bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5705: Analytical validation of Guardant360 v2.10
Authors: Vowles James , Odegaard Justin , Mortimer Stefanie , Fairclough Stephen , Sikora Marcin , Abdueva Diana , Mokhtari Reza , Baca Arthur , Talasaz AmirAli
Published: July 2017

Guardant360 is a cell-free circulating tumor DNA (ctDNA) test that genotypes all guideline-recommended solid tumor somatic genomic treatment targets from a single non-invasive blood draw. The new version, v2.10, was redesigned to enhance sensitivity and specificity across 73 cancer-related genes. It detects all four major variant classes (single nucleotide variants, SNVs, in all 73 genes; indels in 23 genes; gene amplifications, CNAs, in 18 genes; and fusions in 6 genes).

Analytical performance was assessed throughout the reportable range via multiple serial dilution studies of orthogonally-characterized contrived and patient samples. Analytical specificity was assessed by calculating the false positive rate in pre-characterized healthy donor sample mixtures serially diluted. Positive predictive value (PPV) was estimated as a function of allelic fraction/copy number from pre-characterized samples and prevalence-adjusted using a cohort of 2,585 consecutive clinical samples. Confirmation was performed using ddPCR.

Analytical specificity was 100% for SNVs, fusions, and CNAs and 96% for indels across 25 defined samples. Relative to Guardant360v2.9, v2.10 demonstrated 20-50% increase in fusion molecule recovery. Retrospective in silico analysis of 2,585 consecutive clinical samples demonstrated a 15% increase in actionable fusion detection, a 6%-15% increase in actionable indel detection (excluding newly reportable indels), and a 3%-6% increase in actionable SNV detection.

AlterationsReportable Range95% Limit of DetectionAllelic Fraction / Copy NumberAnalytical SensitivityAllelic Fraction / Copy numberPPVSNVs≥0.04%0.25%≥0.25%>99.9%≥0.25%98.7%0.05–0.25%63.8%<0.25%92.3%Indels≥0.02%0.2%≥0.25%>99.9%≥0.25%98.4%0.05–0.25%67.8%<0.25%88.5%Fusions≥0.04%0.4%≥0.3%100%any100%<0.3%83.0%CNAs≥2.12 copies2.24-2.93 copies2.3 copies95.0%any100%

Guardant360 analytical performance characteristics based on standard cfDNA input (30ng). Analytical sensitivity/limit of detection estimates are provided for clinically actionable variants and may vary by sequence context and cfDNA input. PPV is estimated across entire reportable panel space (PPV was 100% for clinically actionable variants).

Conclusion: Guardant360 v2.10 comprehensively detects all adult solid tumor guideline-recommended somatic genomic variants with unparalleled sensitivity, accuracy, and specificity.

Citation Format: James Vowles, Justin Odegaard, Stefanie Mortimer, Stephen Fairclough, Marcin Sikora, Diana Abdueva, Reza Mokhtari, Arthur Baca, AmirAli Talasaz. Analytical validation of Guardant360 v2.10 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5705. doi:10.1158/1538-7445.AM2017-5705
Abstract 5782: A novel method for high-throughput drug screening in 3D tumor organoids
Authors: Phan Nhan , Huang Jessica , Eisenberg David , Memarzadeh Sanaz , Soragni Alice
Published: July 2017

3D tumor organoids are pivotal tools to study tumor development, propagation and invasive properties (Tanner and Gottesman, 2015). They closely recapitulate many characteristics of the tumor of origin in terms of structure, heterogeneity, complexity and response to drug treatment. As such, they are considered an accurate pre-clinical model for drug discovery and development (Nyga et al, 2011; Tanner and Gottesman, 2015; Shroyer, 2016). Nevertheless, reliable methods to perform high-throughput drug screenings using tumor organoids are limited and require extensive manipulation such as sample transfer from plate to plate. In addition, thick 3D matrix supports may hinder drug penetration.

Here we introduce a robust method to generate, treat and assay 3D tumor organoids in standard, uncoated 96- or 384-well plates. By plating cells around the rim of the wells we generate an ultrathin layer of Matrigel that allows penetration of small molecules and bulkier peptide drugs. Media change is easily performed from the center of the well without perturbing the organoids. After a 2-day treatment course, organoids are released by dispase treatment and assayed in the same well by performing a CellTiter-Glo assay (Promega). The method exhibits low plate-to-plate, experiment-to-experiment and operator-to-operator variability.

In addition, it can be extended to patient-derived tumor organoids (PDTO) generated from primary tumors. We show how PDTO from high-grade serous ovarian carcinoma (HGSOC) specimens are generated with a success rate >95%. Given the low number of cells required and high assay sensitivity, we can seed 200 wells from 1Mio cells derived from a tumor biopsy, and test over 60 different drugs/concentrations in triplicates. Moreover, we show how drug combination studies are feasible and allow assessing treatment synergies. As an example, we combined a peptidic inhibitor of p53 aggregation, ReACp53 (Soragni et al, 2016), with carboplatin in PDTO HGSOC established from a panel of patients harboring different p53 mutations. Drug interaction studies are performed across a range of ReACp53 and carboplatin concentrations and cell viability assessed at day 2 is used to compute drug-response curves, EC50 values and combination indexes.

