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Immune checkpoint inhibitors have significantly improved the clinical outcomes of many cancer types, but they induce a range of immune-related adverse events (irAEs). Although adrenal insufficiency (AI) is a rare irAE, it can lead to serious consequences. This study aimed to determine the clinical features of patients with advanced non-small-cell lung cancer (NSCLC) who developed AI following pembrolizumab treatment.We retrospectively reviewed and analyzed the clinical data of all patients with NSCLC treated with pembrolizumab at Juntendo University Hospital from February 2017 to December 2020. The diagnosis of AI was established based on the Endocrine Emergency Guidance for the acute management of endocrine complications of checkpoint inhibitor therapy in the UK and the clinical practice guidelines of the Japan Endocrine Society.AI was clinically suspected in 59 out of 186 patients treated with pembrolizumab, and 10 (5.4%) cases were confirmed. The symptoms included hyponatremia (n = 9), fatigue (n = 8), and loss of appetite (n = 6). All patients had low adrenocorticotropic hormone (ACTH) levels, and five patients were diagnosed with isolated ACTH deficiency. All patients completely recovered with corticosteroid replacement. The median time to onset of AI was 8.0 (range 3.8-15.2) months. The median progression-free survival in these patients was 22.4 (95% confidence interval 11.2-not reached) months.The incidence of AI among patients treated with pembrolizumab is more frequent than previously reported. In addition, secondary AI, especially isolated ACTH deficiency, is a major form of AI induced by pembrolizumab.

Supplementary Figure S5 shows role of MFGE8 in MPM.

Supplementary Figure Legends

Supplementary Table S1 shows the selected genes with statistically significant changes in transcriptome analysis using two different shRNA constructs.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that has a low overall survival; however, no significant treatment advances have been made in the past 15 years. Large-scale molecular studies have identified a poor prognostic subset of MPM linked to the epithelial–mesenchymal transition (EMT) that may contribute toward resistance to chemotherapy, suggesting that EMT could be targeted to treat patients with MPM. Previously, we reported that histone modifiers regulating EMT could be therapeutic targets; therefore, in this study, we investigated whether targeting lysine-specific demethylase 1 (LSD1/KDM1), a histone-modifying enzyme responsible for demethylating histone H3 lysine 4 and lysine 9, could represent a novel therapeutic strategy for MPM. We suppressed LSD1 and investigated the EMT phenotype using EMT marker expression and wound-healing assay; and chemosensitivity using apoptosis assay. We found that suppressing LSD1 induces an epithelial phenotype in sarcomatoid MPM cells, while attenuating the mesenchymal phenotype sensitized MPM cells to cisplatin-induced apoptosis. Subsequent genome-wide identification, comprehensive microarray analysis, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to assess genome-wide changes in chromatin accessibility suggested that LSD1 directly regulates milk fat globulin protein E8 (MFGE8), an integrin ligand that is involved in the FAK pathway. Furthermore, we found that LSD1 regulates the mesenchymal phenotype and apoptosis by activating the FAK–AKT–GSK3β pathway via a positive feedback loop involving MFGE8 and Snail expression, thereby leading to cisplatin resistance.Implications:This study suggests that LSD1 regulates the mesenchymal phenotype and apoptosis, and that LSD1 inhibitors could be combined with the cisplatin as a novel therapy for patients with MPM.

Background Zinc‐finger E‐box‐binding homeobox 1 (ZEB1) is an important regulator of epithelial‐mesenchymal transition (EMT) and is involved in the maintenance of cancer stem cells (CSCs) via miR‐200c and BMI1 pathway. Recent studies revealed that ZEB1 contributes to the EMT‐mediated acquired resistance to gefitinib in EGFR‐mutant non‐small cell lung cancer (NSCLC). However, the precise role of ZEB1 in the maintenance of lung CSCs that lead to acquired resistance to gefitinib remains unclear. Methods PC9 and HCC827 NSCLC cell lines were treated with high concentrations of gefitinib, and surviving cells were referred to as “gefitinib‐resistant persisters” (GRPs). ZEB1 knockdown or overexpression was performed to determine the biological significance of ZEB1 in the CSC features of GRPs, and animal models were studied for in vivo validation. Expression of ZEB1, BMI1, and ALDH1A1 was analyzed by immunohistochemistry in tumor specimens from NSCLC patients with acquired resistance to gefitinib. Results GRPs had characteristic features of mesenchymal and CSC phenotypes with high expression of ZEB1 and BMI1, and decreased miR‐200c, in vitro and in vivo. ZEB1 silencing attenuated the suppression of miR‐200c, resulting in the reduction in BMI1 and reversed the mesenchymal and CSC features of GRPs. Furthermore, ZEB1 overexpression induced EMT and increased the levels of CD133‐ and BMI1‐positive GRPs in vitro and gefitinib resistance in vivo. Finally, ZEB1, BMI1, and ALDH1A1 were highly expressed in tumor specimens from EGFR‐mutant NSCLC patients with gefitinib resistance. Conclusions ZEB1 plays an important role in gefitinib‐resistant lung CSCs with EMT features via regulation of miR‐200c and BMI1., Gefitinib‐resistant persisters (GRPs) of EGFR‐mutant NSCLC cells have characteristic features of mesenchymal and CSC phenotypes. ZEB1 is highly expressed in GRPs and involved in the maintenance of lung CSCs resistant to gefitinib via miR‐200c‐BMI1 pathway.

