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Supplementary tables Table S1: Patient characteristics. Table S2: Non-synonymous mutations for paired cohort. Table S3: Non-synonymous mutations for unpaired cohort. Table S4: Copy number variants. Table S5: Correlation of principal components with gene expression. Table S6: Pathway enrichment analysis by principal component. Table S7: Gene signature evaluated in this study. Table S8: Tumor genes correlated with EBV genes. Table S9: Pathway enrichment of EBV-correlated genes.

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.

Supplementary Figures Figure S1: Subtype identification using random sub-sampling. Figure S2: Subtype robustness. Figure S3: Morphological evaluation of tumors. Figure S4: % Tumor content by subtype. Figure S5: Principle components analysis. Figure S6: Microenvironment signatures. Figure S7: Cytotoxic to T-cell signature ratio and IgD to IgG expression ratio by subtype. Figure S8: Correlation of EBV and tumor gene expression. Figure S9: Mutation and copy number variation for paired and unpaired cohorts. Figure S10: Identification of stromal and intratumoral TILs. Figure S11: Mutation load vs. tumor content.

Ovarian cancer cell lines do not reproduce the clinical association between BRCA loss of function and sensitivity to olaparib, but patient-derived ovarian tumor xenografts do.

Ovarian PDX raw growth and sequencing data.

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.

Mean tumor volume change difference between best monotherapy and each of 9 combinations. Hazard ratio for combination versus independent action simulation PFS.

Supplementary Material PDF, including supplementary methods and supplementary figures 1-4

Compound dose, dosing schedule, administration route, target and drug type.

Ovarian PDX response category by therapy.

Gene therapy with LentiGlobin for sickle cell disease (bb1111, lovotibeglogene autotemcel) consists of autologous transplantation of a patient's hematopoietic stem cells transduced with the BB305 lentiviral vector that encodes the β

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.

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. 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, e.g. emergence of resistant clones. We demonstrate the functionality of OncoThreads using a cohort of 23 glioma patients sampled at 2-4 timepoints. OncoThreads is freely available at http://oncothreads.gehlenborglab.org and implemented in Javascript using the cBioPortal web API as a backend.

