Our findings also suggest that conventional cytology combined with cellblock immunocytochemistry can increase the accuracy of chordoma cell detection in the serosal cavity.

A comparative study of the Parker Flex-Tip tube versus standard portex tube for oral fiberoptic intubation in bronchoscopy performed by pulmonologists with limited experience.

BACKGROUND: Fiberoptic tracheal intubation (FTI) in bronchoscopy is widely performed with a conventional Portex tracheal tube (PTT). Occasionally, it is difficult for pulmonologists with limited experience to insert a tube beyond the vocal cords and advance it into the trachea. A new endotracheal tube, the Parker Flex-Tip tube (PFT), was recently designed. We compared the usefulness and safety of PFT versus PTT for FTI in bronchoscopy performed by pulmonologists with limited experience. METHODS: Forty consecutive patients were enrolled and randomly assigned to either the PFT group (n = 20) or PTT group (n = 20). The time required for the tip of the endotracheal tube to pass from the mouth to the carina, the number of vomiting reflexes, the number of attempts to pass the tube through the vocal cords during intubation, complications, and technical difficulty of intubation were evaluated. RESULTS: Both the PFT and PTT groups exhibited high intubation success rates (100% vs. 90%, respectively). The PFT group was intubated faster than the PTT group (11.5 [5-45] s vs. 22.5 [8-270] s, respectively, p < 0.01). The PFT group showed fewer vomiting reflexes and tube impingements than the PTT group (p < 0.05). Operators felt it was easier to intubate with PFT versus PTT (p < 0.01). Complications were not significantly different between the two groups. CONCLUSION: For pulmonologists with limited experience who perform FTI in bronchoscopy, intubation using PFT versus PTT is faster and easier, without an increase in complications.

A case of lymphocytic interstitial pneumonia presenting with a ground glass nodule as an initial finding.

A 65-year-old woman had a ground glass nodule, which was suspicious for lung cancer, in her right lung S6 by chest computed tomography. For diagnosis, video-assisted thoracoscopic surgery was performed, and the specimen showed a pathological pattern of lymphocytic interstitial pneumonia (LIP). Four years after surgery, new localized ground glass shadows gradually increased on the base of the lung. However, because she had no respiratory symptoms and had normal respiratory function, she was observed with no medication. Subsequently, no other underlying diseases associated with LIP developed. The ground glass nodule was the initial lesion of LIP.

Inflammasome-Independent and Atypical Processing of IL-1ß Contributes to Acid Aspiration-Induced Acute Lung Injury.

Inflammation plays a pivotal role in the pathophysiology of gastric aspiration-induced acute lung injury (ALI). However, its mechanism remains unclear. In this study, we investigated the role of NLRP3 inflammasome-driven IL-1ß production in a mouse model of acid aspiration-induced inflammation and ALI. Acid aspiration-induced inflammatory responses and ALI in wild-type mice were significantly attenuated in IL-1ß-/- mice, but not NLRP3-/- mice. In vitro experiments revealed that severe acidic stress (pH 1.75) induced the processing of pro-IL-1ß into its 18-kDa mature form (p18-IL-1ß), which was different from the caspase-1-processed 17-kDa form (p17-IL-1ß), in human THP-1 macrophages and primary murine macrophages. Deficiency of NLRP3 and caspase-1 had no effect on acidic stress-produced IL-1ß. The production of IL-1ß by severe acidic stress was prevented by inhibitors of serine proteases [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], but not of cysteine proteases (E-64), cathepsin G, or inflammasome. The cathepsin D inhibitor pepstatin A inhibited IL-1ß production induced by mild acidic stress (pH 6.2) or lactic acid, but not severe acidic stress. Using mass spectrometry and processing-site mutants of pro-IL-1ß, we identified D109 as a novel cleavage site of pro-IL-1ß in response to severe acidic stress and calculated the theoretical molecular mass of the mature form to be 18.2 kDa. The bioactivity of acidic stress-produced IL-1ß was confirmed by its ability to promote p38 phosphorylation and chemokine upregulation in alveolar epithelial cells. These findings demonstrate a novel mechanism of acid-induced IL-1ß production and inflammation independent of NLRP3 inflammasome and provide new insights into the therapeutic strategies for aspiration pneumonitis and ALI.

Pazopanib Confers a Progression-free Survival in a Patient with Ewing's Sarcoma/Primitive Neuroectodermal Tumor of the Lung.

