Ryosuke Satomi

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.

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.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.Systemic administration of a SCD1 inhibitor in mouse tumor models enhanced production of CCL4 by cancer cells through reduction of Wnt/β-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.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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly since 2019, and the number of reports regarding long COVID has increased. Although the distribution of long COVID depends on patient characteristics, epidemiological data on Japanese patients are limited. Hence, this study aimed to investigate the distribution of long COVID in Japanese patients. This study is the first nationwide Japanese prospective cohort study on long COVID. This multicenter, prospective cohort study enrolled hospitalized COVID-19 patients aged ≥18 years at 26 Japanese medical institutions. In total, 1200 patients were enrolled. Clinical information and patient-reported outcomes were collected from medical records, paper questionnaires, and smartphone applications. We collected data from 1066 cases with both medical records and patient-reported outcomes. The proportion of patients with at least one symptom decreased chronologically from 93.9% (947/1009) during hospitalization to 46.3% (433/935), 40.5% (350/865), and 33.0% (239/724) at 3, 6, and 12 months, respectively. Patients with at least one long COVID symptom showed lower quality of life and scored higher on assessments for depression, anxiety, and fear of COVID-19. Female sex, middle age (41–64 years), oxygen requirement, and critical condition during hospitalization were risk factors for long COVID. This study elucidated the symptom distribution and risks of long COVID in the Japanese population. This study provides reference data for future studies of long COVID in Japan.

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.

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.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.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).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.

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.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.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).CLDN1 is a novel target of miR-375, and high miR-375 expression shortens survival in NSCLC.

Clinical microarray datasets were analyzed to search for new therapeutic targets and prognostic markers of non-small cell lung cancer (NSCLC).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.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.The expression status of FOXD1 is a novel prognostic factor and may lead to new treatment strategies for NSCLC.

Abstract Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) show antitumor activity in a subset of non–small cell lung cancer (NSCLC) patients. However, the initial tumor response is followed by recurrence. Several studies have suggested the importance of other receptor tyrosine kinases (RTK) and downstream kinases as potential targets in the treatment of NSCLC. We used the multiple-RTK inhibitor AEE788, which inhibits EGFR, vascular endothelial growth factor receptor, and human epidermal growth factor receptor 2, with and without the downstream kinase inhibitor RAD001 (an inhibitor of mammalian target of rapamycin). AEE788 inhibited cell growth more effectively than did erlotinib in three NSCLC cell lines examined (A549, H1650, and H1975). However, in the EGFR-TKI–resistant cell line H1975 harboring T790M resistance mutation, cell growth inhibition by AEE788 was only mild, and the phosphorylation of its leading targets such as EGFR and vascular endothelial growth factor receptor 2 was not inhibited. In H1975, AEE788 induced significantly greater cell growth inhibition when combined with RAD001 than when used alone. This cooperative effect was not seen with the combination of erlotinib and RAD001. We found that c-Met was highly phosphorylated in this cell line, and the phosphorylated c-Met was inhibited effectively by AEE788. Using a phospho-RTK array, the phosphorylation of c-Met and insulin-like growth factor-I receptor was inhibited by AEE788. These results suggest that upstream RTK inhibitor overcomes the acquired resistance to EGFR-TKI when combined with downstream kinase inhibitor. Thus, the combined inhibition of upstream and downstream RTKs is a promising strategy for the treatment of NSCLC. Mol Cancer Res; 8(8); 1142–51. ©2010 AACR.

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.

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.

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.

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.

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.

Epigenetic gene regulation plays essential roles in differentiation of embryonic and tissue stem cells. In these benign undifferentiated cells, some polycomb targeted genes are kept in a state of DNA hypomethylation and they have a distinct chromatin signature termed bivalent chromatin structure to maintain their plasticity. We hypothesized that cancer stem cells (CSC), the malignant counterpart of these cells, are also under the control of epigenetics like benign stem cells. We compared the DNA methylation and chromatin structure in 10 tumor suppressor genes between CSC and differentiated cancer cells of MCF7 and Huh7 cells. We found that the level of DNA methylation was indeed significantly lower in CSC, while surprisingly, the bivalent chromatin structure was more ubiquitously seen in differentiated cancer cells compared to CSC. However, repressive marks of chromatin structure, namely H3K27me3 and EZH2, were significantly lower in CSC. As a consequence, CSC remained in a higher transcriptionally active chromatin state compared to differentiated cancer cells. We found that the differentiation of CSCs is also epigenetically regulated. These findings could help towards a comprehensive understanding of CSC, and also improve the development of eradicative therapies against human malignancies.

