Cough and sputum in long COVID are associated with severe acute COVID-19: a Japanese cohort study.

BACKGROUND: Multiple prolonged symptoms are observed in patients who recover from acute coronavirus disease 2019 (COVID-19), defined as long COVID. Cough and sputum are presented by patients with long COVID during the acute and post-acute phases. This study aimed to identify specific risk factors for cough and sputum in patients with long COVID. METHODS: Hospitalized patients with COVID-19 aged 18 years were enrolled in a multicenter cohort study at 26 medical institutions. Clinical data during hospitalization and patient-reported outcomes after discharge were collected from medical records, paper-based questionnaires, and smartphone apps. RESULTS: At the 3-, 6-, and 12-month follow-ups, there were no differences in the incidence rates of wet and dry coughs. In contrast, the proportion of patients presenting sputum without coughing increased over time compared to those with sputum and coughing. Univariate analyses of cough and sputum at all follow-up visits identified intermittent mandatory ventilation (IMV), smoking, and older age as risk factors for prolonged symptoms. At the 12-month follow-up, persistent cough and sputum were associated with the characteristics of severe COVID-19 based on imaging findings, renal and liver dysfunction, pulmonary thromboembolism, and higher serum levels of LDH, KL-6, and HbA1C. The Kaplan-Meier curves showed that the severity of acute COVID-19 infection was correlated with prolonged cough and sputum production. Multivariable logistic regression analysis showed that IMV ventilator management were independent risk factors for prolonged cough and sputum at 12 months. CONCLUSIONS: In a Japanese population with long COVID, prolonged cough and sputum production were closely associated with severe COVID-19. These findings emphasize that a preventive approach including appropriate vaccination and contact precaution and further development of therapeutic drugs for COVID-19 are highly recommended for patients with risk factors for severe infection to avoid persistent respiratory symptoms.

Diagnostic and prognostic biomarkers for progressive fibrosing interstitial lung disease.

No biomarkers have been identified in bronchoalveolar lavage fluid (BALF) for predicting fibrosis progression or prognosis in progressive fibrosing interstitial lung disease (PF-ILD). We investigated BALF biomarkers for PF-ILD diagnosis and prognosis assessment. Overall, 120 patients with interstitial pneumonia who could be diagnosed with PF-ILD or non PF-ILD were enrolled in this retrospective study. PF-ILD was diagnosed according to Cottin's definition. All patients underwent bronchoscopy and BALF collection. We evaluated blood and BALF parameters, high-resolution computed tomography (HRCT) patterns, and spirometry data to identify factors influencing PF-ILD diagnosis and prognosis. On univariate logistic analysis, age, sex, the BALF white blood cell fraction (neutrophil, lymphocyte, eosinophil, and neutrophil-to-lymphocyte ratio), BALF flow cytometric analysis (CD8), and an idiopathic pulmonary fibrosis/usual interstitial pneumonia pattern on HRCT were correlated with PF-ILD diagnosis. Multivariate logistic regression analysis revealed that sex (male), age (cut-off 62 years, area under the curve [AUC] 0.67; sensitivity 0.80; specificity 0.47), white blood cell fraction in BALF (NLR, neutrophil, and lymphocyte), and CD8 in BALF (cut-off 34.2; AUC 0.66; sensitivity, 0.74; specificity, 0.62) were independent diagnostic predictors for PF-ILD. In BALF, the NLR (cut-off 8.70, AUC 0.62; sensitivity 0.62; specificity 0.70), neutrophil count (cut-off 3.0, AUC 0.59; sensitivity 0.57; specificity 0.63), and lymphocyte count (cut-off 42.0, AUC 0.63; sensitivity 0.77; specificity 0.53) were independent diagnostic predictors. In PF-ILD patients (n = 77), lactate dehydrogenase (cut-off 275, AUC 0.69; sensitivity 0.57; specificity 0.78), Krebs von den Lungen-6 (cut-off 1,140, AUC 0.74; sensitivity 0.71; specificity 0.76), baseline forced vital capacity (FVC) (cut-off 1.75 L, AUC 0.71; sensitivity, 0.93; specificity, 0.46), and BALF neutrophil ratio (cut-off 6.0, AUC 0.72; sensitivity 0.79; specificity 0.80) correlated with death within 3 years. The BALF cellular ratio, particularly the neutrophil ratio, correlated with the diagnosis and prognosis of PF-ILD. These findings may be useful in the management of patients with interstitial pneumonia.