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: B-cell maturation antigen (BCMA) is primarily expressed by malignant and normal plasma cells, making it an attractive target for the treatment of multiple myeloma (MM). bb21217 is a BCMA-directed chimeric antigen receptor (CAR) T cell therapy that uses the same CAR molecule as idecabtagene vicleucel (ide-cel, bb2121), but adds the PI3K inhibitor bb007 during manufacturing to enrich the drug product (DP) for memory-like T cells, thereby reducing the proportion of highly differentiated or senescent T cells. We conducted correlative analyses to investigate the mechanistic hypothesis that CAR+ T cells with memory like phenotypes may persist and function longer, which may be one determinant of duration of response (DOR). Methods: An ongoing phase I clinical study (CRB-402; NCT03274219) is assessing safety and efficacy of bb21217 in relapsed/refractory MM patients. A total of 44 patients had PBMCs, collected from apheresis, and DP characterized by RNA sequencing (RNAseq) and mass Cytometry (CyTOF). The correlation of T cell phenotype with peak expansion, response and DOR per IMWG Uniform Response Criteria was explored. P-values were determined by Wilcoxon test, Spearman correlation, or Cox PH regression on DOR with categorical marker values (high/low). Results: In this patient population, substantial cross patient heterogeneity in T cell phenotypes was observed both in PBMCs and DP. Late differentiation/senescent markers in PBMCs were negatively correlated with clinical response. In particular, patients whose DP had higher expression of CD57 had lower peak expansion (p2x105copies/ug), consistent with the CYTOF findings. Early activation markers (CD38 p Conclusion: As seen with other CAR T cell studies, the quality of incoming PBMCs, in particular the fraction of T cells with a late differentiation/senescent phenotype, influences initial and sustained clinical response. The analyses reported here support the mechanistic hypothesis of bb21217, suggesting the presence of early memory like T cells in PBMC and/or DP may contribute to high peak expansion and prolonged DOR, while presence of highly differentiated or senescent T cells may negatively impact these measures. Further clinical evaluation of bb21217 and robust correlative analyses will be important to help contextualize the influence of patient and product characteristics on clinical outcomes. Disclosures Finney: bluebird bio: Current Employment, Current equity holder in publicly-traded company. Yeri:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Mao:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Pandya:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Alonzo:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hopkins:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hymson:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hu:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Foos:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Bhadoriya:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hintzen:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Gioia:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Timm:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Massaro:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hause:Bristol-Myers Squibb Company: Current Employment, Current equity holder in publicly-traded company. Kaiser:BMS: Current Employment, Current equity holder in publicly-traded company. Martin:BMS: Current Employment, Current equity holder in publicly-traded company. Shah:BMS, Janssen, Bluebird Bio, Sutro Biopharma, Teneobio, Poseida, Nektar: Research Funding; GSK, Amgen, Indapta Therapeutics, Sanofi, BMS, CareDx, Kite, Karyopharm: Consultancy. Raje:Caribou: Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Consultancy; Karyopharm: Consultancy; Janssen: Consultancy; Celgene: Consultancy; BMS: Consultancy; Immuneel: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Bluebird, Bio: Consultancy, Research Funding; Amgen: Consultancy. Berdeja:Novartis: Research Funding; Lilly: Research Funding; Legend: Consultancy; Takeda: Consultancy, Research Funding; Servier: Consultancy; Teva: Research Funding; Vivolux: Research Funding; Bluebird: Research Funding; Acetylon: Research Funding; Amgen: Consultancy, Research Funding; Abbvie: Research Funding; BMS: Consultancy, Research Funding; Bioclinica: Consultancy; CRISPR Therapeutics: Consultancy, Research Funding; Constellation: Research Funding; Cellularity: Research Funding; Celgene: Consultancy, Research Funding; Glenmark: Research Funding; Genentech, Inc.: Research Funding; EMD Sorono: Research Funding; CURIS: Research Funding; Kite Pharma: Consultancy; Kesios: Research Funding; Karyopharm: Consultancy; Janssen: Consultancy, Research Funding; Prothena: Consultancy; Poseida: Research Funding. Grande:bluebird bio: Current Employment, Current equity holder in publicly-traded company. 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. Friedman:bluebird bio: 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.

