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Author summaryImmune cells have the ability to attack tumor cells upon the identification of tumor-specific peptides, i.e., epitopes, that are presented by the major histocompatibility complex (MHC). New cancer immunotherapies that help trigger this process provide a promising therapeutic strategy. One crucial aspect for their success is the ability to determine the molecular properties of a tumor that are informative about the effectiveness of the therapy. Alterations in the way genes are processed to express RNA molecules could lead to the production of new peptides, with some of them potentially being presented as tumor epitopes and facilitate the attack of immune cells. It is therefore essential to facilitate the identification of these splicing-derived epitopes. In this work, we describe a computational pipeline that performs a comprehensive identification of splicing alterations in a tumor and the potential epitopes that they would produce. Analysis of tumor samples with our pipeline show that responders and non-responders to immune therapy do not show differences in the number of splicing-derived epitopes, but splicing neoepitopes have higher affinity to the MHC complex in responders. Our new pipeline facilitates the genome-scale analysis of the role of splicing alterations in shaping the molecular properties that influence response to immunotherapy. Immunotherapies provide effective treatments for previously untreatable tumors and identifying tumor-specific epitopes can help elucidate the molecular determinants of therapy response. Here, we describe a pipeline, ISOTOPE (ISOform-guided prediction of epiTOPEs In Cancer), for the comprehensive identification of tumor-specific splicing-derived epitopes. Using RNA sequencing and mass spectrometry for MHC-I associated proteins, ISOTOPE identified neoepitopes from tumor-specific splicing events that are potentially presented by MHC-I complexes. Analysis of multiple samples indicates that splicing alterations may affect the production of self-epitopes and generate more candidate neoepitopes than somatic mutations. Although there was no difference in the number of splicing-derived neoepitopes between responders and non-responders to immune therapy, higher MHC-I binding affinity was associated with a positive response. Our analyses highlight the diversity of the immunogenic impacts of tumor-specific splicing alterations and the importance of studying splicing alterations to fully characterize tumors in the context of immunotherapies. ISOTOPE is available at https://github.com/comprna/ISOTOPE.

Lentiviral vectors are powerful tools for gene expression studies. Here we report the construction of pTIJ, a vector for inducible gene expression. pTIJ was generated from pTRIPZ backbone, which is designed for the inducible expression of shRNA sequences, by the introducing of a multiple cloning site upstream of the Tet promoter and the removal of miR30 flanking sequences. To evaluate pTIJ as a tool for the inducible expression of genes of interest, we introduced MYC cDNA into pTIJ and infected two small cell lung cancer cell lines, H209 and H345. Induction of MYC expression by doxycycline was detectable in both cell lines by real-time PCR and western blot analysis. This study highlights the relevance of pTIJ vector to allow the inducible expression of any gene of interest. In our belief, pTIJ will be an extremely useful tool to simplify the generation of genetically engineered cell lines for the inducible expression of cDNA sequences in biological studies. Furthermore, we report the generation of a pTIJ-MYC vector for the inducible expression of the oncogene MYC.

Purpose: We aimed to maximize the performance of detecting genetic alterations in lung cancer using high-throughput sequencing for patient-derived xenografts (PDXs). Experimental Design: We undertook an integrated RNA and whole-exome sequencing of 14 PDXs. We focused on the genetic and functional analysis of beta 2-microglobulin (B2M), a component of the HLA class-I complex. Results: We identified alterations in genes involved in various functions, such as B2M involved in immunosurveillance. We extended the mutational analysis of B2M to about 230 lung cancers. Five percent of the lung cancers carried somatic mutations, most of which impaired the correct formation of the HLA-I complex. We also report that genes such as CALR, PDIA3, and TAP1, which are involved in the maturation of the HLA-I complex, are altered in lung cancer. By gene expression microarrays, we observed that restitution of B2M in lung cancer cells upregulated targets of IFN alpha/IFN gamma. Furthermore, one third of the lung cancers lacked the HLA-I complex, which was associated with lower cytotoxic CD8(+) lymphocyte infiltration. The levels of B2M and HLA-I proteins correlated with those of PD-L1. Finally, a deficiency in HLA-I complex and CD8(+) infiltration tended to correlate with reduced survival of patients with lung cancer treated with anti-PD-1/anti-PD-L1. Conclusions: Here, we report recurrent inactivation of B2M in lung cancer. These observations, coupled with the mutations found at CALR, PDIA3, and TAP1, and the downregulation of the HLA-I complex, indicate that an abnormal immunosurveillance axis contributes to lung cancer development. Finally, our observations suggest that an impaired HLA-I complex affects the response to anti-PD-1/anti-PD-L1 therapies. (C) 2016 AACR.