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.

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.

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.

&lt;div&gt;Abstract&lt;p&gt;Patients with non-small cell lung cancer (NSCLC) that harbors epidermal growth factor receptor (&lt;i&gt;EGFR&lt;/i&gt;) 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. &lt;i&gt;Mol Cancer Res; 11(7); 759–67. ©2013 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Patients with non-small cell lung cancer (NSCLC) that harbors epidermal growth factor receptor (&lt;i&gt;EGFR&lt;/i&gt;) 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. &lt;i&gt;Mol Cancer Res; 11(7); 759–67. ©2013 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Supplementary Figure S1 - PDF file 154K, Increased expressions of p-MET and t-MET in HCC827 GR cells&lt;/p&gt;

&lt;p&gt;Supplementary Figure S6 - PDF file 180K, A, B. Viability curves of PC9 GR cells (A) and PC9 gr1 cells (B) transfected with siRNA against FGFR1 (FGFR1 siRNA #1), FGFR3, or with the negative control mix at indicated concentrations of gefitinib. Representative data from three independent experiments are shown. Data represent the mean plus-minus SD. Values are expressed as mean plus-minus SD. *P&lt;0.05, significant difference by Student's t test between control siRNA and FGFR1 siRNA #1 at 1microM gefitinib&lt;/p&gt;

&lt;p&gt;Supplementary Figure S5 - PDF file 109K, Efficient knockdown of FGFR1, FGFR3, and FGF2 with each siRNA confirmed by quantitative RT-PCR&lt;/p&gt;

&lt;p&gt;Supplementary Figure S7 - PDF file 110K, The relative expression profiles of mRNA of FGFRs by microarray analysis. The log2 of fold change to PC9 na was calculated for each sample by using Gene Spring GX software. Each group of columns represents the result from 1 microarray probe&lt;/p&gt;

&lt;p&gt;Supplementary Figure S3 - PDF file 139K,The relative expression profiles of mRNA of FGFs by microarray analysis in PC9 GR, PC9 gr1 and PC9 gr3 cells normalized to mRNA extracted from PC9 na cells. None of FGFs other than FGF2 was elevated in gefitinib resistant cell lines. Each group of columns represents the result from one microarray probe&lt;/p&gt;

&lt;p&gt;Supplementary Figure S2 - PDF file 411K,Sequencing results of EGFR exon 20 of the genomic DNA extracted from PC9 and HCC827 cells. There was no T790M second mutation of EGFR gene&lt;/p&gt;

&lt;p&gt;Supplementary Figure S4 - PDF file 107K, DNA quantification for FGFR1 by quantitative-PCR. DNA copy number was not increased in PC9 GR and PC9 gr1 cells&lt;/p&gt;

&lt;p&gt;Supplementary Figure S2 - PDF file 411K,Sequencing results of EGFR exon 20 of the genomic DNA extracted from PC9 and HCC827 cells. There was no T790M second mutation of EGFR gene&lt;/p&gt;

&lt;p&gt;Supplementary Figure S1 - PDF file 154K, Increased expressions of p-MET and t-MET in HCC827 GR cells&lt;/p&gt;

&lt;p&gt;Supplementary Figure S6 - PDF file 180K, A, B. Viability curves of PC9 GR cells (A) and PC9 gr1 cells (B) transfected with siRNA against FGFR1 (FGFR1 siRNA #1), FGFR3, or with the negative control mix at indicated concentrations of gefitinib. Representative data from three independent experiments are shown. Data represent the mean plus-minus SD. Values are expressed as mean plus-minus SD. *P&lt;0.05, significant difference by Student's t test between control siRNA and FGFR1 siRNA #1 at 1microM gefitinib&lt;/p&gt;