Inhibition of stearoyl-CoA desaturase 1 (SCD1) enhances the antitumor T cell response through regulating ß-catenin signaling in cancer cells and ER stress in T cells and synergizes with anti-PD-1 antibody.

BACKGROUND: Understanding the mechanisms of non-T cell inflamed tumor microenvironment (TME) and their modulation are important to improve cancer immunotherapies such as immune checkpoint inhibitors. The involvement of various immunometabolisms has recently been indicated in the formation of immunosuppressive TME. In this study, we investigated the immunological roles of stearoyl-CoA desaturase 1 (SCD1), which is essential for fatty acid metabolism, in the cancer immune response. METHODS: We investigated the roles of SCD1 by inhibition with the chemical inhibitor or genetic manipulation in antitumor T cell responses and the therapeutic effect of anti-programmed cell death protein 1 (anti-PD-1) antibody using various mouse tumor models, and their cellular and molecular mechanisms. The roles of SCD1 in human cancers were also investigated by gene expression analyses of colon cancer tissues and by evaluating the related free fatty acids in sera obtained from patients with non-small cell lung cancer who were treated with anti-PD-1 antibody. RESULTS: Systemic administration of a SCD1 inhibitor in mouse tumor models enhanced production of CCL4 by cancer cells through reduction of Wnt/ß-catenin signaling and by CD8+ effector T cells through reduction of endoplasmic reticulum stress. It in turn promoted recruitment of dendritic cells (DCs) into the tumors and enhanced the subsequent induction and tumor accumulation of antitumor CD8+ T cells. SCD1 inhibitor was also found to directly stimulate DCs and CD8+ T cells. Administration of SCD1 inhibitor or SCD1 knockout in mice synergized with an anti-PD-1 antibody for its antitumor effects in mouse tumor models. High SCD1 expression was observed in one of the non-T cell-inflamed subtypes in human colon cancer, and serum SCD1 related fatty acids were correlated with response rates and prognosis of patients with non-small lung cancer following anti-PD-1 antibody treatment. CONCLUSIONS: SCD1 expressed in cancer cells and immune cells causes immunoresistant conditions, and its inhibition augments antitumor T cells and therapeutic effects of anti-PD-1 antibody. Therefore, SCD1 is an attractive target for the development of new diagnostic and therapeutic strategies to improve current cancer immunotherapies including immune checkpoint inhibitors.

A solitary pulmonary nodule caused by Mycobacterium avium with pleural effusion and pleuritis after transbronchial biopsy: a case report.

BACKGROUND: Pleural effusion and pleuritis are uncommon manifestations of Mycobacterium avium complex pulmonary disease. Pleuritis caused by Mycobacterium avium complex pulmonary disease presenting as a solitary pulmonary nodule is extremely rare. The pathogenesis of Mycobacterium avium complex pleuritis has not been elucidated. However, it has been suggested that secondary spontaneous pneumothorax from Mycobacterium avium complex pulmonary disease is one of the causes of Mycobacterium avium complex pleuritis. CASE PRESENTATION: A 67-year-old Japanese woman who presented with a solitary pulmonary nodule developed a transient pneumothorax after transbronchial biopsy. A definitive diagnosis of solitary pulmonary nodule could not be made on bronchoscopy, so video-assisted thoracoscopic surgery was performed 1 month after bronchoscopy. On the day of hospitalization for the procedure, a left-sided pleural effusion appeared on a chest radiograph. Thickening of the parietal and visceral pleura and numerous scattered white small granules were seen on thoracoscopy. Histologic examination of the resected left lower lobe and a biopsy of the parietal pleura showed Mycobacterium avium complex solitary pulmonary nodule and Mycobacterium avium complex pleuritis. CONCLUSION: Iatrogenic pneumothorax can be a cause of pleuritis in a patient with Mycobacterium avium complex pulmonary disease. Clinicians should watch for the appearance of secondary pleuritis after transbronchial biopsy even in a patient with localized disease such as Mycobacterium avium complex solitary pulmonary nodule.

