Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

INTRODUCTION: Unresectable or recurrent thymic epithelial tumors (TETs) have a poor prognosis, and treatment options are limited. This study aimed to investigate the immunologic significance of CD80/CD86 or major histocompatibility complex class II (MHC-II) expression in TETs, as potential predictive biomarkers for immune checkpoint inhibitors (ICIs). METHODS: We analyzed CD80, CD86, MHC class I (MHC-I), and MHC-II expression in TETs using immunohistochemistry and investigated their association with T-cell infiltration or ICI efficacy. In addition, we generated CD80- or MHC-II-expressing mouse tumors, evaluated the effects of ICIs, and analyzed tumor-infiltrating lymphocytes. We also performed tumor-rechallenge experiments in vivo. RESULTS: We found that approximately 50% and 30% of TETs had high expression of CD80/CD86 and MHC-II in tumor cells, respectively, and that this expression was related to T-cell infiltration in clinical samples. In mouse models, both CD80 and MHC-II increase the effects of ICIs. In addition, senescent T cells and long-lived memory precursor effector T cells were significantly decreased and increased, respectively, in tumor-infiltrating lymphocytes from CD80-expressing tumors, and rechallenged tumors were completely rejected after the initial eradication of CD80-expressing tumors by programmed cell death protein 1 blockade. Indeed, patients with CD80-high thymic carcinoma had longer progression-free survival with anti-programmed cell death protein 1 monoclonal antibody. CONCLUSIONS: Half of the TETs had high expression of CD80/CD86 or MHC-II with high T-cell infiltration. These molecules could potentially increase the effects of ICIs, particularly inducing a durable response. CD80/CD86 and MHC-II can be predictive biomarkers of ICIs in TETs, promoting the development of drugs for such TETs.

DOI： 10.1016/j.jtocrr.2023.100573

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Language：English   Publisher：(一社)日本癌学会  

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Intracranial metastases are common in nonsmall-cell lung cancer (NSCLC) patients, whose prognosis is very poor. In addition, intracranial progression is common during systemic treatments due to the inability to penetrate central nervous system (CNS) barriers, whereas the intracranial effects of cancer immunotherapies remain unclear. We analyzed clinical data to evaluate the frequency of intracranial progression in advanced NSCLC patients treated with PD-1 blockade therapies compared with those treated without PD-1 blockade therapies, and found that the frequency of intracranial progression in advanced NSCLC patients treated with PD-1 blockade therapies was significantly lower than that in patients treated with cytotoxic chemotherapies. In murine models, intracranial rechallenged tumors after initial rejection by PD-1 blockade were suppressed. Accordingly, long-lived memory precursor effector T cells and antigen-specific T cells were increased by PD-1 blockade in intracranial lesions. However, intracranial rechallenged different tumors are not suppressed. Our results indicate that cancer immunotherapies can prevent intracranial progression, maintaining long-term effects intracranially as well as systemically. If intracranial recurrence occurs during the treatment with PD-1 blockade therapies, aggressive local therapies could be worthwhile.

DOI： 10.1002/ijc.34700

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Hydrogen peroxide (H2 O2 ) induces oxidative stress and cytotoxicity, and can be used for treating cancers in combination with radiotherapy. A product comprising H2 O2 and sodium hyaluronate has been developed as a radiosensitizer. However, the effects of H2 O2 on antitumor immunity remain unclear. To investigate the effects of H2 O2 , especially the abscopal effect when combined with radiotherapy (RT), we implanted murine tumor cells simultaneously in two locations in mouse models: the hind limb and back. H2 O2 mixed with sodium hyaluronate was injected intratumorally, followed by irradiation only at the hind limb lesion. No treatment was administered to the back lesion. The H2 O2 /RT combination significantly reduced tumor growth at the noninjected/nonirradiated site in the back lesion, whereas H2 O2 or RT individually did not reduce tumor growth. Flow cytometric analyses of the tumor-draining lymph nodes in the injected/irradiated areas showed that the number of dendritic cells increased significantly with maturation in the H2 O2 /RT combination group. In addition, analyses of tumor-infiltrating lymphocytes showed that the number of CD8+ (cluster of differentiation 8) T cells and the frequency of IFN-γ+ (interferon gamma) CD8+ T cells were higher in the noninjected/nonirradiated tumors in the H2 O2 /RT group compared to those in the other groups. PD-1 (programmed death receptor 1) blockade further increased the antitumor effect against noninjected/nonirradiated tumors in the H2 O2 /RT group. Intratumoral injection of H2 O2 combined with RT therefore induces an abscopal effect by activating antitumor immunity, which can be further enhanced by PD-1 blockade. These findings promote the development of H2 O2 /RT therapy combined with cancer immunotherapies, even for advanced cancers.

DOI： 10.1111/cas.15911

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IFNγ signaling pathway defects are well-known mechanisms of resistance to immune checkpoint inhibitors. However, conflicting data have been reported, and the detailed mechanisms remain unclear. In this study, we have demonstrated that resistance to immune checkpoint inhibitors owing to IFNγ signaling pathway defects may be primarily caused by reduced MHC-I expression rather than by the loss of inhibitory effects on cellular proliferation or decreased chemokine production. In particular, we found that chemokines that recruit effector T cells were mainly produced by immune cells rather than cancer cells in the tumor microenvironment of a mouse model, with defects in IFNγ signaling pathways. Furthermore, we found a response to immune checkpoint inhibitors in a patient with JAK-negative head and neck squamous cell carcinoma whose HLA-I expression level was maintained. In addition, CRISPR screening to identify molecules associated with elevated MHC-I expression independent of IFNγ signaling pathways demonstrated that guanine nucleotide-binding protein subunit gamma 4 (GNG4) maintained MHC-I expression via the NF-κB signaling pathway. Our results indicate that patients with IFNγ signaling pathway defects are not always resistant to immune checkpoint inhibitors and highlight the importance of MHC-I expression among the pathways and the possibility of NF-κB-targeted therapies to overcome such resistance.

DOI： 10.1158/2326-6066.CIR-22-0815

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Combination therapy with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and anti-programmed death-1 (PD-1) monoclonal antibodies (mAbs) has dramatically improved the prognosis of patients with multiple types of cancer, including renal cell carcinoma (RCC). However, more than half of RCC patients fail to respond to this therapy. Regulatory T cells (Treg cells) are a subset of highly immunosuppressive CD4+ T cells that promote the immune escape of tumors by suppressing effector T cells in the tumor microenvironment (TME) through various mechanisms. CTLA-4 is constitutively expressed in Treg cells and is regarded as a key molecule for Treg cell-mediated immunosuppressive functions, suppressing antigen-presenting cells by binding to CD80/CD86. Reducing Treg cells in the TME with an anti-CTLA-4 mAb with antibody-dependent cellular cytotoxicity (ADCC) activity is considered an essential mechanism to achieve tumor regression. In contrast, we demonstrated that only CTLA-4 blockade without ADCC activity enhanced CD28 costimulatory signaling pathways in Treg cells and promoted Treg-cell proliferation in mouse models. CTLA-4 blockade also augmented CTLA-4-independent immunosuppressive functions, including cytokine production, leading to insufficient antitumor effects. Similar results were also observed in human peripheral blood lymphocytes and tumor-infiltrating lymphocytes from patients with RCC. Our findings highlight the importance of Treg-cell depletion to achieve tumor regression in response to CTLA-4 blockade therapies.

DOI： 10.1111/cas.15756

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BACKGROUND: Identifying biomarkers to predict immune checkpoint inhibitor (ICI) efficacy is warranted. Considering that somatic mutation-derived neoantigens induce strong immune responses, patients with a high tumour mutational burden reportedly tend to respond to ICIs. However, there are several conflicting data. Therefore, we focused on the original function of neoantigenic mutations and their impact on the tumour microenvironment (TME). METHODS: We evaluated 88 high-frequency microsatellite instability (MSI-H) colorectal cancers and analysed the function of the identified neoantigenic mutations and their influence on programmed cell death 1 (PD-1) blockade efficacy. The results were validated using The Cancer Genome Atlas (TCGA) datasets. RESULTS: We identified frameshift mutations in RNF43 as a common neoantigenic gene mutation in MSI-H tumours. However, loss-of-function RNF43 mutations induced noninflamed TME by activating the WNT/β-catenin signalling pathway. In addition, loss of RNF43 function induced resistance to PD-1 blockade even in neoantigen-rich tumours. TCGA dataset analyses demonstrated that passenger rather than driver gene mutations were related to the inflamed TME in diverse cancer types. CONCLUSIONS: We propose a novel concept of "paradoxical neoantigenic mutations" that can induce noninflamed TME through their original gene functions, despite deriving neoantigens, suggesting the significance of qualities as well as quantities in neoantigenic mutations.

DOI： 10.1038/s41416-023-02165-6

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DOI： 10.1038/s41375-022-01772-1

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Immune checkpoint inhibitors (ICIs) have contributed significantly to the treatment of various types of cancer, including skin cancer. However, not all patients respond; some patients do not respond at all (primary resistance), while others experience recurrence after the initial response (acquired resistance). Therefore, overcoming ICI resistance is an urgent priority. Numerous ICI resistance mechanisms have been reported. They are seemingly quite complex, varying from patient to patient. However, most involve T-cell activation processes, especially in the tumor microenvironment (TME). ICIs exert their effects in the TME by reactivating suppressed T cells through inhibition of immune checkpoint molecules, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1). Thus, this review focuses on the resistance mechanisms based on the T-cell activation process. Here, we classify the main mechanisms of ICI resistance into three categories based on (1) antigen recognition, (2) T-cell migration and infiltration, and (3) effector functions of T cells. By identifying and understanding these resistance mechanisms individually, including unknown mechanisms, we seek to contribute to the development of novel treatments to overcome ICI resistance.

DOI： 10.1111/exd.14716

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In T cell biology, 'exhaustion' was initially described as a hyporesponsive state in CD8 + T cells during chronic infections. Recently, exhaustion has been recognized as a T-cell dysfunctional state in the tumor microenvironment (TME). The term 'exhaustion' is used mainly to refer to effector T cells with a reduced capacity to secrete cytokines and an increased expression of inhibitory receptors. The upregulation of exhaustion-related inhibitory receptors, including programmed cell death protein 1 (PD-1), in such T cells has been associated with the development of tumors, prompting the development of immune checkpoint inhibitors. In addition to CD8 + T cells, CD4 + T cells, including the regulatory T (Treg) cell subset, perform a wide variety of functions within the adaptive immune system. Upregulation of the same inhibitory receptors that are associated with CD8 + T-cell exhaustion has also been identified in CD4 + T cells in chronic infections and cancers, suggesting a similar CD4 + T-cell exhaustion phenotype. For instance, high expression of PD-1 has been observed in Treg cells in the TME, and such Treg cells can play an important role in the resistance to PD-1 blockade therapies. Furthermore, recent progress in single-cell RNA sequencing has shown that CD4 + T cells with cytotoxic activity are also vulnerable to exhaustion. In this review, we will discuss novel insights into various exhausted T-cell subsets, which could reveal novel therapeutic targets and strategies to induce a robust antitumor immune response.

DOI： 10.1093/intimm/dxac013

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Language：Japanese   Publisher：(公社)日本皮膚科学会  

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Immune checkpoint inhibitors (ICIs) are effective for various types of cancer, and their application has led to paradigm shifts in cancer treatment. While many patients can obtain clinical benefits from ICI treatment, a large number of patients are primarily resistant to such treatment or acquire resistance after an initial response. Thus, elucidating the resistance mechanisms is warranted to improve the clinical outcomes of ICI treatment. ICIs exert their antitumor effects by activating T cells in the tumor microenvironment. There are various resistance mechanisms, such as insufficient antigen recognition by T cells, impaired T-cell migration and/or infiltration, and reduced T-cell cytotoxicity, most of which are related to the T-cell activation process. Thus, we classify them into three main mechanisms: resistance mechanisms related to antigen recognition, T-cell migration and/or infiltration, and effector functions of T cells. In this review, we summarize these mechanisms of resistance to ICIs related to the T-cell activation process and progress in the development of novel therapies that can overcome resistance.

DOI： 10.1111/cas.15497

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Language：English   Publisher：(一社)日本癌学会  

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Language：English   Publisher：(一社)日本癌学会  

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Language：English   Publisher：(一社)日本癌学会  

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：American Association for Cancer Research (AACR)  

Abstract

Some patients experience mixed response to immunotherapy, whose biological mechanisms and clinical impact have been obscure. We obtained two tumor samples from lymph node (LN) metastatic lesions in a same patient. Whole exome sequencing for the both tumors and single-cell sequencing for the both tumor-infiltrating lymphocytes (TILs) demonstrated a significant difference in tumor clonality and TILs’ characteristics, especially exhausted T cell clonotypes, although a close relationship between the tumor cell and T cell clones were observed as a response of an overlapped exhausted T cell clone to an overlapped neoantigen. To mimic the clinical setting, we generated a mouse model of several clones from a same tumor cell line. Similarly, differential tumor clones harbored distinct TILs, and one responded to PD-1 blockade but the other did not in this model. We further conducted cohort study (n = 503) treated with PD-1 blockade monotherapies to investigate the outcome of mixed response. Patients with mixed responses to PD-1 blockade had a poor prognosis in our cohort. Particularly, there were significant differences in both tumor and T cell clones between the primary and LN lesions in a patient who experienced tumor response to anti-PD-1 mAb followed by disease progression in only LN metastasis. Our results underscore that intertumoral heterogeneity alters characteristics of TILs even in the same patient, leading to mixed response to immunotherapy and significant difference in the outcome.

DOI： 10.1158/2767-9764.crc-22-0050

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PD-1 blockade exerts clinical efficacy against various types of cancer by reinvigorating T cells that directly attack tumor cells (tumor-specific T cells) in the tumor microenvironment (TME), and tumor-infiltrating lymphocytes (TILs) also comprise nonspecific bystander T cells. Here, using single-cell sequencing, we show that TILs include skewed T cell clonotypes, which are characterized by exhaustion (Tex) or nonexhaustion signatures (Tnon-ex). Among skewed clonotypes, those in the Tex, but not those in the Tnon-ex, cluster respond to autologous tumor cell lines. After PD-1 blockade, non-preexisting tumor-specific clonotypes in the Tex cluster appear in the TME. Tumor-draining lymph nodes (TDLNs) without metastasis harbor a considerable number of such clonotypes, whereas these clonotypes are rarely detected in peripheral blood. We propose that tumor-infiltrating skewed T cell clonotypes with an exhausted phenotype directly attack tumor cells and that PD-1 blockade can promote infiltration of such Tex clonotypes, mainly from TDLNs.

DOI： 10.1016/j.celrep.2022.110331

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The balance of programmed death-1 (PD-1)-expressing CD8+ T cells and regulatory T (Treg) cells in the tumor microenvironment (TME) determines the clinical efficacy of PD-1 blockade therapy through the competition of their reactivation. However, factors that determine this balance remain unknown. Here, we show that Treg cells gain higher PD-1 expression than effector T cells in highly glycolytic tumors, including MYC-amplified tumors and liver tumors. Under low-glucose environments via glucose consumption by tumor cells, Treg cells actively absorbed lactic acid (LA) through monocarboxylate transporter 1 (MCT1), promoting NFAT1 translocation into the nucleus, thereby enhancing the expression of PD-1, whereas PD-1 expression by effector T cells was dampened. PD-1 blockade invigorated the PD-1-expressing Treg cells, resulting in treatment failure. We propose that LA in the highly glycolytic TME is an active checkpoint for the function of Treg cells in the TME via upregulation of PD-1 expression.

DOI： 10.1016/j.ccell.2022.01.001

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BACKGROUND: Preoperative chemoradiotherapy (CRT) and surgical resection are standard treatment for locally advanced rectal cancer (LARC). Combining immune-checkpoint inhibitors with radiation suggests a promising approach for enhancing efficacy. We investigated the efficacy of CRT followed by nivolumab and surgery in patients with LARC. METHODS: In phase I, we investigated the feasibility of sequentially-combined CRT, 5 cycles of nivolumab, and radical surgery. In phase II, patients with microsatellite stable (MSS) and microsatellite instability-high (MSI-H) LARC were evaluated. RESULTS: Three patients in phase I received full courses of CRT and nivolumab without dose modification; the schedule was recommended for phase II. A pathological complete response (pCR) was centrally confirmed in 30% (11/37; 90% CI, 18 %-44%) and 60% (3/5) of the MSS and exploratory MSI-H cohorts, respectively. While immune-related severe adverse events were observed in 3 patients, no treatment-related deaths were observed. In 38 patients with MSS who underwent surgery, pCR rates of 75% (6/8) and 17% (5/30) (p=0.004, Fisher's exact test) were observed in those with PD-L1 tumor proportion score {greater than or equal to}1% and <1%, respectively; immunohistochemical staining was performed using pre-CRT samples. In 24 patients with MSS, pre-CRT samples were analyzed by flow cytometry; pCR rates of 78% (7/9) and 13% (2/15) (p=0.003, Fisher's exact test) were observed for CD8+-T cell / effector regulatory-T cell (CD8/eTreg) ratios of {greater than or equal to} 2.5 and < 2.5, respectively, in tumor-infiltrating lymphocytes. CONCLUSIONS: CRT followed by consolidation nivolumab could increase pCR. PD-L1 expression and an elevated CD8/eTreg ratio were positive predictors in patients with MSS LARC.

DOI： 10.1158/1078-0432.CCR-21-3213

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DOI： 10.1016/j.jtho.2021.10.019

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Although transcriptome alteration is an essential driver of carcinogenesis, the effects of chromosomal structural alterations on the cancer transcriptome are not yet fully understood. Short-read transcript sequencing has prevented researchers from directly exploring full-length transcripts, forcing them to focus on individual splice sites. Here, we develop a pipeline for Multi-Sample long-read Transcriptome Assembly (MuSTA), which enables construction of a transcriptome from long-read sequence data. Using the constructed transcriptome as a reference, we analyze RNA extracted from 22 clinical breast cancer specimens. We identify a comprehensive set of subtype-specific and differentially used isoforms, which extended our knowledge of isoform regulation to unannotated isoforms including a short form TNS3. We also find that the exon-intron structure of fusion transcripts depends on their genomic context, and we identify double-hop fusion transcripts that are transcribed from complex structural rearrangements. For example, a double-hop fusion results in aberrant expression of an endogenous retroviral gene, ERVFRD-1, which is normally expressed exclusively in placenta and is thought to protect fetus from maternal rejection; expression is elevated in several TCGA samples with ERVFRD-1 fusions. Our analyses provide direct evidence that full-length transcript sequencing of clinical samples can add to our understanding of cancer biology and genomics in general.

DOI： 10.1038/s42003-021-02833-4

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[Figure: see text].

DOI： 10.1126/sciimmunol.abc6424

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BACKGROUND: Patients with cancer benefit from treatment with immune checkpoint inhibitors (ICIs), and those with an inflamed tumor microenvironment (TME) and/or high tumor mutation burden (TMB), particularly, tend to respond to ICIs; however, some patients fail, whereas others acquire resistance after initial response despite the inflamed TME and/or high TMB. We assessed the detailed biological mechanisms of resistance to ICIs such as programmed death 1 and/or cytotoxic T-lymphocyte-associated protein 4 blockade therapies using clinical samples. METHODS: We established four pairs of autologous tumor cell lines and tumor-infiltrating lymphocytes (TILs) from patients with melanoma treated with ICIs. These tumor cell lines and TILs were subjected to comprehensive analyses and in vitro functional assays. We assessed tumor volume and TILs in vivo mouse models to validate identified mechanism. Furthermore, we analyzed additional clinical samples from another large melanoma cohort. RESULTS: Two patients were super-responders, and the others acquired resistance: the first patient had a non-inflamed TME and acquired resistance due to the loss of the beta-2 microglobulin gene, and the other acquired resistance despite having inflamed TME and extremely high TMB which are reportedly predictive biomarkers. Tumor cell line and paired TIL analyses showed high CD155, TIGIT ligand, and TIGIT expression in the tumor cell line and tumor-infiltrating T cells, respectively. TIGIT blockade or CD155-deletion activated T cells in a functional assay using an autologous cell line and paired TILs from this patient. CD155 expression increased in surviving tumor cells after coculturing with TILs from a responder, which suppressed TIGIT+ T-cell activation. Consistently, TIGIT blockade or CD155-deletion could aid in overcoming resistance to ICIs in vivo mouse models. In clinical samples, CD155 was related to resistance to ICIs in patients with melanoma with an inflamed TME, including both primary and acquired resistance. CONCLUSIONS: The TIGIT/CD155 axis mediates resistance to ICIs in patients with melanoma with an inflamed TME, promoting the development of TIGIT blockade therapies in such patients with cancer.

DOI： 10.1136/jitc-2021-003134

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BACKGROUND AND AIMS: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated. METHODS: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers (MSI-H CRCs) using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data. RESULTS: We detected 101 truncating mutations in 57 (50%) tumors and loss of 61 alleles in 21 (18%) tumors. Based on the integrated analysis that enabled the immunological subclassification of MSI-H CRCs, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes. CONCLUSIONS: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy.

DOI： 10.1053/j.gastro.2021.10.010

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Premalignant clonal expansion of human T-cell leukemia virus type-1 (HTLV-1)-infected cells occurs before viral carcinogenesis. Here we characterize premalignant cells and the multicellular ecosystem in HTLV-1 infection with and without adult T-cell leukemia/lymphoma (ATL) by genome sequencing and single-cell simultaneous transcriptome and T/B-cell receptor sequencing with surface protein analysis. We distinguish malignant phenotypes caused by HTLV-1 infection and leukemogenesis and dissect clonal evolution of malignant cells with different clinical behavior. Within HTLV-1-infected cells, a regulatory T-cell phenotype associates with premalignant clonal expansion. We also delineate differences between virus- and tumor-related changes in the nonmalignant hematopoietic pool, including tumor-specific myeloid propagation. In a newly generated conditional knockout mouse model recapitulating T-cell-restricted CD274 (encoding PD-L1) gene lesions found in ATL, we demonstrate that PD-L1 overexpressed by T cells is transferred to surrounding cells, leading to their PD-L1 upregulation. Our findings provide insights into clonal evolution and immune landscape of multistep virus carcinogenesis. Significance: Our multimodal single-cell analyses comprehensively dissect the cellular and molecular alterations of the peripheral blood in HTLV-1 infection, with and without progression to leukemia. This study not only sheds light on premalignant clonal expansion in viral carcinogenesis, but also helps to devise novel diagnostic and therapeutic strategies for HTLV-1-related disorders.

DOI： 10.1158/2643-3230.BCD-21-0044

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Patients with colorectal cancers (CRCs) generally exhibit improved survival through intensive lymph node (LN) dissection. However, recent progress in cancer immunotherapy revisits the potential importance of regional LNs, where T cells are primed to attack tumor cells. To elucidate the role of regional LN, we investigated the immunological status of non-metastatic regional LN lymphocytes (LNLs) in comparison with those in the tumor microenvironment (tumor-infiltrating lymphocytes; TILs) using flow cytometry and next-generation sequencing. LNLs comprised an intermediate level of the effector T cell population between peripheral blood lymphocytes (PBLs) and TILs. Significant overlap of the T-cell receptor (TCR) repertoire was observed in microsatellite instability-high (MSI-H)/mismatch repair deficient (dMMR) CRCs with high tumor mutation burden (TMB), although limited TCRs were shared between non-metastatic LNs and primary tumors in microsatellite stable (MSS)/MMR proficient (pMMR) CRC patients with low TMB. In line with the overlap of the TCR repertoire, an excessive LN dissection did not provide a positive impact on long-term prognosis in our MSI-H/dMMR CRC cohort (n =130). We propose that regional LNs play an important role in antitumor immunity, particularly in MSI-H/dMMR CRCs with high TMB, requiring to be careful of excessive non-metastatic LN dissection in MSI-H/dMMR CRC patients.