&lt;p&gt;Supplementary Figure S7 - PDF file 110K, The relative expression profiles of mRNA of FGFRs by microarray analysis. The log2 of fold change to PC9 na was calculated for each sample by using Gene Spring GX software. Each group of columns represents the result from 1 microarray probe&lt;/p&gt;

&lt;p&gt;Supplementary Figure S5 - PDF file 109K, Efficient knockdown of FGFR1, FGFR3, and FGF2 with each siRNA confirmed by quantitative RT-PCR&lt;/p&gt;

&lt;p&gt;Supplementary Figure S4 - PDF file 107K, DNA quantification for FGFR1 by quantitative-PCR. DNA copy number was not increased in PC9 GR and PC9 gr1 cells&lt;/p&gt;

&lt;p&gt;Supplementary Figure S3 - PDF file 139K,The relative expression profiles of mRNA of FGFs by microarray analysis in PC9 GR, PC9 gr1 and PC9 gr3 cells normalized to mRNA extracted from PC9 na cells. None of FGFs other than FGF2 was elevated in gefitinib resistant cell lines. Each group of columns represents the result from one microarray probe&lt;/p&gt;

Abstract 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 for COVID-19 is highly recommended for patients with risk factors for severe infection to avoid persistent respiratory symptoms.

Abstract 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.

Although cancer immunotherapy has recently demonstrated durable responses even in patients with advanced cancer, not all patients or cancer types respond to the therapy. Pretreatment immune status varies among cancer patients and is correlated with responses to immunotherapy. Immune conditions may be defined by the balance of positive and negative pathways in the anti-tumor immune responses, which are regulated by both cancer cell characteristics and patients’ immune-reactivity along with various environmental factors. Gene alterations and signal activation define the immunological characteristics of cancer cells; tumor specific peptides derived from passenger mutations induce anti-tumor T-cells and oncogene activation (e.g. driver mutations, overexpression: MAPK, STAT3, NF-κB, β-catenin) rather promote immunosuppression. Oncogene/signal activation in cancer cells triggers multiple immunosuppressive cascades involving various immunosuppressive molecules and cells (e.g. TGF-β, IL10, IL6, VEGF, Treg, MDSC). Signal inhibitors are able to augment anti-tumor T-cell responses through multiple mechanisms including inhibition of cancer-induced immunosuppression, immunogenic cancer cell death, and enhancement of immune cell functions. Since the oncogene-signal activation status is different among patients, personalized immunotherapy combined with appropriate signal inhibitors may be considered for the development of effective immunotherapy.

Lung cancer is leading cause of cancer death in many advanced countries and one of the challenging malignancies because of poor prognosis. Lung cancer is traditionally divided into two major categories, so called small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) because of distinctive prognostic and treatment strategies between them. On the other hand, there is a spectrum of tumors called pulmonary neuroendocrine (NE) tumors that are thought to originate from neuroendocrine cells in the pulmonary and bronchial epithelium. Until recently, pulmonary NE tumors were classified into three categories, i.e., typical carcinoid (TC), atypical carcinoid (AC), and SCLC. Large cell neuroendocrine carcinoma (LCNEC) of the lung was officially identified by Travis et al. in 1991 as a fourth category, a unique higher grade NSCLC existing between TC and SCLC (Travis et al., 1991). It is often difficult to diagnose LCNEC with small biopsy specimens because accurate diagnosis needs morphological and immunohistochemical information. Although earlier reports mainly focused on prognosis after surgical procedures, several recent studies reported on the efficacy of chemotherapy for advanced LCNEC. Because of the limited numbers of cases (in surgical series, LCNEC represents ~3% of lung cancers), large scale prospective studies have not been reported. Standard treatment for LCNEC, especially if advanced, is not established although LCNEC is included in NSCLC in the treatment algorithm in many guidelines. However, accumulating data including recent retrospective studies have suggested that there is similarity in the prognosis and treatment response between LCNEC and SCLC. In this review, we will focus on the treatment of advanced LCNEC for the better selection of chemotherapeutic regimens for the patients with this relatively rare lung cancer.

