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PDF file, 80K, Level of EML4-ALK mRNA in NSCLC cells. Total RNA extracted from the indicated cell lines was subjected to RT and real-time PCR analysis for determination of the abundance of EML4-ALK mRNA. Data were normalized by the amount of GAPDH mRNA and then expressed relative to the corresponding value for H3122 cells, and they are means from three independent experiments.

Cox proportional hazard model for PFS

Effect of using a combination of targeting B7-H3 and PD-L1 on body weight in the murine tumor model

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Purpose: Anti-programmed-death-1 (PD-1) immunotherapy improves survival in non–small cell lung cancer (NSCLC), but some cases are refractory to treatment, thereby requiring alternative strategies. B7-H3, an immune-checkpoint molecule, is expressed in various malignancies. To our knowledge, this study is the first to evaluate B7-H3 expression in NSCLCs treated with anti-PD-1 therapy and the therapeutic potential of a combination of anti-PD-1 therapy and B7-H3 targeting.Experimental Design: B7-H3 expression was evaluated immunohistochemically in patients with NSCLC (n = 82), and its relationship with responsiveness to anti-PD-1 therapy and CD8+ tumor-infiltrating lymphocytes (TILs) was analyzed. The antitumor efficacy of dual anti-B7-H3 and anti-programmed death ligand-1 (PD-L1) antibody therapy was evaluated using a syngeneic murine cancer model. T-cell numbers and functions were analyzed by flow cytometry.Results: B7-H3 expression was evident in 74% of NSCLCs and was correlated critically with nonresponsiveness to anti-PD-1 immunotherapy. A small number of CD8+ TILs was observed as a subpopulation with PD-L1 tumor proportion score less than 50%, whereas CD8+ TILs were still abundant in tumors not expressing B7-H3. Anti-B7-H3 blockade showed antitumor efficacy accompanied with an increased number of CD8+ TILs and recovery of effector function. CD8+ T-cell depletion negated antitumor efficacy induced by B7-H3 blockade, indicating that improved antitumor immunity is mediated by CD8+ T cells. Compared with a single blocking antibody, dual blockade of B7-H3 and PD-L1 enhanced the antitumor reaction.Conclusions: B7-H3 expressed on tumor cells potentially circumvents CD8+-T-cell–mediated immune surveillance. Anti-B7-H3 immunotherapy combined with anti-PD-1/PD-L1 antibody therapy is a promising approach for B7-H3–expressing NSCLCs. Clin Cancer Res; 24(11); 2653–64. ©2018 AACR.

PDF file - 95KB, Supplementary Table S3-A. Human tumor pY proteome. S3-B. Comparison analysis of unique pTyr sites iden_fy from di_erent samples.

PDF file - 104KB, Supplementary Figure 1A-B: PC9GR cells acquired drug resistance to both reversible and irreversible EGFR-TKI. A: PC9 and PC9GR cells were treated for 72 h with increasing concentrations of erlotinib or CL387,785. Data generated by cell viability assay (CellTiter-Glo) are expressed as a percentage of the value for untreated cells. Determinations were done in triplicate. B: PC9 and PC9GR cells were incubated for 1 hour in the absence or presence of erlotinib (1 �M) as indicated. Cell lysates were subjected to protein expression analysis with antibodies to pEGFR, pSrc, pAkt, and pErk along with antibodies to β-actin as a loading control. C-D: Gene status in PC9 and PC9GR cells. C: PC9GR cells acquired T790M while retaining exon 19 deletion as determined by PCR-invader assay. Values of red bars above zero represent positive results with the detection probe for indicated mutation sequences of EGFR, while values of light blue bars are quality controls showing good PCR quality, as previously described (1). The value of red bars = (fluorescence of the samples with detection probe for mutation sequences) - (fluorescence of the normal control with the same probe � 2). The value of light blue bars = (fluorescence of the samples with detection probe for wild type sequences) - (fluorescence of the normal control with the same probe � 0.8). DEL1 represents E746-A750del type1 (2235-2249del GGAATTAAGAGAAGC). DEL2 represents E746-A750del type2 (2236-2250del GAATTAAGAGAAGCA). DEL3 represents L747-P753del insS. D: FISH analysis (CEP7 (green)/D7S522 (red)) was done in PC9 and PC9GR cells as previously described (2). No additional copy of D7S522 compared to CEP 7 was found in these cells, explaining that neither PC9 nor PC9GR cells has MET amplification.