Lentiviral vectors are powerful tools for gene expression studies. Here we report the construction of pTIJ, a vector for inducible gene expression. pTIJ was generated from pTRIPZ backbone, which is designed for the inducible expression of shRNA sequences, by the introducing of a multiple cloning site upstream of the Tet promoter and the removal of miR30 flanking sequences. To evaluate pTIJ as a tool for the inducible expression of genes of interest, we introduced MYC cDNA into pTIJ and infected two small cell lung cancer cell lines, H209 and H345. Induction of MYC expression by doxycycline was detectable in both cell lines by real-time PCR and western blot analysis. This study highlights the relevance of pTIJ vector to allow the inducible expression of any gene of interest. In our belief, pTIJ will be an extremely useful tool to simplify the generation of genetically engineered cell lines for the inducible expression of cDNA sequences in biological studies. Furthermore, we report the generation of a pTIJ-MYC vector for the inducible expression of the oncogene MYC.

PTEN is inactivated in a subset of lung cancer; therefore, we investigated the involvement of PTEN inactivation in invasiveness of lung cancer cells. AKT at Ser473 was phosphorylated in several lung cancer cell lines with loss of PTEN expression. Therefore, we created a tetracycline inducible expression system of wild-type PTEN (PTEN-WT) as well as catalytically (PTEN-G129R) and lipid phosphatase (PTEN-G129E) inactive PTEN mutants using the PC14, PC9 and PC3 lung adenocarcinoma cell lines, in which endogenous PTEN expression was not detected and AKT at Ser473 was phosphorylated by Western blot analysis. Induction of PTEN-WT reduced phosphorylation of AKT and inhibited the transcriptional activity of NFkB, whereas PTEN mutants did not, suggesting that PTEN inactivation results in the activation of the AKT/NFkB pathway in PC14, PC9 and PC3 cells. Furthermore, overexpression of PTEN-WT suppressed anchorage independent growth in soft agar and reduced invasiveness in a trans-well chamber assay of PC14 cells. Neither PTEN-G129R nor PTEN-G129E had suppressive effects on anchorage independent growth and invasiveness. Augmentation of invasiveness by constitutively active AKT was also shown in mouse NIH3T3 cells. Therefore, it was strongly indicated that activation of the PI3K/AKT/NFkB pathway by PTEN inactivation results in augmented invasiveness in lung cancer cells and lipid phosphatase activity of PTEN plays a key role in this process. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Objective and Background: Although short-duration, high-dose glucocorticoid therapy is reportedly ineffective for early-phase acute respiratory distress syndrome (ARDS), steroid pulse therapy is often used in Japan for life-threatening cases because of its beneficial effects on oxygenation. Thus, we evaluated the effect of steroid pulse therapy on oxygenation for early-phase ARDS. Method: Cases of early-phase ARDS treated with steroid pulse therapy in intensive care units of four critical care medical centers in Japan from 2000 to 2003 were investigated. Clinical course and serial changes in PaO2/FIO2 ratio were evaluated. Results: Twenty-nine patients received methylprednisolone 1 g/day for 3 days; 20 patients subsequently received prolonged methylprednisolone treatment. Causes of ARDS were pneumonia (n=23), extrapulmonary sepsis (n=4) and other factors (n=2). The overall mortality rate was 24.1%. PaO2/FIO2 ratios improved significantly after initiation of steroid pulse therapy, in comparison to pretreatment values (day 0: 119.8 ± 30.6, day 1: 172.1 ± 63.7*, day 2: 196.4 ± 90.0*, day 3: 218.4 ± 92.0*, mean ± SD, *p < 0.05 vs. day 0). Significant improvement in oxygenation was observed in both survivors and non-survivors, but the PaO2/FIO2 ratio in non-survivors subsequently deteriorated. In seven patients with life-threatening hypoxemia (PaO2/FIO2 ratio < 100 at day 0), oxygenation improved significantly after pulse treatment; five of these patients survived. Conclusions: Three day steroid pulse therapy can improve oxygenation in patients with progressive ARDS even in the early phase. Steroid therapy for early-phase ARDS should be reconsidered.