Abstract Many of the patients with non-small cell lung cancer (NSCLC) with sensitive epidermal growth factor receptor (EGFR)-mutation who initially responded well to EGFR-tyrosine kinase inhibitors (TKIs) eventually relapse. In spite of many studies over the last few years to elucidate this mechanism of acquired resistance to EGFR-TKIs, approximately 30% of the mechanisms of acquired resistance are still unknown. Recently autocrine signaling of fibroblast growth factors (FGFs) and their receptors (FGFRs) has been demonstrated in NSCLC cell lines. And several studies suggest that the FGF-FGFR autocrine growth pathway could be an important mechanism for intrinsic resistance to EGFR-TKIs in NSCLC cell lines with wild-type EGFR. But until now, no report has clarified the role of FGF-FGFR pathway in acquired resistance to EGFR-TKIs in NSCLC cell lines with sensitive EGFR mutations. We have established a gefitinib-resistant cell line (PC9 GR), by serial exposure of gefitinib to PC9, an originally gefitinib-sensitive lung cancer cell line (PC9 na). We confirmed that these cell lines did not harbor two well-known EGFR-TKI resistance mechanisms, the second mutation in the EGFR gene itself, EGFR T790 and the amplification of the MET oncogene. We collected total RNA from both PC9 na and PC9 GR and examined mRNA expression profile, by using cDNA microarray analysis. We found the expressions of FGFR1 and FGF2 were increased in PC9 GR compared to in PC9 na. The growth of PC9 GR cells was inhibited either by PD173074 (inhibitors of FGFRs) or knocking down of FGFR1 or FGF2 by siRNA in combination with gefitinib. FACS analysis revealed that the combination treatment with PD173074 and gefitinib induced apoptosis more efficiently in PC9 GR cells compared to gefitinib alone. PC9 na cells and PC9 GR cells did not show any change in the proportion of apoptotic cells after treatment with PD173074 alone. To further investigate how FGF2-FGFR1 pathway affects resistance to gefitinib in these cell lines, the downstream targets of EGFR signaling including the MEK-ERK and PI3K-AKT pathways were examined. In PC9 na cells, the phosphorylation of EGFR, ERK, and AKT was efficiently inhibited by gefitinib alone. On the other hand, in PC9 GR cells, the phosphorylation of ERK and AKT was not efficiently inhibited by gefitinib alone. However, the inhibition of phosphorylation of ERK was completely and AKT was less efficiently rescued by gefitinib and PD173074 combination therapy. In conclusion, these data suggest the activation of FGF2-FGFR1 signaling pathway contributes to the gefitinib resistance in PC9 GR. FGF2-FGFR1 pathway will be a therapeutic target for a subset of NSCLC that acquires EGFR-TKI resistance. Citation Format: Hideki Terai, Kenzo Soejima, Katsuhiko Naoki, Hiroyuki Yasuda, Ryosuke Satomi, Sohei Nakayama, Satoshi Yoda, Shinnosuke Ikemura, Takashi Sato, Kota Ishioka, Daisuke Arai, Keiko Ohgino, Tetsuo Tani, Aoi Kuroda, Junko Hamamoto, Tomoko Betsuyaku. Activation of FGF2-FGFR1 pathway in EGFR-mutant lung cancer cell line with long-term gefitinib exposure. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5652. doi:10.1158/1538-7445.AM2013-5652