PDF file - 139KB, Effects of ligands exposure with or without EGFR-TKI in PC9 and PC9GR cells. A-F: PC9 (A, C, and E) or PC9GR cells (B, D, and F) were incubated for 1 hour in the absence or presence of 100 nM erlotinib (A, C, and E) or afatinib (B, D, and F) with indicated concentrations of HGF (A and B), IGF1 (C and D), or Gas6 (E and F). Cell lysates were subjected to protein expression analysis with antibodies to pMET (A and B), IGF1R (C and D), or pAXL (E and F) along with pEGFR, pAkt, pErk, and antibodies to β-actin as a loading control.

Purpose: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI) such as crizotinib show marked efficacy in patients with non–small cell lung cancer positive for the echinoderm microtubule-associated protein–like 4 (EML4)–ALK fusion protein. However, acquired resistance to these agents has already been described in treated patients, and the mechanisms of such resistance remain largely unknown.Experimental Design: We established lines of EML4-ALK–positive H3122 lung cancer cells that are resistant to the ALK inhibitor TAE684 (H3122/TR cells) and investigated their resistance mechanism with the use of immunoblot analysis, ELISA, reverse transcription and real-time PCR analysis, and an annexin V binding assay. We isolated EML4-ALK–positive lung cancer cells (K-3) from a patient who developed resistance to crizotinib and investigated their characteristics.Results: The expression of EML4-ALK was reduced at the transcriptional level, whereas phosphorylation of epidermal growth factor receptor (EGFR), HER2, and HER3 was upregulated, in H3122/TR cells compared with those in H3122 cells. This activation of HER family proteins was accompanied by increased secretion of EGF. Treatment with an EGFR-TKI induced apoptosis in H3122/TR cells, but not in H3122 cells. The TAE684-induced inhibition of extracellular signal–regulated kinase (ERK) and STAT3 phosphorylation observed in parental cells was prevented by exposure of these cells to exogenous EGF, resulting in a reduced sensitivity of cell growth to TAE684. K-3 cells also manifested HER family activation accompanied by increased EGF secretion.Conclusions: EGF-mediated activation of HER family signaling is associated with ALK-TKI resistance in lung cancer positive for EML4-ALK. Clin Cancer Res; 18(22); 6219–26. ©2012 AACR.

PDF file - 54KB, A: Example of peak area (EIC) for EGFR pY 1172. B: Example of peak area (EIC) for MET pY 1234.

Purpose: Irreversible EGFR-tyrosine kinase inhibitors (TKI) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gatekeeper mutations in non–small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M.Experimental Design: We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI–resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass spectrometry–based identification/quantification. Profiles of erlotinib perturbations were examined.Results: We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib–treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced antitumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M-mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib.Conclusions: Our results identified both codrivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in patients with NSCLC with acquired T790M. Clin Cancer Res; 20(15); 4059–74. ©2014 AACR.

PDF file - 91KB, A: Effects of dasatinib combined with afatinib on apoptosis in HCC4006-T790M and HCC827-T790M cells. Apoptosis assay was carried out using PE-conjugated caspase-3 antibody, following incubation of HCC4006-T790M or HCC827-T790M cells for 72 hours with DMSO, erlotinib (E), dasatinib (D), afatinib (A), and afatinib + dasatinib. Values are expressed as a percentage of caspase-3-positive cells. Determinations were done in triplicate. Bars, SD. *P < 0.001 versus DMSO or each single agent (Student's t-test). B: Validation of active pTyr sites from PC9 cell lines on lung cancer patient. Venn diagram was used to compare the pTyr sites identified from PC9 and human lung tumor tissue with positive EGFR mutation. Total 83 overlap active pTyr sites were obtained.

PDF file - 387KB, Effects of ligands exposure with or without EGFR-TKI in PC9 and PC9GR cells. A-F: PC9 (A, C, and E) or PC9GR cells (B, D, and F) were incubated for 1 hour in the absence or presence of 100 nM erlotinib (A, C, and E) or afatinib (B, D, and F) with indicated concentrations of HGF (A and B), IGF1 (C and D), or Gas6 (E and F). Cell lysates were subjected to protein expression analysis with antibodies to pMET (A and B), IGF1R (C and D), or pAXL (E and F) along with pEGFR, pAkt, pErk, and antibodies to β-actin as a loading control.

PDF file - 67KB, Supplementary Table S1: Quantitation of pY changes in between PC9 and PC9GR cell lines with and without erlotinib treatment. The cutoff was set as P value1.5; Supplementary Table S2: Cell viability IC50 in PC9GR cells and H1975 cells.