Background Regimens combining pemetrexed (PEM) and immune checkpoint inhibitors (ICIs) targeting programmed cell death-1 (PD-1) or programmed death-ligand 1 (PD-L1) are widely used for the treatment of advanced non-squamous non-small-cell lung cancer (NSq-NSCLC). Recently, PEM was shown to induce immunogenic cell death (ICD) and to enhance immune-regulatory genes. Some patients demonstrate an extremely long-term response to PEM. It is possible that the continued response in these patients is dependent on not only the pharmacological induction of cytotoxic cell death but also antitumor immunity. However, factors that can predict outcomes associated with long-term PEM administration using blood test results have not yet been elucidated. We investigated the clinical characteristics and predictive factors in patients with advanced NSq-NSCLC who underwent long-term PEM maintenance therapy. Methods In total, 504 patients with advanced NSq-NSCLC who received PEM combination therapy/monotherapy (n = 414) or paclitaxel (PTX) combination therapy (n = 90) between January 2010 and November 2019 were recruited; 381 patients were retained for the final analysis. Patients treated with PEM (n = 301) were divided into subgroups according to the total cycles of PEM (≥ 17 [n = 25] for the long-term administration group and ≤ 16 [n = 276] for the intermediate/short-term group) and compared with another population (n = 80) treated with PTX combination regimen. We investigated clinical features and predictive biomarkers, focusing on immune-regulatory factors, absolute lymphocyte count (ALC), neutrophil-to-lymphocyte ratio (NLR), and PD-1 and PD-L1 expression, to predict long-term response to PEM. Results The long-term PEM administration group exhibited a higher ALC and a lower NLR than the shorter-term group did. Both these markers displayed greater association with progression-free survival and overall survival in the PEM combination therapy group than in the PTX combination therapy group. Increased PD-1 lymphocytes were associated with the long-term PEM response group, as PD-L1 expression in tumors was associated with a high incidence of immune-related adverse effects following ICI administration. Conclusions ALC, NLR, and PD-1 expression are PEM-mediated predictive biomarkers that are indirectly related to tumor immunity and can provide useful predictive information on the long-term response to PEM in patients with NSq-NSCLC.

Background Afatinib is a second-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI). Combination therapies with first-generation EGFR-TKIs and bevacizumab have been reported to prolong progression-free survival (PFS). However, there are few data on the combination of afatinib and bevacizumab. Methods In this phase I trial, we evaluated the safety of afatinib plus bevacizumab in patients with advanced non-squamous non-small cell lung cancer (NSCLC) harboring EGFR mutations. This study consisted of two cohorts. In the dose-finding cohort, enrolled patients were treated with afatinib at a dose of 20, 30, or 40 mg/day (days 1-21) plus bevacizumab at a dose of 15 mg/kg (day 1) in 21-day cycles. This cohort was performed according to a 3 + 3 manner. In the expansion cohort, enrolled patients received the recommended dose (RD) based on the results of the dose-finding cohort. The serum trough concentration of afatinib was determined at the steady state. Results Seventeen patients were enrolled in this study (6 patients in the dose-finding cohort and 11 patients in the expansion cohort). There were no dose-limiting toxicities (DLTs) with afatinib at a dose of 30 mg/day. With afatinib at a dose of 40 mg/day, two of two patients experienced DLTs (grade 3 diarrhea) in cycle 1. With these results, afatinib at a dose of 30 mg/day plus bevacizumab at a dose of 15 mg/kg was determined as the RD. Eleven patients in the expansion cohort were treated with the RD. Common treatment-related adverse events (AEs) with the RD were diarrhea (79%), rash (71%), perionychia (64%), and stomatitis (50%). Grade 3 AEs with the RD were diarrhea (7%), perionychia (7%), and hypertension (7%). There were no grade 4/5 AEs or cases of interstitial lung disease. Dose-proportional increases in serum afatinib trough concentrations at steady state were not observed. The response rates (RRs) and disease control rates were 55% and 100% in EGFR-TKI-naive patients. Re-biopsy was performed in eight patients after progressive disease following the study treatment, and three patients acquired a T790M mutation. Conclusions Afatinib at a dose of 30 mg/day plus bevacizumab at a dose of 15 mg/kg q3w is well tolerated.