Abstract Introduction: Recent advances in the treatment of non-small-cell lung cancer (NSCLC) with new agents require accurate histological subtyping at diagnosis in order to avoid the higher risk of an adverse response and obtain a maximum therapeutic response. However, interobserver variability, tumor heterogeneity and the degree of differentiation may influence the decision concerning a pathological diagnosis of NSCLC. We therefore in this study attempted to identify specific microRNAs as standardized biomarkers with high sensitivity and specificity for distinguishing between squamous cell carcinoma (SCC) and adenocarcinoma (AC). Methods: Quantitative real time polymerase chain reaction (qRT-PCR) based microRNA array was performed to identify microRNAs differentially expressed between SCC and AC using 86 resected NSCLC samples as well as adjacent normal tissues. The results were confirmed by independent qRT-PCR assays with the same test samples and an additional 88 validation samples and the usefulness of the identified microRNAs as biomarkers to distinguish between SCC and AC was evaluated. Results: Three microRNAs (hsa-miR-196b, hsa-miR-205 and hsa-miR-375) were identified. Discriminant analysis combining all 3 microRNAs appeared to distinguish SCC from AC accurately in the test samples and the validation samples, showing sensitivity and specificity of 76% and 80%, and 85% and 83%, respectively. Conclusions: 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 microRNAs may prove to be highly valuable molecular markers for the classification of NSCLC histologic subtypes, and could potentially contribute to the pathogenesis of each subtype of NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4956. doi:10.1158/1538-7445.AM2011-4956

e18013 Background: Incidence of elderly advanced non-small cell lung cancer (NSCLC) is increasing and the establishment of optimal treatment for the elderly patients is desired. Currently, as the elderly are underrepresented in clinical trials, only limited data exist. Standard chemotherapy by platinum doublet of elderly patients with advanced NSCLC is still controversial. This study has been conducted to assess the efficacy and toxicity of biweekly carboplatin plus paclitaxel in elderly patients with advanced NSCLC. Methods: Patients ≥70 years old with cytological or histological confirmation of advanced (stage IIIB/IV) NSCLC were included. Other eligibility criteria were PS≤2, measurable lesions according RECIST criteria, adequate organ function. Patients with prior systemic therapy for NSCLC and/or symptomatic brain metastases were not allowed. Patients received biweekly paclitaxel 90 mg/m2 and carboplatin 2.5 AUC on days 1 and 14 of each 28-day cycle. The primary endpoint was the overall response rate (ORR), and secondary endpoints were progression-free survival (PFS), overall survival, and the toxicity profile. Results: We enrolled 47 pts from multiple institutions (Jan. 2007 – Oct. 2010). Males were 76.6%, median age was 77 years (range 70-85). PS was 0-1 in 95.7%. The median number of treatment cycle was 3 (1-6). The objective responses were CR 0; PR 10; SD 12; PD 14; and NE 11, resulting in an ORR of 21.3% (95% confidence interval [CI], 9.6- 33.0). The overall disease control rate was 46.8% (95% CI, 32.5-61.1). Median PFS was 4.17 month (95% CI: 2.18- 6.16). Hematological toxicities of grade 3/4 included neutropenia (28%), leucopenia (19%) and anemia (11%). Nonhematological toxicities (all grade 3) included infusion reaction (2%), anorexia (2%), infection (13%), thrombosis (2%), fatigue (2%), diarrhea (2%) and gastrointestinal bleeding (2%). No grade 4 nonhematological toxicity was observed. There was 1 possible treatment-related death due to interstitial pneumonia. Conclusions: The combination of biweekly carboplatin and paclitaxel is an active first line treatment with a tolerable toxicity profile for advanced NSCLC in elderly patients.