Trends of concerns from diagnosis in patients with advanced lung cancer and their family caregivers: A 2-year longitudinal study.

BACKGROUND: Both advanced cancer patients and their family caregivers experience distress and have a range of concerns after cancer diagnosis. However, longitudinal studies on this topic have been lacking. AIM: To investigate concerns in both patients with advanced lung cancer and their family caregivers longitudinally from diagnosis. DESIGN: A multi-center prospective questionnaire-based study. SETTING/PARTICIPANTS: We recruited patients with newly diagnosed advanced lung cancer and their family caregivers at 16 hospitals in Japan. We prospectively assessed the prevalence of their concerns using the Concerns Checklist and investigated the associations between their concerns and mental status as well as quality of life until 24 months after diagnosis. RESULTS: A total of 248 patients and their 232 family caregivers were enrolled. The prevalence of serious concerns was highest at diagnosis (patients: 68.3%, family caregivers: 65.3%). The most common serious concern was concern about the future in both groups at diagnosis (38.2% and 40.5%, respectively) and this remained high in prevalence over time, while the high prevalence of concern about lack of information improved 3 months after diagnosis in both groups. Approximately one-third of patient-family caregiver dyads had discrepant reports of serious concerns. The presence of serious concerns was significantly associated with anxiety and depression continuously in both groups. CONCLUSIONS: The majority of advanced lung cancer patients and their family caregivers have serious concerns from diagnosis, which is associated with their psychological distress. The spectrum of concerns alters over the disease trajectory, warranting efficient tailored care and support for both groups immediately after diagnosis.

A phase II trial of induction of erlotinib followed by cytotoxic chemotherapy for EGFR mutation-positive non-squamous non-small cell lung cancer patients.

BACKGROUND: No consensus has been reached regarding the treatment order and timing of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) and cytotoxic chemotherapy administration for EGFR mutation-positive non-small cell lung cancer (NSCLC) patients. METHODS: In this phase II trial, chemotherapy-naïve patients harboring activating EGFR mutations with stage IIIB/IV or post-surgical recurrent non-squamous NSCLC were enrolled. Patients were treated with erlotinib induction at 150 mg/day for 3 months. This was followed by cytotoxic chemotherapy with platinum plus pemetrexed, with or without bevacizumab, when the induction erlotinib achieved a CR or PR. The primary end point was the 1-year progression-free survival (PFS) rate, while the secondary end points were the response rate (RR), PFS, safety, and overall survival (OS). RESULTS: Twenty patients were enrolled in this study. The median age was 63 years. Eighteen patients had stage IV disease, and 2 patients had recurrent disease. Eleven patients achieved a PR after induction of erlotinib and 9 out of 11 patients were switched to chemotherapy. The 1-year PFS rate was 45.0% (90% CI 26.8-63.2), the overall RR was 55.0%, and the median PFS was 10.7 months in the intention-to-treat (ITT) population. Grade 3-4 adverse events were reported for 40% of the patients, including patients with leukopenia (10%), neutropenia (20%), and interstitial pneumonitis, bacterial pneumonia, rash, and nausea (all 5%). CONCLUSIONS: The primary end point of this study was not achieved. However, the therapy was well tolerated and may be a treatment option for a future study with patients responsive to short-term erlotinib treatment. CLINICAL TRIALS REGISTRATION NUMBER: UMIN ID: 000013125.

An Adult Case of Pulmonary Artery Sling Accompanied by Tracheobronchomalacia.