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PDF file - 303K, L704N or I941R cis mutation disrupts constitutive dimerization of mutant EGFR (S1); Tyrosine-phosphorylation is abrogated on L704N or I941R compound mutants of dimerization-dependent mutants (S2); Wild-type EGFR, but not Ex20Ins mutant are dependent on asymmetric dimerization for oncogenic transformation (S3); Tyrosine-phosphorylation on dimerization-independent mutants is significantly impaired by introduction of receiver-impairing or activator-impairing mutation (S4); Cell surface expression levels of various EGFR mutants in NIH-3T3 cells (S5); Activities of receiver monomers and activator monomers of wild-type EGFR (S6); Cell surface expression of mutant EGFR in stable cell lines (S7); Differential pharmacological effects of gefitinib and cetuximab against distinct mutant EGFR (S8); Generation and characterization of bitransgenic mice with inducible expression of EGFR Ex20Ins in lung compartments (S9); Cetuximab downregulates tyrosine phosphorylation on mutant EGFR in mouse tumors (S10).

Tumour burden (TB) is implicated in resistance to programmed cell death-1/PD-L1 inhibitor (immune checkpoint inhibitor [ICI]) therapy. However, whether TB contributes to such resistance in non-small-cell lung cancer (NSCLC) has remained unknown.A total of 260 treatment-naïve patients with advanced NSCLC who started ICI monotherapy (ICI cohort), platinum-doublet therapy (Chemo cohort) or ICI and platinum-doublet therapy (ICI+Chemo cohort) as first-line treatment were consecutively included. TB was estimated on the basis of the sum of the diameters of measurable target lesions as per Response Evaluation Criteria in Solid Tumours. Progression-free survival (PFS) in the ICI cohort was evaluated as per TB as a preplanned primary objective, with the analysis based on propensity score-weighted survival curves and estimation of restricted mean survival time (RMST). The Chemo cohort served as a control to determine whether TB is predictive of ICI treatment outcomes. The ICI+Chemo cohort was exploratory. The relation of TB to tumour immune status was assessed by immune-related gene expression profiling (irGEP) of pretreatment tumour tissue.In the ICI cohort, patients with a low TB showed a significantly longer PFS than did those with a high TB (median, 17.9 vs 4.3 months; weighted hazard ratio, 0.32 [95% confidence interval, 0.19-0.53]). No such difference was apparent in the other cohorts. A significant difference in overall survival was also observed only in the ICI cohort. RMST-based analysis confirmed these results. The irGEP analysis implicated M2-type macrophages, angiogenesis and transforming growth factor-β as well as protumourigenic signalling pathways in ICI resistance conferred by a high TB.A high TB was associated with a poor outcome of ICI therapy for advanced NSCLC as a result of immunosuppressive phenotypes. Development of combination or novel treatment strategies for such disease is thus warranted.

Combining cryopreservation of germline stem cells (GSCs) with their subsequent transplantation into recipient fish is a powerful tool for long-term preservation of genetic resources of endangered fishes. However, application of this technique has been limited because endangered species sometimes have small gonads and do not supply enough GSCs to be used for transplantation. This limitation could be overcome by expanding GSCs in vitro, though this has been difficult due to the complexity of reconstructing the gonadal microenvironment that surrounds GSCs. Here, we describe a novel method of in vitro expansion of rainbow trout GSCs using a feeder layer derived from Sertoli cells and a culture medium containing trout plasma. A transplantation assay demonstrated that the in vitro-expanded GSCs exhibited stem cell activity and potency to produce functional eggs, sperm, and eventually healthy offspring. In vitro expansion of GSCs can aid in rescuing fishes that are on the verge of extinction., Yoshiko Iwasaki-Takahashi et al. report the production of fertilization-capable eggs and sperm from rainbow trout spermatagonia, for application to endangered species preservation. Their method avoids the need for limited numbers of germline stem cells for transplantation, instead allowing in vitro expansion of these cells using a Sertoli cell-derived feeder layer.