Ewing's sarcoma (ES)/primitive neuroectodermal tumors (PNETs) are highly malignant neoplasms that usually affect the bones and soft tissues in children and young adults. ES/PNET of the lung is very rare and is associated with a poor prognosis. We herein report a case of ES/PNET of the left lung in a 45-year-old man. He was treated with neoadjuvant chemotherapy and pneumonectomy, but unfortunately his disease recurred 1.5 months after surgery. He was started on pazopanib, which resulted in a five-month progression-free survival. To our knowledge, this is the first demonstration of pazopanib efficacy in ES/PNET of the lung.

Clinical significance of changes in Torque teno virus DNA titer after chemotherapy in patients with primary lung cancer.

BACKGROUND: Several studies have reported that viral infections are related to lung cancer. We previously reported the involvement of Torque teno virus (TTV) in patients with lung cancer and idiopathic pulmonary fibrosis. However, the role of TTV in lung cancer growth, and its influence on changes in TTV DNA titers due to idiopathic pulmonary fibrosis (IPF) in lung cancer patients are poorly understood. METHODS: Serum TTV DNA titers were measured in serum samples obtained from patients with lung cancer. Forty-eight patients with primary lung cancer, including 8 patients with IPF, were enrolled. Serum TTV DNA titers were quantitated before and after chemotherapy. In addition, patients were classified into two groups according to the presence or absence of IPF, and clinical characteristics were compared between these two groups. RESULTS: Among the 33 patients with partial response to treatment or stable disease in the lung cancer, the mean TTV DNA titer in 28 patients without IPF had significantly decreased after chemotherapy. In contrast, the mean TTV DNA titer in the 5 patients with IPF tended to increase after chemotherapy. In the 15 patients with progressive lung cancer, TTV DNA titers were significantly elevated in those with and without IPF. CONCLUSION: In lung cancer patients without IPF, changes in TTV titers may be correlated with tumor growth. However, in lung cancer patients with IPF, TTV titers were not consistently associated with chemotherapy responses. Therefore, IPF may have an influence on changes in TTV DNA titers.

Nephrotic Syndrome and a Retroperitoneal Mass: A Case Report of a Patient with Recurrent Invasive Thymoma.

A 68-year-old man was admitted to our hospital to undergo an examination for nephrotic syndrome while concurrently complicated with recurrent thymoma in the parietal pleura and retroperitoneum. He had been diagnosed with invasive thymoma and had undergone thymo-thymectomy seven years previously. Based on the renal biopsy findings, his nephrotic syndrome was ascribed to minimal change disease. He was treated with corticosteroid monotherapy, which resulted in complete remission six months later, despite the fact that the recurrent thymoma remained. The role of thymoma in the pathogenesis of paraneoplastic glomerulopathy and the therapeutic concerns that emerged in this case are also discussed.

Influence of Smoking in Interstitial Pneumonia Presenting with a Non-Specific Interstitial Pneumonia Pattern.

Objective The influence of smoking on the pathogenesis and clinical course of interstitial pneumonia has recently attracted attention. To clarify the influence of smoking on the clinical patient characteristics and therapeutic effects in patients with interstitial pneumonia presenting with a non-specific interstitial pneumonia (NSIP) pattern, we compared the clinical patient characteristics and therapeutic effects in smokers and nonsmokers in this study. Methods We divided 31 NSIP (16 idiopathic nonspecific interstitial pneumonia and 15 collagen vascular disease-associated nonspecific interstitial pneumonia) patients into smoker and non-smoker groups for each case. The patient characteristics, pulmonary function tests, Krebs von den Lungen 6 (KL-6), surfactant protein D (SP-D), bronchoalveolar lavage fluid findings, and clinical courses for two years were compared between the smoker and non-smoker groups. Results The smoking subgroup (n=15) of NSIP patients had a significantly lower % diffusing capacity for carbon monoxide/ alveolar ventilation (DLCO/VA) and tended to have higher SP-D values than the nonsmoking subgroup (n=16). Although no difference was observed regarding the prognosis, 5 of 6 cases with NSIP, which had worsening of lung disease were heavy smokers with a pack-year history of 40 or greater. Conclusion Smoking is thus suggested to negatively influence the diffusing capacity caused by damage to alveolar epithelial cells. In addition, smoking may also be potentially related to resistance to therapy in NSIP cases.

Multicentric Castleman's disease developing during follow-up of sarcoidosis.

Pulmonary sarcoidosis is reported to have complication of lymphoproliferative disease such as malignant lymphoma, but the complication of multicentric Castleman's disease (MCD) is rarely reported. In our case of a 60-year-old woman, bilateral hilar lymphadenopathy was noted in her chest X-ray. We performed a transbronchial lung biopsy. She was diagnosed as having pulmonary sarcoidosis (Stage II). The shadow on chest X-ray disappeared without treatment. However, after 8 years, swelling of the mediastinal and abdominal lymph node, thickened bronchovascular bundle, and multiple nodular shadows were identified, and a thoracoscopic lung biopsy was performed. Based on the histopathological findings and elevated serum interleukin-6 level (75.7 pg/mL), she was diagnosed with pulmonary sarcoidosis complicated by MCD. When a change in chest X-ray findings are found during monitoring of pulmonary sarcoidosis, it is important to proceed with a thoracoscopic lung biopsy, because of the possibility of the rare complication of MCD.