Pulmonary artery (PA) sling is a congenital disease in which the left PA abnormally arises from the right PA and is usually diagnosed during the infantile period. We present an adult case of PA sling accompanied by tracheobronchomalacia found in a 49-year-old woman with a history of recurrent pneumonia. Computed tomography of the chest showed that the left lung was nourished by two aberrant PAs. Bronchoscopy demonstrated achondroplasia of the trachea and the right bronchus, which we speculate to have resulted in their stenosis. The recurrent pneumonia was attributable to these tracheobronchial structural abnormalities; we therefore stress the importance of focusing on the anatomic abnormalities in such cases.

Real-world Efficacy and Safety of Nivolumab for Advanced Non-Small-cell Lung Cancer: A Retrospective Multicenter Analysis.

BACKGROUND: Nivolumab, an immune checkpoint inhibitor, is now a standard treatment for previously treated advanced non-small-cell lung cancer based on the results from phase III clinical trials. We evaluated the real-world efficacy and safety of nivolumab in a nonselected population and identified the clinical characteristics that influence efficacy. MATERIALS AND METHODS: A total of 142 patients with advanced non-small-cell lung cancer who were administered nivolumab at Keio University and affiliated hospitals in Japan from January to July 2016 were enrolled. The treatment efficacy and adverse events were retrospectively reviewed, and the clinical characteristics associated with the nivolumab response were evaluated using univariate and stratified analyses and the Cochran-Mantel-Haenszel test. RESULTS: The objective response rate was 17.0% (95% confidence interval [CI], 12.0%-24.0%), the median progression-free survival (PFS) was 58 days (95% CI, 50-67 days), and the proportion of patients with adverse events of any grade was 45.0%. EGFR/ALK mutation status was inversely associated with the treatment response (P < .05), and the difference in PFS for the mutation-positive versus mutation-negative patients was statistically significant (49 vs. 63 days; hazard ratio, 1.9; 95% CI, 1.1-5.2; P = .029). Previous radiotherapy also had a positive association with the treatment response (P = .012). CONCLUSION: The objective response rate, PFS, and adverse event profiles were comparable to those observed in previous clinical trials. EGFR/ALK mutation-negative status and previous radiotherapy might be key clinical characteristics associated with a positive treatment response. Our findings could aid in the efficient immunotherapeutic management of lung cancer.

Erlotinib as second- or third-line treatment in elderly patients with advanced non-small cell lung cancer: Keio Lung Oncology Group Study 001 (KLOG001).

The aim of this study was to assess the efficacy and safety of erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), as second- or third-line treatment for elderly Japanese patients with non-small-cell lung cancer (NSCLC). The patients eligible for this phase II trial were aged ≥70 years, had stage III/IV or recurrent NSCLC, and had previously received 1 or 2 chemotherapy regimens that did not include EGFR-TKIs. The patients received erlotinib at a dose of 150 mg/day. The primary endpoint was overall response rate (ORR), and the secondary endpoints were progression-free survival (PFS), overall survival (OS) and toxicity. A total of 38 patients with a median age of 76 years were enrolled. The majority of the patients were men (66%), had an Eastern Cooperative Oncology Group performance status of 1 (58%), stage IV disease (66%) and adenocarcinoma (74%). Of the 35 patients, 13 (34%) had tumors with EGFR mutations. The ORR was 26.3% (95% confidence interval: 12.1-40.5%) and the disease control rate was 47.4%. The median PFS was 3.7 months and the median OS was 17.3 months. The grade 3 adverse events observed included rash (13%), diarrhea (5%), interstitial pneumonitis (5%), anorexia (3%) and gastrointestinal bleeding (3%). Grade 4 or 5 adverse events were not observed. The median OS did not differ significantly between patients aged <75 years (14.9 months) and those aged ≥75 years (19.0 months; P=0.226). Therefore, erlotinib was found to be effective and well-tolerated in elderly patients with previously treated NSCLC.

Intimal Sarcoma of the Pulmonary Artery Treated with Pazopanib.