Purpose Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix (ECM) protein in the lungs. Transforming growth factor (TGF) β-induced ECM protein synthesis contributes to the development of IPF. Tranilast, an anti-allergy drug, suppresses TGFβ expression and inhibits interstitial renal fibrosis in animal models. However, the beneficial effects of tranilast or its mechanism as a therapy for pulmonary fibrosis have not been clarified. Methods We investigated the in vitro effect of tranilast on ECM production and TGFβ/SMAD2 pathway in TGFβ2-stimulated A549 human alveolar epithelial cells, using quantitative polymerase chain reaction, Western blotting, and immunofluorescence. In vitro observations were validated in the lungs of a murine pulmonary fibrosis model, which we developed by intravenous injection of bleomycin. Results Treatment with tranilast suppressed the expression of ECM proteins, such as fibronectin and type IV collagen, and attenuated SMAD2 phosphorylation in TGFβ2-stimulated A549 cells. In addition, based on a wound healing assay in these cells, tranilast significantly inhibited cell motility, with foci formation that comprised of ECM proteins. Histological analyses revealed that the administration of tranilast significantly attenuated lung fibrosis in mice. Furthermore, tranilast treatment significantly reduced levels of TGFβ, collagen, fibronectin, and phosphorylated SMAD2 in pulmonary fibrotic tissues in mice. Conclusion These findings suggest that tranilast inhibits pulmonary fibrosis by suppressing TGFβ/SMAD2-mediated ECM protein production, presenting tranilast as a promising and novel anti-fibrotic agent for the treatment of IPF.

Background and Purpose In breast cancer patients receiving neoadjuvant chemotherapy (NAC), immediate breast reconstruction (IBR) is controversial. We retrospectively investigated whether IBR after mastectomy influenced the short and long-term outcome of patients receiving NAC. Patients and Methods Between April 2007 and December 2017, 525 breast cancer patients received NAC at our hospital of which 186 patients underwent total mastectomy alone, 55 underwent total mastectomy followed by IBR, and 284 underwent breast conserving surgery (BCS). Patients receiving IBR were compared to patients who did not receive IBR. The NAC regimen was 4 cycles of epirubicin, 5-fluorouracil and cyclophosphamide followed by 4 cycles of docetaxel. Decision making for post mastectomy radiotherapy (PMRT) followed national comprehensive cancer network guidelines. PMRT is not contraindication for patients receiving IBR, however risk such as capsular contracture was informed to patients. Tissue expander was replaced to silicon breast implant within two to three months, and radiotherapy started within 4 to 6 weeks after surgery. In other surgical-procedure, such as direct-to-implant breast reconstruction, BCS or mastectomy alone, radiotherapy started 6-8 weeks later. Post-mastectomy radiation was applied equally with the same selection criteria to both groups. Results Patients in the IBR and BCS groups were on average younger than patients in the mastectomy alone group (p After a median of 76.4 month, 2 patients (4.2%) in the IBR group, 13 patients (4.5%) in the BCS group and 17 patients (9.1%) showed locoregional recurrences. Five-year locoregional recurrence-free survival was 96.1% in the IBR group, 96.8% in the BCS group and 90.1% in the mastectomy alone group (p= 0.032). Five-year distant disease-free survival was 92.9 % in the IBR group, 93.9% in the BCS group and 85.3 % in the mastectomy alone group (p= 0.0008). Five-year overall survival was 98.0 % in the IBR group, 98.4% in the BCS group and 92.3 % in the mastectomy alone group (p= 0.0004). Multivariate analysis showed that IBR was not associated with worse prognosis. Transverse rectus abdominis muscle flap necrosis was seen in one patient. Any breast implant or tissue expander related complications including infection and capsular contracture were not encountered. Conclusions IBR after total mastectomy can be performed safely and was not associated with worse prognosis in patients receiving NAC and therefore IBR may be the preferred strategy in select cases. Citation Format: Hiroko Nogi, Makiko Kamio, Takashi Kazama, Naoko Shimada, Takayuki Ishigaki, Hisashi Shioya, Yasuo Toriumi, Eijiro Nagasaki, Hiroshi Takeyama. Impact of immediate breast reconstruction after mastectomy on the short and long-term outcome of patients receiving neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-14-08.

hnRNP A1 is a member of the hnRNPs (heterogeneous nuclear ribonucleoproteins) family of proteins that play a central role in regulating genes responsible for cell proliferation, DNA repair, apoptosis, and telomere biogenesis. Previous studies have shown that hnRNPA1 had reduced protein levels and increased cytoplasmic accumulation in senescent human diploid fibroblasts. The consequence of reduced protein expression and altered cellular localization may account for the alterations in gene expression observed during senescence. There is limited information for gene targets of hnRNP A1 as well as its in vivo function. In these studies, we performed RNA co-immunoprecipitation experiments using hnRNP A1 as the target protein to identify potential mRNA species in ribonucleoprotein (RNP) complexes. Using this approach, we identified the human double minute 2 (HDM2) mRNA as a binding target for hnRNP A1 in young and senescent human diploid fibroblasts cells. It was also observed that alterations of hnRNP A1 expression modulate HDM2 mRNA levels in young IMR-90 cells. We also demonstrated that the levels of HDM2 mRNA increased with the downregulation of hnRNP A1 and decrease with the overexpression of hnRNP A1. Although we did not observe a significant decrease in HDM2 protein level, a concomitant increase in p53 protein level was detected with the overexpression of hnRNP A1. Our studies also show that hnRNP A1 directly interacts with HDM2 mRNA at a region corresponding to its 3′ UTR (untranslated region of a gene). The results from this study demonstrate that hnRNP A1 has a novel role in participating in the regulation of HDM2 gene expression.