Purpose: Anti-programmed-death-1 (PD-1) immunotherapy improves survival in non–small cell lung cancer (NSCLC), but some cases are refractory to treatment, thereby requiring alternative strategies. B7-H3, an immune-checkpoint molecule, is expressed in various malignancies. To our knowledge, this study is the first to evaluate B7-H3 expression in NSCLCs treated with anti-PD-1 therapy and the therapeutic potential of a combination of anti-PD-1 therapy and B7-H3 targeting. Experimental Design: B7-H3 expression was evaluated immunohistochemically in patients with NSCLC (n = 82), and its relationship with responsiveness to anti-PD-1 therapy and CD8+ tumor-infiltrating lymphocytes (TILs) was analyzed. The antitumor efficacy of dual anti-B7-H3 and anti-programmed death ligand-1 (PD-L1) antibody therapy was evaluated using a syngeneic murine cancer model. T-cell numbers and functions were analyzed by flow cytometry. Results: B7-H3 expression was evident in 74% of NSCLCs and was correlated critically with nonresponsiveness to anti-PD-1 immunotherapy. A small number of CD8+ TILs was observed as a subpopulation with PD-L1 tumor proportion score less than 50%, whereas CD8+ TILs were still abundant in tumors not expressing B7-H3. Anti-B7-H3 blockade showed antitumor efficacy accompanied with an increased number of CD8+ TILs and recovery of effector function. CD8+ T-cell depletion negated antitumor efficacy induced by B7-H3 blockade, indicating that improved antitumor immunity is mediated by CD8+ T cells. Compared with a single blocking antibody, dual blockade of B7-H3 and PD-L1 enhanced the antitumor reaction. Conclusions: B7-H3 expressed on tumor cells potentially circumvents CD8+-T-cell–mediated immune surveillance. Anti-B7-H3 immunotherapy combined with anti-PD-1/PD-L1 antibody therapy is a promising approach for B7-H3–expressing NSCLCs. Clin Cancer Res; 24(11); 2653–64. ©2018 AACR.

Several types of coumarins have been isolated from Peucedanum japonicum Thunb (PJT) and we previously reported the anti-obesity activity of pteryxin (PTX), as an active compound of PJT. However, during our research, we identified another variety of PJT, which does not contain any PTX. Here, we prepared concentrates from the two varieties of PJT and demonstrated that dietary PJT concentrates both with and without PTX significantly decreased the relative weights of white adipose tissue (WAT) and mRNA levels of lipogenic genes in epididymal WAT. Furthermore, we compared the inhibitory effects of dihydropyranocoumarins of PJT on lipid accumulation of differentiated 3T3-L1 adipocytes. All of the dihydropyranocoumarins used in this study significantly inhibited lipid accumulation and lipogenic-related gene expressions in 3T3-L1 adipocytes. However, the magnitudes of the effects were distinctly different. The present study demonstrated that several dihydropyranocoumarins in PJT possess anti-obesity activities, which mainly act by inhibiting lipogenesis in adipocytes.

The aims of this dose-escalating phase I study were to determine the maximum tolerable dose (MTD) and recommended dose (RD) of 5-fluorouracil (5-FU), docetaxel, and nedaplatin (UDON) combination therapy for future phase II studies, and to evaluate the safety and efficacy of this regimen in patients with untreated recurrent or metastatic esophageal cancer. Patients were administered 5-FU on days 1–5, docetaxel on days 1 and 15, and nedaplatin on day 1 at 4-week intervals. The dose levels of 5-FU/docetaxel/nedaplatin were escalated as follows (mg/m2): level 1, 800/30/80; level 2, 800/30/90; and level 3, 800/35/90. Toxicity was evaluated using Common Terminology Criteria for Adverse Events version 4.0. Overall, nine patients were enrolled in this study. All patients had an Eastern Cooperative Oncology Group performance status of 0 or 1 and were diagnosed with squamous cell carcinoma. No dose-limiting toxicity was observed at any level, and planned dose escalation was completed without reaching the MTD. No grade 4 or higher toxicity was observed in this study. The observed grade 3 hematological toxicities included neutropenia in five patients (55.6 %) and leukopenia in three patients (33.3 %). None of the patients developed febrile neutropenia, and no grade 3 or 4 non-hematological toxicities were observed. The overall response rate was 77.8 %, including two complete responses, and the disease control rate was 100 %. The RD of UDON was identified as level 3. The good tolerability and high antitumor efficacy of this regimen warrant further evaluation in this setting.

Porous diaphragm syndrome describes a defect in the diaphragm in which substances pass from the peritoneal cavity to the pleural space. Defects may be congenital or acquired. Acquired defects are caused by the thinning and eventual splitting of collagen fibres in the tendinous part of the diaphragm. We report a case of porous diaphragm syndrome with recurrent thymoma that presented with massive ascites. Increasing intra-abdominal pressure by ascites and diaphragmatic thinning due to malnutrition by malignancies resulted in the formation of an artificial hole. Thoracentesis changed the balance of hydrostatic pressure, which initiated the influx of a large volume of ascites to the pleural cavity through a hole in the diaphragm.