In conclusion, we are introducing an inexpensive, robust, easily automated method to perform high-throughput drug screenings in 3D organoids and PDTO and demonstrate its utility in determining drug sensitivities and optimizing multi-drug combinations in vitro.

Citation Format: Nhan Phan, Jessica Huang, David Eisenberg, Sanaz Memarzadeh, Alice Soragni. A novel method for high-throughput drug screening in 3D tumor organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5782. doi:10.1158/1538-7445.AM2017-5782
Abstract 5891: DCIS to invasive progression in breast cancer is delayed by restoring CCN5
Authors: Sarkar Sandipto , Ghosh Arnab , Maity Gargi , Banerjee Snigdha , Banerjee Sushanta
Published: July 2017

Malignant progression of breast cancer from pre-invasive to invasive lesions remains a mechanistically unknown event and a major challenge in medical research. By revealing the mechanism of action, our new and substantially different approach aims to demonstrate that CCN5/WISP2 might play a role in negative regulation of progression of pre-invasive lesion ductal carcinoma in-situ (DCIS) to invasive carcinoma (IC). DCIS to IC transition results primarily from the loss of the myoepithelial cell (MEC) layer surrounding the breast ducts & lobules and basement membrane (BM) degradation followed by invasion of cancer cells into the surrounding stromal tissue and vasculature. It has been recently discovered that CCN5, a matricellular protein, is highly expressed in DCIS patient specimens and facilitates regression of aggressive phenotypes. Our in-vitro studies with myoepithelial cell lines (MECs) indicate that CCN5 may prevent the DCIS to IC transition through the protection of the MEC layer. CCN5 performs it’s protective role by regulating sonic hedgehog (SHh) expression in MECs. It has been previously shown in separate studies that Neuropilin1 (Nrp1) positively regulates expression of SHh and Nrp1 is exclusively expressed in MEC layer in breast tissues. An extension of our studies indicate that CCN5 might regulate the integrity of the mammary ductal architecture by protecting the MEC layer through a novel Nrp1-SHh signaling axis. Collectively, our studies indicate that regulating CCN5 expression level in breast cancer tissues might help us controlling the rate of progression of the disease from DCIS to an invasive stage.

Citation Format: Sandipto Sarkar, Arnab Ghosh, Gargi Maity, Snigdha Banerjee, Sushanta Banerjee. DCIS to invasive progression in breast cancer is delayed by restoring CCN5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5891. doi:10.1158/1538-7445.AM2017-5891
Abstract 5924: Necrotic cells promote microglia infiltration in glioblastoma through regulating MCP-1 and MIP-3α expression
Authors: Jung Yieun , Ahn So-Hee , Park Hyunju , Lim Jiwoo , Kang Jihee Lee , Choi Youn-Hee , Kim Eun Ju
Published: July 2017

Glioblastoma multiform (GBM), a grade IV astrocytoma, is most lethal and common adult primary intracranial tumor. GBM is characterized by diffuse infiltration into normal brain parenchyma, rapid growth and the presence of necrosis and microglia/macrophage infiltration. Among these properties of GBM, necrosis has been implicated to be a strong predictor of poor prognosis; however, the effect of necrosis on GBM progression is poorly understood at present. In this study, we examined the effect of necrosis on glioblastoma cells by exploring molecular mechanisms underlying gene expression and chemokine expression. Data obtaining from chemokine array and ELISA showed that CRT-MG human gliomablastoma cells secreted several chemokines including MCP-1 and MIP-3α in response to necrotic cells. Expression levels of mRNA and protein of MCP-1 and MIP-3α were also increased by treatment with necrotic cells in CRT-MG in a dose-dependent manner. Necrotic cells induced NF-κB/AP-1 activation and their binding to the MCP-1 and MIP-3α promoter, leading to enhanced MCP-1 and MIP-3α production in GBM cells. Finally, transwell migration assay showed that incubation with necrotic cells significantly enhanced the migration of microglia. Our data demonstrate that when GBM cells are exposed to and stimulated by necrotic cells, the expression and secretion of MCP-1 and MIP-3α are enhanced in GBM cells and microglia infiltration/migration to the tumor site are facilitated.

Citation Format: Yieun Jung, So-Hee Ahn, Hyunju Park, Jiwoo Lim, Jihee Lee Kang, Youn-Hee Choi, Eun Ju Kim. Necrotic cells promote microglia infiltration in glioblastoma through regulating MCP-1 and MIP-3α expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5924. doi:10.1158/1538-7445.AM2017-5924
Abstract 634: RNAi-mediated β-catenin inhibition promotes T-cell infiltration and anti-tumor activity in combination with immune checkpoint blockade
Authors: Ganesh Shanthi , Shui Serena , Wang Weimin , Brown Bob D. , Abrams Marc
Published: July 2017