Background The prognosis of patients with NSCLC harboring oncogenic driver gene alterations, such as EGFR gene mutations or ALK fusion, has improved dramatically with the advent of corresponding molecularly targeted drugs. As patients were followed up for about five years in most clinical trials, the long-term outcomes beyond 5 years are unclear. The objectives of this study are to explore the clinical course beyond five years of chemotherapy initiation and to investigate factors that lead to long-term survival. Methods One hundred and seventy-seven patients with advanced, EGFR-mutated or ALK-rearranged NSCLC who received their first chemotherapy between December 2008 and September 2015 were included. Kaplan Meier curves were drawn for the total cohort and according to subgroups of patients’ characteristics. Results Median OS in the total cohort was 40.6 months, the one-year survival rate was 89%, the three-year survival rate was 54%, and the five-year survival rate was 28%. Median OS was 36.9 months in EGFR-mutated patients and 55.4 months in ALK-rearranged patients. The OS curve seemed to plateau after 72 months, and most of the patients who were still alive after more than five years are on treatment. Female sex, age under 75 years, an ECOG PS of 0 to 1, ALK rearrangement, postoperative recurrence, and presence of brain metastasis were significantly associated with longer OS. Conclusions A tail plateau was found in the survival curves of patients with advanced, EGFR-mutated and ALK-rearranged NSCLC, but most were on treatment, especially with EGFR-mutated NSCLC.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that has a low overall survival; however, no significant treatment advances have been made in the past 15 years. Large-scale molecular studies have identified a poor prognostic subset of MPM linked to the epithelial–mesenchymal transition (EMT) that may contribute toward resistance to chemotherapy, suggesting that EMT could be targeted to treat patients with MPM. Previously, we reported that histone modifiers regulating EMT could be therapeutic targets; therefore, in this study, we investigated whether targeting lysine-specific demethylase 1 (LSD1/KDM1), a histone-modifying enzyme responsible for demethylating histone H3 lysine 4 and lysine 9, could represent a novel therapeutic strategy for MPM. We suppressed LSD1 and investigated the EMT phenotype using EMT marker expression and wound-healing assay; and chemosensitivity using apoptosis assay. We found that suppressing LSD1 induces an epithelial phenotype in sarcomatoid MPM cells, while attenuating the mesenchymal phenotype sensitized MPM cells to cisplatin-induced apoptosis. Subsequent genome-wide identification, comprehensive microarray analysis, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to assess genome-wide changes in chromatin accessibility suggested that LSD1 directly regulates milk fat globulin protein E8 (MFGE8), an integrin ligand that is involved in the FAK pathway. Furthermore, we found that LSD1 regulates the mesenchymal phenotype and apoptosis by activating the FAK–AKT–GSK3β pathway via a positive feedback loop involving MFGE8 and Snail expression, thereby leading to cisplatin resistance. Implications: This study suggests that LSD1 regulates the mesenchymal phenotype and apoptosis, and that LSD1 inhibitors could be combined with the cisplatin as a novel therapy for patients with MPM.

Background There are few studies that directly compare the effects of osimertinib on patients with non‐small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) T790M mutation between the generation of prior EGFR tyrosine kinase inhibitors (TKIs). Methods We retrospectively reviewed clinical data from the medical records of consecutive patients with advanced NSCLC who had developed resistance to first‐ or second‐generation EGFR‐TKIs due to EGFR T790M mutation and were subsequently treated with osimertinib at Juntendo University Hospital. In patients with available tumor samples, target amplicon sequencing analyses were performed to explore the genetic biomarkers. Results A total of 38 patients with NSCLC harboring EGFR T790M mutation were treated with osimertinib. Eight patients were classified into group A (afatinib followed by osimertinib) and 30 patients were classified into group B (first‐generation EGFR‐TKI followed by osimertinib). Progression‐free survival (PFS) was significantly longer in group A than in group B (median PFS; not reached vs. 11.0 months, P = 0.018). Fourteen patients had available tissue samples collected before osimertinib treatment for target sequencing. In group A we found no additional mutations, other than EGFR T790M mutation. On the other hand, there were three samples in which other mutations emerged, in addition to EGFR T790M mutation, in group B. Conclusions PFS of osimertinib was significantly longer in patients with NSCLC harboring EGFR T790M mutation after treatment with afatinib than in patients after treatment with first generation EGFR‐TKIs. Additional mutations other than EGFR T790M may affect the efficacy of osimertinib treatment. Key points Significant findings of the study: PFS of osimertinib was significantly longer in patients with NSCLC harboring EGFR T790M mutation after treatment with afatinib than in patients after treatment with first generation EGFR‐TKIs. What this study adds: Additional mutations other than EGFR T790M may affect the efficacy of osimertinib treatment., There are few studies that directly compare the difference between the generation of prior EGFR tyrosine kinase inhibitors (TKIs) before osimertinib treatment in patients with non‐small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) T790M mutation. In our retrospective study, progression‐free survival of osimertinib was significantly longer in patients with NSCLC harboring EGFR T790M mutation after treatment with afatinib than in patients after treatment with first generation EGFR‐TKIs.