Recent evidence from several relatively small nested case-control studies in prospective cohorts shows an association between longer telomere length measured phenotypically in peripheral white blood cell (WBC) DNA and increased lung cancer risk. We sought to further explore this relationship by examining a panel of seven telomere-length associated genetic variants in a large study of 5,457 never-smoking female Asian lung cancer cases and 4,493 never-smoking female Asian controls using data from a previously reported genome-wide association study. Using a group of 1,536 individuals with phenotypically measured telomere length in WBCs in the prospective Shanghai Women's Health study, we demonstrated the utility of a genetic risk score (GRS) of seven telomere-length associated variants to predict telomere length in an Asian population. We then found that GRSs used as instrumental variables to predict longer telomere length were associated with increased lung cancer risk (OR = 1.51 (95% CI = 1.34-1.69) for upper vs. lower quartile of the weighted GRS, p value = 4.54 × 10(-14) ) even after removing rs2736100 (p value = 4.81 × 10(-3) ), a SNP in the TERT locus robustly associated with lung cancer risk in prior association studies. Stratified analyses suggested the effect of the telomere-associated GRS is strongest among younger individuals. We found no difference in GRS effect between adenocarcinoma and squamous cell subtypes. Our results indicate that a genetic background that favors longer telomere length may increase lung cancer risk, which is consistent with earlier prospective studies relating longer telomere length with increased lung cancer risk.

Human papillomavirus (HPV) is the etiologic risk factor for cervical cancer. Some studies have suggested an association with a subset of lung tumors, but the etiologic link has not been firmly established. We performed an international pooled analysis of crosssectional studies (27 datasets, n = 3249 patients) to evaluate HPV DNA prevalence in lung cancer and to investigate viral presence according to clinical and demographic characteristics. HPV16/18 were the most commonly detected, but with substantial variation in viral prevalence between geographic regions. The highest prevalence of HPV16/18 was observed in South and Central America, followed by Asia, North America and Europe (adjusted prevalence rates = 22, 5, 4 and 3%, respectively). Higher HPV16 prevalence was noted in each geographic region compared with HPV18, except in North America. HPV16/18-positive lung cancer was less likely observed among White race (adjusted odds ratio [OR] = 0.33, 95% confidence interval [CI] = 0.12-0.90), whereas no associations were observed with gender, smoking history, age, histology or stage. Comparisons between tumor and normal lung tissue show that HPV was more likely to be present in lung cancer rather than normal lung tissues (OR = 3.86, 95% CI = 2.87-5.19). Among a subset of patients with HPV16-positive tumors, integration was primarily among female patients (93%, 13/14), while the physical status in male cases (N = 14) was inconsistent. Our findings confirm that HPV DNA is present in a small fraction of lung tumors, with large geographic variations. Further comprehensive analysis is needed to assess whether this association reflects a causal relationship. © The Author 2014. Published by Oxford University Press. All rights reserved.