DOI： 10.1172/jci.insight.137365

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Aging leads to numerous changes that affect many components of the immune system, called "immunosenescence". Indeed, elderly individuals exhibit dysregulated immune responses against pathogens, poor responses to vaccination, and increased susceptibility to many diseases including cancer, autoimmune disorders, and other chronic inflammatory diseases. Despite progressed understanding of immunosenescence, its detailed mechanisms are still not fully understood. With advances in medicine, the population of older cancer patients is expected to rapidly increase in the coming years. Cancer immunotherapies, including immune checkpoint inhibitors (ICIs), have been shown to be effective for multiple cancer types, whereas to date, few specific data for elderly individuals have been published. Some systemic reviews have demonstrated that ICIs exhibit similar efficacy in older cancer patients, but they seem to be less effective in very old patients. In addition, toxicities might be more frequently observed in such patients. Here, we provide a summary to better understand immunosenescence and an overview of its relationship with cancer and antitumor immunity, including the efficacy and toxicity of ICIs.

DOI： 10.1007/s10147-021-01913-z

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BACKGROUND: Patients with non-small cell lung cancer (NSCLC) harboring activating EGFR mutations are sensitive to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) but inevitably develop resistance to the inhibitors mostly through acquisition of the secondary T790M mutation. Although third-generation EGFR-TKIs overcome this resistance by selectively inhibiting EGFR with EGFR-TKI-sensitizing and T790M mutations, acquired resistance to third-generation EGFR-TKIs invariably develops. METHODS: Next-generation sequencing (NGS) and fluorescence in situ hybridization (FISH) analysis were performed in an EGFR T790M-mutated NSCLC patient who had progressed after a third-generation EGFR-TKI, TAS-121. EGFR-mutated cell lines were subjected to a cell proliferation assay and western blotting analysis with EGFR-TKIs and a heat shock protein 90 (HSP90) inhibitor. RESULTS: NGS and FISH analysis revealed EGFR amplification in the resistant cancer cells. While EGFR L858R/T90M-mutated cell line was sensitive to osimertinib or TAS-121 in vitro, EGFR-overexpressing cell lines displayed resistance to these EGFR-TKIs. Western blot analysis showed that EGFR phosphorylation and overexpression of EGFR in cell lines was not suppressed by third-generation EGFR-TKIs. In contrast, an HSP90 inhibitor reduced total and phosphorylated EGFR and inhibited the proliferation of resistant cell lines. CONCLUSIONS: EGFR amplification confers resistance to third-generation EGFR-TKIs which can be overcome by HSP90 inhibition. The results provide a preclinical rationale for the use of HSP90 inhibitors to overcome EGFR amplification-mediated resistance.

DOI： 10.1111/1759-7714.13839

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Single-cell level analysis is powerful tool to assess the heterogeneity of cellular components in tumor microenvironments (TME). In this study, we investigated immune-profiles using the single-cell analyses of endoscopically- or surgically-resected tumors, and peripheral blood mononuclear cells from gastric cancer patients. Furthermore, we technically characterized two distinct platforms of the single-cell analysis; RNA-seq-based analysis (scRNA-seq), and mass cytometry-based analysis (CyTOF), both of which are broadly embraced technologies. Our study revealed that the scRNA-seq analysis could cover a broader range of immune cells of TME in the biopsy-resected small samples of tumors, detecting even small subgroups of B cells or Treg cells in the tumors, although CyTOF could distinguish the specific populations in more depth. These findings demonstrate that scRNA-seq analysis is a highly-feasible platform for elucidating the complexity of TME in small biopsy tumors, which would provide a novel strategies to overcome a therapeutic difficulties against cancer heterogeneity in TME.

DOI： 10.1038/s41598-020-79385-w

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Two cases of pediatric lung cancer (in 23-month-old and 6-year-old boys) resulting from mother-to-infant transmission of uterine cervical tumors were incidentally detected during routine next-generation sequencing of paired samples of tumor and normal tissue. Spontaneous regression of some lesions in the first child and slow growth of the tumor mass in the second child suggested the existence of alloimmune responses against the transmitted tumors. Immune checkpoint inhibitor therapy with nivolumab led to a strong regression of all remaining tumors in the first child. (Funded by the Japan Agency for Medical Research and Development and others; TOP-GEAR UMIN Clinical Trials Registry number, UMIN000011141.).

DOI： 10.1056/NEJMoa2030391

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DOI： 10.1016/j.jdermsci.2020.09.004

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Immune checkpoint blockade has provided a paradigm shift in cancer therapy, but the success of this approach is very variable; therefore, biomarkers predictive of clinical efficacy are urgently required. Here, we show that the frequency of PD-1+CD8+ T cells relative to that of PD-1+ regulatory T (Treg) cells in the tumor microenvironment can predict the clinical efficacy of programmed cell death protein 1 (PD-1) blockade therapies and is superior to other predictors, including PD ligand 1 (PD-L1) expression or tumor mutational burden. PD-1 expression by CD8+ T cells and Treg cells negatively impacts effector and immunosuppressive functions, respectively. PD-1 blockade induces both recovery of dysfunctional PD-1+CD8+ T cells and enhanced PD-1+ Treg cell-mediated immunosuppression. A profound reactivation of effector PD-1+CD8+ T cells rather than PD-1+ Treg cells by PD-1 blockade is necessary for tumor regression. These findings provide a promising predictive biomarker for PD-1 blockade therapies.

DOI： 10.1038/s41590-020-0769-3

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Importance: Although profiling of gene expression and gene alterations by next-generation sequencing (NGS) to predict the primary tumor site and guide molecularly targeted therapy might be expected to improve clinical outcomes for cancer of unknown primary site (CUP), to our knowledge, no clinical trial has previously evaluated this approach. Objective: To assess the clinical use of site-specific treatment, including molecularly targeted therapy based on NGS results, for patients with CUP. Design, Setting, and Participants: This phase 2 clinical trial was conducted at 19 institutions in Japan and enrolled 111 previously untreated patients with the unfavorable subset of CUP between March 2015 and January 2018, with 97 patients being included in the efficacy analysis. Eligibility criteria included a diagnosis of unfavorable CUP after mandatory examinations, including pathological evaluation by immunohistochemistry, chest-abdomen-pelvis computed tomography scans, and a positron emission tomography scan. Interventions: RNA and DNA sequencing for selected genes was performed simultaneously to evaluate gene expression and gene alterations, respectively. A newly established algorithm was applied to predict tumor origin based on these data. Patients received site-specific therapy, including molecularly targeted therapy, according to the predicted site and detected gene alterations. Main Outcomes And Measures: The primary end point was 1-year survival probability. Secondary end points included progression-free survival (PFS), overall survival (OS), objective response rate, safety, efficacy according to predicted site, and frequency of gene alterations. Results: Of 97 participants, 49 (50.5%) were women and the median (range) age was 64 (21-81) years. The cancer types most commonly predicted were lung (21 [21%]), liver (15 [15%]), kidney (15 [15%]), and colorectal (12 [12%]) cancer. The most frequent gene alterations were in TP53 (45 [46.4%]), KRAS (19 [19.6%]), and CDKN2A (18 [18.6%]). The 1-year survival probability, median OS, and median PFS were 53.1% (95% CI, 42.6%-62.5%), 13.7 months (95% CI, 9.3-19.7 months), and 5.2 months (95% CI, 3.3-7.1 months), respectively. Targetable EGFR mutations in tumor specimens were detected in 5 patients with predicted non-small-cell lung cancer (5.2%), 4 of whom were treated with afatinib; 2 of these patients achieved a durable PFS of longer than 6 months. Conclusions and Relevance: This study's findings suggest that site-specific treatment, including molecularly targeted therapy based on profiling gene expression and gene alterations by NGS, can contribute to treating patients with the unfavorable subset of CUP. Trial Registration: UMIN Identifier: UMIN000016794.

DOI： 10.1001/jamaoncol.2020.4643

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Classic Hodgkin lymphoma (cHL) responds markedly to PD-1 blockade therapy, and the clinical responses are reportedly dependent on expression of major histocompatibility complex class II (MHC-II). This dependence is different from other solid tumors, in which the MHC class I (MHC-I)/CD8+ T-cell axis plays a critical role. In this study, we investigated the role of the MHC-II/CD4+ T-cell axis in the antitumor effect of PD-1 blockade on cHL. In cHL, MHC-I expression was frequently lost, but MHC-II expression was maintained. CD4+ T cells highly infiltrated the tumor microenvironment of MHC-II-expressing cHL, regardless of MHC-I expression status. Consequently, CD4+ T-cell, but not CD8+ T-cell, infiltration was a good prognostic factor in cHL, and PD-1 blockade showed antitumor efficacy against MHC-II-expressing cHL associated with CD4+ T-cell infiltration. Murine lymphoma and solid tumor models revealed the critical role of antitumor effects mediated by CD4+ T cells: an anti-PD-1 monoclonal antibody exerted antitumor effects on MHC-I-MHC-II+ tumors but not on MHC-I-MHC-II- tumors, in a cytotoxic CD4+ T-cell-dependent manner. Furthermore, LAG-3, which reportedly binds to MHC-II, was highly expressed by tumor-infiltrating CD4+ T cells in MHC-II-expressing tumors. Therefore, the combination of LAG-3 blockade with PD-1 blockade showed a far stronger antitumor immunity compared with either treatment alone. We propose that PD-1 blockade therapies have antitumor effects on MHC-II-expressing tumors such as cHL that are mediated by cytotoxic CD4+ T cells and that LAG-3 could be a candidate for combination therapy with PD-1 blockade.

DOI： 10.1182/bloodadvances.2020002098

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Immune checkpoint inhibitors may enhance the efficacy of radiotherapy (RT) in cancer treatment but the effect remains unknown in metastatic gastric cancer (mGC). This study aimed to compare the tumor shrinkage by palliative RT for mGC patients with or without previous exposure to anti-PD-1 therapy. METHODS: Data of 36 mGC patients who had received palliative RT from April 2013 to May 2019 were analyzed. Primary tumor responses were evaluated through a volumetric measurement-based method using computed tomography (CT) and endoscopic responses were evaluated in patients who underwent endoscopy before and after RT. Tumor microenvironment (TME) immune status was investigated by analyzing tumor-infiltrating lymphocytes by flow cytometry. RESULTS: Among 36 patients, 18 had previous exposure to anti-PD-1 before RT showing no significant differences in baseline characteristics with the other 18 patients without exposure to anti-PD-1 treatment. Tumor responses were observed in 28% (5/18) and none (0/18) in the anti-PD-1-exposed vs. naïve group, respectively (P = 0.045). Five out of eight patients in the anti-PD-1-exposed group, who underwent endoscopy after RT showed partial response, but none in the anti-PD-1-naïve patients showed response (P = 0.026). Increase in the CD8+ T cell/effector regulatory T cell ratio in TILs after anti-PD-1 therapy was noted in three responders to RT, but not in the other three non-responders. CONCLUSIONS: Prior exposure to anti-PD-1 therapy increases tumor response to RT. Immune profiling suggests that anti-PD-1 therapy may enhance the efficacy of RT by immunoactivation in the TME.

DOI： 10.1007/s10120-020-01058-4

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PURPOSE: This is a phase 1/2 trial of napabucasin plus pembrolizumab for metastatic colorectal cancer (mCRC). EXPERIMENTAL DESIGN: Phase 1 was conducted to determine the recommended phase 2 dose (RP2D) in a dose escalation design of napabucasin (240mg to 480 mg twice daily) with 200mg pembrolizumab every 3 weeks. Phase 2 included cohort A (n = 10, microsatellite instability high) and cohort B (n = 40, microsatellite stable). The primary endpoint was immune-related objective response rate (irORR). PD-L1 combined positive score (CPS), genomic profiles and the consensus molecular subtypes (CMS) of colorectal cancer were assessed. RESULTS: A total of 55 patients were enrolled in this study. In phase 1, no patients experienced dose-limiting toxicities and napabucasin 480 mg was determined as RP2D. The irORR was 50.0% in cohort A and 10.0% in cohort B. In cohort B, the irORR was 0%, 5.3%, and 42.9% in CPS <1, 1≤ CPS <10 and CPS ≥10, respectively. Patients with objective response tended to have higher tumor mutation burden than those without. Of evaluable 18 patients for CMS classification in cohort B, the irORR was 33.3%, 0%, 33.3% and 33.3% in CMS1, CMS2, CMS3 and CMS4, respectively. The common grade 3 or higher treatment-related adverse events included fever (10.0%) in cohort A and decreased appetite (7.5%) and diarrhea (5.0%) in cohort B. CONCLUSIONS: Napabucasin with pembrolizumab showed anti-tumor activity with acceptable toxicities for MSS mCRC patients as well as MSI-H mCRC, although it did not meet the primary end point.

DOI： 10.1158/1078-0432.CCR-20-1803

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Only a small percentage of patients afflicted with gastric cancer (GC) respond to immune checkpoint blockade (ICB). To study the mechanisms underlying this resistance, we examined the immune landscape of GC. A subset of these tumors was characterized by high frequencies of regulatory T (Treg) cells and low numbers of effector T cells. Genomic analyses revealed that these tumors bore mutations in RHOA that are known to drive tumor progression. RHOA mutations in cancer cells activated the PI3K-AKT-mTOR signaling pathway, increasing production of free fatty acids that are more effectively consumed by Treg cells than effector T cells. RHOA mutant tumors were resistant to PD-1 blockade but responded to combination of PD-1 blockade with inhibitors of the PI3K pathway or therapies targeting Treg cells. We propose that the metabolic advantage conferred by RHOA mutations enables Treg cell accumulation within GC tumors, generating an immunosuppressive TME that underlies resistance to ICB.

DOI： 10.1016/j.immuni.2020.06.016

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OBJECTIVES: Mutational signatures associated with tobacco smoking (mutational smoking signatures: SS) are characterized mainly by C > A mutations. The aim of this study was to characterize the association between the tumor immune microenvironment and the SS in lung adenocarcinoma. METHODS: Lung adenocarcinomas surgically resected from 96 patients, for which whole exome sequencing data was available, were included in the study. We extracted the SS from whole exome sequencing data, calculated the weights of SS using deconstructSigs, and compared the clinicopathological features of SS positive (SS+) and negative (SS-) adenocarcinomas. We selected 18 tumor pairs from SS + and SS- adenocarcinomas (sex, EGFR mutation, and tumor size-matched) and examined the expression of five immune markers (CD20, CD8, FOXP3, CD204, and PD-L1) by immunohistochemistry. RESULTS: Of 96 specimens, there were 33 (34 %) SS + adenocarcinoma tumors. The smoking index significantly correlated with the weight of the SS (R = 0.43). Between SS + and SS- tumors, there was no significant difference in clinicopathological factors excluding smoking history. Immunohistochemistry revealed that the number of FOXP3 + T cells in SS + adenocarcinomas was significantly higher than that in the SS- adenocarcinomas (median number 58 vs. 36, p < 0.01). Also, the number of CD20 + B cells in SS + adenocarcinomas was significantly higher than that in the SS- adenocarcinomas (median number 77 vs. 29, p < 0.01); however; these phenomena could not be confirmed when stratified by smoking history. CONCLUSION: In lung adenocarcinoma, SS is associated with an immunosuppressive tumor microenvironment.

DOI： 10.1016/j.lungcan.2020.06.029

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PURPOSE: This is a phase Ib trial of regorafenib plus nivolumab for gastric and colorectal cancer. PATIENTS AND METHODS: Enrolled patients received regorafenib plus nivolumab in a dose-finding part to estimate the maximum tolerated dose. Additional patients were enrolled in a dose-expansion part. Regorafenib of 80-160 mg was administered once daily for 21 days on/7 days off with nivolumab 3 mg/kg every 2 weeks. The primary end point was dose-limiting toxicity (DLT) during the first 4 weeks to estimate the recommended dose. RESULTS: Fifty patients (25 each with gastric and colorectal cancer) were enrolled. All patients had received ≥ 2 previous lines of chemotherapy, including anti-angiogenetic inhibitors in 96% of patients. Seven patients with gastric cancer had previously been treated with immune checkpoint inhibitors. One patient had microsatellite instability-high colorectal cancer, whereas the remaining patients had microsatellite stable or mismatch repair-proficient tumors. Three DLTs (grade 3 colonic perforation, maculopapular rash, and proteinuria) were observed with regorafenib 160 mg; none were observed with 80 or 120 mg. During the dose-expansion part, regorafenib dose was reduced from 120 to 80 mg because of frequent maculopapular rash. The common grade ≥ 3 treatment-related adverse events were rash (12%), proteinuria (12%), and palmar-plantar erythrodysesthesia (10%). Objective tumor response was observed in 20 patients (40%), including 11 with gastric cancer (44%) and 9 with colorectal cancer (36%). Median progression-free survival was 5.6 and 7.9 months in patients with gastric and colorectal cancer, respectively. CONCLUSION: The combination of regorafenib 80 mg plus nivolumab had a manageable safety profile and encouraging antitumor activity in patients with gastric and colorectal cancer, which warrants additional investigations in larger cohorts.

DOI： 10.1200/JCO.19.03296

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BACKGROUND: The standard treatment for patients with unresectable locally advanced esophageal squamous cell carcinoma (ESCC) is definitive chemoradiotherapy (CRT) using 5-FU plus cisplatin. However, complete response (CR) rates are low at 11-25%, resulting in 9-10 months of median overall survival (OS). An improved therapeutic efficacy by combining immunotherapy with radiation has been reported in patients with locally advanced non-small cell lung cancer. The results using ESCC cell lines suggest sequential treatment with anti-PD-L1 agents soon after completion of CRT is the most effective combination. METHODS: TENERGY trial is a multicenter, phase II, proof-of-concept study to assess the efficacy and safety of atezolizumab following definitive CRT in patients with locally advanced ESCC. The main inclusion criteria are unresectable locally advanced ESCC without distant metastasis, completion of 60 Gy of radiation plus two concomitant cycles of chemotherapy (cisplatin 70 mg/m2 on day 1 and 5-FU 700 mg/m2 on days 1-4, every 28 days), and adequate organ function. Within 6 weeks after CRT, participants will start taking 1200 mg of atezolizumab every three weeks and continue until 12 months or disease progression. The primary endpoint is the confirmed CR rate by the investigator's assessment. Secondary endpoints include overall response rate, progression-free survival (PFS), OS, adverse events, and confirmed CR rate by central assessment. We will enroll 50 patients (40 with primary locally advanced ESCC and 10 with postoperative locoregionally recurrent ESCC). We will obtain biopsies from the primary site and will collect blood at 3 time points (before CRT, after CRT, and four weeks after the start of atezolizumab) for an exploratory biomarker study. We will analyze the phenotype of immune-competent cells, neoantigens, tumor mutational burden, PD-L1 status, and Human Leukocyte Antigen haplotyping. DISCUSSION: The synergistic efficacies of the sequential combination of CRT and atezolizumab should improve the CR rate, resulting in survival improvement for patients with unresectable locally advanced ESCC. Because CRT is a standard treatment option for patients with early stage to locally advanced ESCC, the sequential combination of CRT and atezolizumab has the potential to change the standard ESCC treatments. TRIAL REGISTRATION: UMIN000034373, 10/04/2018 and EPOC1802.

DOI： 10.1186/s12885-020-06716-5

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Biliary tract cancer (BTC) is an aggressive cancer with a poor prognosis partially due to the limited success in developing novel therapies, including molecularly targeted therapies and immunotherapies. Programmed cell death-1 (PD-1) blockade therapy is less effective against BTCs, necessitating further studies to understand the detailed immunological status of the tumor microenvironment (TME) in BTC. Here, we examined the immunological status of the TME in 37 BTCs with early- to late-stage disease, especially focusing on PD-1+CD8+ T cells. PD-1+CD8+ T cells, which are reportedly associated with the clinical response to PD-1 blockade therapy, were frequently observed in early-stage BTC and decreased with disease progression. Imaging mass cytometry for representative PD-1+CD8+TIL-high and -low patients demonstrated that tumor-infiltrating PD-1+CD8+ T cells were localized adjacent to tumor cells, whereas PD-1-CD8+ T cells were detected mainly in the stroma of the TME. In a mouse model, PD-1 expression by tumor-infiltrating CD8+ T cells was higher in smaller tumors and decreased with tumor growth. Consequently, large tumors became resistant to PD-1 blockade, while small tumors containing higher numbers of PD-1+CD8+ T cells were sensitive. We propose the important role of tumor-infiltrating PD-1+CD8+ T cells in anti-tumor immunity and the potential application of PD-1 blockade therapy for early-stage BTC.

DOI： 10.1093/intimm/dxz080

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The clinical efficacy of anti-PD-1 (programmed cell death-1) monoclonal antibody (mAb) against cancers with oncogenic driver gene mutations, which often harbor a low tumor mutation burden, is variable, suggesting different contributions of each driver mutation to immune responses. Here, we investigated the immunological phenotypes in the tumor microenvironment (TME) of epidermal growth factor receptor (EGFR)-mutated lung adenocarcinomas, for which anti-PD-1 mAb is largely ineffective. Whereas EGFR-mutated lung adenocarcinomas had a noninflamed TME, CD4+ effector regulatory T cells, which are generally present in the inflamed TME, showed high infiltration. The EGFR signal activated cJun/cJun N-terminal kinase and reduced interferon regulatory factor-1; the former increased CCL22, which recruits CD4+ regulatory T cells, and the latter decreased CXCL10 and CCL5, which induce CD8+ T cell infiltration. The EGFR inhibitor erlotinib decreased CD4+ effector regulatory T cells infiltration in the TME and in combination with anti-PD-1 mAb showed better antitumor effects than either treatment alone. Our results suggest that EGFR inhibitors when used in conjunction with anti-PD-1 mAb could increase the efficacy of immunotherapy in lung adenocarcinomas.

DOI： 10.1126/sciimmunol.aav3937

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Authorship：Lead author   Language：English   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1535-7163.TARG-19-C113

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Publishing type：Research paper (scientific journal)   Publisher：BMJ  

DOI： 10.1186/s40425-019-0720-z

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Other Link： http://link.springer.com/article/10.1186/s40425-019-0720-z/fulltext.html

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Patients treated with immune checkpoint blockade (ICB) sometimes experience immune-related adverse events (irAEs), requiring immuno-suppressive drugs such as corticosteroids despite the possibility that immunosuppression may impair the antitumor effects of ICB. Here, we address the dilemma of using corticosteroids for the treatment of irAEs induced by ICB. ICB augments neoantigen-specific CD8+ T cell responses, resulting in tumor regression. In our model, simultaneous, but not late, administration of corticosteroids impaired antitumor responses with reduction of CD8+ T cell proliferation. Secondary challenge using tumors with/without the neoantigen showed selective progression in tumors lacking the neoantigen when corticosteroids were administered. Corticosteroids decreased low- but not high-affinity memory T cells by suppressing fatty acid metabolism essential for memory T cells. In a small cohort of human melanoma patients, overall survival was shorter after treatment with CTLA-4 blockade in patients who received early corticosteroids or had low tumor mutation burden. Together, low-affinity memory T cells are dominantly suppressed by corticosteroids, necessitating careful and thoughtful corticosteroid use.