Purpose Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix (ECM) protein in the lungs. Transforming growth factor (TGF) β-induced ECM protein synthesis contributes to the development of IPF. Tranilast, an anti-allergy drug, suppresses TGFβ expression and inhibits interstitial renal fibrosis in animal models. However, the beneficial effects of tranilast or its mechanism as a therapy for pulmonary fibrosis have not been clarified. Methods We investigated the in vitro effect of tranilast on ECM production and TGFβ/SMAD2 pathway in TGFβ2-stimulated A549 human alveolar epithelial cells, using quantitative polymerase chain reaction, Western blotting, and immunofluorescence. In vitro observations were validated in the lungs of a murine pulmonary fibrosis model, which we developed by intravenous injection of bleomycin. Results Treatment with tranilast suppressed the expression of ECM proteins, such as fibronectin and type IV collagen, and attenuated SMAD2 phosphorylation in TGFβ2-stimulated A549 cells. In addition, based on a wound healing assay in these cells, tranilast significantly inhibited cell motility, with foci formation that comprised of ECM proteins. Histological analyses revealed that the administration of tranilast significantly attenuated lung fibrosis in mice. Furthermore, tranilast treatment significantly reduced levels of TGFβ, collagen, fibronectin, and phosphorylated SMAD2 in pulmonary fibrotic tissues in mice. Conclusion These findings suggest that tranilast inhibits pulmonary fibrosis by suppressing TGFβ/SMAD2-mediated ECM protein production, presenting tranilast as a promising and novel anti-fibrotic agent for the treatment of IPF.

Skin-sparing mastectomy (SSM) enables a radical cure of breast cancer while overcoming the cosmetic issues related to surgery. We review our experience of performing SSMs and assess whether preservation of the nipple–areola complex (NAC) could have been an option for some patients who underwent SSM. The subjects of this retrospective study were women who underwent SSM that utilized four incision types; namely, the so-called tennis racket incision, a periareolar and midaxillary incision, an areola-sparing and midaxillary incision, and a small transverse elliptical incision. We assessed whether preservation of the NAC would have been an option in SSM, based on histologic examination of three serial cut surfaces of the specimen around the nipple, ruling out the option when evidence of the malignant lesion/s was found in at least one of the following locations: in the nipple, within a 1-cm radius from the base of the nipple, or within 1 cm from the surface of the NAC. We performed 193 SSMs. The cumulative 10-year local disease-free survival rate was 98%, with 89% of patients reporting levels of satisfaction with the reconstructed breast, of excellent, very good, or good. We evaluated that 70 of the 193 procedures could have been performed as nipple-sparing mastectomy (NSM). The outcomes of SSM in this series were excellent and NSM might have been an option for about one-third of the patients.