Intimal sarcoma is a rare disease with a poor prognosis. We herein report the case of a 71-year-old man with intimal sarcoma of the pulmonary artery treated with pazopanib. The tumor showed regression after 1 month of treatment. Hand-foot syndrome led to cessation of pazopanib, which triggered a disease flare. Pazopanib should be considered in patients with intimal sarcoma of the pulmonary artery that is unresectable or recurrent after surgery or cytotoxic chemotherapy. We must be careful about drug cessation, as it can lead to a disease flare.

A phase II study of biweekly paclitaxel and carboplatin in elderly patients with advanced non-small cell lung cancer.

PURPOSE: The number of elderly patients with advanced non-small cell lung cancer (NSCLC) is increasing. Although several studies have suggested the benefit of chemotherapy with a platinum doublet for elderly patients with advanced NSCLC, this treatment is still controversial in this age group. To evaluate the efficacy and tolerability of combination chemotherapy with biweekly paclitaxel and carboplatin for elderly patients with advanced NSCLC, we conducted a multicenter, non-randomized, open label, phase II trial. METHODS: We recruited patients aged ≥70 years with clinical stage IIIB and IV NSCLC and ECOG performance status (PS) of 0-2. Patients received paclitaxel (90 mg/m(2)) and carboplatin (AUC = 2.5) on day 1 and 15, every 4 weeks. The primary endpoint was overall response rate (ORR), and the secondary endpoints were progression-free survival (PFS), overall survival (OS), and safety. RESULTS: Sixty-five patients (median age 79 years; range 70-87 years) were enrolled. Forty-nine patients were men, and 48 were stage IV. The PS was 0, 1, and 2 in 28, 33, and 4 patients, respectively. The histological type of NSCLC was non-squamous in 69.3 % and squamous cell carcinoma in 30.7 % of patients. The median number of treatment cycles was 3 (range 1-6). The response rate was 29.4 % (95 % CI 18.7-43.0), and the disease control rate was 78.0 % (95 % CI 64.8-87.2). Median PFS and OS were 3.8 months (95 % CI 1.9-5.3) and 17.3 months (95 % CI 10.4-25.1), respectively. The most common grade 3 or 4 toxicities were neutropenia (27 %), leukopenia (15 %), infection (10 %), and anemia (8 %). CONCLUSION: The combination of biweekly paclitaxel and carboplatin was effective and well tolerated in elderly patients with advanced NSCLC.

FOXD1 expression is associated with poor prognosis in non-small cell lung cancer.

AIM: Clinical microarray datasets were analyzed to search for new therapeutic targets and prognostic markers of non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Microarray datasets from 90 lung cancer specimens, were analyzed with focus on the FOXD1 gene. Levels of FOXD1 mRNA were assessed in lung cancer cell lines and these levels were correlated with survival. RESULTS: FOXD1-knockdown led to suppression of cell proliferation. Moreover, patients with high FOXD1 expression survived for a significantly shorter time than those with low FOXD1 expression. CONCLUSION: The expression status of FOXD1 is a novel prognostic factor and may lead to new treatment strategies for NSCLC.

Methylation-induced downregulation of TFPI-2 causes TMPRSS4 overexpression and contributes to oncogenesis in a subset of non-small-cell lung carcinoma.