Abstract Clinical studies for immunotherapy targeting various types of carcinoma, including lung carcinoma, are currently underway, yet the clinical effectiveness of these therapies is very limited. Recent findings demonstrate that cancer immune evasion is a major contributing factor to this problem. Cancer cells produce a variety of immunosuppressive molecules that inhibit the host cell's immune response, leading to conditions such as cachexia that decrease patients’ quality of life. EGFR mutations in lung carcinoma are known to lead to processes characteristic of malignancies, such as cell cycle progression, metastasis, and angiogenesis. Based on these observations, we tested the hypothesis that EGFR activation leads to immune evasion by cancer cells via the production of various immunosuppressive molecules. In this study, 6 lung adenocarcinoma cell lines were used, namely A549, RELF-LC-OK, PC-9, HCC-827, NCI-H1650, and NCI-H1975. We demonstrate that these cells secrete immunosuppressive cytokines such as IL-6, TGF-beta and VEGF, which inhibit the maturation of dendritic cells and prevent them to activate T cells. Then, we show that Gefitinib administration at sub-lethal concentration inhibits the production of immunosuppressive cytokines in EGFR-TKI sensitive PC-9 and HCC-827 cells those with EGFR mutations as well as in EGFR-TKI resistant NCI-H1650 cells that have an EGFR mutation but lack the PTEN gene. While, cytokine production is not inhibited by Gefitinib in A549 and RELF-LC-OK cells with wild type EGFR genes and NCI-H1975 cells that have both EGFR-TKI sensitive and resisitant mutations. Additionally, EGFR-TKI administration in PC-9, HCC827, and NCI-H1650 cells restores dendritic cell function, which was initially reduced by the supernatant of tumor cells. These results show that lung adenocaricinoma harboring EGFR mutations evade immune response through secreteting immunosuppressive agents and that the immune response is restored by Gefitinib administration. Thus, the combination of Gefitinib and immunotherapy may have synergistic effect and will be a promising treatment of lung cancer patients harboring EGFR mutation, including certain population of EGFR-TKI resistant cases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5302.

e21067 Background: Serum tumor markers (TMs) have been long used to distinguish malignant from benign lung tumors. While 18F-fluoro-2-deoxy-D-glucose (FDG)-PET is currently widely used and considered as a useful imaging method for the diagnosis of lung carcinoma. Although the diagnostic accuracy of FDG-PET was initially reported to be quite high, it has been revealed that its accuracy is not as high as expected. The purpose of this study is to reevaluate and compare the accuracy of FDG-PET and TMs for the diagnosis of lung carcinoma. Methods: We evaluated 386 cases underwent PET/CT scan from April 2003 to March 2008 in our institution, retrospectively. We selected 146 cases of primary lung carcinoma and 55 cases of benign pulmonary diseases including tuberculosis, cryptococcosis, etc. whose diagnosis was finally confirmed. The study was approved by the IRB of Keio University. We also evaluated clinical parameters including age, gender, smoking history and nodule diameter as well as TMs (CEA, CYFRA and ProGRP). We calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) by FDG-PET and TMs or in combination with other parameters using multiple logistic regression analysis. Results: The PET positive rate in the primary lung carcinomas and the benign diseases was 84.9% (124/146) and 48.1% (26/55), respectively. The PET possessed higher sensitivity but lower specificity compared to TM for all stages, especially in early stages (I + II) (Table). Additionally, the multiple logistic regression analysis revealed that the nodule diameter and the FDG uptake were identified as significant predicting factors (p<0.05) and these factors had predictive value for the early stage lung carcinoma as a sensitivity of 77.3%. Combination with tumor markers and FDG uptake were not able to lead better prediction formula. Conclusions: The FDG-PET is useful to diagnose lung carcinoma, especially in the early stage compared to TMs. Parameters All stages Stage I + II Stage III + IV Sensitivity (%) FDG-PET 84.9 71.6 96.2 TM 62.1 43.9 77.2 Specificity (%) FDG-PET 52.7 52.7 52.7 TM 90.4 90.4 90.4 PPV (%) FDG-PET 82.7 64.9 74.5 TM 94.1 85.3 92.4 NPV (%) FDG-PET 56.9 60.4 93.5 TM 43.6 56.0 75.8 No significant financial relationships to disclose.

A 76-year-old woman with multiple bone metastases from lung adenocarcinoma was admitted due to a pathological femoral fracture. On the night after admission, her consciousness deteriorated rapidly and she developed progressive respiratory failure. Computed tomography of the chest revealed diffuse ground glass opacities in both lungs, and magnetic resonance imaging of the brain showed multiple acute infarctions. Her condition improved after several days of supportive treatment with oxygen, corticosteroids and diuretics. Fat embolism syndrome should be considered as a differential diagnosis if consciousness disturbance and respiratory failure occur in patients with metastatic bone carcinoma and pathological long bone fractures.

Abstract 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.