DOI： 10.1084/jem.20190738

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Major histocompatibility complex class I loss due to the abnormality of β2-microglobulin gene is one of the mechanisms underlying delayed relapses in melanoma patients long after the initial positive responses to anti-PD-1 therapy. However, the tumor-specific reactivity of tumor-infiltrating lymphocytes in tumor lesions that lost major histocompatibility complex class I expression has not been well evaluated. We report the case of a 55-year-old woman with two metastatic melanoma lesions. After a 12-month period of successful tumor suppression by anti-PD-1 antibody therapy, one lesion started to grow again. We resected both lesions and examined the tumor cells and tumor-infiltrating lymphocytes. The shrinking lesion consisted of necrotic tissue and macrophages, and the enlarged lesion consisted of both necrotic tissue and viable tumor cells. The tumor cells completely lost major histocompatibility complex class I expression, but it was restored upon retroviral transduction of the normal β2-microglobulin gene. When we checked the tumor-specific reactivity of tumor-infiltrating lymphocytes derived from the relapsing lesion, we found that these tumor-infiltrating lymphocytes failed to recognize the native tumor cells derived from the lesion, but strongly recognized the major histocompatibility complex class-I-recovered cells by β2-microglobulin transduction. Our report emphasizes the limitations of T-cell-based immunotherapy and highlights the importance of developing alternative strategies for such cases.

DOI： 10.1016/j.jid.2019.01.007

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Authorship：Corresponding author  

DOI： 10.1158/1078-0432.CCR-18-3991

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PD-1 blockade is a cancer immunotherapy effective in various types of cancer. In a fraction of treated patients, however, it causes rapid cancer progression called hyperprogressive disease (HPD). With our observation of HPD in ∼10% of anti-PD-1 monoclonal antibody (mAb)-treated advanced gastric cancer (GC) patients, we explored how anti-PD-1 mAb caused HPD in these patients and how HPD could be treated and prevented. In the majority of GC patients, tumor-infiltrating FoxP3highCD45RA-CD4+ T cells [effector Treg (eTreg) cells], which were abundant and highly suppressive in tumors, expressed PD-1 at equivalent levels as tumor-infiltrating CD4+ or CD8+ effector/memory T cells and at much higher levels than circulating eTreg cells. Comparison of GC tissue samples before and after anti-PD-1 mAb therapy revealed that the treatment markedly increased tumor-infiltrating proliferative (Ki67+) eTreg cells in HPD patients, contrasting with their reduction in non-HPD patients. Functionally, circulating and tumor-infiltrating PD-1+ eTreg cells were highly activated, showing higher expression of CTLA-4 than PD-1- eTreg cells. PD-1 blockade significantly enhanced in vitro Treg cell suppressive activity. Similarly, in mice, genetic ablation or antibody-mediated blockade of PD-1 in Treg cells increased their proliferation and suppression of antitumor immune responses. Taken together, PD-1 blockade may facilitate the proliferation of highly suppressive PD-1+ eTreg cells in HPDs, resulting in inhibition of antitumor immunity. The presence of actively proliferating PD-1+ eTreg cells in tumors is therefore a reliable marker for HPD. Depletion of eTreg cells in tumor tissues would be effective in treating and preventing HPD in PD-1 blockade cancer immunotherapy.

DOI： 10.1073/pnas.1822001116

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The efficacy of programmed cell death-1 (PD-1) blockade in patients with non-small cell lung cancer (NSCLC) positive for epidermal growth factor receptor (EGFR) gene mutations has been found to be limited, but the underlying mechanisms for this poor response have remained obscure. Given that the recognition by T cells of tumor antigens presented by major histocompatibility complex class I (MHC-I) molecules is essential for an antitumor immune response, we examined the effects of EGFR tyrosine kinase inhibitors (TKIs) on MHC-I expression in NSCLC cell lines. Appropriate EGFR-TKIs increased MHC-I expression at the mRNA and cell surface protein levels in NSCLC cells positive for EGFR mutations including those with the T790M secondary mutation. Trametinib, an inhibitor of the extracellular signal-regulated kinase (ERK) kinase MEK, also increased MHC-I expression, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor buparlisib did not, suggesting that the MEK-ERK pathway mediates the down-regulation of MHC-I expression in response to EGFR activation. Immunohistochemical analysis of EGFR-mutated NSCLC specimens obtained before and after EGFR-TKI treatment also revealed down-regulation of phosphorylated forms of EGFR and ERK in association with up-regulation of MHC-I, an increased number of infiltrating CD8+ T cells, and increased PD-1 ligand 1 expression after such treatment. Our results thus suggest that mutational activation of EGFR inhibits MHC-I expression through the MEK-ERK pathway in NSCLC and thereby contributes to the poor response of such tumors to immunotherapy. Further studies are warranted to evaluate the relation between EGFR-MEK-ERK signaling in and the immune response to EGFR-mutated NSCLC. .

DOI： 10.1111/cas.13860

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Authorship：Lead author   Language：English   Publisher：NATURE PORTFOLIO  

Regulatory T (T-reg) cells, an immunosuppressive subset of CD4(+) T cells characterized by the expression of the master transcription factor forkhead box protein P3 (FOXP3), are a component of the immune system with essential roles in maintaining self-tolerance. In addition, T-reg cells can suppress anticancer immunity, thereby hindering protective immunosurveillance of neoplasia and hampering effective antitumour immune responses in tumour-bearing hosts, thus promoting tumour development and progression. Identification of the factors that are specifically expressed in T-reg cells and/or that influence T-reg cell homeostasis and function is important to understanding cancer pathogenesis and to identifying therapeutic targets. Immune-checkpoint inhibitors (ICIs) have provided a paradigm shift in the treatment of cancer. Most immune-checkpoint molecules are expressed in T-reg cells, but the effects of ICIs on T-reg cells, and thus the contributions of these cells to treatment responses, remain unclear. Notably, evidence indicates that ICIs targeting programmed cell death 1 (PD-1) might enhance the immunosuppressive function of T-reg cells, whereas cytotoxic T lymphocyte antigen 4 (CTL A-4) inhibitors might deplete these cells. Thus, although manipulation of T-reg cells is a promising anticancer therapeutic strategy, approaches to controlling these cells require further research. Herein, we discuss novel insights into the roles of T-reg cells in cancer, which can hopefully be used to develop T-reg cell-targeted therapies and facilitate immune precision medicine.

DOI： 10.1038/s41571-019-0175-7

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Language：English   Publisher：John Wiley & Sons Australia, Ltd  

非小細胞肺癌の細胞株や患者由来検体を実験材料とし、上皮成長因子受容体(EGFR)チロシンキナーゼ阻害薬(TKI)がMHCクラスIの発現に及ぼす影響について調査した。EGFR遺伝子にT790M二次的突然変異などの変異がある細胞株を適切なEGFR-TKIで処理するとMHCクラスIのmRNA/蛋白質発現は亢進した。細胞外シグナル調節キナーゼ(ERK)キナーゼであるMEKの阻害薬で処理した場合もMHCクラスI発現が亢進したことから、EGFR活性化に応答してみられるMHCクラスI発現の下方制御はMEK-ERK経路が媒介していることが示唆された。EGFR-TKI治療を施行したEGFR変異非小細胞肺癌患者から得た検体の免疫組織化学解析からも、疾患進行後においては、リン酸化EGFRやリン酸化ERKの下方制御が、MHC-Iの上方制御、CD8+浸潤T細胞の増加、およびPD-1リガンド1発現亢進と関連していることが明らかになった。これらの結果から、非小細胞癌においてEGFRが変異活性化するとMEK-ERK経路を通じてMHCクラスI発現が阻害され、免疫療法に不応となるのに寄与することが示唆された。

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Several studies have established a correlation between the VEGF-VEGFR2 axis and an immunosuppressive microenvironment; this immunosuppression can be overcome by anti-angiogenic reagents, such as ramucirumab (RAM). However, little is known about the immunological impact of anti-angiogenic reagents within the tumor microenvironment in human clinical samples. This study aimed at investigating the effects of RAM on the tumor microenvironmental immune status in human cancers. METHODS: We prospectively enrolled 20 patients with advanced gastric cancer (GC) who received RAM-containing chemotherapy. We obtained paired samples from peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) in primary tumors both pre- and post-RAM therapy to assess immune profiles by immunohistochemistry and flow cytometry. RESULTS: Within the tumor microenvironment, both PD-L1 expression and CD8+ T-cell infiltration increased after RAM-containing therapies. In addition, CD45RA-FOXP3highCD4+ cells (effector regulatory T cells [eTreg cells]) and PD-1 expression by CD8+ T cells were significantly reduced in TILs compared with PBMCs after RAM-containing therapies. Patients with partial response and longer progression-free survival had significantly higher pre-treatment eTreg frequencies in TILs than those with progressive disease. In in vitro analysis, VEGFR2 was highly expressed by eTreg cells. Further, VEGFA promoted VEGFR2+ eTreg cell proliferation, and this effect could be inhibited by RAM. CONCLUSIONS: This study suggests that the frequency of eTreg cells in TILs could be a biomarker for stratifying clinical responses to RAM-containing therapies. Further, we propose that RAM may be employed as an immuno-modulator in combination with immune checkpoint blockade.

DOI： 10.1186/s40425-018-0403-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Tokyo  

Background: Recently, the U.S. Food and Drug Administration approved pembrolizumab for patients (pts) with PD-L1-positive metastatic gastric cancer (MGC) based on 22C3 immunohistochemistry (IHC) assay. However, little is known about detailed clinicopathological features of 22C3 PD-L1 expression in MGC. Patients and methods: Pts with histologically confirmed MGC were eligible for this prospective observational study. PD-L1 expression (22C3) on tumor cell (TC) or immune cell (IC) and mismatch repair (MMR) were analyzed by IHC. Epstein–Barr virus (EBV) was detected by in situ hybridization. The expressions of tyrosine kinase receptors (RTKs) and cancer genome alterations were evaluated by IHC or next-generation sequencing. Results: A total of 225 pts were analyzed in this study. PD-L1 expression on TC, PD-L1 on IC, MMR-deficient (D-MMR), and EBV positivity were identified in 8.4, 65.3, 6.2, and 6.2% cases, respectively. PD-L1 expression in TC was more frequently observed in pts with D-MMR (P &lt
0.001), PIK3CA mutation (P = 0.020), and KRAS mutation (P = 0.002), and PD-L1 on IC was associated with EBV positivity (P = 0.034), and lymph-node metastasis (P &lt
0.001). PD-L1 expression on either IC or TC was less frequently observed in pts with peritoneal metastasis and Borrmann Type 4. A significant association was not observed between PD-L1 expression and RTKs expression or presence of other gene alterations. PD-L1 expression on either TC or IC was not prognostic factor. Conclusions: 22C3 PD-L1 expression in MGC was associated with distinct clinicopathological features, but was not a prognostic factor.

DOI： 10.1007/s10120-018-0843-9

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DOI： 10.1200/JCO.2018.36.5_suppl.98

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Language：English   Publisher：NATURE PUBLISHING GROUP  

Adenosine produced by apoptotic regulatory T cells (T-reg cells) has a more important immunosuppressive role in the tumor microenvironment than that of live Treg cells. This discovery raises the possibility of novel strategies for cancer immunotherapy.

DOI： 10.1038/ni.3870

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Background: The efficacy of programmed death-1 blockade in epidermal growth factor receptor gene (EGFR) mutation-positive non-small-cell lung cancer (NSCLC) patients with different mechanisms of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) is unknown. We retrospectively evaluated nivolumab efficacy and immune-related factors in such patients according to their status for the T790M resistance mutation of EGFR.
Patients and methods: We identified 25 patients with EGFR mutation-positive NSCLC who were treated with nivolumab after disease progression during EGFR-TKI treatment (cohort A). Programmed death-ligand 1 (PD-L1) expression and tumor-infiltrating lymphocyte (TIL) density in tumor specimens obtained after acquisition of EGFR-TKI resistance were determined by immunohistochemistry. Whole-exome sequencing of tumor DNA was carried out to identify gene alterations. The relation of T790M status to PD-L1 expression or TIL density was also examined in an independent cohort of 60 patients (cohort B).
Results: In cohort A, median progression-free survival (PFS) was 2.1 and 1.3 months for T790M-negative and T790M-positive patients, respectively (P = 0.099; hazard ratio of 0.48 with a 95% confidence interval of 0.20-1.24). Median PFS was 2.1 and 1.3 months for patients with a PD-L1 expression level of &gt;= 1% or &lt;1%, respectively (P = 0.084; hazard ratio of 0.37, 95% confidence interval of 0.10-1.21). PFS tended to increase as the PD-L1 expression level increased with cutoff values of &gt;= 10% and &gt;= 50%. The proportion of tumors with a PD-L1 level of &gt;= 10% or &gt;= 50% was higher among T790M-negative patients than among T790M-positive patients of both cohorts A and B. Nivolumab responders had a significantly higher CD8(+) TIL density and nonsynonymous mutation burden.
Conclusion: T790M-negative patients with EGFR mutation-positive NSCLC are more likely to benefit from nivolumab after EGFR-TKI treatment, possibly as a result of a higher PD-L1 expression level, than are T790M-positive patients.

DOI： 10.1093/annonc/mdx183

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Akt2 is an isoform of Akt, and an association between Akt2 and resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been suggested in pancreatic cancer (PC) in vitro. In this study, we investigated the association between Akt2 expression as evaluated using immunohistochemistry and the outcome of patients with advanced PC who had received treatment with erlotinib (an EGFR-TKI). Although the difference was not significant, patients with high levels of Akt2 expression tended to have a poorer response and a shorter progression-free survival period after treatment with erlotinib plus gemcitabine than those with low expression levels (P=0.16 and 0.19, respectively). In vitro, an Akt2-amplified PC cell line and Akt2-overexpressed cell lines exhibited resistance to anti-EGFR therapies, including erlotinib, but combined treatment with BYL719 (a PI3K inhibitor) cancelled this resistance. Our findings suggest that Akt2 might be associated with the resistance to anti-EGFR therapies, especially the use of erlotinib against PC, and that this resistance can be overcome by combined treatment with a PI3K inhibitor. Akt2 expression could become a predictive biomarker for erlotinib resistance in PC.

DOI： 10.3892/ijo.2017.3961

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Background: Non-small cell lung cancer (NSCLC) harboring common epidermal growth factor receptor (EGFR) gene mutations (exon 19 deletion or exon 21 L858R) respond to EGFR tyrosine kinase inhibitors (EGFR-TKIs). The secondary T790 M mutation in exon 20 of the EGFR gene is the most common type of acquired resistance mutation. Several reports have also shown that other secondary mutations (L747S, D761Y and T854A), while uncommon, can induce acquired resistance to first-generation EGFR-TKIs. However, little is known about the anticancer activities of second-or third-generation EGFR-TKIs.
Methods: Uncommon secondary mutations were introduced into Ba/F3 cells along with the sensitive EGFR L858R mutation (Ba/F3-L858R/L747S, Ba/F3-L858R/D761Y, and Ba/F3-L858R/T854A), and the sensitivities to various EGFR-TKIs were then investigated.
Results: Both the Ba/F3-L858R/L747S and Ba/F3-L858R/D761Y cell lines exhibited weak resistances to first-generation reversible EGFR-TKIs, while the Ba/F3-L858R/T854A cell line exhibited a strong resistance. In contrast, irreversible EGFR-TKIs, especially third-generation EGFR-TKIs, were capable of overcoming these resistances. Western blot analyses demonstrated that gefitinib (first-generation) inhibited the phosphorylation of EGFR to a lesser extent in cells with these secondary mutations than in cells with the sensitive L858R mutation alone. In contrast, afatinib and osimertinib (second-and third-generation) inhibited the phosphorylation of EGFR in cells with these secondary mutations to a similar extent as that seen in cells with the sensitive L858R mutation alone.
Conclusions: Our experimental findings suggest that irreversible EGFR-TKIs, especially third-generation EGFR-TKIs, can be effective against uncommon secondary mutations and that switching to third-generation EGFR-TKIs could be a promising treatment strategy for patients with acquired resistance because of these uncommon secondary mutations.

DOI： 10.1186/s12885-017-3263-z

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Lung cancers harboring common EGFR mutations respond to EGFR tyrosine kinase inhibitors (TKI). We previously reported that tumors with exon 18 mutations are particularly sensitive to irreversible second-generation (2G) afatinib compared with firstgeneration TKIs (1G-TKI). However, data on the mechanisms of acquired resistance to afatinib are limited. We established afatinib-resistant cells by transfecting Ba/F3 cells with common or exon 18 (G719A and Del18) mutations and subjecting them to chronic exposure to increasing concentrations of afatinib. Afatinib-resistant clones were separately established through N-ethylN-nitrosourea (ENU) mutagenesis and exposure to fixed concentrations of afatinib. Rebiopsy samples from patients whose tumors acquired resistance to afatinib were analyzed. Afatinibresistant cells with Del19, L858R, or G719A developed T790M, whereas those with Del18 acquired novel L792F mutation. ENU mutagenesis screening established 84 afatinib-resistant clones. All Del19 clones and most of the other clones acquired only T790M. However, C797S occurred in subsets of L858R, G719A, and Del18 clones. In addition, subsets of Del18 clones acquired L792F. C797S-acquired cells were sensitive to 1G erlotinib. L792F demonstrated intermediate resistance between T790M and C797S to both 1G-and 3G-TKIs, whereas L792F was the least resistant to 2G-TKIs, particularly dacomitinib. Chronic exposure of Del18-L792F cells to dacomitinib induced additional T790M. T790M was detected in one of four clinical samples. In conclusion, L792F and C797S, in addition to the major T790M, can develop in afatinib-resistant cells particularly using a low dose of afatinib, and these minor mutations appear to exhibit sensitivity to dacomitinib and erlotinib, respectively. These secondary mutations should be tested in clinical practice.

DOI： 10.1158/1535-7163.MCT-16-0407

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Although fibroblast growth factor (FGF) signals are strongly associated with malignancy, limited information is available regarding the role of the FGF9 signal in colorectal cancer (CRC). In this study, we investigated the frequency of FGF9 amplification in CRC clinical specimens and the association between the FGF9 gene and resistance to anti-EGFR therapies. In clinical samples, an FGF9 copy number gain of &gt
5 copies was observed at a frequency of 8/145 (5.5%) and tended to be related to wild-type KRAS (7/96, 7.3%). Furthermore, FGF9 amplification was not observed in any of the samples from the 15 responders to anti-EGFR therapies but was observed in one sample from the seven non-responders with wild-type KRAS, and two samples from non-responders also had high FGF9 mRNA expression levels. FGF9 amplification was validated using a fluorescence in situ hybridization (FISH) analysis, and FGF9-amplified sections showed readily detectable signals originating from FGF9 protein when examined using immunohistochemistry. In both the in vitro and in vivo experiments using FGF9-overexpressing CRC cell lines, FGF9 overexpression induced strong resistance to anti-EGFR therapies via the enforced FGFR signal, and this resistance was cancelled by the application of an FGFR inhibitor. Considering these results, the FGF9 gene may play an important role in resistance to anti-EGFR therapies in patients with CRC, and such resistance might be overcome by combined treatment with an anti-FGFR inhibitor. These findings strongly encourage the development of FGFR-targeted therapy for CRC patients with FGF9 gene upregulation. © 2016 Wiley Periodicals, Inc.

DOI： 10.1002/mc.22476

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Although fibroblast growth factor (FGF) signals are strongly associated with malignancy, limited information is available regarding the role of the FGF9 signal in colorectal cancer (CRC). In this study, we investigated the frequency of FGF9 amplification in CRC clinical specimens and the association between the FGF9 gene and resistance to anti-EGFR therapies. In clinical samples, an FGF9 copy number gain of 5 copies was observed at a frequency of 8/145 (5.5%) and tended to be related to wild-type KRAS (7/96, 7.3%). Furthermore, FGF9 amplification was not observed in any of the samples from the 15 responders to anti-EGFR therapies but was observed in one sample from the seven non-responders with wild-type KRAS, and two samples from non-responders also had high FGF9 mRNA expression levels. FGF9 amplification was validated using a fluorescence in situ hybridization (FISH) analysis, and FGF9-amplified sections showed readily detectable signals originating from FGF9 protein when examined using immunohistochemistry. In both the in vitro and in vivo experiments using FGF9-overexpressing CRC cell lines, FGF9 overexpression induced strong resistance to anti-EGFR therapies via the enforced FGFR signal, and this resistance was cancelled by the application of an FGFR inhibitor. Considering these results, the FGF9 gene may play an important role in resistance to anti-EGFR therapies in patients with CRC, and such resistance might be overcome by combined treatment with an anti-FGFR inhibitor. These findings strongly encourage the development of FGFR-targeted therapy for CRC patients with FGF9 gene upregulation. (c) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/mc.22476

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CD4(+) regulatory T cells (Tregs) are a highly immune-suppressive subset of CD4(+) T cells, characterized by expression of the master regulatory transcription factor FOXP3. Tregs are proven to play central roles in the maintenance of self-tolerance in healthy individuals. Tregs are involved in maintaining immune homeostasis: they protect hosts from developing autoimmune diseases and allergy, whereas in malignancies, they promote tumor progression by suppressing anti-tumor immunity. Elucidating factors influencing Treg homeostasis and function have important implications for understanding disease pathogenesis and identifying therapeutic opportunities. Thus, the manipulating Tregs for up-or down-regulation of their suppressive function is a new therapeutic strategy for treating various diseases including autoimmune disorders and cancer. This review will focus on recent advances in how Tregs integrate extracellular and intracellular signals to control their survival and stability. Deeper mechanistic understanding of disease-specific Treg development, maintenance, and function could make disease-specific Treg-targeted therapy more effective, resulting in an increase of efficacy and decrease of side effects related to manipulating Tregs.

DOI： 10.1007/82_2017_58

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Several receptor tyrosine kinases (RTKs) including EGFR, ALK, and MET have been identified as therapeutic targets in non-small cell lung cancer (NSCLC). Among the downstream pathways of RTKs, the MAPK pathway is particularly important for cancer cell proliferation, differentiation, and survival. In this study, the effects of MEK inhibitors (trametinib and PD0325901) in several NSCLC cell lines with driver gene alterations, especially RTK genes, were tested in vitro using an MTT assay, and a wide range of sensitivities was found. In particular, all the EGFR-mutated cell lines were resistant to MEK inhibitors, whereas all the MET-amplified cell lines were sensitive. A bioinformatics technique and western blot analyses showed that the PI3K/AKT pathway is more activated in EGFRmutated NSCLC than in MET-amplified NSCLC, and a PI3K inhibitor enhanced the sensitivity to trametinib in the EGFR-mutated cell lines, suggesting that this pathway is associated with resistance to MEK inhibitors. Although the HCC827 cell line (EGFR mutation) was resistant to MEK inhibitors, the HCC827CNXR cell line, whose driver gene shifts from EGFR to MET, exhibited enhanced sensitivity to MEK inhibitors, indicating the biological importance of the MAPK pathway for MET-amplified NCSLC. Furthermore, a synergistic effect of crizotinib (a MET inhibitor) and trametinib was observed in MET-amplified NCLC cell lines. Our findings indicate that the MAPK pathway is biologically important for MET-amplified NSCLC and strongly encourage the development of combination therapy with a MET inhibitor and a MEK inhibitor against MET-amplified NSCLC.

