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Graphical abstract, Genetic heterogeneity within a tumor arises by clonal evolution, and patients with highly heterogeneous tumors are more likely to be resistant to therapy and have reduced survival. Clonal evolution also occurs when a subset of cells leave the primary tumor to form metastases, which leads to reduced genetic heterogeneity at the metastatic site. Although this process has been observed in human cancer, experimental models which recapitulate this process are lacking. Patient-derived tumor xenografts (PDX) have been shown to recapitulate the patient’s original tumor’s intra-tumor genetic heterogeneity, as well as its genomics and response to treatment, but whether they can be used to model clonal evolution in the metastatic process is currently unknown. Here, we address this question by following genetic changes in two breast cancer PDX models during metastasis. First, we discovered that mouse stroma can be a confounding factor in assessing intra-tumor heterogeneity by whole exome sequencing, thus we developed a new bioinformatic approach to correct for this. Finally, in a spontaneous, but not experimental (tail-vein) metastasis model we observed a loss of heterogeneity in PDX metastases compared to their orthotopic “primary” tumors, confirming that PDX models can faithfully mimic the clonal evolution process undergone in human patients during metastatic spreading.

Background Immune therapies have had a major impact in multiple myeloma (MM). Responses induced in relapsed and/or refractory MM (RRMM) patients (pts) are unprecedented; however the key challenge is their durability. Most immunotherapeutic approaches rely on the fitness of pts’ T cells but exposure to multiple therapies may alter immune cell numbers and function. Here we analyzed and compared the transcriptome and phenotype of T cells from healthy donors (HD), newly diagnosed MM (NDMM) and RRMM pts. Methods Peripheral Blood Mononuclear Cells (PBMCs) from age-matched HD, NDMM and RRMM pts were collected before the start of a new line of treatment. Single cell RNA sequencing (scRNAseq) was performed and proportion of cell types were inferred using a reference PBMC dataset with Seurat. Cell type proportions were also estimated with CyTOF and conventional flow cytometry Results PBMCs from 10 HDs (median age 69, range 53-79), 20 NDMM pts (median age 71, range 54-88), 20 RRMM pts (median age 71, range 54-80) were analyzed. RRMM pts were exposed to 1 (n=8), 2 (n=6) or 3 (n=6) lines of treatment. All RRMM pts were exposed to a proteasome inhibitor, 75% were exposed to an immunomodulatory drug, and 65% received autologous stem cell transplantation. 48 out of 50 samples were processed by scRNAseq, 45 by CyTOF and 33 by flow cytometry. NDMM pts demonstrated a trend towards lower frequency of CD4 T cells compared to HD. This decreased number was accompanied by a decrease in the expression of genes involved in mitochondrial oxidative phosphorylation and an increase in expression of immediate early response genes (e.g. FOS, JUN, JUNB). In the CD8 compartment, CD8 naive cells were lower in NDMM compared to HD. No differences were found on CD8 central memory, effector memory and/or terminal effector populations In RRMM, the proportion of naive CD4 cells was significantly lower compared to NDMM (p Conclusion T cell phenotypes in MM pts are altered, especially after treatment. These findings may provide the rationale for the investigation of immunotherapy in NDMM and less heavily pretreated MM. Ongoing functional studies will demonstrate whether these phenotypic and transcriptomic changes lead to less fit T cells with consequent impact on effectiveness of T cell directed therapies.

Motivation Molecular profiling of patient tumors and liquid biopsies over time with next-generation sequencing technologies and new immuno-profile assays are becoming part of standard research and clinical practice. With the wealth of new longitudinal data, there is a critical need for visualizations for cancer researchers to explore and interpret temporal patterns not just in a single patient but across cohorts. Results To address this need we developed OncoThreads, a tool for the visualization of longitudinal clinical and cancer genomics and other molecular data in patient cohorts. The tool visualizes patient cohorts as temporal heatmaps and Sankey diagrams that support the interactive exploration and ranking of a wide range of clinical and molecular features. This allows analysts to discover temporal patterns in longitudinal data, such as the impact of mutations on response to a treatment, for example, emergence of resistant clones. We demonstrate the functionality of OncoThreads using a cohort of 23 glioma patients sampled at 2-4 timepoints. Availability and implementation Freely available at http://oncothreads.gehlenborglab.org. Implemented in Java Script using the cBioPortal web API as a backend. Supplementary information Supplementary data are available at Bioinformatics online.

Advanced ovarian cancers are a leading cause of cancer-related death in women and are currently treated with surgery and chemotherapy. This standard of care is often temporarily successful but exhibits a high rate of relapse, after which, treatment options are few. Here we investigate whether biomarker-guided use of multiple targeted therapies, including small molecules and antibody–drug conjugates, is a viable alternative. A panel of patient-derived ovarian cancer xenografts (PDX), similar in genetics and chemotherapy responsiveness to human tumors, was exposed to 21 monotherapies and combination therapies. Three monotherapies and one combination were found to be active in different subsets of PDX. Analysis of gene expression data identified biomarkers associated with responsiveness to each of the three targeted therapies, none of which directly inhibits an oncogenic driver. While no single treatment had as high a response rate as chemotherapy, nearly 90% of PDXs were eligible for and responded to at least one biomarker-guided treatment, including tumors resistant to standard chemotherapy. The distribution of biomarker positivity in The Cancer Genome Atlas data suggests the potential for a similar precision approach in human patients. Significance: This study exploits a panel of patient-derived xenografts to demonstrate that most ovarian tumors can be matched to effective biomarker-guided treatments.

Advanced ovarian cancers are a leading cause of cancer-related death in women. Such cancers are currently treated with surgery and chemotherapy which is often temporarily successful but exhibits a high rate of relapse after which treatment options are few. Here we assess the responses of a panel of patient-derived ovarian cancer xenografts (PDXs) to 19 mono and combination therapies, including small molecules and antibody-drug conjugates. The PDX panel aimed to mimic the heterogeneity of disease observed in patients, and exhibited a distribution of responsiveness to standard of care chemotherapy similar to human clinical data. Three monotherapies and one drug combination were found to be active in different subsets of PDXs. By analyzing gene expression data we identified gene expression biomarkers predictive of responsiveness to each of three novel targeted therapy regimens. While no single treatment had as high a response rate as chemotherapy, nearly 90% of PDXs were eligible for and responded to at least one biomarker-guided treatment, including tumors resistant to standard chemotherapy. Biomarker frequency was similar in human patients, suggesting the possibility of a new therapeutic approach to ovarian cancer and demonstrating the potential power of PDX-based trials in broadening the reach of precision cancer medicine.

Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus (EBV) associated cancer characterized by a poor prognosis and a high level of lymphocyte infiltrate. Genetic hallmarks of NPC are not completely known but include deletion of the p16 (CDKN2A) locus and mutations in NF-κB pathway components, with a relatively low total mutational load. To better understand the genetic landscape, an integrated genomic analysis was performed using a large clinical cohort of treatment-naïve NPC tumor specimens. This genomic analysis was generally concordant with previous studies; however, three subtypes of NPC were identified by differences in immune cell gene expression, prognosis, tumor cell morphology, and genetic characteristics. A gene expression signature of proliferation was poorly prognostic and associated with either higher mutation load or specific EBV gene expression patterns in a subtype-specific manner. Finally, higher levels of stromal tumor-infiltrating lymphocytes associated with good prognosis and lower expression of a WNT and TGFβ pathway activation signature. Implications: This study represents the first integrated analysis of mutation, copy number, and gene expression data in NPC and suggests how tumor genetics and EBV infection influence the tumor microenvironment in this disease. These insights should be considered for guiding immunotherapy treatment strategies in this disease. Mol Cancer Res; 15(12); 1722–32. ©2017 AACR.

Chronic myeloid leukaemia (CML) is driven by the activity of the BCR-ABL1 fusion oncoprotein. ABL1 kinase inhibitors have improved the clinical outcomes for patients with CML, with over 80% of patients treated with imatinib surviving for more than 10 years. Second-generation ABL1 kinase inhibitors induce more potent molecular responses in both previously untreated and imatinib-resistant patients with CML. Studies in patients with chronic-phase CML have shown that around 50% of patients who achieve and maintain undetectable BCR-ABL1 transcript levels for at least 2 years remain disease-free after the withdrawal of treatment. Here we characterize ABL001 (asciminib), a potent and selective allosteric ABL1 inhibitor that is undergoing clinical development testing in patients with CML and Philadelphia chromosome-positive (Ph +) acute lymphoblastic leukaemia. In contrast to catalytic-site ABL1 kinase inhibitors, ABL001 binds to the myristoyl pocket of ABL1 and induces the formation of an inactive kinase conformation. ABL001 and second-generation catalytic inhibitors have similar cellular potencies but distinct patterns of resistance mutations, with genetic barcoding studies revealing pre-existing clonal populations with no shared resistance between ABL001 and the catalytic inhibitor nilotinib. Consistent with this profile, acquired resistance was observed with single-agent therapy in mice; however, the combination of ABL001 and nilotinib led to complete disease control and eradicated CML xenograft tumours without recurrence after the cessation of treatment. Refereed/Peer-reviewed

Introduction: Identifying prior therapy exposures that affect the patient or their peripheral blood mononuclear cell (PBMC) material is one strategy to optimize outcomes to CAR T cell therapy. Alkylating agents commonly used in multiple myeloma management, such as cyclophosphamide, have been reported to impair the proliferative capacity of T lymphocytes and to blunt their functional activity (Ercolini et al. J Exp Med. 2005;201:1591; Banissi et al. Cancer Immunol Immunother. 2009;58:1627; Litterman et al. J Immunol. 2013;190:6259). In the pivotal phase 2 KarMMa trial (NCT03361748) investigating the BCMA-directed CAR T cell therapy idecabtagene vicleucel (ide-cel, bb2121) in triple-class exposed patients with RRMM, 80% of patients had a history of prior anticancer treatment with ≥1 alkylating agents. In this retrospective analysis, patient and PBMC characteristics associated with time from last dose of alkylating agent(s) until apheresis of PBMCs for CAR T cell manufacture were identified. Methods: PBMCs isolated from patient apheresis material, which serves as starting material for CAR T cell manufacturing, were immunophenotyped by polychromatic flow cytometry for markers associated with T cell differentiation, memory, senescence, and exhaustion. Data from relevant prespecified clinical and exploratory endpoints were collected, and a novel implementation of left-censored time-to-event analysis (Ware et al. Biometrics. 1976;32:459) was used to identify statistically significant relationships between washout time after prior alkylator exposure (encompassing 14 individual drugs) and patient and PBMC variables. Dose intensity of prior alkylators was not considered due to sparse annotations in the patient histories. Optimal cutpoints were identified for each variable that maximized the proportional hazard of receiving an alkylator between patients above and below the cutpoint, and P values were adjusted for testing multiple cutpoints. Relationships were verified by nonparametric correlation, in which alkylator washout was encoded as 1/log(−washout). Results: More recent exposure to an alkylating agent (after diagnosis but before apheresis) was associated with patients receiving more prior therapies per year to manage their disease (hazard ratio [HR]=2.63, ρ=−0.54, P Conclusions: Associations between patient characteristics and alkylator washout suggest that patients who more recently received alkylating agents to manage their myeloma had a more aggressive disease course, having progressed more quickly through prior regimens, and had lower weight and elevated systemic inflammation. Although these factors suggest a suboptimal patient profile, the depletion of T cells by alkylator therapy may be especially disadvantageous for autologous CAR T cell therapies (Wang et al. Mol Ther Oncolytics. 2016;3:16015; Perica et al. Biol Blood Marrow Transplant. 2018;24:1135). Our analysis found that the use of alkylators prior to CAR T cell therapy exhibits a detrimental effect on the apheresis PBMC material up to 6-9 months after the last dose. Figure 1 Disclosures Rytlewski: Adaptive Biotechnologies: Current equity holder in publicly-traded company; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Madduri:Kinevant: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Speaking Engagement, Speakers Bureau; Legend: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Speaking Engagement, Speakers Bureau; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Speaking Engagement, Speakers Bureau; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Foundation Medicine: Consultancy, Honoraria. Fuller:BMS: Current Employment. Campbell:Bristol-Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Mashadi-Hossein:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company; NanoString Technologies: Ended employment in the past 24 months. Thompson:Bristol-Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Jiang:Bristol Myers Squibb: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol Myers Squibb company: Current Employment. Martin:BMS: Current Employment, Current equity holder in publicly-traded company. Sangurdekar:bluebird bio: Current Employment, Current equity holder in publicly-traded company; Biogen: Ended employment in the past 24 months. Finney:bluebird bio: Current Employment, Current equity holder in publicly-traded company; Seattle Childrens Research Institute: Ended employment in the past 24 months. Bitter:Novartis: Ended employment in the past 24 months; Novartis AG: Patents & Royalties; bluebird bio: Current Employment, Current equity holder in publicly-traded company; F Hofmann-La Roche: Patents & Royalties; Predicant Biosciences: Patents & Royalties; Biospect: Patents & Royalties. Agarwal:BMS: Current Employment, Current equity holder in publicly-traded company. Kaiser:BMS: Current Employment, Current equity holder in publicly-traded company. Hege:Arcus Biosciences (Former Board of Directors): Divested equity in a private or publicly-traded company in the past 24 months; Mersana Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Celgene (acquired by Bristol Myers Squibb): Ended employment in the past 24 months; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Patents & Royalties: numerous, Research Funding. Hause:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