We identified transmembrane protease, serine 4 (TMPRSS4) as a putative, druggable target by screening surgically resected samples from 90 Japanese non-small-cell lung cancer (NSCLC) patients using cDNA microarray. TMPRSS4 has two druggable domains and was upregulated in 94.5% of the lung cancer specimens. Interestingly, we found that TMPRSS4 expression was associated with tissue factor pathway inhibitor 2 (TFPI-2) expression in these clinical samples. In contrast to TMPRSS4, TFPI-2 expression was downregulated in NSCLC samples. The in vitro induction of TFPI-2 in lung cancer cell lines decreased the expression of TMPRSS4 mRNA levels. Reporter assay showed that TFPI-2 inhibited transcription of TMPRSS4, although partially. Knockdown of TMPRSS4 reduced the proliferation rate in several lung cancer cell lines. When lung cancer cell lines were treated with 5-aza-2'-deoxycytidine or trichostatin A, their proliferation rate and TMPRSS4 mRNA expression levels were also reduced through the upregulation of TFPI-2 by decreasing its methylation in vitro. The TFPI-2 methylation level in the low TMPRSS4 group appeared to be significantly low in NSCLC samples (P = 0.02). We found a novel molecular mechanism that TFPI-2 negatively regulates cell growth by inhibiting transcription of TMPRSS4. We suggest that TMPRSS4 is upregulated by silencing of TFPI-2 through aberrant DNA methylation and contributes to oncogenesis in NSCLC.

Claudin-1 is a novel target of miR-375 in non-small-cell lung cancer.

OBJECTIVES: We previously reported low expression of miR-375 in squamous-cell carcinoma (SCC) and high expression in adenocarcinoma (AC) of the lung. miR-375's target genes and its function in non-small-cell lung cancer (NSCLC) have not been elucidated. Therefore, the present study was designed to identify the targets of miR-375 and to characterize its function in NSCLC. MATERIALS AND METHODS: Candidate targets of miR-375 were determined using a prediction database and previous data on differential gene expression between SCC and AC. We evaluated miR-375 and target-gene expression levels in 12 NSCLC cell lines. The effect of miR-375 overexpression and knockdown was evaluated in NSCLC cell lines by transfecting them with an miR-375 precursor or inhibitor. A luciferase-reporter assay was performed to confirm a direct interaction between miR-375 and its target gene. Further, a wound-healing assay was performed to evaluate the effect of miR-375 overexpression on the migration of SK-MES-1 cells. Finally, to assess the clinical relevance, 63 clinical NSCLC samples were analyzed. RESULTS: Claudin-1 (CLDN1) has 4 putative miR-375 target sites in its 3'-untranslated region, and this gene was determined to be a target of miR-375. CLDN1 messenger RNA and protein expression were attenuated by overexpression of miR-375 and increased by knockdown of miR-375 in NSCLC cell lines. In a luciferase-reporter assay, miR-375 overexpression resulted in a 3-fold repression of luciferase activity (P<0.001). Cell migration was promoted by miR-375 overexpression, suggesting a high potential for invasion and metastasis in NSCLC expressing high levels of miR-375. In clinical NSCLC samples, there was a negative correlation between miR-375 and CLDN1 expression (r=-0.35, P=0.005). In addition, high miR-375 expression was correlated with a shorter survival time among the clinical samples (P=0.043). CONCLUSION: CLDN1 is a novel target of miR-375, and high miR-375 expression shortens survival in NSCLC.

Identification of microRNAs differentially expressed between lung squamous cell carcinoma and lung adenocarcinoma.

Recent advances in the treatment of non-small cell lung cancer (NSCLC) with new agents require accurate histological subtyping at diagnosis to avoid the higher risk of an adverse response and to obtain the maximum therapeutic response. However, interobserver variability, tumor heterogeneity and the degree of differentiation may affect the decision concerning a pathological diagnosis of NSCLC. Therefore, the aim of this study was to identify specific microRNAs (miRNAs) as standardized biomarkers with high sensitivity and specificity in order to distinguish between squamous cell carcinoma (SCC) and adenocarcinoma (AC). Quantitative polymerase chain reaction (qPCR)­based miRNA array analysis was performed to identify microRNAs differentially expressed between SCC and AC using 86 resected NSCLC samples in addition to adjacent normal tissues. The results were confirmed by independent qRT-PCR assays with the same test samples and 88 additional validation samples, and from this we evaluated the usefulness of the identified miRNAs as biomarkers to distinguish between SCC and AC. Three miRNAs (hsa-miR-196b, hsa-miR-205 and hsa-miR-375) were identified. Discriminant analysis combining the three miRNAs appeared to distinguish SCC from AC accurately in the test and validation samples, demonstrating a sensitivity and specificity of 76 and 80%, and 85 and 83%, respectively. hsa-miR-196b, hsa-miR-205 and hsa-miR-375 were identified as biomarkers capable of distinguishing between lung SCC and lung AC. These newly identified miRNAs may prove to be highly valuable molecular markers for the classification of NSCLC histological subtypes and may contribute to the pathogenesis of each subtype of NSCLC.