Recent research strongly suggests an important role for Wnt/β-catenin signaling in mediating cancer immune evasion and resistance to immune checkpoint therapy, including anti-PD-1, anti-PD-L1, and anti CTLA-4 antibodies. The mechanism by which Wnt/β-catenin signaling causes checkpoint resistance is believed to involve blocking of specific cytokines which trigger immune cell recruitment to the tumor, resulting in the phenomenon of T-cell exclusion and rendering the tumor to a non-inflamed state. Inhibition of β-catenin may be an effective strategy for increasing the low response rate to these effective medicines in numerous cancer populations. DCR-BCAT is a potent and specific chemically-optimized RNA interference (RNAi) trigger targeting CTNNB1, the gene which encodes β-catenin, formulated in a tumor-selective EnCore lipid nanoparticle. In syngeneic mouse tumor models, β-catenin inhibition promoted T-cell infiltration and significantly improved the sensitivity of the tumors to checkpoint inhibition. The combination of DCR-BCAT and IO therapy yielded significant tumor growth inhibition compared to monotherapy in B16F10 melanoma, 4T1 mammary carcinoma, Neuro2A neuroblastoma and Renca renal adenocarcinoma. Importantly, a significant increase in CD8+ T-cells after DCR-BCAT monotherapy was observed in all models evaluated, including genetically-driven spontaneous MMTV-Wnt1 tumors. These data support clinical development of these combination approaches for this first-in-class RNAi therapeutic.

Citation Format: Shanthi Ganesh, Serena Shui, Weimin Wang, Bob D. Brown, Marc Abrams. RNAi-mediated β-catenin inhibition promotes T-cell infiltration and anti-tumor activity in combination with immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 634. doi:10.1158/1538-7445.AM2017-634
Abstract 639: Morphological changes in mitochondria induced by CD137 (4-1BB) co-stimulation on CD8 T cells
Authors: Labiano Sara , Teijeira Álvaro , Azpilikueta Arantza , Aznar Ángela , Bolaños Elixabet , Melero Ignacio
Published: July 2017

Metabolism has emerged as a fundamental biological process in the control of immunological functions of T cells, playing essential roles in survival and differentiation of these immune cells. Mitochondria are key organelles in controlling these processes by performing fundamental steps in aerobic respiration, fatty acid metabolism and in the control of apoptosis. Recently, Carl June and colleagues showed that CARs engineered with the cytoplasmic tail of 4-1BB were able to promote oxidative phosphorylation in T cells as a result of enhanced mitochondrial biogenesis. CD137 (4-1BB) is an immunostimulatory receptor of the TNFR family expressed on the surface of antigen-activated T cells. Treatment with agonist monoclonal antibodies targeting 4-1BB is showing promising results for immunotherapy of cancer.

In this work, we observed that stimulation of 4-1BB with agonistic mAbs was able to raise the mitochondrial membrane potential on human CD8 T cells. 4-1BB co-stimulated CD8 T cells showed enlarged mitochondria (up to 3μm diameter) with a round shape when observed by confocal or transmission electron microscopy. We observed that CD8 T cells from DLNs (drainig lymph nodes) of tumors in mice treated with agonistic anti-4-1BB antibodies also contained enlarged mitochondria.

By double fluorescence labeling of mitochondria prior and after activation, we could ascertain that enlarged mitochondria were a product of preexisting mitochondria rather than de novo generated organelles. This observation prompted us to study the regulation of mitochondrial dynamics by 4-1BB receptor. 4-1BB stimulation induced an increase in expression of the mitochondrial fusion protein OPA-1. By Super-resolution microscopy with AiryScan detectors we observed more pronounced co-localization of OPA-1 with mitochondrial membrane in 4-1BB co-stimulated T cells than in T cells activated only by CD3 crosslinking.

Our results indicate an important role of 4-1BB in the regulation of mitochondrial morphology and performance in such a way that can be relevant for various T cell functions.

Citation Format: Sara Labiano, Álvaro Teijeira, Arantza Azpilikueta, Ángela Aznar, Elixabet Bolaños, Ignacio Melero. Morphological changes in mitochondria induced by CD137 (4-1BB) co-stimulation on CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 639. doi:10.1158/1538-7445.AM2017-639
Abstract 654: The epigenetic landscape of T cell exhaustion
Authors: Sen Debattama
Published: July 2017

Exhausted T cells in cancer and chronic viral infection have distinctive patterns of gene expression, including sustained expression of the inhibitory receptor PD-1, but the regulation of gene expression in exhausted T cells is poorly understood. Here we define the accessible chromatin landscape in mouse and human exhausted CD8+ T cells and show that it is profoundly different from functional memory CD8+ T cells. Exhausted CD8+ T cells in a mouse model of chronic viral infection acquire an extensive, state-specific pattern of enhancers, which are organized into functional modules. One enhancer, -23.8kb from the Pdcd1 locus, is found only in exhausted T cells and other lymphocytes with sustained PD-1 expression. Genome editing shows it to be required for high PD-1 expression. Cas9-mediated in situ saturation mutagenesis of the enhancer pinpoints critical minimal sequences that correspond to bound transcription factor motifs for RAR, T-bet and Sox3 in exhausted CD8+ T cells. State-specific enhancer profiles identified in mouse exhausted CD8+ T cells are conserved in human exhausted antigen-specific CD8+ T cells responding to HIV and HCV infection. Detailed functional enhancer maps of T cell exhaustion reveal state-specific regulatory sequences and offer targets for genome editing that could alter gene expression preferentially in exhausted CD8+ T cells.