Introduction: Chimeric antigen receptor (CAR) T cell therapy directed against B-cell maturation antigen (BCMA) has shown promising results for the treatment of relapsed refractory multiple myeloma (RRMM). bb21217 is an anti-BCMA CAR T cell therapy that uses the same CAR molecule as idecabtagene vicleucel (bb2121), but adds the PI3K inhibitor bb007 during ex vivo culture to enrich the drug product (DP) for memory-like T cells, thereby reducing the proportion of highly differentiated or senescent T cells. To investigate whether DP properties correlate with clinical outcomes including duration of response (DOR), we conducted extensive molecular characterization of patient DPs. Methods: CRB-402 (NCT03274219) is an ongoing, multi-center phase 1 dose escalation trial of bb21217 in RRMM patients who received ≥3 prior regimens, including proteasome inhibitor and immunomodulatory agent, or are double-refractory to both classes. In the expansion cohort, patients additionally required prior exposure to an anti-CD38 antibody and were required to be refractory to last line. Planned enrollment is 74 patients, including 50 in the expansion cohort. Patients undergo lymphodepletion with fludarabine (30 mg/m2) and cyclophosphamide (300 mg/m2) daily for 3 days, then receive a single infusion of bb21217 at 150, 300 or 450 x 106 CAR+ T cells. The primary outcome measure is incidence of adverse events (AEs), including dose-limiting toxicities (DLTs). Additional outcome measures include overall response rate and DOR by IMWG Uniform Response Criteria. We profiled DP and apheresis starting material (PBMC) by RNAseq and cyTOF and correlated expression of memory/senescence markers from apheresis to DP with clinical outcomes, including DOR. Results: Asof March 1, 2020, 46 patients (median age 62 [33-74]) received bb21217, 24 in escalation (12 at 150, 6 at 300 and 6 at 450) and 22 in expansion (8 at 300 and 14 at 450); median follow up for all patients is 8.5 ( Conclusions: The adverse events observed are consistent with known toxicities of CAR T cell therapies. Initial efficacy results with bb21217 are encouraging, with 48% of patients treated across target dose levels of 150-450 obtaining ≥VGPR. The presence of T cell markers associated with memory and the absence of T cells markers associated with differentiation/senescence in DP correlated positively with peak expansion and DOR. These preliminary correlative data support the mechanistic hypothesis that enrichment for memory like T cells in bb21217 DP may result in improved clinical outcomes. Data for approximately 14 additional patients at the 450 target dose will be presented. Disclosures Alsina: Janssen: Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria; Amgen: Honoraria, Speakers Bureau; BMS: Consultancy, Research Funding. Shah:GSK, Amgen, Indapta Therapeutics, Sanofi, BMS, CareDx, Kite, Karyopharm: Consultancy; BMS, Janssen, Bluebird Bio, Sutro Biopharma, Teneobio, Poseida, Nektar: Research Funding. Raje:Janssen: Consultancy; Celgene: Consultancy; Immuneel: Membership on an entity's Board of Directors or advisory committees; Caribou: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Astrazeneca: Consultancy; Bluebird, Bio: Consultancy, Research Funding; Karyopharm: Consultancy; BMS: Consultancy; Amgen: Consultancy. Jagannath:Karyopharm: Consultancy, Honoraria; Legend Biotech: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Madduri:Janssen: Consultancy; BMS: Consultancy; Takeda: Consultancy; Legend: Consultancy; Sanofi: Consultancy; GSK: Consultancy; Kinevant: Consultancy; Foundation Medicine: Consultancy. Kaufman:Janssen: Consultancy, Honoraria; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Tecnopharma: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi/Genyzme: Consultancy, Honoraria. Siegel:Amgen: Consultancy, Honoraria, Speakers Bureau; Merck: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Karyopharma: Consultancy, Honoraria; Celulatiry: Consultancy. Munshi:BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company; Janssen: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy. Lin:Novartis: Consultancy; Celgene: Consultancy, Research Funding; Bluebird Bio: Consultancy, Research Funding; Juno: Consultancy; Legend BioTech: Consultancy; Merck: Research Funding; Takeda: Research Funding; Gamida Cells: Consultancy; Sorrento: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vineti: Consultancy; Janssen: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding. Jakubowiak:AbbVie, Amgen, BMS/Celgene, GSK, Janssen, Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive, Juno: Consultancy, Honoraria. Timm:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Turka:bluebird bio: Current Employment. Mao:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Martin:BMS: Current Employment, Current equity holder in publicly-traded company. Campbell:BMS: Current Employment, Current equity holder in publicly-traded company. Hege:Arcus Biosciences: Divested equity in a private or publicly-traded company in the past 24 months; Mersana Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; BMS: Current Employment, Current equity holder in publicly-traded company, Other: Travel, accommodations, expenses, Patents & Royalties: Numerous, Research Funding. Bitter:Novartis AG, Predicant Biosciences, Biospect, F Hofmann-La Roche: Ended employment in the past 24 months; bluebird bio: Current Employment, Current equity holder in publicly-traded company; Novartis: Ended employment in the past 24 months, Patents & Royalties. Petrocca:bluebird, bio: Current Employment, Current equity holder in publicly-traded company. Berdeja:Celgene: Consultancy, Research Funding; Cellularity: Research Funding; Constellation: Research Funding; CRISPR Therapeutics: Consultancy, Research Funding; CURIS: Research Funding; EMD Sorono: Research Funding; Takeda: Consultancy, Research Funding; Servier: Consultancy; Teva: Research Funding; Prothena: Consultancy; Poseida: Research Funding; Novartis: Research Funding; Lilly: Research Funding; Legend: Consultancy; Kite Pharma: Consultancy; Kesios: Research Funding; Karyopharm: Consultancy; Janssen: Consultancy, Research Funding; Glenmark: Research Funding; Genentech, Inc.: Research Funding; BMS: Consultancy, Research Funding; Bioclinica: Consultancy; Bluebird: Research Funding; Acetylon: Research Funding; Amgen: Consultancy, Research Funding; Abbvie: Research Funding; Vivolux: Research Funding.