Background Cryoablation (CA) is a nonsurgical focal therapy for small tumours. To detect residual or relapsed tumour after CA of renal cancer, contrast - enhanced imaging is generally used to identify tumour blood flow, but no definitive criteria are established for such follow-up after CA of breast cancer. Aims The aim of this study was to compare the usefulness of contrast-enhanced ultrasonography (CEUS) and magnetic resonance (MR) imaging for assessing residual tumours and local relapse following CA of small breast cancers. Methods We enrolled 4 patients treated by CA at our institution between January 2015 and December 2016 for luminal A breast cancer with maximum tumour size of 1.5cm and neither distant metastasis nor metastatic findings in sentinel lymph node biopsy, who underwent CEUS and MR imaging before CA. In addition to our standard postoperative follow-up for breast cancer, these patients underwent CEUS every 3 months and MR imaging every 6 months after CA. Results Six months after CA, no patient showed enhancement at the lesion site on MR imaging, but there were two with continued enhancement on CEUS. They underwent vacuum -assisted breast biopsy under US guidance followed by histopathological examination of tissue that identified no malignancy. Conclusion Our findings of focal enhancement within ablated breast tissue in CEUS after CA is likely attributable to the much higher sensitivity of CEUS to that of other modalities to even slight vascularization. Further investigation in more patients is needed to clarify the utility of CEUS to detect residual or relapsed tumour after CA of small breast cancer.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor characterized by rapid progression. The mechanisms that lead to a shift from initial therapeutic sensitivity to ultimate therapeutic resistance are poorly understood. Although the SCLC genomic landscape led to the discovery of promising agents targeting genetic alterations that were already under investigation, results have been disappointing. Achievements in targeted therapeutics have not been observed for over 30 years. Therefore, the underlying disease biology and novel targets urgently require a better understanding. Epigenetic regulation is deeply involved in the cellular plasticity that could shift tumor cells to the malignant phenotype. We have focused on a histone modifier, LSD1, that is overexpressed in SCLC and is a potent therapeutic target. Interestingly, the LSD1 splice variant LSD1+8a, the expression of which has been reported to be restricted to neural tissue, was detected and was involved in the expression of neuroendocrine marker genes in SCLC cell lines. Cells with high expression of LSD1+8a were resistant to CDDP and LSD1 inhibitor. Moreover, suppression of LSD1+8a inhibited cell proliferation, indicating that LSD1+8a could play a critical role in SCLC. These findings suggest that LSD1+8a should be considered a novel therapeutic target in SCLC., Highlights • LSD1+8a, which was reported to be restricted to neural tissue, is expressed in SCLC. • LSD1+8a is involved in the expression of neuroendocrine marker genes. • SCLC cell lines with high expression of LSD1+8a were resistant to chemotherapy.

Highlights • We report an extremely rare case of breast metastasis from RCC. • Including the present case, only 23 cases of breast metastasis from RCC have been described in the literature. • It is important that the differential diagnosis of primary breast cancer and metastatic breast cancer be de determined by histological examination. • Minimally invasive surgery or nonsurgical ablation should be performed in accordance with the prognosis., Introduction The breast is a rare site for metastatic disease. We report a rare case of breast metastasis 9 years after nephrectomy for renal cell carcinoma (RCC) and include a review of the relevant literature. Presentation of case An 82-year-old woman who developed an RCC underwent left nephrectomy in 2005. In October 2014, computed tomography (CT) revealed a mass of approximately 1 cm in the lateral portion of the right breast. Breast ultrasonography (US) revealed a well-circumscribed, hypoechoic mass at the same site. Fine needle aspiration (FNA) was performed, but the sample was inadequate because it did not capture breast duct epithelial cells. In June 2015, follow-up US revealed enlargement of the mass, and core needle biopsy (CNB) was performed to confirm the diagnosis. Histological examination resulted in the diagnosis of breast metastasis from an RCC. The patient underwent surgery for partial mastectomy in November 2015. The patient was asymptomatic and free of detectable disease at 18-month follow-up. Discussion The diagnosis of breast metastasis by imaging examination is difficult, and the results of FNA examination are often inconclusive because of the absence of breast duct epithelial cells. Only 22 cases of breast metastasis from RCC have been described in the literature. In almost all the reported cases, lumpectomy or partial mastectomy was performed. Conclusion It is important that histological diagnosis be determined by CNB and by other methods if the patient has a history of malignancy, and minimally invasive therapy should be performed in accordance with the prognosis.

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung's structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Background: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is the current major modality for the diagnosis of sarcoidosis with hilar and mediastinal lymphadenopathy because of its higher diagnostic yield and safety; however, predictors for the pathological diagnosis of sarcoidosis by EBUS-TBNA remain uncertain. The objective of this study was to determine a novel predictor for the pathological diagnosis of sarcoidosis by EBUS-TBNA. Methods: Patients with pathological and/or clinical diagnosis of sarcoidosis were identified from patients who underwent EBUS-TBNA between February 2010 and December 2017, retrospectively. We extracted data on age, sex, stage of disease, number of punctured lymph nodes, number of punctures per procedure and target lymph node, and size of punctured lymph node. Next, we divided patients into groups of pathological positive and negative by EBUS-TBNA, and multivariate logistic regression analysis was performed following univariate analyses to evaluate the efficacy of these parameters as a predictive factor of the pathological diagnosis of sarcoidosis by EBUS-TBNA. Results: We selected 89 patients involving 115 mediastinal and hilar lymph nodes. The diagnostic yield of sarcoidosis by EBUS-TBNA was 74/89 (83.1%). There were no significant differences in the size of lymph node and number of punctures between the groups, there was a significant difference in age by univariate analyses. In addition, multivariate logistic regression revealed that age was significantly associated with pathological diagnosis of sarcoidosis by EBUS-TBNA [5 years = 1 unit, odds ratio (OR), 0.79; 95% CI, 0.64–0.97; P=0.03]. Conclusions: The diagnostic yield of sarcoidosis by EBUS-TBNA was higher in younger than older patients. Therefore, age may be a novel independent predictor for the pathological diagnosis of sarcoidosis by EBUS-TBNA.