Background: Patients (pts) with relapsed and refractory multiple myeloma (RRMM) experience unsatisfactory outcomes with established treatment modalities. In the pivotal phase 2 KarMMa study (NCT03361748), idecabtagene vicleucel (ide-cel, bb2121) demonstrated frequent, deep, and durable responses in triple-class exposed pts with RRMM, with an overall response rate (ORR) of 73% and a complete response rate of 33% (Munshi et al. J Clin Oncol. 2020;38[suppl, abstr]:8503). The overall safety profile of ide-cel was also manageable. The median time to onset and duration of cytokine release syndrome (CRS) were 1 d and 5 d, respectively, and the median time to onset and duration of neurotoxicity (NT) were 2 d and 3 d; the frequencies of higher-grade CRS and NT were low. Evaluated here are the T cell phenotypes, soluble factors, and cytokines associated with ide-cel activation, CRS, NT, and tumor responses over time in pts who received ide-cel in the KarMMa study. Methods: After longitudinal sampling of peripheral blood post-ide-cel infusion in the KarMMa study (N=128), plasma was analyzed for levels of proinflammatory cytokines and inflammation-related soluble factors; serum was evaluated for soluble BCMA (sBCMA) as a peripheral surrogate measure of tumor burden, and peripheral blood mononuclear cells were assessed by flow cytometry for memory phenotypes of CAR T cells. Associations between these features over time after ide-cel infusion were evaluated in the context of ORR, ongoing response at 9 mo, and grade ≥2 CRS and NT. Response at 9 mo was selected for analysis because this visit was proximal to the median progression-free survival (PFS) reported in all ide-cel-treated pts in KarMMa. The 9-mo responders were defined as pts with assessments >8 mo postinfusion and no progression before 10 mo postinfusion. Results: The levels of both CD4+ and CD8+ populations of CAR T cells increased to a greater degree postinfusion in responders and were skewed towards a higher fraction of CD8+ cells through peak expansion. Cell expansion in responders was characterized by an increased proportion of TEM in CAR T cells (CCR7−/CD45RA−) for both CD4+ and CD8+ subsets. Congruent with dominant TEM expansion, characteristic TEM-associated proinflammatory cytokines, such as IFN-ɣ and IL-6, were consistently upregulated early after infusion. Peak IFN-ɣ and IL-6 levels occurred a median of 4 d postinfusion, and 90% of pts (5th−95th percentile) had IFN-ɣ and IL-6 peaks 21 d and 15 d postinfusion, respectively, which was in line with the observed early onset of CRS and NT. Pharmacodynamic responses, shown by decreases in sBCMA after infusion, also occurred consistently early after infusion, and the sBCMA nadir occurred in 90% of pts 7 d−85 d postinfusion (5th−95th percentile; median, 31 d). Early sBCMA clearance below the limit of detection of the assay was associated with longer responses, and median PFS was significantly longer in pts with undetectable sBCMA vs detectable sBCMA at 2 mo (12.3 mo [95% CI, 11.6−17.7] vs 2.9 mo [95% CI, 1.9−3.1]; P Conclusions: Postinfusion expansion of ide-cel was characterized by a TEM-dominant phenotype with expansion of both CD4+ and CD8+ CAR T cell populations. The relative magnitudes of the increase in both populations were substantially greater in responders vs nonresponders, with a bias toward a higher proportion of CD8+ CAR T cells in responders. The induction of effector cytokines followed by return to baseline levels occurred predictably and early after infusion and was consistent with both the observed early onset and resolution of CRS and early sBCMA clearance in pts who received ide-cel in the KarMMa study. Disclosures Martin: BMS: Current Employment, Current equity holder in publicly-traded company. Thompson:Bristol-Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Brown:Bristol-Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Finney:bluebird bio: Current Employment, Current equity holder in publicly-traded company; Seattle Childrens Research Institute: Ended employment in the past 24 months. Rytlewski:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company; Adaptive Biotechnologies: Current equity holder in publicly-traded company. Jiang:Juno Therapeutics, a Bristol Myers Squibb company: Current Employment; Bristol Myers Squibb company: 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. Wang:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. 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. Hause:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Campbell:Bristol-Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hege: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; Celgene (acquired by Bristol Myers Squibb): Ended employment 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; Arcus Biosciences (Former Board of Directors): Divested equity in a private or publicly-traded company in the past 24 months. Kaiser:BMS: 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.

ViralZone (http://viralzone.expasy.org) is a knowledge repository that allows users to learn about viruses including their virion structure, replication cycle and host–virus interactions. The information is divided into viral fact sheets that describe virion shape, molecular biology and epidemiology for each viral genus, with links to the corresponding annotated proteomes of UniProtKB. Each viral genus page contains detailed illustrations, text and PubMed references. This new update provides a linked view of viral molecular biology through 133 new viral ontology pages that describe common steps of viral replication cycles shared by several viral genera. This viral cell-cycle ontology is also represented in UniProtKB in the form of annotated keywords. In this way, users can navigate from the description of a replication-cycle event, to the viral genus concerned, and the associated UniProtKB protein records.

Large collections of immortalized cancer cell lines have been widely used as model systems for cancer research and drug discovery. Some of these models however display more fibroblast-like than cancer-like characteristics based on their genetic and genomic characterization. The correct annotation of these cell lines remains a challenge. Here, we report the outcome of our analysis on a large cancer cell line collection, where we found a subset of cell lines misclassified.