Tolerance to self-antigens prevents the elimination of cancer by the immune system1,2. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45RO–CD8+ T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1–CD8+ CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies. An IL-6/STAT3 signature and memory CD8 T cell subset in preinfusion chimeric antigen receptor–expressing T cells associate with response in patients with high-risk chronic lymphocytic leukemia.

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19(-) leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.

We identified genetic mutations in CD19 and loss of heterozygosity at the time of CD19- relapse to chimeric antigen receptor (CAR) therapy. The mutations are present in the vast majority of resistant tumor cells and are predicted to lead to a truncated protein with a nonfunctional or absent transmembrane domain and consequently to a loss of surface antigen. This irreversible loss of CD19 advocates for an alternative targeting or combination CAR approach.

Profiling candidate therapeutics with limited cancer models during preclinical development hinders predictions of clinical efficacy and identifying factors that underlie heterogeneous patient responses for patient-selection strategies. We established ∼1,000 patient-derived tumor xenograft models (PDXs) with a diverse set of driver mutations. With these PDXs, we performed in vivo compound screens using a 1 × 1 × 1 experimental design (PDX clinical trial or PCT) to assess the population responses to 62 treatments across six indications. We demonstrate both the reproducibility and the clinical translatability of this approach by identifying associations between a genotype and drug response, and established mechanisms of resistance. In addition, our results suggest that PCTs may represent a more accurate approach than cell line models for assessing the clinical potential of some therapeutic modalities. We therefore propose that this experimental paradigm could potentially improve preclinical evaluation of treatment modalities and enhance our ability to predict clinical trial responses.

Emphysema in chronic obstructive pulmonary disease (COPD) can be characterized by high-resolution chest computed tomography (HRCT); however, the repeated use of HRCT is limited because of concerns regarding radiation exposure and cost.To evaluate biomarkers associated with emphysema and COPD-related clinical characteristics, and to assess the relationships of soluble receptor for advanced glycation endproducts (sRAGE), a candidate systemic biomarker identified in this study, with single-nucleotide polymorphisms (SNPs) in the gene coding for RAGE (AGER locus) and with clinical characteristics.Circulating levels of 111 biomarkers were analyzed for association with clinical characteristics in 410 patients with COPD enrolled in the TESRA study. sRAGE was also measured in the ECLIPSE cohort in 1,847 patients with COPD, 298 smokers and 204 nonsmokers. The association between 21 SNPs in the AGER locus with sRAGE levels and clinical characteristics was also investigated.sRAGE was identified as a biomarker of diffusing capacity of carbon monoxide and lung density in the TESRA cohort. In the ECLIPSE cohort, lower sRAGE levels were associated with increased emphysema, increased Global Initiative for Chronic Obstructive Lung Disease stage, and COPD disease status. The associations with emphysema in both cohorts remained significant after covariate adjustment (P0.0001). One SNP in the AGER locus, rs2070600, was associated with circulating sRAGE levels both in TESRA (P = 0.0014) and ECLIPSE (7.07 × 10(-16)), which exceeded genome-wide significance threshold. Another SNP (rs2071288) was also associated with sRAGE levels (P = 0.01) and diffusing capacity of carbon monoxide (P = 0.01) in the TESRA study.Lower circulating sRAGE levels are associated with emphysema severity and genetic polymorphisms in the AGER locus are associated with systemic sRAGE levels. Clinical trial registered with www.clinicaltrials.gov (NCT 00413205 and NCT 00292552).

Introduction Increasing evidence links COPD pathogenesis with pulmonary capillary apoptosis. We previously demonstrated that plasma levels of circulating microparticles released from endothelial cells (EMPs) due to apoptosis are elevated in smokers with normal spirometry but low diffusion capacity, that is, with early evidence of lung destruction. We hypothesised that pulmonary capillary apoptosis persists with the development of COPD and assessed its reversibility in healthy smokers and COPD smokers following smoking cessation. Methods Pulmonary function and high-resolution CT (HRCT) were assessed in 28 non-smokers, 61 healthy smokers and 49 COPD smokers; 17 healthy smokers and 18 COPD smokers quit smoking for 12 months following the baseline visit. Total EMP (CD42b − CD31 + ), pulmonary capillary EMP (CD42b − CD31 + ACE + ) and apoptotic EMP (CD42b − CD62E + /CD42b − CD31 + ) levels were quantified by flow cytometry. Results Compared with non-smokers, healthy smokers and COPD smokers had elevated levels of circulating EMPs due to active pulmonary capillary endothelial apoptosis. Levels remained elevated over 12 months in healthy smokers and COPD smokers who continued smoking, but returned to non-smoker levels in healthy smokers who quit. In contrast, levels remained significantly abnormal in COPD smokers who quit. Conclusions Pulmonary capillary apoptosis is reversible in healthy smokers who quit, but continues to play a role in COPD pathogenesis in smokers who progressed to airflow obstruction despite smoking cessation. Trial registration number NCT00974064; NCT01776398.

Approximately 350 million individuals live with CHB, and over 500,000 people die each year due to HBV-associated liver diseases, such as cirrhosis and HCC (1). Pegylated interferon-α (IFN-α) and various nucleos(t)ides are currently licensed for the treatment of CHB, but while these therapies reduce viremia and improve long-term outcome, they rarely lead to cure (2). There is, therefore, an urgent need to develop novel antiviral therapies to achieve this goal. Due to the difficulty in obtaining liver biopsy specimens from chronically infected patients, much of what has been learned about human HBV has been determined in animal models. Studies of acute infection in transgenic (3, 4) and hydrodynamic (5) mouse models and also in chimpanzees (6) have provided useful insights into immunological mechanisms that may contribute to the outcome of HBV infection. However, the immune correlates of cure of chronic HBV infection, and the role of host-virus interactions in persistent infection, are still ill defined. Characterization of these complex immunological phenomena has been hampered by the lack of a well characterized, immunocompetent small animal model of CHB. This therefore remains an important goal, since identification of the mechanism(s) by which HBV escapes or circumvents the host immune response will likely contribute to the identification of novel targets with potential to lead to new antiviral therapies. The Eastern woodchuck (Marmota monax) is naturally infected with WHV, a hepadnavirus which is genetically closely related to human HBV, and has a disease course similar to that in HBV-infected persons (7). As such, the woodchuck has been used to study viral pathogenesis, and to evaluate antiviral and anticancer therapeutics (7). However, a notable limitation of this model is that transcriptional analysis of the woodchuck is restricted to a few hundred sequenced gene segments. To address this shortcoming, we report here the sequencing, assembly and annotation of the woodchuck transcriptome, together with the generation of woodchuck microarrays. By using this new platform, we describe the first global transcriptional analysis of persistent WHV infection and WHV-induced HCC.

Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8–12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB., Author Summary Approximately 250 million people are chronically infected with HBV, and over 500,000 people die every year because of associated liver diseases. IFN-α has been used to treat patients with chronic HBV infection for over 20 years, but it is not well understood why some patients respond to treatment and others do not. In large part, this is because it is not practicable to obtain liver samples to characterize the intrahepatic response to IFN-α in patients with different treatment outcomes. In this study we used the woodchuck model of chronic HBV infection to study how IFN-α changes gene expression patterns in the liver during treatment. Surprisingly, we found that the treatment response did not correlate with the expression of antiviral effector genes that have previously been shown to mediate the direct antiviral effects of IFN-α in vitro. Instead, we found that the response to IFN-α treatment was associated with the presence of select immune cells (natural killer cells and T cells) in the liver. Our work also indicates that these immune cells inhibit the virus by killing infected cells, as well as in ways that do not require killing of liver cells. Altogether, our study suggests that new therapies that stimulate these immune cells in the liver may hold promise for the treatment of chronic HBV infection.

Biomarkers for patient selection are essential for the successful and rapid development of emerging targeted anti-cancer therapeutics. In this study, we report the discovery of a novel patient selection strategy for the p53–HDM2 inhibitor NVP-CGM097, currently under evaluation in clinical trials. By intersecting high-throughput cell line sensitivity data with genomic data, we have identified a gene expression signature consisting of 13 up-regulated genes that predicts for sensitivity to NVP-CGM097 in both cell lines and in patient-derived tumor xenograft models. Interestingly, these 13 genes are known p53 downstream target genes, suggesting that the identified gene signature reflects the presence of at least a partially activated p53 pathway in NVP-CGM097-sensitive tumors. Together, our findings provide evidence for the use of this newly identified predictive gene signature to refine the selection of patients with wild-type p53 tumors and increase the likelihood of response to treatment with p53–HDM2 inhibitors, such as NVP-CGM097. DOI: http://dx.doi.org/10.7554/eLife.06498.001, eLife digest Stress from daily activities and exposure to chemicals or UV radiation can all damage cells. Damaged cells may develop into cancerous tumors if unchecked. Normally, a protein called p53 helps to repair or eliminate damaged cells and prevent tumors from forming. The p53 protein does this by switching on or off genes that control DNA repair, cell division, and cell death. But half of all cancerous tumors have mutations that prevent p53 from doing its job. Another protein called HDM2 keeps p53 in check by binding to p53 and preventing it from switching on and off genes after the stress passes. In cancers that have normal p53, sometimes HDM2 is overly active and prevents p53 from suppressing tumor formation and growth. Scientists are developing anticancer drugs that work by targeting HDM2; this frees p53 and allows it to wipe out cancerous cells. However, it is not always clear which patients with cancer are the most likely to benefit from anti-HDM2 therapy. Jeay et al. screened hundreds of cancer cells to determine which ones are sensitive to HDM2-targeting drugs. As expected, the screen revealed that cancer cells that have mutations in the gene encoding p53 are insensitive to the anti-HDM2 drug because there is no working p53 to free up. But about 60% of the cancer cells that have normal p53 proteins also did not respond to the anti-HDM2 therapy. This finding indicates that the presence of normal p53 protein is necessary but not sufficient for tumor cells to respond to anti-HDM2 therapy. Next, Jeay et al. compared the patterns of gene expression in the cancer cells that responded to an anti-HDM2 drug with those in cells that didn't respond. The analysis showed that a group of 13 genes are expressed more in the cells that responded to the drug. All 13 genes are unexpectedly direct targets of p53, suggesting that p53 remains active in these tumor cells, even if it is not working optimally. To verify these results, Jeay et al. grew human tumors in mice and found that tumors with high expression of the 13 genes are sensitive to the anti-HDM2 drug (called NVP-CGM097). The experiments strongly suggest that this 13-gene signature can be used to determine if a patient with cancer will respond to anti-HDM2 therapy. Following on from this work, researchers have already launched an early clinical trial with the anti-HDM2 drug and will test whether this gene signature is indeed useful in a real clinical setting. DOI: http://dx.doi.org/10.7554/eLife.06498.002

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.

137 Background: The adoptive transfer of autologous T cells genetically modified to express a CD19-specific, 4-1BB/CD3ζ-signaling CAR (CTL019) has shown remarkable activity and induces long-term remissions in a subset of patients with relapsed/refractory chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In ALL, CTL019 induces a complete response (CR) in over 90% of patients while in CLL 25% of patients obtain a CR. It is not fully understood why only certain patients respond to therapy. Methods: We employed next generation sequencing of RNA (RNAseq) to identify predictive indicators of response to CTL019. We performed RNAseq on leukapheresis and manufactured product T cells prior to re-infusion from 35 CLL and 7 pediatric ALL patients with heavily pre-treated and high-risk disease. To characterize potency, we performed RNAseq on the infusion product after stimulation with the CAR. Results: We find that durable remission in CLL is associated with gene expression signatures of early memory and T-effector cells, while T cells from non-responding patients are enriched in signatures of T-regulatory cells, terminal differentiation, and exhaustion. In following the results from CLL, we find that pediatric ALL manufactured T cells are significantly enriched for an early memory, naïve T cell state and all achieved a CR. In parallel in vitro experiments, stimulation of the infusion product further demonstrated that CTL019 cells from CRs have an increased capacity for activation upon stimulation. We tested if we could extend these observations to identify a phenotype of T cells that is predictive of response prior to CTL019 manufacturing and find that the signatures predictive of response at the pre-infusion stage are also observed at the earlier leukapheresis time point. Conclusions: These findings suggest that intrinsic T cell fitness dictates response to CAR T cells. These gene expression signatures, along with additional immunological biomarkers, may be used to identify which patients are most likely to respond to adoptive transfer strategies and suggest manufacturing modifications that might potentiate the generation of maximally efficacious infusion products. Clinical trial information: NCT01029366, NCT01747486, NCT01626495.