Citation Format: Debattama Sen. The epigenetic landscape of T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 654. doi:10.1158/1538-7445.AM2017-654
Abstract 661: Evaluating "harmonization" of PD-L1 assays using image analysis
Authors: Martin Nathan T. , Black Joshua C. , Pollack Zachary , Aeffner Famke , Krueger Joseph
Published: July 2017

The current diagnostics landscape for therapeutics that target the PD1/PD-L1 pathway is highly complex. Four different companion or complementary diagnostics have been developed for pembrolizumab in NSCLC, nivolumab in NSCLC and melanoma, and atezolizumab in urothelial carcinoma. The need to reconcile diagnostics for this class of targeted therapies has been recognized by the creation of the FDA-AACR-ASCO “PD-L1 Blueprint” working group to explore means to “harmonize” PD-L1 testing in tissue based IHC assays. The results reported from this working group noted similarities, but also several important discrepancies, between the current assays. Since each test uses a specific interpretation for each assay and indication, this creates a highly complex diagnostic landscape, which is likely to continue to increase in complexity as more PD-1/PD-L1 therapeutics and potentially novel diagnostics continue to be approved in additional indications.

To address the need for adaptive, sustainable harmonization for PD-L1 diagnostics, Flagship Biosciences evaluated the utility of image analysis-based methods to harmonize multiple PD-L1 tests. We executed a proof-of-concept study utilizing a cohort of serial tissue sections from the same NSCLC patients, stained with the FDA approved Dako PD-L1 tests (28-8 and 22C3 clones), and our in-house PD-L1 assays (SP142 and E1L3N clones) for comparison. We digitized the tissue slides using a whole slide scanner, and evaluated the samples with our tissue Image Analysis (tIA™) technology. As expected, the patient samples stained with the separate PD-L1 assays yielded differences in staining and, thus, the reported scores for PD-L1 expression based on each test used, despite serial sections being derived from the same patient. To attempt to harmonize the scoring approaches for each test, we leveraged our computational Tissue Analysis (cTA™) platform to create a mathematically-derived “virtual slide score” for each sample, which enabled calibration of the various tests to deliver cross PD-L1 comparative scores. Based on the proof-of-concept demonstrated in this study, the cTA™ approaches could enable harmonization of the various PD-L1 tests through use of a digital pathology platform. The data presented provides a foundation for potential application of the cTA™ platform in the clinical laboratory setting to achieve harmonization of multiple PD-L1 tests.

Citation Format: Nathan T. Martin, Joshua C. Black, Zachary Pollack, Famke Aeffner, Joseph Krueger. Evaluating "harmonization" of PD-L1 assays using image analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 661. doi:10.1158/1538-7445.AM2017-661
Abstract 663: Clinical performance and utilization of 3 PD-L1 immunohistochemical assays
Authors: Anderson Steven M. , Horten Bruce , McCune Bryan , Paler Ronald
Published: July 2017

Three different PD-L1 immunohistochemical (IHC) assays have recently been approved to help guide treatment decisions regarding anti-PD-1 (Keytruda®, Opdivo®) and anti-PD-L1 (Tecentriq®) immuno-oncology based therapies. The three assays differ in the primary monoclonal antibodies used, the assay platform (Dako, Ventana), scoring and interpretation criteria, and intended use. Our clinical laboratories currently offer all 3 assays (pharmDx™ 22C3, pharmDx™ 28-8, and SP142) because they are associated with different intended uses, companion and complementary diagnostics for non-small-cell lung cancer (NSCLC), and complementary diagnostic for urothelial carcinomas (UC). In the NSCLC samples evaluated in the first year since the approval of the two pharmDx™ assays, we have noted a higher percentage of positive samples with the pharmDx™ 28-8 assay (50%) compared to the pharmDx™ 22C3 assay (30%). This difference is mainly due to the use of different assay cutoffs associated with the two assays, as there is good correlation between the overall staining patterns, including the distribution of the percentage of tumor cells exhibiting positive staining. For the SP142 assay tested on UC samples, 22% of the cases were considered positive. All 3 assays are robust, with a limited number of samples considered inconclusive (<10%), mainly due to an inadequate number of tumor cells. The companion diagnostic PD-L1 assay (pharmDx™ 22C3) has had the greatest utilization of the 3 assays, as it is required for the use of the Keytruda® in NSCLC samples. The performance features of the 3 different PD-L1 assays in the clinical laboratory setting has shown that all assays are robust and can be readily scored and interpreted by the pathology team. The utilization of the 3 assays correlates with respective intended use, tumor type and companion versus complementary diagnostic application, for each assay.

Citation Format: Steven M. Anderson, Bruce Horten, Bryan McCune, Ronald Paler. Clinical performance and utilization of 3 PD-L1 immunohistochemical assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 663. doi:10.1158/1538-7445.AM2017-663
Abstract 694: TK-216: a novel, first-in-class, small molecule inhibitor of EWS-FLI1 in early clinical development, for the treatment of Ewing Sarcoma
Authors: Selvanathan Saravana P. , Moseley Eric , Graham Garrett T. , Jessen Katti , Lannutti Brian , Üren Aykut , Toretsky Jeffrey A.
Published: July 2017