Cell autonomous cancer dependencies are now routinely identified using CRISPR loss-of-function viability screens. However, a bias exists that makes it difficult to assess the true essentiality of genes located in amplicons, since the entire amplified region can exhibit lethal scores. These false-positive hits can either be discarded from further analysis, which in cancer models can represent a significant number of hits, or methods can be developed to rescue the true-positives within amplified regions. We propose two methods to rescue true positive hits in amplified regions by correcting for this copy number artefact. The Local Drop Out (LDO) method uses the relative lethality scores within genomic regions to assess true essentiality and does not require additional orthogonal data (e.g. copy number value). LDO is meant to be used in screens covering a dense region of the genome (e.g. a whole chromosome or the whole genome). The General Additive Model (GAM) method models the screening data as a function of the known copy number values and removes the systematic effect from the measured lethality. GAM does not require the same density as LDO, but does require prior knowledge of the copy number values. Both methods have been developed with single sample experiments in mind so that the correction can be applied even in smaller screens. Here we demonstrate the efficacy of both methods at removing the copy number effect and rescuing hits from some of the amplified regions. We estimate a 70–80% decrease of false positive hits with either method in regions of high copy number compared to no correction., Author summary Cancer vulnerabilities have been identified by systematically disrupting individual genes in cancer cells and observing the resulting effect on cell proliferation. In recent years, a new gene editing technique called CRISPR has made it easier and cheaper to disrupt genes by precisely and completely suppressing the function of individual genes by cutting through its DNA. However, an artefact of the approach yields false positives: using CRISPR to target genes in regions of the genome which are abnormally repeated, called copy number alterations (CNA), has been shown to kill the investigated cells irrespective of the true essentiality of the amplified genes. This artefact is a particular issue when studying tumours, since CNAs are common in cancer. Additionally cancer-specific genes are known to selectively drive amplification, making the ability to assess the essentiality of genes in these regions even more important. Here we describe and provide the code to computationally correct for this artefact and recover the true essentiality of CNA genes.

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.

Gene expression data from microarrays are being applied to predict preclinical and clinical endpoints, but the reliability of these predictions has not been established. In the MAQC-II project, 36 independent teams analyzed six microarray data sets to generate predictive models for classifying a sample with respect to one of 13 endpoints indicative of lung or liver toxicity in rodents, or of breast cancer, multiple myeloma or neuroblastoma in humans. In total, >30,000 models were built using many combinations of analytical methods. The teams generated predictive models without knowing the biological meaning of some of the endpoints and, to mimic clinical reality, tested the models on data that had not been used for training. We found that model performance depended largely on the endpoint and team proficiency and that different approaches generated models of similar performance. The conclusions and recommendations from MAQC-II should be useful for regulatory agencies, study committees and independent investigators that evaluate methods for global gene expression analysis.

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

In order to identify biological processes relevant for cell death and survival in the brain following stroke, the postischemic brain transcriptome was studied by a large-scale cDNA array analysis of three peri-infarct brain regions at eight time points during the first 24 h of reperfusion following middle cerebral artery occlusion in the rat. K-means cluster analysis revealed two distinct biphasic gene expression patterns that contained 44 genes (including 18 immediate early genes), involved in cell signaling and plasticity (i.e. MAP2K7, Sprouty2, Irs-2, Homer1, GPRC5B, Grasp). The first gene induction phase occurred at 0-3 h of reperfusion, and the second at 9-15 h, and was validated by in situ hybridization. Four gene clusters displayed a progressive increase in expression over time and included 50 genes linked to cell motility, lipid synthesis and trafficking (i.e. ApoD, NPC1, G3P-dehydrogenase1, and Choline kinase) or cell death-regulating genes such as mitochondrial CLIC. We conclude that a biphasic transcriptional up-regulation of the brain-derived neurotrophic factor (BDNF)-G-protein coupled receptor (GPCR)-mitogen-activated protein (MAP) kinase signaling pathways occurs in surviving tissue, concomitant with a progressive and persistent activation of cell proliferation signifying tissue regeneration, which provide the means for cell survival and postischemic brain plasticity.