Even after quitting smoking, the risk of the development of chronic obstructive pulmonary disease (COPD) and lung cancer remains significantly higher compared to healthy nonsmokers. Based on the knowledge that COPD and most lung cancers start in the small airway epithelium (SAE), we hypothesized that smoking modulates miRNA expression in the SAE linked to the pathogenesis of smoking-induced airway disease, and that some of these changes persist after smoking cessation. SAE was collected from 10th to 12th order bronchi using fiberoptic bronchoscopy. Affymetrix miRNA 2.0 arrays were used to assess miRNA expression in the SAE from 9 healthy nonsmokers and 10 healthy smokers, before and after they quit smoking for 3 months. Smoking status was determined by urine nicotine and cotinine measurement. There were significant differences in the expression of 34 miRNAs between healthy smokers and healthy nonsmokers (p1.5), with functions associated with lung development, airway epithelium differentiation, inflammation and cancer. After quitting smoking for 3 months, 12 out of the 34 miRNAs did not return to normal levels, with Wnt/β-catenin signaling pathway being the top identified enriched pathway of the target genes of the persistent dysregulated miRNAs. In the context that many of these persistent smoking-dependent miRNAs are associated with differentiation, inflammatory diseases or lung cancer, it is likely that persistent smoking-related changes in SAE miRNAs play a role in the subsequent development of these disorders.

The adoptive transfer of autologous T cells genetically modified to express a CD19-specific, 4-1BB/CD3z-signaling CAR (CTL019) has shown remarkable activity and induce long-term remissions in a subset of patients with relapsed/refractory chronic lymphocytic leukemia (CLL). To date, little is known about predictive indicators of efficacy. This study was designed to evaluate biomarkers of clinical response to CTL019 in CLL. We studied forty-one patients with advanced, heavily pre-treated and high-risk CLL who received at least one dose of CTL019 cells. We show that in vivo expansion and persistence are key quality attributes of CTL019 cells in CLL patients who have complete responses to therapy; in 2 patients responses are sustained beyond five years and accompanied by the persistence of functional CTL019 cells. Furthermore, durable remissions were associated with transcriptomic signatures of early memory T cells, while T cells from non-responding patients were enriched in genes belonging to known pathways of terminal differentiation and exhaustion. Polychromatic flow cytometry also demonstrated a significantly higher level of T cell exhaustion markers on the infused CAR T cells and reduced CD27 expression in non-responding patients. Accordingly, the combined assessment of PD1 and CD27 expression on CD8+ CTL019 cells in the infusion product accurately predicted response to treatment. Restimulation of the infusion product through the CAR further demonstrated that CTL019 cells from complete responders secreted significantly higher levels of several cytokines, including CCL20, IL-21, IL-22, IL-17, and IL-6, suggesting that the STAT3 signaling pathway may play a role in potentiating the enhanced potency of CTL019 cells. To identify a phenotype of T cells that is predictive of response prior to CTL019 manufacturing, we initially retrospectively evaluated the proportions of naïve, stem cell memory, central memory, effector memory and effector cells at the time of leukapheresis and observed either marginally significant or no significant correlations with clinical outcome. A systematic, unbiased analysis of the same biomarker panel revealed that the frequency of CD27+CD45RO- cells in the CD8+ T cell population correlated significantly with complete and durable responses to this therapy. Analysis of the infusion products using the same flow cytometric panel showed that most (>95%) of T cells expressed CD45RO at the end of the manufacturing run; CD27 expression frequencies, however, were maintained at the same level as in the leukapheresis. Together, these findings suggest that intrinsic T cell fitness dictates both response and resistance to highly active engineered CAR T cells. Thus, enrichment of T cells with optimal differentiation potential and proliferative capacity by timing of collection or culture modification might potentiate the generation of maximally efficacious infusion products. These data and additional immunological biomarkers may be used to identify which patients are most likely to respond to adoptive transfer strategies, leading to an enhanced personalized approach to cellular therapy. Disclosures Fraietta: Novartis: Patents & Royalties: Novartis, Research Funding. Lacey:Novartis: Research Funding. Wilcox:Novartis: Research Funding. Bedoya:Novartis: Research Funding. Chen:Novartis: Research Funding. Orlando:Novartis: Employment. Brogdon:Novartis: Employment. Hwang:Novartis: Research Funding. Frey:Novartis: Research Funding; Amgen: Consultancy. Pequignot:Novartis: Research Funding. Ambrose:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding; GE Healthcare Bio-Sciences: Consultancy. Bitter:Novartis: Employment. Porter:Genentech: Employment; Novartis: Patents & Royalties, Research Funding. Xu:Novartis: Research Funding. June:Immune Design: Consultancy, Equity Ownership; University of Pennsylvania: Patents & Royalties; Celldex: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder ; Pfizer: Honoraria; Johnson & Johnson: Research Funding. Melenhorst:Novartis: Patents & Royalties: Novartis, Research Funding.

Background & Aims The interaction between HBV replication and immune modulatory effects mediated by IFNα therapy is not well understood. We characterized the impact of HBV DNA replication on the early IFNα-induced immunomodulatory mechanisms. Methods We interrogated the transcriptional, serum cytokine/chemokine and cellular immune profiles of 28 patients with HBeAg+ chronic HBV infection (CHB) randomly assigned to one of 4 treatment cohorts (untreated n=5, weekly dosing of 360μg Pegasys® [PegIFNα] n=11, daily dose of 300mg Viread® [tenofovir disoproxil fumarate, TDF] n=6, or a combination of both n=6). Samples were characterized at multiple early time points through day 14 of therapy, after which all patients were given standard of care (180μg Pegasys® injected subcutaneously, weekly). Results PegIFNα induced a distinct and rapid up-regulation of IFN signaling pathway that coincided with increase detection of distinct serum cytokines/chemokines (IL-15, IL-6, and CXCL-10) and the up-regulation of the frequency of proliferating NK and activated total CD8+ T cells. IFNα treatment alone did not result in rapid decay of HBV replication and was not able to restore the defective HBV-specific T cell response present in CHB patients. In addition, the IFNα immune-stimulatory effects diminished after the first dose, but this refractory effect was reduced in patients where HBV replication was simultaneously inhibited with TDF. Conclusions We present here the first comprehensive description of the early effects of IFNα treatment on immune and viral biomarkers in HBeAg+ CHB patients. Our results show that PegIFNα-induced innate immune activation directly benefits from the suppression of HBV replication.