DOI： 10.3892/ijo.2016.3736

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Fibroblast growth factor receptor (FGFR) gene alterations are relatively frequent in lung squamous cell carcinoma (LSCC) and are a potential targets for therapy with FGFR inhibitors. However, little is known regarding the clinicopathologic features associated with FGFR alterations. The angiokinase inhibitor nintedanib has shown promising activity in clinical trials for non-small cell lung cancer. We have now applied next-generation sequencing (NGS) to characterize FGFR alterations in LSCC patients as well as examined the antitumor activity of nintedanib in LSCC cell lines positive for FGFR1 copy number gain (CNG). The effects of nintedanib on the proliferation of and FGFR signaling in LSCC cell lines were examined invitro, and its effects on tumor formation were examined invivo. A total of 75 clinical LSCC specimens were screened for FGFR alterations by NGS. Nintedanib inhibited the proliferation of FGFR1 CNG-positive LSCC cell lines in association with attenuation of the FGFR1-ERK signaling pathway invitro and invivo. FGFR1 CNG (10.7%), FGFR1 mutation (2.7%), FGFR2 mutation (2.7%), FGFR4 mutation (5.3%), and FGFR3 fusion (1.3%) were detected in LSCC specimens by NGS. Clinicopathologic features did not differ between LSCC patients positive or negative for FGFR alterations. However, among the 36 patients with disease recurrence after surgery, prognosis was significantly worse for those harboring FGFR alterations. Screening for FGFR alterations by NGS warrants further study as a means to identify patients with LSCC recurrence after surgery who might benefit from nintedanib therapy.

DOI： 10.1111/cas.13071

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Comprehensive genomic profiling for non-small cell lung cancer (NSCLC) is likely to identify more patients with rare genetic alterations including uncommon epidermal growth factor receptor gene (EGFR) mutations. It remains unclear how such patients should be treated, however. We here report a case of NSCLC positive for two uncommon mutations of EGFR and a KRAS mutation, including its treatment with the second-generation EGFR tyrosine kinase inhibitor (TKI) afatinib. Tumor specimen obtained by a NSCLC patient with no smoking history was analyzed by next-generation sequencing. Comprehensive genomic profiling revealed that the patient harbored the EGFR mutations G719C and S768I as well as the E49K mutation of KRAS. Treatment with afatinib was clinically effective as confirmed by PET-CT scans of bone metastases and by a marked decrease in the serum concentration of carcinoembryonic antigen. Afatinib was the most effective among seven EGFR-TKIs tested in inhibiting the growth of Ba/F3 cells expressing EGFR(S768I), showing an efficacy similar to that apparent with cells expressing the common EGFR mutant L858R, whereas first- and third-generation EGFR-TKIs were markedly less effective against EGFR(S7681) than against EGFR(L858R). These data suggest that EGFR-TKIs differ in their activity toward cells expressing EGFR(S7681) in vitro. Consistently, afatinib was clinically effective for the treatment of NSCLC harboring G719C and S768I mutations of EGFR. Further studies are warranted to determine the most appropriate EGFR-TKI for treatment of NSCLC harboring uncommon EGFR mutations. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.lungcan.2016.09.001

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Most patients with non-small cell lung cancer (NSCLC) harboring common epidermal growth factor receptor (EGFR) mutations, such as deletions in exon 19 or the L858R mutation in exon 21, respond dramatically to EGFR tyrosine kinase inhibitors (EGFR-TKI), and their sensitivities to various EGFR-TKI have been well characterized. Our previous article showed the in vitro sensitivities of EGFR exon 18 mutations to EGFR-TKI, but little information regarding the sensitivities of other uncommon EGFR mutations is available. First, stable transfectant Ba/F3 cell lines harboring EGFR L858R (Ba/F3-L858R), L861Q (Ba/F3-L861Q) or S768I (Ba/F3-S768I) mutations were created and their drug sensitivities to various EGFR-TKI were examined. Both the Ba/F3-L861Q and Ba/F3-S768I cell lines were less sensitive to erlotinib, compared with the Ba/F3-L858R cell line, but their sensitivities to afatinib were similar to that of the Ba/F3-L858R cell line. The Ba/F3-L861Q cell line was similarly sensitive and the Ba/F3-S768I cell line was less sensitive to osimertinib, compared with the Ba/F3-L858R cell line. The results of western blot analyses were consistent with these sensitivities. Next, similar experiments were also performed using the KYSE270 (L861Q) and KYSE 450 (S768I) cell lines, and their results were compatible with those of the transfectant Ba/F3 cell lines. Our findings suggest that NSCLC harboring the EGFR L861Q mutation might be sensitive to afatinib or osimertinib and that NSCLC harboring the EGFR S768I mutation might be sensitive to afatinib. Overall, afatinib might be the optimal EGFR-TKI against these uncommon EGFR mutations.

DOI： 10.1111/cas.12980

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The prognosis for patients with advanced esophageal or head-and-neck squamous cell carcinoma (ESCC or HNSCC) remains poor, and the identification of additional oncogenes and their inhibitors is needed. In this study, we evaluated the sensitivities of several ESCC and HNSCC cell lines to HER inhibitors (cetuximab, erlotinib, and afatinib) in vitro and found two cell lines that were hypersensitive to afatinib. Sequence analyses for the afatinib-targeted HER family genes in the two cell lines revealed that one cell line had a previously reported activating EGFR L861Q mutation, whereas the other had an HER4 G1109C mutation of unknown function. No amplification of HER family genes was found in either of the two cell lines. The phosphorylation level of HER4 was elevated in the HER4 G1109C mutation-overexpressed HEK293 cell line, and the mutation had a transforming potential and exhibited tumorigenicity in an NIH3T3 cell line, indicating that this HER4 mutation was an activating oncogenic mutation. Afatinib dramatically reduced the phosphorylation level of EGFR or HER4 and induced apoptosis in the two cell lines. In vivo, tumor growth was also dramatically decreased by afatinib. In a database, the frequencies of HER family gene mutations in ESCC or HNSCC ranged from 0% to 5%. In particular, HER4 mutations have been found relatively frequently in HNSCC. Considering the addiction of cancer cells to activating oncogenic EGFR or HER4 mutations for proliferation, HNSCC or ESCC with such oncogenic mutations might be suitable for targeted therapy with afatinib. (C) 2016 AACR.

DOI： 10.1158/1535-7163.MCT-15-0737

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Purpose: This study investigated whether mutations of receptor tyrosine kinase (RTK) genes detected using next-generation sequencing (NGS) are suitable therapeutic targets.
Experimental design: Fifty surgically resected non-small cell lung cancer (NSCLC) samples were target resequenced using NGS. We then investigated the functions of the identified RTK gene mutations, including their oncogenic potential, in vitro.
Results: Mutations in RTK genes were found in 20 samples (EGFR, 15; ERBB4, 1; ALK, 1; DDR2, 2; FGFR1, 1), mutations in MAPK pathway genes were found in nine samples (KRAS, 7; NRAS, 1; BRAF, 2), and mutations in PI3K pathway genes were found in three samples (PIK3CA, 1; PTEN, 3). Among the mutations in RTKs, the functions of four mutations were unclear (ERBB4 D245G; DDR2 H246R and E655K; FGFR1 A263V). These mutations did not exhibit any transformational activities. Neither the phosphorylation nor the protein expressions of RTKs were changed by the DDR2 H246R, ERBB4 D245G, and FGFR1 A263V mutations, although the expression level of the DDR2 protein harboring the E655K mutation was particularly low. Collagen stimulation decreased cellular proliferation through p38 activation in the DDR2 wild-type-overexpressed cell lines, whereas the growth-suppressive effect was weakened in DDR2 E655K-overex-pressed cell lines. Furthermore, the DDR2 E655K protein strongly bound to ubiquitin ligase E3 (Cbl-b), and the mutant protein expression was increased after treatment with a proteasome inhibitor.
Conclusions: Our experimental findings suggest that RTK mutations are not always suitable as therapeutic targets. The DDR2 E655K mutation can play a role in cancer progression by reducing the growth-inhibitory effect of collagen. (C) 2016 AACR.

DOI： 10.1158/1078-0432.CCR-15-2093

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Purpose: Lung cancers harboring common EGFR mutations respond to EGFR tyrosine kinase inhibitors (TKI), whereas exon 20 insertions (Ins20) are resistant to them. However, little is known about mutations in exon 18.
Experimental Design: Mutational status of lung cancers between 2001 and 2015 was reviewed. Three representative mutations in exon 18, G719A, E709K, and exon 18 deletion (Del18: delE709_T710insD) were retrovirally introduced into Ba/F3 and NIH/3T3 cells. The 90% inhibitory concentrations (IC(90)s) of first-generation (1G; gefitinib and erlotinib), second-generation (2G; afatinib, dacomitinib, and neratinib), and third-generation TKIs (3G; AZD9291 and CO1686) were determined.
Results: Among 1,402 EGFR mutations, Del19, L858R, and Ins20 were detected in 40%, 47%, and 4%, respectively. Exon 18 mutations, including G719X, E709X, and Del18, were present in 3.2%. Transfected Ba/F3 cells grew in the absence of IL3, and NIH/3T3 cells formed foci with marked pile-up, indicating their oncogenic abilities. IC(90)s of 1G and 3G TKIs in G719A, E709K, and Del18 were much higher than those in Del19 (by &gt; 11-50-fold), whereas IC(90)s of afatinib were only 3- to 7-fold greater than those for Del19. Notably, cells transfected with G719A and E709K exhibited higher sensitivity to neratinib (by 5-25-fold) than those expressing Del19. Patients with lung cancers harboring G719X exhibited higher response rate to afatinib or neratinib (similar to 80%) than to 1G TKIs (35%-56%) by compilation of data in the literature.
Conclusions: Lung cancers harboring exon 18 mutations should not be overlooked in clinical practice. These cases can be best treated with afatinib or neratinib, although the currently available in vitro diagnostic kits cannot detect all exon 18 mutations. (C) 2015 AACR.

DOI： 10.1158/1078-0432.CCR-15-1046

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Background: MET splice site mutations resulting in an exon 14 deletion have been reported to be present in about 3% of all lung adenocarcinomas. Patients with lung adenocarcinoma and a MET splice site mutation who have responded to MET inhibitors have been reported. The CRISPR/Cas9 system is a recently developed genome-engineering tool that can easily and rapidly cause small insertions or deletions.
Materials and methods: We created an in vitro model for MET exon 14 deletion using the CRISPR/Cas9 system and the HEK293 cell line. The phenotype, which included MET inhibitor sensitivity, was then investigated in vitro. Additionally, MET splice site mutations were analyzed in several cancers included in The Cancer Genome Atlas (TCGA) dataset.
Results: An HEK293 cell line with a MET exon 14 deletion was easily and rapidly created; this cell line had a higher MET protein expression level, enhanced MET phosphoiylation, and prolonged MET activation. In addition, a direct comparison of phenotypes using this system demonstrated enhanced cellular growth, colony formation, and MET inhibitor sensitivity. In the TCGA dataset, lung adenocarcinomas had the highest incidence of MET exon 14 deletions, while other cancers rarely carried such mutations. Approximately 10% of the lung adenocarcinoma samples without any of driver gene alterations carried the MET exon 14 deletion.
Conclusions: These findings suggested that this system may be useful for experiments requiring the creation of specific mutations, and the present experimental findings encourage the development of MET-targeted therapy against lung cancer carrying the MET exon 14 deletion. (c) 2015 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.lungcan.2015.10.020

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BACKGROUND: Although colorectal mucinous adenocarcinomas (MCs) are conventionally regarded as exhibiting high-grade differentiation, they can be divided by differentiation into 2 groups according to the glandular appearance: low-grade mucinous adenocarcinoma (low-MC) and high-grade mucinous adenocarcinoma (high-MC). METHODS: Patients with colorectal cancer (CRC) who underwent surgical resection between 2000 and 2012 were enrolled in this study. Among the cases with MC, the clinicopathological and genetic differences between low-MC and high-MC were investigated with next-generation sequencing. RESULTS: A total of 1373 patients with CRC were analyzed. Forty patients (2.9%) had MC, and 13 patients had high-MC. Patients with MC had significantly shorter disease-free survival (DFS) and overall survival (OS) periods than those with nonmucinous carcinoma. When low-MC patients and high-MC patients were compared, those with high-MC had significantly shorter DFS and OS periods than those with low-MC. Multivariate analyses revealed that high-MC was significantly associated with both shorter DFS and shorter OS, but low-MC was not. A genome analysis revealed that low-MC had a considerably larger number of mutations than high-MC, and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations and adenomatous polyposis coli mutations were particularly frequently found in low-MC. In contrast, SMAD family member 4 (SMAD4) mutations were frequently found in high-MC. CONCLUSIONS: High-MC is an independent prognostic factor in CRC (but low-MC is not), and it is genetically different from other CRCs, including low-MC. Both the clinicopathological differences and the genetic differences suggest that low-MC and high-MC should be distinguished in clinical settings. (C) 2015 American Cancer Society.

DOI： 10.1002/cncr.29676

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Acute kidney injury (AKI) is a common and serious adverse effect of cisplatin-based chemotherapy. However, traditional markers of kidney function, such as serum creatinine, are suboptimal, because they are not sensitive measures of proximal tubular injury. We aimed to determine whether the new urinary biomarkers such as kidney injury molecule-1 (KIM-1), monocyte chemotactic protein-1 (MCP-1), and neutrophil gelatinase-associated lipocalin (NGAL) could detect cisplatin-induced AKI in lung cancer patients in comparison with the conventional urinary proteins such as N-acetyl-beta-d-glucosaminidase (NAG) and beta 2-microglobulin.
We measured KIM-1, MCP-1, NGAL, NAG, and beta 2-microglobulin concentrations in urine samples from 11 lung cancer patients, which were collected the day before cisplatin administration and on days 3, 7, and 14. Subsequently, we evaluated these biomarkers by comparing their concentrations in 30 AKI positive (+) and 12 AKI negative (-) samples and performing receiver operating characteristic (ROC) curve analyses.
The urinary levels normalized with urine creatinine of KIM-1 and MCP-1, but not NGAL, NAG, and beta 2-microglobulin in AKI (+) samples were significantly higher than those in AKI (-) samples. In addition, ROC curve analyses revealed that KIM-1 and MCP-1, but not NGAL, could detect AKI with high accuracy (area under the curve [AUC] = 0.858, 0.850, and 0.608, respectively). The combination of KIM-1 and MCP-1 outperformed either biomarker alone (AUC = 0.871).
Urinary KIM-1 and MCP-1, either alone or in combination, may represent biomarkers of cisplatin-induced AKI in lung cancer patients.

DOI： 10.1007/s00280-015-2880-y

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Since the prognosis of unresectable advanced gastric cancer remains poor, novel therapeutic strategies are needed. Somatic MEKI gene mutations have been reported as oncogenic activating mutations in gastric cancer, and MEK inhibitors can be effective against such gastric cancers. In the present study, however, activated EGFR and HER2 signals after treatment with a MEK inhibitor (trametinib) were found in a MEK1-mutated gastric cancer cell line (OCUM-1 cell line) using a phospho-receptor tyrosine kinase array. The phosphorylation of EGFR and HER2 reactivated ERK1/2, which had been inhibited by trametinib, and EGF stimulation led to resistance to trametinib in this cell line. Lapatinib, an EGFR and an HER2 inhibitor, reversed the activation of ERK1/2 by inhibiting the phosphorylation of EGFR and HER2 and cancelled the resistance. The combination of trametinib and lapatinib synergistically inhibited the cell growth of the OCUM-1 cell line and strongly induced apoptosis by inhibiting the activated EGFR and HER2 signals. These results suggest that the EGFR and HER2 signals play a salvage role and are related to resistance to MEK inhibitors in MEK1-mutated gastric cancer. Moreover, combination therapy with trametinib and lapatinib can exhibit a synergistic effect and may contribute to overcoming the resistance to MEK inhibitors.

DOI： 10.3892/ijo.2015.3050

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DOI： 10.1093/annonc/mdv240

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Gemcitabine is a key drug for the treatment of pancreatic cancer; however, with its limitation in clinical benefits, the development of another potent therapeutic is necessary. Vascular endothelial growth factor receptor 2 is an essential target for tumor angiogenesis, and we have conducted a phase I clinical trial using gemcitabine and vascular endothelial growth factor receptor 2 peptide (elpamotide). Based on the promising results of this phase I trial, a multicenter, randomized, placebo-controlled, double-blind phase II/III clinical trial has been carried out for pancreatic cancer. The eligibility criteria included locally advanced or metastatic pancreatic cancer. Patients were assigned to either the Active group (elpamotide+gemcitabine) or Placebo group (placebo+gemcitabine) in a 2:1 ratio by the dynamic allocation method. The primary endpoint was overall survival. The Harrington-Fleming test was applied to the statistical analysis in this study to evaluate the time-lagged effect of immunotherapy appropriately. A total of 153 patients (Active group, n=100; Placebo group, n=53) were included in the analysis. No statistically significant differences were found between the two groups in the prolongation of overall survival (Harrington-Fleming P-value, 0.918; log-rank P-value, 0.897; hazard ratio, 0.87, 95% confidence interval [CI], 0.486-1.557). Median survival time was 8.36months (95% CI, 7.46-10.18) for the Active group and 8.54months (95% CI, 7.33-10.84) for the Placebo group. The toxicity observed in both groups was manageable. Combination therapy of elpamotide with gemcitabine was well tolerated. Despite the lack of benefit in overall survival, subgroup analysis suggested that the patients who experienced severe injection site reaction, such as ulceration and erosion, might have better survival.
Phase II/III trial of elpamotide was performed to evaluate the clinical effect for advanced pancreatic cancer. Despite the lack of benefit in OS, sub-group analysis suggested that the patients with severe ISR might have better survival.

DOI： 10.1111/cas.12674

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Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (alpha = 2 x 10(-5)). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.

DOI： 10.1371/journal.pone.0121891

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Background: Some of patients with non-small cell lung cancer (NSCLC) harboring somatic activating mutations of the epidermal growth factor receptor gene (EGFR mutations) show poor responses to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) treatment. Cigarette smoking is the strongest documented risk factor for the development of lung cancer. Nicotine, while not carcinogenic by itself, has been shown to induce proliferation, angiogenesis, and the epithelial-mesenchymal transition; these effects might be associated with EGFR-TKI resistance.
Materials and methods: PC-9 and 11-18 cell lines (EGFR-mutated NSCLC cell lines) were cultured with 1 mu M nicotine for 3 months and were designated as PC-9/N and 11-18/N cell lines, respectively. The sensitivities of these cell lines to EGFR-TKI were then tested in vitro. Moreover, the association between the smoking status and the progression-free survival (PFS) period was investigated in patients with EGFR-mutated NSCLC who were treated with gefitinib.
Results: The PC-9/N and 11-18/N cell lines were resistant to EGFR-TKI, compared with controls. The phosphorylation of EGFR in these cell lines was reduced by EGFR-TKI to a smaller extent than that observed in controls, and a higher concentration of EGFR-TKI was capable of further decreasing the phosphorylation. Clinically, smoking history was an independent predictor of a poor PFS period on gefitinib treatment.
Conclusions: Chronic nicotine exposure because of cigarette smoking mediates resistance to EGFR-TKI via an EGFR signal. Smoking cessation is of great importance, while resistance may be overcome through the administration of high-dose EGFR-TKI. (c) 2015 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.lungcan.2015.01.027

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT INST ANTICANCER RESEARCH  

Background: A recent pooled analysis of the LUX-LUNG3 and LUX-LUNG6 trials suggested that afatinib (an irreversible epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)) is especially effective against non-small cell lung cancer (NSCLC) carrying an EGFR exon 19 deletion. Materials and Methods: Stable viral transfectant HEK293 cell lines carrying an exon 19 deletion (HEK293/19 del) or exon 21 L858R mutation (HEK293/L858R)) were created and their drug sensitivities to AG1478 (a reversible EGFR-TKI) and afatinib were examined using an MTT assay. Western blot analyses were performed to estimate the phosphorylation of EGFR. Results: In the HEK293/19 del, the 50% inhibitory concentration (IC50) of afatinib was significantly lower than that in the HEK293/L858R. In addition, afatinib inhibited the phosphorylation of EGFR to a greater degree in the HEK293/19 del than in the HEK293/L858R. Conclusion: Our experimental findings suggest that afatinib is especially effective against NSCLC carrying an EGFR exon 19 deletion.

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Non-small cell lung cancer (NSCLC) carrying echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangements is hypersensitive to ALK inhibitors, including crizotinib and alectinib. Crizotinib was initially designed as a MET inhibitor, whereas alectinib is a selective ALK inhibitor. The MET signal, which is inhibited by crizotinib but not by alectinib, is dysregulated in many human cancers. However, the role of the MET signal in ALK-positive NSCLC remains unclear. In this study, we found that hepatocyte growth factor (HGF), ligand of MET, mediated the resistance to alectinib, but not to crizotinib, via the MET signal in ALK-positive NSCLC cell lines (H3122 and H2228 cell lines). In addition, alectinib activated the MET signal even in the absence of HGF and the inhibition of the MET signal enhanced the efficacy of alectinib. These findings suggest that activated MET acts as a salvage signal in ALK-positive NSCLC. This novel role of the MET signal in ALK-positive NSCLC may pave the way for further clinical trials examining MET inhibitors.

DOI： 10.3892/ijo.2014.2797

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Intra- and inter-tumor heterogeneity may hinder personalized molecular-target treatment that depends on the somatic mutation profiles. We performed mutation profiling of formalin-fixed paraffin embedded tumors of multi-regional colon cancer and characterized the consequences of intra- and inter-tumor heterogeneity and metastasis using targeted re-sequencing.
We performed targeted re-sequencing on multiple spatially separated samples obtained from multi-regional primary colon carcinoma and associated metastatic sites in two patients using nextgeneration sequencing. In Patient 1 with four primary tumors (P1-1, P1-2, P1-3, and P1-4) and one liver metastasis (H1), mutually exclusive pattern of mutations was observed in four primary tumors. Mutations in primary tumors were identified in three regions; KARS (G13D) and APC (R876*) in P1-2, TP53 (A161S) in P1-3, and KRAS (G12D), PIK3CA (Q546R), and ERBB4 (T272A) in P1-4. Similar combinatorial mutations were observed between P1-4 and H1. The ERBB4 (T272A) mutation observed in P1-4, however, disappeared in H1. In Patient 2 with two primary tumors (P2-1 and P2-2) and one liver metastasis (H2), mutually exclusive pattern of mutations were observed in two primary tumors. We identified mutations; KRAS (G12V), SMAD4 (N129K, R445*, and G508D), TP53 (R175H), and FGFR3 (R805W) in P2-1, and NRAS (Q61K) and FBXW7 (R425C) in P2-2. Similar combinatorial mutations were observed between P2-1 and H2. The SMAD4 (N129K and G508D) mutations observed in P2-1, however, were nor detected in H2. These results suggested that different clones existed in primary tumors and metastatic tumor in Patient 1 and 2 likely originated from P1-4 and P2-1, respectively.
In conclusion, we detected the muti-clonalities between intra- and inter-tumors based on mutational profiling in multi-regional colon cancer using next-generation sequencing. Primary region from which metastasis originated could be speculated by mutation profile. Characterization of inter- and inter-tumor heterogeneity can lead to underestimation of the tumor genomics landscape and treatment strategy of personal medicine. (C) 2015 Published by Elsevier Inc.