Background Chemotherapy is a standard treatment for patients with advanced non-small cell lung cancer (NSCLC); however, it occasionally causes adverse events. Serious adverse events (SAEs) are defined as any untoward medical occurrence that is related to drug use and results in life-threatening experiences, prolonged or initial hospitalization, or significant or persistent disability. However, as few studies have reported on the risk factors for SAEs, we aimed to identify the factors that could predict SAEs in NSCLC. Patients and methods We retrospectively reviewed the medical records of patients treated with pemetrexed plus cisplatin (PC), paclitaxel plus carboplatin plus bevacizumab (BVCP), docetaxel monotherapy (DTX), or pemetrexed monotherapy (PEM) at Juntendo University Hospital between January 2010 and March 2012. Two investigators reviewed the clinical records and judged SAEs. Multivariate analyses were performed to identify independent risk factors for SAEs among the following factors: gender, age, performance status, line of chemotherapy, preexisting interstitial lung disease (ILD), smoking status, and chemotherapeutic regimen. Results A total of 252 patients received chemotherapy (male/female, 162/90; median age [range], 66 years [36–92 years]; stage III/stage IV/postoperative recurrence, 53/145/54; adenocarcinoma/squamous cell carcinoma/not otherwise specified, 211/24/17; PC/BVCP/PEM/DTX, 50/ 51/ 67/ 84). Of these, 30 (11.9%) patients experienced SAEs. The SAEs were anorexia/nausea in 10 patients, febrile neutropenia (FN) in eight, drug-induced ILD in six, infection (sepsis, pleural infection, soft tissue infection) in three, elevated creatinine level in one, pneumothorax in one, and gastric hemorrhage in one. Treatment-related death was noted in four patients, two with drug-induced ILD, one with FN, and one with infection. Multivariate analysis revealed that preexisting ILD (odds ratio=5.06; p=0.0012) and the chemotherapeutic regimen (p=0.00-0.03) were significantly associated with SAEs. Conclusions Preexisting ILD and the chemotherapeutic regimen were risk factors for the prediction of SAEs in the treatment of NSCLC in clinical practice.

A 64-year-old man with the bone marrow metastasis due to malignant pleural mesothelioma (MPM) was diagnosed with anemia, leukoerythroblastosis, thrombocytopenia, and lower back pain. A bone marrow biopsy demonstrated infiltrative malignant mesothelioma lesions in the bone marrow. The patient died within 15 days of the detection of the bone marrow involvement. Physicians should consider performing a bone marrow biopsy to diagnose bone marrow metastasis and treat patients with palliative chemotherapy at an earlier phase of the disease. To our knowledge, this is the first report of an MPM patient having bone marrow metastasis with anemia, leukoerythroblastosis, and thrombocytopenia.

Insulin-like growth factor 1 receptor (IGF1R) is expressed in many types of solid tumors including non-small cell lung cancer (NSCLC), and enhanced activation of IGF1R is thought to reflect cancer progression. Epithelial-mesenchymal transition (EMT) has been established as one of the mechanisms responsible for cancer progression and metastasis, and microenvironment conditions, such as hypoxia, have been shown to induce EMT. The purposes of this study were to address the role of IGF1R activation in hypoxia-induced EMT in NSCLC and to determine whether inhibition of IGF1R might reverse hypoxia-induced EMT. Human NSCLC cell lines A549 and HCC2935 were exposed to hypoxia to investigate the expression of EMT-related genes and phenotypes. Gene expression analysis was performed by quantitative real-time PCR and cell phenotypes were studied by morphology assessment, scratch wound assay, and immunofluorescence. Hypoxia-exposed cells exhibited a spindle-shaped morphology with increased cell motility reminiscent of EMT, and demonstrated the loss of E-cadherin and increased expression of fibronectin and vimentin. Hypoxia also led to increased expression of IGF1, IGF binding protein-3 (IGFBP3), and IGF1R, but not transforming growth factor β1 (TGFβ1). Inhibition of hypoxia-inducible factor 1α (HIF1α) with YC-1 abrogated activation of IGF1R, and reduced IGF1 and IGFBP3 expression in hypoxic cells. Furthermore, inhibition of IGF1R using AEW541 in hypoxic condition restored E-cadherin expression, and reduced expression of fibronectin and vimentin. Finally, IGF1 stimulation of normoxic cells induced EMT. Our findings indicated that hypoxia induced EMT in NSCLC cells through activation of IGF1R, and that IGF1R inhibition reversed these phenomena. These results suggest a potential role for targeting IGF1R in the prevention of hypoxia-induced cancer progression and metastasis mediated by EMT.