One of the most significant challenges in creating more potent, less toxic treatments for patients is to identify new cancer therapeutic targets that distinguish the malignant from normal cells. EWS-FLI1 is a well-established Ewing sarcoma (ES) oncogene that has the potential to be an ideal therapeutic target by directly impacting malignant cells. We have previously reported the discovery and characterization of YK-4-279, an enantiomer-specific inhibitor of EWS-FLI1, which has been demonstrated to induce apoptosis, inhibit EWS-FLI1 transcription, block RNA helicase A co-immunoprecipitation with EWS-FLI1, and result in alternative splicing to mimic EWS-FLI1 knockdown. Continuous efforts in structure-guided medicinal chemistry has yielded TK-216, an analog of YK-4-279 inhibitor of EWS-FLI1, which is 3-4 fold more potent with excellent drug-like properties. TK-216 potently inhibits the proliferation of ES cells. Induces apoptosis in a dose -dependent manner as measured by caspase-3 activity in multiple ES cell lines with distinct translocation variants. The effects of TK-216 on alternative splicing (AS) were further validated using genes including ARID1A, CLK1, CASP3, PPFIBP1 and RUNX2. The splicing pattern was similar between TK-216 and YK-4-279. In addition to the in vitro activity of TK-216 , we show that TK-216 displays anti-tumor activity in a number of ES xenograft models. In summary, TK-216, a novel, first-in-class therapeutic which directly inhibits EWS-FLI1, offers a promising approach for the treatment of Ewing Sarcoma and is currently in Phase 1 clinical trials in patients with relapsed or refractory Ewing Sarcoma (clinicaltrials.gov - NCT02657005).

Citation Format: Saravana P. Selvanathan, Eric Moseley, Garrett T. Graham, Katti Jessen, Brian Lannutti, Aykut Üren, Jeffrey A. Toretsky. TK-216: a novel, first-in-class, small molecule inhibitor of EWS-FLI1 in early clinical development, for the treatment of Ewing Sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 694. doi:10.1158/1538-7445.AM2017-694
Abstract 982: Development of homogeneous dual-drug ADCs: Application to the co-delivery of auristatin payloads with complementary antitumor activities
Authors: Levengood Matthew R. , Zhang Xinqun , Emmerton Kim K. , Hunter Joshua H. , Senter Peter D.
Published: July 2017

A common theme in treating cancer is the use of combination chemotherapy, where multiple drugs with different mechanisms of action are combined to elicit synergistic activity or overcome differential drug sensitivities. Antibody-drug conjugates (ADCs) have emerged as a powerful approach for treating cancer, combining the tumor targeting specificity of monoclonal antibodies with the potent cell-killing activity of cytotoxic drugs. Like other therapies, these agents are increasingly being tested in combination with unconjugated, clinically approved anticancer agents. In addition, emerging data demonstrates that insensitivity to a particular ADC can be overcome through delivery of a different payload using the same antibody. For these reasons, the development of ADCs that can deliver two complementary payloads to a tumor would likely be a significant advancement in ADC technology. To enable dual-drug conjugation, we utilized a multiplexing drug carrier that contains cysteine residues with orthogonal protecting groups and identified novel conditions for utilization of these protecting groups on a folded protein. Sequential cysteine unmasking enables discrimination between conjugation sites to allow for site-specific drug conjugation. This strategy provides homogeneous ADCs bearing 16 total drugs per antibody, split evenly between the two drug linkers. Importantly, this strategy is flexible, as it does not require engineered antibodies or custom enzymes for drug-linker conjugation. To demonstrate the potential benefits of ADC dual drug delivery, this strategy was applied to the construction of ADCs bearing two classes of auristatin drug linkers that have different physiochemical properties and complementary anti-cancer activities. Dual-auristatin ADCs were tested in cell line and xenograft models that have differential sensitivities to the individual auristatin components, including those with heterogeneous antigen expression or high levels of drug efflux transporters. The data from these studies demonstrate that the dual-auristatin ADCs were active on cells and tumors that are refractory to treatment with either of the individual component drugs. This work highlights the potential for delivering two synergistic or complementary payloads on a single ADC and presents a flexible method for constructing dual-drug ADCs with site-specific and homogeneous drug loading.

Citation Format: Matthew R. Levengood, Xinqun Zhang, Kim K. Emmerton, Joshua H. Hunter, Peter D. Senter. Development of homogeneous dual-drug ADCs: Application to the co-delivery of auristatin payloads with complementary antitumor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 982. doi:10.1158/1538-7445.AM2017-982
Abstract CT041: The clinical study on CD19-directed chimeric antigen receptor-modified T cells in patient with Richter Syndrome
Authors: Xia Leiming , Chen Qian , Li Qiao , Li Tan , Wang Yi , Bao Yangyi
Published: July 2017

Richter syndrome (RS) indicates the transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma (mostly DLBCL). RS is a rare complication with unfavorable clinical outcome. There is no effective treatment for it currently. As a new type of cell-based immune therapy, chimeric antigen receptor modified T (CAR-T) cell treatment is now widely used in treating hematological malignancies, especially in CD19+ B cell malignancy. Therefore, CD19-directed chimeric antigen receptor modified T cells (CART-19) treatment is worthwhile to be developed as a new method for Richter Syndrome’s patients. In this study, the patient with Richter Syndrome expressing high level of CD19 was enrolled in a clinical trial. The patient had received a series of standard treatments with no therapeutic effect before 4.55 x 108 autologous CART-19 cells were infused, of which approximately 50% were transduced with the CAR. After CART-19 infusion, the main side effects were repeated fever, pancytopenia and significant rises of serum cytokines, including IFN-γ, IL-6 and IL-10. However, before discharge, the level of cytokines reduced to normal level. In addition, we detected K+, Ca2+, creatinine and glutamic-pyruvic transaminase, and found all of these were normal, which suggested that there was no significant tumor necrosis after treatment. The treatment included two courses. During the first course, the patient was infused with 4 x 107 CART-19 cells twice, followed by 1 x 108 cells in the 3rd infusion and then 1x106 cells in the 4th infusion. After these infusions, the proportion of B cells in the patient’s peripheral blood decreased from 72% to 40%, accompanied by reduced size of cervical, mediastinal and axillary lymph nodes. Eighteen days later, the patient was infused in the second course with 3.7x107 CART-19 cells twice, followed by 1x108 cells twice. After the second course, the proportion of B cells maintained at approximate 85%, and the lymph nodes enlargement was observed as disease progression. Based on the recent therapeutic landscape for hematological malignancies including B cell lymphomas, CD19-CAR T cell therapy may offer a new therapeutic option for RS, which aims to reduce the patients’ tumor burden, prolong their survival time, and provide opportunities for combined therapy with chemotherapy and bone marrow transplantation.