DOI： 10.1016/j.bbrc.2015.01.064

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Introduction: To investigate the impact of pre-existing radiological interstitial lung disease (ILD) findings on the incidence of radiation pneumonitis (RP) and clinical outcomes after stereotactic body radiation therapy (SBRT) for stage I non-small-cell lung cancer.
Methods: We included 157 consecutive patients who underwent SBRT alone for stage I non-small-cell lung cancer and whose pretreatment lung computed tomography images were available for retrospective review. The pretreatment computed tomography images were evaluated retrospectively for the presence of ILD. The incidence of RP, overall survival (OS) rate, and the incidence of disease progression and local progression were evaluated between patients with ILD (ILD[+]) and without ILD (ILD[-]).
Results: Pre-existing ILD was identified in 20 patients. The median follow-up period was 39.5 months. The incidences of RP worse than grade 2 (&gt;= Gr2 RP) and worse than grade 3 (&gt;= Gr3 RP) were significantly higher in ILD(+) than ILD(-) (1 year &gt;= Gr2 RP rate, 55.0% versus 13.3%; p &lt; 0.001 and 1year &gt;= Gr3 RP rate 10.0% versus 1.5%; p = 0.020). Multivariate analysis also indicated that ILD(+) was a risk factor for &gt;= Gr2 and &gt;= Gr3 RP, and the volume of the irradiated lung. The OS rate tended to be worse in ILD(+) than ILD(-) (3-year OS, 53.8% versus 70.8%; p = 0.28). No difference was observed in the disease progression or local progression rates.
Conclusions: Pre-existing ILD was a significant risk factor for symptomatic and severe RP. Prescreening for ILD findings is important for determining the radiation pneumonitis risk when planning SBRT.

DOI： 10.1097/JTO.0000000000000359

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

POU5F1B (POU domain class 5 transcription factor 1B), a processed pseudogene that is highly homologous to OCT4, was recently shown to be transcribed in cancer cells, but its clinical relevance and biological function have remained unclear. We now show that POU5F1B, which is located adjacent to MYC on human chromosome 8q24, is frequently amplified in gastric cancer (GC) cell lines. POU5F1B, but not OCT4, was also found to be expressed at a high level in GC cell lines and clinical specimens. In addition, the DNA copy number and mRNA abundance for POU5F1B showed a positive correlation in both cancer cell lines and GC specimens. Overexpression of POU5F1B in GC cells promoted colony formation in vitro as well as both tumorigenicity and tumor growth in vivo, and these effects were enhanced in the additional presence of MYC overexpression. Furthermore, knockdown of POU5F1B expression with a short hairpin RNA confirmed a role for the endogenous pseudogene in the promotion of cancer cell growth in vitro and tumor growth in vivo. POU5F1B overexpression induced upregulation of various growth factors in GC cells as well as exhibited mitogenic, angiogenic and antiapoptotic effects in GC xenografts. Finally, amplification of POU5F1B was detected in 17 (12%) of 145 cases of GC and was a significant predictor of poor prognosis in patients with stage IV disease. In conclusion, we found that the POU5F1B pseudogene is amplified and expressed at a high level in, as well as confers an aggressive phenotype on, GC, and that POU5F1B amplification is associated with a poor prognosis in GC patients.

DOI： 10.1038/onc.2013.547

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER INTERNATIONAL PUBLISHING AG  

We compared the performance of the 3D-Gene (R) mutation assay (3D-Gene (R) KRAS mutation assay kit) with the Scorpion-ARMS (therascreen (R) KRAS RGQ PCR Kit) and Luminex (MEBGEN (TM) KRAS kit) assays for the detection of KRAS mutations in formalin-fixed, paraffin-embedded tissue samples from 150 patients diagnosed with colorectal cancer. DNA was extracted from the paraffin-embedded tissue samples with or without macrodissection under hematoxylin and eosin staining and the KRAS mutation status was independently determined using these assays. Discordant results were re-analyzed by Sanger sequencing. Mutation detection analysis was successfully performed in all 150 specimens using the 3D-Gene (R) mutation assay without an invalid case. The concordance rate between the 3D-Gene (R) mutation assay and Scorpion-ARMS or Luminex was 98.7% (148/150). KRAS mutations were detected at a frequency of 35.3% (53/150) in colorectal cancer specimens. Three discrepant cases were found between the three assays. Overall, our results demonstrate a high concordance rate of between the 3D-Gene (R) mutation assay and the two existing in-vitro diagnostics kits. All three assays proved to be validated methods for detecting clinically significant KRAS mutations in paraffin-embedded tissue samples.

DOI： 10.1186/2193-1801-4-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

We previously reported that activin produces a signal with a tumor suppressive role in pancreatic cancer (PC). Here, the association between plasma activin A and survival in patients with advanced PC was investigated. Contrary to our expectations, however, patients with high plasma activin A levels had a significantly shorter survival period than those with low levels (median survival, 314 days vs. 482 days, P = 0.034). The cellular growth of the MIA PaCa-2 cell line was greatly enhanced by activin A via non-SMAD pathways. The cellular growth and colony formation of an INHBA (beta subunit of inhibin)overexpressed cell line were also enhanced. In a xenograft study, INHBA-overexpressed cells tended to result in a larger tumor volume, compared with a control. The bodyweights of mice inoculated with INHBA-overexpressed cells decreased dramatically, and these mice all died at an early stage, suggesting the occurrence of activin-induced cachexia. Our findings indicated that the activin signal can promote cancer progression in a subset of PC and might be involved in cachexia. The activin signal might be a novel target for the treatment of PC. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.canlet.2014.10.037

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Introduction: Patients with non-small-cell lung cancer (NSCLC) with somatic activating mutations of the epidermal growth factor receptor gene (EGFR mutations) generally respond to EGFR tyrosine kinase inhibitors (EGFR-TKIs). beta-Catenin is a key component of the Wnt/beta-Catenin signal and is an important oncogene that is involved in the pathogenesis and progression of malignant tumors, especially cancer stem cells.
Methods and Results: We found that EGFR-mutated NSCLC cell lines exhibited a high expression level of beta-Catenin, compared with cell lines with the wild-type EGFR gene, and XAV939 (a beta-Catenin inhibitor) enhanced the sensitivities to EGFR-TKI in EGFR-mutated NSCLC cell lines. In EGFR-mutated NSCLC cell lines with the acquired resistance threonine-to-methionine mutation in codon 790 (T790M) mutation, XAV939 enhanced the sensitivity of the cells to an irreversible EGFR-TKI but not a reversible EGFR-TKI. The combination of XAV939 and EGFR-TKIs strongly inhibited the beta-Catenin signal and strongly decreased the phosphorylation of EGFR, compared with the use of EGFR-TKIs alone, suggesting an interaction between EGFR and the beta-Catenin signal. The stem cell-like properties of the EGFR-mutated cell line carrying the T790M mutation were inhibited by XAV939 and BIBW2992 (an irreversible EGFR-TKI). Furthermore, the stem cell-like properties were strongly inhibited by a combination of both the agents. A xenograft study demonstrated that beta-Catenin knockdown enhanced the antitumor effect of BIBW2992 in the EGFR-mutated NSCLC cell line carrying the T790M mutation.
Conclusion: Our findings indicate that beta-Catenin might be a novel therapeutic target in EGFR-mutated NSCLC carrying the T790M mutation.

DOI： 10.1097/JTO.0000000000000353

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC INTERNAL MEDICINE  

Objective Information regarding multiple primary malignancies is important, as it has the potential to clarify etiological factors and may indicate the need to refine patient follow-up to include screening for associated malignancies. Upper aerodigestive tract cancer often develops in patients with smoking-related lung cancer; however, little is known about the frequencies or types of other primary malignancies in patients with non-small cell lung cancer (NSCLC) without a history of smoking.
Methods We retrospectively evaluated the records of patients examined and/or treated for NSCLC at the Institute of Biomedical Research and Innovation between January 2007 and June 2012. Patients In total, 938 patients, including 599 men (never-smoker/ever-smoker: 35/564) and 339 women (never-smoker/ever-smoker: 236/103), were analyzed.
Results Among the 209 patients (22.3%) with multiple primary malignancies, 151 had a history of smoking and 58 were never-smokers. The most common cancers were gastric (43 cases), colorectal (33 cases), and prostate (29 cases) cancer. Smoking-related cancer was more common in current smokers and ex-smokers for both men and women. Among women with NSCLC, never-smokers were more likely to have thyroid cancer than those with a history of smoking (5.1% vs. 0%, p= 0.021).
Conclusion In this study, several differences in malignancies were observed between never-smokers and patients with a history of smoking. Thyroid cancer and NSCLC co-existed in some women without a history of smoking, implicating predisposing factors other than tobacco smoke in the onset of these cancers.

DOI： 10.2169/internalmedicine.54.2921

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC CANCER RESEARCH  

The prognosis for patients with unresectable advanced or recurrent gastric cancer remains poor. The identification of additional oncogenes with influences similar to those of epidermal growth factor receptor gene mutations, upon which the growth of cancer cells is dependent, is needed. In this study, we evaluated sensitivity to MEK inhibitors (GSK1120212 and PD0325901) in several gastric cancer cell lines in vitro and found three poorly differentiated gastric cancer cell lines that were hypersensitive to the inhibitors. The sequence analyses in these three cell lines revealed that one cell line had a novel MEK1 mutation, while the other two had previously reported KRAS and MEK1 mutations, respectively; the gene statuses of the other resistant cell lines were all wild-type. Experiments using MEK1 expression vectors demonstrated that the MEK1 mutations induced the phosphorylation of ERK1/2 and had a transforming potential, enhancing the tumorigenicity. The MEK inhibitor dramatically reduced the phosphorylation of ERK1/2 and induced apoptosis in the cell lines with MEK1 mutations. In vivo, tumor growth was also dramatically decreased by an inhibitor. One of the 46 gastric cancer clinical samples that were examined had a MEK1 mutation; this tumor had a poorly differentiated histology. Considering the addiction of cancer cells to active MEK1 mutations for proliferation, gastric cancer with such oncogenic MEK1 mutations might be suitable for targeted therapy with MEK inhibitors. (C)2014 AACR.

DOI： 10.1158/1535-7163.MCT-14-0429

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

Patients with non-small cell lung cancer (NSCLC) with echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangements generally respond to ALK inhibitors such as crizotinib. However, some patients with EML4-ALK rearrangements respond poorly to crizotinib. Hypoxia is involved in the resistance to chemotherapeutic treatments in several cancers, and we investigated the association between the responses to ALK inhibitors and hypoxia. Sensitivity of the H3122 NSCLC cell line (EML4-ALK rearrangement) to ALK inhibitors (crizotinib or alectinib) was investigated during a normoxic or hypoxic state using an MTT assay. We found that the cell line was resistant to the inhibitors during hypoxia. Hypoxia mediated morphologic changes, including cell scattering and the elongation of the cell shape, that are characteristic of the epithelial-mesenchymal transition (EMT). A migration assay demonstrated that the number of migrating cells increased significantly during hypoxia, compared with during normoxia. Regarding EMT-related molecules, the expressions of slug, vimentin, and fibronectin were increased while that of E-cadherin was decreased by hypoxia. In addition, hypoxia inducible factor 1A-knockdown cancelled the hypoxia-induced EMT and resistance. Our findings indicate that hypoxia induces resistance to ALK inhibitors in NSCLC with an EML4-ALK rearrangement via the EMT.

DOI： 10.3892/ijo.2014.2574

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IMPACT JOURNALS LLC  

The KIAA1199 gene was first discovered to be associated with non-syndromic hearing loss. Recently, several reports have shown that the up-regulation of KIAA1199 is associated with cancer cell migration or invasion and a poor prognosis. These findings indicate that KIAA1199 may be a novel target for cancer therapy. Therefore, we explored in detail the function of KIAA1199 in cancer cells. In this study, we investigated the interaction of KIAA1199 protein with intracellular proteins in cancer cells. To this end, we expressed KIAA1199-MBP fusion protein and performed a pull-down assay. In addition, KIAA1199-overexpressing cancer cell lines were constructed using a retroviral vector and were used for further experiments. A pull-down analysis showed that the glycogen phosphorylase kinase beta-subunit (PHKB) interacted with the C-terminal region of KIAA1199 protein. Furthermore, we observed the interaction of KIAA1199 with glycogen phosphorylase brain form (PYGB) under serum-free conditions. The interaction promoted glycogen breakdown and cancer cell survival. Our findings indicate that KIAA1199 plays an important role in glycogen breakdown and cancer cell survival and that it may represent a novel target for cancer therapy.

DOI： 10.18632/oncotarget.2220

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER INTERNATIONAL PUBLISHING AG  

Triple-negative breast cancer (TNBC) is associated with a higher incidence of recurrence and distant metastasis and a poor prognosis, whereas effective treatment strategies remain to be established. Finding an effective treatment for TNBC has become imperative. We examined the effect of the combination of S-1 (or 5-FU in an in vitro study) and eribulin in TNBC cell lines. The in vitro effect of the combination was examined in four TNBC cell lines (MDA-MB-231, MDA-MB-468, BT-549 and MX-1) using a combination index and isobolograms. In addition, we assessed the effect of the combination in an MDA-MB-231 tumor xenograft model. A synergistic effect was observed in three TNBC cell lines (MDA-MB-231, MDA-MB-468, and MX-1), and in an in vivo study, the combination of S-1 and eribulin resulted in significantly higher antitumor effects compared with S-1 or eribulin alone. 5-FU induced epithelial-mesenchymal transition (EMT) change in the TNCB cell line, as supported by the decreased expression of epithelial marker and the increased expression of mesenchymal markers. Meanwhile, TGF-beta induced EMT changes in a TNBC cell line and decreased the sensitivity to 5-FU. This result suggests that 5-FU-induced EMT changes reduce the sensitivity to 5-FU. In contrast, eribulin induced a mesenchymal-epithelial transition (MET) in a TNBC cell line. The EMT phenotype induced by 5-FU was also canceled by eribulin. We demonstrate that the combination of S-1 (5-FU) and eribulin exerts a synergistic effect for TNBC cell lines through MET-induction by eribulin. Therefore, this combination therapy may be a potential treatment option for TNBC.

DOI： 10.1186/2193-1801-3-417

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Chromosomal band 11q13 seems to be one of the most frequently amplified lesions in human cancer, including esophageal squamous cell cancer (ESCC). The oral cancer overexpressed 1 (ORAOV1) gene has been identified within this region, but its detailed biological function in human ESCC remains largely unclear. In our clinical samples of stage III ESCC, ORAOV1 amplification was observed in 49 of 94 cases (53%). ORAOV1 amplification was significantly associated with a poorly differentiated histology and tumors located in the upper or middle esophagus. Patients with ORAOV1 amplification tended to have a shorter survival period, although the difference was not significant. To investigate the function of ORAOV1, we created ORAOV1-overexpressed ESCC cell lines that exhibited increased cellular proliferation and colony formation, compared with in vitro controls. In vivo, ORAOV1-overexpressed cells exhibited a significantly increased tumorigenicity and a significantly larger tumor volume and poorer differentiation than controls. The peptide mass fingerprinting technique demonstrated that ORAOV1 bound to pyrroline-5-carboxylate reductase (PYCR), which is associated with proline metabolism and reactive oxygen species (ROS) production. Then, ORAOV1-overexpressed cell lines were resistant to stress treatment, which was cancelled by PYCR-knockdown. In addition, the ORAOV1-overexpressed cell line had a higher intracellular proline concentration and a lower ROS level. Our findings indicate that the ORAOV1 gene is frequently amplified in ESCC, enhances tumorigenicity and tumor growth, and is associated with a poorly differentiated tumor histology via proline metabolism and ROS production. ORAOV1 could be a novel target for the treatment of ESCC.

DOI： 10.18632/oncotarget.1561

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Transforming growth factor, beta (TGFB) signal is considered to be a tumor suppressive pathway based on the frequent genomic deletion of the SMAD4 gene in pancreatic cancer (PC); however; the role of the activin signal, which also belongs to the TGFB superfamily, remains largely unclear.
Methods and results: We found a homozygous deletion of the activin A receptor, type IB (ACVR1B) gene in 2 out of 8 PC cell lines using array-comparative genomic hybridization, and the absence of ACVR1B mRNA and protein expression was confirmed in these 2 cell lines. Activin A stimulation inhibited cellular growth and increased the phosphorylation level of SMAD2 and the expression level of p21(CIP1/WAF1) in the Sui66 cell line (wild-type ACVR1B and SMAD4 genes) but not in the Sui68 cell line (homozygous deletion of ACVR1B gene). Stable ACVR1B-knockdown using short hairpin RNA cancelled the effects of activin A on the cellular growth of the PC cell lines. In addition, ACVR1B-knockdown significantly enhanced the cellular growth and colony formation abilities, compared with controls. In a xenograft study, ACVR1B-knockdown resulted in a significantly elevated level of tumorigenesis and a larger tumor volume, compared with the control. Furthermore, in clinical samples, 6 of the 29 PC samples (20.7%) carried a deletion of the ACVR1B gene, while 10 of the 29 samples (34.5%) carried a deletion of the SMAD4 gene. Of note, 5 of the 6 samples with a deletion of the ACVR1B gene also had a deletion of the SMAD4 gene.
Conclusion: We identified a homozygous deletion of the ACVR1B gene in PC cell lines and clinical samples and proposed that the deletion of the ACVR1B gene may mediate an aggressive cancer phenotype in PC. Our findings provide novel insight into the role of the activin signal in PC.

DOI： 10.1186/1476-4598-13-126

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Molecular markers for predicting or monitoring the efficacy of bevacizumab in patients with metastatic colorectal cancer (mCRC) remain to be identified. We have now measured the serum concentrations of 25 angiogenesis-related molecules with antibody suspension bead array systems for 25 mCRC patients both before and during treatment in a previously reported phase II trial of FOLFIRI chemotherapy plus bevacizumab. The serum concentration of vascular endothelial growth factor-A (VEGF-A) decreased after the onset of treatment (P < 0.0001), whereas that of placental growth factor increased (P < 0.0001). Significant differences in the levels of several factors (such as VEGF-A, soluble VEGF receptor-2, and interleukin-8) were apparent between responders and nonresponders during treatment. The rapid and pronounced decrease in serum VEGF-A level after treatment onset was apparent in all subjects and was independent of the baseline concentration. However, four of nine nonresponders showed a subsequent early increase in the serum VEGF-A level. Our results thus suggest that an early increase in the serum VEGF-A concentration after the initial decrease is a potential predictive marker of a poor response and reactive resistance to bevacizumab plus chemotherapy.

DOI： 10.18632/oncotarget.1811

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The tissue distribution and function of hemoglobin or myoglobin are well known; however, a newly found cytoglobin (CYGB), which also belongs to the globin family, remains to be characterized. To assess its expression in human malignancies, we sought to screen a number of cell lines originated from many tissues using northern blotting and real time PCR techniques. Unexpectedly, we found that several, but not all, melanoma cell lines expressed CYGB mRNA and protein at much higher levels than cells of other origins. Melanocytes, the primary origin of melanoma, also expressed CYGB at a high level. To verify these observations, immunostaining and immunoblotting using anti-CYGB antibody were also performed. Bisulfite-modified genomic sequencing revealed that several melanoma cell lines that abrogated CYGB expression were found to be epigenetically regulated by hypermethylation in the promoter region of CYGB gene. The RNA interference-mediated knockdown of the CYGB transcript in CYGB expression-positive melanoma cell lines resulted in increased proliferation in vitro and in vivo. Flow cytometric analysis using 2'-, 7'-dichlorofluorescein diacetate (DCFH-DA), an indicator of reactive oxygen species (ROS), revealed that the cellular ROS level may be involved in the proliferative effect of CYGB. Thus, CYGB appears to play a tumor suppressive role as a ROS regulator, and its epigenetic silencing, as observed in CYGB expression-negative melanoma cell lines, might function as an alternative pathway in the melanocyte-to-melanoma transition.

DOI： 10.1371/journal.pone.0094772

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Language：English   Publisher：DOVE MEDICAL PRESS LTD  

Gefitinib, an epidermal growth factor tyrosine kinase inhibitor (EGFR-TKI), has been approved in Japan for the treatment of patients with advanced non-small-cell lung cancer (NSCLC) based on Phase II clinical trials since 2002. Erlotinib, another EGFR-TKI, was also approved a few years thereafter. In 2004, activating mutations in the EGFR gene were discovered to be a predictive biomarker for EGFR-TKI treatment, and gefitinib, which is not effective for patients with EGFR wild-type NSCLC, has since been used only in patients with EGFR-mutated NSCLC. In contrast, erlotinib is potentially effective for the treatment of EGFR wild-type NSCLC. Similar to gefitinib, erlotinib is also effective for EGFR-mutated NSCLC and has been used as an initial treatment for patients with advanced EGFR-mutated NSCLC. Both gefitinib and erlotinib can be used in a Japanese clinical setting. The approved daily dose of erlotinib (150 mg) is equal to the maximum tolerated dose of erlotinib. In contrast, the daily dose of gefitinib has been set at 250 mg, which is approximately one-third of the maximum tolerated dose of gefitinib. Accordingly, a higher serum concentration can be achieved using erlotinib, compared with gefitinib. This advantage can be applied to the treatment of central nervous system metastases (brain metastasis and carcinomatous meningitis), the treatment of which is complicated by the difficulty drugs have penetrating the blood-brain barrier. Although patients with EGFR-mutated NSCLC respond dramatically to EGFR-TKIs, some patients have a poor response and the majority eventually undergo disease progression. To overcome such resistance, several novel treatment strategies, such as combination therapy and next-generation EGFR-TKIs, have been attempted.