The preclinical model of bleomycin-induced lung fibrosis, used to investigate mechanisms related to idiopathic pulmonary fibrosis (IPF), has incorrectly predicted efficacy for several candidate compounds suggesting that it may be of limited value. As an attempt to improve the predictive nature of this model, integrative bioinformatic approaches were used to compare molecular alterations in the lungs of bleomycin-treated mice and patients with IPF. Using gene set enrichment analysis we show for the first time that genes differentially expressed during the fibrotic phase of the single challenge bleomycin model were significantly enriched in the expression profiles of IPF patients. The genes that contributed most to the enrichment were largely involved in mitosis, growth factor, and matrix signaling. Interestingly, these same mitotic processes were increased in the expression profiles of fibroblasts isolated from rapidly progressing, but not slowly progressing, IPF patients relative to control subjects. The data also indicated that TGFβ was not the sole mediator responsible for the changes observed in this model since the ALK-5 inhibitor SB525334 effectively attenuated some but not all of the fibrosis associated with this model. Although some would suggest that repetitive bleomycin injuries may more effectively model IPF-like changes, our data do not support this conclusion. Together, these data highlight that a single bleomycin instillation effectively replicates several of the specific pathogenic molecular changes associated with IPF, and may be best used as a model for patients with active disease.

The woodchuck model of hepatitis B virus (HBV) infection displays many characteristics of human infection and has particular value for characterizing the host immune responses during the development of chronic infection. Using the newly developed custom woodchuck microarray platform, we compared the intrahepatic transcriptional profiles of neonatal woodchucks with self-limiting woodchuck hepatitis virus (WHV) infection to those woodchucks progressing to persistent WHV infection. This revealed that WHV does not induce significant intrahepatic gene expression changes during the early-acute stage of infection (8 weeks), suggesting it is a stealth virus. At the mid-acute phase of infection (14 weeks), resolution was associated with induction of a prominent cytotoxic T-cell signature. Strikingly, this was accompanied by high-level expression of PD-1 and various other inhibitory T-cell receptors, which likely act to minimize liver damage by cytotoxic T cells during viral clearance. In contrast to the expression of perforin and other cytotoxic effector genes, the interferon-γ (IFN-γ) signaling response in the mid-acute phase was comparable to that in chronically infected adult animals. The absence of a strong IFN-α/β transcriptional response indicated that type I IFN is not a critical mediator of self-limiting infection. Nevertheless, a number of antiviral genes, including viperin, were differentially expressed during resolving infection, suggesting that a subset of IFN-stimulated genes (ISG) may play a role in the control of WHV replication. Conclusion: We identified new immune pathways associated with the clearance of hepadnavirus infection revealing novel molecular targets with potential for the therapeutic treatment of chronic hepatitis B. (HEPATOLOGY 2013)

9064Background: To better understand genetic determinants of response to ceritinib, tumor biopsies from NSCLC pts treated with ceritinib in ASCEND-1 (NCT01283516) were analyzed by next-generation s...

The identification and validation of biomarkers to support the assessment of novel therapeutics for COPD continues to be an important area of research. The aim of the current study was to identify systemic protein biomarkers correlated with measures of COPD severity, as well as specific protein signatures associated with comorbidities such as metabolic syndrome. 142 protein analytes were measured in serum of 140 patients with stable COPD, 15 smokers without COPD and 30 non-smoking controls. Seven analytes (sRAGE, EN-RAGE, NGAL, Fibrinogen, MPO, TGF-α and HB-EGF) showed significant differences between severe/very severe COPD, mild/moderate COPD, smoking and non-smoking control groups. Within the COPD subjects, univariate and multivariate analyses identified analytes significantly associated with FEV(1), FEV(1)/FVC and DLCO. Most notably, a set of 5 analytes (HB-EGF, Fibrinogen, MCP-4, sRAGE and Sortilin) predicted 21% of the variability in DLCO values. To determine common functions/pathways, analytes were clustered in a correlation network by similarity of expression profile. While analytes related to neutrophil function (EN-RAGE, NGAL, MPO) grouped together to form a cluster associated with FEV(1) related parameters, analytes related to the EGFR pathway (HB-EGF, TGF-α) formed another cluster associated with both DLCO and FEV(1) related parameters. Associations of Fibrinogen with DLCO and MPO with FEV(1)/FVC were stronger in patients without metabolic syndrome (r = -0.52, p = 0.005 and r = -0.61, p = 0.023, respectively) compared to patients with coexisting metabolic syndrome (r = -0.25, p = 0.47 and r = -0.15, p = 0.96, respectively), and may be driving overall associations in the general cohort. In summary, our study has identified known and novel serum protein biomarkers and has demonstrated specific associations with COPD disease severity, FEV(1), FEV(1)/FVC and DLCO. These data highlight systemic inflammatory pathways, neutrophil activation and epithelial tissue injury/repair processes as key pathways associated with COPD.

Background: Genetic alterations in the phosphatidylinositol 3-kinase (PI3K) pathway are frequent in endometrial carcinoma (EC; 59% PIK3CA and 66% PTEN alterations; Kandoth C et al., Nature 2013), thus providing a rationale for testing PI3K inhibitors in this tumor type. A Phase II, single-arm clinical trial of buparlisib, an oral pan-PI3K inhibitor that inhibits all four class I PI3K isoforms (α, β, δ, γ), as second-line therapy, was conducted in advanced EC (N=70). All patients (pts) received buparlisib 100 mg/day until disease progression or discontinuation due to an adverse event. The primary objective was objective response rate (RECIST v1.1) in pts with PI3K-activated tumors (PIK3CA and/or PTEN alteration) or in all pts. The activity of single-agent buparlisib was marginal and PI3K pathway activation was not associated with a better outcome in this pt population (Teneriello MG et al., ICACT 2013). Retrospective molecular characterization was undertaken to assess a potential alternate contribution to PI3K pathway activation in EC and the impact on response to treatment. Methods: Next-generation sequencing data were generated for 51 pts (serous n=17, endometrioid n=28, unknown n=6) and targeted coding regions of approximately 400 genes. Using standard bioinformatics tools, short nucleotide variants (SNVs) were called. To generate signatures of PI3K pathway activation, data from the TCGA endometrial cohort (Kandoth C et al., Nature 2013) were first leveraged. Levels of pAKT (pT308 and pS473) and pS6 (pS235 and pS240), proxies for PI3K pathway activation, were modeled using SNVs in the TCGA endometrial cohort. Specifically, elastic net models predicting pAKT and pS6 were optimized using somatic SNVs for endometrioid (6-gene model) and serous (3-gene model) subtypes separately. The TCGA models were then used to create pathway activation scores for each pt based on their molecular profile. Results: No significant relationships were found for pts with the serous subtype, hence the results focus on the endometrioid subtype. Subgroup analysis was performed to assess trends in progression-free survival (PFS) using Cox proportional hazards regression. Pts with the lowest PI3K pathway activation scores, based on the pAKT TCGA model, including pts with PTEN wildtype (n=5), trended towards a lower risk of PFS event compared with pts with moderate or high activation (n=23). In addition, pts with the highest pathway activation scores, based on the pS6 TCGA model, including pts with alterations in mTOR (n=5), trended towards a higher risk of PFS event compared with pts with moderate or low activation (n=23). When combining the two signatures, pts with high pS6 activation (n=5) had a higher risk of PFS event followed by pts with low pS6 and high pAKT scores (n=19) compared with pts with low pS6 and low pAKT scores (n=4). PIK3CA mutations were unrelated to pAKT in the TCGA model for the endometrioid subtype, but PIK3CA-mutated tumors in the TCGA model had slightly higher levels of pS6. Conclusion: In both models, pts with the lowest levels of PI3K pathway activation trended towards a lower risk of PFS event compared with pts with moderate or high levels of PI3K activation. A possible explanation is that the high levels of PI3K pathway activation observed in EC may represent a challenge for single-agent inhibition. Alternatively, for this single-arm study in EC, we can only elucidate the prognostic value of the biomarker and not the contribution of the treatment per se. Further validation with larger sample sizes is warranted to support this hypothesis further. Citation Format: Nadia Solovieff, Angad Pal Singh, Hans Bitter, Markus Riester, Michael Teneriello, Amit Oza, Bradley Monk, Douglas Robinson, Lucia Trandafir, Cristian Massacesi, Emmanuelle di Tomaso, R. Wendel Naumann. Biomarkers of drug response to buparlisib: Results of next-generation sequencing in a phase II trial of advanced endometrial carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A02.