Note: This abstract was not presented at the meeting.

Citation Format: Leiming Xia, Qian Chen, Qiao Li, Tan Li, Yi Wang, Yangyi Bao. The clinical study on CD19-directed chimeric antigen receptor-modified T cells in patient with Richter Syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT041. doi:10.1158/1538-7445.AM2017-CT041
Abstract DDT01-02: AZD5991: A potent and selective macrocyclic inhibitor of Mcl-1 for treatment of hematologic cancers
Authors: Hird Alexander W. , Secrist J. Paul , Adam Ammar , Belmonte Matthew A. , Gangl Eric , Gibbons Frank , Hargreaves David , Johannes Jeffrey W. , Kazmirski Stephen L. , Kettle Jason G. , Kurtz Stephen E. , Lamb Michelle L. , Packer Martin J. , Peng Bo , Stewart Craig R. , Tyner Jeffrey W. , Yang Wenzhan , Ye Qing , Zheng XiaoLan , Clark Edwin A.
Published: July 2017

Mcl-1, a member of the Bcl/Mcl family, is a key protein involved in evasion of apoptosis in a wide variety of tumors. Its amplification and overexpression have also been implicated in innate and acquired resistance to anticancer drugs. Mcl-1 is capable of preventing induction of apoptosis, both by binding and inactivating the pro-apoptotic executioner Bcl-2 protein, Bak, as well as by sequestering other pro-apoptotic BH3-only proteins such as Bim and Noxa. AZD5991 is a rationally designed macrocycle with sub-nanomolar affinity for Mcl-1. It demonstrates all the hallmarks of a true Mcl-1 inhibitor: 1. potent, selective, and rapid apoptosis in Mcl-1-dependent cell lines (e.g., GI50 as low as 10 nM in multiple myeloma cell lines); 2. loss of activity upon overexpression of Bcl-xL or siRNA-mediated knockout of Bak; 3. Mcl-1:Bak complex disruption as demonstrated by co-immunoprecipitation. AZD5991 is active in vivo, with complete (100%) tumor regression demonstrated in several mouse xenograft models after a single tolerated dose. We have also demonstrated synergistic in vivo efficacy in combination with standard-of-care agents. Analysis of ex vivo activity in primary samples from leukemia patients indicates that a high percentage of leukemia patients should respond to drug treatment, which supports our plan for a phase I trial of AZD5991 in patients with hematologic cancers.

Citation Format: Alexander W. Hird, J. Paul Secrist, Ammar Adam, Matthew A. Belmonte, Eric Gangl, Frank Gibbons, David Hargreaves, Jeffrey W. Johannes, Stephen L. Kazmirski, Jason G. Kettle, Stephen E. Kurtz, Michelle L. Lamb, Martin J. Packer, Bo Peng, Craig R. Stewart, Jeffrey W. Tyner, Wenzhan Yang, Qing Ye, XiaoLan Zheng, Edwin A. Clark. AZD5991: A potent and selective macrocyclic inhibitor of Mcl-1 for treatment of hematologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr DDT01-02. doi:10.1158/1538-7445.AM2017-DDT01-02
Abstract LB-033: The transcriptional landscape of p53 signaling pathway
Authors: Matsuda Koichi , Tanikawa Chizu
Published: July 2017

Although recent cancer genomics studies have identified a large number of genes that were mutated in human cancers, p53 remains as the most frequently mutated gene. To further elucidate the p53-signalling network, we performed transcriptome analysis on 24 tissues in p53+/+ or p53-/- mice after whole-body X-ray irradiation. Here we found transactivation of a total of 3,551 genes in one or more of the 24 tissues only in p53+/+ mice, while 2,576 genes were downregulated. p53 mRNA expression level in each tissue was significantly associated with the number of genes upregulated by irradiation. Annotation using TCGA (The Cancer Genome Atlas) database revealed that p53 negatively regulated mRNA expression of several cancer therapeutic targets or pathways such as BTK, SYK, and CTLA4 in breast cancer tissues. In addition, stomach exhibited the induction of Krt6, Krt16, and Krt17 as well as loricrin, an epidermal differentiation marker, after the X-ray irradiation only in p53+/+ mice, implying a novel mechanism to protect damaged tissues by rapid induction of differentiation. Our comprehensive transcriptome analysis elucidated tissue specific roles of p53 and its signalling networks in DNA-damage response that will enhance our understanding of cancer biology.