DOI： 10.2147/DDDT.S50358

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ADIS INT LTD  

Erlotinib shows large inter-patient pharmacokinetic variability, but the impact of early drug exposure and genetic variations on the clinical outcomes of erlotinib remains fully investigated. The primary objective of this study was to clarify the population pharmacokinetics/pharmacodynamics of erlotinib in Japanese patients with non-small cell lung cancer (NSCLC). The secondary objective was to identify genetic determinant(s) for the cerebrospinal fluid (CSF) permeability of erlotinib and its active metabolite OSI-420.
A total of 88 patients treated with erlotinib (150 mg/day) were enrolled, and CSF samples were available from 23 of these patients with leptomeningeal metastases. Plasma and CSF concentrations of erlotinib and OSI-420 were measured by high-performance liquid chromatography with UV detection. Population pharmacokinetic analysis was performed with the nonlinear mixed-effects modelling program NONMEM. Germline mutations including ABCB1 (1236C &gt; T, 2677G &gt; T/A, 3435C &gt; T), ABCG2 (421C &gt; A), and CYP3A5 (6986A &gt; G) polymorphisms, as well as somatic EGFR activating mutations if available, were examined. Early exposure to erlotinib and its safety/efficacy relationship were evaluated.
The apparent clearance of erlotinib and OSI-420 were significantly decreased by 24 and 35 % in patients with the ABCG2 421A allele, respectively (p &lt; 0.001), while ABCB1 and CYP3A5 polymorphisms did not affect their apparent clearance. The ABCG2 421A allele was significantly associated with increased CSF penetration for both erlotinib and OSI-420 (p &lt; 0.05). Furthermore, the incidence of grade a parts per thousand yen2 diarrhea was significantly higher in patients harboring this mutant allele (p = 0.035). A multivariate logistic regression model showed that erlotinib trough (C-0) levels on day 8 were an independent risk factor for the development of grade a parts per thousand yen2 diarrhea (p = 0.037) and skin rash (p = 0.031). Interstitial lung disease (ILD)-like events occurred in 3 patients (3.4 %), and the median value of erlotinib C-0 levels adjacent to these events was approximately 3 times higher than that in patients who did not develop ILD (3253 versus 1107 ng/mL; p = 0.014). The objective response rate in the EGFR wild-type group was marginally higher in patients achieving higher erlotinib C-0 levels (a parts per thousand yen1711 ng/mL) than that in patients having lower erlotinib C-0 levels (38 versus 5 %; p = 0.058), whereas no greater response was observed in the higher group (67 %) versus the lower group (77 %) within EGFR mutation-positive patients (p = 0.62).
ABCG2 can influence the apparent clearance of erlotinib and OSI-420, and their CSF permeabilities in patients with NSCLC. Our preliminary findings indicate that early exposure to erlotinib may be associated with the development of adverse events and that increased erlotinib exposure may be relevant to the antitumor effects in EGFR wild-type patients while having less of an impact on the tumor response in EGFR mutation-positive patients.

DOI： 10.1007/s40262-013-0058-5

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End-stage cancer patients frequently receive continuous morphine infusion (CMI) to alleviate the various symptoms associated with cancer progression or adverse events; however, there have been a limited number of studies concerning such patients. We conducted a retrospective analysis of 79 end-stage lung cancer patients who received CMI at the Kyoto University Hospital, Kyoto, Japan between 2008 and 2010. Thirty-one patients (39%) received CMI intravenously and 48 (61%) received it subcutaneously. The patients were divided into four groups based on the indications for CMI: group A (uncontrolled pain; n=9), group B (dyspnea; n=44), group C (both dyspnea and pain; n=13) and group D (an inability to take oral medicine; n=13). The median maximum dose of morphine in groups A-D was 60.0, 25.0, 50.0 and 15.0 mg/day, respectively. The median survival time from the start of CMI was 4 days (range 0-136). In our limited experience, pain, dyspnea and the inability to take oral medicine were identified as indications for CMI in end-stage lung cancer patients, with dyspnea being the major indication for CMI. Patients in group B (dyspnea) required a lower dose of morphine for alleviation compared with those in groups A (uncontrolled pain) and C (both dyspnea and pain). The survival time from the initiation of CMI was markedly shorter in patients with dyspnea (groups B and C) than in patients without dyspnea (group A). Further studies are required to facilitate the effective and appropriate use of CMI in end-stage lung cancer patients. Dyspnea was the major indication for CMI in end-stage lung cancer patients, and the survival time was extensively limited in such patients.

DOI： 10.3892/ol.2012.1101

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Pneumocystis jiroveci pneumonia (PCP) has long been recognized as a cause of mortality in immuno-compromised populations, including those with advanced lung cancer. Although Pneumocystis colonization has only recently been described due to the development of more sensitive molecular techniques, including polymerase chain reaction (PCR), it is unknown whether Pneumocystis colonization leads to the development of PCP. In the present study, we aimed to determine the prevalence of Pneumocystis colonization in advanced lung cancer patients. Furthermore, the association between PCP and Pneumocystis colonization was also investigated. Advanced lung cancer patients with no indication of PCP were evaluated to determine the prevalence of Pneumocystis colonization. We analyzed their oral wash (OW) samples and retrospectively evaluated advanced lung cancer patients with PCP by analyzing their sections of formalin-fixed, paraffin-embedded lung tissues obtained following a diagnosis of lung cancer. Pneumocystis colonization was determined by a PCR test for Pneumocystis jiroveci (P. jiroveci). No P. jiroveci was detected by PCR in the OW samples of 47 advanced lung cancer patients with no indication of PCP, or in the lung tissues of four advanced lung cancer patients with PCP. These results indicate that PCP is not associated with Pneumocystis colonization in advanced lung cancer patients, although this study is limited since this was a cross-sectional and retrospective study.

DOI： 10.3892/ol.2012.1052

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INTRODUCTION: This study aimed to evaluate the association between BCL2 single-nucleotide polymorphisms and survival outcome in advanced non-small cell lung cancer (NSCLC). METHODS: One hundred and sixty-eight patients with advanced NSCLC who were treated with anti-cancer drugs and could be evaluated for therapeutic response between April 2005 and March 2010 at Kyoto University Hospital were enrolled. DNA was extracted from peripheral blood samples. The BCL2 polymorphisms -938 C→A (rs2279115) and +21 A→G (rs1801018) were genotyped using the 5'-nuclease assay. The univariate relationship between each independent clinicopathologic variable and BCL2 genotype was examined using Fisher's exact test. To evaluate risk factors associated with prognosis, a Cox proportional hazards regression model with a step-down procedure was used. RESULTS: The median survival time of patients with the -938 AA and AC genotypes were significantly shorter than those with the -938 CC genotype (p = 0.027 by log-rank test). Based on multivariate analysis, poor performance status [hazard ratio (HR) 2.424, 95% confidence interval (CI) 1.727-3.262; p < 0.0001], non-adenocarcinoma histology (HR 1.512, 95% CI 1.167-1.938; p = 0.0048) and the BCL2 -938 AA + AC genotype (HR 1.219, 95% CI, 1.024-1.456; p = 0.0256) were significant independent prognostic factors for survival. CONCLUSIONS: Polymorphisms in BCL2 may be associated with survival in advanced-stage NSCLC patients who received chemotherapy.

DOI： 10.1159/000342854

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

Mutation of the epidermal growth factor receptor gene (EGFR mutation) is a very important marker in the treatment for non-small cell lung cancer. Since signaling from this receptor induces tumor-associated angiogenesis, we hypothesized that lung cancers with EGFR mutations tend to develop locally with increased angiogenesis. Thus, the association between vascular-poor area of primary tumors and EGFR status was retrospectively investigated in advanced lung adenocarcinomas. To assess vascular-poor area, contrast-enhanced computed tomography scans taken before initial treatment for lung cancer were analyzed, together with primary tumor location (peripheral or central) and size. We analyzed 178 patients with advanced lung adenocarcinoma. EGFR mutations were detected in 95 of the 178 patients (53.4 %). EGFR mutation was found to be significantly related to women (P = 0.0070), never-smokers (P &lt; 0.0001), and tumors without vascular-poor area (P &lt; 0.0001). Based on a multivariate analysis, presence of EGFR mutations was independently associated with never-smokers (P = 0.0046), lack of vascular-poor area (P = 0.0001), and tumor size &gt; 30 mm (P = 0.0080). EGFR mutations were found in 41 of 51 never-smokers without vascular-poor area (80.4 %), 19 of 36 never-smokers with vascular-poor area (52.8 %), 19 of 37 current or former-smokers without vascular-poor area (51.4 %), and 16 of 54 current or former-smokers with vascular-poor area (29.6 %). This study showed an association between vascular-poor area of primary tumors and EGFR status. As a consequence, evaluation using a combination of smoking status and vascular-poor area allows us to predict presence of EGFR mutations at a high frequency.

DOI： 10.1007/s12032-012-0235-7

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A study of patients with advanced non-squamous non-small cell lung cancer (NSCLC) evaluated epidermal growth factor receptor (EGFR) mutation status and serum hepatocyte growth factor (HGF) for their associations with response to gefitinib therapy and prognostic impact. An enzyme-linked immunosorbent assay was used to determine levels of HGF in serum from 96 Japanese patients with advanced non-squamous NSCLC. The peptic nucleic acid-locked nucleic acid clamp method was used to determine their EGFR somatic mutation status. We evaluated the relationship between each independent clinicopathological variable and the response to gefitinib therapy and risk factors associated with prognosis. HGF-positive serum status (hazard ratio, 1.536; 95% confidence interval, 1.042-2.400; P = 0.0295) had a significant and independent negative effect on progression-free survival among patients with wild-type EGFR. We demonstrate that having HGF-positive serum is predictive of a negative response to gefitinib therapy in patients with advanced NSCLC who harbor wild-type EGFR.

DOI： 10.1007/s12032-011-0009-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CIG MEDIA GROUP, LP  

The insulin-like growth factor (IGF) pathway plays an important role in tumor progression. We examined immunohistochemical expression of both IGF receptor-1 (IGF-1R) and IGF binding protein 3 in 68 patients with advanced non-small-cell lung cancer and found that IGF-1R expression was significantly more frequent in squamous cell carcinoma (100%) than in adenocarcinoma (44%) (P &lt; .001). IGF-1R antibody may be useful in the pathologic diagnosis in non-small-cell lung cancer.
Background: The insulin-like growth factor (IGF) pathway plays an important role in cell proliferation, differentiation, and apoptosis, and IGF induces those effects mainly through IGF receptor-1 (IGF-1R). The activities of IGF are strictly regulated by a family of IGF binding proteins (IGFBP), especially IGFBP3, a major serum carrier protein for IGF. Patients and Methods: Between January 2006 and February 2009, in our hospital, 191 patients were histologically diagnosed as having non-small-cell lung cancer (NSCLC), and 74 patients were treated by chemotherapy alone. We examined immunohistochemical expression of both IGF-1R and IGFBP3 in 68 patients who were definitively diagnosed as having adenocarcinoma or squamous cell carcinoma among the 74 patients. Results: The clinical characteristics of the included patients were as follows: median age was 68 years (range, 29-86 years); men vs. women, 40 vs. 28; stage III vs. IV, 18 vs. 50; performance status 0-1 vs. 2-4, 58 vs. 10; smoker vs. non-smoker, 44 vs. 24; and squamous cell carcinoma vs. adenocarcinoma, 13 vs. 55. Expression of IGF-1R and IGFBP3 was observed in 37 (54%) and 11 patients (16%), respectively. IGF-1R expression was detected more frequently in patients with squamous cell carcinoma (100%) than in patients with adenocarcinoma (44%) (P &lt; .001), although IGFBP3 expression was not significantly associated with any clinical variables. Among all factors, including IGF-1R and IGFBP3 expression, IGF-1R was significantly associated with response to chemotherapy (P = .028) and performance status was significantly associated with overall survival (P &lt; .001). Conclusions: High sensitivity of IGF-1R to squamous cell carcinoma (100%) in this study and another study encourages the use of IGF-1R antibody in the pathologic diagnosis between squamous cell and non-squamous cell carcinoma when using small biopsy specimens.

DOI： 10.1016/j.cllc.2011.11.009

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Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

DOI： 10.1161/CIRCULATIONAHA.112.105304

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Several cases have been reported in which central nervous system (CNS) metastases of non-small cell lung cancer (NSCLC) resistant to gefitinib were improved by erlotinib. However, there has been no study in which cerebrospinal fluid (CSF) concentrations of gefitinib and erlotinib are directly compared. Thus, we aimed to compare them.
We examined 15 Japanese patients with NSCLC and CNS metastases with epidermal growth factor receptor gene mutations who received CSF examinations during epidermal growth factor receptor-tyrosine kinase inhibitors treatment (250 mg daily gefitinib or 150 mg daily erlotinib). Plasma and CSF concentrations were determined using high-performance liquid chromatography with tandem mass spectrometry.
The concentration and penetration rate of gefitinib (mean +/- A standard deviation) in the CSF were 3.7 +/- A 1.9 ng/mL (8.2 +/- A 4.3 nM) and 1.13 +/- A 0.36 %, respectively. The concentration and penetration rate of erlotinib in the CSF were 28.7 +/- A 16.8 ng/mL (66.9 +/- A 39.0 nM) and 2.77 +/- A 0.45 %, respectively. The CSF concentration and penetration rate of erlotinib were significantly higher than those of gefitinib (P = 0.0008 and &lt; 0.0001, respectively). The CNS response rates of patients with erlotinib treatment were preferentially (but not significantly) higher than those with gefitinib treatment. (1/3 vs. 4/7, respectively). Leptomeningeal metastases in one patient, which were refractory to gefitinib, dramatically responded to erlotinib.
This study suggested that higher CSF concentration could be achieved with erlotinib and that erlotinib could be more effective for the treatment for CNS metastases, especially leptomeningeal metastases, than gefitinib.

DOI： 10.1007/s00280-012-1929-4

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Language：English   Publisher：ELSEVIER IRELAND LTD  

The most serious adverse reaction associated with treatment with epidermal growth factor receptortyrosine kinase inhibitors (EGFR-TKIs) is drug-induced interstitial lung disease (ILD). Because EGFR-TKIs are key drugs for patients with non-small cell lung cancer who have somatic activating mutations of the epidermal growth factor receptor gene (EGFR mutations), several cases of retreatment with EGFR-TKIs after ILD induced by these drugs have been reported. Here, we present a 68-year-old man with lung adenocarcinoma and leptomeningeal metastases having an EGFR mutation who was retreated with erlotinib after erlotinib-induced ILD. He suffered no ILD recurrence and his leptomeningeal metastases dramatically improved. In addition to the present case, reports of nine patients who were retreated with EGFR-TKIs after ILD were found in the literature. Only one patient had recurrence of ILD (although seven were retreated at a reduced dose of EGFR-TKIs, including the patient with recurrence). In contrast, three patients had no recurrence of ILD even without dose-reduction. These reports suggest that dose-reduction plays a limited role in preventing recurrence. Many patients received corticosteroids during retreatment, but not the one with recurrence of ILD. This may suggest that corticosteroids can prevent recurrence due to their antiinflammatory properties. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.lungcan.2012.04.013

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Subgroup analyses of randomized studies have consistently shown that pemetrexed is exclusively effective in non-small-cell lung cancer (NSCLC) other than squamous cell carcinoma and the combination of pemetrexed and platinum agents is recommended for first-line chemotherapy in advanced non-squamous NSCLC; however, there have been few prospective studies of a selected population.
This was a single-arm phase II study of carboplatin and pemetrexed in Japanese patients with chemo-naive advanced non-squamous NSCLC. Patients received six cycles of pemetrexed (500 mg/m(2)) combined with carboplatin (area under the curve: AUC 6) every 3 weeks. Maintenance chemotherapy with pemetrexed was permitted in patients whose disease did not progress after combination chemotherapy. The primary endpoint was the response rate, and secondary endpoints were safety and survival.
Fifty-one patients were enrolled between November 2009 and March 2011, and 49 patients were evaluable for both safety and efficacy. All but one patient had adenocarcinoma histology. Forty-four (90 %) patients completed four cycles, and 33 (67 %) completed six cycles of chemotherapy. Partial response was achieved in 25 patients (response rate: 51 %) and stable disease in 18 patients (37 %). Median progression-free survival (PFS) and overall survival (OS) were 6.3 months and 24.3 months, respectively. The median PFS and OS were 7.9 months and 24.3 months in patients with epidermal growth factor receptor (EGFR) mutation, and 6.3 months and 21.0 months in patients with EGFR wild type or unknown. There were no statistical differences between EGFR mutants and non-mutants for both PFS (p = 0.09) and OS (p = 0.23). Grade 3/4 neutropenia and thrombocytopenia were observed in 16 (33 %) and 9 (18 %) patients, respectively. Non-hematologic toxicities were generally mild, and there were no treatment-related deaths.
The combination of carboplatin and pemetrexed was safe and effective in advanced non-squamous NSCLC. Although the sample size was small, our results indicate that pemetrexed is a key drug for advanced non-squamous NSCLC, irrespective of the EGFR mutation status (UMIN-CTR number 000002451).

DOI： 10.1007/s00280-012-1910-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CIG MEDIA GROUP, LP  

We retrospectively investigated patients with small-cell lung cancer with or without interstitial lung disease (ILD). Response rates and median progression-free survival of first-line chemotherapy in patients with or without preexisting ILD was not significantly different. However, pneumonitis associated with chemotherapy was significantly increased in patients with preexisting ILD, and preexisting ILD is an independent prognostic factor for poorer survival.
Background: In Japan, iatrogenic acute exacerbation of interstitial lung disease (ILD) is a serious complication in patients with lung cancer and simultaneous ILD. Results of some reports suggest that patients with ILD and small-cell lung cancer (SCLC) might benefit from chemotherapy, but the influence of ILD on prognosis is unclear. Patients and Methods: Retrospective study of patients with SCLC with or without ILD. Between April 2006 and March 2011, 122 patients with SCLC who were receiving platinum-based combination chemotherapy participated. Results: Twenty-eight patients (23.0%) had ILD at diagnosis. Pneumonitis associated with chemotherapy, including acute exacerbation-ILD was significantly increased in patients with preexisting ILD (8/28 vs. 2/94; P = .0001). In patients receiving chemotherapy alone, response rates and median progression-free survival of first-line chemotherapy in patients with or without preexisting ILD was not significantly different (P = .26; 20/26 vs. 52/60 and P = .089; 4.4 months vs. 5.4 months, respectively). The median overall survival of all patients was 15.5 months, but those without preexisting ILD survived significantly longer (P = .0010; 17.8 months vs. 10.7 months). Multivariate analysis revealed that performance status of 0 or 1 (hazard ratio [HR] 0.19 [95% confidence interval {CI}, 0.10-0.37]; P &lt; .0001) limited disease (HR 0.42 [95% CI, 0.23-0.73]; P = .0017), and no preexisting ILD (HR 0.36 [95% CI, 0.19-0.69]; P = .0027) were significantly associated with longer overall survival. Conclusion: Patients with SCLC and ILD might benefit from chemotherapy, but preexisting ILD is an independent prognostic factor for poorer survival.

DOI： 10.1016/j.cllc.2011.11.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

Pemetrexed (PEM) is a novel, multitargeted, antifolate, antineoplastic agent for the treatment of non-small cell lung cancer and malignant pleural mesothelioma. Additional effects of nitric oxide (NO) donors on the chemosensitivity of cancers have been reported. However, the effects of an NO donor on PEM-induced cytotoxicity remain unknown. In this study, we investigated the effects of the NO donors, NOC-18 on the cytotoxicity in A549 cells in vitro and of nitroglycerin (GTN), on the tumor growth of Lewis lung carcinoma cells in a murine syngraft model treated with PEM. The effects of NO donors on the expression of proteins associated with PEM metabolism, including thymidylate synthase (TS), reduced folate carrier 1 (RFC1), folylpolyglutamate synthase (FPGS), gamma-glutamyl hydrolase (GGH) and multidrug resistance-related protein (MRP)5, and the effects of cyclic guanosine monophosphate (cGMP) signaling on these proteins were examined in A549 cells. Treatment with 100 nM NOC-18 for 3 days significantly enhanced PEM-induced cytotoxicity and increased the expression of RFC1 and FPGS in A549 cells. Treatment with 10 nM 8-bromo-cGMP (8-Br-cGMP) for 3 days also increased the expression of RFC1 and FPGS in A549 cells. 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 mu m) significantly reversed the increase in RFC1 and FPGS expression induced by 100 nM NOC-18 in A549 cells. Combination therapy with GTN and PEM significantly reduced tumor growth compared with PEM alone in the syngraft model. The enhanced antitumor effect of GTN plus PEM was significantly reversed by the concomitant addition of ODQ. These findings suggest that NO donors, such as NOC-18 and GTN, enhance the anticancer effects of PEM by increasing the RFC1 and FPGS expression and stimulating cGMP signaling pathways in cancer cells.

DOI： 10.3892/ijo.2012.1461

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Previous reports have shown that circulating endothelial progenitor cells (CEPs) are released in response to cytotoxic chemotherapy. We investigate the relationship between the kinetics of CEPs during one cycle of chemotherapy and the response to cytotoxic chemotherapy and prognostic impacts. Previously untreated patients (n = 38) receiving cytotoxic chemotherapy for non-small-cell lung cancer were included. Blood sampling was carried out on day 1, day 8, and just before the second cycle of chemotherapy. The mononuclear cell fraction was analyzed for CEPs by FACS analysis. We evaluated the relationship between the kinetics of CEPs, each independent clinicopathological variable, the response to chemotherapy, and the risk factors associated with prognosis. On the eighth day after chemotherapy, a significant decrease in CEPs was observed. In contrast, CEP counts before the second cycle of chemotherapy were significantly increased. The high percentage change in CEPs between day 1 and before the second cycle of chemotherapy is an independent predictive factor for response to chemotherapy. However, the change in CEP levels did not predict progression-free survival. These findings indicate that the late release of CEPs is a common phenomenon after chemotherapeutic treatment. The correlation with clinical response to chemotherapy provides further support for the biologic relevance of these cells in patients' prognosis and highlights the potential use of CEPs as therapeutic targets. (Cancer Sci 2012; 103: 10651070)

DOI： 10.1111/j.1349-7006.2012.02249.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

A standard, valid assay of comorbidities for elderly patients with advanced non-small-cell lung cancer (NSCLC) who have received antitumor therapy is needed to provide useful prognostic information. The aim of this study was to analyze prognostic factors and validate classic Charlson comorbidity index (CCI) and comorbidity scores in elderly patients with advanced NSCLC treated with chemotherapy or epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). A retrospective analysis was conducted on 162 patients with advanced NSCLC over 70 years old at diagnosis, who were treated with cytotoxic chemotherapy or with EGFR-TKIs between April 2003 and April 2009 at Kyoto University Hospital. Collected data included clinical assessments, treatments, toxicities, and outcomes. Survival was estimated using the Kaplan-Meier method. Prognostic factors were evaluated with log-rank and Cox regression tests. Based on multivariate analysis, unspecified NSCLC histology [Hazard ratio (HR), 1.631; 95% Confidence interval (CI), 1.184-2.263; P = 0.0016], more than 3 comorbidities (HR, 1.317; 95% CI, 1.020-2.675; P = 0.0350), and a CCI of more than 3 (HR, 1.321; 95% CI, 1.031-1.664; P = 0.0285) were significant independent negative prognostic factors for survival. Our results indicate that CCI and the number of comorbidities are independent predictors of survival in elderly patients undergoing systemic chemotherapy including EGFR-TKIs for advanced NSCLC. These factors should be taken into consideration in the pretreatment assessment as important factors predicting survival outcome.

DOI： 10.1007/s12032-010-9764-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

This study of patients with advanced non-squamous non-small cell lung cancer (NSCLC) evaluated epidermal growth factor receptor (EGFR) mutation status and two serum markers, serum insulin-like growth factor 1 (IGF1) and IGF binding protein 3 (IGFBP3), for their associations to response to gefitinib therapy and for their prognostic impact. An immunoradiometric assay determined levels of IGF1 and IGFBP3 in serum from 68 patients with advanced non-squamous NSCLC. The peptic nucleic acid locked nucleic acid clamp method determined their EGFR somatic mutation status. We evaluated the relationship between each independent clinicopathological variable and the response to gefitinib therapy and the risk factors associated with prognosis. Having IGF1-positive serum as determined by the 75th percentile and having wild-type EGFR were both independent negative predictive factors for geftinib treatment by multivariate logistic regression model analysis. Both having serum positive for IGF1 as determined by the 25th percentile and having wild-type EGFR were significant independent negative prognostic factors for survival based on multivariate analysis. We demonstrated that having IGF1-positive serum predicts a negative response to gefitinib therapy independent of EGFR mutational status. We also demonstrated that both IGF1-positive serum and wild-type EGFR were independent poor prognostic factors in patients with non-squamous NSCLC who received gefitinib therapy.