Mutations in the EGFR gene are critical determinants of treatment with EGFR tyrosine kinase inhibitors (TKIs) for non-small cell lung cancer (NSCLC) patients. DNA isolation from tumor samples usually requires surgery; therefore, we wanted to isolate DNA from circulating tumor cells by using the serum of NSCLC patients. This protocol was recently published. DNA was isolated from the serum of 52 Turkish NSCLC patients and their EGFR mutation status was examined by pyrosequencing. EGFR mutations were detected in 25 of the 52 patients (48.1%): 17 patients with delE746-A750, 2 with delE747-A750insP, and 6 with L858R. All mutations detected by pyrosequencing were confirmed by dideoxy sequencing, and the presence of the same mutations in the tumors was verified by using paraffin embedded tissues of all the patients. Mutations were detected more frequently in adenocarcinomas (24 of 36, 66.7%) than in squamous cell carcinomas (1 of 16, 6.3%) (P

本研究の目的は,脳死後長期間にわたり循環を維持した際の造血能を検討することである。対象は外出血のない単独頭部外傷に起因する脳死例で,血液像の変化とともにエリスロポエチン,骨髄像を検討した。なお,脳死後の循環はarginine vasopressin(以下ADHと略す)とcatecholamine(以下CAと略す)で平均21.6±15.0日間維持した。Hb値は脳死前に比べ約4g/dlの低下が認められ,その後も低値のままで推移した。エリスロポエチン値は平均50.2±19.4mU/mlと正常より高値であり,他の原因による貧血患者とほぼ同等の分泌が認められた。網状赤血球数は脳死後第20病日までは(8.8±4.3)×104/mm3とあまり増加しないものの,第21病日以降には(25.5±14.6)×104/mm3と著しく増加した。一方,赤血球より産生反応が早い血小板は,脳死後いったん低下するものの,脳死後第10病日には脳死前値にまで回復し,その後正常以上となった。骨髄像では肺炎像を呈した感染群と非感染群のうち,脳死後1週間以内施行群で骨髄球が有意に増加していた。成熟した分節核球は感染群と非感染群の脳死後1週間以降群で有意な増生を認めた。脳死後の循環維持のために,脳死直後に2,000ml/day以上の体液負荷がなされており,この希釈性因子と赤血球産生反応に時間を要することが脳死後の貧血持続の原因と考えられた。以上より,脳死患者の骨髄機能は正常に保たれていると結論した。

PTEN is inactivated in a subset of lung cancer; therefore, we investigated the involvement of PTEN inactivation in invasiveness of lung cancer cells. AKT at Ser473 was phosphorylated in several lung cancer cell lines with loss of PTEN expression. Therefore, we created a tetracycline inducible expression system of wild-type PTEN (PTEN-WT) as well as catalytically (PTEN-G129R) and lipid phosphatase (PTEN-G129E) inactive PTEN mutants using the PC14, PC9 and PC3 lung adenocarcinoma cell lines, in which endogenous PTEN expression was not detected and AKT at Ser473 was phosphorylated by Western blot analysis. Induction of PTEN-WT reduced phosphorylation of AKT and inhibited the transcriptional activity of NFkB, whereas PTEN mutants did not, suggesting that PTEN inactivation results in the activation of the AKT/NFkB pathway in PC14, PC9 and PC3 cells. Furthermore, overexpression of PTEN-WT suppressed anchorage independent growth in soft agar and reduced invasiveness in a trans-well chamber assay of PC14 cells. Neither PTEN-G129R nor PTEN-G129E had suppressive effects on anchorage independent growth and invasiveness. Augmentation of invasiveness by constitutively active AKT was also shown in mouse NIH3T3 cells. Therefore, it was strongly indicated that activation of the PI3K/AKT/NFkB pathway by PTEN inactivation results in augmented invasiveness in lung cancer cells and lipid phosphatase activity of PTEN plays a key role in this process.