Citation Format: Koichi Matsuda, Chizu Tanikawa. The transcriptional landscape of p53 signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-033. doi:10.1158/1538-7445.AM2017-LB-033
Abstract LB-053: Next-generation sequencing of T-cell receptor-beta gene rearrangements reveals dramatic expansion of T-cell clonotypes after CART-EGFRvIII therapy for glioblastoma
Authors: O'Rourke Donald M. , Chen Fang , Binder Zev A. , Kulikovskaya Irina , Gupta Minnal , Finklestein Jeffrey , Ambrose David E. , Nasrallah MacLean P. , June Carl H. , Lacey Simon F. , Melenhorst J. Joseph
Published: July 2017

We are interested in the T cell repertoire in glioblastoma (GBM) patients who have been administered peripherally infused CART-EGFRvIII adoptive T cell therapy for recurrent EGFRvIII-positive GBM. On neuropathologic evaluation following GBM tumor resection, we noticed that several subjects had a robust lymphocytic infiltrate in their tumors following CART-EGFRvIII infusion, which was out of proportion to the level of CART-EGFRvIII genomic sequences observed. We sought to quantify the T cell infiltrate in five subjects whose tumors were resected at different time points following CART infusion (days 6-104). In these five subjects, we analyzed the T cell clonotypic repertoire in the infusion product and in pre- and post-infusion brain tumor specimens by deep sequencing the T cell receptor (TCR) Vβ chain of tumor-infiltrating T cells. Data analysis was performed using ImmunoSeq and exported data were further examined using Graphpad Prism. The infusion products in these patients were highly polyclonal with no clones represented above 1%. We found that in all five of these subjects, the number and frequency of unique TCRVβ clonotypes increased by several thousand (~20-fold) in post-infusion GBM specimens, and only a small portion of these unique TCRs were shared with pre-existing tumor-infiltrating specific T cells (TILs). A consistent finding in these patients was that the clonotype compositional diversity of T cells in the tumor bed post-infusion was considerably larger than the pre-CART vs. infusion product sharing frequency. These data indicate there was a significant increase in the number and clonotypic diversity of tumor-infiltrating T cells following intravenous CART-EGFRvIII infusion, but only a small proportion appears to be derived from the infusion product. Collectively, these results show that CART infusion can trigger accumulation of TILs. Whether or not these TILs respond to GBM neoantigens remains to be investigated.

Citation Format: Donald M. O'Rourke, Fang Chen, Zev A. Binder, Irina Kulikovskaya, Minnal Gupta, Jeffrey Finklestein, David E. Ambrose, MacLean P. Nasrallah, Carl H. June, Simon F. Lacey, J. Joseph Melenhorst. Next-generation sequencing of T-cell receptor-beta gene rearrangements reveals dramatic expansion of T-cell clonotypes after CART-EGFRvIII therapy for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-053. doi:10.1158/1538-7445.AM2017-LB-053
Abstract LB-178: The impact of the gut microbiome on the antitumor effects of radiotherapy
Authors: Facciabene Andrea , Rafail Stavros , Uribe-Herranz Mireia , Pierini Stefano , Bittinger Kile , Verginadis Iannis , Koumenis Costas , Maity Amit
Published: July 2017

The sheer enormity of the microbial biomass in the human intestinal tract, the co-evolution between humans and the microbiota, and the established function of the gut microbes in regulating normal host physiologic functions, are all consistent with the idea that alterations in gut microbial ecology play a role in the pathophysiology of several conditions including cancer. Radiotherapy (RT) is an established curative and palliative cancer treatment regimen, with approximately half of cancer patients with solid tumors receiving RT some time during their disease. Mounting evidence also suggests that high-dose (hypofractionated) radiation exerts potent immune modulatory effects, prompting immunological active tumor cell death inducing tumor associated antigen (TAA) cross priming with elicitation of anti-tumor CD8+ T cells, and abscopal effects. Although there have been groundbreaking responses to immunotherapy in certain malignancies such as lung cancer and melanomas, so far, immunotherapy is effective only in a portion of patients19. This raises the issue of whether there are additional important regulators of T cell function that are relevant to tumor control and could be harnessed to enhance radiotherapy. In support of this hypothesis, we have generated preliminary results demonstrating that: a) Treatment with vancomycin, an antibiotic with action in the gut mostly against Gram+ bacteria, significantly increased the presence of overall tumor infiltrating T cells or cytotoxic CD8+ cells in tumors from mice treated with RT/vancomycin combination compared to RT alone; b) vancomycin caused a significant enhancement of the tumor inhibitory effect of targeted radiation. c) Vancomycin enhanced the ability of RT to increase the expression of IFN-g in CD8+ infiltrating T cells and antigen presentation in the tumor draining lymph nodes and d) the observed synergy between vancomycin and RT in eliciting an anti-tumor immune response and inhibiting tumor growth was abrogated in IFN-g KO animals or by CD8+ cell depletion.