DOI： 10.3892/or.2011.1354

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Purpose: The maximum tolerated dose (MTD) of erlotinib (150 mg) is the approved daily dose. In contrast, the approved daily dose of gefitinib (250 mg) is only one-third of its MID. Significantly different adverse events have been associated with gefitinib and erlotinib.
Experimental design: A retrospective investigation examining the adverse events and tolerances of 250 mg daily gefitinib and 150 mg daily erlotinib in Japanese patients with non-small cell lung cancer (NSCLC) was performed. Adverse events were assessed according to Common Terminology Criteria for Adverse Events version 3.0. To determine tolerance for each agent, failure was defined as dose reduction or discontinuation of the drug due to adverse events, and early failure as dose reduction or discontinuation due to adverse events before the first evaluation of response.
Results: More adverse events including skin disorders, diarrhea, oral mucositis, asthenic conditions, anorexia, nausea, vomiting, and gastrointestinal bleeding were observed in the erlotinib group. Liver function test abnormalities and pneumonitis did not differ between the two groups. Based on multivariate analysis, failure, early failure, and discontinuation due to adverse events were independently associated with erlotinib use.
Conclusion: Our data show that 150 mg daily erlotinib was associated with more toxicity and less tolerability than 250 mg daily gefitinib. (C) 2011 Published by Elsevier Ireland Ltd.

DOI： 10.1016/j.lungcan.2011.01.022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Purpose Recent reports indicate that refractory central nervous system (CNS) metastases of non-small cell lung cancer (NSCLC) are improved by high-dose gefitinib or erlotinib administration. Wedescribe a Japanese woman with NSCLC and CNS metastases who was resistant to 75 mg daily erlotinib, but the metastases were improved by 150 mg daily erlotinib. We investigated the plasma and CSF concentrations of erlotinib at each dose as well as the correlation between the plasma and CSF concentrations of erlotinib.
Methods Including this patient, we administered 150 mg erlotinib daily to nine NSCLC patients with CNS metastases and measured the plasma and CSF concentrations just before administration on day 8. The concentrations were determined using high-performance liquid chromatography with ultraviolet detection.
Results The plasma and CSF concentrations of erlotinib at a dose of 75 mg were 433 and 14 nM, respectively. The plasma and CSF concentrations of erlotinib at a dose of 150 mg were increased to 1,117 and 44 nM, respectively. The mean +/- standard deviation of CSF concentrations and penetration rates were 106 +/- 59 nM and 4.5 +/- 1.5%, respectively. There was a good correlation (R(2) = 0.84) between plasma and CSF concentrations (P = 0.0005).
Conclusions This study indicates that CSF concentrations of erlotinib depend on its plasma concentration. As seen in this patient, high CSF concentrations of erlotinib can be achieved by high-dose administration, and this finding suggests the efficacy of high-dose administration, especially to refractory CNS metastases of NSCLC patients.

DOI： 10.1007/s00280-011-1691-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CIG MEDIA GROUP, LP  

Background: Erlotinib is orally active and selectively inhibits the tyrosine kinase activity of the epidermal growth factor receptor. The pleural space penetration and exposure of erlotinib is poorly understood. Thus, we investigated the pharmacokinetics (PK) of erlotinib and its active metabolite OSI-420 in non-small-cell lung cancer (NSCLC) of malignant pleural effusion (MPE). Patients and Methods: We analyzed the PK of erlotinib and OSI-420 on days 1 and 8 after beginning erlotinib therapy in 9 patients with MPE. Their concentrations were determined by high-performance liquid chromatography with ultraviolet detection. Blood samples were obtained five times per day: before administration, and 2, 4, 8, and 24 hours after administration. Pleural effusions were obtained once per day, 2 hours after administration on day 1, and before administration on day 8. The exceptions were cases 2 and 4, which had pleural effusions obtained just before drug administration, and 2, 4, 8, and 24 hours after administration. Results: The mean percentage of penetration from plasma to pleural effusion for erlotinib was 18% on day 1 and 112% on day 8, while these values for OSI-420 were 9.5% on day 1 and 131% on day 8. The area under the drug concentration-time curve of pleural fluid for erlotinib was 28,406 ng-hr/mL for case 2 and 45,906 ng-hr/mL for case 4. Conclusions: There seems to be a significant accumulation of both erlotinib and OSI-420 in MPE with repeated dosing. Although larger studies will be necessary to determine the true impact of erlotinib MPE accumulation on plasma PK and safety, erlotinib can be administered safely to patients with MPE with respect to efficacy and side effects.

DOI： 10.1016/j.cllc.2011.06.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

Internal jugular vein thrombosis is much less common than deep venous thrombosis of lower limbs and is generally caused by an indwelling venous catheter or otological infection. Several cases of internal jugular vein thrombosis associated with malignancy have been also reported. Bevacizumab, a monoclonal neutralizing antibody against vascular endothelial growth factor, has shown benefits in the treatment of many types of malignancy and its use is increasing. Serious adverse effects, however, are associated with the use of bevacizumab, including venous thromboembolism. In this article, we present a rare case of non-small cell lung cancer complicated by pulmonary embolism due to internal jugular vein thrombosis associated with bevacizumab.

DOI： 10.1007/s10147-010-0148-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CLINICAL ONCOLOGY  

DOI： 10.1200/JCO.2010.34.0216

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Cancer-associated retinopathy is a rare paraneoplastic syndrome that is often associated with small-cell lung cancer. It is caused by an autoantibody to the 23 kDa photoreceptor protein, recoverin. A small number of reports have described effective treatment for the disease. We report two cases of cancer-associated retinopathy with small-cell lung cancer whose visual symptom preceded the diagnosis of cancer. Their visual acuity and visual field were slightly improved after steroid and anticancer therapy. Steroid therapy was effective, although the period from visual symptom onset to therapy was comparative longer. When cancer-associated retinopathy is suspected, a comparatively large quantity of steroids and anticancer treatment should be combined immediately.

DOI： 10.1093/jjco/hyr025

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The prognosis of small-cell lung cancer remains poor and it is speculated that small-cell lung cancer patients with end-stage renal failure undergoing hemodialysis have a poorer prognosis. In this article, we present a Japanese woman with extensive small-cell lung cancer with end-stage renal failure undergoing hemodialysis who received multiple courses of chemotherapy. Although she achieved long-term survival of more than 2 years, the last-line chemotherapy, consisting of irinotecan, induced grade 4 febrile neutropenia and her performance status deteriorated even with a reduced dose according to the analysis of UDP-glucuronosyltransferase 1A1. We also conducted a pharmacokinetic analysis of irinotecan, the resulting values of which were much higher than in previous reports. Although further studies are needed, chemotherapy for small-cell lung cancer patients should not be withheld just because they are undergoing hemodialysis; however, chemotherapy should be performed more carefully because more severe toxicities can occur than expected.

DOI： 10.1093/jjco/hyq244

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

Somatic mutations of epidermal growth factor receptor (EGFR) are the strongest predictive markers for the response to EGFR-tyrosine kinase inhibitors (TKIs). Patients with EGFR mutations generally receive EGFR-TKI treatment, and their survival has been significantly improved compared with that before the development of EGFR-TKIs. This study aimed to clarify the impact of EGFR mutational status on the survival of patients with non-small cell lung cancer (NSCLC) receiving cytotoxic agents, but not EGFR-TKIs, as their first-line chemotherapy. In addition, we analyzed patients with EGFR mutations to determine whether the timing of EGFR-TKI administration affects overall survival (OS). A total of 83 NSCLC patients with stage IIIB/IV who received chemotherapy alone and whose EGFR mutational status was known were investigated. Univariate and multivariate analysis for OS was performed using parameters such as age, gender, performance status (PS), histology, disease stage, smoking status, EGFR mutational status and administration of a first-line regimen. Among the 52 patients with EGFR mutations who received EGFR-TKIs, OS between those who received EGFR-TKIs as their first-line treatment and after chemotherapy were similar. Among the 83 patients who received cytotoxic agents as their first-line chemotherapy, the multivariate analysis showed OS to be significantly associated with PS (p &lt; 0.001), histology (p=0.039) and EGFR mutational status (p=0.040). OS was almost similar among the 52 patients with EGFR mutations who received EGFR-TKIs in a first- and second-line setting (25.6 vs. 26.8 months, p=0.914). The EGFR mutational status had a significant impact on the survival of NSCLC patients, although these patients did not receive EGFR-TKIs as their first-line chemotherapy. In future randomized trials, even when EGFR-TKIs are not included in experimental regimens, patients may need to be stratified by EGFR mutational status in order that study results be evaluated appropriately.

DOI： 10.3892/ol.2011.240

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

This study aimed to investigate the relationship between clinicopathological factors and plasma brain natriuretic peptide (BNP) levels in non-small cell lung cancer (NSCLC) patients. A total of 133 patients with advanced NSCLC were included in this study. The level of BNP was determined at the time of diagnosis. The BNP plasma concentration was measured using a chemiluminescent enzyme immunoassay kit. The univariate relationship between each independent clinicopathological variable and plasma BNP was examined using the Chi-square test. The survival curves were determined using the Kaplan-Meier method. According to the cut-off value of plasma BNP levels (11.5 and 22.4 pg/ml), plasma BNP negatively correlated with the presence of metastases (Chi-square test, p=0.0374 and p=0.0098, respectively). However, no significant association between patient survival time and plasma BNP levels was found. Reduced plasma BNP levels in advanced NSCLC patients with metastases were noted and the possibility was raised that BNP decreases distant metastases of advanced NSCLC patients.

DOI： 10.3892/ol.2011.247

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

DOI： 10.1097/JTO.0b013e3182037c80

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

The aims of this study are to: (a) confirm the prognostic significance of the procoagulant molecules D dimer, thrombin-antithrombin complex (TAT), and plasmin-alpha 2-plasmin inhibitor complex (PIC); (b) to evaluate hemostatic activation in patients with advanced non-small cell lung cancer (NSCLC); and (c) to delineate the relationships between markers of hemostasis and other clinical characteristics. In this study, a low PIC/TAT ratio and poor PS were significant independent negative prognostic factors for survival in patients with advanced NSCLC. The PIC/TAT ratio may become a surrogate marker for treatment with anticoagulants in the future.

DOI： 10.1007/s12032-010-9454-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The objectives of this phase I trial were to evaluate the toxicity of the nedaplatin/gemcitabine regimen, determine the maximum tolerated doses (MTDs) of these agents, and observe the anti-tumor effects of this regimen on advanced squamous cell lung cancer.
Patients with previously untreated advanced squamous cell lung cancer were eligible if they had a performance status of 0 or 1 with adequate organ function. The doses of gemcitabine (days 1 and 8) and nedaplatin (day 8) studied were 800/70, 1,000/80, 1,000/90, and 1,000/100 (mg/m(2)), repeated every 3 weeks.
Toxicity and response could be assessed in all 13 patients enrolled. The patients included 12 men and one woman with a median age of 69 years (range 57-81 years). Three patients had stage IIIB disease and 10 patients had stage IV disease. The MTDs were reached at 1,000 mg/m(2) gemcitabine and 80 mg/m(2) nedaplatin. The most frequent toxic effects were thrombocytopenia and neutropenia; grade 3 or 4 thrombocytopenia was observed in 23% of patients, and grade 3 or 4 neutropenia was seen in 46% of patients. Non-hematologic toxicities were mild. Grade 3 fatigue, nausea/vomiting, and appetite loss occurred in two patients. The overall response rate was 62%.
We recommend doses of 800 mg/m(2) gemcitabine and 70 mg/m(2) nedaplatin for phase II study. This combination chemotherapeutic regimen is active and well tolerated.

DOI： 10.1007/s00280-010-1321-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

Transforming growth factor beta (TGF-beta) signaling can inhibit tumor growth in developing tumors. However, it promotes tumor invasiveness and metastasis in late-stage tumors. A number of TGF-beta gene polymorphisms have been identified that can affect the survival of patients with advanced non-small cell lung cancer (NSCLC). In this study, we investigated the association of the TGF-beta 1 polymorphism, C-509T, with survival in patients with advanced NSCLC. Japanese patients who were treated for unresectable advanced NSCLC between April 2003 and March 2008 at Kyoto University Hospital, were enrolled in this study. Analyses of genotype associations with survival outcomes were performed using statistical tests. The median survival of patients with the TT genotype was shorter, although not significantly, than that of patients with either the CT or CC genotype. Based on both univariable and multivariable analyses, the TGF-B1 polymorphism, C-509T, was not associated with prognosis. In patients with a smoking status of &lt;40 pack-years, the median survival was significantly shorter with the TT genotype than with the CT or CC genotype. Based on univariable analysis, stage IV cancer and the TT genotype had a significant prognostic effect on survival. Based on multivariable analysis, the TT genotype was a significantly independent prognostic factor for survival. There was no association between the TGF-beta 1 polymorphism, C-509T, and survival in patients with advanced NSCLC. In patients with a smoking status of &lt;40 pack-years, however, the TGF-beta 1 polymorphism, C-509T, was significantly associated with the prognosis of advanced NSCLC, and the TT genotype was an independent prognostic factor for poor survival.

DOI： 10.3892/or.2010.1098

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

BACKGROUND: Although the association of multiple pulmonary metastases, and particularly miliary metastases, with response to gefitinib treatment in patients with nonsmall cell lung cancer has been reported, the association of miliary pulmonary metastases with epidermal growth factor receptor gene (EGFR) mutations remains unclear. METHODS: The authors retrospectively investigated the association of diffuse, random pulmonary metastases in patients with lung adenocarcinoma. The study included 163 Japanese patients who had unresectable, advanced lung adenocarcinoma diagnosed between April 2003 and March 2010. Computed tomography scans that were obtained at the time of diagnosis were analyzed by 2 investigators. For the purposes of this study, diffuse, random pulmonary metastases were defined as multiple nodules (n = 50; &lt;= 3 cm in greatest dimension) distributed diffusely and randomly throughout the lungs. RESULTS: Of 163 patients, 55 had pulmonary metastases, and EGFR mutations were detected in 22 of those 55 patients. The mutations were identified preferentially among women (P = .15) and were identified significantly among patients who had a smoking history of &lt;10 pack-years (P = .0057). Diffuse, random pulmonary metastases were identified in 11 of 22 patients who had EGFR mutations and in 4 of 33 patients who had the wildtype EGFR (P = .0043). On the basis of multivariate analyses, EGFR mutations were associated independently with a smoking history of &lt;10 pack-years (P = .026) and with diffuse, random pulmonary metastases (P = .012). CONCLUSIONS: When patients with lung adenocarcinomas who had EGFR mutations developed pulmonary metastases, they tended to be diffuse and random, including military metastases. However, such metastases were much less common in patients who had lung adenocarcinomas with wild-type EGFR. Cancer 2011;117:819-25. (C) 2070 American Cancer Society

DOI： 10.1002/cncr.25618

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CIG MEDIA GROUP, LP  

Background: The aim of this study was to analyze the factors independent of epidermal growth factor (EGFR) gene mutations that affect the progression-free survival (PFS) of patients with advanced non-small-cell lung cancer (NSCLC) after gefitinib therapy. Patients and Methods: Eighty patients with advanced NSCLC between January 2003 and April 2010 at Kyoto University Hospital were analyzed for EGFR somatic mutations and treated with gefitinib. We adopted the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method for determination of EGFR mutation status. To evaluate risk factors associated with PFS, Cox proportional hazards regression model with a step-down procedure was used. Proportional hazards assumptions were checked and satisfied; only those variables with statistically significant results in univariate analysis were included in a multivariate analysis. Results: The median PFS of patients with EGFR mutations were significantly longer than in patients with wild-type EGFR. The median PFS of patients after first-line gefitinib therapy was significantly longer than those who received treatment as a second-line therapy. The median PFS of patients over 75 years of age was significantly longer than in younger patients. Based on multivariate analysis, wild-type EGFR status and age &lt; 75 years were significant and independent negative factors that affect PFS after gefitinib therapy. Conclusion: In this study, we showed the EGFR mutants and age &gt; 75 years were good predictive factors for PFS after gefitinib therapy, suggesting that first-line gefitinib treatment for older patients is efficacious regardless of EGFR mutational status.

DOI： 10.3816/CLC.2011.n.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TOHOKU UNIV MEDICAL PRESS  

Fibrosing mediastinitis (FM) is a rare benign disorder that is characterized by excessive fibrotic reactions in the mediastinum. FM is associated with various diseases, including Histoplasma capsulatum infection and IgG4-related disease, and may compromise the airways, great vessels, and other mediastinal structures. Chylothorax is not a common manifestation of FM, and there is no standard treatment for FM or chylothorax. Recently, however, somatostatin and octreotide, a somatostatin analogue, were successfully used for the treatment of chylothorax due to various causes, and they are considered as putative therapeutic interventions for chylothorax. Here, we present a 28-year-old Japanese man with chylothorax due to idiopathic FM, who was successfully treated with octreotide. The patient visited our hospital because of dyspnea on exertion. On admission, chest computed tomography revealed pericardial effusion, bilateral pleural effusion, and a mass in the mediastinum. The right pleural effusion appeared chylous, with the triglyceride level of 253 mg/dl. The biopsy specimen from the mediastinal mass showed collagenous fibers and fibroblasts with moderate infiltration of lymphocytes. Neither fungi nor bacteria were cultured from the biopsy specimen. Steroid therapy was not effective. The patient was then treated with subcutaneous octreotide (100 mu g three times daily). Five days after starting the treatment, the drained pleural fluid was decreased to similar to 150 ml/day from similar to 1,000 ml/day. The mediastinal mass decreased in size 2 weeks after the initiation of octreotide treatment. After discharge, the patient has received octreotide treatment for 6 months without serious adverse events. We suggest octreotide as a treatment option for FM.

DOI： 10.1620/tjem.222.51

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

The study aimed to determine the diagnostic utility of procalcitonin (PCT) in order to discriminate between infective fever and fever due to inflammation in febrile advanced lung cancer patients treated with cytotoxic chemotherapy. A total of 121 patients with advanced lung cancer, treated with a cytotoxic chemotherapy regimen between September 2007 and September 2008 at Kyoto University Hospital, were recruited. Blood samples were obtained on the first day of the fever. Serum c-reactive protein (CRP) and PCT levels were measured. At least two blood cultures were performed, and sputum was taken for Gram staining and culture. There were 71 episodes in 61 patients in the 12 months of the study, representing 50.4% of our study population. A total of 41 patients (57.7%) were diagnosed with, pneumonia using imaging modalities, 6 (8.5%) with bacteremia using blood culture and 4 (5.6%) with urinary tract infections using urine culture. Among the 41 pneumonia cases, culture from sputum revealed pathologic bacteria in 21 (51.2%) and fungal disease in 14 (34.1%) cases. Among the 71 febrile episodes, serum procalcitonin and CRP were measured in 50 episodes.. Serum procalcitonin-positive patients showed poor outcomes on antibiotics therapy (Fisher's exact test, p=0.042). Furthermore, serum procalcitonin positivity was able to discriminate infective fever from fever due to inflammation (Chi-square test, p=0.001). We showed the causative organisms of febrile advanced lung cancer patients who received cytotoxic chemotherapy, as well as the possibility of PCT to discriminate infective fever from fever due to inflammation.

DOI： 10.3892/ol_00000150

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Background: Although there have been several reports in which central nervous system (CNS) metastases of non-small cell lung cancer (NSCLC) were improved by erlotinib, cerebrospinal fluid (CSF) penetration of erlotinib in such patients has not been reported. We investigated CSF concentrations of erlotinib and its active metabolite OSI-420.
Method: We administered 150 mg erlotinib daily to four patients with NSCLC who had CNS metastases, and we investigated plasma pharmacokinetics of erlotinib and OSI-420 on days 1 and 8. In addition, we measured the concentrations of erlotinib and OSI-420 in CSF just before administration of erlotinib on day 8.
Results: In all cases except for one case, plasma pharmacokinetics data on day 8 were similar to those previously reported. The mean +/- SD CSF concentrations of erlotinib and OSI-420 were 54 +/- 30 ng/ml and 10.8 +/- 8.2 ng/ml, respectively. The mean +/- SD CSF penetration rates of erlotinib and OSI-420 were 5.1% +/- 1.9% and 5.8% +/- 3.6%, respectively. CSF concentrations of erlotinib exceeded median inhibitory concentration (IC(50)) of erlotinib in intact tumor cells with wild-type epidermal growth factor receptor gene.
Conclusion: The CSF penetrations of erlotinib and OSI-420 in patients with NSCLC who had CNS metastases were approximately 5.1% and 5.8%, respectively. This indicates that erlotinib can become a treatment option for CNS metastases of NSCLC.

DOI： 10.1097/JTO.0b013e3181e2138b

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Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

DOI： 10.1097/JTO.0b013e3181dab0dd

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Introduction: Although erlotinib, an orally active and selective tyrosine kinase inhibitor of epidermal growth factor receptor, is mainly metabolized in the liver, its effectiveness and safety for patients with chronic renal failure (CRF) undergoing hemodialysis (HD) has not been reported. Thus, we investigated the pharmacokinetics (PK) of erlotinib and its active metabolite OSI-420 in such patients with nonsmall cell lung cancer (NSCLC).
Method: We administered 150 mg erlotinib daily to three patients with NSCLC and CRF undergoing HD (HD group) and five patients with NSCLC and normal organ function (control group) and analyzed the PK of erlotinib and OSI-420. In the HD group, PK analyses were performed on day 1 (off HD), day 8 (off HD), and day 9 (on HD) after starting administration of erlotinib, and in the control group, they were performed on day 1 and day 8.
Results: In the HD group, there were little differences in the PK data between day 8 and day 9. The PK data on day 1 and day 8 of the HD group were also similar to those of the control group. There were no serious adverse events in any cases, and one of the HD patients achieved partial response.
Conclusion: Erlotinib was hardly affected by renal function and HD, which confirms the effectiveness and safety of erlotinib treatment in patients with NSCLC and CRF undergoing HD. Erlotinib can become one treatment option for such patients.

DOI： 10.1097/JTO.0b013e3181d32287

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Recently, 2 small molecule kinase inhibitors (TKIs), targeting epidermal growth factor receptor (EGFR), have proven effective in the treatment of non-small cell lung cancer. However, it is unknown whether the EGFR double activating mutation of L858R in exon 21 and the in-frame deletion in exon 19 is a predictor of the effectiveness of EGFR-TKIs. We report for the first time a case of non-small cell lung cancer with central nervous system metastases harboring a rare EGFR double activating mutation who showed a good clinical response to erlotinib, regardless of his poor performance status, as swallowing is not possible. Therefore, we suggest that erlotinib may become a therapeutic choice in cases of central nervous system metastases even with poor performance status.