Drug-induced Hypersensitivity Syndrome Accompanied by Pulmonary Lesions Exhibiting Centrilobular Nodular Shadows.

A 51-year-old woman diagnosed with Crohn's disease developed drug-induced hypersensitivity syndrome (DIHS) 12 and six weeks after starting the oral intake of mesalazine and trimethoprim/sulfamethoxazole, respectively. Chest CT showed centrilobular nodular shadows and a transbronchial lung biopsy (TBLB) revealed infiltration of inflammatory cells predominantly in the small pulmonary artery walls and bronchiolar walls. Regarding pulmonary lesions of DIHS, infiltrative shadows have sometimes been reported, whereas nodular shadows have rarely been documented. This is a valuable case report for considering the mechanism underlying the development of pulmonary lesions in case of DIHS.

Serum torque teno virus DNA titer in idiopathic pulmonary fibrosis patients with acute respiratory worsening.

OBJECTIVE: Acute respiratory worsening is defined as the unexpected rapid deterioration of idiopathic pulmonary fibrosis (IPF), and idiopathic acute respiratory worsening is known as an acute exacerbation of IPF. Torque teno virus (TTV) is a circular single-stranded DNA virus whose pathological significance remains unclear. The aim of the present study was to investigate the prevalence and titer of TTV DNA in IPF patients with acute respiratory worsening. METHODS: The serum TTV DNA titer was measured using real-time PCR in nine IPF patients (two treated with steroids and immunosuppressants; seven treated without steroids or immunosuppressants) who developed acute worsening, including five patients with acute exacerbation. The serum TTV DNA titer was also measured in eight stable IPF cases and four IPF cases of lung cancer. In addition, in order to examine time course changes in the TTV DNA titer, the titer was measured more than once, with an interval of four weeks or longer, in eight patients. RESULTS: Among the nine IPF patients with acute worsening, the TTV DNA titer was above 1×10(6) copies/mL in two subjects without acute exacerbation who had been continuously treated with steroids and immunosuppressants. Meanwhile, the mean TTV DNA titer was 2.4±2.6 (×10(4) copies/mL) in the five patients with acute exacerbation and 3.1±3.4 (×10(4) copies/mL) in the eight patients with stable IPF. Moreover, the TTV DNA titers were increased in all three IPF patients who started treatment with steroids and immunosuppressants. CONCLUSION: Our results suggest that it is unlikely that TTV is directly involved in the onset of acute exacerbation of IPF and that the serum TTV DNA titer potentially reflects the immunosuppressive state of the host due to treatment.

Physical activity in patients with idiopathic pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: Physical activity is an important parameter in patients with chronic obstructive pulmonary disease, but has not been studied in detail in patients with interstitial lung disease. This study aimed to evaluate physical activity in patients with idiopathic pulmonary fibrosis (IPF). METHODS: Physical activity was monitored in 31 stable IPF patients using an accelerometer for 1 month. The following factors reflecting physical activity were measured: the number of steps, walking distance, the time spent at magnitude of movement (MM) 1-6, physical activity-related energy expenditure (PAEE) and total energy expenditure. We also measured the following clinical parameters: the modified Medical Research Council (MRC) scale, Krebs von den Lungen-6 (KL-6), pulmonary function parameters, 6-min walk test (6MWT) results and high-resolution computed tomography (HRCT) findings of the chest. We determined the relationship between these parameters and physical activity. RESULTS: We recorded 24 days of physical activity data. The time spent at MM < 1 was more than 10 h per day, whereas that at MM > 1 was approximately 1 h per day. The modified MRC scale, serum KL-6 levels, 6MWT distance, and the extent of honeycomb and reticular abnormality on HRCT were associated with several facets of physical activity. In particular, lower KL-6 levels were correlated with higher physical activity based on the number of steps, walking distance, the time spent at MM 1-4 and PAEE. CONCLUSIONS: The modified MRC scale, 6MWT distance, extent of fibrosis on HRCT and serum KL-6 levels are strongly associated with physical activity.

NLRP3 protein deficiency exacerbates hyperoxia-induced lethality through Stat3 protein signaling independent of interleukin-1ß.