Cancer-induced immunosuppressive cascades and their reversal by molecular-targeted therapy.

Immunological status in tumor tissues varies among patients. Infiltration of memory-type CD8(+) T cells into tumors correlates with prognosis of patients with various cancers. However, the mechanism of the differential CD8(+) T cell infiltration has not been well investigated. In general, tumor-associated microenvironments, including tumor and sentinel lymph nodes, are under immunosuppressive conditions such that the immune system is not able to eliminate cancer cells without immune-activating interventions. Constitutive activation of various signaling pathways in human cancer cells triggers multiple immunosuppressive cascades that involve various cytokines, chemokines, and immunosuppressive cells. Signaling pathway inhibitors could inhibit these immunosuppressive cascades by acting on either cancer or immune cells, or both. In addition, common signaling mechanisms are often utilized for multiple hallmarks of cancer (e.g., cell proliferation/survival, invasion/metastasis, and immunosuppression). Therefore, targeting these common signaling pathways may be an attractive strategy for cancer therapy including immunotherapy.

Activation of the FGF2-FGFR1 autocrine pathway: a novel mechanism of acquired resistance to gefitinib in NSCLC.

Patients with non-small cell lung cancer (NSCLC) that harbors epidermal growth factor receptor (EGFR) mutations initially respond to EGFR-tyrosine kinase inhibitors (TKI) but eventually experience relapse. Acquired resistance to EGFR-TKIs is strongly associated with patient mortality. Thus, elucidation of the mechanism of acquired resistance to EGFR-TKIs is of great importance. In this study, gefitinib-resistant cell line models were established by long-term exposure to gefitinib using the gefitinib-sensitive lung cancer cell lines, PC9 and HCC827. Expression analyses indicated that both FGFR1 and FGF2 were increased in PC9 gefitinib-resistant (PC9 GR) cells as compared with PC9 naïve (PC9 na) cells. Importantly, proliferation of gefitinib-resistant cells was dependent on the FGF2 -FGFR1 pathway. Mechanistically, inhibition of either FGF2 or FGFR1 by siRNA or FGFR inhibitor (PD173074) restored gefitinib sensitivity in PC9 GR cells. These data suggest that FGF2 -FGFR1 activation through an autocrine loop is a novel mechanism of acquired resistance to EGFR-TKIs.

The PCR-invader method (structure-specific 5' nuclease-based method), a sensitive method for detecting EGFR gene mutations in lung cancer specimens; comparison with direct sequencing.

BACKGROUND: Several sensitive assays, including the PCR-invader method (structure-specific 5' nuclease-based method), have been used to detect EGFR mutations in non-small-cell lung cancer (NSCLC). However, validation has not been reported. We assessed the detection rate of EGFR mutation by the PCR-invader method and direct sequencing using same clinical specimens. PATIENTS AND METHODS: EGFR mutations were analyzed with the PCR-invader method and compared with direct sequencing using paraffin tissues and pleural and pericardial effusions from NSCLC patients. The relationships between the treatment responses and mutations were evaluated retrospectively. RESULTS: Fifty-four samples from 42 NSCLC patients were studied. EGFR mutations were identified in 52% of the patients and 52% of the samples with the PCR-invader method, but only in 43% of the patients and in 35% of the samples by direct sequencing. In the samples obtained from the same patients at different sites and different times, EGFR mutations were coincident in nine out of ten patients by the PCR-invader method but in six out of ten patients by direct sequencing. Seventeen patients with EGFR mutations were treated with gefitinib; the response rate (RR) and disease control rate (DCR) were 41 and 94%, and median treatment duration was more than 6 months. Seven EGFR mutation-negative patients were treated with gefitinib; the RR and DCR were 0 and 14%, and median treatment duration was 1 month. CONCLUSION: The PCR-invader method was useful for detecting EGFR mutations in clinical lung cancer specimens and is more sensitive than direct sequencing.