DOI： 10.1159/000310830

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BACKGROUND: Spinal metastases of patients with advanced stage lung cancer are an important target for palliative therapy, because their incidence is high, and they often cause severe symptoms and worsen the quality of life. Surgery is one of the most effective treatment options, but the indication of surgery is unclear as the procedure is invasive and patients with spinal metastasis have a rather short life expectancy. Furthermore, there have been few studies that have focused on lung cancer with poor prognosis. METHODS: We reviewed all of the cases of lung cancer from January 1999 to July 2007 in the Department of Respiratory Medicine, Kyoto University Hospital, Japan. Thirteen patients with metastatic spinal tumor of lung cancer underwent surgery, and all of them had a poor performance status score (3 or 4). RESULTS: Neurological improvement by at least 1 Frankel grade was seen in 10 of 14 cases (71%). Improvement of the movement capacity was noted in 9 of 14 cases (64%), and pain improvement was noted in 12 of 14 (86%). Median postoperative survival was 5 months (1-25 months). In particular, the group with a good postoperative performance status score (0-2) was shown to have a better median postoperative survival of 13 months. CONCLUSIONS: Surgical treatment for symptomatic metastatic spinal tumor of lung cancer can improve quality of life in a substantially high percentage of patients. Surgery should be considered even if preoperative performance status is poor.

DOI： 10.1159/000299385

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Purpose: We examined patients with advanced nonsquamous, non-small cell lung cancer (NSCLC) to evaluate epidermal growth factor receptor (EGFR) mutation status and serum C-reactive protein (CRP) for their associations with response to gefitinib therapy and for prognostic impacts. Methods: Serum levels of CRP from 79 Japanese patients with advanced nonsquamous NSCLC were measured before the start of gefitinib. We used the peptic nucleic acid-locked nucleic acid clamp method to determine their EGFR somatic mutation status. We evaluated the relationship between each independent clinicopathological variable and the response to gefitinib therapy and the risk factors associated with prognosis. Results: Having CRP-positive serum and having wild-type EGFR were both independent negative predictive factors for the response to gefitinib treatment by multivariate logistic regression model analysis. Having CRP-positive serum and having wild-type EGFR were significant independent negative prognostic factors for survival based on multivariate analysis. Conclusions: Having CRP-positive serum predicted a lack of response to gefitinib therapy independent of EGFR mutational status. Both CRP-positive serum and wild-type EGFR were independent poor prognostic factors in patients with nonsquamous NSCLC who received gefitinib therapy. Copyright (C) 2011 S. Karger AG, Basel

DOI： 10.1159/000323486

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Language：Japanese  

DOI： 10.11406/rinketsu.49.664

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J-GLOBAL

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2008&ichushi_jid=J01540&link_issn=&doc_id=20080903310019&doc_link_id=10021932175&url=http%3A%2F%2Fci.nii.ac.jp%2Fnaid%2F10021932175&type=CiNii&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00003_1.gif

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J-GLOBAL

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC INTERNAL MEDICINE  

This report describes a Japanese family with vessel and craniofacial abnormalities. Although the clinical findings of the patient's father fulfilled the diagnostic criteria for Marfan syndrome, arterial tortuosity, aneurysms, hypertelorism and a bifid uvula were noted in both the patient and his father. These findings were compatible with the clinical manifestations that were previously reported in Loeys-Dietz syndrome. A molecular genetic analysis demonstrated a heterozygous missense mutation of the transforming growth factor-beta receptor II gene in both the patient and his father, which thus caused Loeys- Dietz syndrome. This is the first Japanese family case report of typical Loeys- Dietz syndrome.

DOI： 10.2169/internalmedicine.46.0467

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Language：Japanese   Publisher：(株)羊土社  

がん細胞は免疫から逃避して自身に有利な腫瘍微小環境を構築する「がん免疫編集」により生存し増殖している。免疫チェックポイント阻害薬は抗腫瘍応答を担う細胞傷害性T細胞を活性化させることでがん細胞を排除して効果を発揮するが、がん細胞もさらに抗腫瘍免疫応答から逃れるために不均一な腫瘍微小環境下で自身に有利なクローンを選択することで進化を重ねている。免疫系もまた不均一な集団だが、免疫チェックポイント阻害薬を使用した際にがん細胞を直接攻撃する細胞傷害性T細胞クローンが拡大することが奏効において重要である。がんと免疫系のクロストークのなかで双方でどのようなクローンが選択され、進化しているかを理解することは、免疫チェックポイント阻害薬の奏効や耐性のメカニズムを解明するうえでも重要であると考えている。(著者抄録)

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Language：English   Publisher：(一社)日本癌学会  

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Language：English   Publisher：(一社)日本癌学会  

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Language：English   Publisher：(一社)日本癌学会  

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Language：English   Publisher：(一社)日本癌学会  

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Language：Japanese   Publisher：(株)癌と化学療法社  

がん免疫療法は多くのがん種で有効性が示されている一方、治療効果予測が難しいなど多くの課題がある。腫瘍微小環境において腫瘍細胞は免疫から逃避し増殖するため、対峙する免疫細胞は腫瘍細胞を攻撃するためにお互いに変化している。双方がどのようなクローンを選択し、進化をしているかを明らかにすることは新規治療効果予測バイオマーカーや新規治療法の開発にもつながる。こういった腫瘍微小環境におけるクローン進展を正確に理解するためには、不均一で多様性が高い細胞集団に対して塊(バルク)での解析ではなく1細胞レベルでの解析が必要である。近年、シングルセルシークエンスの登場により網羅的な遺伝子発現を解析できるだけでなく、T細胞受容体配列やB細胞受容体配列など特定の領域に絞って解析することも可能になった。さらに組織の位置情報を保持したまま1細胞レベルに近い形で解析する技術も登場するなど技術革新が進んでいる。最近では、同一患者で治療前後のサンプルを用いるなど時系列的な腫瘍微小環境の変化を追い、腫瘍細胞自体やそれに対峙する免疫細胞のクローン進展を解析することで新たな知見が明らかになっている。(著者抄録)

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Other Link： https://search-tp.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2022&ichushi_jid=J00296&link_issn=&doc_id=20220920380001&doc_link_id=%2Fab8gtkrc%2F2022%2F004909%2F001%2F0907b0913%26dl%3D3&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fab8gtkrc%2F2022%2F004909%2F001%2F0907b0913%26dl%3D3&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_4.gif

Language：Japanese   Publisher：(有)科学評論社  

CiNii Books

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Language：Japanese   Publisher：(有)科学評論社  

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Language：Japanese   Publisher：(有)科学評論社  

CiNii Books

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Language：Japanese   Publisher：(株)篠原出版新社  

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Language：Japanese   Publisher：日本がん免疫学会  

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Language：English   Publisher：(一社)日本リンパ網内系学会  

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Language：Japanese   Publisher：(公社)日本臨床細胞学会  

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Language：Japanese   Publisher：医歯薬出版(株)  

腫瘍微小環境でがん細胞は複雑なネットワークを介して自己に有利な環境を形成している。がん免疫療法はがん細胞に有利な腫瘍微小環境をがん細胞にとって不利な環境に変化させて免疫細胞による抗腫瘍効果を発揮させるが、これらのメカニズムを詳細に解明することは耐性化の克服や新規治療法の開発につながる。安価で簡便に使用できるフローサイトメトリーはシングルセルレベルで解析するツールとして従来から広く使用されており、さらに40分子程度のタンパク質を同時に解析できるマスサイトメトリーが登場したことで、より詳細な分析が可能になり、これらは腫瘍微小環境の解析でも使用されている。近年は腫瘍微小環境で抗腫瘍免疫応答の中心的役割を果たすexhausted T細胞の詳細や、CD4陽性T細胞を含む他の細胞とCD8陽性T細胞の関連性が徐々に解明されてきている。シングルセルレベルでの解析手法が発展することで、より詳細な腫瘍微小環境の解明が期待される。(著者抄録)

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腫瘍微小環境でがん細胞は複雑なネットワークを介して自己に有利な環境を形成している。がん免疫療法はがん細胞に有利な腫瘍微小環境をがん細胞にとって不利な環境に変化させて免疫細胞による抗腫瘍効果を発揮させるが、これらのメカニズムを詳細に解明することは耐性化の克服や新規治療法の開発につながる。安価で簡便に使用できるフローサイトメトリーはシングルセルレベルで解析するツールとして従来から広く使用されており、さらに40分子程度のタンパク質を同時に解析できるマスサイトメトリーが登場したことで、より詳細な分析が可能になり、これらは腫瘍微小環境の解析でも使用されている。近年は腫瘍微小環境で抗腫瘍免疫応答の中心的役割を果たすexhausted T細胞の詳細や、CD4陽性T細胞を含む他の細胞とCD8陽性T細胞の関連性が徐々に解明されてきている。シングルセルレベルでの解析手法が発展することで、より詳細な腫瘍微小環境の解明が期待される。(著者抄録)

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Language：Japanese   Publisher：(一財)日本消化器病学会  

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Language：Japanese   Publisher：Okayama Medical Association  

DOI： 10.4044/joma.133.151

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Language：Japanese   Publisher：岡山医学会  

腫瘍微小環境の解析から明らかになっている免疫チェックポイント阻害剤の効果予測バイオマーカーについて、以下に分けて概説した。1)PD-L1発現、2)腫瘍組織の遺伝子発現、3)1細胞レベルの解析、4)腫瘍浸潤エフェクターT細胞の1細胞レベルの解析、5)腫瘍微小環境に存在するエフェクターT細胞以外の免疫細胞(制御性T細胞、B細胞、腫瘍関連マクロファージ、骨髄由来抑制細胞)の解析、として述べた。

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DOI： 10.5227/skincancer.36.127

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がん免疫療法の有効性が多くのがん種で証明されたが、その治療効果は不十分である。効果予測バイオマーカーやより有効性の高い治療を求めて、抗腫瘍免疫応答の本質を解明するために腫瘍微小環境の解析がさかんに行われているが、免疫細胞は非常に不均一で多様な集団であり、塊(バルク)での解析では本質に迫ることは難しい。そこで、シングルセルレベルでの解析が必要であるが、タンパク質レベルの解析として抗体に蛍光色素をラベルして解析するフローサイトメトリーを代表に、よりマーカーを増やすために金属をラベルした抗体を用いるマスサイトメトリーも使用され、40分子程度までシングルセルレベルで腫瘍微小環境の解析が可能である。さらに網羅的な遺伝子発現をシングルセルレベルで解析できるようにもなっている。また、抗体やMHCマルチマー、抗原ペプチドにオリゴヌクレオチドをラベルしてシークエンスする技術や、T細胞・B細胞受容体配列、オープンクロマチン領域、ゲノム自体も解析することがシングルセルレベルで可能である。加えて、位置情報もシングルセルレベルに近い形で解析できる技術も登場し、次々と腫瘍微小環境に関する新たな知見が明らかになっている。(著者抄録)

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER  

DOI： 10.1016/j.annonc.2021.05.724

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制御性T細胞(Treg)は、自己に対する免疫応答や過度な免疫応答を制御する自己免疫寛容にかかわる免疫細胞である。また、Tregは抗腫瘍免疫応答を抑制することで腫瘍の進展に寄与しているが、不明な点も多い。Tregの機能分類と抑制メカニズム、腫瘍微小環境におけるTregの役割、Tregと免疫チェックポイント分子阻害剤との関係、Tregを標的とした治療法の開発などについて概説した。

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DOI： 10.1200/JCO.2021.39.3_suppl.100

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免疫チェックポイント阻害剤の有効性が多くのがん種で証明されたが、単剤での効果は50%以下と限定的であることから、その耐性メカニズムの解明が有効性を高めるうえでも重要である。初期耐性に加えて、一度効果がみられたあとに耐性化する獲得耐性も問題となっている。免疫チェックポイント阻害剤は細胞傷害性T細胞を活性化させることで有効性を発揮するため、このT細胞活性化のプロセスが耐性メカニズムを考えるうえでも非常に重要で、それに沿ったような耐性メカニズムがさまざまに報告されている。免疫チェックポイント阻害剤の有効性を高めるためにも、それらを明らかにしたうえで、克服するような新たな治療開発が進められている。(著者抄録)

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Language：Japanese   Publisher：(株)羊土社  

免疫チェックポイント阻害剤の有効性が多くのがん種で証明されたが、単剤での効果は50%以下と限定的であることから、その耐性メカニズムの解明が有効性を高めるうえでも重要である。初期耐性に加えて、一度効果がみられたあとに耐性化する獲得耐性も問題となっている。免疫チェックポイント阻害剤は細胞傷害性T細胞を活性化させることで有効性を発揮するため、このT細胞活性化のプロセスが耐性メカニズムを考えるうえでも非常に重要で、それに沿ったような耐性メカニズムがさまざまに報告されている。免疫チェックポイント阻害剤の有効性を高めるためにも、それらを明らかにしたうえで、克服するような新たな治療開発が進められている。(著者抄録)

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Language：Japanese   Publisher：(一社)日本呼吸器学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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不均一な集団である免疫細胞の解析は、1細胞レベルでCD4やCD8といったlineageマーカーや既知のPD-1などに対するモノクローナル抗体を利用してフローサイトメトリーといった機器でなされてきた。しかしながら、抗体や色素に依存してしまうため、既知の分子でかつ抗体が存在するものに限られ、解析できるマーカー数にも限界がある。T細胞やB細胞は多様な抗原を認識するために遺伝子再構成や体細胞超突然変異という機構でその受容体の多様性を獲得している。一方で、その多様性がゆえにT細胞受容体(T cell receptor:TCR)やB細胞受容体(B cell receptor:BCR)の解析を正確に行うことは難しく、従来は存在する限られたモノクローナル抗体を利用してフローサイトメトリーで解析する方法や、塊(バルク)の検体をシークエンスして受容体のレパートリー(レパトア)から頻度や多様性で論じる方法しかなかった。これらの方法では多様な受容体のα/β鎖や重/軽鎖を正確にペアで解析することや、どういったTCRやBCRをもつ細胞がどのような表現型を呈しているのか?といったことを正確に解析することは難しかった。ところが、最近の技術革新によってシングルセルシークエンスが可能となり、遺伝子発現だけでなくTCRやBCRも1細胞ずつ解析できるようになった。網羅的な遺伝子発現と一緒に解析することによってクローンごとの表現型も詳細に明らかにできるようになっている。将来的にはさまざまな疾患での病態解明や個別化療法・バイオマーカー開発といった「個別化免疫医療」につながる可能性がある。(著者抄録)

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Language：Japanese   Publisher：(株)羊土社  

不均一な集団である免疫細胞の解析は、1細胞レベルでCD4やCD8といったlineageマーカーや既知のPD-1などに対するモノクローナル抗体を利用してフローサイトメトリーといった機器でなされてきた。しかしながら、抗体や色素に依存してしまうため、既知の分子でかつ抗体が存在するものに限られ、解析できるマーカー数にも限界がある。T細胞やB細胞は多様な抗原を認識するために遺伝子再構成や体細胞超突然変異という機構でその受容体の多様性を獲得している。一方で、その多様性がゆえにT細胞受容体(T cell receptor:TCR)やB細胞受容体(B cell receptor:BCR)の解析を正確に行うことは難しく、従来は存在する限られたモノクローナル抗体を利用してフローサイトメトリーで解析する方法や、塊(バルク)の検体をシークエンスして受容体のレパートリー(レパトア)から頻度や多様性で論じる方法しかなかった。これらの方法では多様な受容体のα/β鎖や重/軽鎖を正確にペアで解析することや、どういったTCRやBCRをもつ細胞がどのような表現型を呈しているのか?といったことを正確に解析することは難しかった。ところが、最近の技術革新によってシングルセルシークエンスが可能となり、遺伝子発現だけでなくTCRやBCRも1細胞ずつ解析できるようになった。網羅的な遺伝子発現と一緒に解析することによってクローンごとの表現型も詳細に明らかにできるようになっている。将来的にはさまざまな疾患での病態解明や個別化療法・バイオマーカー開発といった「個別化免疫医療」につながる可能性がある。(著者抄録)

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がん免疫療法の有効性が多くのがん種で証明されたが、治療効果が限定的であることから抗腫瘍免疫応答の本質に迫るための免疫細胞の解析が注目されている。免疫細胞は不均一な集団であり、塊(バルク)での解析では限界がありシングルセルレベルでの解析が必要である。タンパク質レベルの解析では従来から利用されてきた抗体に蛍光色素をラベルして解析するフローサイトメトリーを代表に、さらにマーカーを増やすために金属をラベルした抗体を用いるマスサイトメトリーも使用され、40分子程度までシングルセルレベルでの解析が可能になっている。また網羅的な遺伝子発現をシングルセルレベルで解析できるようにもなり(シングルセルRNAシークエンス)、最大で10,000遺伝子程度まで解析可能になっている。また、抗体やMHCマルチマーにオリゴヌクレオチドをラベルしてシークエンスする技術や、T細胞・B細胞受容体配列、オープンクロマチン領域も解析することがシングルセルレベルで可能となっている。さらには位置情報も含めた解析技術も登場してきて、次々と新たな知見が明らかになっている。(著者抄録)

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がん免疫療法の有効性が多くのがん種で証明されたが、治療効果が限定的であることから抗腫瘍免疫応答の本質に迫るための免疫細胞の解析が注目されている。免疫細胞は不均一な集団であり、塊(バルク)での解析では限界がありシングルセルレベルでの解析が必要である。タンパク質レベルの解析では従来から利用されてきた抗体に蛍光色素をラベルして解析するフローサイトメトリーを代表に、さらにマーカーを増やすために金属をラベルした抗体を用いるマスサイトメトリーも使用され、40分子程度までシングルセルレベルでの解析が可能になっている。また網羅的な遺伝子発現をシングルセルレベルで解析できるようにもなり(シングルセルRNAシークエンス)、最大で10,000遺伝子程度まで解析可能になっている。また、抗体やMHCマルチマーにオリゴヌクレオチドをラベルしてシークエンスする技術や、T細胞・B細胞受容体配列、オープンクロマチン領域も解析することがシングルセルレベルで可能となっている。さらには位置情報も含めた解析技術も登場してきて、次々と新たな知見が明らかになっている。(著者抄録)

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Language：English   Publisher：日本癌学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2019-CT024

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DOI： 10.1200/JCO.2019.37.15_suppl.3606

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DOI： 10.1200/JCO.2019.37.15_suppl.TPS4141

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DOI： 10.20837/12018081831

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DOI： 10.1200/JCO.2018.36.15_suppl.3011

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DOI： 10.1200/JCO.2018.36.15_suppl.4106

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DOI： 10.1200/JCO.2018.36.15_suppl.TPS3117

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CLINICAL ONCOLOGY  

DOI： 10.1200/JCO.2018.36.4_suppl.TPS878

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Language：Japanese   Publisher：SGH財団  

CiNii Article

CiNii Books

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

血中循環腫瘍細胞、血中遊離DNA、エクソソームなどの、主に血液由来の液性検体はリキッドバイオプシーと総称される。これらを用いた、腫瘍由来DNAの分子異常の検出は、低侵襲であり複数回の実施が可能であるため、治療選択、モニタリング、proof of conceptのために期待される。デジタルPCR、次世代シーケンサーの登場により、高感度、マルチ解析が可能となり、実用化が近づいた。また低頻度変異アレル検出のための技術革新が進行中である。血中遊離DNAは、遺伝子導入、免疫への作用などの生物学的な働きも明らかになりつつあり、これらの知見は癌治療への応用へとつながる。肺癌領域では、血漿サンプルによるEGFR遺伝子変異検査が追加承認され、実用化された。リキッドバイオプシーは、腫瘍不均一性によるバイアスを克服する手段として期待される。今後はリキッドバイオプシーを用いたモニタリングにより、adaptive treatmentへのパラダイムシフトが訪れる。(著者抄録)

DOI： 10.2482/haigan.57.733

CiNii Article

J-GLOBAL

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Language：Japanese   Publisher：(株)日本医事新報社  

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Language：Japanese   Publishing type：Book review, literature introduction, etc.   Publisher：Japan Lung Cancer Society  

Cell free DNA, circulating tumor cell, and exosomes are collectively called liquid biopsy. The detection of molecular abnormalities in a liquid biopsy is a low-invasive technique that allows us to perform analyses and monitoring multiple times. Digital polymerase chain reaction and next-generation sequencing have improved the sensitivity and multiplicity of liquid biopsy analyses. Efforts to improve the detection of low-frequency mutation alleles further are now underway. Biological understanding of the cell free DNA with regard to aspects such as gene transfer and immune reactions have led to the development of cancer treatment regimens using liquid biopsies. In the clinical setting of lung cancer patients, serum EGFR mutation kits are available to identify patients indicated for third-generation EGFR-tyrosine kinase inhibitors. Liquid biopsies may eliminate the sampling bias caused by tumor heterogeneity and bring a paradigm shift to the adaptive treatment of lung cancer.

DOI： 10.2482/haigan.57.733

Scopus

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2017&ichushi_jid=J01244&link_issn=&doc_id=20171127490002&doc_link_id=%2Fec7jaluc%2F2017%2F005706%2F002%2F0733-0738%26dl%3D0&url=http%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fec7jaluc%2F2017%2F005706%2F002%2F0733-0738%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

Language：Japanese  

DOI： 10.20837/5201711023

J-GLOBAL

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2017&ichushi_jid=J03229&link_issn=&doc_id=20171115250004&doc_link_id=10.20837%2F5201711023&url=https%3A%2F%2Fdoi.org%2F10.20837%2F5201711023&type=%88%E3%8F%91.jp_%83I%81%5B%83%8B%83A%83N%83Z%83X&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00024_2.gif

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publisher：日本癌学会  

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CLINICAL ONCOLOGY  

DOI： 10.1200/JCO.2017.35.15_suppl.TPS3623

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CLINICAL ONCOLOGY  

DOI： 10.1200/JCO.2017.35.15_suppl.4040

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CLINICAL ONCOLOGY  

DOI： 10.1200/JCO.2017.35.15_suppl.e15570

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE INC  

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J-GLOBAL

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J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

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Language：English   Publisher：日本癌学会  

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Language：Japanese   Publisher：(株)メディカルレビュー社  

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Language：English   Publisher：日本癌学会  

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Language：English   Publisher：日本癌学会  

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Language：English   Publisher：日本癌学会  

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Language：English   Publisher：日本癌学会  

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Language：English   Publisher：日本癌学会  

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Language：English   Publisher：日本癌学会  

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Language：English   Publisher：日本癌学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2016-954

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Language：Japanese   Publisher：(株)メディカルレビュー社  

上皮成長因子受容体(EGFR)遺伝子の活性型変異(exon 19欠失変異もしくはexon 21 L858R変異など)を有する非小細胞肺がん(NSCLC)ではEGFRチロシンキナーゼ阻害薬(TKI)が著効する。しかしながら、大多数はいずれ耐性化してしまい、その機序としては2次変異としてゲートキーパー変異であるexon 20のT790M変異が50%程度関与する。そこで、T790M変異を有する場合でも阻害できる第2・3世代EGFR-TKIが開発されてきた。第2世代EGFR-TKIは、T790M変異を有する場合の臨床効果は限定的であるが、ほかのヒト上皮成長因子受容体(HER)ファミリーを阻害できる効果や稀な変異(exon 18変異、S768I変異、L861Q変異)への効果が期待されている。第3世代EGFR-TKIは、T790M変異を有する場合でも有効性が証明されているが、新たな耐性変異であるC797S変異がすでに報告されている。それぞれのEGFR-TKIの特徴を理解することで、より効果的な使い分け、すなわち「EGFR遺伝子変異肺がんの個別化医療」が可能となるであろう。(著者抄録)

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2016-2014

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Language：Japanese   Publisher：(一社)日本外科学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publisher：日本癌学会  

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Language：English   Publisher：日本癌学会  

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Language：English   Publisher：日本癌学会  

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

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Language：English   Publisher：日本癌学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCI LTD  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCI LTD  

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