Supplemental oxygen inhalation is frequently used to treat severe respiratory failure; however, prolonged exposure to hyperoxia causes hyperoxic acute lung injury (HALI), which induces acute respiratory distress syndrome and leads to high mortality rates. Recent investigations suggest the possible role of NLRP3 inflammasomes, which regulate IL-1ß production and lead to inflammatory responses, in the pathophysiology of HALI; however, their role is not fully understood. In this study, we investigated the role of NLRP3 inflammasomes in mice with HALI. Under hyperoxic conditions, NLRP3(-/-) mice died at a higher rate compared with wild-type and IL-1ß(-/-) mice, and there was no difference in IL-1ß production in their lungs. Under hyperoxic conditions, the lungs of NLRP3(-/-) mice exhibited reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, as well as increased and decreased expression of MMP-9 and Bcl-2, respectively. NLRP3(-/-) mice exhibited diminished expression and activation of Stat3, which regulates MMP-9 and Bcl-2, in addition to increased numbers of apoptotic alveolar epithelial cells. In vitro experiments revealed that alveolar macrophages and neutrophils promoted Stat3 activation in alveolar epithelial cells. Furthermore, NLRP3 deficiency impaired the migration of neutrophils and chemokine expression by macrophages. These findings demonstrate that NLRP3 regulates Stat3 signaling in alveolar epithelial cells by affecting macrophage and neutrophil function independent of IL-1ß production and contributes to the pathophysiology of HALI.

A patient with sarcoidosis who developed heterochronic involvements in different organs from initial organs during 7 years.

A 59-year-old woman, who was given a diagnosis of sarcoidosis by supraclavicular lymph node biopsy 5 years previously, was admitted for further examination following abnormal radiologic findings. Nodular pulmonary and abdominal lesions were observed by computed tomography, and liver biopsy was performed and showed epithelioid cell granulomas. She was asymptomatic and was followed up with no therapy. At 1 year follow-up, the pulmonary and abdominal lesions had nearly complete resolution. Nodular pulmonary and abdominal lesions in patients with sarcoidosis can mimic metastatic disease, lymphoma, and infection, and can reappear during disease activity. Therefore, differential diagnosis and continual follow-up are important.

[Clinical evaluation of inpatients with tuberculosis diagnosed after admission--comparison of characteristics of patients diagnosed with and without the acid-fast bacillus test].

PURPOSE: To evaluate the clinical characteristics of patients in whom tuberculosis was not suspected before admission but was diagnosed after admission. METHODS: We enrolled 39 inpatients who were diagnosed with tuberculosis between April 2007 and March 2011 at Jichi Medical University Hospital. The patients were divided into 2 groups as follows: those who underwent a preadmission acid-fast bacillus (AFB) test (AFB group) and those who did not (non-AFB group). We retrospectively evaluated the clinical characteristics of these 2 groups of patients. RESULTS: A total of 22 patients and 17 patients comprised the non-AFB and AFB groups, respectively. The prevalence rates of malignancy (9 vs. 2 patients, p = 0.04), extrapulmonary tuberculosis without pulmonary tuberculosis (9 vs. 2 patients, p = 0.04), and smear-negative tuberculosis (16 vs. 7 patients, p = 0.04) were higher in the non-AFB group than in the AFB group. In contrast, the computed tomographic findings of the patients with pulmonary tuberculosis revealed that compared with the AFB group, the non-AFB group had less consolidation (2 vs. 11 patients, p < 0.01) and fewer cavitary lesions (0 vs. 6 patients, p < 0.01) and more nodular lesions (8 vs. 2 patients, p < 0.01). We then divided the inpatients into groups with and without malignancy and compared their clinical characteristics. The mean interval from admission to diagnosis of tuberculosis was significantly longer in the patients with malignancy as an underlying disease than in those without malignancy (23.5 vs. 10.5 days; p < 0.01). CONCLUSION: Malignancy was the most frequent underlying disease in the tuberculosis inpatients in the non-AFB group and could be the reason for the delayed diagnosis of tuberculosis.

A rare case of asymptomatic diffuse pulmonary ossification detected during a routine health examination.

A 26-year-old man presented at our hospital in 2008 to undergo detailed investigations as part of a routine health examination. Chest computed tomography (CT) showed linear and reticular opacities with, in part, diffuse calcification in the lung fields bilaterally. A surgical lung biopsy was performed and the histological findings were compatible with a diagnosis of diffuse pulmonary ossification (DPO) of the dendriform type. DPO usually occurs as a secondary disease. As the histological changes in interstitial fibrosis were minimal rather than diffuse and not significant enough to be regarded as interstitial pneumonia, we considered this to be an idiopathic case. However, the findings appear to suggest that inflammation and fibrosis were associated with ossification.