Smoking and COPD are associated with decreased mucociliary clearance, and healthy smokers have shorter cilia in the large airway than nonsmokers. We hypothesized that changes in cilia length are consistent throughout the airway, and we further hypothesized that smokers with COPD have shorter cilia than healthy smokers. Because intraflagellar transport (IFT) is the process by which cilia of normal length are produced and maintained, and alterations in IFT lead to short cilia in model organisms, we also hypothesized that smoking induces changes in the expression of IFT-related genes in the airway epithelium of smokers and smokers with COPD. To assess these hypotheses, airway epithelium was obtained via bronchoscopic brushing. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject and Affymetrix microarrays were used to evaluate IFT gene expression in nonsmokers and healthy smokers in 2 independent data sets from large and small airway as well as in COPD smokers in a data set from the small airway. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. The gene expression data confirmed that a set of 8 IFT genes were down-regulated in smokers in both data sets; however, no differences were seen in COPD smokers compared to healthy smokers. These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies.

Blood consists of different cell populations with distinct functions and correspondingly, distinct gene expression profiles. In this study, global miRNA expression profiling was performed across a panel of nine human immune cell subsets (neutrophils, eosinophils, monocytes, B cells, NK cells, CD4 T cells, CD8 T cells, mDCs and pDCs) to identify cell-type specific miRNAs. mRNA expression profiling was performed on the same samples to determine if miRNAs specific to certain cell types down-regulated expression levels of their target genes. Six cell-type specific miRNAs (miR-143; neutrophil specific, miR-125; T cells and neutrophil specific, miR-500; monocyte and pDC specific, miR-150; lymphoid cell specific, miR-652 and miR-223; both myeloid cell specific) were negatively correlated with expression of their predicted target genes. These results were further validated using an independent cohort where similar immune cell subsets were isolated and profiled for both miRNA and mRNA expression. miRNAs which negatively correlated with target gene expression in both cohorts were identified as candidates for miRNA/mRNA regulatory pairs and were used to construct a cell-type specific regulatory network. miRNA/mRNA pairs formed two distinct clusters in the network corresponding to myeloid (nine miRNAs) and lymphoid lineages (two miRNAs). Several myeloid specific miRNAs targeted common genes including ABL2, EIF4A2, EPC1 and INO80D; these common targets were enriched for genes involved in the regulation of gene expression (p

Kinases are heavily pursued pharmaceutical targets because of their mechanistic role in many diseases. Small molecule kinase inhibitors (SMKIs) are a compound class that includes marketed drugs and compounds in various stages of drug development. While effective, many SMKIs have been associated with toxicity including chromosomal damage. Screening for kinase-mediated toxicity as early as possible is crucial, as is a better understanding of how off-target kinase inhibition may give rise to chromosomal damage. To that end, we employed a competitive binding assay and an analytical method to predict the toxicity of SMKIs. Specifically, we developed a model based on the binding affinity of SMKIs to a panel of kinases to predict whether a compound tests positive for chromosome damage. As training data, we used the binding affinity of 113 SMKIs against a representative subset of all kinases (290 kinases), yielding a 113×290 data matrix. Additionally, these 113 SMKIs were tested for genotoxicity in an in vitro micronucleus test (MNT). Among a variety of models from our analytical toolbox, we selected using cross-validation a combination of feature selection and pattern recognition techniques: Kolmogorov-Smirnov/T-test hybrid as a univariate filter, followed by Random Forests for feature selection and Support Vector Machines (SVM) for pattern recognition. Feature selection identified 21 kinases predictive of MNT. Using the corresponding binding affinities, the SVM could accurately predict MNT results with 85% accuracy (68% sensitivity, 91% specificity). This indicates that kinase inhibition profiles are predictive of SMKI genotoxicity. While in vitro testing is required for regulatory review, our analysis identified a fast and cost-efficient method for screening out compounds earlier in drug development. Equally important, by identifying a panel of kinases predictive of genotoxicity, we provide medicinal chemists a set of kinases to avoid when designing compounds, thereby providing a basis for rational drug design away from genotoxicity., Author Summary Small molecule kinase inhibitors (SMKIs) are a class of chemicals that have successfully been used for the treatment of a number of oncological diseases that are now being pursued by the pharmaceutical industry for inflammatory diseases, such as rheumatoid arthritis. SMKIs are generally designed to specifically inhibit one kinase, but this is challenging due to the structural similarity of the ATP binding pocket amongst different members of the kinase family. The inability to selectively inhibit just one kinase can be problematic, as kinases play key roles in a number of cellular processes. Thus the unwanted inhibition of additional kinases can lead to undesirable toxicities that may halt drug development. One type of toxicity often observed with this class of compounds is damage to chromosomes, which can occur when kinases involved with cell cycle progression or chromosome dynamics are inhibited. Here we demonstrate that mathematical modeling can be used to identify kinases that correlate with chromosome damage, information which can assist medicinal chemists in avoiding certain kinases when synthesizing new chemicals. Generation of this type of information is one of the first steps in beginning to reduce toxicity-based attrition for this class of compounds.