A phase I study of S-1 and irinotecan combination therapy in previously treated advanced non-small cell lung cancer patients.

BACKGROUND: This phase I study was conducted to evaluate the feasibility and to determine the recommended doses of the combination therapy of S-1 and irinotecan (CPT-11) in patients with advanced non-small cell lung cancer (NSCLC) as second-line treatment. METHODS: Patients with NSCLC who were previously treated with one chemotherapy regimen and had a performance status of 0 or 1 were eligible. CPT-11 was administered at 60 mg/m² (level 1), 80 mg/m² (level 2) on days 1 and 8, and oral S-1 was administered at 80 mg/day for body surface area (BSA) less than 1.25 m², 100 mg/day for BSA 1.25-1.5 m², and 120 mg/day for BSA more than 1.5 m² on days 1-14 every 3 weeks. The dose-limiting toxicity (DLT) was defined as grade 4 leukocytopenia or neutropenia, grade ≥ 3 neutropenia with fever over 38°C, grade ≥ 3 thrombocytopenia, or grade ≥ 3 major nonhematological toxicities. RESULTS: Nine patients were enrolled in the study. None of 3 patients enrolled in level 1 had any DLT. Of 6 patients in level 2, 2 patients had grade 3 diarrhea and one had grade 3 interstitial pneumonia. Level 1 was declared as the recommended dose. CONCLUSION: The feasibility of the combination therapy of S-1 and CPT-11 was shown in the second-line setting for the treatment of advanced NSCLC. The recommended dose of CPT-11 was 60 mg/m² combined with standard dose of S-1 for phase II trials of pretreated advanced NSCLC patients.

Bronchoscopic microsampling is a useful complementary diagnostic tool for detecting lung cancer.

PURPOSE: Bronchoscopic microsampling (BMS) is a novel and direct method with which to obtain epithelial lining fluid (ELF) from the lungs. Analysis of DNA hypermethylation of tumor suppressor genes (TSGs) is expected to be a sensitive tool for the early detection of lung cancer. It has been reported that the existence of EGFR mutations and EML4-ALK gene rearrangements are related to the sensitivity of corresponding kinase inhibitors. We aimed to evaluate the suitability of ELF as a sample for analyzing molecular changes specific for lung cancer. PATIENTS AND METHODS: We collected ELF from 61 lung cancer patients by BMS from the airway close to the peripheral lung nodule and purified the nucleic acids. We performed methylation specific PCR in each ELF as well as matched serum and tumor tissue for TSGs for DNA methylation analysis. We also examined EGFR mutations and EML4-ALK rearrangement. RESULTS: The sensitivity for detecting DNA hypermethylation in ELF vs serum was 74.1% vs 18.5%. We found 60.1% of patients had at least one hypermethylation in ELF, while only 27.9% had it in serum. Of note, DNA hypermethylation was detected even in stage I patients (60.0%) and the detection rate was almost the same level in each stage. We also found the sensitivity for detecting EGFR mutation in ELF vs serum was 58.3% vs 8.3%. We detected an EML4-ALK fusion gene using ELF in one patient. CONCLUSIONS: BMS is an alternative method to detect cancer specific genetic and epigenetic alterations and will be a useful complementary diagnostic tool for lung cancer. SUMMARY: Investigation of genetic and epigenetic changes associated with lung cancer has clinical importance for its diagnosis and management. The clinical usefulness of bronchoscopic microsampling (BMS) in lung cancer has not yet been evaluated. This study demonstrates that BMS could be useful for detecting lung cancer specific molecular changes and valuable for early diagnosis and determination of treatment options for lung cancer.