Numerous variants of unknown significance (VUS) have been identified through large-scale cancer genome projects, although their functional relevance remains uninvestigated. We developed a mixed-all-nominated-mutants-in-one (MANO) method to evaluate the transforming potential and drug sensitivity of oncogene VUS in a high-throughput manner and applied this method to 101 nonsynonymous epidermal growth factor receptor (EGFR) mutants. We discovered a number of mutations conferring resistance to EGFR tyrosine kinase inhibitors (TKIs), including gefitinib- and erlotinib-insensitive missense mutations within exon 19 and other gefitinib-resistant mutations, such as L833V, A839T, V851I, A871T, and G873E. L858R -positive tumors (12.8%) harbored compound mutations primarily in the cis allele, which decreased the gefitinib sensitivity of these tumors. The MANO method further revealed that some EGFR mutants that are highly resistant to all types of TKIs are sensitive to cetuximab. Thus, these data support the importance of examining the clinical relevance of uncommon mutations within EGFR and of evaluating the functions of such mutations in combination. This method may become a foundation for the in vitro and in vivo assessment of variants of cancer-related genes and help customize cancer therapy for individual patients.

Numerous variants of unknown significance (VUS) have been identified through large-scale cancer genome projects, although their functional relevance remains uninvestigated. We developed a mixed-all-nominated-mutants-in-one (MANO) method to evaluate the transforming potential and drug sensitivity of oncogene VUS in a high-throughput manner and applied this method to 101 nonsynonymous epidermal growth factor receptor (EGFR) mutants. We discovered a number of mutations conferring resistance to EGFR tyrosine kinase inhibitors (TKIs), including gefitinib- and erlotinib-insensitive missense mutations within exon 19 and other gefitinib-resistant mutations, such as L833V, A839T, V851I, A871T, and G873E.

Background Interstitial lung diseases induced by anticancer agents (ILD-AA) are rare adverse effects of anticancer therapy. However, prognostic biomarkers for ILD-AA have not been identified in patients with advanced lung cancer. Our aim was to analyze the association between serum biomarkers sialylated carbohydrate antigen Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D), and clinical characteristics in patients diagnosed with ILD-AA. Methods Between April 2011 and March 2016, 1224 advanced lung cancer patients received cytotoxic agents and epidermal growth factor receptor tyrosine kinase inhibitors at Juntendo University Hospital and Juntendo University Urayasu Hospital. Of these patients, those diagnosed with ILD-AA were enrolled in this case control study. ΔKL-6 and ΔSP-D were defined as the difference between the levels at the onset of ILD-AA and their respective levels prior to development of ILD-AA. We evaluated KL-6 and SP-D at the onset of ILD-AA, ΔKL-6 and ΔSP-D, the risk factors for death related to ILD-AA, the chest high resolution computed tomography (HRCT) findings, and survival time in patients diagnosed with ILD-AA. Results Thirty-six patients diagnosed with ILD-AA were enrolled in this study. Among them, 14 patients died of ILD-AA. ΔSP-D in the patients who died was significantly higher than that in the patients who survived. However, ΔKL-6 did not differ significantly between the two groups. Moreover, ΔSP-D in patients who exhibited diffuse alveolar damage was significantly higher than that in the other patterns on HRCT. Receiver operating characteristic curve analysis was used to set the optimal cut off value for ΔSP-D at 398 ng/mL. Survival time for patients with high ΔSP-D (≥ 398 ng/mL) was significantly shorter than that for patients with low ΔSP-D. Multivariate analysis revealed that ΔSP-D was a significant prognostic factor of ILD-AA. Conclusions This is the first research to evaluate high ΔSP-D (≥ 398 ng/mL) in patients with ILD-AA and to determine the risk factors for ILD-AA in advanced lung cancer patients. ΔSP-D might be a serum prognostic biomarker of ILD-AA. Clinicians should evaluate serum SP-D during chemotherapy and should carefully monitor the clinical course in patients with high ΔSP-D.

The plasma membrane Na(+)/H(+) exchanger 1 (NHE1) is rapidly activated in response to various stimuli. The membrane-proximal cytoplasmic region (∼60 residues), termed the lipid-interacting domain (LID), is an important regulatory domain of NHE1. Here, we used a pharmacological approach to further characterize the role of LID in the regulation of NHE1. Pharmacological analysis using staurosporine-like indolocarbazole and bisindolylmaleimide compounds suggested that the phorbol ester- and receptor agonist-induced activation of NHE1 occurs through a protein kinase C-independent mechanism. In particular, only indolocarbazole compounds that inhibited NHE1 activation were able to interact with the LID, suggesting that the inhibition of NHE1 activation is achieved through the direct action of these compounds on the LID. Furthermore, in addition to phorbol esters and a receptor agonist, okadaic acid and hyperosmotic stress, which are known to activate NHE1 through unknown mechanisms, were found to promote membrane association of the LID concomitant with NHE1 activation; these effects were inhibited by staurosporine, as well as by a mutation in the LID. Binding experiments using the fluorescent ATP analog trinitrophenyl ATP revealed that ATP and the NHE1 activator phosphatidylinositol 4,5-bisphosphate bind competitively to the LID. These findings suggest that modulation of NHE1 activity by various activators and inhibitors occurs through the direct binding of these molecules to the LID, which alters the association of the LID with the plasma membrane.

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1.