Citation Format: Andrea Facciabene, Stavros Rafail, Mireia Uribe-Herranz, Stefano Pierini, Kile Bittinger, Iannis Verginadis, Costas Koumenis, Amit Maity. The impact of the gut microbiome on the antitumor effects of radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-178. doi:10.1158/1538-7445.AM2017-LB-178
Association Between High‐Sensitivity Cardiac Troponin I and Cardiac Events in Elderly Women
Authors: Lewis Joshua R. , Lim Wai H. , Wong Germaine , Abbs Samuel , Zhu Kun , Lim Ee M. , Thompson Peter L. , Prince Richard L.
Published: July 2017

Background

Elderly women are at high risk of coronary heart disease (
CHD
) and heart failure. High‐sensitivity assays allow detection of cardiac troponin I (hsTnI) well below diagnostic cutoffs for acute coronary syndrome. We investigated the association between these levels with future cardiac events in community‐based ambulant white women aged over 70 years initially recruited for a 5‐year randomized, controlled trial of calcium supplements.

Methods and Results

This was a prospective study of 1081 elderly women without clinical
CHD
at baseline (1998) or hsTnI above the diagnostic cutoffs for acute coronary syndrome with 14.5‐year follow‐up hospitalization and mortality (events). Two hundred forty‐three (22%) women had
CHD
events, 163 (15%) myocardial infarction or
CHD
death (hard
CHD
), and 109 (10%) heart failure. In 99.6% of available serum samples, hsTnI was above the level of detection (median, 4.5 ng/L; interquartile range, 3.6–5.8). After adjusting for Framingham risk factors, each
SD
natural log‐transformed hsTnI increase was associated with an increased hazard for
CHD
(hazard ratio, 1.34; 95%
CI
, 1.18–1.53;
P
<0.001) hard
CHD
(hazard ratio, 1.51; 95%
CI
, 1.29–1.76;
P
<0.001), and heart failure (hazard ratio, 1.65; 95%
CI
, 1.36–1.99;
P
<0.001). Step‐wise increases in relative hazards were observed with increasing quartiles of hsTnI (
P
for trend, <0.001), whereas the addition of hsTnI to conventional risk factors modestly improved discrimination indices: Harrell's c‐statistic, net reclassification, and integrated discrimination (
P
<0.05).

Conclusions

Cardiac troponin I is independently associated with future cardiac events in elderly women without apparent clinical manifestations. The addition of cardiac troponin I to conventional risk factors may modestly improve risk prediction in this setting.
Using the Artificial Tracer e90 to Examine Present and Future UTLS Tracer Transport in WACCM
Authors: Abalos Marta , Randel William J. , Kinnison Douglas E. , Garcia Rolando R.
Published: July 2017

Large-scale tracer transport in the upper troposphere and lower stratosphere (UTLS) is investigated using simulations of the Whole Atmosphere Community Climate Model (WACCM) over the period 1955–2099. The analyses are based on e90, an artificial passive tracer with constant emissions and atmospheric loss rates. The separate contributions of advection by the residual circulation, eddy mixing, and subgrid convection to total transport are explicitly evaluated. The results highlight distinct large-scale transport regimes in the tropics, characterized by efficient vertical tracer transport, and the extratropics, dominated by isentropic mixing. One novel result is the important role of vertical eddy mixing in the tropical upper troposphere. It is shown that interannual variability in e90 is largely driven by El Niño–Southern Oscillation and the quasi-biennial oscillation. The long-term trends emphasize a strong impact of a rising tropopause with climate change on UTLS dynamics and tracer transport. The analyses directly attribute the e90 trends to changes in the different transport components. Stronger residual circulation in the future leads to increased tracer concentrations in the tropical lower stratosphere. Enhanced eddy mixing increases e90 in the extratropical lowermost stratosphere, linked to an upward shift of wave dissipation tied to the tropopause rise. In the troposphere, reduced concentrations in the future are due to weaker convective transport out of the boundary layer and weaker extratropical isentropic eddy mixing.
Evaluating Outer Tropical Cyclone Size in Reanalysis Datasets Using QuikSCAT Data
Authors: Schenkel Benjamin A. , Lin Ning , Chavas Daniel , Oppenheimer Michael , Brammer Alan
Published: July 2017

The present study examines the fidelity of outer tropical cyclone (TC) size and wind field structure in four atmospheric reanalysis datasets to evaluate whether reanalyses can be used to derive a long-term TC size dataset. Specifically, the precision and accuracy of reanalysis TC size for the North Atlantic (NA) and western North Pacific (WNP) basins are analyzed through comparison with a recently developed QuikSCAT TC size dataset (2000–09). Both outer TC size and structure in reanalyses closely match QuikSCAT data as revealed by strong correlations, similar standard deviations, and generally small biases. Of the TC size metrics examined, the radii of 6–8 m s−1 winds in the NA and radii of 6–10 m s−1 winds in the WNP are generally most comparable to QuikSCAT data. Compared to WNP TCs, NA TC size and structure are represented with greater fidelity. Among the four reanalyses examined, the National Centers for Environmental Prediction Climate Forecast System Reanalysis and the Japan Meteorological Agency Japanese 55-year Reanalysis represent TC size and structure with the greatest fidelity for both basins. Differences between reanalysis and QuikSCAT TC size increase with increasing QuikSCAT TC size in both basins and with decreasing TC latitude in the WNP. Finally, comparison of the distribution of reanalysis TC size during the satellite era with the distribution of QuikSCAT TC size suggests that reanalysis TC size is represented with reasonable fidelity throughout the satellite era and, thus, may be useful for constructing a multidecadal TC size dataset.